Modified letrozole vs GnRH antagonist protocols in ovarian aging women for IVF: an open-label, multicenter, randomized controlled trial

Domain: biology
Published: 2026-03-20
Editorial outcome: major_revision

Abstract
For women with diminished ovarian reserve or of advanced age, controlled ovarian stimulation presents a significant challenge during in vitro fertilization cycles. This multi-center, open-label, randomized controlled trial enrolled 318 women with diminished ovarian reserve (defined as an antral follicle count < 5 or anti-Müllerian hormone level of 0.1-1.1 ng/mL) or advanced age (40-45 years) between 2020 and 2023. Participants were assigned to either a modified letrozole protocol (mLP, n = 159) or a gonadotropin-releasing hormone antagonist protocol (n = 159). Primary outcomes, cumulative clinical pregnancy rate and cumulative live birth rate, were analyzed using both the full analysis set and per-protocol set. Secondary outcomes including live birth rate, clinical pregnancy rate and pregnancy loss rates were analyzed using per-protocol set. Results from the full analysis set showed comparable cumulative clinical pregnancy rates (32.1% vs. 34.0%; RR 0.94, 95% CI 0.69-1.29) and cumulative live birth rates (24.5% vs. 22.6%; RR 1.08, 95% CI 0.73-1.61) between the two groups. The per-protocol analysis also demonstrated comparable cumulative clinical pregnancy rates (33.3% vs. 36.0%; RR 0.93, 95% CI 0.68-1.27). Notably, the mLP was associated with a significantly higher clinical pregnancy rate among patients with diminished ovarian reserve who underwent dual cleavage-stage fresh embryo transfers (65.8% vs. 36.4%; RR 1.81, 95% CI 1.15-2.85). Although primary outcomes were similar between protocols, the mLP improved clinical pregnancy rates in fresh embryo transfers for women with diminished ovarian reserve, suggesting its potential to enhance in vitro fertilization efficacy in this population. Fresh transfers demonstrated non-significant difference of mLP for live birth rate (34.0% vs. 22.2%; RR 1.53, 95% CI 0.96-2.43; p = 0.164), non-significant reduced biochemical pregnancy loss (22.0% vs. 34.3%; RR 0.59, 95% CI 0.29-1.21; p = 0.229) and miscarriage rate (20.0% vs. 26.7%; RR 0.75, 95% CI 0.32-1.77). Trial registration: ChiCTR2000029272.

Introduction
The progressive decline in oocyte quality and quantity associated with diminished ovarian reserve (DOR) poses significant challenges in assisted reproduction1,2, affecting 25%-50% of couples undergoing in vitro fertilization-embryo transfer (IVF-ET)3. Women under 40 years with DOR experience 10-15% reductions in clinical pregnancy rates (CPR) and 5-17% live birth rates (LBR) per cycle compared to those with normal ovarian reserve3-8. This challenge intensifies in women aged 40-45 years, resulting in LBRs below 10% in many cohorts9. While diverse controlled ovarian stimulation (COS) protocols have been explored, no consensus exists due to inconsistent efficacy reports, particularly for advanced maternal age populations10-13. The lack of evidence-based protocols tailored to women with DOR or advanced age with 40-45 years-a demographic experiencing both accelerated ovarian aging and time-sensitive fertility decline-underscores the critical need for optimized COS strategies that balance ovarian response efficiency with minimized treatment burden.

Letrozole, a third-generation aromatase inhibitor, enhances antral follicle recruitment through androgen priming-elevating intraovarian androstenedione and testosterone levels, thereby increasing follicle stimulating hormone (FSH) sensitivity. This pharmacodynamic profile positions letrozole as a promising agent for optimizing COS in DOR populations. Our prior clinical investigations demonstrated that follicular-phase serum testosterone surges correlate with 2.3-fold higher CPR14. And our retrospective cohort study revealed that the modified letrozole protocol (mLP), which strategically combines letrozole administration with gonadotropin supplementation, significantly improved both CPR (42.1% vs. 28.3%, p = 0.023) and LBR (31.6% vs. 18.9%, p = 0.038) in women ≥ 40 years or < 40 years with DOR15. These results suggest that mLP’s dual mechanism-amplifying androgen-mediated follicular recruitment while maintaining physiological estrogen synthesis-may address the dual challenges of poor ovarian response and endometrial receptivity in DOR patients. To establish robust evidence for this innovative approach, we conducted this first multicenter randomized controlled trial (RCT) comparing mLP with conventional gonadotrophin-releasing hormone antagonist (GnRH-ant) protocols in infertile patients < 40 years with DOR, or advanced age with 40-45 years. This study not only evaluates mLP’s efficacy among these populations but also pioneers the development of a standardized, patient-tailored COS protocol for this clinically challenging cohort.

