# Caesarean section scar: Histological analysis on hysterectomy specimen. A pilot study

Constance Maudot, Elodie Debras, Astrid Laurent-Bellue, Margot Dupeux, Suzanne Chartier, Sophie Prevost, Victor Beaucoté, Pascale Chavatte-Palmer, Perrine Goussault Capmas

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Constance Maudot, Elodie Debras, Astrid Laurent-Bellue, Margot Dupeux, Suzanne Chartier, et al. Caesarean section scar: Histological analysis on hysterectomy specimen. A pilot study. European Journal of Obstetrics & Gynecology and Reproductive Biology, 2024, 303, pp.236-243. 10.1016/j.ejogrb.2024.10.048. hal-04770282

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Full length article

# Caesarean section scar: Histological analysis on hysterectomy specimen. A pilot study

<figure><img src="image_3.png" /></figure>

Constance Maudot<sup>a,1</sup>, Elodie Debras<sup>a,d,1</sup>, Astrid Laurent-Bellue<sup>a,b</sup>, Margot Dupeux<sup>a</sup>, Suzanne Chartier<sup>a</sup>, Sophie Prevost<sup>a,b</sup>, Victor Beaucoté<sup>a</sup>, Pascale Chavatte-Palmer<sup>d</sup>, Perrine Goussault Capmas<sup>a,b,c,*</sup>

## ARTICLE INFO

### Keywords:

- Caesarean section

- Uterus wound healing

- Caesarean scar

- Isthmocele

- Hysterectomy

- Pathology

## ABSTRACT

**Introduction:** In recent years, caesarean section (CS) rate has risen worldwide. Complications associated with CS scars have risen too, such as scar dehiscences and uterine ruptures. Uterine healing is a complex phenomenon still poorly understood. The aim of this study is to carry out a comparative histological analysis of healthy and scarred uterus.

**Material and methods:** Women who underwent hysterectomy for benign pathology were included prospectively and divided into two groups: previous CS (group 1) versus control (group 2). Hysterectomy specimen were analyzed histologically and immunohistochemically.

**Results:** Sixty women were included: 30 women per group. In group 1, only 19 women could be analyzed. Median total thickness at the thinnest site of the scar is significantly thinner (4.34 mm; IQR [2.76-9.45]) than that of adjacent healthy isthmus (12.70 mm; IQR [10.45-14.95]) (p < 0.001). It is also thinner than in group 2 (13.45 mm; IQR [11.03-16.90]) (p < 0.001). Median myometrial thickness within the scar in group 1 was also thinner (1.14 mm; IQR [0.30-2.69]) than that of the adjacent healthy isthmus (8.90 mm; IQR [8.18-10.08]) (p < 0.001) and that in group 2 (10.00 mm; IQR [8.38-13.35]) (p < 0.001). There was a significant increase in fibrosis in the scar (55.01%; IQR [35.71-63.46]) compared with adjacent tissue (17.41%; IQR [15.08-24.78]) (p < 0.001) and with healthy uterus (33.91%; IQR [18.93-46.53]) (p = 0.006).

**Conclusion:** In uterus with previous CS scar, total thickness of the wall and thickness of the myometrium are reduced and proportion of fibrosis is significantly increased. This study shows that the thickness of the wall remains reduced in scarred uterus, even very long after CS. Further studies are currently in progress to understand its pathophysiology within the uterus using animal models.

## Introduction

Caesarean section (CS) is one of the most common surgical procedures in the world, leading to a reduction in maternal and neonatal morbidity and mortality and should be universally accessible. In WHO recommendations, the optimal rate of CS is around 10–15 % [1]; it has, however, risen worldwide [2,3]. In France, this rate is around 20 %, relatively stable since 2010 [4]. This increase leads to an increase in surgical complications, mainly dehiscence of the CS scars (isthmocedes)

and uterine ruptures [5,6].

Isthmocele or scar dehiscence is defined by the presence of a “niche” within the anterior myometrium on the lower segment of the uterus, reflecting a discontinuity in the myometrium at the site of a previous caesarean section. Prevalence of isthmocele is estimated between 56 and 84 % [7–10]. Most isthmocedes are asymptomatic. In symptomatic women, the most frequent symptoms are delayed retentional abnormal uterine bleeding, pelvic pain and secondary infertility [8]. Hysterosonography is the gold standard for diagnosis. It defines isthmocele as a defect of at least 2 mm deep [11–13].

