Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase 1/2 trial

Domain: biology
Published: 2026-01-02
Editorial outcome: major_revision

Abstract
Autologous CD133+ bone marrow-derived stem cell (BMDSC) therapy has been designated as an Orphan Drug by the EMA and FDA for the treatment of Asherman Syndrome (AS). This phase 1/2, non-randomized, open-label, single-arm trial assessed the safety and efficacy of this novel therapy in 20 infertile women with moderate to severe AS, unresponsive to prior hysteroscopic treatments. Primary endpoints were safety and tolerability over 15 months follow-up, including during pregnancy and after live birth. The therapy was well tolerated with a mean dosage of 125.41×10^6 cells, with no treatment-related serious adverse events and only reversible events such as arm pain, headache, and nausea. In pregnant patients, minor obstetric complications were reflux-related cough (n=1), gestational diabetes (n=2), cervical shortening requiring pessary placement (n=2), and postpartum placenta accreta (n=1). No preterm labor occurred, and all six newborns remained free of significant adverse events. Our findings suggest that autologous CD133+ BMDSC therapy is a safe and effective treatment for AS. Clinical trial registration (EudraCT): 2016-003975-23

Introduction
Asherman Syndrome (AS) is an acquired pathological condition affecting women’s reproductive health, triggered by the substitution of a bona fide endometrium with intrauterine adhesions (IUAs) that cause the uterine walls to adhere to each other. Clinically, AS results in menstrual abnormalities (amenorrhea or oligomenorrhea), pelvic pain, infertility, recurrent miscarriage and abnormal placentation1. The onset of AS is mainly caused by procedures such as postpartum curettage of the uterine cavity, after miscarriage and/or infection. These events can disrupt the endometrial niche where progenitor/stem cells may reside2 causing a significant reduction in endometrial epithelial compartment and a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment3.

Hysteroscopic adhesiolysis is the current standard treatment with limited clinical results, especially in the moderate and severe stages4–6. Advanced cell therapies have emerged as a potential therapy for AS aiming to treat with cells what cannot be treated by drugs7. Specifically, autologous CD133+ bone marrow derived stem cells (BMDSCs) were initially used to treat AS in a murine model of AS8,9. Then, a proof-of-concept trial provided preliminary evidence of the safety and therapeutic benefit of this investigational product in patients with moderate/severe AS refractory to operative hysteroscopy treatments10. As a result, autologous CD133+ BMDSCs were granted Orphan Drug designation for the treatment of AS by both the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA)11.

We present a 15-month follow-up study from a non-randomized, open-label, phase 1/2 trial for 20 patients experiencing clinical symptoms including infertility, due to moderate or severe AS according to the IUAs ASRM classification12, those who are refractory to operative hysteroscopy treatments. Here, we describe the safety, effectiveness in terms of therapeutic outcomes, and biological plausibility at single cell level of autologous CD133+ BMDSC treatment.

Results
Participant identification and baseline characteristics
This is a prospective, single-arm, open-label, non-randomized, phase 1/2 trial, in which we enrolled infertile patients with a hysteroscopic diagnosis of moderate or severe AS according to the IUAs ASRM classification12. This clinical trial involved administration of an advanced therapy medicinal product (ATMP) during the simulated secretory phase (5 days after progesterone administration), where the use of autologous CD133+ BMDSCs was approved by the Spanish Agency of Medicines and Medical Devices (AEMPS, April 20th, 2020) and the Clinical Research Ethics Committee at the Hospital Universitari Vall D’Hebron Barcelona, Spain (April 17th, 2020, IGX1-ENT-XS-16-01).

Following diagnostic hysteroscopy, hematopoietic stem cells were mobilized, harvested through leukapheresis, and selected for CD133+ before intra-endometrial spiral arterioles instillation. Although the minimum accepted range of mobilized cells was at least 30 million cells with a >70% purity and >50% viability, the average of CD133+ instilled cells was 125.41×10^6 cells with a purity of 89.54±8.29%, and a viability of 99.7±0.27%, exceeding minimum thresholds. An overview of the study design is described in Fig. 1A.

Between September 7th, 2020 and September 19th, 2022, 24 patients were screened for eligibility, 20 were enrolled and received autologous CD133+ BMDSC therapy (Fig. 1B). A detailed summary of clinical characteristics of participants pre- and post-AS therapy is presented in Table 1.

The mean age of patients was 38.3 years (IQR 37.5–40). The average number of previously attempted reparative operative hysteroscopies was 3.2 (range 1 to 11). Despite previous surgical treatments, no patient reported significant improvement of their endometrial thickness, volume, hysteroscopic scores, and none had achieved pregnancy after AS was diagnosed (Table 1).

Primary endpoints
The primary endpoints of this study were to evaluate safety and tolerability of this autologous cell therapy with up to 15 months follow-up, during pregnancy and in cases of live birth. The therapy was well tolerated at doses ranging from 37 to 166.1×10^6 cells (mean dosage 125.41×10^6 cells). No serious adverse events related to the medicinal product were observed and no haemopoietic malignancies were reported. The most common adverse events after stem cell administration included arm pain, headache and nausea, which in all cases were mild and reversible. Arm pain was mostly related to catheterization and was solved with local ice administration. A detailed list of adverse events is presented in Table 2.

Among the pregnant patients, no serious adverse events were observed beyond common obstetric complications. One patient experienced a persistent cough attributed to gastroesophageal reflux. Two patients developed gestational diabetes, which was successfully managed through dietary interventions. Cervical shortening requiring pessary placement occurred in two cases. Additionally, one patient presented placenta accreta after delivery, requiring manual placental extraction and blood transfusion. Notably, there were no cases of preterm labor and no significant adverse events were reported in the six newborns during the first month of life.

Secondary endpoints
The secondary endpoints included endometrial morphometry, thickness, volume, uterine hysteroscopic scores, and endometrial function evaluated by menstruation recovery and pregnancy outcomes. Endometrial morphometry was obtained from the biopsies extracted analyzed by digital analysis of H&E-stained endometrial biopsy sections before and one month after therapy (Supplementary Fig. 1A). After cell therapy, we observed a 62% increase in gland percentage, a 64% reduction in fiber-rich stroma, and a 73% increase in vessel percentage within the stroma. Additionally, a statistically significant decrease in gland area and gland lumen was noted (Supplementary Fig. 1A).

Analysis on endometrial thickness showed an improvement in all patients analyzed (Fig. 2A) from the maximal value of 4.90 mm (range 3–4.9 mm) with a mean of 3.80±0.95 mm pre-AS therapy to a maximal thickness post-AS therapy of 6.50 mm (range 4-6.5 mm) with a mean value of 5.29±0.77 mm (p<0.05) (Fig. 2B, Table 1). Endometrial volume as assessed by 3D ultrasound also increased in all patients (Fig. 2A) with a mean of 1.26 cm³ (range 0.27-3.5 cm³) before, versus 2.78 cm³ (range 0.48–4.9 cm³) after cell therapy (p<0.05) (Fig. 2C, Table 1).

Hysteroscopic scores according to the IUAs ASRM classification12 performed before (pre-AS therapy) and approximately one month after cell therapy (post-AS therapy), revealed improvements in the endometrium and the uterine cavity for the patients (Fig. 3A). IUAs score pre-AS therapy was 8.0±3.17, whereas post-AS therapy was reduced to 4.42±2.09 (p<0.0001, Fig. 3B). More specifically, 6 out of 8 severe AS cases improved to moderate stage, 1 improved to mild stage and 1 remained as severe but with a better score. From the rest of the 12 moderate AS patients, 8 improved to a mild stage and 4 improved their IUA to a moderate score.

Endometrial function post-AS therapy was assessed clinically by the recovery of menstrual function in terms of pictorial blood loss assessment, as previously reported13. We used a validated menstrual pictogram to measure total menstrual blood loss as described in methods based on the number and staining of tampons and/or menstrual pads used per menstrual period. Each score is comparable to a milliliter of blood loss. Most patients did not have their period prior to the treatment and so menstrual blood records were prospectively assessed after therapy (1–6 months), as stated per protocol. Menstrual blood loss records showed an increase from 29.60±45.40 mL in the first month post-therapy to 56.66±107.41 mL after 2 months post-therapy. Data showed then a decrease in the records, with 27.66±19.79 mL in the last month after treatment (M6) (Table 3).

As per the reproductive outcomes among the 20 treated patients, one live birth resulted from a spontaneous pregnancy post-therapy and 5 live births resulted after ARTs. In total, we performed a total of 41 single embryo transfers in 19 patients yielding an implantation rate of 31.7% per embryo transferred (13/41). Out of these transfers, as stated above, we obtained 5 live births corresponding to a 12.19% of life birth rate per transfer attempt. When analyzed per patient, the cumulative pregnancy rate was 55% (11/20), and the cumulative live birth rate was 30% (6/20) (Table 1). Interestingly, the patients who achieved a live birth had been diagnosed with moderate stage AS, suggesting that treatment efficacy may be influenced by disease severity. However, given the small sample size and data heterogeneity—the unequal distribution of moderate and severe cases—this observation should be considered preliminary.

The analysis of associations between pre- and post-AS therapy changes in endometrial volume, thickness, IUA scores, and morphometry revealed statistically significant differences between patients who achieved live birth (LB) and those who experienced miscarriage. Specifically, LB was significantly associated with post-AS therapy increases in endometrial volume and thickness (Supplementary Fig. 1B). In contrast, the miscarriage group exhibited significant changes in endometrial thickness and IUA scores (Supplementary Fig. 1C). These findings suggest that achieving an endometrial thickness of approximately 5.46±0.89 mm and an endometrial volume of around 3.62±0.85 cm³ may be associated with improved live birth outcomes in this study. Although we showed a potential association between enhanced endometrial characteristics and improved reproductive outcomes, it is important to note that live birth outcomes are also influenced by embryo transfer–related factors, such as embryo quality, number of transfers, timing, etc. These elements likely interact to determine overall success and need to be taken into consideration.

Associations between obstetrical outcomes revealed that the time range to achieve a successful pregnancy was 77 to 313.7 days (75% CI) (Supplementary Fig. 1D). Regarding dosage, no statistically significant differences were observed between pregnant and non-pregnant patients in terms of the number of CD133+ BMDSC cells administered, which ranged from 37 to 166.1×10^6 cells (75% CI) (Supplementary Fig. 1E).

As per endometrial functionality obtained (obstetrical outcomes), three out of six deliveries were performed by cesarean section and there were 3 vaginal deliveries. All deliveries were at term (range 37–38.6 W). All the newborns had normal weights (mean 3399 gr; range 3040–3970 gr) and normal development at one month. None displayed any signs of infection and there were no recorded congenital abnormalities. One of the participants experienced a first-trimester miscarriage that required hysteroscopic removal of the retained products of conception.

Exploratory endpoints
Single-cell RNA sequencing of the AS endometrium pre- and post-AS therapy. Previously, we published the AS endometrial cell atlas at single-cell resolution describing syndrome-associated alterations in cell types, cell-to-cell communication and gene expression profiles that supported a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment3. We had previously characterized the transcriptomic profile of AS (pre-AS therapy) and identified up to 18 cell types, including two unique cell types only present in AS (AS epithelium-SLPI+ and a population of smooth muscle cells). These same patients, who received CD133+ stem cell treatment during the simulated secretory phase, are now presented in the current study with both pre- and post-AS therapy endometrial biopsies (nine out of the 20 enrolled patients, Supplementary Fig. 2A). In parallel, we also performed single cell RNA-sequencing (scRNA-seq) profiling of the CD133+ product, which revealed a heterogeneous composition consistent with mobilized progenitor cell populations (Supplementary Fig. 2B).

For the endometrial biopsies, scRNA-seq yielded 123,250 cells (40,336 pre-AS therapy vs. 82,914 post-AS therapy, Fig. 4A, Supplementary Fig. 2C). We integrated matched pre-treatment (pre-AS therapy) and post-treatment (post-AS therapy) samples into the same dimensional reduction depiction. Overall, we observed a tendency towards an increase on the epithelial, stromal, endothelial and perivascular compartments (Fig. 4A, Supplementary Fig. 2D, E), a significantly reduced AS-characteristic epithelium (0.22% vs. 0.017%), and decreased macrophage populations (0.61% vs. 0.21%) (Fig. 4A, C). Notably, we previously showed that AS-epithelial population expresses high levels of SLPI3. When comparing the SLPI signal in pre and post-AS therapy samples, we observed that fluorescence was markedly reduced in post-AS therapy samples, which may account for the decrease in this cell population following CD133+ BMDSC treatment (Supplementary Fig. 2F).

We also determined the differential gene expression between pre- and post-AS therapy in different cell types related to a gene set associated to endometrial function (see Methods). The results revealed that epithelial, AS-epithelial, stromal, endothelial, and PV cells presented a higher expression of the gene set after CD133+ BMDSCs treatment, suggesting that cell treatment reactivates endometrial functionality (Fig. 4B). Further differential gene expression evaluation between pre- and post-AS therapy in epithelial cells revealed an increase in post-AS therapy samples of SCGB1D2 (related to functional secretory activity), PDGFRA and IGF1, associated with extracellular matrix remodeling and regeneration. We also observed the reduced expression of S100A10 and genes related to inflammation and immune response in post-treatment epithelial cells. These gene expression differences suggest that autologous cell therapy in the AS endometrium induces the appearance of an epithelium with reduced immune activity and inflammation and restores secretory and regenerative functions. The stromal cell cluster in the AS endometrium after autologous cell therapy exhibited the upregulated expression of genes related to angiogenesis and vascular development such as IGFBP2 and PTN (Supplementary Fig. 3).

Analysis of the endothelial compartment indicated a transcriptomic shift from a pro-inflammatory profile characterized by cell stress to a more homeostatic state (Supplementary Fig. 3). We observed the downregulation of genes involved in leukocyte/myeloid trans-endothelial migration (ICAM), antigen presentation (HLA-DRA and HLA-DRB5), cell proliferation (FOS and JUNB) or heat shock proteins (HSPH1 and HSPA1B). Furthermore, IGFBP2 overexpression suggested the enhanced formation of new blood vessels, and the expression of different collagen genes (e.g., COL1A1, COL3A1) indicated ECM cell adhesion and the development of capillaries from vessels surroundings.

Cell-to-cell communication (CCC) analysis related to immune response and pro-inflammatory pathways revealed a post-AS therapy enrichment in TWEAK, CX3C, IGF and GDF (Fig. 4D) and a reduced in strength after CD133+ BMDSC administration in RESISTIN, IL4, ICAM and LIGHT (Fig. 4D). Within the LIGHT pathway, we observed a disruption in established CCC between CD8+ T cells and various cell types (including stroma, epithelium, endothelium, PV, and immune cells), which was accompanied by a decline in LIGHT signaling between MAIT and stromal cells (Supplementary Fig. 4A). Pre-AS therapy samples also displayed IL4 and RESISTIN signaling pathway activation, which suggested a strong immune activity AS endometrium. Importantly, CD133+ BMDSC administration prompted the loss of these signaling pathways, contributing to the restoration of a homeostatic environment in the endometrium (Supplementary Fig. 4B, C).

Endometrial regeneration after damage has been linked to the activity of the canonical and non-canonical WNT pathways. In this case, CD133+ BMDSC administration prompted the recovery of CCC between the stroma, epithelium, and contractile cells in the WNT signaling pathway (Supplementary Fig. 4D).

Angiogenic pathways involving ANGPTL showed notable changes in CCC interactions following CD133+ BMDSC therapy (Supplementary Fig. 4E). Specifically, we observed a shift toward increased ANGPTL2-mediated interactions in the post-AS therapy group which was validated by immunofluorescence (Supplementary Fig. 4E, F). Given that a balance between ANGPTL1 and ANGPTL2 is critical for angiogenesis and vascular regeneration regulation, the increased ANGPTL2 signaling may reflect a regenerative response induced by the therapy.

Overall, CCC data suggested that CD133+ BMDSC therapy partially restores epithelial populations to a healthier state, enhances cell functionality by reducing oxidative stress and inflammation and promotes angiogenesis.

Lineage tracing differentiation of engrafted CD133+ cells. To track grafting and differentiation of infused CD133+ BMDSCs after cell therapy, we identified unique mitochondrial DNA (mtDNA) variants present in CD133+ BMDSCs before therapy, which were subsequently detected in various differentiated endometrial cell lineages post-therapy. Mitochondrial alteration enrichment from single-cell transcriptomes (MAESTER) was utilized for this analysis to establish relatedness14. Since the MAESTER technique requires reamplification of residual cDNA from single-cell RNA sequencing libraries, we had sufficient high-quality cDNA to perform the analysis in only three patients (N7, N9, and N15). Comprehensive profiling in these three patients revealed 32, 9 and 2 shared mtDNA variants in the endometrium post-treatment, respectively, all of which were absent before the stem cell therapy. RNA-based UMAP clustering post-treatment demonstrated the presence of CD133+ mtDNA clones within diverse endometrial cell populations (Fig. 4E), confirming the grafting and multilineage differentiation capacity of CD133+ BMDSC post-cell therapy.

Single-cell RNA sequencing of endometrial epithelial organoids (EEOs) derived from endometrial biopsies pre- and post-AS therapy. We also profiled endometrial epithelial organoids (EEOs) from pre- and post-AS therapy and controls at the single-cell level to compare this model with our in vivo data. We collected pre- and post-therapy endometrial samples from three AS patients and three controls (see supplementary data 1), generated EEOs and passaged them twice to remove stromal cell contamination (Fig. 5A). Data showed a significant enhancement in organoid-forming capacity (measured as the number of EEOs generated from the same number of cells) in control and post-AS therapy EEOs compared to pre-AS therapy EEOs (p<0.05). After passage 2, the organoid-forming capacity of cells from the three conditions displayed no significant differences, suggesting the positive selection of organoid-forming cells after 2 passages (Fig. 5A).

scRNA-seq of progesterone-treated EEOs yielded transcriptomic profiles from 49,609 cells in total: 14,459, pre-AS therapy, 19,556, post-AS therapy and 15,594 for controls. Seven populations were identified, consistent with previously characterized AS-derived epithelial organoids3, including glandular, glandular secretory, ciliated, cycling cells (cycling org), proliferative KRT19+, proliferative EMT and KRT17+ cells (Fig. 5B).

In terms of cell percentage, post-AS therapy EEOs showed a higher proportion of proliferative EMT and KRT9 cells than control EEOs and pre-AS therapy EEOs, which could suggest an enhanced proliferative capacity after therapy, although the trend was non-significant. Notably, the ratio glandular secretory/glandular was similar for post-AS therapy EEOs and control EEO (ratio 3.22 and 3.69, respectively), whereas the ratio remained lower for pre-AS therapy (ratio 1.03). Although the proportions of post-AS therapy do not fully mirror those of healthy controls, the shift in epithelial glandular composition after treatment may indicate a trend toward functional remodeling, including an enrichment of cells with a potentially more receptive-like profile (Fig. 5C). These results suggest that EEOs represent a useful in vitro model for studying AS, demonstrating partial recovery of AS EEOs after CD133+ BMDSC therapy to a state resembling control EEOs.

Methods
Study design and patients
This research complies with all relevant ethical regulations. The trial was registered at the European Union Clinical Trials Register (EU-CTR, EudraCT: 2016-003975-23; Registration date: April 1stst, 2020; https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-003975-23/ES), and the Spanish Clinical Studies Registry (REec, https://reec.aemps.es/reec/estudio/2016-003975-23). The investigational medical product (IMP) autologous CD133+ Bone Marrow Derived Stem Cells (BMDSC) was designated as an Orphan Drug (OD) by the EMA on April 20th, 2017 (EMA/OD/313/16)11 and by the FDA on February 1st, 2019 (#DRU-2017-6131). For the design of this clinical trial, scientific advice and protocol assistance were obtained from the AEMPS (February 10th, 2017) and the EMA (September 1st, 2017), respectively. The clinical research ethics committee at Vall d’Hebron Hospital approved this research in March 2020.

Out of 24 eligible patients, we enrolled 20 patients that met inclusion criteria and provided written informed consent. Patients were referred by clinics worldwide based on their refractoriness to previous operative hysteroscopy treatments and provided written consent. The first and last patient inclusions took place on Sept 9th, 2020 and Sept 19th, 2022, respectively. A detailed summary of clinical characteristics of participants pre- and post-treatment is presented in Table 1.

Study outcomes
The primary endpoints of the trial were safety and tolerability of the autologous CD133+ BMDSC therapy by identifying the incidence, prevalence, and frequency of adverse events occurring:
1. During the study period up to 15 months post-treatment.
2. In the event of a term pregnancy
3. In a case of live birth

Secondary endpoints were the efficacy of the therapy in achieving an improvement in morphological and clinical features such as endometrial histology through digital image analysis, endometrial thickness and volume by 2D and 3D vaginal ultrasound, hysteroscopic grading scores (IUAs), menstrual function recorded monthly based on the duration (days), volume (pads/day) and frequency (days) of menstrual bleeding with the Pictorial Blood Assessment Chart13, and reproductive outcomes after treatment measured by pregnancy, miscarriage, ongoing and live birth rates. The interim results of the trial were supervised by an independent Data Monitoring Committee (DMC) with special focus on the safety data.

Exploratory endpoints included the study of endometrial biopsies before and after stem cell treatment to compare differences in endometrial structure and tissue regeneration mechanisms, as the case of single cell RNA sequencing.

Procedures
Patients were managed as previously described10 with hormonal replacement therapy (HRT) to establish an endometrium with functional equivalence to the proliferative (estrogen dominant) and secretory (progesterone dominant) phases of a normal menstrual cycle before and after stem cell therapy. This was necessary as study patients experienced a lack of regular menstruation or negligible blood loss at menses. During the simulated “secretory phase” (5 days after progesterone administration) pre-treatment and post-treatment endometrial status was monitored by histological digital image analysis, 2D/3D ultrasound (Samsung, South Korea) and hysteroscopic assessment according to ASRM classification of IUAs12 using a 5 mm Betocchi hysteroscope (Karl-Storz, Germany). Hysteroscopic diagnostice valuation was used to grade the severity of AS; however, no operative hysteroscopy was performed. In the four patients for whom images were unavailable, ASRM scoring was verified through direct visualization by both surgeons (X.S. and J.T) during hysteroscopy, with detailed operative notes recorded. Although this represents an unavoidable limitation, we sought to minimize potential bias by applying predefined scoring criteria and maintaining consistent documentation practices.

Endometrial biopsies were performed under hysteroscopic vision from the posterior wall of the uterus at least 1cm away from any visually identified IUA using a semi-rigid double-action biopsy and grasping forceps (Karl-Storz, Germany). The endometrial specimens were processed for histology, ERA26 and EMMA27 to assess endometrial structure, receptivity and microbiome to rule out the existence of dysbiosis, respectively. Single cell RNA seq of biopsies and endometrial organoids were also derived from these endometrial biopsies.

Eligible patients were assessed by the hematology department at Vall Hebron (Barcelona, Spain). Requirements for participation in the study included a patient age of 18–44 years old, a BMI of 18-30, normal liver, heart, and kidney function, the absence of pregnancy, HIV, Hepatitis B or C, syphilis or any psychiatric pathology, and a willingness to complete the study.

BMDSC mobilization was performed using granulocyte-CSF (G-CSF) injection at 10 mcgr/kg for 5 days. CD133+ BMDSCs were collected through peripheral blood apheresis and subsequently isolated. Patients were excluded if they did not reach the minimal requirements for mobilized cells of at least 30 million cells, a purity above 70% and a viability >50%, presented an unstable medical condition, or refused a central venous catheter when required. Isolation of mononuclear cells was then performed by apheresis through peripheral venous access using the CobeSpectra separator (Terumo BCT, Lakewood, CO, USA) according to the manufacturer instructions. Subsequently CD133+ cell selection and isolation were performed using the cell sorter CliniMACS Pro (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). A mean of 267 min and 2.83 volemias in the apheresis were required to obtain a product with an average viability of a 99.7±0.27% and a mean purity of 89.54±8.29%. The minimum of CD133+ cells to be obtained by selection was 30 million cells that were diluted into 15–30 cc of saline solution and transported in a sterile bag with a syringe to the Interventional Radiology Department at Vall Hebron Hospital for delivery.

Finally, isolated CD133+ BMDSCs were delivered into the spiral arterioles of the patient using a minimal invasive radiology intervention through the left brachial artery, using the Seldinger technique28. The method consisted of placing a 4F introducer into the artery and catheterizing using a 2-curve Cobra angiographic catheter and a 0.035” Terumo guide wire. Through the Cobra catheter, a 2.5F microcatheter with a 0.014” guide wire was inserted and used to catheterize both uterine arteries up to the most distal level allowed by the microcatheter. The procedure was carried out under procedural sedation. Once the catheter was stabilized and its position verified, approximately 15-20 mL containing a mean CD133+ cell dose of 122×10^6±48×10^6 cells (range 42–200×10^6 cells) were administered.

All the diagnostic procedures (2D/3D ultrasound, diagnostic hysteroscopy and endometrial biopsies) before and one month after autologous CD133+ BMDSC administration in an outpatient setting were performed by the same surgeon (X.S.). In the four patients for whom IUAs images were unavailable, ASRM scoring was verified through direct visualization by both surgeons (X.S. and J.T) during hysteroscopy. Afterwards, patients were invited to undergo single embryo transfer (SET). All safety and effectiveness outcomes were recorded during the 15 months following treatment.

Pictorial Blood Assessment Chart (PBAC)
Endometrial function after cell therapy was assessed clinically by the recovery of menstrual function in terms of blood loss with a validated menstrual pictogram7. This method uses a score based on the number and staining of tampons and/or menstrual pads used per menstrual period. Each score number is comparable to a milliliter of blood loss. Menstrual pictograms were provided after the hysteroscopy following stem cell therapy, and the patient was requested to monitor menstrual cycles for six months if any menstrual cycles occurred (See Table 3).

Immunohistochemistry
Endometrial samples were fixed with 4% paraformaldehyde overnight and embedded in paraffin for hematoxylin and eosin (H&E) staining or immunofluorescence. For immunofluorescence, 10 μm-thick paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by heating the slides in 10 mM sodium citrate buffer (pH 8-9) at 95–100°C for 20 min, followed by cooling at room temperature for 20 min. Sections were then permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked for 1 h at room temperature in PBS containing 3% BSA and 0.1% Tween-20. Samples were incubated overnight at 4°C with goat anti-ANGPTL2 primary antibody (R&D Systems, Cat# AF623) or goat anti-SLPI (R&D Systems Cat# AF1274), both diluted 1:100 in blocking buffer. After washing with PBS, sections were incubated for 1 h at room temperature in the dark with donkey anti-goat Alexa Fluor 488-conjugated secondary antibody (Invitrogen, 1:400). Nuclei were counterstained with DAPI (1 μg/mL) for 5 min. Slides were mounted with antifade mounting medium and imaged using a Confocal fluorescence microscope Stellaris 5.

Digital image analysis
Endometrial biopsy sections stained with hematoxylin and eosin (H&E) were digitized with a VENTANA iScan HT slide scanner (Roche) at 20X and analyzed in QuPath29. Samples selected had intact glands and extracellular matrix (ECM), excluding regions with necrosis or hemorrhage. Gland sections from 13 patients (pre- and post-treatment) were carefully segmented using the brush and wand QuPath annotation tools and luminal epithelium was excluded to obtain a specific annotation for the gland epithelium.

Epithelial cell nuclei from the gland were then detected using StarDist, a deep-learning extension script for star-convex nuclear detection based on pre-trained datasets of H&E images (http://github.com/qupath/qupath-extension-stardist). Morphometric data on lumen, gland, and gland epithelium were gathered, calculating lumen percentage within glands and epithelial cell density. Stromal areas were using StarDist and ECM-related features (collagen, ground substance, and vessels) such as ingurgitated vessels and small diffuse hemorrhagic area included were obtained by training a pixel classification-based Artificial Neural Network with full resolution (0.25 μm/px) in QuPath. The resulting classifier corresponding to each ECM component was then applied to all samples and a percentage relative to the stroma area was obtained. Percentage of hematoxylin-stained cell nuclei was obtained by subtracting the area occupied by the ECM components to the stroma annotation. Cases with the existence of ECM underlying the uterine lumen lining epithelium with evidenced stroma decidualization were qualitatively evaluated by an expert pathologist.

Processing and dissociation of endometrial biopsies for single cell analysis
A two-stage dissociation protocol separated endometrial biopsies into stromal fibroblasts and epithelial-enriched single-cell suspensions. Before dissociation, tissue was rinsed with PBS in a petri dish to remove blood and mucus, and excess PBS was removed after rinsing. The tissue was then minced into small pieces and dissociated with 3 mL collagenase mix (Collagenase V, Sigma, USA), RPMI+10% fetal bovine serum, and DNase I (Sigma, USA) at 37°C under continuous shaking at 175 rpm for 25 min in a 15 mL Falcon tube in a horizontal position. This primary enzymatic step mostly dissociates stromal fibroblasts into single cells while leaving epithelial glands and lumen mostly undigested. The contents were transferred to a 50 mL tube in RPMI media and filtered with a 100 μm cell strainer. The tissue remaining on the filter was used for the epithelial enrichment cell isolation by resuspending and incubating in 10 mL Trypsin mix (Trypsin-EDTA (0.25%) phenol and DNase I) for 10 min. The resulting two contents were transferred to 50 mL tubes with 20 mL RPMI, filtered with a 100 μm cell strainer, centrifuged, and then resuspended in 1 mL RPMI. Dead cells were removed using the MACS dead cell removal kit (Miltenyi Biotec, Germany). Live-cell suspensions were loaded into a Chromium Next GEM Chip G (10X Genomics, USA) to obtain cDNA from individual cells.

10x Genomics Chromium library preparation and sequencing
Cells suspension was loaded into a Chromium Next GEM Chip G (10x Genomics) and run in a Chromium Controller (10x Genomics). cDNA synthesis and amplification were performed using the Single Cell 3’ Reagent Kit v3.1 (10x Genomics). 25% of the resulting cDNA was used for enzymatic fragmentation, end repair, A-tailing, adapter ligation, and amplification (with an expected yield of 0.1 ng/μl and a peak size between 200 and 9000 bp as determined in a TapeStation using HSD1000 assay reagents), serving as a quality control. Libraries were then quantified in a TapeStation using HSD1000 assay reagents.