Results
Participants, Baseline, and IVF Laboratory Characteristics
The clinical trial has been completed currently. Between March 2020 and February 2023, we screened a total of 437 women. Of these women, 318 who met the inclusion criteria were provided informed consent. Then they underwent randomization (with 159 women assigned to each group). The trial profile was shown in Figure 1. Follow-up for all pregnancies was finalized by November 2023. Among them, eight participants stopped halfway since preovulation or absent follicular development, and seven participants withdrew or deviated from the protocols. Throughout the study duration, no adverse events (AEs) were observed. 153 women in the mLP group and 150 women in the GnRH-ant group who finished the study were secondary analyzed by per protocol. Baseline characteristics between two groups were comparable analyzed by the Full Analysis Set (FAS) (Table 1). Ovarian stimulation outcomes and laboratory parameters for both groups are detailed in Table 2 analyzed by per-protocol set (PPS) and in Supplementary Table 3 by FAS, with the detailed data regarding the cancellation of oocyte retrieval or fresh embryo transfer presented in Supplementary Table 4. The generalized linear mixed model (GLMM) yielded consistent results with the primary analysis and estimated treatment effects as odds ratios (ORs) with 95% CIs.

The mLP group exhibited lower endometrial thickness on hCG trigger day (9.2 ± 2.5 vs. 10.3 ± 3.0 mm, p = 0.001), fewer metaphase II (MII) oocytes retrieved (2.4 ± 2.3 vs. 3.1 ± 2.6, p = 0.028), and fewer available embryos (2.0 ± 1.4 vs. 2.7 ± 1.9, p < 0.001). The mLP group demonstrated significantly shorter ovarian stimulation duration (6.6 ± 2.8 vs. 9.4 ± 2.1 days, p < 0.0001) and less total Gn consumption (2006.0 ± 797.3 vs. 2652.8 ± 657.7 IU, p < 0.0001) compared to the GnRH-ant group, with statistically significant differences (Table 2). But no significant intergroup differences were observed in follicle output rate (FOR) or the number of high-quality embryos (p > 0.05). There is no significant difference regarding to the fertilization method between two groups (Supplementary Table 5).

Primary outcomes
A formal protocol amendment was implemented during the trial. The original primary outcome was the cumulative clinical pregnancy rate (CCPR) in one COS. Analysis of the full analysis set (FAS, n = 159 per group) showed comparable CCPR between the mLP and GnRH-ant groups (32.1% vs. 34.0%; RR 0.94, 95% CI 0.69-1.29; p = 0.283). This finding was consistent with the per-protocol set (PPS) analysis per COS cycle (33.3% vs. 36.0%; RR 0.93, 95% CI 0.68-1.27; p = 0.275) and the CCPR per embryo-available cycle (45.5% vs. 41.2%; RR 1.10, 95% CI 0.82-1.47; p = 0.794).

The amended primary outcomes included both the CCPR and the Cumulative live birth rate (CLBR) in one COS. FAS analysis demonstrated no significant difference in CLBR between the mLP and GnRH-ant groups (24.5% vs. 22.6%; RR 1.08, 95% CI 0.73-1.61; p = 0.991). Similarly, the PPS analysis (mLP: n = 153; GnRH-ant: n = 150) showed comparable results (25.5% vs. 24.0%; RR 1.06, 95% CI 0.72-1.57; p = 0.991). For CLBR per embryo-available COS cycle, the mLP group also showed numerically elevated rates compared with the GnRH-ant group (34.8% vs. 27.5%; RR 1.27, 95% CI 0.87-1.85; p = 0.508), though these differences lacked statistical significance (Table 3). Sensitivity analyses using age and center-adjusted odds ratios corroborated the primary findings (Supplementary Table 6). Sensitivity analyses adjusting for clinical center effects through generalized linear mixed models (ICC < 0.1) confirmed minimal between-center variability (p > 0.20 for center interaction) (Supplementary Table 7).

