Cord blood myostatin concentrations by gestational diabetes mellitus and fetal sex.

Introduction: Myostatin is a member of the transforming growth factor ß superfamily, and is mainly secreted from skeletal muscle. Animal studies have demonstrated that deficiency in myostatin promotes muscle growth and protects against insulin resistance. In humans, gestational diabetes mellitus (GDM) affects fetal insulin sensitivity. Females are more insulin resistant and weigh less than males at birth. We sought to assess whether cord blood myostatin concentrations vary by GDM and fetal sex, and the associations with fetal growth factors. Methods: In a study of 44 GDM and 66 euglycemic mother-newborn dyads, myostatin, insulin, proinsulin, insulin-like growth factor (IGF)-1, IGF-2 and testosterone were measured in cord blood samples. Results: Cord blood myostatin concentrations were similar in GDM vs. euglycemic pregnancies (mean ± SD: 5.5 ± 1.4 vs. 5.8 ± 1.4 ng/mL, P=0.28), and were higher in males vs. females (6.1 ± 1.6 vs. 5.3 ± 1.0 ng/mL, P=0.006). Adjusting for gestational age, myostatin was negatively correlated with IGF-2 (r=-0.23, P=0.02), but not correlated with IGF-1 (P=0.60) or birth weight (P=0.23). Myostatin was strongly correlated with testosterone in males (r=0.56, P<0.001), but not in females (r=-0.08, P=0.58) (test for difference in r, P<0.001). Testosterone concentrations were higher in males vs. females (9.5 ± 6.4 vs. 7.1 ± 4.0 nmol/L, P=0.017), and could explain 30.0% (P=0.039) of sex differences in myostatin concentrations. Discussion: The study is the first to demonstrate that GDM does not impact cord blood myostatin concentration, but fetal sex does. The higher myostatin concentrations in males appear to be partly mediated by higher testosterone concentrations. These findings shed novel insight on developmental sex differences in insulin sensitivity regulation relevant molecules.

Maternal blood EBF1-based microRNA transcripts as biomarkers for detecting risk of spontaneous preterm birth: a nested case-control study.

OBJECTIVE: Both genetic variants and maternal blood mRNA levels of EBF1 gene have been linked to sPTB. Animal and human studies suggest that specific EBF1-based miRNAs are involved in various physiological and pathophysiological processes. However, to date, we did not find any reports of EBF1-based miRNAs or miRNA transcripts in relation to sPTB. We therefore aimed to examine whether maternal blood early B cell factor 1 (EBF1) gene-based microRNA (miRNA) transcripts can be used for detecting risk of spontaneous preterm birth (sPTB). METHODS: We conducted a nested case-control study within a Canadian cohort consisting of 1878 singleton pregnancies enrolled from May 2008 to December 2010 in Calgary, Alberta, Canada. We used a public gene expression dataset (GSE59491) derived from maternal blood in trimesters 2-3 that included women with sPTB (n = 51) and term births (n = 106) matched for maternal age, race/ethnicity, pre-pregnancy body mass index, smoking during pregnancy, and parity within the Canadian cohort. Two bioinformatics tools, miRWalk and STarMirDB, with different algorithms were applied to retrieve miRNA transcripts that putatively target the EBF1 gene (i.e. EBF1-based). Limma moderated t-tests were used to examine differentially expressed (DE) miRNA transcripts (sPTB vs term) within trimesters. Logistic regression models with miRNA transcript tertiles were applied to assess threshold associations between candidate miRNA transcripts' levels and sPTB. Receiver operating characteristic (ROC) analyses were used to identify the maximum Youden Index and its corresponding optimal sensitivity/specificity cut-point of EBF1-based miRNA transcripts for classifying sPTB, and to compare the classification performance of a linear combination (score) of miRNA transcripts with that of individual miRNA transcripts. A five-fold cross-validation was applied to examine the possible overfitting problem of the final ROC model. RESULTS: Four maternal blood EBF1-based miRNA transcripts (MIR4266, MIR1251, MIR601, MIR3612) in the 3rd trimester were significantly associated with sPTB. The odds ratios (95%CIs) for highest versus lowest tertile of the four miRNA transcripts were 3.01-5.25(1.21-13.14, p ≤ .018). The combined 4-miRNA transcripts' score significantly improved the classification of sPTB compared to individual miRNA transcripts (AUC increased from 0.65-0.69 to 0.82, p ≤ .0034) and showed a sensitivity for sPTB of 0.81 and a specificity of 0.72. The final ROC model of the EBF1-based 4 miRNA transcripts' score in cases and controls had no significant overfitting issue. CONCLUSIONS: Maternal blood EBF1-based miRNA transcripts may, along with other biomarkers, be useful in screening for sPTB risk in 3rd trimester. Our results also provide clues for further study of potential molecular mechanisms underlying the relationship between EBF1 gene and sPTB, e.g. connecting genetic variants, mRNA expression, and miRNA regulation.