10x-Genomics v3 libraries were prepared as per the manufacturer’s instructions. Libraries were sequenced, aiming at a minimum coverage of 50,000 raw reads per cell, on an Illumina NovaSeq 6000 S2 v1.5 300 cycles at a mean 40x coverage (paired-end; read 1: 26 cycles; i7 index: 8 cycles, i5 index: 0 cycles; read 2: 98 cycles).

Alignment and quantification of 10X scRNA-seq data
scRNA-seq data was processed using Cell Ranger v3.1.0 and Seurat v4. These versions were maintained to ensure consistency with a previously analyzed dataset and to enable direct comparability3. While newer versions include improvements in cell calling, UMI correction, doublet detection, and annotation, we applied the same pipeline and stringent quality controls across all samples to minimize technical variability. Core Seurat steps—normalization, dimensionality reduction, clustering, and visualization—also improved across versions. Nevertheless, we consider our biological interpretations robust, while acknowledging possible limitations in sensitivity and cross-study compatibility with newer software releases.

Raw reads were demultiplexed using the mkfastq wrapper command for bcl2fastq (Illumina). Libraries were aligned to the GRCh38-3.0.0 reference genome (based on GRCh38.p12, NCBI: GCA_000001405.27), as provided by 10x Genomics. Gene expression count matrices were generated per sample using the count pipeline with default parameters. This pipeline includes read alignment using the STAR aligner, UMI counting, barcode calling, and filtering of empty droplets. The filtering step relies on a Good-Turing smoothing model of background gene expression to distinguish low-RNA-content cells from ambient RNA.

Quality control filtering of cells and doublet detection
Low-quality cells were filtered out based on quality control metrics. Specifically, cells with a low number of detected genes or a high proportion of mitochondrial reads were excluded. All quality control procedures and downstream analyses were performed using R (v4.1). Doublet detection was performed with two methods: DoubletFinder (v2.0.3) and scds (v1.6.0) packages. The expected doublet formation rate was fixed for each sample according to the cell count recovery given per sample by Cell Ranger. The hybrid approach from the scds package was used to avoid removing false-positive doublets. Cells marked as doublets by both algorithms were removed from the samples.

Integration of scRNA-seq data across different conditions and cell clustering
Individual samples were aggregated, processed, and integrated with functions from the Seurat (v4.0.1) package. First, sample-to-sample aggregation for each condition dataset was performed with the merge function. Next, each aggregated dataset (pre-AS and post-AS therapy) was processed with the SCTransform. The SCTransform pipeline integrates different study conditions by applying a regularized negative binomial regression to allow the detection of condition-to-condition shared cell identities and the performance of differential expression analysis. Mitochondrial ratios and cell cycle phase were regressed out from integrated datasets. The 3000 most variable genes present in combined datasets using the SelectIntegrationFeatures function were used. PrepSCTIntegration and FindIntegrationAnchors were applied to identify the anchoring vectors across datasets and integration steps performed with IntegrateData (normalization method set to SCT). In the dimensional reduction step, the first 30 dimensions of PCA and UMAP were used to visualize cell clusters on the nearest-neighbor graphs. The clustree (0.4.4) package was used to evaluate the optimal resolution number in the FindCluster function; multiple clusters with a range of resolution from 0.5 to 3 were generated, with increased steps of 0.1. A manual review of the generated UMAPs and clustree output determined an optimal resolution of 0.8 to label the different clusters. The integration step was validated by evaluating the conservation of canonical markers across pre- and post-conditions.

Annotation of cell types in scRNA-seq datasets
Cell type identification was conducted by examining the differentially expressed genes of each cluster in comparison to the others. The Wilcoxon Rank Sum test was used, and the p-values were adjusted using the false discovery rate (FDR) method. Cluster cell identity was evaluated by comparison of cluster marker genes that were compared to annotated major cell types reported in published endometrial atlases10,24.

Analysis of significant changes in cell population sizes
Cell ratio analysis across different experimental groups was performed using a pseudobulk strategy. A count matrix of the different cell types detected per sample was created, and the edgeR (v3.34.0) package was used to compute a differential abundance (DA) test across cell types. The FDR p-value adjusting method was applied.

Cell-to-cell communication network analysis
To study the possible signaling interactions between cells, total interaction probabilities and communication information flows were determined with the CellChat (v1.1.3) package23. The first measure indicates the probability of communication between two cell types; the total probability of interaction is split into two parts: the probability of interaction between the sender and receiver and sender cell, considering the number of interactions (ligand-receptor or L-R pairs expressed) and the level of expression (strength of the interaction). A database of 229 families of signaling pathways was used to obtain information regarding interactions; this curated list of molecules considers the known structure of L-R pairs, including multimeric complexes, soluble agonists and antagonists, stimulators, and inhibitory co-receptors. The second measure, the communication information flow, assesses the amount of information exchanged between several cells through various interactions. A higher value of communication information flow between cell populations indicates a more complex communication network. The population.size argument in the computeCommunProb function was used to correct the communication weight of each cell population. The cell-to-cell communication (CCC) networks were filtered to exclude those with fewer than ten cells to achieve robust results. The rankNet function with a significance threshold of 0.05 was used to test for network differences between AS endometrial samples pre- and post-treatment with CD133+ BMDSCs. These comparisons included only common cell types, excluding cycling cells from the analysis. Significantly altered signaling pathways between groups are highlighted in the bar graphs of the flow of information. To explore these plots further, interaction probability scatter plots can be used to identify the receiving and sending cells. The contribution of each L-R pair to a pathway is also shown in interaction probability plots.

MAESTER library preparation, sequencing and analysis
The methodology was applied as previously described14. Briefly, MAESTER consists of two PCRs, whereby we would include 10x Genomics 3’ v3 cDNA from where the CB and UMI of the transcripts would be retained. In the first PCR, twelve primers were used to tile across the whole mitochondrial transcriptome at 1 μM each together with a specific barcode GoT-P5-i5-BCXX for each sample14. The mixture was as follows: 20 μL of KAPA HiFi Hotstart ReadyMix (Thermofisher Scientific), 16 μL of cDNA product, (diluted to achieve at least 1 ng/uL) along with 3 μL of primer mix 10 μM and 1 μL of Got-P5-i5-BCXX (10 μM). The PCR was set to initial denaturation at 95°C for 3 min followed by 10 cycles of 98°C for 20 s, 65°C for 15 s and 72°C for 3 min, with a final extension at 72°C for 5 min. After amplification, each individual primer generated 12 reactions, and so these were pooled together at a ratio of: mix 1: 32 μL; mix 2: 40 μL; mix 3: 40 μL; mix 4: 40 μL; mix 5: 40 μL; mix 6: 40 μL; mix 7: 40 μL; mix 8: 40 μL; mix 9: 8 μL; mix 10: 8 μL; mix 11: 8 μL and mix 12: 16 μL.

The pooled products were purified with 1× AMPure XP beads (Beckman Coulter, USA) as per protocol instructions. After AMPure XP purification, the pooled PCR 1 product was eluted in 20 μl H2O RNAse free. Thereafter, the PCR 2 was performed by adding 18 μl of the purified product from PCR 1, with 2 μl of a 5 μM primer mix (P5-generic + GoT-P7-i7-BCXX)14 and 20 μl KAPA HiFi Hotstart ReadyMix (Thermo Fisher Scientific).

The parameters used for PCR 2 were an initial denaturation at 95°C for 3 min; six cycles of 98°C for 20 s, 60°C for 30 s and 72°C for 3 min, followed by a final extension at 72°C for 5 min. The resultant PCR was purified with 0.8X AMPure XP beads. The DNA was eluted in 20 μL TE, quantified and sequenced accordingly. The libraries contained 0.6–50 ng/μL with sizes ranging from 300 to 1,500 bp and were sequenced on the Illumina NovaSeq X 10B sequencing (paired-end; read 1: 28 cycles; read 2: 256 cycles, 2× 8-bp index barcodes).

Raw sequencing data was pre-processed according to the pipeline previously described14, up to the generation of BAM files containing mitochondrial reads from individual cells in the respective scRNA-Seq samples. An additional preprocessing step was implemented to trim 50 nucleotides from the 3’ ends and remove poly-A tails, using Fastp v0.23.430. Then, BAM files were split by cell barcodes, and each subset was processed independently. Duplicated reads were removed using fgbio v2.1.0 (fulcrumgenomics/fgbio: 2.4.0 (2.4.0)). To exclude low-quality cells, the total number of covered bases per cell was calculated using samtools v1.19.231 and bedtools v2.31.132. Only cells with coverage exceeding the overall median minus one standard deviation were retained for downstream analyses. Variant calling was performed with LoFreq v2.1.533 following base recalibration, which was conducted using the GATK toolkit v4.5.0.034 (DePristo, 2011) and LoFreq. The resulting VCF files were normalized using bcftools v1.1831 and filtered. Selected variants included those detected in the CD133 population of the CD133 samples and shared with S2/E2 samples while excluding variants identified in S1/E1 samples. Additionally, variants were retained only if they exhibited a variant allele frequency exceeding 10% per cell and were detected in at least five cells, but excluded if present in ≥90% of total cells. Filtered variants were manually reviewed using IGV v2.12.335. Variants with more than 10 reads in S1/E1 were discarded. The final set of variants was added to the metadata of the scRNA-seq objects using the Seurat (v5.1.0) package36 in R v4.2.2.

Differential expression and gene signatures score
The FindMarkers function from the Seurat package was used to perform differential expression analysis across the treatment stages and the different cell types reported in the dataset. The Wilcoxon Rank Sum test was used, and the p-values were corrected using the FDR method. Genes with FDR values under 0.05 were considered significant. Gene set signature scoring was performed using escape (v1.6.0) package37 and UCell package to study menstrual cycle function in the secretory phase between treatment groups. The AddModuleScore function from UCell package was used to compute the score for each cell present in the dataset. Finally, the scored differences between treatment stages in the endothelium, epithelium, AS epithelium, perivascular (PV), and stromal cell fractions were evaluated. The getSignificance function was used to perform statistical testing using a Wilcoxon test; p-values were corrected using the FDR method. The following gene set related to menstrual cycle function in the secretory phase was used38: SCGB1D2, MT1F, MT1X, MT1E, MT1G, CXCL14, WNT5A, SFRP1, ZFYVE21, CILP, SLF2, MATN2, and S100A4.

Organoid culture from endometrial biopsies
Organoids were generated from AS patients’ endometrium pre- and post-treatment and from healthy control endometrium. After enzymatic dissociation of the endometrial biopsies, epithelial and stromal cells were obtained and total cell numbers were counted. After centrifugation at 300g, the pellet was resuspended in 70% Matrigel (Corning, 356231) and 30% DMEM/F12 (Thermo Fisher Scientific, 11330032) and supplemented with the ROCK inhibitor Y-27632 (1/500). The suspension was cultured in 20-μL droplets containing 25,000 cells, deposited in prewarmed 48-well plates, and allowed to form a gel at 37°C and 5% CO2 before adding culture medium. Organoid culture medium was prepared as previously described39,40. Matrigel droplets were cultured for 14-16 days in the first passage (P0) with medium changes every two days. Organoids were then grown for seven days before the following passage, with media changes every two days.

Organoids were recovered for passaging by liquifying the Matrigel droplets with ice-cold DMEM/F12 (without any growth factors, serum, or enzymes), followed by repeated pipetting to ensure maximum recovery. Organoids were then dissociated by incubation with TrypLE supplemented with Y-27632 (1/1000) and mechanically triturated. The resulting cell suspension was centrifuged at 300g, and the pellets were resuspended in 70% Matrigel and 30% DMEM/F12 supplemented with Y-27632 and placed as droplets in 48-well plates. Two passages were performed to remove the contaminating stromal cells from the epithelial organoids. After seven days of P2 culture, organoids were dissociated for single-cell transcriptomic analysis following the same protocol described for passaging but with an additional cell counting step for the single-cell transcriptomics protocol as per manufacturer’s instructions (10x Genomics).

Organoid counting
Multiple microscopy images (Thermo Fisher, EVOS M5000) were taken to count organoids present in each Matrigel droplet. Images were taken by dividing each drop into two depth sections and each section into four regions. Two images were counted diagonally from each section, and the mean from these images was calculated. This mean was multiplied by the number of divisions of each section (4) and the number of sections (2) to infer the total organoid number per drop. For each condition, two experimental replicates were measured. For organoid counting we conducted a two-way analysis of variance (ANOVA) followed by a Tukey’s HSD post-hoc tests to assess pairwise comparisons between the control, CD133+ BMDSC pre-treatment and post-treatment AS EEOs per passage.

Organoids scRNA-seq
Single-cell RNA-seq data from organoids were aggregated, processed, and integrated using a similar approach to that described for the patient-derived samples. Briefly, integration across pre-treatment, post-treatment, and control datasets was performed using the SCTransform function. Dimensionality reduction was conducted based on the 750 most highly variable genes using the first 30 principal components, and cell clusters were visualized using UMAP on the nearest-neighbor graph. Finally, major cell types were identified and annotated by analyzing differentially expressed genes in each cluster, using previously described cell-type-specific marker genes10,24.

Statistical analysis
Statistical analysis of clinical design. The study sample size was estimated based on the hypothesis that autologous CD133+ BMDSCs instillation in the uterine arteries improves endometrial function in terms of reproductive outcomes, specifically live birth (LB) rate. It was designed as a paired cohort study “before and after”, consisting of the same patient undergoing embryo transfer after treatment. Our hypothesis was that improvement would involve a transition from 0% prior to stem cell treatment to 28% LB rate after treatment (α 0.05, power=80%) which is the average LB rate reported in the context of assisted reproduction treatments by the European IVF-Monitoring Consortium41. Based on these assumptions, it was estimated that 18 patients would be required for the protocol study. Additionally, a 20% loss to follow-up was anticipated, resulting in a required total of 20 patients. The final analysis was conducted after all patients completed a 15-month follow-up after treatment. However, since the actual drop-out rate was much lower than expected (only 5%), we successfully completed 19 per protocol patients out of 20 recruited, surpassing our pre-specified target of 18 per protocol patients. Given that recruiting patients with this rare condition was challenging, and upon consultation and agreement with our Ethics Committee, we concluded recruitment once the necessary number of patients had been successfully treated and completed follow-up.

Statistical analysis of clinical outcomes. For the secondary endpoints, a convenience sample was studied. Endometrial volume, thickness, IUA score from Figs. 2, 3 and Supplementary Fig. 1B, C was presented as mean±standard deviation. Statistical significance between endometrial volume and thickness was evaluated using a parametric paired t-test. Normality assumption was confirmed by applying the Shapiro–Wilk test to assess the distribution of differences between pre and post treatment for each parameter. For the IUA score, non-parametric Wilcoxon signed-rank test was applied.

Data from digital analyses presented in Supplementary Fig. 1A is presented as mean±standard error of the mean (S.E.M). Statistical significance between general characteristics of gland and stroma in paired samples grouped based on reproductive outcomes was evaluated using a parametric paired t-test whereas differences between specific characteristics of gland and stroma were assessed using a Wilcoxon rank sum test. Statistical significance within pre- and post-AS therapy between these groups was evaluated using non-parametrical Kruskal Wallis with Dunn’s test for multiple comparisons.

Statistical significance was considered when the p-value was less than 0.05 and was indicated as follows: p<0.05 (*), p<0.01 (**), p<0.005 (***), and p<0.0001 (****). Data was analyzed and represented using GraphPad Prism 8.0.1 and 10.2.0 software.

Statistical analysis for single cell data of in vivo biopsies. Statistical analyses were performed in software R (v4.1 and v4.2, https://www.r-project.org). Data from Fig. 4B, displaying boxplots of gene signature enrichment scores (UCellScore), were statistically tested using the Wilcoxon test implemented via the getSignificance function from the escape (v1.6.0) package. P-values were adjusted using the FDR correction method.

Data from Fig. 4C, showing cell type ratios boxplots, were analyzed using a negative binomial generalized linear model (NB-GLM) implemented in the edgeR (v3.34.0) package. Statistical significance was defined as FDR<0.05 (**), and FDR<0.1 (*).

For the data shown in Fig. 4D, we conducted a differential CCC analysis between pre-treatment (Pre-AS therapy) and post-treatment (Post-AS therapy) samples using the rankNet function from the CellChat (v1.1.3) package. Statistical significance was assessed with a Wilcoxon test, applying a significance threshold of 0.001 and a p-value cutoff of 0.01. P-values were adjusted for multiple testing using the FDR correction method.

Data from Supplementary Fig. 3, displaying dot plots of the differential expression between Pre-AS therapy and Post-AS therapy across different cell types, were statistically tested using the Wilcoxon test implemented via the findMarkers function from the Seurat (v4.0.1) package, with p-values corrected for multiple comparisons using the FDR method.

Supplementary Fig. 4, showing cellular communication through chord plots and bar plots, was generated using the netVisual_aggregate and netAnalysis_contribution functions from the CellChat (v1.1.3) package, respectively. A p-value threshold of 0.05 was applied to define significant interactions and contributions.