Secondary outcomes
In fresh embryo transfer per-protocol analyses, secondary outcomes showed no statistically significant differences: live birth rates (LBR, 34.0% vs. 22.2%; RR 1.53, 95%CI 0.96-2.43, p = 0.164), clinical pregnancy rates (CPR, 42.6% vs. 30.3%; RR 1.40, 95%CI 0.96-2.05, p = 0.753), biochemical pregnancy loss rates (22.0% vs. 34.3%; RR 0.59, 95%CI 0.29-1.21, p = 0.244) and miscarriage rate (20.0% vs. 26.7%; RR 0.75, 95% CI 0.32-1.77); which remained non-significant after multiplicity adjustment (p adjusted > 0.05) (Table 4). Both groups maintained comparable embryo transfer profiles, with cleavage-stage transfers predominating (mLP: 97.9% [92/94] plus 2.1% D5 blastocysts vs. GnRH-ant: 99.0% [98/99] plus 1.0% D5 blastocysts, p = 0.615) and similar single/double cleavage embryo utilization rates (single: 23.4% vs. 25.3%, p = 0.873; double: 72.3% vs. 73.7%, p = 0.892).

The prespecified subgroup analyses indicated numerical, though not statistically significant, improvements in live birth rates with the mLP among women with DOR under 40 years (45.1% vs. 29.1%; RR 1.55, 95% CI 0.93-2.59; p = 0.132) and those aged 40-45 years (20.9% vs. 13.6%; RR 1.53, 95% CI 0.60-3.93; p = 0.408). Notably, DOR patients receiving two cleavage-stage embryos by using mLP achieved significantly higher clinical pregnancy rates compared to GnRH-ant (65.8% vs. 36.4%; RR 1.81, 95% CI 1.15-2.85; p = 0.015) (Table 4), though blastocyst transfers were infrequent (mLP: 2/94; GnRH-ant: 1/99) with no successful pregnancies. Safety analyses showed numerically lower multiple pregnancy rates with mLP when transferring two cleavage-stage embryos (8.0% vs. 12.5%; RR 0.64, 95% CI 0.14-2.88; p = 0.615), with no significant interaction effect among the age and embryo types detected (p > 0.05) (Table 4).

Post Hoc Subgroup Analyzes
Stratified analyses by serum AMH thresholds (<1.2 vs. ≥ 1.2 ng/ml) and maternal age deciles revealed no statistically significant intergroup disparities in CPR, LBR, or CLBR (Fig. 2A-C). However, all ART outcome metrics exhibited a non-significant numerical decline paralleling advancing age, with CLBR decreasing from 28.4% (age < 35 years) to 14.7% (age ≥ 40 years), independent of ovarian reserve status. In addition, the impact of the mLP among the women with repeated IVF cycles was also evaluated, presented in Supplementary Table 8. For those undergoing more than one cycle, the mLP group demonstrated a non-significantly higher LBR (41.4% vs. 16.7%), for a rate ratio of 2.48 (95% CI 1.0-6.17; p = 0.071), and a significantly higher CPR (51.7% vs. 23.3%), for a rate ratio of 2.22 (95% CI 1.06-4.64; p = 0.047). Regarding those women with the endometrial thickness of 6-7 mm, there were 9 and 4 cases in mLP and GnRH-ant groups, respectively. There is no live birth among them (Supplementary Table 9).

Hormonal dynamics and relation with follicle growth during COS
Hormonal profiling revealed distinct pharmacodynamic responses between protocols (Fig. 2D-H). The mLP group exhibited sustained TT elevation during folliculogenesis (1.3 → 1.8 nmol/L vs. GnRH-ant 1.0 → 1.3 nmol/L; p < 0.05), with per-follicle TT concentrations at terminal maturation ( ≥ 16-18 mm) tripling controls (1.5 vs. <1.0 nmol/L; p < 0.001). This androgen surge paralleled mid-cycle LH spikes at critical follicular milestones (11-12 mm and ≥ 18 mm diameters), while concurrently suppressing E production (trigger-day: 516.2 vs. 1428.7 pg/mL; per-follicle: 200-300 vs. >600 pg/mL; p < 0.0001). FSH trajectories remained comparable between groups despite these divergent steroidogenic patterns.