EBF1-Correlated Long Non-coding RNA Transcript Levels in 3rd Trimester Maternal Blood and Risk of Spontaneous Preterm Birth.

Biomarkers associated with spontaneous preterm birth (sPTB) before labor onset could aid in prediction, triage, and stratification for testing interventions. In this study we examined maternal blood EBF1-correlated long non-coding RNAs (lncRNAs) in relation to sPTB. We retrieved all lncRNA transcripts from a public gene expression dataset (GSE59491) derived from maternal blood in trimesters 2 and 3 from a Canadian cohort with a matched set of sPTB (n = 51) and term births (n = 106). LncRNA transcripts differentially expressed (limma moderated t-tests) in sPTB vs. term were tested for correlations (Pearson) with EBF1 mRNA levels in the same blood samples. Using logistic regression, EBF1-correlated lncRNAs were divided into tertiles and assessed in relation to odds of sPTB. Two lncRNA transcripts in the 3rd trimester maternal blood were differentially expressed between sPTB and term births (all p < 0.001 and FDR < 0.250) and positively and negatively correlated with EBF1 mRNA levels. They were as follows: (1) LINC00094 r = 0.196 (95% CI: 0.039 to 0.344), p = 0.015, and BH adjusted p = 0.022 and (2) LINC00870 r = - 0.303 (95% CI: - 0.441 to - 0.152), p < 0.001, and BH adjusted p < 0.001. As compared with term births, sPTBs were more likely to be in the highest tertile of LINC00870 (odds ratio (OR) = 4.08 (95% CI 1.60, 10.40), p = 0.003) and the lowest tertile of LINC00094 (OR = 5.16 (95% CI 1.96, 13.61), p < 0.001). Two sPTB-associated EBF1-correlated lncRNAs (LINC00870 and LINC00094) had multiple potential enhancers containing EBF1 binding site(s). Our current findings, along with previous reports linking EBF1 and sPTB, motivate additional research on the EBF1 gene-related gene expression and regulation in relation to sPTB within other cohorts and within laboratory-based models.

Expression of severe acute respiratory syndrome coronavirus 2 cell entry genes, angiotensin-converting enzyme 2 and transmembrane protease serine 2, in the placenta across gestation and at the maternal-fetal interface in pregnancies complicated by preterm birth or preeclampsia.