<footnote><sup>a</sup> AP-HP, GHU-Sud, Hospital Bicêtre, Department of Gynecology and Obstetrics, 78 rue du Général Leclerc, 94270 Le Kremlin Bicêtre, France</footnote>

<footnote><sup>b</sup> Faculty of medicine, University Paris-Sud Saclay, 63 rue Gabriel Péri, 94270 Le Kremlin Bicêtre, France</footnote>

<footnote><sup>c</sup> INSERM, UMR1018, CESP, Hopital Paul Brousse, 12 avenue Paul Vaillant Couturier, 94800 Villejuif, France</footnote>

<footnote><sup>d</sup> University of Paris-Saclay, UVSQ, INRAE, BREED, Jouy-en-Josas, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: ferrine.capmas@aphp.fr (P. Goussault Capmas).</footnote>

<footnote n="1">¹ Co first-authors.</footnote>
<table><thead><tr><th colspan="2">Nomenclature</th></tr></thead><tbody><tr><td colspan="2">Acronyms</td></tr><tr><td>CS</td><td>ceasection</td></tr><tr><td>HES</td><td>hematoxylin-eosin-safran</td></tr><tr><td>DES</td><td>3,3'-Diaminobenzidine tetrahydrochloride hydrate</td></tr><tr><td>RO</td><td>oetrogen receptor</td></tr><tr><td>RP</td><td>progesterone receptor</td></tr><tr><td>CKPAN</td><td>pancytokeratin</td></tr><tr><td>α-SMA</td><td>alpha smooth muscle actin</td></tr></tbody></table>

previous CS, while the second one (control group) included women with no previous CS. Pre-operative clinical data such as age, BMI, smoking status, history of diabetes, history of CS and its modalities, symptoms and preoperative pelvic ultrasound +/- hysterosonography were collected. Women underwent vaginal, abdominal laparoscopic, laparotomic or vaginal laparoscopic surgery (using the V-NOTES® device).

## Histology

Hysterectomy specimens were sent fresh to the pathological unit. Macroscopic examination by a pathologist with a great experience in gynecology, enabled identification and sampling of the isthmic zone, with or without scar, which was then fixed in formalin and embedded in

<figure><img src="image_4.png" /><figcaption>Fig. 1. Flow chart.</figcaption></figure>

Wound healing is a highly studied phenomenon in the skin [14,15] unlike in uterus. Histological aspects of uterine healing have been studied in murine models, where a key role has been described for collagen and smooth muscle tissue differentiation [16]. Human uterine healing is complex and involved multiple cellular and molecular mechanisms that are still poorly understood, there are few immunohistochemical data on uterine scar tissue in the literature [17-20]. Papping et al. showed that the high collagen content in uterine scars results from an ongoing overexpression of collagen I and III [19]. In their study, Pollio et al. showed that in the case of uterine dehiscence, the scarred lower uterine segment has a higher collagen content, a reduction of several transforming growth factor, a reduction of connective tissue growth factor and an increase in basic fibroblast growth factor [17].

Very few studies report pathological characteristics of CS scar dehiscence [21-23]. Karpathiou et al. suggest a standardized methodology to describe uterine scar tissue, including the following criteria: type of mucosa, type of stroma, presence of thickened vessels, myometrium, adipose tissue, electrocoagulation artifacts, hemorrhagic and inflammatory remodeling, polyp, adenomyosis, implantation site or microcalcification [22].

The main aim of this study is to explore morphological characteristics of uterine scarring in humans by carrying out a comparative histological analysis of healthy and scarred uteri.

## Material and methods

### Participants

Women who underwent hysterectomy for benign pathology were included prospectively between January and June 2023. Women were included into two groups: the first one included women with at least one

paraffin.

For each sample, a minimum of 10 serial sections 3.5 µm thick were cut using a Leica® RM2245 manual paraffin microtome. Paraffin sections were systematically dehydrated in Ethanol baths and deparaffinized in Xylene baths, then stained with hematoxylin-eosin-safran on a Leica® ST5020 machine. Sections were digitalized using the Nano-zoomer Digital Pathology – Hamamatsu® slide scanner.

Histological analysis focused on the following parameters: macroscopic scar marker or presence of isthmocele, thickness of scar or anterior uterine wall in isthmic situation, respect of the three layers (endometrium, myometrium, serosa), type of mucosa, presence of thickened vessels, myometrium, adipose tissue, inflammatory infiltrate and fibrosis as described by Karpathiou et al. [22].