Methods
Trial Design and Oversight
We conducted a multicenter, randomized controlled trial across six academic fertility centers in China. This was an open-label trial; therefore, participants and treating clinicians were not blinded to the treatment allocation. However, outcome assessors and the statistician remained blinded throughout the study to minimize assessment and analytical bias. All data provided to them were coded to conceal group identities. The trial is registered at www.chictr.org.cn (ChiCTR2000029272) (Original study protocol V1.0, 2019-12-20; Final study protocol V2.0, 2023-12-01; Supplementary material). Approval was granted by the ethics committees of Sun Yat-sen Memorial Hospital (2019-KY-067), Guangdong Provincial People’s Hospital (2020062H (R1)) and other participating hospitals including Affiliated Hospital of Guizhou Medical University (2022[269]), The Tenth Affiliated Hospital of Southern Medical University (DRYA2021-016-A1) , Shenzhen Maternal and Child Health Hospital (SFYLS[2020]063) and Zhejiang Provincial People’s Hospital (2020-KY-001). An Independent Data Monitoring Committee (IDMC) supervised by Sun Yat-sen University’s Office of Scientific Research and Development monitored the study. The trial was funded by the 5010 clinical research grants from Sun Yat-sen University and inspected annually by IDMC (2021-2022).

Participants
Participants were infertile women under 40 years of age with DOR with anti-mullerian hormone (AMH) < 1.2ng/ml or antral follicles count (AFC) < 5, or aged 40-45 years, scheduled for less than four IVF or intracytoplasmic sperm injection (ICSI) cycles. Exclusion criteria were a history of ovarian surgery, significant male factor infertility (e.g. a low male spermatogenic function, non-obstructive azoospermia, or AZF gene microdeletion), unilateral or bilateral hydrosalpinx, uterine abnormalities (e.g. single horn uterus, uterine adenomyosis, intrauterine adhesions, untreated endometrial polyps, and submucous myoma) and abnormal parental karyotype.

Randomization
After written informed consent, participants were randomly assigned using a block randomization scheme with 1:1 allocation to the mLP and GnRH-ant group with predefined stratification for age (< 40 years vs. ≥ 40 years) and study center (six academic fertility centers). Within each stratum, a separate block randomization sequence was generated with a block size of 4 and an allocation ratio of 1:1, with block sequences concealed from study recruiters. The allocation sequence was implemented via a centralized computer-generated randomization system, developed and managed by an independent biostatistics team from School of Public Health at Sun Yat-sen University This system ensured strict allocation concealment, wherein treatment assignment was only revealed after participant enrollment. Recruitment occurred from March 2020 to February 2023, with follow-up completed on November 2023. Participants provided written informed consent prior to randomization.

Interventions
Standardized procedures were implemented per respectively predefined standard operating procedures (SOPs) (Supplementary Table 1) to minimize interoperator variability. The mLP group initiated letrozole (5 mg/day) on menstrual day 2 for five days alongside gonadotropins (Gn) (300 IU/day from day 5); and the GnRH-ant group received Gn (300 IU/day from day 2). Both groups were added daily antagonist (0.25 mg) from day 6 until trigger. The trigger timing of mLP is determined by follicular maturity (≥ 50% ratio of ≥ 18 mm to ≥ 14 mm follicles); and that of GnRH-ant protocol is activated upon achieving ≥ 2 follicles ≥ 17 mm or 1 follicle ≥ 18 mm. Dual trigger 10,000 IU human chorionic gonadotropin (HCG) and 0.1 mg GnRH-agonist were given on the trigger day. All cycles underwent daily ultrasound monitoring using 3D automated follicle tracking (Voluson E8), with hormonal assessments (FSH, luteinizing hormone [LH], estradiol [E ], progesteron [P], and total testosterone [TT] measured by chemiluminescent immunoassay) regularly.

Oocyte pick-up (OPU) was conducted 34-36 hours post-trigger, with embryo transfer mainly with one or two cleavage embryos, few with blastocyst. Luteal-phase support was provided using vaginal progesterone gel at a dosage of 90 mg daily, or Utrogestan at 0.2 g administered three times daily, starting immediately post-OPU and continuing until the day serum HCG levels were measured.