BACKGROUND: Although there is some evidence that severe acute respiratory syndrome coronavirus 2 can invade the human placenta, limited data exist on the gestational age-dependent expression profile of the severe acute respiratory syndrome coronavirus 2 cell entry mediators, angiotensin-converting enzyme 2 and transmembrane protease serine 2, at the human maternal-fetal interface. There is also no information as to whether the expression of these mediators is altered in pregnancies complicated by preeclampsia or preterm birth. This is important because the expression of decidual and placental angiotensin-converting enzyme 2 and transmembrane protease serine 2 across gestation may affect the susceptibility of pregnancies to vertical transmission of severe acute respiratory syndrome coronavirus 2. OBJECTIVE: This study aimed to investigate the expression pattern of specific severe acute respiratory syndrome coronavirus 2 cell entry genes, angiotensin-converting enzyme 2 and transmembrane protease serine 2, in the placenta across human pregnancy and in paired samples of decidua and placenta in pregnancies complicated by preterm birth or preeclampsia compared with those in term uncomplicated pregnancies. STUDY DESIGN: In this study, 2 separate cohorts of patients, totaling 87 pregnancies, were included. The first cohort was composed of placentae from first- (7-9 weeks), second- (16-18 weeks), and third-trimester preterm (26-31 weeks) and third-trimester term (38-41 weeks) pregnancies (n=5/group), whereas the second independent cohort included matched decidua and placentae from pregnancies from term uncomplicated pregnancies (37-41 weeks' gestation; n=14) and pregnancies complicated by preterm birth (26-37 weeks' gestation; n=11) or preeclampsia (25-37 weeks' gestation; n=42). Samples were subjected to quantitative polymerase chain reaction and next-generation sequencing or RNA sequencing for next-generation RNA sequencing for angiotensin-converting enzyme 2 and transmembrane protease serine 2 mRNA expression quantification, respectively. RESULTS: In the first cohort, angiotensin-converting enzyme 2 and transmembrane protease serine 2, exhibited a gestational age-dependent expression profile, that is, angiotensin-converting enzyme 2 and transmembrane protease serine 2 mRNA was higher (P<.05) in the first-trimester placenta than in second-trimester, preterm birth, and term placentae (P<.05) and exhibited a negative correlation with gestational age (P<.05). In the second cohort, RNA sequencing demonstrated very low or undetectable expression levels of angiotensin-converting enzyme 2 in preterm birth, preeclampsia, and term decidua and in placentae from late gestation. In contrast, transmembrane protease serine 2 was expressed in both decidual and placental samples but did not change in pregnancies complicated by either preterm birth or preeclampsia. CONCLUSION: The increased expression of these severe acute respiratory syndrome coronavirus 2 cell entry-associated genes in the placenta in the first trimester of pregnancy compared with those in later stages of pregnancy suggests the possibility of differential susceptibility to placental entry to severe acute respiratory syndrome coronavirus 2 across pregnancy. Even though there is some evidence of increased rates of preterm birth associated with severe acute respiratory syndrome coronavirus 2 infection, we found no increase in mRNA expression of angiotensin-converting enzyme 2 or transmembrane protease serine 2 at the maternal-fetal interface.

Combinatorial extracellular matrix microarray identifies novel bioengineered substrates for xeno-free culture of human pluripotent stem cells.

Stem cells in their microenvironment are exposed to a plethora of biochemical signals and biophysical forces. Interrogating the role of each factor in the cell microenvironment, however, remains difficult due to the inability to study microenvironmental cues and tease apart their interactions in high throughput. To address this need, we developed an extracellular matrix (ECM) microarray screening platform capable of tightly controlling substrate stiffness and ECM protein composition to screen the effects of these cues and their interactions on cell fate. We combined this platform with a design of experiments screening strategy to identify optimal conditions that can maintain human pluripotent stem cell (hPSC) pluripotency in chemically defined, xeno-free conditions. Combinations of ECM proteins (fibronectin, vitronectin, laminin-521, and collagen IV) were deposited on polydimethylsiloxane substrates with elastic moduli ranging from ~1 to 60 kPa using a high throughput protein plotter. Through our screening approach, we identified several non-intuitive protein-protein and protein-stiffness interactions and developed three novel culture substrates. hPSCs grown on these novel culture substrates displayed higher proliferation rates and pluripotency marker expression than current gold-standard culture substrates Geltrex- and vitronectin-coated plastic. This ECM microarray and screening approach is not limited to the factors studied here and can be broadly applied to other cell types to systematically screen microenvironmental conditions to optimally guide cell phenotype.

Maternal malnutrition impacts placental morphology and transporter expression: an origin for poor offspring growth.

The placenta promotes fetal growth through nutrient transfer and selective barrier systems. An optimally developed placenta can adapt to changes in the pregnancy environment, buffering the fetus from adverse exposures. We hypothesized that the placenta adapts differently to suboptimal maternal diets, evidenced by changes in placental morphology, developmental markers and key transport systems. Mice were fed a control diet (CON) during pregnancy, undernourished (UN) by 30% of control intake from gestational day (GD) 5.5-18.5 or fed 60% high-fat diet (HF) 8 weeks before and during pregnancy. At GD18.5, placental morphometry, development and transport were assessed. Junctional and labyrinthine areas of UN and HF placentae were smaller than CON by >10%. Fetal blood space area and fetal blood space:fetal weight ratios were reduced in HF vs. CON and UN. Trophoblast giant cell marker Ctsq mRNA expression was lower in UN vs. HF, and expression of glycogen cell markers Cx31.1 and Pcdh12 was lower in HF vs. UN. Efflux transporter Abcb1a mRNA expression was lower in HF vs. UN, and Abcg2 expression was lower in UN vs. HF. mRNA expression of fatty acid binding protein Fabppm was higher in UN vs. CON and HF. mRNA and protein levels of the lipid transporter FAT/CD36 were lower in UN, and FATP4 protein levels were lower in HF vs. UN. UN placentae appear less mature with aberrant transport, whereas HF placentae adapt to excessive nutrient supply. Understanding placental adaptations to common nutritional adversities may reveal mechanisms underlying the developmental origins of later disease.