Statistical analysis for organoids. For organoid counting we conducted a two-way analysis of variance (ANOVA) followed by a Tukey’s HSD post-hoc tests to assess pairwise comparisons between the groups. Data was analyzed and represented using GraphPad Prism 8.0.1 and 10.2.0 software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review of the article by Santamaria et al “Autologous cell therapy with CD133+ bone marrow derived stem cells for Asherman Syndrome: a phase 1/2 trial’. The authors are reporting a non-randomized prospective open label trial on autologous hematopoetic stem cell therapy for Asherman syndrome cases resistant to existing therapies. The group has been working on the possibility of such therapy for over a decade now, and while this approach is cumbersome, expensive and possibly not available for many, it is an important pathophysiology driven treatment that has a potential. I want to commend on use of lineage tracing experiments showing the engraftment of the CD133+ cells, and on demonstrated changes in the cell populations by single-cell RNA seq studies. Please see comments below. Comments: 1. Line 114, “the most frequent treatment-emergent adverse event…”. What do you mean? You mean this was the most serious adverse event? It was only in 1 patient, and what about chest pain or bleeding? 2. I suggest adding “infection” to the causes of AS. 3. I recommend presenting the data on menstrual pattern and its recovery (before and after the treatment) in the text clearer and in Table 1 (or separately). It is an important clinical information. Provide reference for menstrual pictogram. 4. Make sure it is clear in the Abstract and text that hysteroscopy was only diagnostic, no lysis of adhesions was performed, to be able to truly evaluate the effect of the therapy. This is really not clear. 5. Table 1 and text: you did not present pregnancy rates and miscarriage rates, just live births. How many transfers were performed per patient and in total to get the 6 live births? 6. Lines 164-166: these conclusions should be softer as clinical significance is not clear, if any. What about difference in delta from pre- to post- in these measures? 7. Line 182: what do you mean by uterine curettage was performed by operative hysteroscopy? Do you want to say that retained products of conception were removed hysteroscopically? I do hope that uterine curettage was not performed in this AS patient. 8. Single-cell RNA seq results: why would AS-epithelial cells increase after cell therapy? Aren’t they being replaced more by epithelial cells? 9. Analysis of unique mtDNA: why only 2 patients? Also, in the text you mention only 2 patient samples, but in Figure 4E and figure legend its 3 patients, unless I am getting it wrong. 10. When you get to organoids, the description of results is more chaotic and you are losing the reader. 11. What are the in vivo controls? 12. I am not sure how you showed the improved receptivity, that’s an overstatement. 13. What are the future directions? Are the authors proposing large clinical trial (RCT) or to move this therapy to clinic now, without phase 3? Not clear how this will look like. Should mention the associated health care costs and alternatives, as well as interdisciplinary approach involved in this treatment. 14. Figure 3: why didn’t you present all 19 patients that completed the treatment and post-treatment evaluations? 15. Who was performing the hysteroscopies, before and after the treatment? Were clinicians blinded to the therapy timing such as did they know that they are doing pre- or post-stem cell therapy cavity assessment? How was their bias managed? 16. When was the endometrial thickness measured, in which cycle phase? I understand that these are moderate-severe AS patients, but if you want this study to be taken seriously, you have to be consistent in when the lining thickness is measured. Where in the menstrual cycle were the 2D and 3D evaluations done, hysteroscopy and then the stem cell treatment? If amenorrhea, cycle phase could have been assessed by scanning ovaries and hormonal testing. 17. Table 1: why NA for endometrial volume assessment in 3 patients? 18. Table 2: I suggest using “false passage instead of “false path”. “Bleeding”- what is it, uterine/vaginal or intraabdominal? Vomiting (not vomit. What is Abdominal swelling, is it hives on the skin, or bloating? You should be more specific about skin rash, just localized or generalized? Also, is arm pain same as pain associated with arm hematoma? What is respiratory infection, just URI or something more serious? 19. Was the brachial artery catheterization done under anesthesia or procedural cedation? Minor comments: 1. Some language revision is recommended for sentence flow, and some typos and missing words. 2. Sentence on lines 76-79: please revise, not too clear. 3. In the Results, please be consistent when writing pre-AS, pre-treatment AS or AS-pre- and -post. Both in the text and Figures. 4. Discussion, line 306: are your references 17-23 or 28 as stated? Reference list goes to 23 only.", "reviewer_2": "The authors reported the safety and efficacy of intrauterine intravascular injection of bone marrow-derived CD133+ stem cells in treating 20 cases of refractory Asherman syndrome. Single-cell sequencing of endometrial tissues from a subset of cases before and after treatment was performed to investigate the stem cell homing and differentiation，and transcriptional profile changes of the administered cells in vivo, as well as organoid formation capacity and associated expression alterations. While the study is interesting and holds significant clinical relevance, several critical aspects require further elucidation to strengthen its translational impact. 1. In the research proposal, this research was described as a prospective, multicenter, and self-controlled study. However, the current report appears to be based on work conducted at a single center. Could you please provide the reasons? 2.Result 1 On line 94, the author states that “Following diagnostic hysteroscopy, hematopoietic stem cells were mobilized, harvested through leukapheresis, and selected for CD133+ and intra-endometrial spiral arterioles instillation (Fig. 1A).” Was surgical separation of intrauterine adhesions required before injection? In which phase of the menstrual cycle should the injection be administered? 3.How was the cell therapy dosage determined? Was it simply based on the amount of cells that could be isolated and obtained? 4.In secondary endpoint, I noticed that the specific location of the endometrial biopsy for comparing histological changes and immunohistochemical staining before and after treatment seems to be missing in the methodology section. Could you please provide more details on this aspect? 5.In the legend of Fig. 2, the sample sizes indicated for the box plots of endometrial volume (n=16) and endometrial thickness (n=19) appear to be inconsistent with the figure itself. It is possible that there might be a typographical error by the author. Why are the sample sizes for the analysis of endometrial thickness and endometrial volume different—one being 16 cases and the other 19 cases—when the total sample size should be 20 cases? 6.Regarding the efficacy, the key efficacy endpoint is the live birth rate. From Table 1, it appears that successful pregnancy mainly depends on the severity of the disease. Could the author address this point in the manuscript? 7.On line 256, the author states that “RNA-based UMAP clustering post-treatment demonstrated the presence of CD133+ mtDNA clones within diverse endometrial cell populations (Figure 4E), confirming the grafting and multilineage differentiation capacity of CD133+ BMDSC post-cell therapy.” However, it may be necessary to have experimental evidence to confirm the differentiation capacity of CD133+ BMDSC, rather than relying solely on the findings from single-cell sequencing. The viability of the cells used for treatment was >50%. Could it be that dead cells entered the body, their mitochondrial DNA was engulfed or phagocytosed by other cells, rather than differentiating into other cell types? 8.The author selected endometrial samples from nine patients before and after treatment for subsequent single-cell sequencing analysis. However, the author did not explain the criteria for selecting these nine cases. The relevant clinical information for these nine patients should be provided. Additionally, the results of the single-cell sequencing need to be validated experimentally. 9.In Single-cell RNA sequencing of endometrial epithelial organoids (EEOs) derived from endometrial biopsies pre- and post-treatment，the clinical information of the patients from whom the organoids were derived should be provided. 10.In discussion part, to facilitate readers' understanding of the progress in stem cell therapy for Asherman's syndrome, the author would provide a brief and objective overview of the published studies. When emphasizing the strengths of this research, the author mentions the use of \"a specific, well-characterized cell\" for treatment. However, based on the \"methods\" section, the purity of the cells used is about 70%. What about the remaining 30%? What types of cells do they consist of, and what is their impact on endometrial regeneration? It would be best if the author could clarify this.", "reviewer_3": "This study titled ‘Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase1/2 trial’ by Xavier Santamaria et al., present promising results on the use of autologous CD133+ bone marrow-derived stem cells (BMDSCs) for treating Asherman Syndrome (AS), a condition with limited treatment options. I have several questions and comments listed below that need to be answered by the authors. My major points are these and further details follows below: Major points: Without a control group/placebo, interpreting the real effectiveness of the intervention is very difficult because you lack a baseline for comparison in stage 2. The main limitation of this study is that the data are limited to only one-month post-transplantation, which is not sufficient to assess the long-term safety and efficacy of the treatment. Extended follow-up, ideally up to the trial’s defined 15-month endpoint is necessary to support the conclusions. Additionally, the absence of a control group makes it difficult to confidently attribute observed effects to the treatment. While the hysteroscopic images provide some information, their value is limited due to variation in sampling location, unknown menstrual cycle phase and inconsistent hormone levels during the first month. These factors introduce variability and make it hard to draw firm conclusions from the histological data. 1. For the 20 patients recruited to the study, they all underwent reparative operative hysteroscopies (range 1 to 11). During how long time was treatment attempted? 2. Authors mentioned that despite the previous surgical treatments, no patients reported improvement of endometrial thickness. Did they not receive conventional hormonal therapy after the previous operation? 3. After the cell transplantation, have you considered administered estrogen supplement? 4. For the secondary endpoints, authors stated that ‘pretreatment patient menstrual blood loss was around 29.6ml increasing to 55.2ml in the first month post-treatment, however, decreased to 31.1ml and 29.9ml in third and fifth month post-treatment. What could be the reason for this? In addition, you recruited moderate to severe AS patients, these patients are most likely amenorrheic or spotting. What is the menstrual conditions pre-treatment for these patients? There is no information in Table 1. 5. Regarding the dose range (37–166.1 × 10⁶ cells): Was this range selected based on prior studies, or was it a dose-escalation design? The rationale for the wide range should be clarified. 6.Reversible Limb Paresthesia: This is an interesting and somewhat unexpected adverse event. Further discussion on the mechanism would be valuable. 7. No haemopoietic malignancies were reported. For how long will the patients be followed and what is known regarding this risk? 8.Endometrial Improvements: authors reported increases in thickness, volume, and hysteroscopic scores are encouraging. Was there a correlation between cell dose and response/duration of effect? 9. The authors mentioned a key outcome about reproductive endometrial function recovery. It includes 6 Live Births and 6 miscarriages. Before the cell transplantation treatment did these patients also receive embryo transfer? 10. How many Et attempts per patient? More than one? 11. The authors added mechanistic Insights by applying Single-cell RNA Sequencing before and after treatment. In the previous paper (Santamaria X, et al. 2023), authors identified two unique cell types only present in AS (AS epithelium and smooth muscle cells). The cells isolated for scRNA-seq most likely came from superficial layer of the endometrium. Do authors think that severe AS are more related to damaged basalis layer? In that case the mechanistic study would need to include basalis tissue to truly reveal the efficacy and underlying mechanism. 12.The finding of \"partial reversion of AS-related changes\" is intriguing but vague. Were there specific gene pathways or cell populations most affected except for the increase of several cell types? What about the post-treatment changes of the two AS unique cell types? 13. For the organoid part, the organoids were hormone-treated in vitro. The three controls—were they age-/fertility-matched to AS patients? If not, differences could reflect baseline variability rather than treatment effects. 14. Regarding the organoid sequencing results, the increase in proliferative EMT cells post-treatment is intriguing but raises questions: Is this proliferative shift a direct effect of CD133+ BMDSCs (e.g., via paracrine signaling) or an indirect consequence of endometrial repair? Could excessive EMT proliferation risk fibrosis or aberrant regeneration? The authors should discuss whether this profile aligns with healthy endometrial repair. 15: Figure 3 presents hysteroscopic images before and after treatment for all participants, which is well appreciated. However, it is not specified on which day of the menstrual cycle these images were taken, nor how many days post-treatment they were obtained. From Table 1, it appears but not clear that participants were not amenorrheic. Could the authors clarify the timing of image acquisition in relation to the menstrual cycle and the treatment timeline? This information is particularly important considering the statement in lines 148–150, which notes an increase in menstrual blood flow during the first month after treatment, returning to pre-treatment levels after three months. Was this temporal pattern in blood flow reflected in the hysteroscopic findings? 16. Considering the transient increase in menstrual blood flow during the first month post-treatment, it would be informative to include hysteroscopic images from three months after treatment in non-pregnant women to assess long-term endometrial changes. 17. In reference to lines 143–150, the authors state that menstrual blood loss increased during the first month post-treatment but returned to pre-treatment levels after three months. Does this suggest that the underlying condition was not fully resolved, given that the menstrual pattern ultimately reverted to its baseline state? If normal menstrual cycling resumed or improved, one might expect a sustained change in menstrual pattern or blood flow, especially considering the reported increase in endometrial thickness after treatment (lines 127–133, Figure 2B, and Table 1). Could the authors clarify how this temporal pattern in menstrual blood loss corresponds to treatment efficacy and the overall resolution of the syndrome? 18: The scale bar in Supplementary Figure 3 for the gene expression data needs to be corrected for accurate interpretation. 19: Tissue samples were collected pre- and post-treatment from nine participants, and the data in Figure 5A suggest that at least three samples were used to generate 3D organoids. For clarity and completeness, it would be helpful to include representative 3D organoid data from all nine participants if performed, or at minimum the three samples that were analyzed and presented in figure 5 A, like the presentation style in Figure 2, to allow assessment of inter-patient variability and enhance data transparency. 20. For the organoid part, the organoids were hormone-treated in vitro. The three controls—were they age-/fertility-matched to AS patients? If not, differences could reflect baseline variability rather than treatment effects. 21. Regarding the organoid sequencing results, the increase in proliferative EMT cells post-treatment is intriguing but raises questions: Is this proliferative shift a direct effect of CD133+ BMDSCs (e.g., via paracrine signaling) or an indirect consequence of endometrial repair? Could excessive EMT proliferation risk fibrosis or aberrant regeneration? The authors should discuss whether this profile aligns with healthy endometrial repair. 22. It is not indicated when the sample were obtained after the treatment for scRNA and 3D organoids culture. 23: The scRNA-seq data in Supplementary Figure 4E indicate upregulation of angiogenic pathways ANGPTL; to strengthen these findings, it would be valuable to include supporting protein-level validation of key pathway components, such as through immunostaining or western blotting. 24: Mitochondrial Variant Tracking: This is a clever approach to assess engraftment. What percentage of endometrial cells post-treatment showed donor mitochondrial DNA? What is the most abundant cell type that bone marrow stem cells may differentiate into? Did engraftment correlate with clinical outcomes? The use of mtDNA for lineage tracing is powerful but not sure how mitochondrial transfer between cells via mechanisms such as tunneling nanotubes, cell fusion, or exosome-mediated exchange affects the clonality. These events can introduce exogenous mtDNA, potentially leading to misinterpretation of clonal relationships. 25. line 196-199 concludes that perivascular cells increase after the treatment and Macrophage decreases, need some comments in discussion about this. It's not clear how pericytes are involved in endometrium but in CNS it's very well accepted that pericytes forms the scar tissue after injury and inhibits regeneration. 26: Protein validation for the genes has not been presented, only RNA data was shown. It would be valuable to present how the AS epithelial cells change after treatment and how the scar/perivascular cells are affected.", "reviewer_4": "Comments on “Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase 1/2 trial” As requested by the Editor, our review will be limited to those aspects that relate to the trial design and statistics of this manuscript. We do not have sufficient experience and knowledge for evaluation of aspects that are more specific to cells and laboratory work. Considering that Asherman Syndrome has a significant impact on the quality of life of many people, and that currently the only possible intervention - which is often not definitive - is operative hysteroscopy, the identification of different approaches could be of great value. This is especially true if these possible new interventions are able to overcome infertility. Therefore, we commend the authors for their commitment to the search for new treatment options. We thus appreciated the authors’ effort in conducting this clinical trial aimed at evaluating the tolerability, safety, and efficacy of autologous CD133+ bone marrow-derived stem cell (BMDSC) therapy in patients with Asherman Syndrome (AS) refractory to surgical interventions. Overall, the methodological components of the study are appropriate for a non-randomised, open-label, single-arm, phase 1/2 study. We are of the opinion that the study design is well-structured, and we do not have any major comments on this aspect. However, considering that the study represents a complex methodological project integrating clinical and molecular approaches, we believe that some clarifications and further details may be useful, both regarding the objectives of the study and the use of statistics. This is all the more important given that the sample consisted of 20 women (one of whom did not complete the study), which is relatively large considering the prevalence of the disease, but relatively small considering the numerous endpoints set by the authors. We reiterate the importance of this study and offer some comments and suggestions to further improve the clarity, transparency and robustness of the analyses. Major comments 1. Partially addressed primary endpoints According to the trial registration on the European Union Clinical Trial Register (EudraCT: 2016-003975-23), three primary endpoints were declared: “1. Identification of incidence, prevalence and frequency of different adverse events that may occur in the patient throughout the study up until Month 15 post-treatment. 2. In the event of a term pregnancy, identification of incidence, prevalence and frequency of different adverse events that may occur during the pregnancy, birth and puerperal period. 3. In case of a Live Birth (LB), identification of incidence, prevalence and frequency of the different adverse events that may be associated with the investigational medicinal product (IMP) until the first month of age.” However, in the present study, only the first primary endpoint appears to have been thoroughly developed, and its results reported. The results of the primary endpoints are reported in the manuscript on lines 109-116 and in Table 2. There is no mention of primary endpoints 2 and 3, i.e. pregnancies and births. Some information on these is provided in secondary endpoints section and in the supplementary material, but as these are primary endpoints - and therefore theoretically the most important ones - they should be clearly presented in the section of the results dealing with the primary endpoints. It would be appropriate for the authors to complete the analysis by including the remaining two primary endpoints as outlined in the registered protocol. Alternatively, the authors should state why the results of the other two primary endpoints were not reported in the primary endpoint results section and were not discussed. For example, the most outstanding result of this study is mentioned by the authors in the abstract: \"...reproductive function was restored, culminating in six live births after embryo transfer\". It is not clear why this striking observation is barely mentioned in the manuscript, neither in the results nor in the discussion, even if one of the primary endpoints was the “identification of incidence, prevalence and frequency of the different adverse events” in case of a life birth. It is clear that the set of secondary and explanatory outcomes is intended to try to understand the biological and molecular reasons behind the regained fertility of some women. Nevertheless, the primary endpoint is in danger of being obscured by all the other analyses. 2. Excessive detailing of secondary and exploratory endpoints Although the presentation of the results and discussion of the secondary and exploratory endpoints is comprehensive and thorough, the length and level of detail may be reconsidered as it seems disproportionate to the primary objectives of the study. A more concise approach could benefit the overall focus and readability of the manuscript. Alternatively, the endpoints of the study could be refocused. 3. Predictive model based on Support Vector Machine (SVM) In the section concerning endometrial epithelial organoids (EEOs), the authors add the use of a Support Vector Machine (SVM) with a kernel radial basis functon(RBF), implemented via the scPred package, to perform transcriptomic comparison with in vivo data. However, although a brief description of the process is provided (10-fold cross-validation and C parameter optimization), several key methodological elements are missing, e.g. the type of learning adopted, the target classes used for model training, the feature selection procedure, the model performance metrics, and the rationale for choosing the RBF kernel, among others. Given the complexity and methodological significance of a machine learning-based analysis, we recommend that a dedicated and detailed methods section be added. Alternatively, since this does not represent one of the study’s primary endpoints, the analysis could be considered for omission. Minor comments 1. Statistical methodology presentation Although the statistical methodology covers a broad range of analyses, it is overall unclear, dispersed, and difficult to follow. The statistical tests used are reported in different sections of the methodology and not always in a systematic manner, making it challenging to obtain a comprehensive view of the analyses performed. We therefore recommend including a more structured and concise section that clearly summarizes all the applied tests, the type of data analysed, and the rationale behind the choice of each statistical test. Alternatively, we suggest including a subsection on the statistics and software used within each section of the methodology. This will allow the reader to better understand the different statistical approaches used. 2. Assumptions behind the use of parametric tests When parametric tests were used instead of non-parametric ones, it is not specified whether the assumption of normality of the data distribution was verified. An example of this is the use of the paired t-test to evaluate the IUA score. Since this is a score, it is likely that nonparametric tests would be more appropriate. Or, if a parametric test is needed, it would be appropriate to state the rationale for such a choice. 3. Choice of statistical test for organoid quantification A two-way ANOVA test was used in the organoid quantification section. It would be useful for the authors to clarify the rationale for using a two-way analysis. A two-way ANOVA analyses the effect of two different categorical independent variables on a continuous dependent variable. It is not clear what the two independent variables are in this case. In addition, it is important to specify whether there were two independent interacting factors. Could it be that a one-way ANOVA would have been more appropriate? 4. Statistical power and sample size calculation The authors calculated the sample size based on a hypothetical increase in live births from 0% to 28%. The hypothesis is probably correct. However, this sample size calculation assumes that the primary objective of the trial is to examine the percentage of women who have a pregnancy that ends in a live birth. For all the other analyses that have been performed, namely those related to secondary and exploratory endpoints, this sample calculation is not as crucial. To give just one example, when it comes to endometrial volume or thickness a sample calculation based on the proportion of life births has no reason to be (even if it is still a paired cohort study). It is sufficient to say that a convenience sample was studied, or the sample size should be computed on a hypothetical difference on volumes or thickness. In any case, in the description of the sample size calculation, the authors do not report the statistical power value used for the estimate. It is recommended that the power and alpha values assumed in the calculation are explicitly reported. 5. Typographical errors. Finally, we suggest a review of, e.g. in the methods section: “Patients were managed as described previously described…”", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "General This phase 1/2 trial assessed the safety, efficacy, and biological plausibility of autologous CD133+ bone marrow-derived stem cell (BMDSC) therapy for treating moderate to severe Asherman Syndrome (AS) in 20 infertile women unresponsive to prior treatments. The therapy was well-tolerated, with reversible limb paresthesia as the most common side effect. Significant improvements were observed in endometrial thickness, volume, and hysteroscopic scores. Clinically, menstruation and reproductive function were restored, leading to six live births following embryo transfer. Single-cell RNA sequencing revealed partial reversion of AS-related changes, and mitochondrial variant tracking showed insights into stem cell engraftment. The results suggest this therapy is safe and effective, warranting further investigation in larger trials. The manuscript is well-written, grammatically sound, and addresses a highly interesting topic, combining stem cell applications as orphan drugs with single-cell technologies. However, some technical aspects related to the bioinformatics analyses, such as the use of older packages (CellRanger, Seurat), as well as the provision of analysis scripts, could benefit from refinement. Major 1. How was the concentration of mobilized cells (at least 30 million cells with >70% purity and >50% viability) determined for this study? Is this concentration commonly used for CD133+ BMDSCs in this application? In cardiac regeneration studies, similar cell collections often use lower concentrations, how does this compare to your approach? 2. Could you explain the rationale behind using CellRanger Software version 3.1.0 (from July 2019) for this analysis, given that the current version is 9.0.1? Additionally, I noticed that Seurat version 4 was used, whereas version 5 is available for quite some time. The use of these older versions, both for CellRanger and Seurat, can potentially result in different analysis outcomes due to improvements or changes in data processing and downstream analysis methods introduced in newer versions. 3. Did the authors also perform quality assessments using tools like MultiQC to aggregate the overall quality of the samples? It might be beneficial to provide these quality reports as supplemental material to account for the sequencing quality. 4. Did the authors also perform dataset integration using Harmony (https://www.nature.com/articles/s41592-019-0619-0) in addition to the SCT transformation, which is commonly considered a standard approach for handling batch effects, particularly when samples have been processed in different batches? While Harmony is mentioned in the methods section, it appears to be described within the machine learning part, rather than in the single-cell processing section. Could you clarify when Harmony was applied in the analysis and why it was not used in all experiments? 5. Could you please include the cell-to-cell communication analyses in the methods section? 6. Providing the relevant scripts in a public repository would greatly enhance the overall reproducibility and depths of the data analysis. Minor 1. The required number of patients is inconsistent between the methods section and the study protocol, 20 versus 22. It may be helpful for the authors and readers to standardize and use a single number throughout the entire work. 2. The authors also may provide the exact version of the genome build used GRCh38-3.0.0. 3. Did the authors used specific databases, tools or scientific literature to annotate the single cell clusters - Reference 3 in the main manuscript (results) might be added also in the methods section in more detail, if the annotation is mainly based on previous works of the authors, including the species under investigation (e.g., murine, cell-based, …). 4. How was the selection of the 9 out of 20 patients was conducted for the single-cell analysis? 5. Please remove the “.” In the middle of the sentence in line 327."}, "author_response": "Reviewer #1 (Remarks to the Author): Review of the article by Santamaria et al “Autologous cell therapy with CD133+ bone marrow derived stem cells for Asherman Syndrome: a phase 1/2 trial’.The authors are reporting a non-randomized prospective open label trial on autologous hematopoetic stem cell therapy for Asherman syndrome cases resistant to existing therapies. The group has been working on the possibility of such therapy for over a decade now, and while this approach is cumbersome, expensive and possibly not available for many, it is an important pathophysiology driven treatment that has a potential. I want to commend on use of lineage tracing experiments showing the engra ftment of the CD133+ cells, and on demonstrated changes in the cell populations by single-cell RNA seq studies. Please see comments below. Comments: 1. Line 114, “the most frequent treatment-emergent adverse event…”. What do you mean? You mean this was the most serious adverse event? It was only in 1 patient, and what about chest pain or bleeding? A1. Thank you for your comment. Indeed, the term “treatment-emergent adverse effect” may seem confusing, and we have modified it “adverse event”. In the same manner, we have noticed that we mentioned limb paresthesia, which was a common effect before stem cell instillation, but the AEs we report here occur after administration. We have now modified the wording and now we refer to the most frequent AE after apheresis, which was nausea, headache and arm pain. We have included it in the text as indeed this was not clear in the current text (see line 118: “The most common adverse events after stem cell administration included arm pain, headache and nauseas, which in all cases were mild and reversible. Arm pain was mostly related to catheterism and was solved with local ice administration”(see table 2). 2. I suggest adding “infection” to the causes of AS. A2. We agree with the reviewer, infection has been added as a possible cause of AS (line 63): “…or after miscarriage and/or infection that disrupts the endometrial niche…”. 3. I recommend presenting the data on menstrual pattern and its recovery (before and after the treatment) in the text clearer and in Table 1 (or separately). It is an important clinical information. Provide reference for menstrual pictogram. A3. Menstrual pattern and its recovery are important clinical indicators. Since the majority of patients presented with amenorrhea or minimal bleeding at baseline, consistent with severe Asherman’s Syndrome, pre-treatment menstrual bleeding data were not recorded. This made unfeasible to apply a menstrual pictogram prior to treatment. Additionally, in accordance with the study protocol, menstrual patterns were prospectively assessed after patient enrollment. Specifically, they were assessed through 6 consecutive months following treatment. This has been now clarified in line 161: “Most patients did not have their period prior to the treatment and so menstrual blood records were prospectively assessed after therapy (1-6 months), as stated per protocol”. Further, to provide a comprehensive overview of the effects of the stem cell intervention on menstrual cycles, we present menstrual pattern data after 6 months treatment in Table 3 Pictorial Blood Assessment Chart (PBAC) Determination of total menstrual blood loss. Fertil Steril 76, 125-131 (2001). The reference for this is mentioned in the text. We refer to reference: Determination of total menstrual blood loss. Fertil Steril 76, 125-131 (2001). 4. Make sure it is clear in the Abstract and text that hysteroscopy was only diagnostic, no lysis of adhesions was performed, to be able to truly evaluate the effect of the therapy. This is really not clear. A4. Thank you for the comment. We have reviewed it through the text to make sure this is clear. Also, to clarify this concept, we have added the following statement in the Abstract (line 34) and the Methods section (line 53-54): “Hysteroscopic diagnostic evaluation was used to grade the severity of Asherman's Syndrome (AS); however, no operative hysteroscopy was performed”. 5. Table 1 and text: you did not present pregnancy rates and miscarriage rates, just live births. How many transfers were performed per patient and in total to get the 6 live births? A5. We have now added two extra columns in Table 1 indicating the number of transfers and clinical outcome (live birth, miscarriage, non-pregnancy). We performed a total of 41 embryo transfers in 19 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41) resulting in 5 live births. In addition, we recorded one spontaneous pregnancy resulting in live birth from patient N11. This has been now clarified in Table 1 and the following sentence has been added in lines 169: “we performed a total of 41 embryo transfers in 19 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41)”. 6. Lines 164-166: these conclusions should be softer as clinical significance is not clear, if any. What about difference in delta from pre- to post- in these measures? A6. Thank you for your comments. The mean change (delta) from pre- to post- measures in endometrial thickness was 1.49 +/- 0.63mm, and for endometrial volume was 1.52 +/0.80cm3 —both statistically significant (p<0.01). (Supplementary Figure 1B and 1C). Nevertheless, we have softened the conclusion and it’s included from line 184 to191: “These findings suggest that achieving an endometrial thickness of approximately 5.46 ± 0.89 mm and an endometrial volume of around 3.62 ± 0.85 cm³ may be associated with improved live birth outcomes in this study Although we show a potential association between enhanced endometrial characteristics and improved reproductive outcomes, it is important to note that live birth outcomes are also influenced by embryo transfer–related factors, such as embryo quality, number of transfers, timing, etc. These elements likely interact to determine overall success and need to be taken into consideration. 7. Line 182: what do you mean by uterine curettage was performed by operative hysteroscopy? Do you want to say that retained products of conception were removed hysteroscopically? I do hope that uterine curettage was not performed in this AS patient. A7. The reviewer is correct. We meant that the retained products of conception were removed hysteroscopically. The sentence has been revised as follows: “One of the participants experienced a first-trimester miscarriage that required hysteroscopic removal of the retained products of conception” (lines 203). 8. Single-cell RNA seq results: why would AS-epithelial cells increase after cell therapy? Aren’t they being replaced more by epithelial cells? A8. Thank you for your insightful question. We believe you are referring to Figure 4B. To clarify, in Figure 4A the cluster corresponding to AS-epithelial cells is reduced post-treatment, and Figure 4C shows that the relative abundance (cell ratio) of AS-epithelial cells decreases significantly after CD133⁺ BMDSC therapy. However, Figure 4B presents a different type of analysis: it shows the enrichment score of a gene set signature associated with endometrial functionality during the secretory phase (including genes such as SCGB1D2, MT1F, MT1X, CXCL14, WNT5A, etc.). This gene set was derived from our previous work identifying markers enriched in the early- and mid-secretory phases. Therefore, the increase observed in Figure 4B for the AS-epithelial population does not reflect a numerical increase in cell count, but rather an upregulation of this functional gene set within the remaining AS-epithelial cells post-treatment. In other words, although the proportion of AS-epithelial cells decreases (Figures 4A and 4C), those that persist exhibit higher expression of genes associated with a receptive, functional endometrium. This suggests that CD133⁺ BMDSC therapy not only shifts the cellular composition but may also reprogram the remaining AS-epithelial cells to a more functional state, contributing to improved endometrial receptivity. 9. Analysis of unique mtDNA: why only 2 patients? Also, in the text you mention only 2 patient samples, but in Figure 4E and figure legend its 3 patients, unless I am getting it wrong. A9. We apologize for the confusion. We indeed performed three of them, so the figure legend is correct (n = 3). We have now updated the text accordingly (line 295: “Comprehensive profiling in three patients revealed 32, 9 and 2 shared mtDNA variants in the endometrium post-treatment respectively…”). As of why only three patients, the MAESTER protocol needs to start with the reamplification of the left over cDNA used to run the single cell RNA seq data. In this case we only had enough good quality cDNA samples left to run MAESTER in these three samples. 