Fibrosis was defined as tissue composed of fibroblasts, myofibroblasts and collagen. It was assessed using “ImageJ” software (version 1.53 t, National Institutes of Health, Bethesda, MD, USA) on HES-stained sections, at x40 magnification, on 3 randomly selected fields in the healthy zone and 3 fields in the scarred zone in order to define a percentage of fibrous tissue (saffron collagen marking). The area ratios of fibrosis were determined using the plugin “color deconvolution” for stain separation “H&E2”. Three images were separated and we used the first purple one for the analysis. The threshold value was defined and the area ratios of the target areas were obtained by the “analyze” function. The mean values obtained in three different areas were used for data analysis. Two observers performed the evaluation and results between them were compared. It was also transcribed according to the following score: 0: no fibrosis, +: <10 % of cells, ++: 10-40 % of cells, +++: >40 % of cells [17].
<table><caption>**Table 1**
Population characteristics.</caption><thead><tr><th></th><th>Group 1: Hysterectomy with previous caesarean section<br>CICATRICAL UTERUS (n = 19)</th><th>Group 2: Hysterectomy without previous caesarean section<br>HEALTHY UTERUS (n = 30)</th></tr></thead><tbody><tr><td>Age (median [IQR])</td><td>47.00 [44.00, 50.50]</td><td>47.00 [45.25, 50.75]</td></tr><tr><td>Number of previous gestation: n (%)</td><td></td><td></td></tr><tr><td>0</td><td>0</td><td>3 (10.0)</td></tr><tr><td>1</td><td>4 (21.1)</td><td>4 (13.3)</td></tr><tr><td>2 and more</td><td>15 (78.9)</td><td>23 (76.7)</td></tr><tr><td>Number of previous deliveries: n (%)</td><td></td><td></td></tr><tr><td>0</td><td>0</td><td>3 (10.0)</td></tr><tr><td>1</td><td>4 (21.1)</td><td>7 (23.3)</td></tr><tr><td>2 and more</td><td>15 (78.9)</td><td>20 (66.7)</td></tr><tr><td>BMI (median [IQR])</td><td>26.40 [23.02, 31.90]</td><td>25.05 [22.75, 29.93]</td></tr><tr><td>Tobacco: n (%)</td><td>0</td><td>6 (20.0)</td></tr><tr><td>Diabetes: n (%)</td><td>0</td><td>2 (6.7)</td></tr><tr><td>Previous caesarean section: n (%)</td><td></td><td></td></tr><tr><td>0</td><td>0</td><td>0</td></tr><tr><td>1</td><td>11 (57.9)</td><td>—</td></tr><tr><td>2 and more</td><td>8 (42.1)</td><td>—</td></tr><tr><td>Time from last caesarean section to care (year)</td><td>14.50 [9.50, 19.50]</td><td>—</td></tr><tr><td>History of pregnancy on scar: n (%)</td><td>0</td><td>—</td></tr><tr><td>History of isthmocoele surgery: n (%)</td><td>0</td><td>—</td></tr><tr><td>Symptoms: n (%)</td><td>12 (63.2)</td><td>26 (86.7)</td></tr><tr><td>Abnormal uterine bleeding (AUB)</td><td>16 (84.2)</td><td>20 (66.7)</td></tr><tr><td>Pelvic pain</td><td>6 (100)</td><td>21 (70.0)</td></tr><tr><td>Infertility</td><td>1 (5.3)</td><td>1 (3.3)</td></tr><tr><td>PMA follow-up</td><td>1 (5.3)</td><td>1 (3.3)</td></tr><tr><td>Pre-op sonography: n (%)</td><td>15 (78.9)</td><td>10 (33.3)</td></tr><tr><td>Visible scar: n (%)</td><td>13 (68.4)</td><td>—</td></tr><tr><td>External orifice-scar distance (median [IQR])</td><td>35.75 [30.75, 40.20]</td><td>—</td></tr><tr><td>Total anterior thickness (median [IQR])</td><td>11.70 [9.71, 13.80]</td><td>—</td></tr><tr><td>Pre-op hysterosonography: n (%)</td><td>10 (52.6)</td><td>—</td></tr><tr><td>Isthmocele: n (%)</td><td>6/10 (60)</td><td>—</td></tr><tr><td>Length (mm) (median [IQR])</td><td>9.80 [6.55, 12.90]</td><td>—</td></tr><tr><td>Width (mm) (median [IQR])</td><td>10.70 [10.10, 12.00]</td><td>—</td></tr><tr><td>Depth (mm) (median [IQR])</td><td>6.85 [5.95, 8.80]</td><td>—</td></tr><tr><td>Residual myometrial thickness (median [IQR])</td><td>2.30 [1.50, 2.50]</td><td>—</td></tr><tr><td>Surgical indication:</td><td></td><td></td></tr><tr><td>Symptomatic polymyomatous uterus</td><td>6 (31.6)</td><td>14 (46.7)</td></tr><tr><td>Symptomatic adenomyotic uterus</td><td>7 (36.8)</td><td>9 (30.0)</td></tr><tr><td>Essure removal request</td><td>5 (26.3)</td><td>6 (20.0)</td></tr><tr><td>Endometriosis</td><td>1 (5.3)</td><td>0</td></tr><tr><td>Other: Prophylactic</td><td></td><td>1(3.3)</td></tr><tr><td>Surgical approach:</td><td></td><td></td></tr><tr><td>Laparotomy</td><td>3 (15.8)</td><td>2 (6.7)</td></tr><tr><td>Laparoscopy</td><td>3 (15.8)</td><td>2 (6.7)</td></tr><tr><td>Vaginal approach</td><td>14 (73.7)</td><td>26 (86.7)</td></tr></tbody></table>