Subject to cancellation under certain conditions including the absence of available embryos, a thin endometrium (endometrial thickness < 6 mm), ongoing endometrial diseases, needle passage through the endometrium during OPU, abnormal vaginal bleeding, a high risk of ovarian hyperstimulation syndrome (OHSS) indicated by the retrieval of ≥ 20 oocytes, elevated progesterone levels (> 2.0 ng/ml) on the trigger day, spontaneous pregnancy, fever (body temperature ≥ 37.5 ℃), and personal reasons.

The endometrial preparation methods for frozen-thawed embryo transfer (FET) included hormone replacement therapy (HRT) cycle, GnRH-a+HRT cycle, natural cycle (NC), and ovulation induction cycle. FET was scheduled with the same luteal-phase support. Two weeks after fresh ET or FET, serum hCG levels were assessed. For those with positive hCG ( ≥ 20 IU/ml on the 14th day) tests, luteal-phase support was extended through the 10th week of gestation. Detailed follow-ups were conducted for all participants until the completion of IVF treatment or determination of the pregnancy outcome. In case of conception, transvaginal ultrasonography was conducted two weeks subsequently to verify clinical pregnancy, with a follow-up scan at 12 weeks to confirm the ongoing pregnancy.

AEs were systematically recorded and defined as any undesirable medical occurrence observed in trial participants from the time of informed consent signing through the study completion.

Outcomes
The primary outcomes were initially set as the cumulative clinical pregnancy rate （CCPR) of one started IVF cycle. Since live birth represents the gold-standard outcome in IVF, the cumulative clinical pregnancy rate (CLBR) was added as a co-primary outcome. The CCPR and CLBR, analyzed through two metrics: (1) per initiated COS cycle, defined as the proportion of women achieving ≥ 1 clinical pregnancy or live birth from fresh and subsequent FETs within a single COS cycle; and (2) per embryo-available COS cycle, calculated as the proportion of women attaining ≥ 1 clinical pregnancy or live birth following any embryo transfer (fresh or FET). Secondary outcomes included live birth rate (LBR, the proportion of women with viable delivery ≥ 28 gestational weeks per fresh transfer), clinical pregnancy rate (CPR, the proportion of women with intrauterine gestational sac confirmed by ultrasound 28-30 days post-transfer), and pregnancy loss rate stratified as pregnancy loss among biochemical pregnancy (the percentage of women with serum hCG ≥ 20 IU/mL at 14 days post-transfer) or miscarriage (with pregnancy loss ≤ 12 weeks post-confirmation in clinical pregnancy) (Definition of outcomes; Supplementary Table 2).

Sample Size Calculation
For the initial sample size calculation, the expected CCPR in one controlled ovarian stimulation COS was 44.44% in mLP group and 27.70% in GnRH-ant group. The superiority margin was set at 5%. A 1:1 allocation ratio between the two groups was used (statistical power 0.8; two-sided significance level 0.025), 261 cases were required per group. Anticipating 10% loss to follow-up or dropout, 580 participants were required. Due to the COVID-19 pandemic, recruitment was much slower than anticipated, and on February 2023, the trial was halted after recruitment of 318 participants. The post-hoc power analysis indicated that this sample size provided 81.1% power to detect the 16.7% difference that was previously determined to be clinically relevant if a superiority margin of 0% is used.

Statistical Analysis
The Full Analysis Set (FAS) was derived by including all subjects who underwent randomization and completed ≥ 1 post-randomization follow-up assessment. The Per-Protocol Set (PPS) constituted a subset of the FAS comprising subjects who satisfied all inclusion/exclusion criteria, demonstrated protocol compliance without major deviations, completed the full treatment regimen, maintained adequate study adherence, had fully documented case report forms (CRFs) without critical omissions in primary efficacy data. Primary analyses were conducted on the FAS, with per-protocol analyses serving as secondary evaluations. Both FAS and per-protocol approaches were applied to primary outcomes (CCPR and CLBR). Post-hoc subgroup analyses were performed exclusively using per-protocol data for hypothesis generation.

For fresh embryo transfer outcomes (LBR, CPR, and pregnancy loss rate), per-protocol analyses (PPS) were conducted as exploratory subgroup assessments. Continuous variables were expressed as mean ± standard deviation and compared using Wilcoxon rank-sum tests. Categorical variables were summarized as counts (percentages) and analyzed with χ² tests or Fisher’s exact tests when expected cell counts fell below five. Treatment effects were reported as rate ratios (RRs) and risk differences (RDs) with 95% confidence intervals calculated via Wald method. Multiple comparisons adjustment for FAS analyses employed the Benjamini-Hochberg procedure. In addition, a prespecified sensitivity analysis was performed using GLMM with random intercepts for clinical centers. This model was added to address a reviewer’s comment on accounting for center effects and clustering by study site. Between-center variability was quantified through intraclass correlation coefficients (ICCs) with 95% CIs derived from parametric bootstrapping (1,000 replicates).