10. When you get to organoids, the description of results is more chaotic and you are losing the reader. We thank the reviewer for this valuable feedback. We understand that clarity and logical flow are crucial, especially in complex sections such as the organoid experiments. To address this point, we have thoroughly revised the corresponding section of the manuscript to improve organization and readability. We believe these changes significantly improve the flow and coherence of the organoid results section and help maintain the reader’s engagement. 11. What are the in vivo controls? A11. The in vivo controls used for the organoid studies referred to endometrium obtained from healthy women that served as controls (healthy endometrium) presented in detail in our previous study by Santamaria, X., Roson, B., Perez-Moraga, R. et al., Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution, Nat Commun 14, 5890 (2023). https://doi.org/10.1038/s41467-023-41656-1 12. I am not sure how you showed the improved receptivity; that’s an overstatement. A12. We think you refer to line 320, where we stated “These results may offer insights into the differences in organoid development after treatment, reinforcing the claim of glandular epithelium differentiation towards a more receptive profile after treatment”, since we do not see any claims on “improved receptivity” throughout the text. If you refer to this part, we have now modified it from line 315-318 to the following statement “Although the proportions of Post-AS therapy do not fully mirror those of healthy controls, the shift in epithelial glandular composition after treatment may indicate a trend toward functional remodeling, including an enrichment of cells with a potentially more receptive-like profile (Figure 5C)”. 13. What are the future directions? Are the authors proposing large clinical trial (RCT) or to move this therapy to clinic now, without phase 3? Not clear how this will look like. Should mention the associated health care costs and alternatives, as well as interdisciplinary approach involved in this treatment. A.13. We are currently in the process of reviewing our protocol for a Phase 3 trial from the European Medicines Agency (EMA) which will start in Q4 2025 involving approximately 30 patients. Since this condition is considered a rare disease, and we have been granted orphan drug designation by both the FDA and EMA, we are hopeful that, upon successful completion of Phase 3 and receipt of the necessary regulatory approvals, this therapy can be advanced to clinical use. Healthcare costs have not yet been calculated, as they will depend on the final approved protocol. 14. Figure 3: why didn’t you present all 19 patients that completed the treatment and post-treatment evaluations? A14. We appreciate the reviewer’s suggestion to include additional hysteroscopy images. However, due to technical limitations at the time of patient follow-up—including temporary unavailability of the hysteroscopy equipment and, in some cases, equipment malfunction—it was not possible to systematically acquire additional endoscopic images beyond those already included in Figure 3A. Although images are not available for all patients, visualization from the side of the clinician at the time of analysis was performed, so that IUA score for all the patients is present (n=19). 15. Who was performing the hysteroscopies, before and after the treatment? Were clinicians blinded to the therapy timing such as did they know that they are doing pre- or post-stem cell therapy cavity assessment? How was their bias managed? A15. All hysteroscopies were performed by the same surgeon (XS), who was not blinded to the treatment allocation. To minimize potential bias, the American Society for Reproductive Medicine (ASRM) classification for intrauterine adhesions (IUAs) was strictly followed, with scoring thoroughly documented through photographic evidence and independently reviewed in a blinded manner by a second surgeon (JT). 16. When was the endometrial thickness measured, in which cycle phase? I understand that these are moderate-severe AS patients, but if you want this study to be taken seriously, you have to be consistent in when the lining thickness is measured. Where in the menstrual cycle were the 2D and 3D evaluations done, hysteroscopy and then the stem cell treatment? If amenorrhea, cycle phase could have been assessed by scanning ovaries and hormonal testing. A16. In the Methods section, under Procedures (lines 43–99) of our original paper, the reviewer can find relevant information. Specifically, during the simulated “secretory phase” (five days after progesterone administration), endometrial status was assessed both before and after treatment. This evaluation included histological digital image analysis, 2D/3D transvaginal ultrasound (Samsung, South Korea), and hysteroscopic assessment using the ASRM classification of intrauterine adhesions (IUAs)¹. Hysteroscopies were performed with a 5 mm Betocchi hysteroscope (Karl-Storz). These procedures were conducted in accordance with the protocol approved by the local ethics committee and the Spanish Agency of Medicines and Medical Devices (AEMPS). 17. Table 1: why NA for endometrial volume assessment in 3 patients? Endometrial volume data is presented for 16 patients rather than the full cohort of 19. This discrepancy is due to a temporary unavailability of the 3D transvaginal probe, which prevented the acquisition of high-quality volumetric imaging for three patients at all required time points. Consequently, 3D imaging data could only be obtained and analyzed for 16 patients. 18. Table 2: I suggest using “false passage instead of “false path”. “Bleeding”- what is it, uterine/vaginal or intraabdominal? Vomiting (not vomit. What is Abdominal swelling, is it hives on the skin, or bloating? You should be more specific about skin rash, just localized or generalized? Also, is arm pain same as pain associated with arm hematoma? What is respiratory infection, just URI or something more serious? We have modified the wording according to the reviewer's suggestion and table 2 is now updated. 19. Was the brachial artery catheterization done under anesthesia or procedural sedation? Just with procedural sedation. This has been added in line 85 of methods doc. Minor comments: 1. Some language revision is recommended for sentence flow, and some typos and missing words. We have revised language throughout the whole text. Thank you 2. Sentence on lines 76-79: please revise, not too clear. Done. This is now line 75-77. 3. In the Results, please be consistent when writing pre-AS, pre-treatment AS or AS-pre- and -post. Both in the text and Figures. Thanks, we have consistently used pre-AS therapy and post- AS therapy throughout manuscript and figures 4. Discussion, line 306: are your references 17-23 or 28 as stated? Reference list goes to 23 only. References are 17-23, thanks, it has already been modified Reviewer #2 (Remarks to the Author): The authors reported the safety and efficacy of intrauterine intravascular injection of bone marrow-derived CD133+ stem cells in treating 20 cases of refractory Asherman syndrome. Single-cell sequencing of endometrial tissues from a subset of cases before and after treatment was performed to investigate the stem cell homing and differentiation，and transcriptional profile changes of the administered cells in vivo, as well as organoid formation capacity and associated expression alterations. While the study is interesting and holds significant clinical relevance, several critical aspects require further elucidation to strengthen its translational impact. 1. In the research proposal, this research was described as a prospective, multicenter, and self-controlled study. However, the current report appears to be based on work conducted at a single center. Could you please provide the reasons? A1. Thank you for the opportunity to clarify this point. As outlined in the original research proposal, the study was designed as a prospective, multicenter, self-controlled clinical trial involving three institutions: Clínica del Pilar, Vall d’Hebron, and Quirón. While all three centers contributed to specific aspects of the study, the majority of patient recruitment and data collection ultimately took place at Clínica del Pilar. This was primarily due to its stronger infrastructure and higher patient flow for the targeted clinical indication, which made it the most efficient site for enrollment. Nonetheless, the other two centers were actively involved in critical components of the protocol. Specifically: Vall d’Hebron participated in hematological assessments and catheterization procedures. Quirón was responsible for embryo transfer procedures. Clínica del Pilar conducted gynecological assessments and hysteroscopies, in addition to leading patient recruitment. Therefore, while the report may appear to reflect single-center activity due to the concentration of patient data from Clínica del Pilar, the study remains a collaborative multicenter effort, with each site contributing to different phases of the clinical workflow. 2.Result 1 On line 94, the author states that “Following diagnostic hysteroscopy, hematopoietic stem cells were mobilized, harvested through leukapheresis, and selected for CD133+ and intra-endometrial spiral arterioles instillation (Fig. 1A).” Was surgical separation of intrauterine adhesions required before injection? In which phase of the menstrual cycle should the injection be administered? A2. Hysteroscopic diagnostic evaluation was used to grade the severity of Asherman's Syndrome; however, no operative hysteroscopy was performed. Surgical separation of intrauterine adhesions was performed to facilitate endoscopic access for diagnostic evaluation. Cell injection was conducted during a simulated mid secretory phase, as all patients were receiving hormonal replacement therapy owing to the absence of spontaneous menstruation in many of them (see Methods section, lines 44-99) 3. How was the cell therapy dosage determined? Was it simply based on the amount of cells that could be isolated and obtained? A3. Thank you for your comment. In this Phase 1–2 study, the administered dose of CD133⁺ BMDSCs was not pre-specified nor determined through a dose-escalation design. Instead, it was based on the total number of CD133⁺ cells that could be isolated and enriched from each individual patient following bone marrow aspiration. The actual cell yield depended on several patient-specific factors, including their response to filgrastim, vascular access quality, and tolerance to apheresis. As a result, the observed dose range (37–166.1 × 10⁶ cells, 75% CI) reflects inter-patient variability in cell yield rather than adherence to a standardized dosing protocol. This pragmatic approach was chosen to assess the feasibility and safety of autologous cell therapy in a real-world clinical setting. 4. In secondary endpoint, I noticed that the specific location of the endometrial biopsy for comparing histological changes and immunohistochemical staining before and after treatment seems to be missing in the methodology section. Could you please provide more details on this aspect? A4. In moderate to severe Asherman syndrome, the location of endometrial biopsies depends on the presence of identifiable areas of bona fide endometrium rather than on a specific topographical site. This is detailed in the Methods section (Procedures subsection lines 55-61.\" Specifically, it says: Endometrial biopsies were performed under hysteroscopic vision from the posterior wall of the uterus at least 1 cm away from any visually identified IUA using a semi-rigid double-action biopsy and grasping forceps (Karl-Storz, Germany). 5. In the legend of Fig. 2, the sample sizes indicated for the box plots of endometrial volume (n=16) and endometrial thickness (n=19) appear to be inconsistent with the figure itself. It is possible that there might be a typographical error by the author. Why are the sample sizes for the analysis of endometrial thickness and endometrial volume different—one being 16 cases and the other 19 cases—when the total sample size should be 20 cases? A5. We thank the reviewer for the comment and appreciate the opportunity to clarify this point-by-point. Total cases — why not 20? Although 20 patients were initially enrolled in the study, one participant (Patient N11) voluntarily withdrew before completing the follow-up assessment and was therefore excluded from the post-treatment analysis. As a result, the total number of cases analyzed was 19. Why is the sample size for endometrial thickness 19, but 16 for endometrial volume? Endometrial thickness was measured using in situ ultrasound imaging during patient appointments. Consequently, we were able to obtain endometrial thickness data for all patients who participated in the studies (n = 19). Regarding endometrial volume, there was a period during the study when the 3D transvaginal ultrasound probe was unavailable for technical reasons. This limitation prevented us from acquiring high-quality volumetric imaging for three (12, 13 and 14) patients at all required time points. Therefore, reliable 3D endometrial volume data are available for only 16 patients. Regarding Figure 2A: Since the purpose of Figure 2A was to compare 2D and 3D measurements, we included only 16 patients for whom both endometrial thickness and volume data were available. This explains why the figure presents data for only 16 individuals. We hope this clarification resolves the reviewer’s concerns 6. Regarding the efficacy, the key efficacy endpoint is the live birth rate. From Table 1, it appears that successful pregnancy mainly depends on the severity of the disease. Could the author address this point in the manuscript? A6. Thank you for this thoughtful observation. At this stage, we believe we do not have sufficiently robust data to support a definitive conclusion regarding the relationship between disease severity and successful pregnancy outcomes. There are several limitations that preclude a meaningful analysis: Sample size: The overall number of patients included in the study is limited (n = 20), which reduces the statistical power to detect significant associations. Severity distribution: The distribution of Asherman syndrome severity among participants is imbalanced, with 12 patients classified as having moderate AS and 8 as severe. Live birth outcomes: Looking at the cases of live birth, it is true that there is a trend towards improvement in moderate cases, however, the small numbers and heterogeneity of the cohort prevent us from drawing reliable conclusions. We acknowledge the importance of this point, and we have included the following paragraph in the manuscript, specifically in lines 175-177: “Interestingly, the patients who achieved a live birth had been diagnosed with moderate-stage AS, suggesting that treatment efficacy may be influenced by disease severity. However, given the small sample size and data heterogeneity—the unequal distribution of moderate and severe cases—this observation should be considered preliminary”. 7.On line 256, the author states that “RNA-based UMAP clustering post-treatment demonstrated the presence of CD133+ mtDNA clones within diverse endometrial cell populations (Figure 4E), confirming the grafting and multilineage differentiation capacity of CD133+ BMDSC post-cell therapy.” However, it may be necessary to have experimental evidence to confirm the differentiation capacity of CD133+ BMDSC, rather than relying solely on the findings from single-cell sequencing. The viability of the cells used for treatment was >50%. Could it be that dead cells entered the body, their mitochondrial DNA was engulfed or phagocytosed by other cells, rather than differentiating into other cell types? A7. We thank the reviewer for their valuable comments. Just to clarify, as this was a Phase 1-2 clinical trial, the product specification was established in agreement with the European Medicines Agency (EMA), permitting a minimum CD133⁺ cell viability threshold of 50%. However, the actual data reported a mean of 99.7±0.27% on the cell viability for the patients, so the proposed scenario seems unlikely. We understand the 50% minimum requirement may rise some questions so to clarify this point we have now added this in methods (line 72-73) and in the draft (line 103-106) the actual results of the purity and viability of the presented patients. Regarding CD133 differentiation, we generated experimental evidence confirming the multilineage differentiation potential of CD133⁺ BMDSCs. These cells were cultured on gelatin-coated plates (~1×10⁵ cells per well) and subjected to lineage-specific induction protocols for osteogenic, adipogenic, and stromal differentiation. Furthermore, we provide proof of concept demonstrating that stromal cells differentiated from CD133⁺ BMDSCs are capable of undergoing decidualization in vitro. This was achieved using a well-established in vitro decidualization protocol, supporting their functional capacity and relevance in downstream applications (see below Figures for the reviewer only). Osteogenic Differentiation Osteogenic induction was carried out using the MesenCult™ Osteogenic Differentiation Kit (STEMCELL Technologies) over a 14-day period as previously described. Post-induction, the cells were fixed and stained for two key markers: alkaline phosphatase (ALP), indicative of early osteoblast commitment and matrix mineralization, and osteocalcin (OCN), a marker of late osteoblast maturation. A subset of cells exposed to the osteogenic differentiation medium displayed cytoplasmic expression of both ALP and OCN, confirming their commitment to the osteogenic lineage (Figure 1, reviewer only) Figure 1 (Reviewer only). Immunofluorescence analysis of CD133⁺ BMDSCs under osteogenic differentiation conditions. Representative images showing nuclear staining with DAPI (blue), alkaline phosphatase (ALP, red), and osteocalcin (OCN, green). CD133⁺ BMDSCs cultured without osteogenic differentiation medium are shown as the control. ALP and OCN expression confirm osteogenic commitment in treated cells. Adipogenic Differentiation CD133⁺ BMDSCs were cultured in MesenCult™ Adipogenic Differentiation Medium (STEMCELL Technologies) for 14 days to induce adipogenic differentiation. Successful differentiation was confirmed by the cytoplasmic expression of adiponectin, a well-established marker of mature adipocytes, which was observed exclusively in treated cells (Figure 2, reviewer only). Figure 2 (reviewer only). Immunofluorescence analysis of CD133⁺ BMDSCs under adipogenic differentiation conditions. Representative images display nuclear staining with DAPI (blue, left panel) and adiponectin expression (green, right panels). Cells cultured in adipogenic differentiation medium exhibit prominent cytoplasmic accumulation of adiponectin, particularly in the bottom right panel, indicating successful adipogenic induction. In contrast, control cells (top right) show minimal or no adiponectin expression Stromal Differentiation To induce stromal differentiation, CD133⁺ BMDSCs were cultured for 7 days in conditioned medium derived from primary human fibroblasts composed of DMEM/F12 with 10% fetal bovine serum (FBS), 5-7ng/ML of FGF 7 (Fibroblast Growth Factor 7) and 1% penicillin–streptomycin incubated at 37 °C in 5% CO . This 2 medium was collected from confluent fibroblast cultures every 24-48h, filtered, and applied to CD133⁺ cells seeded on gelatin-coated plates. Immunofluorescence analysis was then performed throughout the 7-day culture period to assess the acquisition of a stromal phenotype. Cells were stained for vimentin, a mesenchymal marker, and cytokeratin, an epithelial marker. A robust vimentin signal was detected in a subset of cells at days 5 and 7, exclusively in the group treated with conditioned medium. Cytokeratin expression was undetectable in both treated and control groups across all time points, confirming a mesenchymal phenotype and lack of epithelial features (Figure 3, reviewer only). Figure 3 (reviewer only). Phenotypic characterization of stromal cells derived from CD133⁺ BMDSCs. Immunofluorescence staining was conducted for vimentin (red), a marker of mesenchymal cells, and cytokeratin (red), a marker of epithelial cells. Nuclei were counterstained with DAPI (blue). Cells cultured with fibroblast-conditioned medium exhibited strong vimentin expression and no detectable cytokeratin, supporting their stromal identity. To further demonstrate their functional capacity, stromal-like cells derived from CD133⁺ BMDSCs were subjected to an in vitro decidualization protocol for 7 days. Cells were treated with 1 mM cyclic AMP (cAMP) and medroxiprogesterone (MPA), following established protocols². Expression of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1), two hallmark decidual markers, was evaluated by immunofluorescence, qRT-PCR, and ELISA. Immunofluorescence analysis revealed a marked increase in IGFBP1 (red) and PRL (magenta) in cAMP-treated cultures compared to untreated controls. Quantitative RT-PCR confirmed these observations, showing a ~2.7-fold upregulation of IGFBP1 and ~16-fold upregulation of PRL. Consistently, ELISA detected elevated levels of secreted prolactin in the conditioned media, with the highest concentrations observed in the decidualized CD133⁺ group. Together, these results provide compelling evidence that stromal cells differentiated from CD133⁺ BMDSCs are competent to undergo decidualization at transcriptomic, protein, and secretory levels (Figure 4, reviewer only). Figure 4 (reviewer only). Decidualization of stromal cells derived from CD133⁺ BMDSCs. Left: Representative immunofluorescence images of control (top row) and decidualized (bottom row) cells, showing increased expression of PRL (magenta) and IGFBP1 (red) in the decidualized group. Right: qRT-PCR analysis shows significant upregulation of IGFBP1 (~2.7-fold) and PRL (~16-fold) in decidualized cells relative to controls. ELISA quantification confirms increased prolactin secretion in the decidualized CD133⁺ stromal group. References 1. Cervelló I, Mas A, Gil-Sanchis C, Peris L, Faus A, Saunders PTK, et al. (2011). Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines. PLoS ONE, 6(6): e21221. https://doi.org/10.1371/journal.pone.0021221 2. Garrido-Gómez T, et al. (2017). Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology. Proc Natl Acad Sci USA, 114: E8468–E8477. 8. The author selected endometrial samples from nine patients before and after treatment for subsequent single-cell sequencing analysis. However, the author did not explain the criteria for selecting these nine cases. The relevant clinical information for these nine patients should be provided. Additionally, the results of the single-cell sequencing need to be validated experimentally. A8. The subgroup of AS patients selected for single-cell RNA sequencing was randomly chosen to ensure enough cells for robust transcriptomic analysis. This subgroup comprises patients 5, 6, 7, 8, 9, 10, 13, 14, and 15, whose pre-treatment results were previously compared against healthy endometrium samples and published in Santamaria, X., Roson, B., Perez-Moraga, R. et al., Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution, Nat Commun 14, 5890 (2023). https://doi.org/10.1038/s41467-023-41656-1. The current publication of the post-treatment results is justified by the fact that exploratory outcomes related to clinical trials cannot be disclosed until the primary and secondary outcomes have been reported. Accordingly, we now present the post treatment findings of this exploratory outcome. Relevant clinical data for these patients is provided in Table 1. 9.In Single-cell RNA sequencing of endometrial epithelial organoids (EEOs) derived from endometrial biopsies pre- and post-treatment，the clinical information of the patients from whom the organoids were derived should be provided. A 9. The patients from whom EEOs were derived correspond to numbers 13, 14 and 15. We have now included relevant clinical information in Supplementary Data and named in line 298. 10.In discussion part, to facilitate readers' understanding of the progress in stem cell therapy for Asherman's syndrome, the author would provide a brief and objective overview of the published studies. When emphasizing the strengths of this research, the author mentions the use of \"a specific, well-characterized cell\" for treatment. However, based on the \"methods\" section, the purity of the cells used is about 70%. What about the remaining 30%? What types of cells do they consist of, and what is their impact on endometrial regeneration? It would be best if the author could clarify this. A10. Thank you for this insightful comment. In line with the answer regarding cell viability, the concern regarding cell purity is indeed well-founded. As this was a phase 1-2 clinical trial, the product specification was established in agreement with the European Medicines Agency (EMA), permitting a minimum CD133⁺ cell purity threshold of 70%. This relatively permissive criterion was considered appropriate given the exploratory nature of the trial, which required balancing manufacturing feasibility with safety and efficacy considerations at this early stage. To better characterize the final cell product administered to patients, we performed fluorescence-activated cell sorting (FACS) on an aliquot from each of the 20 manufactured batches. This analysis revealed a mean CD133⁺ cell content of 89.54%, with a standard deviation of 8.29%, demonstrating that the vast majority of preparations exceeded the predefined minimum purity threshold by a substantial margin. We have now indicated this detail in both methods and manuscript to clarify it (lines 97-100 of manuscript and line 79 of methods). Additionally, we evaluated the composition of the CD133- (negative) BMDSC fraction using FACS with lineage-specific markers. The mean expression levels and standard deviations for non-target cell populations were as follows: • CD56⁺ (Natural Killers): 0.02± 0.041% • CD66b⁺ (granulocytes): 0.465±0.449% • CD14⁺ (monocytes): 1.87±2.62% • CD19⁺ (B cells): 0.385±0.221% • CD3⁺ (T cells): 0.055±0.076% • CD34⁺ (hematopoietic progenitors): 8.66±11.77% • CD45⁺ (leukocytes): 12.42±11.17% These results confirm that the administered product was consistently enriched for CD133⁺ cells while maintaining minimal contamination by other hematopoietic lineages. Reviewer #3 (Remarks to the Author): This study titled ‘Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase1/2 trial’ by Xavier Santamaria et al., present promising results on the use of autologous CD133+ bone marrow-derived stem cells (BMDSCs) for treating Asherman Syndrome (AS), a condition with limited treatment options. I have several questions and comments listed below that need to be answered by the authors. My major points are these and further details follows below: Major points: Without a control group/placebo, interpreting the real effectiveness of the intervention is very difficult because you lack a baseline for comparison in stage 2. The main limitation of this study is that the data are limited to only one-month post-transplantation, which is not sufficient to assess the long-term safety and efficacy of the treatment. Extended follow-up, ideally up to the trial’s defined 15-month endpoint is necessary to support the conclusions. Additionally, the absence of a control group makes it difficult to confidently attribute observed effects to the treatment. While the hysteroscopic images provide some information, their value is limited due to variation in sampling location, unknown menstrual cycle phase and inconsistent hormone levels during the first month. These factors introduce variability and make it hard to draw firm conclusions from the histological data. 1. For the 20 patients recruited to the study, they all underwent reparative operative hysteroscopies (range 1 to 11). During how long time was treatment attempted? A1. We thank the reviewer for this important observation. All of these infertile patients had undergone reparative operative hysteroscopies between 1 and 7 years prior to enrolling in this Phase 1–2 study, reflecting the real-world heterogeneity commonly encountered among patients with Asherman's Syndrome. 2. Authors mentioned that despite the previous surgical treatments, no patients reported improvement of endometrial thickness. Did they not receive conventional hormonal therapy after the previous operation? A2. We thank the reviewer for this pertinent question. Indeed, all patients received conventional hormonal replacement therapy (HRT) prior to undergoing the study intervention. Importantly, all diagnostic and follow-up hysteroscopies—both before and after treatment—were performed under HRT, specifically timed during the window of implantation phase, to ensure consistency and comparability of endometrial assessments across patients. In addition to HRT, most patients had also undergone other therapeutic interventions, including intrauterine devices (IUDs), platelet-rich plasma (PRP) infusions, and various intrauterine barrier methods. Despite these conventional and adjunctive treatments, no significant or sustained improvement in endometrial thickness was observed prior to enrollment in the study. This context underscores the refractory nature of the patient population, highlighting the unmet clinical need that this study aims to address. 3. After the cell transplantation, have you considered administered estrogen supplement? A3. Yes, we administered not only estrogen but also progesterone as part of the hormonal replacement therapy (HRT). All patients received HRT both before and after the AS treatment (now included in line 45 from methods document). These procedures were carried out in accordance with the protocol approved by the local ethics committee and the Spanish Agency of Medicines and Medical Devices (AEMPS). 4. For the secondary endpoints, authors stated that ‘pretreatment patient menstrual blood loss was around 29.6ml increasing to 55.2ml in the first month post-treatment, however, decreased to 31.1ml and 29.9ml in third and fifth month post-treatment. What could be the reason for this? In addition, you recruited moderate to severe AS patients, these patients are most likely amenorrheic or spotting. What is the menstrual conditions pre-treatment for these patients? There is no information in Table 1. A4. We agree that menstrual pattern and its recovery are important clinical indicators. In accordance with the study protocol, menstrual patterns were prospectively assessed after patient enrollment, specifically, these were assessed 6 consecutive months following treatment. We have now added a new table (table 3), providing the menstrual pattern data using a Pictorial Blood Assessment Chart (PBAC). Regarding the menstrual conditions pre-treatment, this data was not recorded, as the majority of patients presented with amenorrhea or minimal bleeding at baseline, consistent with severe Asherman’s Syndrome. This made it unfeasible to apply a menstrual pictogram prior to treatment so the data presented is based after 1-6 months after stem cell therapy. We have also clarified this in line 162-166 to avoid confusion: “Most patients did not have their period prior to the treatment and so menstrual blood records were prospectively assessed after therapy (1-6 months), as stated per protocol. Menstrual blood loss records showed an increase from 29.60±45.40 mL in the first month post-therapy to 56.66±107.41 mL after 2 months post-therapy. Data showed then a decrease in the records, with 27.66±19.79 mL in the last month after treatment (M6) (Table 3)”. Regarding the decrease in blood loss, in our proof-of-concept study (Santamaria et al., Hum Reprod, 2016), we reported comparable clinical findings: menstrual cycles resumed with hormone replacement therapy (HRT) during the first month following cell therapy. However, both the duration and intensity of menstruation progressively decreased. The mean duration fell from 5.06 days (range: 3–7) in the first month to 3.25 days (range: 1–3) by the third month post-treatment. Similarly, menstrual volume, as measured by the number of sanitary pads used per day, declined from a mean of 2.69 (range: 1–5) to 1.75 (range: 1–4) pads per day by the third month. Our current clinical experience indicates that subsequent cell injections produce a similar clinical effect, supporting the hypothesis that the observed effects result from temporal engraftment of the injected cells rather than permanent integration. 5. Regarding the dose range (37–166.1 × 10⁶ cells): Was this range selected based on prior studies, or was it a dose-escalation design? The rationale for the wide range should be clarified. A5. Thank you for your comment. In this Phase 1–2 study, the administered dose of CD133⁺ BMDSCs was not pre-specified or based on a dose-escalation design. Instead, it reflected the total number of CD133⁺ cells that could be isolated and enriched from each individual patient following bone marrow aspiration. Therefore, the optimal observed dose range (37–166.1 × 10⁶ cells, CI 75%) reflects inter-patient variability in cell yield rather than a predefined dosing strategy. This approach was chosen to assess feasibility and safety in a real-world autologous setting. 6.Reversible Limb Paresthesia: This is an interesting and somewhat unexpected adverse event. Further discussion on the mechanism would be valuable. A6. Thank you for the comment, we have now updated this part in the manuscript (please see line 127) to clarify this part. Limb paresthesia was the most common AE before stem cell instillation (CD133 collection), which was likely due to transient hypocalcemia from the apheresis. After stem cell instillation (CD133 administration), the most common AEs are reported in Table 2, and these include nausea, headache and arm pain. To avoid confusion, we rewrote it in the text (line 119) so we only mention the AEs after CD133 administration, which are those present in Table 2. 7. No haemopoietic malignancies were reported. For how long will the patients be followed and what is known regarding this risk? A7. We appreciate the reviewer’s concern regarding the risk of haemopoietic malignancies. We confirm that no haemopoietic malignancies have been reported during a follow-up period of 15 months in our study population. All patients are being monitored in accordance with our established safety follow-up protocol, which includes routine hematological evaluations and clinical assessments. Further, the therapy employed is fundamentally analogous to an autologous bone marrow transplant—a well-established medical procedure first performed in 1957 with a strong safety record. Given that the infused cells are autologous and minimally manipulated, the theoretical risk of malignant transformation is considered extremely low and comparable to standard autologous cell therapies. 8.Endometrial Improvements: authors reported increases in thickness, volume, and hysteroscopic scores are encouraging. Was there a correlation between cell dose and response/duration of effect? A8. At this stage, no correlation was observed between the administered cell dose and either the treatment response or the duration of the effect. This absence of association is illustrated in Supplementary Figure 1E. 9. The authors mentioned a key outcome about reproductive endometrial function recovery. It includes 6 Live Births and 6 miscarriages. Before the cell transplantation treatment did these patients also receive embryo transfer? A9. Thank you for your comments. Prior to undergoing cell transplantation treatment, these patients also received embryo transfers, with a mean of 1.95 transfers (range: 0–8). 10. How many Et attempts per patient? More than one? A10. The embryo transfers (ET) performed during the study were often more than one. This information has now been included in a new column in Table 1. 11. The authors added mechanistic Insights by applying Single-cell RNA Sequencing before and after treatment. In the previous paper (Santamaria X, et al. 2023), authors identified two unique cell types only present in AS (AS epithelium and smooth muscle cells). The cells isolated for scRNA-seq most likely came from superficial layer of the endometrium. Do authors think that severe AS are more related to damaged basalis layer? In that case the mechanistic study would need to include basalis tissue to truly reveal the efficacy and underlying mechanism. A11. The cells used for single-cell RNA sequencing (scRNA-seq) were isolated from endometrial biopsies that encompassed both the functionalis and basalis layers of the endometrium, as confirmed by the H&E staining of a representative sample (see figure below). In this figure, we can see the presence of the myometrium in the H&E images, which provides clear evidence that the biopsy extended through the full thickness of the endometrium, including the basalis layer. In cases of severe AS, the endometrial lining is markedly thinned due to fibrosis. Given this thinning and the histological presence of myometrium, we are certain that the basalis was sampled. Therefore, the scRNA-seq data likely captures the cellular composition of both superficial and basal compartments, even in the context of a fibrotic endometrium. Figure for reviewer only. Representative images of H&E endometrium tissue collected from AS patients. (E: endometrium, M: myometrium). 12.The finding of \"partial reversion of AS-related changes\" is intriguing but vague. Were there specific gene pathways or cell populations most affected except for the increase of several cell types? What about the post-treatment changes of the two AS unique cell types? A12. Thank you for your insightful comments. We have now specified more the cell populations involved and have extended some of the key gene pathways. Below, we provide a more extensive version of which specific gene pathways and cell populations were most affected by treatment, as well as the behavior of the two AS-unique cell types. Cellular composition changes: Following CD133+ BMDSC treatment, we observed a expansion trend of key endometrial populations, including epithelial, ciliated epithelial, endothelial, and cycling cells, accompanied by a marked reduction in the AS-specific epithelial cluster and macrophages. These changes suggest a shift toward a more regenerative and less inflammatory tissue state. Gene expression and pathway changes: In Figure 4B, we evaluated a gene set signature associated with endometrial function during the early- and mid-secretory (WOI) phases of the menstrual cycle, previously identified by our group. This set includes: SCGB1D2, MT1F, MT1X, MT1E, MT1G, CXCL14, WNT5A, SFRP1, ZFYVE21, CILP, SLF2, MATN2, and S100A4. After treatment, this gene set showed increased expression in epithelial, AS-epithelial, stromal, endothelial, and perivascular (PV) cells, suggesting enhanced endometrial functionality and potential receptivity to implantation. Notably, although the AS-epithelial population was reduced in abundance, the remaining cells were enriched in this gene set. Differential expression analysis (Supplementary Figure 3): • Epithelial cells showed increased expression of SCGB1D2 (secretory function), LGALS1 (immune modulation), and PDGFRA/IGF1 (ECM remodeling and regeneration), along with decreased expression of pro-inflammatory and immune-related genes such as S100A10, IL6ST, MIF, and DEFB1. • Stromal cells exhibited upregulation of genes associated with angiogenesis and migration (IGFBP2, PTN, SPARCL1), and downregulation of proliferation (FOS, JUN), stress response (SOD2, HSPA1A/B), and anti-angiogenic genes (IGFBP3). • Endothelial cells shifted from a stressed, pro-inflammatory profile to a more homeostatic one, with decreased expression of ICAM, HLA-DRA/DRB5, FOS, JUNB, and increased collagen-related ECM genes (COL1A1, COL3A1, COL6A1, COL6A3) and IGFBP2. • The PV cell, contractile PV cell, and SMC populations also displayed reduced cellular stress response (with decreased expression of S100A6 after CD133+ BMDSC administration. Overall, the transcriptomic profiles of these populations suggest that the PV and contractile PV cell populations exhibit a loss of muscular characteristics, which results in increased proliferation of PV cells and differentiation towards endothelial cells. Cell–cell communication (Figure 4D and Supplementary Figure 4): Signaling pathways associated with immune response and inflammation (e.g., CCL, CXCL, MHC-I/II, LIGHT, NECTIN, CD22, TNF, and RESISTIN) were reduced after therapy, as was the ITGB2 pathway associated with fibrosis. In contrast, epithelial development pathways (CADM) and angiogenesis pathways (PTPRM, FGF) were enriched. We also observed an increased prevalence of ANGPTL2 signaling, highlighting a shift toward vascular regeneration, in line with the balanced activity of ANGPTL1/2 known to support angiogenesis. Taken together, these findings suggest that CD133+ BMDSC therapy promotes endometrial regeneration by enhancing epithelial secretory capacity, suppressing pro-inflammatory and pro-fibrotic signaling, promoting angiogenesis through ANGPTL pathway activity, and partially reprogramming AS-specific cell types, including reduction and functional reactivation of AS-epithelial cells. We have now amended the discussion to add some of these key points (lines 408-438). 