Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (<P10) of the cell lines revealing there were no genetic drift over the passages. Karyotyping also revealed no chromosomal anomalies. Creation of these cell lines can standardize experimental approaches, eliminate subject to subject variabilities, and benefit the reproductive biological studies on pregnancies by using these cells.

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

Current intervention strategies have not been successful in reducing the risks of adverse pregnancy complications nor maternal and fetal morbidities associated with pregnancy complications. Improving pregnancy and neonatal outcomes requires a better understanding of drug transport mechanisms at the feto-maternal interfaces, specifically the placenta and fetal membrane (FM). The role of several solute carrier uptake transporter proteins (TPs), such as the organic anion transporting polypeptide 2B1 (OATP2B1) in transporting drug across the placenta, is well-established. However, the mechanistic role of FMs in this drug transport has not yet been elucidated. We hypothesize that human FMs express OATP2B1 and functions as an alternate gatekeeper for drug transport at the feto-maternal interface. We determined the expression of OATP2B1 in term, not-in-labor, FM tissues and human FM cells [amnion epithelial cell (AEC), chorion trophoblast cell (CTC), and mesenchymal cells] using western blot analyses and their localization using immunohistochemistry. Changes in OATP2B1 expression was determined for up to 48 h after stimulation with cigarette smoke extract (CSE), an inducer of oxidative stress. The functional role of OATP2B1 was determined by flow cytometry using a zombie violet dye substrate assay. After OATP2B1 gene silencing, its functional relevance in drug transport through the feto-maternal interface was tested using a recently developed feto-maternal interface organ-on-a-chip (OOC) system that contained both FM and maternal decidual cells. Propagation of a drug (Rosuvastatin, that can be transported by OATP2B1) within the feto-maternal interface OOC system was determined by mass spectrometry. FMs express OATP2B1 in the CTC and AEC layers. In FM explants, OATP2B1 expression was not impacted by oxidative stress. Uptake of the zombie violet dye within AECs and CTCs showed OATP2B1 is functionally active. Silencing OATP2B1 in CTCs reduced Rosuvastatin propagation from the decidua to the fetal AEC layer within the feto-maternal interface-OOC model. Our data suggest that TPs in FMs may function as a drug transport system at the feto-maternal interface, a function that was previously thought to be performed exclusively by the placenta. This new knowledge will help improve drug delivery testing during pregnancy and contribute to designing drug delivery strategies to treat adverse pregnancy outcomes.

Clinical Profile and Feto-Maternal Outcome of Preterm Prelabour Rupture of The Membrane in a Tertiary Level Hospital

Background: The rupture of fetal membrane before onset of labour at less than 37 completed weeks of gestation. Incidence of Preterm prelabour rupture of membrane (PPROM) ranges from 3.0-10.0% of all deliveries and causes around 25-30% of all preterm deliveries. Objective: To see the clinical profile, maternal and fetal outcome of preterm prelabour rupture of the membrane. Materials and Methods: One hundred fifteen pregnant women with 28-37 weeks of gestation and diagnosed as PPROM admitted in different units selected by purposive sampling, fulfilled the inclusion and exclusion criteria were enrolled as study population in this study. Results: The mean age was 24.65(±3.68) years. Majority (75.65%) were primi para. The mean gestational age was 32.34(±2.86) weeks, 79.13% had gestational age between 30-36 weeks and 20.87% had up to 30 wks. The common risk factors of the study population were history of coitus, CPD, infection and history of abortion which were 42.61%, 9.57%, 5.22% and 4.35% respectively. Majority 55.65% women had vaginal delivery, 44.35% had caesarian section. Majority of the babies born to PPROM group were in the very low birth weight category (53 cases 62.3%), whereas only 32 cases (37.6%) were of normal birth weight. Infection and perinatal mortality was significantly associated with PPROM. Conclusion: PPROM can be prevented avoiding the certain risk factors by proper ante natal checkup, strict follow up, good obstetrical care and perinatal care , making mass awareness of the sequele. KYAMC Journal. 2021;12(3): 166-171

Minimally Invasive Precise Application of Bioadhesives to Prevent IPPROM on a Pregnant Sheep Model

<b><i>Introduction:</i></b> Iatrogenic preterm premature rupture of the membrane remains the Achille’s heel of fetoscopy. The aim of this study was to show in vivo feasibility of fetal membrane (FM) defect sealing by the application of tissue glues with umbrella-shaped receptors. <b><i>Methods:</i></b> First, we adapted our previously described ex vivo strategy and evaluated the adhesion strength of different tissue glues, Histoacryl® and Glubran2®, by bonding polytetrafluoroethylene or silicone encapsulated nitinol glue receptor to human FM. Then, we exposed pregnant sheep uterus through a laparotomy and placed a 10-French trocar into the amniotic cavity through which the umbrella-shaped glue receptor (<i>n</i> = 9) was inserted and fixated onto the FM with the tissue glues (<i>n</i> = 8). The tightness of the sealed defects was assessed 4 h post-surgery. <b><i>Results:</i></b> Both tissue glues tested resulted in adhesion of the glue receptors to the FM ex vivo. In vivo, all glue receptors opened in the amniotic cavity (<i>n</i> = 9) and all successfully placed glue receptors sealed the FM defect (<i>n</i> = 8). Four hours post-surgery, 2 treatment sites showed minimal leakage whereas the negative control without glue (<i>n</i> = 1) showed substantial leakage. <b><i>Discussion:</i></b> This in vivo study confirms that fetoscopically induced FM defects can be sealed by the application of tissue adhesives.

Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues

High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue (‘bulk’) RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.

Outcome of induction and associated factors among induced labours in public Hospitals of Harari Regional State, Eastern Ethiopia: A two years’ retrospective analysis

Background Induction of labor (IOL) is an essential intervention to reduce adverse maternal and neonatal outcomes. It is also improved pregnancy outcomes, especially in resource-limited countries, where maternal and perinatal mortality is unacceptably high. However, there is a scarcity of evidence regarding the outcome of induction of labor and its predictors in low-income countries like Sub-Saharan Africa. Therefore, this study was aimed at assessing the outcome of induction of labor and associated factors among mothers who underwent labor induction in public Hospitals of Harari Regional State, Estern Ethiopia. Methods A facility-based cross-sectional study was conducted from 1 to 30 March, 2019 in Harari Regional State, Eastern Ethiopia. A total of 717 mothers who underwent induction of labor in public Hospitals of Harari Regional State, Eastern Ethiopia from January 2017 to December 2018 were enrolled in the study. Data were collected using a pretested structured questionnaire. The collected data were entered into Epi-data version 3.1 and exported to SPSS version 24 (IBM SPSS Statistics, 2016) for further analysis. A multivariable logistic regression analysis was performed to estimate the effects of each predictor variable on the outcome of induction of labor after controlling for potential confounders. Statistical significance was declared at p-value <0.05. Results Overall, the prevalence of success of induction of labor was 65% [95% CI (61.5, 68.5)]. Pre-eclampsia/eclampsia was found to be the most common indication for induction of labor (46.70%) followed by pre-labor rupture of fetal membrane (33.5%). In the final model of multivariable analysis, predictors such as: maternal age < 24 years old [AOR = 1.93, 95%CI(1.14, 3.26)], nulliparity[AOR = 0.34, 95%CI(0.19, 0.59)], unfavorable Bishop score [AOR = 0.06, 95%CI(0.03, 0.12)], intermediate Bishop score [AOR = 0.08, 95%CI(0.04, 0.14)], misoprostol only method [AOR = 2.29, 95%CI(1.01, 5.19)], nonreassuring fetal heart beat pattern [AOR = 0.14, 95%CI (0.07, 0.25)] and Birth weight 3500 grams and above[AOR = 0.32, 95% CI (0.17, 0.59)] were statistically associated with the successful outcome of induction of labor. Conclusion The prevalence of successful of induction of labor was relatively low in this study area because only two-thirds of the mothers who underwent induction of labor had a successful of induction. Therefore, this result calls for all stakeholders to give more emphasis on locally available induction protocols and guidelines. In addition, pre-induction conditions must be taken into consideration to avoid unwanted effect of failed induction of labour.

IL-27 Mediates Pro-Inflammatory Effects via the ERK Signaling Pathway During Preterm Labor

Preterm labor (PTL) is a multifactorial syndrome that results in birth prior to 37 weeks of gestation. However, the specific molecular mechanisms underlying this condition have yet to be elucidated. Previous research demonstrated that the abnormal expression of IL-27, and its receptors, played a role in the pathophysiology of preterm labor. In the present study, we established a Lipopolysaccharide (LPS)-stimulated, infection-induced, preterm mouse model based on wild-type C57BL/6 mice and WSX-1-/-C57BL/6 mice. WSX-1 knockdown led to a significant delay in birth by 11.32 ± 2.157h. In addition, compared with wild-type C57B/6 mice, the expression levels of IFN-γ, IL-1β, IL-6, TNF-α, and CXCL10, in the fetal membrane and myometrium of WSX-1-/-mice were significantly lower, particularly in the myometrium. We also confirmed similar pro-inflammatory effects arising from IL-27 in human amniotic cell line (WISH) and human myometrial smooth muscle cell line (HMSMC). Once stimulated by LPS, the pro-inflammatory action exhibited a synergistic effect and appeared to be time-dependent. Finally, we demonstrated that LY3214996, an inhibitor of the ERK pathway, significantly inhibited the pro-inflammatory effect mediated by IL-27. Overall, our data confirmed that the inflammatory effect mediated by the IL-27/IFN-r/ERK axis is involved in preterm labor. Our findings, therefore, provide an enhancement in our etiological understanding of the mechanisms underlying PTL.