EBF1 Gene mRNA Levels in Maternal Blood and Spontaneous Preterm Birth.

Genetic variants of six genes (EBF1, EEFSEC, AGTR2, WNT4, ADCY5, and RAP2C) have been linked recently to gestational duration and/or spontaneous preterm birth (sPTB). Our goal was to examine sPTB in relation to maternal blood mRNA levels of these genes. We used a public gene expression dataset (GSE59491) derived from maternal blood in trimesters 2 and 3 that included women with sPTB (n = 51) and term births (n = 106) matched for maternal age, race/ethnicity, pre-pregnancy body mass index, smoking during pregnancy, and parity. T tests were used to examine mRNA mean differences (sPTB vs term) within and across trimesters, and logistic regression models with mRNA quartiles were applied to assess associations between candidate gene mRNA levels and sPTB. Based on these analyses, one significant candidate gene was used in a Gene Set Enrichment Analysis (GSEA) to identify related gene sets. These gene sets were then compared with the ones previously linked to sPTB in the same samples. Our results indicated that among women in the lowest quartile of EBF1 mRNA in the 2nd or 3rd trimester, the odds ratio for sPTB was 2.86 (95%CI 1.08, 7.58) (p = 0.0349, false discovery rate (FDR) = 0.18) and 4.43 (95%CI 1.57, 12.50) (p = 0.0049, FDR = 0.06), respectively. No other candidate gene mRNAs were significantly associated with sPTB. In GSEA, 24 downregulated gene sets were correlated with 2nd trimester low EBF1 mRNA and part of previous sPTB-associated gene sets. In conclusion, mRNA levels of EBF1 in maternal blood may be useful in detecting increased risk of sPTB as early as 2nd trimester. The potential underlying mechanism might involve maternal-fetal immune and cell cycle/apoptosis pathways.

MicroRNA-transcriptome networks in whole blood and monocytes of women undergoing preterm labour.

Preterm birth is attributed to neonatal morbidity as well as cognitive and physiological challenges. We have previously identified significant differences in mRNA expression in whole blood and monocytes, as well as differences in miRNA concentration in blood plasma, extracellular vesicles (EV) and EV-depleted plasma in women undergoing spontaneous preterm labour (sPTL). The goal of this analysis was to identify differences in miRNA expression within whole blood (WB) and peripheral monocytes (PM) from the same population of women undergoing sPTL compared with non-labouring controls matched by gestational age. We performed single-end small RNA sequencing in whole blood and peripheral monocytes from women undergoing sPTL with active contractions (24-34 weeks of gestation, N = 15) matched for gestational age to healthy pregnant non-labouring controls (>37 weeks gestation, N = 30) who later delivered at term as a part of the Ontario Birth Study (Toronto, Ontario CA). We identified significant differences in expression of 16 miRNAs in PMs and nine miRNAs in WB in women undergoing sPTL. In PMs, these miRNAs were predicted targets of 541 genes, including 28 previously associated with sPTL. In WB, miRNAs were predicted to target 303 genes, including nine previously associated with sPTL. These genes were involved in a variety of immune pathways, including interleukin-2 signalling. This study is the first to identify changes in miRNA expression in WB and PMs of women undergoing sPTL. Our results shed light on potential mechanisms by which miRNAs may play a role in mediating systemic inflammatory response in pregnant women that deliver prematurely.

P-Glycoprotein (P-gp)/ABCB1 plays a functional role in extravillous trophoblast (EVT) invasion and is decreased in the pre-eclamptic placenta.