## Immunochemistry

Sections were deparaffined and rehydrated, then incubated with hydrogen peroxide to quench endogenous peroxidase activity. A user-supplied specific primary monoclonal mouse anti-human antibody was applied. The substrate chromogen, 3,3'-Diaminobenzidine

tetrahydrochloride hydrate (DAB), visualizes the complex via a brown precipitate (BOND Polymer Refine Detection, Leica®). The sections were counterstained with hematoxylin.

Anti-RO antibody (1:60 dilution; clone 6F11; Leica®), anti-RP antibody (1:120 dilution; clone 16, Leica®), anti-CD10 (no dilution; clone 56C6, Leica®), anti-CD31 (1:80 dilution; clone JC70A; Agilent®), anti-CKPAN (1:200 dilution; clone AE1, AE3, 5D3; Zytomed®), anti-α-SMA antibody (1:15000 dilution; clone 1A4; Sigma-aldrich®) and anti-Ki67 (1:500 dilution, clone SP6 (MA5-14520), ThermoScientific®) were used as primary antibodies.

## Ethical approval

The ethics committee approval was obtained (CEROG 2022-GYN-120). No funding was obtained for this study and the full protocol can be obtained on demand.

## Statistics

Demographic and histological data were analyzed using non-parametric statistics due to the non-normal distribution of variables and the limited number of samples. Results are presented in median and IQR [interquartile ranges]. Continuous variables were compared between study groups in pairs using the Wilcoxon Mann-Whitney test. Statistical tests were performed at a significance level of 5 % (p < 0.05) to assess significant differences between groups. To take account of the inflation of alpha risk induced by the multiplication of statistical tests, we chose to adjust data using the Benjamini-Hochberg method. Correlation tests between measurements of two observers were carried out using Spearman's coefficient and the Bland-Altman diagram. The Jonckheere-Terpstra test was used to assess the trend in quantitative variables across several ordered groups. For qualitative variables, the Cochran-Armitage trend test was employed. All analyses were performed using R software (version 4.2.3).

## Results

Between January and May 2023, 30 women were included with a previous CS (group 1) and 30 without previous CS (group 2). In group 1, 11 women were excluded because scar has not been searched and identified by the technician on macroscopic histological examination and therefore has not been sampled (Fig. 1). Group 1 is subdivided into 2 parts: women with isthmocele and women without isthmocele, isthmocele being defined by a defect at least 2 mm deep measured on HES stained section [21].

Characteristics of women are described in Table 1. There was no significant difference between groups. The median age was 47 years [44,51], with only non-menopausal women in both groups. The main surgical indications were symptomatic uterine myoma (40.81 %), suspected adenomyosis or endometriosis (34.69 %), and requests for removal of Essure© intra-tubal devices (22.45 %). We considered that all women with a previous caesarean section had a simple overjet uterine suture, in line with standard caesarean section practice in France. In group 1, none of the women were treated for symptomatic isthmocele. Fifteen out of 19 (78.9 %) women had a pre-operative ultrasound scan, 9 out of 19 (47.3 %) had a hysterosonography, and for 6 out of 19 (31.6 %) we had a pre-operative diagnosis of isthmocele.

## Histology

Histological slides were studied by two observers, as the correlation calculations between these two observers were acceptable (Spearman's correlation coefficient: $\rho$ = 0.98 (p < 0.001) Fig Sup 3), it was decided to carry out the statistical analysis using quantitative data from only one of them.

The tissue architecture in the healthy isthmus consists of the isthmic
<caption>**Table 2**</caption>

Histological and immunohistochemical characteristics of scarred and healthy uterine.