13. For the organoid part, the organoids were hormone-treated in vitro. The three controls—were they age-/fertility-matched to AS patients? If not, differences could reflect baseline variability rather than treatment effects. A13. We appreciate the reviewer’s observation regarding potential baseline differences due to unmatched controls. Indeed, age or fertility status may contribute to variability in some organoid-derived endpoints. Unfortunately, control patients cannot be matched with AS patients, since control patients referred to healthy patients with natural menstrual cycle and no previous issues for pregnancy or pathologies, whereas AS patients are often infertile and lack menstrual period. Nevertheless, the primary comparisons for the organoid study focused on pre- versus post-treatment organoids, which are derived from the same AS patients, whereas the control samples were included to provide a baseline reference framework consistent with the methodological design validated in our earlier publication (Santamaria et al., Nature Communications, 2023) 14. Regarding the organoid sequencing results, the increase in proliferative EMT cells post-treatment is intriguing but raises questions: Is this proliferative shift a direct effect of CD133+ BMDSCs (e.g., via paracrine signaling) or an indirect consequence of endometrial repair? Could excessive EMT proliferation risk fibrosis or aberrant regeneration? The authors should discuss whether this profile aligns with healthy endometrial repair. A14. Thank you for this insightful comment. We agree that the potential role of proliferative EMT cells in endometrial regeneration requires careful consideration. In our dataset, we observed a modest increase in the EMT-like population post-treatment; however, this trend did not reach statistical significance. While such a shift could suggest a regenerative response, possibly mediated by paracrine effects of CD133⁺ BMDSCs, the limited magnitude and statistical power preclude firm conclusions. Importantly, these observations were made in an in vitro organoid model, which—despite capturing key features of the epithelial compartment—does not fully recapitulate the multicellular complexity of the endometrial environment, particularly the stromal-epithelial interactions involved in fibrosis or tissue remodelling. For this reason, and to avoid speculative interpretation, we chose to remove explicit references to the EMT population from the main text. We consider this an interesting finding that warrants further investigation in more physiologically integrated models and larger cohorts. 15. Figure 3 presents hysteroscopic images before and after treatment for all participants, which is well appreciated. However, it is not specified on which day of the menstrual cycle these images were taken, nor how many days post-treatment they were obtained. From Table 1, it appears but not clear that participants were not amenorrheic. Could the authors clarify the timing of image acquisition in relation to the menstrual cycle and the treatment timeline? This information is particularly important considering the statement in lines 148–150, which notes an increase in menstrual blood flow during the first month after treatment, returning to pre-treatment levels after three months. Was this temporal pattern in blood flow reflected in the hysteroscopic findings? A15. Thank you for this comment. We have previously clarified in your previous point the menstrual blood data and we hope this is now more comprehensive. Regarding the timings of image acquisition, in the Methods section, under Procedures (lines 44–99), the reviewer can find relevant information. Specifically, during the simulated “secretory phase” (five days after progesterone administration), endometrial status was assessed both before and after treatment. This evaluation included histological digital image analysis, 2D/3D transvaginal ultrasound (Samsung, South Korea), and hysteroscopic assessment using the ASRM classification of intrauterine adhesions (IUAs)¹. Hysteroscopies were performed with a 5 mm Betocchi hysteroscope (Karl-Storz). The post treatment images were taken one month after therapy, as stated in the figure legend. These images then would correspond to menstrual blood data one month after therapy. The hysteroscopic findings clearly reflected the increased blood flow, as evidenced by the images after the first menstruation following therapy (Figure 3A), and a statistically significant decrease in the IUAs score (Figure 3B). 16. Considering the transient increase in menstrual blood flow during the first month post-treatment, it would be informative to include hysteroscopic images from three months after treatment in non-pregnant women to assess long-term endometrial changes. A16. Thank you for your comment. Although they would surely be informative, according to the protocol approved by the Ethics Committee and AEMPS (see protocol), hysteroscopies were performed immediately before cell therapy and one month after cell therapy (see Methods, Procedures section). Monthly hysteroscopies were not planned as part of the study protocol; therefore, corresponding images are not available. 17. In reference to lines 143–150, the authors state that menstrual blood loss increased during the first month post-treatment but returned to pre-treatment levels after three months. Does this suggest that the underlying condition was not fully resolved, given that the menstrual pattern ultimately reverted to its baseline state? If normal menstrual cycling resumed or improved, one might expect a sustained change in menstrual pattern or blood flow, especially considering the reported increase in endometrial thickness after treatment (lines 127–133, Figure 2B, and Table 1). Could the authors clarify how this temporal pattern in menstrual blood loss corresponds to treatment efficacy and the overall resolution of the syndrome? A17. Thank you for your comment. This is a trend we have observed throughout this and previous studies. Actually, in our proof-of-concept study (Santamaria et al., Hum Reprod, 2016), we reported comparable clinical findings: menstrual cycles resumed with hormone replacement therapy (HRT) during the first month following cell therapy. However, both the duration and intensity of menstruation progressively decreased. The mean duration fell from 5.06 days (range: 3–7) in the first month to 3.25 days (range: 1–3) by the third month post-treatment. Similarly, menstrual volume, as measured by the number of sanitary pads used per day, declined from a mean of 2.69 (range: 1–5) to 1.75 (range: 1–4) pads per day by the third month. Our current clinical experience also indicates that subsequent cell injections produce a similar clinical effect, supporting the hypothesis that the observed effects result from temporal engraftment of the injected cells rather than permanent integration. Having said this, we have not found how this menstrual pattern blood loss is correlated to the treatment efficacy. Although we have used a validated method for the bloss, it is important to note that there is a high inter and intra observation variability that difficult drawing robust correlations between the blood patterns and the actual treatment efficacy. 18. The scale bar in Supplementary Figure 3 for the gene expression data needs to be corrected for accurate interpretation. A18. Thank you for your comment regarding the scale bar in Supplementary Figure 3. However, we do not quite understand where the correction is needed. The figure uses a commonly used dual encoding to represent gene expression data, where the dot color indicates the average gene expression, with a continuous color scale ranging from blue (low expression) to red (high expression), whereas the dot size reflects the percentage of cells within each cluster expressing the gene, with larger dots indicating a higher proportion of cells expressing the gene. To clarify this for readers, we have included a more detailed explanation in the figure legend stating:“Dot color indicates average gene expression (log-normalized), while dot size corresponds to the percentage of cells expressing the gene within each cell type cluster. Both color and size scales are consistent across all panels.” If you require us to change the actual display of the data, please let us know. 19. Tissue samples were collected pre- and post-treatment from nine participants, and the data in Figure 5A suggest that at least three samples were used to generate 3D organoids. For clarity and completeness, it would be helpful to include representative 3D organoid data from all nine participants if performed, or at minimum the three samples that were analyzed and presented in figure 5 A, like the presentation style in Figure 2, to allow assessment of inter-patient variability and enhance data transparency. A19. We thank the reviewer for this helpful suggestion. As correctly noted, 3D organoid experiments were performed on tissue samples from three participants, not all nine, as this constitutes an exploratory objective of the study. We acknowledge that this could have been made clearer in the original manuscript and have now explicitly stated this in the revised text (line 297-298). In addition, Figure 5A has been added including images of all three patient-derived organoids, as requested by the reviewer. 20. For the organoid part, the organoids were hormone-treated in vitro. The three controls—were they age-/fertility-matched to AS patients? If not, differences could reflect baseline variability rather than treatment effects. This comment is a repetition from point 13 that has been answered above 21. Regarding the organoid sequencing results, the increase in proliferative EMT cells post-treatment is intriguing but raises questions: Is this proliferative shift a direct effect of CD133+ BMDSCs (e.g., via paracrine signaling) or an indirect consequence of endometrial repair? Could excessive EMT proliferation risk fibrosis or aberrant regeneration? The authors should discuss whether this profile aligns with healthy endometrial repair. This comment is a repetition from point 14 that has been answered above 22. It is not indicated when the sample were obtained after the treatment for scRNA and 3D organoids culture. A22. The reviewer can find this information in our original submission in Methods, procedures section, lines 44-99: “During the simulated “secretory phase” (5 days after progesterone administration) pre-treatment and post-treatment endometrial status was monitored by histological digital image analysis, 2D/3D ultrasound (Samsung, South Korea) and hysteroscopic assessment according to ASRM classification of IUAs1 using a 5mm Betocchi hysteroscope (Karl-Storz)”. We have also added in line 60 of methods this part: Single cell RNA seq and endometrial organoids were also derived from these endometrial biopsies 23. The scRNA-seq data in Supplementary Figure 4E indicate upregulation of angiogenic pathways ANGPTL; to strengthen these findings, it would be valuable to include supporting protein-level validation of key pathway components, such as through immunostaining or western blotting. A23. In response to the reviewer’s request, we have validated at the protein level the scRNA-seq findings related to the upregulation of angiogenic pathways, specifically, the ANGPTL2 marker, which showed a higher expression in post-AS therapy samples (Figure 4E). To this end, we performed immunostaining analysis of ANGPTL2 expression in pre and post tissue samples and we have generated a representative Figure (Figure 4F) that supports scRNA-seq findings. The results confirm the upregulation observed at the transcriptomic level, supporting the involvement of angiogenic mechanisms. We also include the image here for the ease of the reviewer. New Supplementary Figure 4F. ANGPTL2 validation for pre and post-AS therapy samples. ANGPTL2: gray, DAPI: blue. 24. Mitochondrial Variant Tracking: This is a clever approach to assess engraftment. What percentage of endometrial cells post-treatment showed donor mitochondrial DNA? What is the most abundant cell type that bone marrow stem cells may differentiate into? Did engraftment correlate with clinical outcomes? The use of mtDNA for lineage tracing is powerful but not sure how mitochondrial transfer between cells via mechanisms such as tunneling nanotubes, cell fusion, or exosome-mediated exchange affects the clonality. These events can introduce exogenous mtDNA, potentially leading to misinterpretation of clonal relationships. A24. We appreciate the reviewer’s detailed question and provide the following point-by-point response: What percentage of endometrial cells post-treatment showed donor mitochondrial DNA? We would like to highlight that our cell therapy is autologous, not allogeneic. Consequently, while mitochondrial variant tracking is a powerful tool for lineage tracing, its utility for quantifying engraftment in our setting is inherently limited, as it only detects cells carrying a mitochondrial variant (a minority of cells). In our study, only 1.12% of post-treatment endometrial cells exhibited such donor-specific mitochondrial DNA (mtDNA). Rather than serving as a quantitative measure of engraftment, this approach was used to characterize the phenotypic and transcriptomic identity of engrafted cells, enabling us to gain insights into their differentiation behavior within the endometrial niche. What is the most abundant cell type that bone marrow stem cells may differentiate into? Our data suggest that CD133⁺ BMDSCs most frequently differentiate into stromal and endothelial cell lineages. This interpretation is supported by transcriptomic shifts related to angiogenesis and extracellular matrix remodeling observed in scRNA-seq analyses before and after stem cell therapy. However, due to the low sample size (3 patients), these conclusions remain hypothesis-generating and should be interpreted with caution. Did engraftment correlate with clinical outcomes? While we demonstrated the presence of mitochondrial variants, it is important to note that the number of detectable variants varied between patients, and the analysis was limited to three individuals. Therefore, although we observed overall clinical improvements across the cohort—including increased endometrial volume, thickness, and live birth rates—we were unable to directly correlate mtDNA-based engraftment levels with individual clinical outcomes. Moreover, as previously stated, the autologous nature of the therapy significantly limits the ability to detect engraftment events and to establish quantitative correlations with clinical endpoints. Concerns about mtDNA transfer mechanisms (e.g., nanotubes, cell fusion, exosomes): Biologically, we believe this remains an unresolved question, as mitochondrial transfer between cells has been described, but its actual frequency and relevance in human endometrial tissue remain unclear. From a bioinformatic perspective, we acknowledge that it is not possible to definitively exclude the possibility that the mitochondrial DNA detected in recipient cells originated from transfer mechanisms. However, we applied stringent thresholds for variant calling, including only high-confidence heteroplasmic variants that were absent in pre-treatment samples and uniquely observed post-treatment, thereby strengthening the specificity of our lineage detection. Miller et al. (Cell, 2022), who developed and validated the MAESTER method, did not report such transfer events as a significant confounding factor in their lineage tracing studies, which could led us to rely on the robustness and interpretability of mtDNA-based clonal inference when applied with appropriate analytical thresholds. 25. line 196-199 concludes that perivascular cells increase after the treatment and Macrophage decreases, need some comments in discussion about this. It's not clear how pericytes are involved in endometrium but in CNS it's very well accepted that pericytes forms the scar tissue after injury and inhibits regeneration. We thank the reviewer for highlighting the relevance of the observed changes in perivascular cells and macrophages following treatment. While it is true that in the central nervous system (CNS), pericytes have been implicated in scar formation and inhibition of regeneration after injury, the role of perivascular cells in the endometrium is distinct and context-dependent. Our data demonstrate a tendency towards increase post-treatment in perivascular cells (putative pericytes), along with a marked reduction in macrophage abundance within the endometrial tissue. In contrast to the central nervous system (CNS), where pericytes have been implicated in the formation of fibrotic scar tissue that hinders regeneration after injury, pericytes in the endometrial context appear to play a distinct, pro-regenerative role. Specifically, endometrial pericytes have been associated with microvascular stability, paracrine support of angiogenesis, and interactions with endothelial and epithelial cells that contribute to cyclical regeneration of the uterine lining (Li et al., 2019). Moreover, perivascular cells in the uterus may also exhibit mesenchymal stem cell-like properties, supporting tissue repair and remodeling rather than fibrotic encapsulation (Spitzer et al., 2012). The expansion of this population post-treatment may thus reflect the activation of a regenerative niche rather than a fibrotic or inhibitory microenvironment. This is consistent with previous reports in reproductive biology suggesting that perivascular stromal cells coordinate angiogenesis and modulate immune responses during endometrial regeneration (Gargett et al., 2016). In parallel, the observed reduction in macrophage abundance—particularly those associated with inflammatory phenotypes—suggests a shift away from active inflammation toward immune resolution and tissue remodeling. This dynamic is similar to processes described in successful tissue repair across organ systems, where a timely reduction of pro-inflammatory macrophages, coupled with increased vascular support and stromal cell activation, marks a transition toward functional healing (Davies et al., 2013). These findings have now been further elaborated in the revised Discussion to clarify the functional implications of the cellular changes we observed and to distinguish the uterine regenerative context from that of CNS injury and scarring. References: Li Z, Yan G, Diao Q, Yu F, et al. Transplantation of human endometrial perivascular cells with elevated CYR61 expression induces angiogenesis and promotes repair of a full-thickness uterine injury in rat. Stem Cell Res Ther. 2019 Jun 18;10(1):179. doi: 10.1186/s13287-019-1272-3. Spitzer, T. L., Rojas, A., Zelenko, Z., et al. (2012). Perivascular human endometrial mesenchymal stem cells express pathways relevant to self-renewal, lineage specification, and functional phenotype. Biology of Reproduction, 86(2), 58. Gargett, C. E., Schwab, K. E., & Deane, J. A. (2016). Endometrial stem/progenitor cells: The first 10 years. Human Reproduction Update, 22(2), 137–163. Davies, L. C., Jenkins, S. J., Allen, J. E., & Taylor, P. R. (2013). Tissue-resident macrophages. Nature Immunology, 14(10), 986–995. 26. Protein validation for the genes has not been presented, only RNA data was shown. It would be valuable to present how the AS epithelial cells change after treatment and how the scar/perivascular cells are affected. A26. From our previous paper (Santamaria et al., 2023), we identified that the stressed epithelial subpopulation of AS-epithelium expressed elevated levels of SLPI. In our previous paper, we presented RNA scope data illustrating the changes in SLPI of AS vs healthy endometrium. Although this was not published, in this case we also studied the changes of SLPI after the treatment (Post-AS therapy), showing that SLPI was transcriptomically expressed mainly in the pre-AS therapy samples (see figure below for reviewer only). New figure for reviewer only. Representative RNAscope images showing the expression of SLPI as a marker of the characteristic AS epithelium cell type in AS patients. Images show an example of SLPI expression in Pre-AS therapy, Post-AS therapy and control endometria. SLPI (green) expression counterstained with DAPI shown merged and in separate channels for clarity. Scale bar = 20 µm. In response to the reviewer’s request, we further analysed this marker at the protein level, so we performed immunofluorescence staining pre and post-AS therapy of SLPI marker. In this case, SLPI protein expression was strongly expressed in pre-AS therapy, with a diffuse and intense staining pattern throughout the epithelial compartments (Figure below, zoom top row). After CD133⁺ BMDSC therapy, SLPI expression was significantly reduced and faintly localized in the glands, coinciding with the reduction of staining in AS epithelium post therapy (Figure below, bottom row). We have now added this figure in supplementary Figure 2F and lines 226-229 of the manuscript: “Notably, we previously showed that AS-epithelial population expresses high levels of SLPI. Interestingly, SLPI expression was markedly reduced in post-AS therapy samples, which may account for the decrease in this cell population following CD133+ BMDSC treatment (Supplementary Figure 2F)”. New supplementary Figure 2F. Representative immunofluorescence images showing the expression of SLPI as a marker of the characteristic AS epithelium cell type in AS patients. Images show an example of SLPI expression in Pre-and Post-AS therapy. SLPI (green) expression counterstained with DAPI shown merged and in separate channels for clarity. Scale bar = 20 µm. Regarding how scar/perivascular cells are affected, we performed a quantitative digital histological analysis using H&E images where we analysed morphological changes before and after therapy (Supplementary Figure 1A). In this analysis, we assessed the percentage of fiber-rich stroma as an indication of the presence of fibrotic tissue pre and post therapy. As shown in Supplementary Figure 1A (see also below), we observed that most samples presented a significant decrease in the percentage of fibrotic/stromal area post-therapy (64% reduction), alongside an increase in the percentage of glands and vessels present in the tissue (62% and 73%, respectively), suggesting a partial resolution of fibrosis and restoration of perivascular architecture, even if not directly marked by specific molecular staining. Supplementary Figure 2F. Reviewer #4 (Remarks to the Author): Comments on “Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase 1/2 trial” As requested by the Editor, our review will be limited to those aspects that relate to the trial design and statistics of this manuscript. We do not have sufficient experience and knowledge for evaluation of aspects that are more specific to cells and laboratory work. Considering that Asherman Syndrome has a significant impact on the quality of life of many people, and that currently the only possible intervention - which is often not definitive - is operative hysteroscopy, the identification of different approaches could be of great value. This is especially true if these possible new interventions are able to overcome infertility. Therefore, we commend the authors for their commitment to the search for new treatment options. We thus appreciated the authors’ effort in conducting this clinical trial aimed at evaluating the tolerability, safety, and efficacy of autologous CD133+ bone marrow-derived stem cell (BMDSC) therapy in patients with Asherman Syndrome (AS) refractory to surgical interventions. Overall, the methodological components of the study are appropriate for a non-randomised, open-label, single-arm, phase 1/2 study. We are of the opinion that the study design is well-structured, and we do not have any major comments on this aspect. However, considering that the study represents a complex methodological project integrating clinical and molecular approaches, we believe that some clarifications and further details may be useful, both regarding the objectives of the study and the use of statistics. This is all the more important given that the sample consisted of 20 women (one of whom did not complete the study), which is relatively large considering the prevalence of the disease, but relatively small considering the numerous endpoints set by the authors. We reiterate the importance of this study and offer some comments and suggestions to further improve the clarity, transparency and robustness of the analyses. A. We sincerely thank Reviewer #4 for the thoughtful and constructive comments, as well as the recognition of the importance of our study and the efforts made by our multidisciplinary team over the past four years. We are especially grateful for your acknowledgment of the significance of developing new therapeutic strategies for Asherman Syndrome (AS), a condition with considerable impact on patients' quality of life and limited treatment options. We appreciate your positive assessment of the trial design and the methodological framework of our non-randomized, open-label, single-arm, phase 1/2 study. Your recognition that the overall structure is appropriate and well-conceived is encouraging and greatly valued. We also acknowledge your suggestion that, given the complexity of our integrated clinical and molecular approach, additional clarifications and details regarding the study objectives and statistical analyses would enhance transparency and rigor—particularly in light of our sample size and the multiple endpoints assessed. We fully agree with this observation and have revised the manuscript accordingly to provide the following: 1. Clarified Study Objectives: We have updated the Methods section to explicitly distinguish between primary, secondary and exploratory objectives (lines 22-41) 2. Statistical Considerations: We have rewritten the statistical section part to include more details on how each analysis was performed as further described below. We hope that these revisions address your concerns and contribute to a more transparent and robust presentation of our findings. Once again, we thank you for your expert evaluation and for your helpful suggestions to improve our manuscript. Major comments 1. Partially addressed primary endpoints According to the trial registration on the European Union Clinical Trial Register (EudraCT: 2016-003975-23), three primary endpoints were declared: “1. Identification of incidence, prevalence and frequency of different adverse events that may occur in the patient throughout the study up until Month 15 post-treatment. 2. In the event of a term pregnancy, identification of incidence, prevalence and frequency of different adverse events that may occur during the pregnancy, birth and puerperal period. 3. In case of a Live Birth (LB), identification of incidence, prevalence and frequency of the different adverse events that may be associated with the investigational medicinal product (IMP) until the first month of age.” However, in the present study, only the first primary endpoint appears to have been thoroughly developed, and its results reported. The results of the primary endpoints are reported in the manuscript on lines 109-116 and in Table 2. There is no mention of primary endpoints 2 and 3, i.e. pregnancies and births. Some information on these is provided in secondary endpoints section and in the supplementary material, but as these are primary endpoints - and therefore theoretically the most important ones - they should be clearly presented in the section of the results dealing with the primary endpoints. It would be appropriate for the authors to complete the analysis by including the remaining two primary endpoints as outlined in the registered protocol. Alternatively, the authors should state why the results of the other two primary endpoints were not reported in the primary endpoint results section and were not discussed. For example, the most outstanding result of this study is mentioned by the authors in the abstract: \"...reproductive function was restored, culminating in six live births after embryo transfer\". It is not clear why this striking observation is barely mentioned in the manuscript, neither in the results nor in the discussion, even if one of the primary endpoints was the “identification of incidence, prevalence and frequency of the different adverse events” in case of a life birth. It is clear that the set of secondary and explanatory outcomes is intended to try to understand the biological and molecular reasons behind the regained fertility of some women. Nevertheless, the primary endpoint is in danger of being obscured by all the other analyses. A1. Thank you for your insightful analysis of the trial design. As noted by the reviewer, we have now provided further information of the incidence, prevalence, and frequency of various adverse events that may occur: These endpoints are now outlined in the section “primary endpoints” in lines 112-129. We have also added further explanation in the discussion (line 338-344). 2. Excessive detailing of secondary and exploratory endpoints Although the presentation of the results and discussion of the secondary and exploratory endpoints is comprehensive and thorough, the length and level of detail may be reconsidered as it seems disproportionate to the primary objectives of the study. A more concise approach could benefit the overall focus and readability of the manuscript. Alternatively, the endpoints of the study could be refocused. A2. Thank you for your comments. We have made every effort to be concise in our presentation of secondary and exploratory endpoints. However, the amount of information extracted from the exploratory (scRNA-seq) analysis to describe the molecular mechanisms of the therapy is very large and even if we have shortened it, it still requires a significant space in the manuscript. Additionally, other reviewers have requested to further emphasize into these findings, so we have had to extend certain parts of this analysis. Besides, it is important to consider the breadth of clinical, biological, and basic research that supports not only the effectiveness but also the mechanism of action of this therapy. Nonetheless, since we have now added more information about the primary end points, we believe there is now a more balanced structure between the primary and secondary/exploratory end points. 3. Predictive model based on Support Vector Machine (SVM) In the section concerning endometrial epithelial organoids (EEOs), the authors add the use of a Support Vector Machine (SVM) with a kernel radial basis functon(RBF), implemented via the scPred package, to perform transcriptomic comparison with in vivo data. However, although a brief description of the process is provided (10-fold cross-validation and C parameter optimization), several key methodological elements are missing, e.g. the type of learning adopted, the target classes used for model training, the feature selection procedure, the model performance metrics, and the rationale for choosing the RBF kernel, among others. Given the complexity and methodological significance of a machine learning-based analysis, we recommend that a dedicated and detailed methods section be added. Alternatively, since this does not represent one of the study’s primary endpoints, the analysis could be considered for omission. A3. As the reviewer correctly notes, this analysis does not represent a primary endpoint of the study and may potentially confuse readers when interpreting the organoid results. Therefore, we have decided to omit it and it has been now removed from Figure 5 and methods section. Minor comments 1. Statistical methodology presentation Although the statistical methodology covers a broad range of analyses, it is overall unclear, dispersed, and difficult to follow. The statistical tests used are reported in different sections of the methodology and not always in a systematic manner, making it challenging to obtain a comprehensive view of the analyses performed. We therefore recommend including a more structured and concise section that clearly summarizes all the applied tests, the type of data analysed, and the rationale behind the choice of each statistical test. Alternatively, we suggest including a subsection on the statistics and software used within each section of the methodology. This will allow the reader to better understand the different statistical approaches used. A1. Thank you for your valuable comment. We have now structured the statistical section into various parts: Statistical analysis of clinical design and data outcomes, statistical analysis for single cell data of in vivo biopsies and statistical analysis for organoids. We believe this organization makes the statistical tests easier to access and understand. 2. Assumptions behind the use of parametric tests When parametric tests were used instead of non-parametric ones, it is not specified whether the assumption of normality of the data distribution was verified. An example of this is the use of the paired t-test to evaluate the IUA score. Since this is a score, it is likely that nonparametric tests would be more appropriate. Or, if a parametric test is needed, it would be appropriate to state the rationale for such a choice. A2. Thank you very much for this helpful comment. We have now updated the Statistical Analysis section to include the normality assessments, which had been omitted in the previous version. Specifically, we used the Shapiro–Wilk test to evaluate the normality assumption for each dataset. Regarding the use of parametric tests in Figures 2 and 3 (endometrial volume, thickness, and IUA score), we initially selected the paired t-test because the data consist of paired measurements from the same patients before and after treatment, and when normality is satisfied, parametric tests provide greater statistical power. To ensure this approach was appropriate, we tested each dataset for normality. For endometrial volume and thickness, the data met the normality assumption and thus justified the use of the paired t-test. However, for the IUA score shown in Figure 4, you are absolutely right—normality was only observed in the post-treatment data. Therefore, we additionally applied the non-parametric Wilcoxon signed-rank test, which yielded the same statistical significance as the paired t-test (p < 0.0001). We agree this is a more appropriate approach, and we have accordingly updated both the graph and the statistical test presented. Just to double check, we did perform now both parametric and non-parametric tests to verify robustness of results and ensure significance for both scenarios. 3. Choice of statistical test for organoid quantification A two-way ANOVA test was used in the organoid quantification section. It would be useful for the authors to clarify the rationale for using a two-way analysis. A two-way ANOVA analyses the effect of two different categorical independent variables on a continuous dependent variable. It is not clear what the two independent variables are in this case. In addition, it is important to specify whether there were two independent interacting factors. Could it be that a one-way ANOVA would have been more appropriate? A3. We employed a two-way ANOVA for organoid quantification to evaluate the effects of two independent categorical variables: (1) experimental group (AS-Pre EEO, AS-Post EEO, and Control EEO) and (2) passage number (P0, P1, P2), on the continuous dependent variable—organoid count. This analysis allowed us to assess both the main effects of group and passage, as well as their interaction, indicating whether the treatment effect varied across passages. This is particularly relevant given that organoids are cultured in a cocktail of growth factors, and repeated passaging may diminish therapeutic effects. The two-way ANOVA thus captured both group differences and passage-dependent dynamics. 