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

ABSTRACT 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 lasermicrodissection (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 humanmaternal uterine scar tissue obtained fromuterotomy 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. METHOD SUMMARY FFPE sample sections weremounted on glass slides and stained with hematoxylin and eosin (HE). A corresponding slide was stained according to Gomori trichrome for orientation. Regions of interest in HE-stained samples weremarked using lasermicrodissection (LMD) and subsequently scratched off the slide with a sterile scalpel. RNA extraction and cDNA synthesis were carried out, and quantitative real-time PCR with TaqMan probes was performed. TWEETABLE ABSTRACT We describe a protocol for maximized RNA output from FFPE tissue delineated by laser microdissection devised to analyze gene expression of human maternal uterine scar tissue obtained from uterotomy scars of prior cesarean deliveries

Charité Caesarean Birth Improves Birth Experience in Planned and Unplanned Caesarean Sections While Maintaining Maternal and Neonatal Safety: A Prospective Cohort Study

Background: In this study, we aimed to assess the safety of a modified caesarean delivery (Charite caesarean birth) in an extended frame of indications, and to examine its impact on parents' birth experience and long-term effects. Methods: This prospective cohort study was performed from January to June 2019. A standardized questionnaire was given to all women who gave birth as an inpatient delivery. Eight months after hospital discharge, all women who gave consent were sent a follow-up questionnaire including questions on current feelings, breast feeding, bonding, and support system, as well as a screening for postnatal depression. Indications for caesarean delivery included preterm birth, fetal malpresentation, fetal malformation, twin pregnancy, and maternal pre-existing conditions. Results: The study cohort included 110 women. The mode of delivery was spontaneous in 49%, per vacuum extraction in 15%, conventional caesarean section in 7%, and Charite caesarean birth in 29%. The groups with Charite versus conventional caesarean delivery did not significantly differ in neonatal admission rates, umbilical cord parameters, maternal blood loss, or duration of surgery. Compared to conventional caesarean delivery, women who underwent a Charite caesarean delivery were significantly more satisfied with their birth experience. At follow-up, the mode of delivery was not associated with significant differences in postnatal depression, breast feeding, or bonding parameters. Conclusions: Outside of emergency situations, Charite caesarean birth improves patients' well-being, without increased maternal and neonatal morbidity

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

Maternal baseline characteristics, birth, and postpartum details of 48 Merino ewes

Maternal baseline characteristics, birth, and postpartum details of 48 Merino ewes are found in the excel sheet.</p