Dysregulation of trophoblast differentiation is implicated in the placental pathologies of intrauterine growth restriction and pre-eclampsia. P-glycoprotein (P-gp encoded by ABCB1) is an ATP-binding cassette transporter present in the syncytiotrophoblast layer of the placenta where it acts as a molecular sieve. In this study, we show that P-gp is also expressed in the proliferating cytotrophoblast (CT), the syncytiotrophoblast (ST) and the extravillous trophoblast (EVT), suggesting our hypothesis of a functional role for P-gp in placental development. Silencing of ABCB1, via siRNA duplex, results in dramatically reduced invasion and migration, and increased tube formation and fusion in the EVT-like HTR8/SVneo cell line. In both EVT and CT explant differentiation experiments, silencing of ABCB1 leads to induction of the fusion markers human hCG, ERVW-1 and GJA1 and terminal differentiation of both trophoblast subtypes. Moreover, P-gp protein levels are decreased in both the villous and the EVT of severe early-onset pre-eclamptic placentas. We conclude that, in addition to its role as a syncytial transporter, P-gp is a key factor in the maintenance of both CT and EVT lineages and that its decrease in severe pre-eclampsia may contribute to the syncytial and EVT placental pathologies associated with this disease.

HIV antiretroviral exposure in pregnancy induces detrimental placenta vascular changes that are rescued by progesterone supplementation.

Adverse birth outcomes are common in HIV-positive pregnant women receiving combination antiretroviral therapy (cART), especially when cART is initiated in early pregnancy. The mechanisms remain poorly understood. Using a mouse model we demonstrate that protease inhibitor based-cART exposure beginning on day 1 of pregnancy was associated with a pro-angiogenic/pro-branching shift in the placenta driven by lower Flt-1 levels and higher Gcm-1 expression. Micro-CT imaging revealed an increase in the number of arterioles in cART-treated placentas, which correlated with fetal growth restriction. Delaying initiation of cART, or supplementing cART-treated mice with progesterone, prevented the pro-angiogenic/pro-branching shift and the associated placenta vascular changes. In agreement with our mouse findings, we observed an increase in the number of terminal-villi capillaries in placentas from HIV-positive cART-exposed women compared to HIV-negative controls. Capillary number was inversely correlated to maternal progesterone levels. Our study provides evidence that cART exposure during pregnancy influences placenta vascular formation that may in turn contribute to fetal growth restriction. Our findings highlight the need for closer investigation of the placenta in HIV-positive pregnancies, particularly for pregnancies exposed to cART from conception, and suggest that progesterone supplementation could be investigated as a possible intervention to improve placenta function in HIV-positive pregnant women.

Comparative analysis of gene expression in maternal peripheral blood and monocytes during spontaneous preterm labor.

BACKGROUND: Preterm birth is the leading cause of newborn death worldwide, and is associated with significant cognitive and physiological challenges in later life. There is a pressing need to define the mechanisms that initiate spontaneous preterm labor, and for development of novel clinical biomarkers to identify high-risk pregnancies. Most preterm birth studies utilize fetal tissues, and there is limited understanding of the transcriptional changes that occur in mothers undergoing spontaneous preterm labor. Earlier work revealed that a specific population of maternal peripheral leukocytes (macrophages/monocytes) play an active role in the initiation of labor. Thus, we hypothesized that there are dynamic gene expression changes in maternal blood leukocytes during preterm labor. OBJECTIVE: Using next-generation sequencing we aim to characterize the transcriptome in whole blood leukocytes and peripheral monocytes of women undergoing spontaneous preterm labor compared to healthy pregnant women who subsequently delivered at full term. STUDY DESIGN: RNA sequencing was performed in both whole blood and peripheral monocytes from women who underwent preterm labor (24-34 weeks of gestation, N = 20) matched for gestational age to healthy pregnant controls (N = 30). All participants were a part of the Ontario Birth Study cohort (Toronto, Ontario, Canada). RESULTS: We identified significant differences in expression of 262 genes in peripheral monocytes and 184 genes in whole blood of women who were in active spontaneous preterm labor compared to pregnant women of the same gestational age not undergoing labor, with 43 of these genes differentially expressed in both whole blood and peripheral monocytes. ADAMTS2 expression was significantly increased in women actively undergoing spontaneous preterm labor, which we validated through digital droplet reverse transcriptase polymerase chain reaction. Intriguingly, we have also identified a number of gene sets including signaling by stem cell factor-KIT, nucleotide metabolism, and trans-Golgi network vesicle budding, which exhibited changes in relative gene expression that was predictive of preterm labor status in both maternal whole blood and peripheral monocytes. CONCLUSION: This study is the first to investigate changes in both whole blood leukocytes and peripheral monocytes of women actively undergoing spontaneous preterm labor through robust transcript measurements from RNA sequencing. Our unique study design overcame confounding based on gestational age by collecting blood samples from women matched by gestational age, allowing us to study transcriptomic changes directly related to the active preterm parturition. We performed RNA profiling using whole genome sequencing, which is highly sensitive and allowed us to identify subtle changes in specific genes. ADAMTS2 expression emerged as a marker of prematurity within peripheral blood leukocytes, an accessible tissue that plays a functional role in signaling during the onset of labor. We identified changes in relative gene expression in a number of gene sets related to signaling in monocytes and whole blood of women undergoing spontaneous preterm labor compared to controls. These genes and pathways may help identify potential targets for the development of novel drugs for preterm birth prevention.