4. Statistical power and sample size calculation The authors calculated the sample size based on a hypothetical increase in live births from 0% to 28%. The hypothesis is probably correct. However, this sample size calculation assumes that the primary objective of the trial is to examine the percentage of women who have a pregnancy that ends in a live birth. For all the other analyses that have been performed, namely those related to secondary and exploratory endpoints, this sample calculation is not as crucial. To give just one example, when it comes to endometrial volume or thickness a sample calculation based on the proportion of life births has no reason to be (even if it is still a paired cohort study). It is sufficient to say that a convenience sample was studied, or the sample size should be computed on a hypothetical difference on volumes or thickness. A4. Thank you for this thoughtful observation. We agree that the original sample size calculation, which was based on an expected increase in live birth rates, primarily supports the trial’s primary objective related to reproductive outcomes. For analyses related to secondary and exploratory endpoints—such as endometrial volume and thickness—we acknowledge that this calculation is not directly applicable. As suggested, we have now clarified in methods section (section Statistical analysis of clinical outcomes) that a convenience sample was used for these secondary analyses. In any case, in the description of the sample size calculation, the authors do not report the statistical power value used for the estimate. It is recommended that the power and alpha values assumed in the calculation are explicitly reported. A4. Thank you for the comment. We have now added the alpha level and statistical power to the Methods section (line 369 of the methods doc inside the subsection of “statistical analysis of clinical design”). Below are the main inputs used for the sample size calculation: • Proportion of events: Group 0 = 0.00% Group 1 = 28.00% • Minimum expected effect size: 28.00% difference • Alpha risk (Type I error): 5% • Power (1 - Type II error): 80% • Test type: One-sided • Expected dropout rate: 20% • Required sample size per group (before dropout adjustment): 18 • Adjusted sample size per group (accounting for 20% dropout): 22 5. Typographical errors. Finally, we suggest a review of, e.g. in the methods section: “Patients were managed as described previously described…” A5. Thank you for the comment. We have amended this section, which now reads: 'Patients were managed as previously described (line 44 of methods section).' Reviewer #5 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts. Reviewer #6 (Remarks to the Author): General This phase 1/2 trial assessed the safety, efficacy, and biological plausibility of autologous CD133+ bone marrow-derived stem cell (BMDSC) therapy for treating moderate to severe Asherman Syndrome (AS) in 20 infertile women unresponsive to prior treatments. The therapy was well-tolerated, with reversible limb paresthesia as the most common side effect. Significant improvements were observed in endometrial thickness, volume, and hysteroscopic scores. Clinically, menstruation and reproductive function were restored, leading to six live births following embryo transfer. Single-cell RNA sequencing revealed partial reversion of AS-related changes, and mitochondrial variant tracking showed insights into stem cell engraftment. The results suggest this therapy is safe and effective, warranting further investigation in larger trials. The manuscript is well-written, grammatically sound, and addresses a highly interesting topic, combining stem cell applications as orphan drugs with single-cell technologies. However, some technical aspects related to the bioinformatics analyses, such as the use of older packages (CellRanger, Seurat), as well as the provision of analysis scripts, could benefit from refinement. Major 1. How was the concentration of mobilized cells (at least 30 million cells with >70% purity and >50% viability) determined for this study? Is this concentration commonly used for CD133+ BMDSCs in this application? In cardiac regeneration studies, similar cell collections often use lower concentrations, how does this compare to your approach? A1. Thank you for this insightful comment. The product specification for the cell therapy—including a minimum of ≥30 million CD133⁺ cells with ≥70% purity and ≥50% viability—was established in agreement with the European Medicines Agency (EMA) during the clinical trial authorization process. As this was a Phase 1–2 trial, these criteria were designed to ensure feasibility, safety, and consistency of the product while allowing for some variability in early-stage manufacturing. Importantly, the cell dose was not arbitrarily selected but was determined by the number of CD133⁺ cells that could be realistically mobilized and recovered from each patient following G-CSF stimulation and apheresis. The threshold of 30 million cells reflected the minimum yield considered necessary for therapeutic delivery, based on preclinical work and prior experience with autologous stem cell therapies. It also accounted for the variability in mobilization capacity across patients. While some cardiac regeneration trials have used lower cell doses, the biological context and clinical indications differ significantly. Cardiac patients typically face life-threatening conditions and are often older, with reduced bone marrow stem cell mobilization capacity. In contrast, our study population comprises younger individuals who are not affected by life-threatening diseases—this is an explicit exclusion criterion of our trial.Therefore, despite administering only minimal doses of filgrastim, our patients generally exhibit higher cell yields. This enhanced mobilization capacity allows for a more robust collection of CD133+ cells. Importantly, this Phase I/II trial was designed to determine the optimal therapeutic dosage. In line with this objective—and owing to the biological advantages mentioned—it is expected that higher cell dosages will be obtained. Our primary goal was to ensure the administration of a therapeutically relevant cell dose, while simultaneously maintaining patient safety and manufacturing feasibility. 2. Could you explain the rationale behind using CellRanger Software version 3.1.0 (from July 2019) for this analysis, given that the current version is 9.0.1? Additionally, I noticed that Seurat version 4 was used, whereas version 5 is available for quite some time. The use of these older versions, both for CellRanger and Seurat, can potentially result in different analysis outcomes due to improvements or changes in data processing and downstream analysis methods introduced in newer versions. A2. Thank you for the comment. The reviewer is correct in noting that newer versions of CellRanger (currently v9.0.1) and Seurat (v5) are available. The rationale behind using CellRanger v3.1.0 (released July 2019) and Seurat v4 lies in the study’s timeline. The experimental protocol was approved and initiated on April 20, 2020, at which point CellRanger v3.1.0 and Seurat v4 were the most stable and supported versions for our pipeline. Importantly, re-analysis with newer versions would introduce inconsistencies between the original pipeline and current computational standards, potentially affecting reproducibility and interpretation. We also note that CellRanger v3.1.0 was last updated in February 2025 for archival maintenance, and Seurat v4 remained the working version throughout our analysis pipeline, even though v5 has been available since November 2023. While newer versions offer improvements, the results and conclusions of this study remain robust and directly comparable within the analytical context under which they were originally generated and published (Santamaria, X., Roson, B., Perez-Moraga, R. et al., Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution, Nat Commun 14, 5890 (2023). https://doi.org/10.1038/s41467-023-41656-1) 3. Did the authors also perform quality assessments using tools like MultiQC to aggregate the overall quality of the samples? It might be beneficial to provide these quality reports as supplemental material to account for the sequencing quality. A3. We appreciate the reviewer’s suggestion regarding MultiQC. Unfortunately, integrating MultiQC is not feasible in this context because the version of CellRanger (3.1.0) used in our study is not fully compatible with MultiQC’s current parsers. Therefore, we provide individual per-sample QC reports generated by CellRanger, which accurately reflect the quality of the data generated using our original pipeline. We believe this is the most appropriate approach and will include these reports as supplemental material. 4. Did the authors also perform dataset integration using Harmony (https://www.nature.com/articles/s41592-019-0619-0) in addition to the SCT transformation, which is commonly considered a standard approach for handling batch effects, particularly when samples have been processed in different batches? While Harmony is mentioned in the methods section, it appears to be described within the machine learning part, rather than in the single-cell processing section. Could you clarify when Harmony was applied in the analysis and why it was not used in all experiments? A4. Thank you for this valuable suggestion. We agree that Harmony is widely recognized as a best-practice tool for batch effect correction in single-cell analysis. While it is referenced in our Methods, its use in our study was limited to exploratory machine learning analyses and was not part of the primary single-cell processing pipeline. Due to another reviewer’s request we do not longer have the machine learning data and so we have actually deleted the part of the methodology were harmony was used. As per your request, we have now performed an Harmony integration and generated UMAPs to compare with our current integration. Both approaches produced visually similar results. However, incorporating Harmony at this stage would introduce subtle shifts in cluster boundaries that could affect cell-type annotations, potentially compromising consistency with our previously published dataset (Santamaria et al. Nat Com 2023) and the current analysis. Therefore, to maintain coherence across studies, we retained the original SCT-based integration for downstream analyses. 5. Could you please include the cell-to-cell communication analyses in the methods section? A5. Thank you. The methods section has now been updated to include the details of the cell-to-cell communication analysis (added in methods section, from line 235 to 258). This section outlines the pipeline used and references the tools applied. 6. Providing the relevant scripts in a public repository would greatly enhance the overall reproducibility and depths of the data analysis. A6. We agree with the importance of reproducibility and transparency. A public repository containing all relevant analysis scripts and documentation will be made available upon acceptance of the paper. The link is provided in the new section named “code availability” Minor 1. The required number of patients is inconsistent between the methods section and the study protocol, 20 versus 22. It may be helpful for the authors and readers to standardize and use a single number throughout the entire work. A1. Thank you for pointing this out. The number of patients has been standardized to 20 throughout the manuscript. The original protocol mentioned 22 patients to account for an anticipated 20% dropout rate. However, the actual dropout rate was only 5%, and with 20 patients, we retained sufficient statistical power to detect significant differences, in line with the original power calculation. 2. The authors also may provide the exact version of the genome build used GRCh38-3.0.0. A2. We used the GRCh38.p12 build, specifically NCBI:GCA_000001405.27, for read alignment and downstream annotation. This has now been added in line 184-185 of the methods section. 3. Did the authors used specific databases, tools or scientific literature to annotate the single cell clusters - Reference 3 in the main manuscript (results) might be added also in the methods section in more detail, if the annotation is mainly based on previous works of the authors, including the species under investigation (e.g., murine, cell-based, …). A3. In response to the reviewer’s comment, we have added a dedicated section in the Methods titled “Annotation of cell types in scRNA-seq datasets”. In this section, we clarify that cell cluster identities were assigned by comparing cluster-specific marker genes to annotated major cell types described in previously published human endometrial single-cell atlases, including Garcia-Alonso et al. (Nat Genet 53, 1698–1711, 2021) and Wang et al. (Nat Med 26, 1644–1653, 2020). 4. How was the selection of the 9 out of 20 patients conducted for the single-cell analysis? A4. The 9 patients selected for single-cell analysis were chosen randomly from the full cohort to ensure sufficient cellular representation and transcriptomic coverage. These correspond to patients 5,7,8,9,11,12,13,14,15 whose pretreatment data were already published in Santamaria et al., Nat Commun, 2023. The post treatment data reported here were generated contemporaneously but are presented separately due to clinical trial reporting constraints. Relevant clinical metadata is included in Table 1. 5. Please remove the “.” In the middle of the sentence in line 327 A5. Thank you, already modified (now line 348). REVIEWER COMMENTS All changes made in response to the reviewers’ comments have been highlighted in yellow throughout the manuscript. Reviewer #1 (Remarks to the Author): The reviewer appreciate details responses not only to my, but other reviewers' comments, including running additional experiments. I have some more questions/comments. 1. Diagnostic hysteroscopy: you mention that surgeries were performed by single non-blinded physician, scoring was thoroughly documented through photographic evidence and independently reviewed in a blinded manner by a second surgeon. But then you also mentioned that you could not document via images or video results of all hysteroscopies due to equipment malfuncton(Although images are not available for all patients, visualization from the side of the clinician at the time of analysis was performed). So, how would second physician review and confirm the ASRM scoring without documentation available? This is pretty important, as assessment should be reliable. The surgeon is also first author of the study, as well as co-founder of the company, so the potential bias is there. A1. Thank you for your comment. We acknowledge the potential limitations in documentation and recognize the importance of rigorous, unbiased assessment, particularly in the context of possible conflicts of interest. Photographic documentation was obtained for most hysteroscopic procedures (n= 15) and independently reviewed by a second experienced surgeon, blinded to both treatment allocation and patient identity. In the remaining four cases where photographic recording was unavailable, the ASRM hysteroscopic score was verified through direct visualization at the time of the procedure by both surgeons, with detailed operative notes recorded contemporaneously. While this represents an unavoidable limitation, we minimized potential bias through predefined scoring criteria and consistent documentation protocols. This limitation has now been explicitly stated in methods (line 51-55) and also mentioned in Figure legend 3 (see below A2) 2. Please explain in the text that you only had enough good quality cDNA samples left to run MAESTER in three samples, hence only n=3 for mtDNA. In general, any missing data should be explained otherwise raises questions (e.g. missing endometrial volumes, or missing hysteroscopic images). A2. Thank you, we have now added an explanation in line 287-289 (highlighted in yellow) as follows: Since the MAESTER technique requires reamplification of residual cDNA from single-cell RNA sequencing libraries, we had sufficient high-quality cDNA to perform the analysis in only three patients (N7, N9, and N15). As per the missing values in endometrial volumes and hysteroscopic images, we have also added a sentence in the associated figure legends indicated below. Figure 2. Effect of CD133+ therapy on the endometrium. A) Effect of the autologous CD133+ BMDSC therapy on the endometrial thickness and volume assessed by 2D and 3D vaginal ultrasound pre- (Pre-AS therapy) and one-month post-therapy (Post-AS therapy). Data shown are from the 16 patients with both 2D and 3D endometrial measurements available. B) Box plots of endometrial thickness (n=19), and (C) endometrial volume (n=16) of AS patients pre- and post-AS therapy representing all data before and after one month of cell therapy. Endometrial volume data for three patients (n=16/19) could not be measured due to unavailability of the 3D transvaginal probe at the time of patient follow-up. Whisker ends on the box plot represent the minimum and the maximum data point. Paired t-test; **** p<0.0001. Figure 3. Effect of CD133+ therapy in the endometrial cavity. A) Representative hysteroscopic images of the effect of the autologous CD133+ BMDSC therapy on the endometrial cavity assessed by hysteroscopic scores performed pre (Pre-AS therapy), and one-month post-therapy (Post-AS therapy) according to the IUA ASRM score. Representative hysteroscopy images of the four remaining patients (n=15/19) could not be illustrated due to technical limitations of hysteroscopy equipment at the time of patient follow-up. Hysteroscopic images consistently show constricted uterine cavities by IUAs with limited space pre-AS therapy while images demonstrate a significantly widened uterine cavity, indicating successful structural remodeling post-AS therapy. B) Box plots of IUAs score from AS patients (n=19) pre-AS therapy (8±3.17) and post-AS therapy (4.42±2.09). Whisker ends on the box plot represent the minimum and the maximum data point. Wilcoxon test; **** p<0.0001. 3. Can you please explain the rationale for doing all evaluation and treatment in mid-secretory phase? How do you explain that incorporation of stem cells into endometrium and subsequent regeneration of endometrium happens when intravascular injection is performed in the presence of progesterone? Have the authors trouble shoot the timing in therapy in prior studies? This needs to be better explained. A3. The rationale for performing stem cell treatment in mid-secretory phase is because progesterone essentially creates a “fertile ground” for the stem cells to recognize, adhere, engraft, and contribute to regeneration. Progesterone transforms the estrogen-primed endometrium into a secretory, receptive state. This includes decidualization of stromal fibroblasts, increased extracellular matrix deposition, and altered immune tolerance. Also, P4 upregulates angiogenic factors such as VEGF and angiopoietins, which make the microvasculature more permeable to circulating cells. In this primed environment, progesterone also induces expression of key chemotactic and adhesion molecules—including CXCL12 (SDF-1), ICAM-1, and selectins—that are critical for guiding stem cells toward the endometrial tissue. Finally, P induces expression of factors like CXCL12 (SDF-1), ICAM-1, and selectins, which guide stem cells toward the endometrial tissue (1-9). Stem cells administered via intravascular injection in uterine microcirculation can reach the spiral arterioles and venules of the endometrium. They express surface receptors such as CXCR4, which respond to progesterone-induced chemokine gradients, facilitating active homing and targeted migration into the stromal compartment. Progesterone-induced endothelial changes further support stem cell adhesion, rolling, and diapedesis into the tissue. Once engrafted, stem cells secrete growth factors (e.g., HGF, IGF-1, bFGF) that enhance epithelial proliferation, angiogenesis, and stromal repair. In some instances, mesenchymal stem cells may differentiate into endometrial stromal-like cells, replenishing damaged areas. Both progesterone and stem cells both promote a locally anti-inflammatory, pro regenerative environment, reducing fibrosis and allowing structural regeneration (10). Importantly, if stem cells were injected without progesterone priming, the endometrium may remain in a proliferative or atrophic state with lower vascular permeability, less chemokine signaling, and reduced receptivity, impairing stem cell engraftment and therapeutic efficacy. The rationale for evaluation in the mid-secretory phase is because this part of the menstrual cycle represents the most informative time point for clinical evaluation of endometrial receptivity improvements following stem cell therapy. This window allows for the assessment of key surrogate markers, including endometrial thickness, volume, and hysteroscopic scores. These parameters provide measurable evidence of structural and functional regeneration. We have previously optimized the therapeutic timing internally and successfully validated the concept in our pilot study (11). 1. Santamaria, X., Cabanillas, S., Cervelló, I., Arbona, C., Raga, F., Ferro, J., & Simón, C. (2023). Autologous stem cell therapy for refractory Asherman’s syndrome and endometrial atrophy: A translational approach. Nature Communications, 14, Article 1284. https://doi.org/10.1038/s41467-023-01284-y 2. Cervelló, I., Gil-Sanchis, C., Mas, A., Delgado-Rosas, F., Martinez-Conejero, J. A., Galán, A., … & Simón, C. (2015).Human endometrial CD140b+ stem cells: Involvement in recurrent miscarriage and infertility.Stem Cells, 33(2), 467–477. https://doi.org/10.1002/stem.1853 3. Gargett, C. E., Schwab, K. E., & Brosens, J. J. (2016). Putting stem cells in context in the human endometrium.Biology of Reproduction, 95(2), 1–10. https://doi.org/10.1095/biolreprod.116.139451 4. Mendoza-Tesarik, R., Mendoza, C., & Tesarik, J. (2019). Improvement of endometrial receptivity by autologous stem cell therapy in infertility patients refractory to conventional treatments: A pilot study.Reproductive Biomedicine Online, 39(6), 836–842.https://doi.org/10.1016/j.rbmo.2019.07.001 5. Gargett, C. E., & Masuda, H. (2010). Adult stem cells in the endometrium. Molecular Human Reproduction, 16(11), 818–834. https://doi.org/10.1093/molehr/gaq061 6. Critchley, H. O. D., Maybin, J. A., & Saunders, P. T. K. (2020). Progesterone receptor and mechanisms of action in the endometrium. Journal of Endocrinology, 246(2), R1–R14. https://doi.org/10.1530/JOE-20-0193 7. Liu, Y., Niu, X., & Sun, Y. (2019). CXCL12/CXCR4 axis in stem cell homing and recruitment: New therapeutic perspectives for regenerative medicine.Histology and Histopathology, 34(11), 1161–1173. https://doi.org/10.14670/HH-18-099 8. Tang, J., Li, W., Ma, Q., & Liu, H. (2018). The role of the SDF-1/CXCR4 axis in endothelial progenitor cell homing in ischemic tissues. 9. Stem Cell Reviews and Reports, 14(4), 584–591. https://doi.org/10.1007/s12015-018-9809-5 10. Moreno, A. Capalbo, A. Mas, T. Garrido-Gomez, B. Roson, M. Poli, E. Dimitriadis, X. Santamaria, F. Vilella, C. Simon, The human periconceptional maternal-embryonic space in health and disease. Physiol Rev, doi: 10.1152/physrev.00050.2021 (2023). 11. Santamaria, X. et al. Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman’s syndrome and endometrial atrophy: a pilot cohort study. Hum Reprod 31, 1087-1096 (2016). 4. Regarding the cycle phase: while in the materials and methods you provided a statement, throughout the text this information was not consistent or clear, hence every reviewer having similar comment. Please revise accordingly. Thank you for your comment. We have reviewed the manuscript to ensure consistency, and information regarding the secretory phase has now been added at lines 89–90 and 213 of the main manuscript, in addition to the description already provided in the Methods section. We hope this is now clear. 5. After the revision it became less clear if the pregnancy outcomes (positive pregnancy rate, clinical pregnancy, miscarriage, live birth) are among secondary outcomes or not. Thank you for your comment. In line 130, under the subsection of “secondary endpoints” we stated: The secondary endpoints included endometrial morphometry, thickness, volume, uterine hysteroscopic scores, and endometrial function evaluated by menstruation recovery and pregnancy outcomes (line 130-135). We have also highlighted in yellow in line 168 the following: As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 embryo transfers in 19 patients… In the Methods section (lines 28–34), we stated: “Secondary endpoints were the efficacy of the therapy in achieving improvements in morphological and clinical features, such as endometrial histology assessed through digital image analysis; endometrial thickness and volume measured by 2D and 3D vaginal ultrasound; hysteroscopic grading scores; menstrual function recorded monthly based on duration (days), volume (pads/day), and frequency (days) of menstrual bleeding using the Pictorial Blood Assessment Chart⁷; and reproductive outcomes after treatment, measured by implantation, pregnancy, miscarriage, ongoing, and live birth rates.” We identified a section in the Discussion where this may not have been entirely clear and have added further clarification (lines 342–345). Our findings indicate that the primary endpoints of the therapy—safety and tolerability of this autologous cell therapy—were met, with the treatment being well tolerated for up to 15 months of follow-up. Further, as a secondary endpoint, the AS patients after cell therapy achieved an implantation rate of 31.7% per embryo transferred (13/41) resulting in 5 live births. In addition, we recorded one spontaneous pregnancy. As per adverse events of live birth and newborns, no serious adverse events were observed beyond common obstetric complications, fulfilling the primary end points of safety and tolerability of the study. We hope this is now clear throughout the manuscript. 6. Results: “We performed a total of 41 embryo transfers in 2019 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41). , and 12 pregnancies (60.0% pregnancy rate)” . I don’t think you should be reporting 60% pregnancy rate per se, but you can say that cumulative pregnancy rate for 20 patients after 41 transfers was 60%. Was your pregnancy rate 12/41 or 13/41? What was the intention to treat analysis for pregnancy rate, after first embryo transfer per patient? A6. For the sake of clarity, we have reworded this paragraph (lines 168–172) as follows: “As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 transfers in 19 patients, yielding an implantation rate of 31.7% per embryo (13/41). Among the 20 treated patients, six live births were recorded—including one from a spontaneous pregnancy post-therapy—and eight miscarriages, obtaining a live birth rate of 30% (Table 1). As per protocol, the intention-to-treat pregnancy rate was assessed over a 15‑month follow-up period. 7. Please have careful language revision done again, as after the revisions there are several typos or missing words, double or extra words or commas, and some sentences are now difficult to read. Maybe I was reading the wrong highlighted version, but it appears as if both deleted and new text are still present. Language revision will also help with proper word usage, such as “catheterization” instead of “catheterism”. A7. Thank you for your comment. Upon review, we realized that the tracked changes version still contained elements that had previously been removed. We have now highlighted these sections in yellow, rather than using track changes, and have thoroughly reviewed the manuscript to ensure accuracy and clarity. Reviewer #2 (Remarks to the Author): The author has revised and supplemented the article. However, I still have some doubts about certain statements, as follows: 1. In secondary endpoints, the manuscript described that compared with before cell therapy, the endometrial fibrosis was significantly reduced after cell therapy, a 62% increase in gland percentage, a 64% 136 reduction in fiber-rich stroma, and a 73% increase in vessel percentage within the stroma were observed (Supplementary Figure 1A). I concerned that how long the author took the sample after cell therapy and it is better to clarified. A1. Thank you for your comment. We agree that clarification is important. As stated in the Methods section, endometrial biopsies were collected one month after cell therapy in all cases. This applies to all analyses, including scRNA-seq, histology, and all other data presented here. We have now also added a clarifying sentence in the Results section (lines 133–135, highlighted in yellow) 2. When the author answer question 4 from reviewer 2, they stated as following: In moderate to severe AS, the location of endometrial biopsies depends on the presence of identifiable areas of bona fide endometrium rather than on a specific topographical site. This is detailed in the Methods section (Procedures subsection lines 55-61.\" Specifically, it says: Endometrial biopsies were performed under hysteroscopic vision from the posterior wall of the uterus at least 1 cm away from any visually identified IUA using a semi-rigid double-action biopsy and grasping forceps (Karl-Storz, Germany)”. I have some concerns because in patients with IUA the degree of endometrial damage may not be consistent throughout the entire endometrium. Some areas may show severe fibrosis, while others may not. If we want to evaluate the effect of the cell therapy through histological changes in the endometrium, it is best to determine the sampling site of the endometrium. I am concerned that sampling the endometrium from different sites before and after therapy may affect the evaluation of the effectiveness of cell therapy. A2. We understand your concern, which addresses an important methodological aspect in evaluating endometrial histology in patients with intrauterine adhesions (IUA). Geographical heterogeneity of endometrial damage is a well-recognized challenge in Asherman’s Syndrome (AS), and standardizing biopsy site selection is essential to ensure reliable interpretation of histological changes. In this study, as well as in our previous related work (Santamaria X et al., Nature Communications, 2023), biopsy site selection was deliberately standardized. Specifically, biopsies were obtained under direct hysteroscopic vision from the posterior wall of the uterus, maintaining a 1 cm margin from any visible intrauterine adhesion. This approach ensured that all samples were collected from bona fide, non-fibrotic endometrium adjacent to the pathological area—tissue most likely to reflect the therapeutic response—while maintaining consistency across timepoints. The posterior wall of the uterine cavity is among the most reproducible and accessible sampling sites, particularly in cases where extensive synechiae may limit access to other regions. Hysteroscopic guidance allowed us to avoid areas of severe fibrosis or necrosis that would preclude meaningful histological evaluation, focusing instead on viable tissue with regenerative potential. Furthermore, the literature acknowledges the existence of different adhesion types (fibrous, muscular, or mixed) that cause variable degrees of damage across the uterine cavity (Foix A et al., Am J Obstet Gynecol, 1966). Our approach accounted for this variability by consistently sampling from the perilesional functional endometrium, which is most relevant for evaluating the impact of cell therapy aimed at tissue regeneration. In summary, while the entire endometrium may be variably affected in IUA, our standardized and targeted biopsy strategy ensured comparability of samples before and after treatment, thereby strengthening the reliability of our histological assessments. Santamaria, X., Roson, B., Perez-Moraga, R. et al. Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution. Nat Commun 14, 5890 (2023). https://doi.org/10.1038/s41467-023-41656-1 Foix A , R O Bruno, T Davison, B Lema. The pathology of postcurettage intrauterine adhesions. Am J Obstet Gynecol. 1966 Dec 1;96(7):1027-33. 3 In line 187, the author stated that “although we showed a potential association between enhanced endometrial characteristics and improved reproductive outcomes, it is important to note that live birth outcomes are also influenced by embryo transfer–related factors, such as embryo quality, number of transfers, timing, etc”. What dose the author mean? The author mentioned above that a single blastocyst was transferred. Could it be that the patients who failed to achieve pregnancy had poor-quality embryos? A3. Thank you for your comment. We agree that this point warrants clarification. In each case, a single blastocyst of documented quality was transferred. The sentence in question was intended to emphasize that live birth outcomes are multifactorial, influenced by numerous factors beyond the endometrial environment and embryo quality alone (for review, see Moreno I et al., Physiol Rev, 2023). Our findings therefore suggest a potential association between improved endometrial features and reproductive outcomes; however, we acknowledge that this association is not strong enough to establish causality, and that other confounding factors may have contributed to the observed outcomes. Moreno, A. Capalbo, A. Mas, T. Garrido-Gomez, B. Roson, M. Poli, E. Dimitriadis, X. Santamaria, F. Vilella, C. Simon, The human periconceptional maternal-embryonic space in health and disease. Physiol Rev, doi: 10.1152/physrev.00050.2021 (2023). 4. One of the characteristic of this study, as the author mentioned, is that single-cell sequencing of the endometrial tissue of patients before and after cell therapy was performed, revealing changes in cell composition and gene expression. If there were validation experiments, it would be more convincing. A4. We conducted validation experiments during the first round of reviews, as requested by another reviewer. Specifically, we validated at the protein level the scRNA-seq findings related to the upregulation of angiogenic pathways, focusing on the ANGPTL2 marker, which showed higher expression in post-AS therapy samples (Figure 4E). To this end, we performed immunostaining analysis of ANGPTL2 expression in paired pre- and post-treatment tissue samples and generated a representative image (Figure 4F) that supports the scRNA-seq results. The findings confirmed the transcriptomic-level upregulation, reinforcing the involvement of angiogenic mechanisms. For the reviewer’s convenience, we also include the image here. New Supplementary Figure 4F. ANGPTL2 validation for pre and post-AS therapy samples. ANGPTL2: gray, DAPI: blue. Additionally, in our previous study (Santamaria et al., 2023), we identified that the stressed epithelial subpopulation in AS epithelium expressed elevated levels of SLPI marker. In the present work, we also examined changes in SLPI expression after treatment (post-AS therapy) and found that SLPI was transcriptomically expressed predominantly in the pre-AS therapy samples (see figure below, provided for reviewer reference only). New figure for reviewer only. Representative RNAscope images showing the expression of SLPI as a marker of the characteristic AS epithelium cell type in AS patients. Images show an example of SLPI expression in Pre-AS therapy, Post-AS therapy and control endometria. SLPI (green) expression counterstained with DAPI shown merged and in separate channels for clarity. Scale bar = 20 µm. We extended our analysis of SLPI to the protein level by performing immunofluorescence staining in paired pre- and post-AS therapy samples. Pre-therapy, SLPI showed strong, diffuse epithelial staining, whereas post-CD133‑ BMDSC therapy, expression was markedly reduced and restricted to glands. This finding, now presented in Supplementary Figure 2F, is described in the revised manuscript as follows: “Notably, we previously showed that the AS-epithelial population expresses high levels of SLPI. Interestingly, SLPI expression was markedly reduced in post-AS therapy samples, which may account for the decrease in this cell population following CD133‑ BMDSC treatment (Supplementary Figure 2F).” New supplementary Figure 2F. Representative immunofluorescence images showing the expression of SLPI as a marker of the characteristic AS epithelium cell type in AS patients. Images show an example of SLPI expression in Pre-and Post-AS therapy. SLPI (green) expression counterstained with DAPI shown merged and in separate channels for clarity. Scale bar = 20 µm. 5. When the author mentioned in the Methods section and answering the question the author stated that hysteroscopy was only used for diagnosis and not for separating intrauterine adhesions, does this mean that cell therapy can make the adhesions disappear? As far as I know, it is very difficult for adhesion bands to disappear in moderate to severe IUA and usually requires surgical separation. A5. Thank you for your sharp comment. As you correctly point out, cell therapy alone does not make intrauterine adhesions disappear, particularly in moderate to severe IUA, where surgical separation is often required. In our study, we documented improvements in ASRM hysteroscopic scores, but not the complete disappearance of all adhesions. The literature recognizes that different adhesion types—fibrous, muscular, or mixed—arise from varying degrees of endometrial damage across the uterine cavity (Foix A et al., Am J Obstet Gynecol, 1966). In our study, we have shown that autologous CD133‑ BMDSC therapy improves endometrial quality at the single-cell level by generating bona fide endometrium. This newly formed functional tissue can progressively replace areas of adhesion and, more importantly, restore endometrial functionality, enabling normal menstruation and supporting pregnancy. Reviewer #3 (Remarks to the Author): Thank you for revising you manuscript and for your detailed answers. I have no further comments Reviewer #4 (Remarks to the Author): Regarding the manuscript's methodology and statistics, we believe the authors responded comprehensively and clearly to our comments. In particular, they have provided a clearer explanation of the difference between primary and secondary endpoints, which was a source of confusion in the previous version. Additionally, the statistics section now appears more organized and systematic. Finally, the paragraph about machine learning that was in the previous version has been omitted. We believe this was an appropriate choice because, although the paragraph did not add essential information, it increased the risk of bias. Reviewer #5 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts. Reviewer #6 (Remarks to the Author): Thank you very much for addressing my comments. 1. While I understand that the use of older software versions (CellRanger 3.1.0 and Seurat 4) was specified in the original protocol from 2020, I would recommend acknowledging this limitation in the manuscript. Specifically, it would be prudent to mention that these older versions were used as per the protocol and to discuss any potential inconsistencies when compared to newer versions, which are now available. Using older software versions could potentially result in inconsistencies or missed improvements in data analysis, which may impact the comparisons with other related works in the field. Additionally, this could limit the ability of other researchers to use the provided cell annotation clusters effectively unless they also use the same older versions, which may introduce additional complications. In the rapidly evolving field of bioinformatics, keeping up with updates is generally recommended to ensure accurate and reproducible results for all researchers, especially considering that the patient data in this study cannot be stored for reanalysis in an open repository. A1. Thank you for your comment. We have added the following clarification to the Methods section (highlighted in yellow, lines 179-189): scRNA-seq data was processed using Cell Ranger v3.1.0 and Seurat v4. These versions were maintained to ensure consistency with a previously analyzed dataset and to enable direct comparability3. While newer versions include improvements in cell calling, UMI correction, doublet detection, and annotation, we applied the same pipeline and stringent quality controls across all samples to minimize technical variability. Core Seurat steps—normalization, dimensionality reduction, clustering, and visualization—also improved across versions. Nevertheless, we consider our biological interpretations robust, while acknowledging possible limitations in sensitivity and cross-study compatibility with newer software releases 2. Regarding the availability of the analysis code, I support the idea of publishing it on GitHub after acceptance. However, for a journal like Nature Communications, I believe it would be beneficial to provide the code during the revision process itself. This would allow reviewers, including myself, to verify the consistency, transparency, and reproducibility of the analysis before final publication. Once again, I appreciate your efforts in addressing these issues, and I believe the manuscript will benefit from further clarification and transparency regarding the software versions used and the availability of the analysis code. A2. Thank you for your suggestion and for the previous comments that made the manuscript improve thoroughly. We fully support transparency and reproducibility, and so here is the link where the code is fully available:https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public REVIEWER COMMENTS All changes made in response to the reviewers’ comments have been highlighted in yellow throughout the manuscript. Reviewer #1 (Remarks to the Author): The reviewer appreciate details responses not only to my, but other reviewers' comments, including running additional experiments. I have some more questions/comments. 