<table><thead><tr><th rowspan="2"></th><th colspan="3">Group 1: Hysterectomy with previous caesarean section<br/>CICATRICAL UTERUS (n = 19)</th><th rowspan="2">Group 2: Hysterectomy without previous<br/>caesarean section<br/>HEALTHY UTERUS (n = 30)</th></tr><tr><th>WITHOUT ISTMOCELE (n<br/>= 9)</th><th>WITH ISTHMOCELE (n<br/>= 9)</th><th>ALL (n = 19)</th></tr></thead><tbody><tr><td><strong>Macroscopic examination of the surgical piece</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Visible scar: n (%)</td><td>10 (100)</td><td>9 (100)</td><td>19 (100)</td><td>0</td></tr><tr><td>Isthmocele: n (%)</td><td>10 (100)</td><td>9 (100)</td><td>9 (47.4)</td><td>0</td></tr><tr><td>Length (mm) (median [IQR])</td><td>—</td><td>6.94 [4.10, 7.19]</td><td>6.94 [4.10, 7.19]</td><td>—</td></tr><tr><td>Depth (mm) (median [IQR])</td><td>—</td><td>5.30 [3.50, 5.47]</td><td>5.30 [3.50, 5.47]</td><td>—</td></tr><tr><td>Residual myometrial thickness (mm) (median [IQR])</td><td>9.45 [4.94, 10.78]</td><td>2.72 [2.50, 4.34]</td><td>4.34 [2.76, 9.45]</td><td>—</td></tr><tr><td><strong>HES</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Uterine adenomyosis: n (%)</td><td>8 (80.0)</td><td>6 (66.7)</td><td>14 (73.7)</td><td>10 (33.3)</td></tr><tr><td><strong>Uterine parietal thickness (median [IQR])</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Scar zone</td><td>9.45 [4.94, 10.78]</td><td>2.72 [2.50, 4.34]</td><td>4.34 [2.76, 9.45]</td><td>—</td></tr><tr><td>Healthy zone</td><td>13.30 [10.65, 17.20]</td><td>11.20 [10.60, 12.80]</td><td>12.70 [10.45, 14.95]</td><td>13.45 [11.03, 16.90]</td></tr><tr><td><strong>Mucosa type: n (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Endometrial</td><td>3 (30.0)</td><td>1 (11.1)</td><td>4 (21.1)</td><td>14 (46.7)</td></tr><tr><td>Cervical</td><td>3 (30.0)</td><td>4 (44.4)</td><td>7 (36.8)</td><td>6 (20.0)</td></tr><tr><td>Mixed endometrial and cervical</td><td>4 (40.0)</td><td>4 (44.4)</td><td>8 (42.1)</td><td>10 (33.3)</td></tr><tr><td><strong>Stroma type: n (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Fibro-muscular</td><td>10 (100)</td><td>9 (100)</td><td>19 (100)</td><td>30 (100)</td></tr><tr><td>Fibro-adipose</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Thick-walled vessels: n (%)</td><td>9 (90.0)</td><td>8 (88.9)</td><td>17 (89.5)</td><td>28 (93.3)</td></tr><tr><td>Myometrium: n (%)</td><td>10 (100.0)</td><td>9 (100.0)</td><td>19 (100.0)</td><td>30 (100,0)</td></tr><tr><td>Adipose tissue: n (%)</td><td>4 (40.0)</td><td>7 (77.8)</td><td>11 (57.9)</td><td>0</td></tr><tr><td>Hemorrhagic remodelling: n (%)</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Inflammatory remodelling: n (%)</td><td>1 (10)</td><td>0</td><td>1 (5,3)</td><td>1 (3.3)</td></tr><tr><td>Null</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Minimal</td><td>1 (100)</td><td>0</td><td>1 (100)</td><td>1 (100)</td></tr><tr><td>Important</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Other: n (%)</td><td>2 (20)</td><td>0</td><td>2 (10.5)</td><td>0</td></tr><tr><td>Polyp</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Adenomyosis</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Trophoblastic implantation site</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Microcalcifications</td><td>2/2(100)</td><td>0</td><td>2/2(100)</td><td>0</td></tr><tr><td><strong>Scar cyst</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Fibrosis (%) (3-field average (SD))</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Scar zone</td><td>55.13 [38.49, 67.34]</td><td>49.97 [36.10, 57.12]</td><td>55.01 [35.71, 63.46]</td><td>—</td></tr><tr><td>Healthy zone</td><td>23.44 [16.61, 43.23]</td><td>15.68 [14.50, 19.33]</td><td>17.43 [15.08, 24.78]</td><td>33.91 [18.93, 46.53]</td></tr><tr><td><strong>Immunohistochemistry</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Smooth muscle actin (α-SMA)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Muscular thickness (median [IQR])</td><td></td><td></td><td></td><td></td></tr><tr><td>Scar zone</td><td>2.31 [0.32, 4.19]</td><td>1.02 [0.40, 1.14]</td><td>1.14 [0.30, 2.69]</td><td>—</td></tr><tr><td>Healthy zone</td><td>9.00 [8.09, 10.24]</td><td>8.90 [8.40, 9.76]</td><td>8.90 [8.18, 10.08]</td><td>10.00 [8.38, 13.35]</td></tr><tr><td><strong>Proliferation index (Ki67) (%)</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Scar zone</td><td></td><td></td><td></td><td></td></tr><tr><td>Glandular epithelium</td><td>3.00 [1.80–9.40]</td><td>13.50 [6.70–23.00]</td><td>7.25 [2.92–21.62]</td><td>—</td></tr><tr><td>Connective tissue</td><td>1.00 [0.50–4.50]</td><td>2.00 [1.00–6.80]</td><td>1.70 [0.85–6.22]</td><td>—</td></tr><tr><td>Healthy zone</td><td></td><td></td><td></td><td></td></tr><tr><td>Glandular epithelium</td><td>9.30 [7.00–15.00]</td><td>14.00 [2.20–28.60]</td><td>9.65 [2.28–16.05]</td><td>5.00 [3.40, 15.20]</td></tr><tr><td>Connective tissue</td><td>2.00 [0.70–3.00]</td><td>2.00 [0.00–3.00]</td><td>2.00 [0.17–3.00]</td><td>2.40 [1.70, 4.00]</td></tr></tbody></table>