Stratified analyses examining treatment effect modification by clinical site and age category were pre-specified. We also performed stratified analysis by DOR with age < 40 years and age of 40-45 years, age (< 35 years, 35-39 years, ≥ 40 years) based on AMH concentration (< 1.2 ng/ml, ≥ 1.2 ng/ml), and the number of IVF cycles (none vs. ≥ 1). And the corresponding stratified analyses were included to evaluate heterogeneity in protocol effects. The pre-specified Statistical Analysis Plan is provided in the supplementary material (Final Statistical Analysis Plan V2.0, dated 2023-12-05). During manuscript preparation and revision, the following changes to the statistical analyses were introduced: Benjamini-Hochberg procedure for multiplicity, GLMM to further estimate the treatment effect, and the ICC calculation to quantify between-center variability. Except those analyses adjusted by BH procedure, all other statistical tests were two-sided with α = 0.05. Analyses were performed using R version 4.4.2.

Data availability
The study protocol and statistical analysis plan are provided as supplementary files; and the analytic codes used to generate figures and tables are accessible at GitHub platform immediately following the paper publication. No end date. ASource data are provided. Individual participant data that underlie the results reported in this article after deidentification (text, tables,figures, and appendices) are available with researchers who provide a proposal with a request to the corresponding authors, subject to ethical and legal restrictions.