1. Diagnostic hysteroscopy: you mention that surgeries were performed by single non-blinded physician, scoring was thoroughly documented through photographic evidence and independently reviewed in a blinded manner by a second surgeon. But then you also mentioned that you could not document via images or video results of all hysteroscopies due to equipment malfuncton(Although images are not available for all patients, visualization from the side of the clinician at the time of analysis was performed). So, how would second physician review and confirm the ASRM scoring without documentation available? This is pretty important, as assessment should be reliable. The surgeon is also first author of the study, as well as co-founder of the company, so the potential bias is there. A1. Thank you for your comment. We acknowledge the potential limitations in documentation and recognize the importance of rigorous, unbiased assessment, particularly in the context of possible conflicts of interest. Photographic documentation was obtained for most hysteroscopic procedures (n= 15) and independently reviewed by a second experienced surgeon, blinded to both treatment allocation and patient identity. In the remaining four cases where photographic recording was unavailable, the ASRM hysteroscopic score was verified through direct visualization at the time of the procedure by both surgeons, with detailed operative notes recorded contemporaneously. While this represents an unavoidable limitation, we minimized potential bias through predefined scoring criteria and consistent documentation protocols. This limitation has now been explicitly stated in methods (line 51-55) and also mentioned in Figure legend 3 (see below A2) 2. Please explain in the text that you only had enough good quality cDNA samples left to run MAESTER in three samples, hence only n=3 for mtDNA. In general, any missing data should be explained otherwise raises questions (e.g. missing endometrial volumes, or missing hysteroscopic images). A2. Thank you, we have now added an explanation in line 287-289 (highlighted in yellow) as follows: Since the MAESTER technique requires reamplification of residual cDNA from single-cell RNA sequencing libraries, we had sufficient high-quality cDNA to perform the analysis in only three patients (N7, N9, and N15). As per the missing values in endometrial volumes and hysteroscopic images, we have also added a sentence in the associated figure legends indicated below. Figure 2. Effect of CD133+ therapy on the endometrium. A) Effect of the autologous CD133+ BMDSC therapy on the endometrial thickness and volume assessed by 2D and 3D vaginal ultrasound pre- (Pre-AS therapy) and one-month post-therapy (Post-AS therapy). Data shown are from the 16 patients with both 2D and 3D endometrial measurements available. B) Box plots of endometrial thickness (n=19), and (C) endometrial volume (n=16) of AS patients pre- and post-AS therapy representing all data before and after one month of cell therapy. Endometrial volume data for three patients (n=16/19) could not be measured due to unavailability of the 3D transvaginal probe at the time of patient follow-up. Whisker ends on the box plot represent the minimum and the maximum data point. Paired t-test; **** p<0.0001. Figure 3. Effect of CD133+ therapy in the endometrial cavity. A) Representative hysteroscopic images of the effect of the autologous CD133+ BMDSC therapy on the endometrial cavity assessed by hysteroscopic scores performed pre (Pre-AS therapy), and one-month post-therapy (Post-AS therapy) according to the IUA ASRM score. Representative hysteroscopy images of the four remaining patients (n=15/19) could not be illustrated due to technical limitations of hysteroscopy equipment at the time of patient follow-up. Hysteroscopic images consistently show constricted uterine cavities by IUAs with limited space pre-AS therapy while images demonstrate a significantly widened uterine cavity, indicating successful structural remodeling post-AS therapy. B) Box plots of IUAs score from AS patients (n=19) pre-AS therapy (8±3.17) and post-AS therapy (4.42±2.09). Whisker ends on the box plot represent the minimum and the maximum data point. Wilcoxon test; **** p<0.0001. 3. Can you please explain the rationale for doing all evaluation and treatment in mid-secretory phase? How do you explain that incorporation of stem cells into endometrium and subsequent regeneration of endometrium happens when intravascular injection is performed in the presence of progesterone? Have the authors trouble shoot the timing in therapy in prior studies? This needs to be better explained. A3. The rationale for performing stem cell treatment in mid-secretory phase is because progesterone essentially creates a “fertile ground” for the stem cells to recognize, adhere, engraft, and contribute to regeneration. Progesterone transforms the estrogen-primed endometrium into a secretory, receptive state. This includes decidualization of stromal fibroblasts, increased extracellular matrix deposition, and altered immune tolerance. Also, P4 upregulates angiogenic factors such as VEGF and angiopoietins, which make the microvasculature more permeable to circulating cells. In this primed environment, progesterone also induces expression of key chemotactic and adhesion molecules—including CXCL12 (SDF-1), ICAM-1, and selectins—that are critical for guiding stem cells toward the endometrial tissue. Finally, P induces expression of factors like CXCL12 (SDF-1), ICAM-1, and selectins, which guide stem cells toward the endometrial tissue (1-9). Stem cells administered via intravascular injection in uterine microcirculation can reach the spiral arterioles and venules of the endometrium. They express surface receptors such as CXCR4, which respond to progesterone-induced chemokine gradients, facilitating active homing and targeted migration into the stromal compartment. Progesterone-induced endothelial changes further support stem cell adhesion, rolling, and diapedesis into the tissue. Once engrafted, stem cells secrete growth factors (e.g., HGF, IGF-1, bFGF) that enhance epithelial proliferation, angiogenesis, and stromal repair. In some instances, mesenchymal stem cells may differentiate into endometrial stromal-like cells, replenishing damaged areas. Both progesterone and stem cells both promote a locally anti-inflammatory, pro regenerative environment, reducing fibrosis and allowing structural regeneration (10). Importantly, if stem cells were injected without progesterone priming, the endometrium may remain in a proliferative or atrophic state with lower vascular permeability, less chemokine signaling, and reduced receptivity, impairing stem cell engraftment and therapeutic efficacy. The rationale for evaluation in the mid-secretory phase is because this part of the menstrual cycle represents the most informative time point for clinical evaluation of endometrial receptivity improvements following stem cell therapy. This window allows for the assessment of key surrogate markers, including endometrial thickness, volume, and hysteroscopic scores. These parameters provide measurable evidence of structural and functional regeneration. We have previously optimized the therapeutic timing internally and successfully validated the concept in our pilot study (11). 1. Santamaria, X., Cabanillas, S., Cervelló, I., Arbona, C., Raga, F., Ferro, J., & Simón, C. (2023). Autologous stem cell therapy for refractory Asherman’s syndrome and endometrial atrophy: A translational approach. Nature Communications, 14, Article 1284. https://doi.org/10.1038/s41467-023-01284-y 2. Cervelló, I., Gil-Sanchis, C., Mas, A., Delgado-Rosas, F., Martinez-Conejero, J. A., Galán, A., … & Simón, C. (2015).Human endometrial CD140b+ stem cells: Involvement in recurrent miscarriage and infertility.Stem Cells, 33(2), 467–477. https://doi.org/10.1002/stem.1853 3. Gargett, C. E., Schwab, K. E., & Brosens, J. J. (2016). Putting stem cells in context in the human endometrium.Biology of Reproduction, 95(2), 1–10. https://doi.org/10.1095/biolreprod.116.139451 4. Mendoza-Tesarik, R., Mendoza, C., & Tesarik, J. (2019). Improvement of endometrial receptivity by autologous stem cell therapy in infertility patients refractory to conventional treatments: A pilot study.Reproductive Biomedicine Online, 39(6), 836–842.https://doi.org/10.1016/j.rbmo.2019.07.001 5. Gargett, C. E., & Masuda, H. (2010). Adult stem cells in the endometrium. Molecular Human Reproduction, 16(11), 818–834. https://doi.org/10.1093/molehr/gaq061 6. Critchley, H. O. D., Maybin, J. A., & Saunders, P. T. K. (2020). Progesterone receptor and mechanisms of action in the endometrium. Journal of Endocrinology, 246(2), R1–R14. https://doi.org/10.1530/JOE-20-0193 7. Liu, Y., Niu, X., & Sun, Y. (2019). CXCL12/CXCR4 axis in stem cell homing and recruitment: New therapeutic perspectives for regenerative medicine.Histology and Histopathology, 34(11), 1161–1173. https://doi.org/10.14670/HH-18-099 8. Tang, J., Li, W., Ma, Q., & Liu, H. (2018). The role of the SDF-1/CXCR4 axis in endothelial progenitor cell homing in ischemic tissues. 9. Stem Cell Reviews and Reports, 14(4), 584–591. https://doi.org/10.1007/s12015-018-9809-5 10. Moreno, A. Capalbo, A. Mas, T. Garrido-Gomez, B. Roson, M. Poli, E. Dimitriadis, X. Santamaria, F. Vilella, C. Simon, The human periconceptional maternal-embryonic space in health and disease. Physiol Rev, doi: 10.1152/physrev.00050.2021 (2023). 11. Santamaria, X. et al. Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman’s syndrome and endometrial atrophy: a pilot cohort study. Hum Reprod 31, 1087-1096 (2016). 4. Regarding the cycle phase: while in the materials and methods you provided a statement, throughout the text this information was not consistent or clear, hence every reviewer having similar comment. Please revise accordingly. Thank you for your comment. We have reviewed the manuscript to ensure consistency, and information regarding the secretory phase has now been added at lines 89–90 and 213 of the main manuscript, in addition to the description already provided in the Methods section. We hope this is now clear. 5. After the revision it became less clear if the pregnancy outcomes (positive pregnancy rate, clinical pregnancy, miscarriage, live birth) are among secondary outcomes or not. Thank you for your comment. In line 130, under the subsection of “secondary endpoints” we stated: The secondary endpoints included endometrial morphometry, thickness, volume, uterine hysteroscopic scores, and endometrial function evaluated by menstruation recovery and pregnancy outcomes (line 130-135). We have also highlighted in yellow in line 168 the following: As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 embryo transfers in 19 patients… In the Methods section (lines 28–34), we stated: “Secondary endpoints were the efficacy of the therapy in achieving improvements in morphological and clinical features, such as endometrial histology assessed through digital image analysis; endometrial thickness and volume measured by 2D and 3D vaginal ultrasound; hysteroscopic grading scores; menstrual function recorded monthly based on duration (days), volume (pads/day), and frequency (days) of menstrual bleeding using the Pictorial Blood Assessment Chart⁷; and reproductive outcomes after treatment, measured by implantation, pregnancy, miscarriage, ongoing, and live birth rates.” We identified a section in the Discussion where this may not have been entirely clear and have added further clarification (lines 342–345). Our findings indicate that the primary endpoints of the therapy—safety and tolerability of this autologous cell therapy—were met, with the treatment being well tolerated for up to 15 months of follow-up. Further, as a secondary endpoint, the AS patients after cell therapy achieved an implantation rate of 31.7% per embryo transferred (13/41) resulting in 5 live births. In addition, we recorded one spontaneous pregnancy. As per adverse events of live birth and newborns, no serious adverse events were observed beyond common obstetric complications, fulfilling the primary end points of safety and tolerability of the study. We hope this is now clear throughout the manuscript. 6. Results: “We performed a total of 41 embryo transfers in 2019 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41). , and 12 pregnancies (60.0% pregnancy rate)” . I don’t think you should be reporting 60% pregnancy rate per se, but you can say that cumulative pregnancy rate for 20 patients after 41 transfers was 60%. Was your pregnancy rate 12/41 or 13/41? What was the intention to treat analysis for pregnancy rate, after first embryo transfer per patient? A6. For the sake of clarity, we have reworded this paragraph (lines 168–172) as follows: “As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 transfers in 19 patients, yielding an implantation rate of 31.7% per embryo (13/41). Among the 20 treated patients, six live births were recorded—including one from a spontaneous pregnancy post-therapy—and eight miscarriages, obtaining a live birth rate of 30% (Table 1). As per protocol, the intention-to-treat pregnancy rate was assessed over a 15‑month follow-up period. 7. Please have careful language revision done again, as after the revisions there are several typos or missing words, double or extra words or commas, and some sentences are now difficult to read. Maybe I was reading the wrong highlighted version, but it appears as if both deleted and new text are still present. Language revision will also help with proper word usage, such as “catheterization” instead of “catheterism”. A7. Thank you for your comment. Upon review, we realized that the tracked changes version still contained elements that had previously been removed. We have now highlighted these sections in yellow, rather than using track changes, and have thoroughly reviewed the manuscript to ensure accuracy and clarity. Reviewer #1 (Remarks on code availability): n/a Reviewer #6 (Remarks to the Author): Thank you for the improvements to the manuscript. The overall organization has clearly progressed, so that analyses for AS patients, organoids, CD133+, CellChat, and differential abundance/expression are now easier to navigate. As a minor addition, a brief, targeted cleanup of the repo could reach a higher level of transparency and reusability that will greatly benefit readers. Here, I would recommend adding a stepwise analysis scheme directly in the README as a small table to anchor timelines and versions for Data collection window, QC/processing window, Figure generation, and Manuscript freeze. This makes it more comprehensible which code/outputs map to the results in the manuscript and helps future users reuse the analyses tied to a specific manuscript freeze (the one from 2023 or the potentially upcoming one). In particular, the repository’s scope (single-cell analysis of AS patients, organoids, CellChat cell–cell communication, differential abundance/expression) overlaps conceptually with the Nature Communications paper “Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution” (21 Sep 2023). Please cite it prominently in the README and in the relevant scripts, and briefly note what was adopted or extended (e.g., “CellChat settings and epithelial subpopulation markers adapted from …”). This avoids ambiguity and situates your work in the existing literature, even if its your own. Please also check again for hard-coded elements that reduce portability, e.g. replace absolute paths, inline parameters, and environment-specific toggles. with a single config.yml (paths, sample IDs, thresholds). (Example found in https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/src/R/AS_Patients/maester/analyze_variants/explore_variants_final.r) You could also commit an environment spec (R: renv.lock via renv::snapshot() or a minimal DESCRIPTION; Python: requirements.txt / environment.yml if applicable). In summary, the repository is already in good shape, and the above points are minimal, pragmatic changes that will substantially increase transparency, reproducibility, and code reuse. I appreciate the progress so far. With these tweaks, the repo will be straightforward to reuse alongside the manuscript and be more valuable for the community. Thank you to reviewer 6 for the comment, we have now improved the transparency and usability of the code repository , specifically we answer step by step to your comments: a) Here, I would recommend adding a stepwise analysis scheme directly in the README as a small table to anchor timelines and versions for Data collection window, QC/processing window, Figure generation, and Manuscript freeze. This makes it more comprehensible which code/outputs map to the results in the manuscript and helps future users reuse the analyses tied to a specific manuscript freeze (the one from 2023 or the potentially upcoming one). In particular, the repository’s scope (single-cell analysis of AS patients, organoids, CellChat cell–cell communication, differential abundance/expression) overlaps conceptually with the Nature Communications paper “Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution” (21 Sep 2023). Please cite it prominently in the README and in the relevant scripts, and briefly note what was adopted or extended (e.g., “CellChat settings and epithelial subpopulation markers adapted from …”). This avoids ambiguity and situates your work in the existing literature, even if its your own. a) Although some single-cell analyses of patients and organoids conceptually overlap with the previous work, this project was developed independently. Consequently, there is no formal freeze or direct mapping to the 2023 article. Some analyses, however, represent extended versions of those in the 2023 paper, and certain parameters and markers were reused. For these cases we have now explicitly indicated in the code and in the table added to the README, which details each analysis step, links the corresponding code to its outputs and figures, and includes an additional column noting any elements adopted or extended from the 2023 article. This can now be found here: https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/README.md b) Please also check again for hard-coded elements that reduce portability, e.g. replace absolute paths, inline parameters, and environment-specific toggles. with a single config.yml (paths, sample IDs, thresholds). (Example found in https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/src/R/AS_Patients/m… b) Thank you for the comment. All previously hard-coded elements, such as paths, sample IDs, parameters, and thresholds, have been now replaced with variables defined in config.yml files specific to each type of analysis. c) You could also commit an environment spec (R: renv.lock via renv::snapshot() or a minimal DESCRIPTION; Python: requirements.txt / environment.yml if applicable). c) Thank you, we have now uploaded a requirements.txt file and an renv.lock file for the MAESTER analysis (_MAESTER), as well as an renv.lock file for the rest of the analyses (see below)"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The reviewer appreciate details responses not only to my, but other reviewers' comments, including running additional experiments. I have some more questions/comments. 1. Diagnostic hysteroscopy: you mention that surgeries were performed by single non-blinded physician, scoring was thoroughly documented through photographic evidence and independently reviewed in a blinded manner by a second surgeon. But then you also mentioned that you could not document via images or video results of all hysteroscopies due to equipment malfuncton(Although images are not available for all patients, visualization from the side of the clinician at the time of analysis was performed). So, how would second physician review and confirm the ASRM scoring without documentation available? This is pretty important, as assessment should be reliable. The surgeon is also first author of the study, as well as co-founder of the company, so the potential bias is there. 2. Please explain in the text that you only had enough good quality cDNA samples left to run MAESTER in three samples, hence only n=3 for mtDNA. In general, any missing data should be explained otherwise raises questions (e.g. missing endometrial volumes, or missing hysteroscopic images). 3. Can you please explain the rationale for doing all evaluation and treatment in mid-secretory phase? How do you explain that incorporation of stem cells into endometrium and subsequent regeneration of endometrium happens when intravascular injection is performed in the presence of progesterone? Have the authors trouble shoot the timing in therapy in prior studies? This needs to be better explained. 4. Regarding the cycle phase: while in the materials and methods you provided a statement, throughout the text this information was not consistent or clear, hence every reviewer having similar comment. Please revise accordingly. 5. After the revision it became less clear if the pregnancy outcomes (positive pregnancy rate, clinical pregnancy, miscarriage, live birth) are among secondary outcomes or not. 6. Results: “We performed a total of 41 embryo transfers in 2019 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41). , and 12 pregnancies (60.0% pregnancy rate)” . I don’t think you should be reporting 60% pregnancy rate per se, but you can say that cumulative pregnancy rate for 20 patients after 41 transfers was 60%. Was your pregnancy rate 12/41 or 13/41? What was the intention to treat analysis for pregnancy rate, after first embryo transfer per patient? 7. Please have careful language revision done again, as after the revisions there are several typos or missing words, double or extra words or commas, and some sentences are now difficult to read. Maybe I was reading the wrong highlighted version, but it appears as if both deleted and new text are still present. Language revision will also help with proper word usage, such as “catheterization” instead of “catheterism”.", "reviewer_2": "The author has revised and supplemented the article. However, I still have some doubts about certain statements, as follows: 1. In secondary endpoints, the manuscript described that compared with before cell therapy, the endometrial fibrosis was significantly reduced after cell therapy, a 62% increase in gland percentage, a 64% 136 reduction in fiber-rich stroma, and a 73% increase in vessel percentage within the stroma were observed (Supplementary Figure 1A). I concerned that how long the author took the sample after cell therapy and it is better to clarified. 2. When the author answer question 4 from reviewer 2, they stated as following: In moderate to severe AS, the location of endometrial biopsies depends on the presence of identifiable areas of bona fide endometrium rather than on a specific topographical site. This is detailed in the Methods section (Procedures subsection lines 55-61.\" Specifically, it says: Endometrial biopsies were performed under hysteroscopic vision from the posterior wall of the uterus at least 1 cm away from any visually identified IUA using a semi-rigid double-action biopsy and grasping forceps (Karl-Storz, Germany)”. I have some concerns because in patients with IUA the degree of endometrial damage may not be consistent throughout the entire endometrium. Some areas may show severe fibrosis, while others may not. If we want to evaluate the effect of the cell therapy through histological changes in the endometrium, it is best to determine the sampling site of the endometrium. I am concerned that sampling the endometrium from different sites before and after therapy may affect the evaluation of the effectiveness of cell therapy. 3 In line 187, the author stated that “although we showed a potential association between enhanced endometrial characteristics and improved reproductive outcomes, it is important to note that live birth outcomes are also influenced by embryo transfer–related factors, such as embryo quality, number of transfers, timing, etc”. What dose the author mean? The author mentioned above that a single blastocyst was transferred. Could it be that the patients who failed to achieve pregnancy had poor-quality embryos? 4. One of the characteristic of this study, as the author mentioned, is that single-cell sequencing of the endometrial tissue of patients before and after cell therapy was performed, revealing changes in cell composition and gene expression. If there were validation experiments, it would be more convincing. 5. When the author mentioned in the Methods section and answering the question the author stated that hysteroscopy was only used for diagnosis and not for separating intrauterine adhesions, does this mean that cell therapy can make the adhesions disappear? As far as I know, it is very difficult for adhesion bands to disappear in moderate to severe IUA and usually requires surgical separation.", "reviewer_3": "Thank you for revising you manuscript and for your detailed answers. I have no further comments", "reviewer_4": "Regarding the manuscript's methodology and statistics, we believe the authors responded comprehensively and clearly to our comments. In particular, they have provided a clearer explanation of the difference between primary and secondary endpoints, which was a source of confusion in the previous version. Additionally, the statistics section now appears more organized and systematic. Finally, the paragraph about machine learning that was in the previous version has been omitted. We believe this was an appropriate choice because, although the paragraph did not add essential information, it increased the risk of bias.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "Thank you very much for addressing my comments. While I understand that the use of older software versions (CellRanger 3.1.0 and Seurat 4) was specified in the original protocol from 2020, I would recommend acknowledging this limitation in the manuscript. Specifically, it would be prudent to mention that these older versions were used as per the protocol and to discuss any potential inconsistencies when compared to newer versions, which are now available. Using older software versions could potentially result in inconsistencies or missed improvements in data analysis, which may impact the comparisons with other related works in the field. Additionally, this could limit the ability of other researchers to use the provided cell annotation clusters effectively unless they also use the same older versions, which may introduce additional complications. In the rapidly evolving field of bioinformatics, keeping up with updates is generally recommended to ensure accurate and reproducible results for all researchers, especially considering that the patient data in this study cannot be stored for reanalysis in an open repository. Regarding the availability of the analysis code, I support the idea of publishing it on GitHub after acceptance. However, for a journal like Nature Communications, I believe it would be beneficial to provide the code during the revision process itself. This would allow reviewers, including myself, to verify the consistency, transparency, and reproducibility of the analysis before final publication. Once again, I appreciate your efforts in addressing these issues, and I believe the manuscript will benefit from further clarification and transparency regarding the software versions used and the availability of the analysis code."}, "author_response": "Reviewer #1 (Remarks to the Author): The reviewer appreciate details responses not only to my, but other reviewers' comments, including running additional experiments. I have some more questions/comments. 1. Diagnostic hysteroscopy: you mention that surgeries were performed by single non-blinded physician, scoring was thoroughly documented through photographic evidence and independently reviewed in a blinded manner by a second surgeon. But then you also mentioned that you could not document via images or video results of all hysteroscopies due to equipment malfuncton(Although images are not available for all patients, visualization from the side of the clinician at the time of analysis was performed). So, how would second physician review and confirm the ASRM scoring without documentation available? This is pretty important, as assessment should be reliable. The surgeon is also first author of the study, as well as co-founder of the company, so the potential bias is there. A1. Thank you for your comment. We acknowledge the potential limitations in documentation and recognize the importance of rigorous, unbiased assessment, particularly in the context of possible conflicts of interest. Photographic documentation was obtained for most hysteroscopic procedures (n= 15) and independently reviewed by a second experienced surgeon, blinded to both treatment allocation and patient identity. In the remaining four cases where photographic recording was unavailable, the ASRM hysteroscopic score was verified through direct visualization at the time of the procedure by both surgeons, with detailed operative notes recorded contemporaneously. While this represents an unavoidable limitation, we minimized potential bias through predefined scoring criteria and consistent documentation protocols. This limitation has now been explicitly stated in methods (line 51-55) and also mentioned in Figure legend 3 (see below A2) 2. Please explain in the text that you only had enough good quality cDNA samples left to run MAESTER in three samples, hence only n=3 for mtDNA. In general, any missing data should be explained otherwise raises questions (e.g. missing endometrial volumes, or missing hysteroscopic images). A2. Thank you, we have now added an explanation in line 287-289 (highlighted in yellow) as follows: Since the MAESTER technique requires reamplification of residual cDNA from single-cell RNA sequencing libraries, we had sufficient high-quality cDNA to perform the analysis in only three patients (N7, N9, and N15). As per the missing values in endometrial volumes and hysteroscopic images, we have also added a sentence in the associated figure legends indicated below. Figure 2. Effect of CD133+ therapy on the endometrium. A) Effect of the autologous CD133+ BMDSC therapy on the endometrial thickness and volume assessed by 2D and 3D vaginal ultrasound pre- (Pre-AS therapy) and one-month post-therapy (Post-AS therapy). Data shown are from the 16 patients with both 2D and 3D endometrial measurements available. B) Box plots of endometrial thickness (n=19), and (C) endometrial volume (n=16) of AS patients pre- and post-AS therapy representing all data before and after one month of cell therapy. Endometrial volume data for three patients (n=16/19) could not be measured due to unavailability of the 3D transvaginal probe at the time of patient follow-up. Whisker ends on the box plot represent the minimum and the maximum data point. Paired t-test; **** p<0.0001. Figure 3. Effect of CD133+ therapy in the endometrial cavity. A) Representative hysteroscopic images of the effect of the autologous CD133+ BMDSC therapy on the endometrial cavity assessed by hysteroscopic scores performed pre (Pre-AS therapy), and one-month post-therapy (Post-AS therapy) according to the IUA ASRM score. Representative hysteroscopy images of the four remaining patients (n=15/19) could not be illustrated due to technical limitations of hysteroscopy equipment at the time of patient follow-up. Hysteroscopic images consistently show constricted uterine cavities by IUAs with limited space pre-AS therapy while images demonstrate a significantly widened uterine cavity, indicating successful structural remodeling post-AS therapy. B) Box plots of IUAs score from AS patients (n=19) pre-AS therapy (8±3.17) and post-AS therapy (4.42±2.09). Whisker ends on the box plot represent the minimum and the maximum data point. Wilcoxon test; **** p<0.0001. 3. Can you please explain the rationale for doing all evaluation and treatment in mid-secretory phase? How do you explain that incorporation of stem cells into endometrium and subsequent regeneration of endometrium happens when intravascular injection is performed in the presence of progesterone? Have the authors trouble shoot the timing in therapy in prior studies? This needs to be better explained. A3. The rationale for performing stem cell treatment in mid-secretory phase is because progesterone essentially creates a “fertile ground” for the stem cells to recognize, adhere, engraft, and contribute to regeneration. Progesterone transforms the estrogen-primed endometrium into a secretory, receptive state. This includes decidualization of stromal fibroblasts, increased extracellular matrix deposition, and altered immune tolerance. Also, P4 upregulates angiogenic factors such as VEGF and angiopoietins, which make the microvasculature more permeable to circulating cells. In this primed environment, progesterone also induces expression of key chemotactic and adhesion molecules—including CXCL12 (SDF-1), ICAM-1, and selectins—that are critical for guiding stem cells toward the endometrial tissue. Finally, P induces expression of factors like CXCL12 (SDF-1), ICAM-1, and selectins, which guide stem cells toward the endometrial tissue (1-9). Stem cells administered via intravascular injection in uterine microcirculation can reach the spiral arterioles and venules of the endometrium. They express surface receptors such as CXCR4, which respond to progesterone-induced chemokine gradients, facilitating active homing and targeted migration into the stromal compartment. Progesterone-induced endothelial changes further support stem cell adhesion, rolling, and diapedesis into the tissue. Once engrafted, stem cells secrete growth factors (e.g., HGF, IGF-1, bFGF) that enhance epithelial proliferation, angiogenesis, and stromal repair. In some instances, mesenchymal stem cells may differentiate into endometrial stromal-like cells, replenishing damaged areas. Both progesterone and stem cells both promote a locally anti-inflammatory, pro regenerative environment, reducing fibrosis and allowing structural regeneration (10). Importantly, if stem cells were injected without progesterone priming, the endometrium may remain in a proliferative or atrophic state with lower vascular permeability, less chemokine signaling, and reduced receptivity, impairing stem cell engraftment and therapeutic efficacy. The rationale for evaluation in the mid-secretory phase is because this part of the menstrual cycle represents the most informative time point for clinical evaluation of endometrial receptivity improvements following stem cell therapy. This window allows for the assessment of key surrogate markers, including endometrial thickness, volume, and hysteroscopic scores. These parameters provide measurable evidence of structural and functional regeneration. We have previously optimized the therapeutic timing internally and successfully validated the concept in our pilot study (11). 1. Santamaria, X., Cabanillas, S., Cervelló, I., Arbona, C., Raga, F., Ferro, J., & Simón, C. (2023). Autologous stem cell therapy for refractory Asherman’s syndrome and endometrial atrophy: A translational approach. Nature Communications, 14, Article 1284. https://doi.org/10.1038/s41467-023-01284-y 2. Cervelló, I., Gil-Sanchis, C., Mas, A., Delgado-Rosas, F., Martinez-Conejero, J. A., Galán, A., … & Simón, C. (2015).Human endometrial CD140b+ stem cells: Involvement in recurrent miscarriage and infertility.Stem Cells, 33(2), 467–477. https://doi.org/10.1002/stem.1853 3. Gargett, C. E., Schwab, K. E., & Brosens, J. J. (2016). Putting stem cells in context in the human endometrium.Biology of Reproduction, 95(2), 1–10. https://doi.org/10.1095/biolreprod.116.139451 4. Mendoza-Tesarik, R., Mendoza, C., & Tesarik, J. (2019). Improvement of endometrial receptivity by autologous stem cell therapy in infertility patients refractory to conventional treatments: A pilot study.Reproductive Biomedicine Online, 39(6), 836–842.https://doi.org/10.1016/j.rbmo.2019.07.001 5. Gargett, C. E., & Masuda, H. (2010). Adult stem cells in the endometrium. Molecular Human Reproduction, 16(11), 818–834. https://doi.org/10.1093/molehr/gaq061 6. Critchley, H. O. D., Maybin, J. A., & Saunders, P. T. K. (2020). Progesterone receptor and mechanisms of action in the endometrium. Journal of Endocrinology, 246(2), R1–R14. https://doi.org/10.1530/JOE-20-0193 7. Liu, Y., Niu, X., & Sun, Y. (2019). CXCL12/CXCR4 axis in stem cell homing and recruitment: New therapeutic perspectives for regenerative medicine.Histology and Histopathology, 34(11), 1161–1173. https://doi.org/10.14670/HH-18-099 8. Tang, J., Li, W., Ma, Q., & Liu, H. (2018). The role of the SDF-1/CXCR4 axis in endothelial progenitor cell homing in ischemic tissues. 9. Stem Cell Reviews and Reports, 14(4), 584–591. https://doi.org/10.1007/s12015-018-9809-5 10. Moreno, A. Capalbo, A. Mas, T. Garrido-Gomez, B. Roson, M. Poli, E. Dimitriadis, X. Santamaria, F. Vilella, C. Simon, The human periconceptional maternal-embryonic space in health and disease. Physiol Rev, doi: 10.1152/physrev.00050.2021 (2023). 11. Santamaria, X. et al. Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman’s syndrome and endometrial atrophy: a pilot cohort study. Hum Reprod 31, 1087-1096 (2016). 4. Regarding the cycle phase: while in the materials and methods you provided a statement, throughout the text this information was not consistent or clear, hence every reviewer having similar comment. Please revise accordingly. Thank you for your comment. We have reviewed the manuscript to ensure consistency, and information regarding the secretory phase has now been added at lines 89–90 and 213 of the main manuscript, in addition to the description already provided in the Methods section. We hope this is now clear. 5. After the revision it became less clear if the pregnancy outcomes (positive pregnancy rate, clinical pregnancy, miscarriage, live birth) are among secondary outcomes or not. Thank you for your comment. In line 130, under the subsection of “secondary endpoints” we stated: The secondary endpoints included endometrial morphometry, thickness, volume, uterine hysteroscopic scores, and endometrial function evaluated by menstruation recovery and pregnancy outcomes (line 130-135). We have also highlighted in yellow in line 168 the following: As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 embryo transfers in 19 patients… In the Methods section (lines 28–34), we stated: “Secondary endpoints were the efficacy of the therapy in achieving improvements in morphological and clinical features, such as endometrial histology assessed through digital image analysis; endometrial thickness and volume measured by 2D and 3D vaginal ultrasound; hysteroscopic grading scores; menstrual function recorded monthly based on duration (days), volume (pads/day), and frequency (days) of menstrual bleeding using the Pictorial Blood Assessment Chart⁷; and reproductive outcomes after treatment, measured by implantation, pregnancy, miscarriage, ongoing, and live birth rates.” We identified a section in the Discussion where this may not have been entirely clear and have added further clarification (lines 342–345). Our findings indicate that the primary endpoints of the therapy—safety and tolerability of this autologous cell therapy—were met, with the treatment being well tolerated for up to 15 months of follow-up. Further, as a secondary endpoint, the AS patients after cell therapy achieved an implantation rate of 31.7% per embryo transferred (13/41) resulting in 5 live births. In addition, we recorded one spontaneous pregnancy. As per adverse events of live birth and newborns, no serious adverse events were observed beyond common obstetric complications, fulfilling the primary end points of safety and tolerability of the study. We hope this is now clear throughout the manuscript. 6. Results: “We performed a total of 41 embryo transfers in 2019 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41). , and 12 pregnancies (60.0% pregnancy rate)” . I don’t think you should be reporting 60% pregnancy rate per se, but you can say that cumulative pregnancy rate for 20 patients after 41 transfers was 60%. Was your pregnancy rate 12/41 or 13/41? What was the intention to treat analysis for pregnancy rate, after first embryo transfer per patient? A6. For the sake of clarity, we have reworded this paragraph (lines 168–172) as follows: “As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 transfers in 19 patients, yielding an implantation rate of 31.7% per embryo (13/41). Among the 20 treated patients, six live births were recorded—including one from a spontaneous pregnancy post-therapy—and eight miscarriages, obtaining a live birth rate of 30% (Table 1). As per protocol, the intention-to-treat pregnancy rate was assessed over a 15‑month follow-up period. 