or upper cervical endometrial mucosa, depending on the sampling site, the muscular layer corresponding to the myometrium of normal thickness and the uterine serosa. The median total thickness of the anterior isthmus was 13.45 mm; IQR [11.03,16.90] (Table 2). Thick-walled vessels could be seen within the stroma and myometrium.

HES and immunohistochemistry enabled to discriminate two types of women in group 1: those with healing without dehiscence and those with a macroscopic and histopathological isthmocele (Fig. 2).

With or without isthmocele, total thickness at the thinnest site of the scar was significantly thinner (median = 4.34 mm; IQR [2.76–9.45]) than that of the adjacent healthy isthmus (median = 12.70 mm; IQR [10.45–14.95]) (p < 0.001) and that of group 2 (median = 13.45 mm; IQR [11.03–16.90]) (p < 0.001).

We performed a subgroup analysis according to the number of

previous CS, the rate of isthmocedes increased with the number of caesarean sections. (27.3 % for one previous CS vs 66.7 % for two previous CS vs 100 % for three previous CS) (p = 0.03) (Table sup1). We also performed an additional subgroup analysis according to the time since the last caesarean section, which showed that the number of isthmocedes was upper in the subgroup with the shortest healing time. (100 % <10 years vs 66.7 % 10–15 years vs 16.7 % >15 years) (p = 0.015). (Table sup2).

When looking separately at women with “normal” healing without dehiscence, the total thickness of the scarred anterior isthmus was still significantly thinner than the thickness of adjacent healthy tissue (median = 9.45 mm; IQR [4.94–10.78]) vs (median 13.30 mm; IQR [10.65–17.20]) (p = 0.011) and than that of healthy isthmus (median = 13.45 mm; IQR [11.03–16.90]) (p = 0.001) (Table 2 and Fig. 2). Tissue
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Sagittal sections of scarred and healthy uterus. A: Macroscopic examination of a scarred uterus with isthmocele. B: Measurement of isthmocele and normal anterior isthmic thickness (HES, x0.33). C: Measurement of myometrial thickness within the scar and adjacent healthy tissue (αSMA, x0,33). D: Macroscopic examination of a scarred uterus without isthmocele. E: Measurement of total scar thickness and normal anterior isthmic thickness (HES, x0.29). F: Measurement of myometrial thickness within the scar and adjacent healthy tissue (αSMA, x0,30). G: Macroscopic examination of a healthy uterus. H: Measurement of normal anterior isthmic thickness (HES, x0.26). I: Measurement of myometrial thickness on healthy isthmus (αSMA, x0,29).</figcaption></figure>

architecture was only slightly altered. Endometrial mucosa covered the inner surface of the scar, with occasional cystic glands trapped in the scar tissue. The cytogenic chorion was fibro-musclar. Myometrium was present within the scar, but thinner and with disorganized muscle fibers. There was an increase of fibrous tissue (55.13 %; IQR [38.49, 67.34] in the scar vs 23.44 %; IQR [16.61, 43.23] in healthy zone).

In women with an **isthmocele**: total thickness at the thinnest point of the scar was significantly thinner (median = 2.72 mm; IQR [2.50-4.34]) than the adjacent healthy isthmus (median = 11.20 mm; IQR

[10.60-12.80]) (p < 0.001) and than the healthy isthmus (median = 13.45 mm; IQR [11.03-16.90]) (p < 0.001) (Table 2 and Fig. 2).