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies in AggreWell Plates.

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. The method described here makes use of commercially available AggreWell 400 plates with prefabricated indentations that cradle each EB. Although these plates are relatively expensive, they may be suitable for some high-throughput experiments. This protocol describes the preparation of embryoid bodies of defined size and shape in a large-scale format (approximately 1200 embryoid bodies per preparation).

P-glycoprotein expression and localization in the rat uterus throughout gestation and labor.

Uterine tissues contain the efflux transporter P-glycoprotein (P-gp, encoded by Abcb1a/1b gene), but little is known about how it changes through gestation. Our aim was to investigate the expression profile and cellular localization of P-gp in the pregnant, laboring and post-partum (PP) rat uterus. We propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial Abcb1a/1b/P-gp. Samples from bilaterally and unilaterally pregnant rats were collected throughout gestation, during labor, and PP (n=4-6/gestational day). RNA and protein were isolated and subjected to quantitative PCR and immunoblotting; P-gp transcript and protein were localized by in situ hybridization and immunohistochemistry. Expression of Abcb1a/1b gene and membrane P-gp protein in uterine tissue (1) increased throughout gestation, peaked at term (GD19-21) and dropped during labor (GD23L); and (2) was upregulated only in gravid but not in empty horn of unilaterally pregnant rats. (3) The drop of Abcb1a/1b mRNA on GD23 was prevented by artificial maintenance of elevated progesterone (P4) levels in late gestation; (4) injection of the P4 receptor antagonist RU486 on GD19 caused a significant decrease in Abcb1 mRNA levels. (5) In situ hybridization and immunohistochemistry indicated that Abcb1/P-gp is absent from myometrium throughout gestation; (6) was expressed exclusively by uterine microvascular endothelium (at early gestation) and luminal epithelium (at mid and late gestation), but was undetectable during labor. In conclusion, ABC transporter protein P-gp in pregnant uterus is hormonally and mechanically regulated. However, its substrate(s) and precise function in these tissues during pregnancy remains to be determined.

Generation and Use of Trophoblast Stem Cells and Uterine Myocytes to Study the Role of Connexins for Pregnancy and Labor.

Transgenic mouse models have demonstrated critical roles for gap junctions in establishing a successful pregnancy. To study the cellular and molecular mechanisms, the use of cell culture systems is essential to discriminate between the effects of different connexin isoforms expressed in individual cells or tissues of the developing conceptus or in maternal reproductive tissues. The generation and analysis of gene-deficient trophoblast stem cell lines from mice clearly revealed the functions of connexins in regulating placental development. This chapter focuses on the use of connexin gene-deficient trophoblast stem cell cultures to reveal the individual role of gap junctions in regulating trophoblast differentiation and proliferation in vitro under controlled conditions. In addition, cultures of primary uterine myocytes, isolated from mice or rats, allow studying the effects of mechanical stretch or ovarian hormones on regulating connexin expression, and thus, to model the molecular mechanisms of uterine growth and development during pregnancy. Here, we describe the derivation of primary uterine myocyte cultures and their use in in vitro stretch experiments to study the mechanisms of myometrial remodeling essential to accommodate the growing fetus throughout gestation.

Quantitative large scale gene expression profiling from human stem cell culture micro samples using multiplex pre-amplification.