Code availability
The R scripts codes used to generate figures and tables have been deposited in a public GitHub repository (https://github.com/zhaoxmiao/Modified-letrozole-versus-GnRH-antagonist-protocols).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a PRCT comparing letrozole plus gonadotropin to traditional antagonist cycles for IVF in DOR.\nIn the abstract you state that “The mLP achieved higher LBRs (DOR: 45.1% vs. 51 29.1%, rate ratio of 1.6, 95% CI, 0.7 to 2.0; P=0.132; 40-\n45 years: 20.9% vs. 13.6%, rate ratio of 1.5, 95% CI, 0.5 to 3.1; P=0.408) after fresh ET, as well as a higher CLBR per 53 woman after one\nfresh or frozen ET (34.8% vs. 27.5%, rate ratio of 1.3; 95% CI, 0.8 to 1.6; 54 P=0.273).” However, none of these were different and this\nneeds to be rephrased. AZs stating that no statistical difference in outcomes were noted. Be careful about calling differences where you\ndid not have one statistically.\nIn the abstract you state that “In fresh cycles with two cleavage embryos transferred, the mLP also showed a 55 significant higher CPR\nthan the GnRH-ant (65.8% vs. 36.4%, rate ratio of 1.8, 95% CI, 0.8 to 2.0; P=0.015)” yet your p value and your CI are inconsistent your CI\ncrosses one and as such should not be statistically different.\nPlease be more specific about the randomization method and how each patient was assigned. This deserves a full paragraph\nfor discussion.\nYou state that you did an intention to treat analysis however, you also state that “. Follow-up for all pregnancies was finalized by June\n2023. Among them, 171 eight participants stopped halfway since preovulation or absent follicular development, and 172 seven\nparticipants withdrew or deviated from the protocols. Finally, 153 women in the 173 mLP group and 150 women in the GnRH-ant group\nwho finished the study were analyzed.” This is not an intention to treat analysis you drop0-ed out patients. For an intention to treat\nanalysis these need to be included in the analysis as not pregnant\nFor baseline demographics please provide the amount of subject in both groups with endometrium 6 to 7 mm at trigger and\nthose with EE under 6 mm\nAnd compare those groups statistically\nAgain in the results you state that “The mLP showed higher LBR after fresh ET in DOR subgroup (45.1% vs. 29.1%, rate ratio, 186 1.6, 95%\nCI, 0.7 to 2.0; P=0.132), as well as in 40-45 years subgroup (20.9% vs. 13.6%, 187 rate ratio, 1.5, 95% CI, 0.5 to 3.1; P=0.408).” this is not\ntrue they were not higher and this should be stated as such\nAgain you erroneously state that “In addition, the mLP group 197 reported a lower frequency of clinical pregnancy loss (20.0% vs.\n26.7%), for a rate ratio of 198 0.8 (95% CI, 0.4 to 2.1; P=0.712)(Table 3). These results highlight the beneficial impact 199 of the mLP\non fresh IVF cycles, particularly among women with DOR” these are not different and need to be stated as such.\nFor results on table 2 be specific in what was different and what was not, in the results text.\nTable 3 in the results text you state LBR was better but again it was not.\nI think that this article should be rejected completely re-written and then send back including many changes in the discussion\nYou cannot claim differences if the p value was greater than 0.05 or the 95%CI crosses 1.\nThis needs to be reflected in the discussion where you discuss the benefits of the letrozole protocol but you did not\ndemonstrate one.\nlastly demonstrating a benefit in clinical pregnancy rates but not live birth or cumulative live birth shows that adding the letrozole\ndid not benefit the patients and you should state this\nLastly, you need to give citations for you power analysis explaining why you chose the differences you did to calculate the number of\nsubjects needed.", "reviewer_2": "I have the pleasure to review the paper by Zhao et al submitted to Nature Communication Journal. The study assessed the results of\novarian stimulation using letrozole vs antagonist protocol in women with diminished ovarian reserve. The study demonstrated better\nLBR and CLBR in the group treated with letrozole vs antagonist even without statistical significance. This RCT proposed the protocol\nwith letrozole as a viable first-line approach, particularly for women with DOR, those over 40 years old individuals with AMH levels ≥1.2\nng/ml or a history of unsuccessful IVF attempts.\nAromatase inhibitors, particularly letrozole (LZ), are now established as first-line ovulation induction agents, offering an effective\novarian stimulation strategy to enhance outcomes of intrauterine insemination. In recent years, they have also emerged as potentially\nvaluable adjuvants to gonadotropin ovarian stimulation for in vitro fertilization, particularly in fertility preservation in women with\nestrogen-responsive cancers. Also, this paper and other shreds of evidence support the role of adjuvant LZ in poor responders. Even\nthough the study represents an interesting topic there are many major concerns to resolve.\n1. The authors randomize 159 patients per group, but then 153 and 150 come in, as shown in Table 3 they will do the transfer (fresh or\nfrozen ET) in 112 and 130. As an RCT the paper should report the analysis for the intention to treat (ITT) including all the patients\nrandomized in the study, another analysis should included \"for protocol\". The authors missed the analysis for ITT and also when they\nanalyzed \"for protocol\" they considered only the patients that received transfer... without considering the patient that had a cancellation\ndue to several reasons as they reported.\n2. It's unclear the causes of cancellation in some cases OHSS is mentioned, but the average number of eggs is less than 5, vaginal bleeding\nis mentioned (?), and spontaneous pregnancy and endometrium are lower than 6 mm. There is not enough information regarding the\nnumber and reasons for cancellations in each group.\n3. It is unclear the protocol used in frozen embryo transfer. Natural cycle, HRT?\n4. The authors reported that the major cases of ET were at the cleavage stage, however, also D5 transfer was included in the study.