There was a complete change in tissue architecture. Endometrial or cervical glandular epithelium persisted within the scar. Cystic glands could be observed within the fibrous scar tissue. Myometrial muscle fibers were disorganized, less numerous and less dense. Most of the scar was composed of fibrous scar tissue made up of fibroblasts, myofibroblasts and collagen. Adipocytes trapped in fibrosis could be seen. Thick-walled vessels were also visible within the remaining myometrium
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Histological findings on uterine scars after HES staining. **A:** Architectural disorganization of muscle fibers, with tissue essentially composed of fibrosis (x20). **B:** Adipose tissue trapped in fibrous tissue in isthmocoele area (x20). **C:** Endometrial glands trapped within fibrous scar tissue (x10). **D:** Thickened vessels in fibrous scar tissue (x10).</figcaption></figure>

(Fig. 3).

Evaluation of fibrosis (+<10%; ++ 10 and 40%; +++>40%) showed an increase in fibrosis within the scar (55.01% [35.71-63.46] or +++ ) compared with adjacent tissue (17.41%, IQR [15.08-24.78] or ++) (p < 0.001) and compared with healthy tissue in group 2 (33.91%, IQR [18.93-46.53]) (p = 0.006) (Supplementary Fig. S1 and Table 2). Fibrosis, however, appeared to be more important in the healthy isthmus in group 2 than in the healthy adjacent scar tissue of scarred uteri, although this was not significant (p = 0.1).

Adenomyosis of the corporeal part of the uterus was observed in 4 women. In all hysterectomy specimens with CS scars, endometrial glands trapped within the fibrous tissue are indeed observed.

Immunochemistry

Immunohistochemical analysis of **healthy isthmus** revealed the presence of epithelium by cytoplasmic staining for pancytokeratin, cytogenic chorion by cytoplasmic staining for CD10 and smooth muscle by cytoplasmic staining for smooth muscle actin (α-SMA). The median thickness of smooth muscle, as measured on α-SMA-stained sections, was 10 mm, IQR [8.38-13.35] (Table 2). Nuclear staining for estrogen and progesterone receptors in epithelium and connective tissue was also consistently observed. CD31 staining of endothelial cells highlighted the presence of vessels.

Finally, Ki67 staining was used to assess cell proliferation, which is physiologically higher in epithelium than in connective tissue, with 5.00%, IQR [3.40-15.20] of epithelial cells engaged in the cell cycle versus 2.40%, IQR [1.70-4.00] (p = 0.05) of connective cells in group 2.

In Groupe 1, apart from measurements made on α-SMA sections, immunostained slide readings are similar for women with normal scarring and those with isthmocele. There was no significant difference

in cell proliferation in the epithelium and connective tissue between the scar and healthy areas (Fig. 4). All women with scared uterus had homogeneous estrogen and progesterone receptor staining in the epithelium and connective tissue. We did not perform quantitative analysis via Image J for these receptors. Cytogenic chorion was present in all specimens, including within the isthmoceles. Vascularization was highlighted homogeneously throughout the scar and adjacent tissue. Pancytokeratin confirmed the presence of epithelium in all specimens, including in the isthmoceles.

Myometrial smooth muscle cells were present in all specimens (Fig sup 2). Myometrial thickness, however, was lower in the scar in group 1 (with or without isthmocèle) (1.14 mm; IQR [0.30-2.69]) than in the adjacent healthy isthmus (8.90 m; IQR [8.18-10.08]) (p < 0.001) and similarly significantly lower than myometrial thickness in group 2 (10 mm; IQR [8.38-13.35] (p < 0.001) (Table2).

In subgroup analyses, smooth muscle thickness in the scar seems to decreased with the number of previous caesarean sections but it is not a statistically significant (p = 0.18) (Table sup1). However, smooth muscle thickness in the scar seems higher when the delay between the last caesarean and the hysterectomy was more than 10 years although it is also not a statistically significant (p = 0.12) (Table sup2).

Discussion

A reduction in total scar thickness was observed in scarring tissue, compared with adjacent healthy uterine tissue or with control, together with a reduction in myometrial thickness and a disorganization of the tissue's usual architecture with increased fibrosis. Similarly, the proportion of muscle in the scar was reduced. Two groups of women were identified according to their type of scarring: "normal healing" or "with isthmocele".
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Statistical analysis of histological and immunohistochemical sections of uterine isthmus in patients with healthy uterus and scar uterus. (*p* < 0.05, **p** < 0.01, ***p* < 0.001). A: Measurement of total uterine parietal thickness (mm) on HES-stained section. B: Measurement of myometrial thickness (mm) on αSMA-stained section. C: Quantification of fibrosis (%) average of 3 random fields studied on HES-stained section (x40). D: Quantification of cell proliferation Ki67(%) on cytogenic epithelial and chorionic cells.</figcaption></figure>