Transcriptional profiling is a powerful tool to study biological mechanisms during stem cell differentiation and reprogramming. Genome-wide methods like microarrays or next generation sequencing are expensive, time consuming, and require special equipment and bioinformatics expertise. Quantitative RT-PCR remains one of today's most widely accepted and used methods for analyzing gene expression in biological samples. However, limitations in the amount of starting materials often hinder the quantity and quality of information that could be obtained from a given sample. Here, we present a fast 4-step workflow allowing direct, column-free RNA isolation from limited human pluripotent stem cell (hPSC) cultures that is directly compatible with subsequent reverse transcription, target specific multiplex pre-amplification, and standard SYBR-Green quantitative PCR (qPCR) analysis. The workflow delivers excellent correlations in normalized gene-expression data obtained from different samples of hPSCs over a wide range of cell numbers (500-50,000 cells). We demonstrate accurate and unbiased target gene quantification in limiting stem cell cultures which allows for monitoring embryoid body differentiation and induced pluripotent stem cell (iPSC) reprogramming. This method highlights a rapid and cost effective screening process, allowing reduction of culture formats and increase of processing throughputs for various stem cell applications.

Physiological roles of connexins and pannexins in reproductive organs.

Reproductive organs are complex and well-structured tissues essential to perpetuate the species. In mammals, the male and female reproductive organs vary on their organization, morphology and function. Connectivity between cells in such tissues plays pivotal roles in organogenesis and tissue functions through the regulation of cellular proliferation, migration, differentiation and apoptosis. Connexins and pannexins can be seen as major regulators of these physiological processes. In the present review, we assembled several lines of evidence demonstrating that these two families of proteins are essential for male and female reproduction.

Connexin31.1 (Gjb5) deficiency blocks trophoblast stem cell differentiation and delays placental development.

The gap junction channel forming connexins (Cx) Cx31 (Gjb3) and Cx31.1 (Gjb5) are co-expressed in the mouse trophoblast lineage. Inactivation of either gene results in partial embryonic loss at mid gestation (60% and 30%, respectively, between embryonic days E10.5and E13.5) caused by placental phenotypes. Cx31 deficiency results in loss of stem cell potential and enhanced trophoblast giant cell (TGC) differentiation, whereas the molecular role of the co-expressed Cx31.1 remained unclear. It was assumed that both isoforms have overlapping functions and can compete for each loss in placentation as both knockout mice show similar survival rates, reduced placental weights, and growth restricted embryos. Instead, here we show that Cx31.1 has opposed functions in regulating trophoblast differentiation. Cx31.1 deficiency causes a shift in placental subpopulations, reduced area of fetal blood spaces, and a reduced number of secondary TGC in the junctional zone, as shown by stereology at E10.5. Cx31.1 is critical for terminal differentiation of trophoblast cells during placentation resulting in a delayed induction of marker genes Tpbpa, Prl3b1/Pl-2, and Ctsq in Cx31.1-deficient placentas. Derivation and analysis of Cx31.1-deficient trophoblast stem lines clearly indicates a delayed trophoblast differentiation manifested by repression of marker genes for placental subpopulations and continued expression of stem cell marker genes Id2 and Ascl2, which is correlated to enhanced proliferation capacity of differentiating stem cells These findings clarify the disparate actions of Cx31.1 and Cx31 that act in opposition to balance the fate of trophoblast cells during differentiation, with Cx31.1 promoting, and Cx31 delaying terminal differentiation.

DREAM mediated regulation of GCM1 in the human placental trophoblast.

The trophoblast transcription factor glial cell missing-1 (GCM1) regulates differentiation of placental cytotrophoblasts into the syncytiotrophoblast layer in contact with maternal blood. Reduced placental expression of GCM1 and abnormal syncytiotrophoblast structure are features of hypertensive disorder of pregnancy--preeclampsia. In-silico techniques identified the calcium-regulated transcriptional repressor--DREAM (Downstream Regulatory Element Antagonist Modulator)--as a candidate for GCM1 gene expression. Our objective was to determine if DREAM represses GCM1 regulated syncytiotrophoblast formation. EMSA and ChIP assays revealed a direct interaction between DREAM and the GCM1 promoter. siRNA-mediated DREAM silencing in cell culture and placental explant models significantly up-regulated GCM1 expression and reduced cytotrophoblast proliferation. DREAM calcium dependency was verified using ionomycin. Furthermore, the increased DREAM protein expression in preeclamptic placental villi was predominantly nuclear, coinciding with an overall increase in sumolylated DREAM and correlating inversely with GCM1 levels. In conclusion, our data reveal a calcium-regulated pathway whereby GCM1-directed villous trophoblast differentiation is repressed by DREAM. This pathway may be relevant to disease prevention via calcium-supplementation.