\nThey do not report in the table the proportion\n5. In the letrozole group the authors reported 65% of cases with DET at D3 vs 36% DET at D3 with antagonist, even the policy to\ntransfer 2 embryos should discouraged for the maternal and fetal implication, we can't exclude that letrozole group have higher live\nbirth rate due to the higher number of DET.\n6.The table lists causes of cancellation, the authors define fever on the day of transfer, “Accidentally fever is defined as the body\ntemperature exceeding 37.3℃ on the embryo transfer day” Is this a cause of cancellation? The authors assume that a temperature of\n37.5 can be a cause for cancellation for what reason?", "reviewer_3": "The authors present findings from a multicentre randomised controlled trial (RCT) evaluating a modified letrozole protocol\n(mLP) versus a GnRH antagonist protocol in women with diminished ovarian reserve (DOR). The study reports a numerical increase in\nlive birth rate (LBR) following fresh embryo transfer and in cumulative live birth rate (CLBR), both assessed in subgroups of participants.\nHowever, the conclusion appears overstated, and the study’s conduct and reporting lack the statistical rigour expected of a high-quality\nRCT, as detailed in the comments below.\nMajor comments\n1. Analysis population (ITT vs. per-protocol):\nThe analyses are stated as intention-to-treat (ITT); however, the primary endpoints appear to be restricted to a subset of participants\n(e.g., only those who underwent embryo transfer), which would correspond to a modified ITT. Furthermore, the CONSORT diagram\nindicates the exclusion of participants with protocol deviations, suggesting a per-protocol approach may have been used. This requires\nclarification and consistency across the manuscript.\n2. Interpretation of main findings:\nThe primary outcomes (LBR and CLBR) show a numerical, but statistically non-significant, increase in the mLP group. As such, the\nconclusion that mLP led to improved reproductive outcomes appears to overstate the treatment effect. Rather, the findings suggest\nthat larger studies are needed to confirm any potential benefit of mLP.\n3. Primary endpoint sample size:\nThe primary endpoints (LBR and CLBR) are evaluated in subgroups of approximately 100 participants per arm, which renders the study\nunderpowered for its primary endpoints. A more appropriate endpoint would have been one assessable in all randomised participants.\n4. Subgroup analyses and endpoint selection:\nThe study focuses heavily on results of subgroup analyses. It would be more appropriate to evaluate the treatment effect on\nthe entire cohort first. Subgroup and interaction analyses, which are exploratory and underpowered, should follow.\n5. Multiple testing:\nNo adjustment for multiple testing is described. At a minimum, correction should be applied for the two co-primary endpoints.\n6. Power and sample size calculation:\nThe sample size calculations are inconsistent and difficult to follow, with two different approaches presented. The initial calculation (n ≈\n300) is more transparent and appears to justify the final sample size. In contrast, the second calculation (n ≈ 120) is based on vague\nassumptions, lacks justification, and appears to assume no treatment effect (confidence interval centred around 1). Moreover, I was\nunable to replicate the numbers provided for this second calculation.\nMinor comments\n7. Abstract:\no Consider mentioning \"advanced maternal age\" in the background.\no Clarify whether analyses were ITT, mITT, or per-protocol.\n8. CONSORT reporting:\no The lack of blinding should be explicitly reported.\no The method of randomisation (stratified by centre and age group) should be described in the main text.\n9. Statistical methods:\no Methods for calculating confidence intervals are not described.\no Method for interaction tests are not reported.\no The significance level is inconsistently reported as alpha = 0.025 (two-sided) in the SAP and p < 0.05 in the main text. Please clarify\nand correct accordingly.\no Adjustment for stratification variables and centre effects (e.g., using mixed-effects models) is not conducted but would be\nappropriate.\no Treatment heterogeneity across sites should be evaluated with appropriate models (e.g., site as a random effect and\nsite*treatment interactions).\n10. Results presentation:\no Please summarise baseline characteristics briefly in the main text.\no Present findings for the entire cohort before subgroup results.\no Report rate ratios with two decimal places consistently.\no Ensure all tables, figures, and supplementary materials are referenced in the main text.\no No adverse events are reported—please clarify whether any were observed.\n11. Supplementary materials:\no Two versions of the protocol and analysis plan are included with no clear version history or reconciliation of differences.\no The SAP adds little beyond the protocol/manuscript and lacks detailed definitions of endpoints.\no The case report of the \"zero patient\" is included in the supplement but not referenced in the manuscript.\nFormatting and language issues\n12. Line 108: Change \"standard operating protocols\" to \"standard operating procedures\".\n13. Line 168: remove “were” in “were provided informed consent”.\n14. Lines 198–199, 209–211, 227–228: statements implying treatment benefit (e.g., “highlight the beneficial impact”) are\ninterpretive and do not belong in the results section—please revise or remove.\n15. Figure 2A–C: add units to y-axes (assumed to be %). Define all abbreviations in the legend.\n16. Figure 2D–F: for skewed continuous data, t-tests and CIs based on normal assumption may not be appropriate. Consider dot/scatter\nplots to show data distributions.\n17. Figure 2G–H: avoid bar plots for continuous data. Use scatterplots to better reflect data variability.\n18. Table 1: avoid testing for baseline differences in RCTs—randomisation ensures differences are due to chance.\n19. Table 2: consider using Poisson regression or log-transformed t-tests for count data rather than standard t-tests.\n20. Table 3: subgroup results and interaction tests should be presented separately for clarity."}, "author_response": "Comment of Reviewer 1"}]

Decision letter