These findings are consistent with the few published studies investigating histology of CS and isthmocœles. Li et al. reported pathological features of uterine surgical scarring in a mouse model: they showed an inhibition of myometrial smooth muscle cell growth but excessive collagen fiber deposition [24]. Donnez et al. [21] also described a significant decrease in muscle density in the residual myometrium of the defect compared with adjacent myometrium. Indeed, in 38 women with CS defect managed by laparoscopy, histological analysis revealed the presence of fibrosis in 30 women (78.9 %),

We also observed a significantly higher rate presence of fibrosis within the scars compared with adjacent healthy tissue in scared uterus. There was a non-significant trend towards higher fibrosis in the isthmus of healthy uteri compared with healthy tissue adjacent to CS scars. This difference may be explained by the location of sampling: in group 2 of healthy uteri, sampling was carried out either on the isthmus or on the high cervix, although it is known that the cervix is physiologically more fibrous than uterine isthmus.

Donnez et al. showed evidence of endometriosis in 8/38 cases (21.1 %), defined as the presence of endometrial glands within the scar, not connected to the endometrial surface on serial sections [14]. Interpretation of endometrial glands within the scar must remain cautious as to its endometriotic origin. Hysterorraphy during CS is mostly performed

using total stitches, that includes endometrium, muscle and serosa. These three layers can therefore be found side by side during healing, resulting in the inclusion of trapped endometrial glands in muscle or connective tissue.

In the same way, the presence of adipose tissue in scars can be explained by bladder detachment during CS, leaving a portion of prevesical adipose tissue on the uterine isthmus, which will then be included in total stitches.

The presence of thickened vessels can be explained by multiparity in most women, meaning that uterus has undergone significant structural changes with repeated vascular remodeling. These observations led us to revise the description proposed by Karpathiou et al [22]. Some criteria appeared less relevant: presence of vessels with thickened walls (also found in healthy isthmuses), presence of adenomyosis within the scar (difficult interpretation of glands included in scar tissue). We therefore think that further studies are needed to solve the question of etiology of these endometrial glands.

In conclusion, we found a reduction in total scar thickness in scarring tissue, compared with adjacent healthy uterine tissue or with control, together with a reduction in myometrial thickness and a disorganization of the tissue's usual architecture with increased fibrosis. This is an original work that provides an initial approach to human uterine
healing, particularly with regard to the histological description of isthmocele.

Lack of power due to small numbers and missing data on previous caesarean section modalities are the main limitations of this work. In the study of uterine healing, the human model has several limitations. First of all, difficulty of access to uterine tissue that prevented the study of healing at its earliest stages. Women included in this study for hysterectomy had a minimal delay of 9 years between their last CS and hysterectomy. Histological analysis was therefore performed a long time after CS and after healing process. This delay did not allow study of factors involved in the early healing process. We would like to continue our work on uterine healing by analyzing biopsies of per-caesarean uterine scars in a future study, in order to deepen our initial results in particular immunohistochemical results with quantitative analysis of selected antibodies, including hormone receptors.

Due to the small number of women, it was not possible to predict the type of healing depending on women's characteristics, with or without isthmocele, looking for factors influencing healing. The histological subgroup analysis seems to confirm the known epidemiological data on the increased risk of complications including isthmocele with the number of previous caesarean sections [5,6]. Interpretation of subgroup analyses must be cautious, given the small number of women per group.

Comparisons to the control group must be cautious, as women were not matched and histological samples of healthy uteri were sampled from the isthmus or upper part of the cervix, as there is no lower segment outside pregnancy.

As it is ethically difficult to study growth factors involved in the early phase of human uterine scarring *in vivo*, animal models remain indispensable to better describe the timing of uterine scarring. Further studies are currently on-going to develop an animal model to study this question.

### CRediT authorship contribution statement

**Constance Maudot:** Writing – original draft, Visualization, Validation, Project administration, Methodology, Data curation. **Elodie Debras:** Writing – review & editing, Supervision, Conceptualization. **Astrid Laurent-Bellue:** Visualization, Validation, Methodology, Formal analysis, Data curation. **Margot Dupeux:** Visualization, Validation, Methodology, Formal analysis, Data curation. **Suzanne Chartier:** Visualization, Validation, Methodology, Formal analysis, Data curation. **Sophie Prevost:** Visualization, Validation, Methodology, Conceptualization. **Victor Beaucoté:** Methodology, Formal analysis. **Pascale Chavatte-Palmer:** Writing – review & editing, Supervision, Conceptualization. **Perrine Goussault Capmas:** Writing – review & editing, Visualization, Supervision, Project administration, Methodology, Formal analysis, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgement

The authors wish to thank @BRIDGe and BREED laboratory team at INRAE Jouy-en-Josas and the anatomopathology laboratory team at Bicêtre hospital, for their help in conducting this research.

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejogrb.2024.10.048.

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