7. Please have careful language revision done again, as after the revisions there are several typos or missing words, double or extra words or commas, and some sentences are now difficult to read. Maybe I was reading the wrong highlighted version, but it appears as if both deleted and new text are still present. Language revision will also help with proper word usage, such as “catheterization” instead of “catheterism”. A7. Thank you for your comment. Upon review, we realized that the tracked changes version still contained elements that had previously been removed. We have now highlighted these sections in yellow, rather than using track changes, and have thoroughly reviewed the manuscript to ensure accuracy and clarity. Reviewer #2 (Remarks to the Author): The author has revised and supplemented the article. However, I still have some doubts about certain statements, as follows: 1. In secondary endpoints, the manuscript described that compared with before cell therapy, the endometrial fibrosis was significantly reduced after cell therapy, a 62% increase in gland percentage, a 64% 136 reduction in fiber-rich stroma, and a 73% increase in vessel percentage within the stroma were observed (Supplementary Figure 1A). I concerned that how long the author took the sample after cell therapy and it is better to clarified. A1. Thank you for your comment. We agree that clarification is important. As stated in the Methods section, endometrial biopsies were collected one month after cell therapy in all cases. This applies to all analyses, including scRNA-seq, histology, and all other data presented here. We have now also added a clarifying sentence in the Results section (lines 133–135, highlighted in yellow) 2. When the author answer question 4 from reviewer 2, they stated as following: In moderate to severe AS, the location of endometrial biopsies depends on the presence of identifiable areas of bona fide endometrium rather than on a specific topographical site. This is detailed in the Methods section (Procedures subsection lines 55-61.\" Specifically, it says: Endometrial biopsies were performed under hysteroscopic vision from the posterior wall of the uterus at least 1 cm away from any visually identified IUA using a semi-rigid double-action biopsy and grasping forceps (Karl-Storz, Germany)”. I have some concerns because in patients with IUA the degree of endometrial damage may not be consistent throughout the entire endometrium. Some areas may show severe fibrosis, while others may not. If we want to evaluate the effect of the cell therapy through histological changes in the endometrium, it is best to determine the sampling site of the endometrium. I am concerned that sampling the endometrium from different sites before and after therapy may affect the evaluation of the effectiveness of cell therapy. A2. We understand your concern, which addresses an important methodological aspect in evaluating endometrial histology in patients with intrauterine adhesions (IUA). Geographical heterogeneity of endometrial damage is a well-recognized challenge in Asherman’s Syndrome (AS), and standardizing biopsy site selection is essential to ensure reliable interpretation of histological changes. In this study, as well as in our previous related work (Santamaria X et al., Nature Communications, 2023), biopsy site selection was deliberately standardized. Specifically, biopsies were obtained under direct hysteroscopic vision from the posterior wall of the uterus, maintaining a 1 cm margin from any visible intrauterine adhesion. This approach ensured that all samples were collected from bona fide, non-fibrotic endometrium adjacent to the pathological area—tissue most likely to reflect the therapeutic response—while maintaining consistency across timepoints. The posterior wall of the uterine cavity is among the most reproducible and accessible sampling sites, particularly in cases where extensive synechiae may limit access to other regions. Hysteroscopic guidance allowed us to avoid areas of severe fibrosis or necrosis that would preclude meaningful histological evaluation, focusing instead on viable tissue with regenerative potential. Furthermore, the literature acknowledges the existence of different adhesion types (fibrous, muscular, or mixed) that cause variable degrees of damage across the uterine cavity (Foix A et al., Am J Obstet Gynecol, 1966). Our approach accounted for this variability by consistently sampling from the perilesional functional endometrium, which is most relevant for evaluating the impact of cell therapy aimed at tissue regeneration. In summary, while the entire endometrium may be variably affected in IUA, our standardized and targeted biopsy strategy ensured comparability of samples before and after treatment, thereby strengthening the reliability of our histological assessments. Santamaria, X., Roson, B., Perez-Moraga, R. et al. Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution. Nat Commun 14, 5890 (2023). https://doi.org/10.1038/s41467-023-41656-1 Foix A , R O Bruno, T Davison, B Lema. The pathology of postcurettage intrauterine adhesions. Am J Obstet Gynecol. 1966 Dec 1;96(7):1027-33. 3 In line 187, the author stated that “although we showed a potential association between enhanced endometrial characteristics and improved reproductive outcomes, it is important to note that live birth outcomes are also influenced by embryo transfer–related factors, such as embryo quality, number of transfers, timing, etc”. What dose the author mean? The author mentioned above that a single blastocyst was transferred. Could it be that the patients who failed to achieve pregnancy had poor-quality embryos? A3. Thank you for your comment. We agree that this point warrants clarification. In each case, a single blastocyst of documented quality was transferred. The sentence in question was intended to emphasize that live birth outcomes are multifactorial, influenced by numerous factors beyond the endometrial environment and embryo quality alone (for review, see Moreno I et al., Physiol Rev, 2023). Our findings therefore suggest a potential association between improved endometrial features and reproductive outcomes; however, we acknowledge that this association is not strong enough to establish causality, and that other confounding factors may have contributed to the observed outcomes. Moreno, A. Capalbo, A. Mas, T. Garrido-Gomez, B. Roson, M. Poli, E. Dimitriadis, X. Santamaria, F. Vilella, C. Simon, The human periconceptional maternal-embryonic space in health and disease. Physiol Rev, doi: 10.1152/physrev.00050.2021 (2023). 4. One of the characteristic of this study, as the author mentioned, is that single-cell sequencing of the endometrial tissue of patients before and after cell therapy was performed, revealing changes in cell composition and gene expression. If there were validation experiments, it would be more convincing. A4. We conducted validation experiments during the first round of reviews, as requested by another reviewer. Specifically, we validated at the protein level the scRNA-seq findings related to the upregulation of angiogenic pathways, focusing on the ANGPTL2 marker, which showed higher expression in post-AS therapy samples (Figure 4E). To this end, we performed immunostaining analysis of ANGPTL2 expression in paired pre- and post-treatment tissue samples and generated a representative image (Figure 4F) that supports the scRNA-seq results. The findings confirmed the transcriptomic-level upregulation, reinforcing the involvement of angiogenic mechanisms. For the reviewer’s convenience, we also include the image here. New Supplementary Figure 4F. ANGPTL2 validation for pre and post-AS therapy samples. ANGPTL2: gray, DAPI: blue. Additionally, in our previous study (Santamaria et al., 2023), we identified that the stressed epithelial subpopulation in AS epithelium expressed elevated levels of SLPI marker. In the present work, we also examined changes in SLPI expression after treatment (post-AS therapy) and found that SLPI was transcriptomically expressed predominantly in the pre-AS therapy samples (see figure below, provided for reviewer reference only). New figure for reviewer only. Representative RNAscope images showing the expression of SLPI as a marker of the characteristic AS epithelium cell type in AS patients. Images show an example of SLPI expression in Pre-AS therapy, Post-AS therapy and control endometria. SLPI (green) expression counterstained with DAPI shown merged and in separate channels for clarity. Scale bar = 20 µm. We extended our analysis of SLPI to the protein level by performing immunofluorescence staining in paired pre- and post-AS therapy samples. Pre-therapy, SLPI showed strong, diffuse epithelial staining, whereas post-CD133‑ BMDSC therapy, expression was markedly reduced and restricted to glands. This finding, now presented in Supplementary Figure 2F, is described in the revised manuscript as follows: “Notably, we previously showed that the AS-epithelial population expresses high levels of SLPI. Interestingly, SLPI expression was markedly reduced in post-AS therapy samples, which may account for the decrease in this cell population following CD133‑ BMDSC treatment (Supplementary Figure 2F).” New supplementary Figure 2F. Representative immunofluorescence images showing the expression of SLPI as a marker of the characteristic AS epithelium cell type in AS patients. Images show an example of SLPI expression in Pre-and Post-AS therapy. SLPI (green) expression counterstained with DAPI shown merged and in separate channels for clarity. Scale bar = 20 µm. 5. When the author mentioned in the Methods section and answering the question the author stated that hysteroscopy was only used for diagnosis and not for separating intrauterine adhesions, does this mean that cell therapy can make the adhesions disappear? As far as I know, it is very difficult for adhesion bands to disappear in moderate to severe IUA and usually requires surgical separation. A5. Thank you for your sharp comment. As you correctly point out, cell therapy alone does not make intrauterine adhesions disappear, particularly in moderate to severe IUA, where surgical separation is often required. In our study, we documented improvements in ASRM hysteroscopic scores, but not the complete disappearance of all adhesions. The literature recognizes that different adhesion types—fibrous, muscular, or mixed—arise from varying degrees of endometrial damage across the uterine cavity (Foix A et al., Am J Obstet Gynecol, 1966). In our study, we have shown that autologous CD133‑ BMDSC therapy improves endometrial quality at the single-cell level by generating bona fide endometrium. This newly formed functional tissue can progressively replace areas of adhesion and, more importantly, restore endometrial functionality, enabling normal menstruation and supporting pregnancy. Reviewer #3 (Remarks to the Author): Thank you for revising you manuscript and for your detailed answers. I have no further comments Reviewer #4 (Remarks to the Author): Regarding the manuscript's methodology and statistics, we believe the authors responded comprehensively and clearly to our comments. In particular, they have provided a clearer explanation of the difference between primary and secondary endpoints, which was a source of confusion in the previous version. Additionally, the statistics section now appears more organized and systematic. Finally, the paragraph about machine learning that was in the previous version has been omitted. We believe this was an appropriate choice because, although the paragraph did not add essential information, it increased the risk of bias. Reviewer #5 (Remarks to the Author): I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts. Reviewer #6 (Remarks to the Author): Thank you very much for addressing my comments. 1. While I understand that the use of older software versions (CellRanger 3.1.0 and Seurat 4) was specified in the original protocol from 2020, I would recommend acknowledging this limitation in the manuscript. Specifically, it would be prudent to mention that these older versions were used as per the protocol and to discuss any potential inconsistencies when compared to newer versions, which are now available. Using older software versions could potentially result in inconsistencies or missed improvements in data analysis, which may impact the comparisons with other related works in the field. Additionally, this could limit the ability of other researchers to use the provided cell annotation clusters effectively unless they also use the same older versions, which may introduce additional complications. In the rapidly evolving field of bioinformatics, keeping up with updates is generally recommended to ensure accurate and reproducible results for all researchers, especially considering that the patient data in this study cannot be stored for reanalysis in an open repository. A1. Thank you for your comment. We have added the following clarification to the Methods section (highlighted in yellow, lines 179-189): scRNA-seq data was processed using Cell Ranger v3.1.0 and Seurat v4. These versions were maintained to ensure consistency with a previously analyzed dataset and to enable direct comparability3. While newer versions include improvements in cell calling, UMI correction, doublet detection, and annotation, we applied the same pipeline and stringent quality controls across all samples to minimize technical variability. Core Seurat steps—normalization, dimensionality reduction, clustering, and visualization—also improved across versions. Nevertheless, we consider our biological interpretations robust, while acknowledging possible limitations in sensitivity and cross-study compatibility with newer software releases 2. Regarding the availability of the analysis code, I support the idea of publishing it on GitHub after acceptance. However, for a journal like Nature Communications, I believe it would be beneficial to provide the code during the revision process itself. This would allow reviewers, including myself, to verify the consistency, transparency, and reproducibility of the analysis before final publication. Once again, I appreciate your efforts in addressing these issues, and I believe the manuscript will benefit from further clarification and transparency regarding the software versions used and the availability of the analysis code. A2. Thank you for your suggestion and for the previous comments that made the manuscript improve thoroughly. We fully support transparency and reproducibility, and so here is the link where the code is fully available:https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public REVIEWER COMMENTS All changes made in response to the reviewers’ comments have been highlighted in yellow throughout the manuscript. Reviewer #1 (Remarks to the Author): The reviewer appreciate details responses not only to my, but other reviewers' comments, including running additional experiments. I have some more questions/comments. 1. Diagnostic hysteroscopy: you mention that surgeries were performed by single non-blinded physician, scoring was thoroughly documented through photographic evidence and independently reviewed in a blinded manner by a second surgeon. But then you also mentioned that you could not document via images or video results of all hysteroscopies due to equipment malfuncton(Although images are not available for all patients, visualization from the side of the clinician at the time of analysis was performed). So, how would second physician review and confirm the ASRM scoring without documentation available? This is pretty important, as assessment should be reliable. The surgeon is also first author of the study, as well as co-founder of the company, so the potential bias is there. A1. Thank you for your comment. We acknowledge the potential limitations in documentation and recognize the importance of rigorous, unbiased assessment, particularly in the context of possible conflicts of interest. Photographic documentation was obtained for most hysteroscopic procedures (n= 15) and independently reviewed by a second experienced surgeon, blinded to both treatment allocation and patient identity. In the remaining four cases where photographic recording was unavailable, the ASRM hysteroscopic score was verified through direct visualization at the time of the procedure by both surgeons, with detailed operative notes recorded contemporaneously. While this represents an unavoidable limitation, we minimized potential bias through predefined scoring criteria and consistent documentation protocols. This limitation has now been explicitly stated in methods (line 51-55) and also mentioned in Figure legend 3 (see below A2) 2. Please explain in the text that you only had enough good quality cDNA samples left to run MAESTER in three samples, hence only n=3 for mtDNA. In general, any missing data should be explained otherwise raises questions (e.g. missing endometrial volumes, or missing hysteroscopic images). A2. Thank you, we have now added an explanation in line 287-289 (highlighted in yellow) as follows: Since the MAESTER technique requires reamplification of residual cDNA from single-cell RNA sequencing libraries, we had sufficient high-quality cDNA to perform the analysis in only three patients (N7, N9, and N15). As per the missing values in endometrial volumes and hysteroscopic images, we have also added a sentence in the associated figure legends indicated below. Figure 2. Effect of CD133+ therapy on the endometrium. A) Effect of the autologous CD133+ BMDSC therapy on the endometrial thickness and volume assessed by 2D and 3D vaginal ultrasound pre- (Pre-AS therapy) and one-month post-therapy (Post-AS therapy). Data shown are from the 16 patients with both 2D and 3D endometrial measurements available. B) Box plots of endometrial thickness (n=19), and (C) endometrial volume (n=16) of AS patients pre- and post-AS therapy representing all data before and after one month of cell therapy. Endometrial volume data for three patients (n=16/19) could not be measured due to unavailability of the 3D transvaginal probe at the time of patient follow-up. Whisker ends on the box plot represent the minimum and the maximum data point. Paired t-test; **** p<0.0001. Figure 3. Effect of CD133+ therapy in the endometrial cavity. A) Representative hysteroscopic images of the effect of the autologous CD133+ BMDSC therapy on the endometrial cavity assessed by hysteroscopic scores performed pre (Pre-AS therapy), and one-month post-therapy (Post-AS therapy) according to the IUA ASRM score. Representative hysteroscopy images of the four remaining patients (n=15/19) could not be illustrated due to technical limitations of hysteroscopy equipment at the time of patient follow-up. Hysteroscopic images consistently show constricted uterine cavities by IUAs with limited space pre-AS therapy while images demonstrate a significantly widened uterine cavity, indicating successful structural remodeling post-AS therapy. B) Box plots of IUAs score from AS patients (n=19) pre-AS therapy (8±3.17) and post-AS therapy (4.42±2.09). Whisker ends on the box plot represent the minimum and the maximum data point. Wilcoxon test; **** p<0.0001. 3. Can you please explain the rationale for doing all evaluation and treatment in mid-secretory phase? How do you explain that incorporation of stem cells into endometrium and subsequent regeneration of endometrium happens when intravascular injection is performed in the presence of progesterone? Have the authors trouble shoot the timing in therapy in prior studies? This needs to be better explained. A3. The rationale for performing stem cell treatment in mid-secretory phase is because progesterone essentially creates a “fertile ground” for the stem cells to recognize, adhere, engraft, and contribute to regeneration. Progesterone transforms the estrogen-primed endometrium into a secretory, receptive state. This includes decidualization of stromal fibroblasts, increased extracellular matrix deposition, and altered immune tolerance. Also, P4 upregulates angiogenic factors such as VEGF and angiopoietins, which make the microvasculature more permeable to circulating cells. In this primed environment, progesterone also induces expression of key chemotactic and adhesion molecules—including CXCL12 (SDF-1), ICAM-1, and selectins—that are critical for guiding stem cells toward the endometrial tissue. Finally, P induces expression of factors like CXCL12 (SDF-1), ICAM-1, and selectins, which guide stem cells toward the endometrial tissue (1-9). Stem cells administered via intravascular injection in uterine microcirculation can reach the spiral arterioles and venules of the endometrium. They express surface receptors such as CXCR4, which respond to progesterone-induced chemokine gradients, facilitating active homing and targeted migration into the stromal compartment. Progesterone-induced endothelial changes further support stem cell adhesion, rolling, and diapedesis into the tissue. Once engrafted, stem cells secrete growth factors (e.g., HGF, IGF-1, bFGF) that enhance epithelial proliferation, angiogenesis, and stromal repair. In some instances, mesenchymal stem cells may differentiate into endometrial stromal-like cells, replenishing damaged areas. Both progesterone and stem cells both promote a locally anti-inflammatory, pro regenerative environment, reducing fibrosis and allowing structural regeneration (10). Importantly, if stem cells were injected without progesterone priming, the endometrium may remain in a proliferative or atrophic state with lower vascular permeability, less chemokine signaling, and reduced receptivity, impairing stem cell engraftment and therapeutic efficacy. The rationale for evaluation in the mid-secretory phase is because this part of the menstrual cycle represents the most informative time point for clinical evaluation of endometrial receptivity improvements following stem cell therapy. This window allows for the assessment of key surrogate markers, including endometrial thickness, volume, and hysteroscopic scores. These parameters provide measurable evidence of structural and functional regeneration. We have previously optimized the therapeutic timing internally and successfully validated the concept in our pilot study (11). 1. Santamaria, X., Cabanillas, S., Cervelló, I., Arbona, C., Raga, F., Ferro, J., & Simón, C. (2023). Autologous stem cell therapy for refractory Asherman’s syndrome and endometrial atrophy: A translational approach. Nature Communications, 14, Article 1284. https://doi.org/10.1038/s41467-023-01284-y 2. Cervelló, I., Gil-Sanchis, C., Mas, A., Delgado-Rosas, F., Martinez-Conejero, J. A., Galán, A., … & Simón, C. (2015).Human endometrial CD140b+ stem cells: Involvement in recurrent miscarriage and infertility.Stem Cells, 33(2), 467–477. https://doi.org/10.1002/stem.1853 3. Gargett, C. E., Schwab, K. E., & Brosens, J. J. (2016). Putting stem cells in context in the human endometrium.Biology of Reproduction, 95(2), 1–10. https://doi.org/10.1095/biolreprod.116.139451 4. Mendoza-Tesarik, R., Mendoza, C., & Tesarik, J. (2019). Improvement of endometrial receptivity by autologous stem cell therapy in infertility patients refractory to conventional treatments: A pilot study.Reproductive Biomedicine Online, 39(6), 836–842.https://doi.org/10.1016/j.rbmo.2019.07.001 5. Gargett, C. E., & Masuda, H. (2010). Adult stem cells in the endometrium. Molecular Human Reproduction, 16(11), 818–834. https://doi.org/10.1093/molehr/gaq061 6. Critchley, H. O. D., Maybin, J. A., & Saunders, P. T. K. (2020). Progesterone receptor and mechanisms of action in the endometrium. Journal of Endocrinology, 246(2), R1–R14. https://doi.org/10.1530/JOE-20-0193 7. Liu, Y., Niu, X., & Sun, Y. (2019). CXCL12/CXCR4 axis in stem cell homing and recruitment: New therapeutic perspectives for regenerative medicine.Histology and Histopathology, 34(11), 1161–1173. https://doi.org/10.14670/HH-18-099 8. Tang, J., Li, W., Ma, Q., & Liu, H. (2018). The role of the SDF-1/CXCR4 axis in endothelial progenitor cell homing in ischemic tissues. 9. Stem Cell Reviews and Reports, 14(4), 584–591. https://doi.org/10.1007/s12015-018-9809-5 10. Moreno, A. Capalbo, A. Mas, T. Garrido-Gomez, B. Roson, M. Poli, E. Dimitriadis, X. Santamaria, F. Vilella, C. Simon, The human periconceptional maternal-embryonic space in health and disease. Physiol Rev, doi: 10.1152/physrev.00050.2021 (2023). 11. Santamaria, X. et al. Autologous cell therapy with CD133+ bone marrow-derived stem cells for refractory Asherman’s syndrome and endometrial atrophy: a pilot cohort study. Hum Reprod 31, 1087-1096 (2016). 4. Regarding the cycle phase: while in the materials and methods you provided a statement, throughout the text this information was not consistent or clear, hence every reviewer having similar comment. Please revise accordingly. Thank you for your comment. We have reviewed the manuscript to ensure consistency, and information regarding the secretory phase has now been added at lines 89–90 and 213 of the main manuscript, in addition to the description already provided in the Methods section. We hope this is now clear. 5. After the revision it became less clear if the pregnancy outcomes (positive pregnancy rate, clinical pregnancy, miscarriage, live birth) are among secondary outcomes or not. Thank you for your comment. In line 130, under the subsection of “secondary endpoints” we stated: The secondary endpoints included endometrial morphometry, thickness, volume, uterine hysteroscopic scores, and endometrial function evaluated by menstruation recovery and pregnancy outcomes (line 130-135). We have also highlighted in yellow in line 168 the following: As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 embryo transfers in 19 patients… In the Methods section (lines 28–34), we stated: “Secondary endpoints were the efficacy of the therapy in achieving improvements in morphological and clinical features, such as endometrial histology assessed through digital image analysis; endometrial thickness and volume measured by 2D and 3D vaginal ultrasound; hysteroscopic grading scores; menstrual function recorded monthly based on duration (days), volume (pads/day), and frequency (days) of menstrual bleeding using the Pictorial Blood Assessment Chart⁷; and reproductive outcomes after treatment, measured by implantation, pregnancy, miscarriage, ongoing, and live birth rates.” We identified a section in the Discussion where this may not have been entirely clear and have added further clarification (lines 342–345). Our findings indicate that the primary endpoints of the therapy—safety and tolerability of this autologous cell therapy—were met, with the treatment being well tolerated for up to 15 months of follow-up. Further, as a secondary endpoint, the AS patients after cell therapy achieved an implantation rate of 31.7% per embryo transferred (13/41) resulting in 5 live births. In addition, we recorded one spontaneous pregnancy. As per adverse events of live birth and newborns, no serious adverse events were observed beyond common obstetric complications, fulfilling the primary end points of safety and tolerability of the study. We hope this is now clear throughout the manuscript. 6. Results: “We performed a total of 41 embryo transfers in 2019 patients, resulting in an implantation rate of 31.7% per embryo transferred (13/41). , and 12 pregnancies (60.0% pregnancy rate)” . I don’t think you should be reporting 60% pregnancy rate per se, but you can say that cumulative pregnancy rate for 20 patients after 41 transfers was 60%. Was your pregnancy rate 12/41 or 13/41? What was the intention to treat analysis for pregnancy rate, after first embryo transfer per patient? A6. For the sake of clarity, we have reworded this paragraph (lines 168–172) as follows: “As per the reproductive outcomes, stated as a secondary endpoint, we performed a total of 41 transfers in 19 patients, yielding an implantation rate of 31.7% per embryo (13/41). Among the 20 treated patients, six live births were recorded—including one from a spontaneous pregnancy post-therapy—and eight miscarriages, obtaining a live birth rate of 30% (Table 1). As per protocol, the intention-to-treat pregnancy rate was assessed over a 15‑month follow-up period. 7. Please have careful language revision done again, as after the revisions there are several typos or missing words, double or extra words or commas, and some sentences are now difficult to read. Maybe I was reading the wrong highlighted version, but it appears as if both deleted and new text are still present. Language revision will also help with proper word usage, such as “catheterization” instead of “catheterism”. A7. Thank you for your comment. Upon review, we realized that the tracked changes version still contained elements that had previously been removed. We have now highlighted these sections in yellow, rather than using track changes, and have thoroughly reviewed the manuscript to ensure accuracy and clarity. Reviewer #1 (Remarks on code availability): n/a Reviewer #6 (Remarks to the Author): Thank you for the improvements to the manuscript. The overall organization has clearly progressed, so that analyses for AS patients, organoids, CD133+, CellChat, and differential abundance/expression are now easier to navigate. As a minor addition, a brief, targeted cleanup of the repo could reach a higher level of transparency and reusability that will greatly benefit readers. Here, I would recommend adding a stepwise analysis scheme directly in the README as a small table to anchor timelines and versions for Data collection window, QC/processing window, Figure generation, and Manuscript freeze. This makes it more comprehensible which code/outputs map to the results in the manuscript and helps future users reuse the analyses tied to a specific manuscript freeze (the one from 2023 or the potentially upcoming one). In particular, the repository’s scope (single-cell analysis of AS patients, organoids, CellChat cell–cell communication, differential abundance/expression) overlaps conceptually with the Nature Communications paper “Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution” (21 Sep 2023). Please cite it prominently in the README and in the relevant scripts, and briefly note what was adopted or extended (e.g., “CellChat settings and epithelial subpopulation markers adapted from …”). This avoids ambiguity and situates your work in the existing literature, even if its your own. Please also check again for hard-coded elements that reduce portability, e.g. replace absolute paths, inline parameters, and environment-specific toggles. with a single config.yml (paths, sample IDs, thresholds). (Example found in https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/src/R/AS_Patients/maester/analyze_variants/explore_variants_final.r) You could also commit an environment spec (R: renv.lock via renv::snapshot() or a minimal DESCRIPTION; Python: requirements.txt / environment.yml if applicable). In summary, the repository is already in good shape, and the above points are minimal, pragmatic changes that will substantially increase transparency, reproducibility, and code reuse. I appreciate the progress so far. With these tweaks, the repo will be straightforward to reuse alongside the manuscript and be more valuable for the community. Thank you to reviewer 6 for the comment, we have now improved the transparency and usability of the code repository , specifically we answer step by step to your comments: a) Here, I would recommend adding a stepwise analysis scheme directly in the README as a small table to anchor timelines and versions for Data collection window, QC/processing window, Figure generation, and Manuscript freeze. This makes it more comprehensible which code/outputs map to the results in the manuscript and helps future users reuse the analyses tied to a specific manuscript freeze (the one from 2023 or the potentially upcoming one). In particular, the repository’s scope (single-cell analysis of AS patients, organoids, CellChat cell–cell communication, differential abundance/expression) overlaps conceptually with the Nature Communications paper “Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution” (21 Sep 2023). Please cite it prominently in the README and in the relevant scripts, and briefly note what was adopted or extended (e.g., “CellChat settings and epithelial subpopulation markers adapted from …”). This avoids ambiguity and situates your work in the existing literature, even if its your own. a) Although some single-cell analyses of patients and organoids conceptually overlap with the previous work, this project was developed independently. Consequently, there is no formal freeze or direct mapping to the 2023 article. Some analyses, however, represent extended versions of those in the 2023 paper, and certain parameters and markers were reused. For these cases we have now explicitly indicated in the code and in the table added to the README, which details each analysis step, links the corresponding code to its outputs and figures, and includes an additional column noting any elements adopted or extended from the 2023 article. This can now be found here: https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/README.md b) Please also check again for hard-coded elements that reduce portability, e.g. replace absolute paths, inline parameters, and environment-specific toggles. with a single config.yml (paths, sample IDs, thresholds). (Example found in https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/src/R/AS_Patients/m… b) Thank you for the comment. All previously hard-coded elements, such as paths, sample IDs, parameters, and thresholds, have been now replaced with variables defined in config.yml files specific to each type of analysis. c) You could also commit an environment spec (R: renv.lock via renv::snapshot() or a minimal DESCRIPTION; Python: requirements.txt / environment.yml if applicable). c) Thank you, we have now uploaded a requirements.txt file and an renv.lock file for the MAESTER analysis (_MAESTER), as well as an renv.lock file for the rest of the analyses (see below)"}, {"round": 3, "reviewer_comments": {"reviewer_1": "Regarding the comment on reporting pregnancy outcomes: it should clearly state if the pregnancy rate and life birth rate is per attempt (per embryo transfer) or if it is cumulative after multiple transfers in same patient. Please consult statistician if needed. Regarding pregnancy outcomes: you should report pregnancy rate per transfer first, and can report cumulative pregnancy rate per patient, and the life birth should be reported per transfer and cumulative per patient. For clarity. (Remarks on code availability) n/a", "reviewer_2": "(Remarks on code availability)", "reviewer_6": "Thank you for the improvements to the manuscript. The overall organization has clearly progressed, so that analyses for AS patients, organoids, CD133+, CellChat, and differential abundance/expression are now easier to navigate. As a minor addition, a brief, targeted cleanup of the repo could reach a higher level of transparency and reusability that will greatly benefit readers. Here, I would recommend adding a stepwise analysis scheme directly in the README as a small table to anchor timelines and versions for Data collection window, QC/processing window, Figure generation, and Manuscript freeze. This makes it more comprehensible which code/outputs map to the results in the manuscript and helps future users reuse the analyses tied to a specific manuscript freeze (the one from 2023 or the potentially upcoming one). In particular, the repository’s scope (single-cell analysis of AS patients, organoids, CellChat cell–cell communication, differential abundance/expression) overlaps conceptually with the Nature Communications paper “Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution” (21 Sep 2023). Please cite it prominently in the README and in the relevant scripts, and briefly note what was adopted or extended (e.g., “CellChat settings and epithelial subpopulation markers adapted from …”). This avoids ambiguity and situates your work in the existing literature, even if its your own. Please also check again for hard-coded elements that reduce portability, e.g. replace absolute paths, inline parameters, and environment-specific toggles. with a single config.yml (paths, sample IDs, thresholds). (Example found in https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/src/R/AS_Patients/maester/analyze_variants/explore_variants_final.r) You could also commit an environment spec (R: renv.lock via renv::snapshot() or a minimal DESCRIPTION; Python: requirements.txt / environment.yml if applicable). In summary, the repository is already in good shape, and the above points are minimal, pragmatic changes that will substantially increase transparency, reproducibility, and code reuse. I appreciate the progress so far. With these tweaks, the repo will be straightforward to reuse alongside the manuscript and be more valuable for the community. (Remarks on code availability)"}, "author_response": "Thank you to reviewer 6 for the comment, we have now improved the transparency and usability of the code repository , specifically we answer step by step to your comments: a) Here, I would recommend adding a stepwise analysis scheme directly in the README as a small table to anchor timelines and versions for Data collection window, QC/processing window, Figure generation, and Manuscript freeze. This makes it more comprehensible which code/outputs map to the results in the manuscript and helps future users reuse the analyses tied to a specific manuscript freeze (the one from 2023 or the potentially upcoming one). In particular, the repository’s scope (single-cell analysis of AS patients, organoids, CellChat cell–cell communication, differential abundance/expression) overlaps conceptually with the Nature Communications paper “Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution” (21 Sep 2023). Please cite it prominently in the README and in the relevant scripts, and briefly note what was adopted or extended (e.g., “CellChat settings and epithelial subpopulation markers adapted from …”). This avoids ambiguity and situates your work in the existing literature, even if its your own. a) Although some single-cell analyses of patients and organoids conceptually overlap with the previous work, this project was developed independently. Consequently, there is no formal freeze or direct mapping to the 2023 article. Some analyses, however, represent extended versions of those in the 2023 paper, and certain parameters and markers were reused. For these cases we have now explicitly indicated in the code and in the table added to the README, which details each analysis step, links the corresponding code to its outputs and figures, and includes an additional column noting any elements adopted or extended from the 2023 article. This can now be found here: https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/README.md b) Please also check again for hard-coded elements that reduce portability, e.g. replace absolute paths, inline parameters, and environment-specific toggles. with a single config.yml (paths, sample IDs, thresholds). (Example found in https://github.com/FCS-BioIT/asherman-trial-cd133-cell-therapy-public/blob/main/src/R/AS_Patients/m… b) Thank you for the comment. All previously hard-coded elements, such as paths, sample IDs, parameters, and thresholds, have been now replaced with variables defined in config.yml files specific to each type of analysis. c) You could also commit an environment spec (R: renv.lock via renv::snapshot() or a minimal DESCRIPTION; Python: requirements.txt / environment.yml if applicable). c) Thank you, we have now uploaded a requirements.txt file and an renv.lock file for the MAESTER analysis (_MAESTER), as well as an renv.lock file for the rest of the analyses (see below)"}, {"round": 4, "reviewer_comments": {"reviewer_1": "Thanks for addressing the comments. The paper is better and clearer now. Best of luck! (Remarks on code availability)", "reviewer_6": "Thank you for the thorough and constructive revision. You have incorporated all outstanding requests and the manuscript now reads clearly, with practical guidance and improved transparency of the code repository. (Remarks on code availability)"}, "author_response": ""}]

Decision letter
Peer Review File Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase 1/2 trial Corresponding Author: Dr Xavier Santamaria This file contains all reviewer reports in order by version, followed by all author rebuttals in order by version.