Uterine scars after caesarean delivery: From histology to the molecular and ultrastructural level.

Uterine rupture during a trial of labor after caesarean delivery (CD) is a serious complication for mother and fetus. The lack of knowledge on histological features and molecular pathways of uterine wound healing has hindered research in this area from evolving over time. We analysed collagen content and turnover in uterine scars on a histological, molecular and ultrastructural level. Therefore, tissue samples from the lower uterine segment were obtained during CD from 16 pregnant women with at least one previous CD, from 16 pregnant women without previous CD, and from 16 non-pregnant premenopausal women after hysterectomy for a benign disease. Histomorphometrical collagen quantification showed, that the collagen content of the scar area in uterine wall specimens after previous CD was significantly higher than in the unscarred myometrium of the same women and the control groups. Quantitative real-time PCR of uterine scar tissue from FFPE samples delineated by laser microdissection yielded a significantly higher COL3A1 expression and a significantly lower COL1A2/COL3A1 ratio in scarred uteri than in samples from unscarred uteri. Histological collagen content and the expression of COL1A2 and COL3A1 were positively correlated, while COL1A2/COL3A1 ratio was negatively correlated with the histological collagen content. Transmission electron microscopy revealed a destroyed myometrial ultrastructure in uterine scars with increased collagen density. We conclude that the high collagen content in uterine scars results from an ongoing overexpression of collagen I and III. This is a proof of concept to enable further analyses of specific factors that mediate uterine wound healing.

Intraoperative ultrasound during repeat cesarean delivery facilitates sampling of uterine scar tissue.

OBJECTIVES: Histological examination of uterine scars provides insight into uterine wound healing and helps to develop prevention methods of uterine wall rupture after previous uterine surgery. Therefore, exact intraoperative scar identification is needed for specimen collection from the actual scar tissue. The aim of this study was to correlate pre- and intraoperative ultrasound measurements of the lower uterine segment (LUS) with histological findings of scar tissue and to evaluate the relevance of intraoperative ultrasound. METHODS: In a prospective observational study, preoperative and intraoperative sonographic measurements of the LUS thickness were performed in 33 women with a history of at least one cesarean delivery. Intraoperative ultrasound with a linear transducer placed directly on the uterus identified the scar area and uterotomy was performed 2 cm cranially. Tissue samples were taken after extraction of the fetus, embedded in paraffin wax, and stained according to Gomori Trichrome to identify scar tissue. Collagen content was evaluated with imaging software Fiji (NIH, Bethesda, USA). Preoperative and intraoperative sonographic measurements were correlated with histologic evidence of scar tissue. RESULTS: Histological evidence of scar tissue was found in 11 out of 33 samples with significantly lower ultrasonographic thickness of the lower uterine segment compared to the other 22 samples, both antepartum (1.4 mm [1.3-1.9] vs. 2.0 mm [1.6-2.6], p=0.03) and intrapartum (1.6 mm [1.3-1.9] vs. 3.7 mm [2.0-4.7], p<0.01). Intraoperative ultrasound had a significantly higher predictive power (AUC difference 0.18 [0.03-0.33], p=0.01). CONCLUSIONS: Intraoperative sonography identifies the uterine wall area with histologically confirmable scar tissue far better than preoperative sonography.

Optimized RNA isolation of FFPE uterine scar tissues for RNA expression analyses delineated by laser microdissection.

Samples for histological analyses are often formalin-fixed paraffin-embedded (FFPE) and slide-mounted, which complicates RNA extraction for many downstream molecular applications. Furthermore, when the region of interest is extremely small due to isolation with laser microdissection (LMD), extracting RNA of adequate quality and quantity is difficult. We describe an optimized protocol for maximizing RNA output from FFPE tissue devised to identify and analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars resulting from prior cesarean deliveries. Gomori trichrome staining allowed for region identification for LMD. Successful RNA isolation, reverse transcription and, importantly, quantitative real-time PCR (qRT-PCR) were performed. This report provides an optimized step-by-step protocol yielding sufficient RNA for qRT-PCR analyses from challenging tissue/LMD-FFPE samples.