The hormonal control of parturition.

Parturition is a complex physiological process that must occur in a reliable manner and at an appropriate gestation stage to ensure a healthy newborn and mother. To this end, hormones that affect the function of the gravid uterus, especially progesterone (P4), 17ß-estradiol (E2), oxytocin (OT), and prostaglandins (PGs), play pivotal roles. P4 via the nuclear P4 receptor (PR) promotes uterine quiescence and for most of pregnancy exerts a dominant block to labor. Loss of the P4 block to parturition in association with a gain in prolabor actions of E2 are key transitions in the hormonal cascade leading to parturition. P4 withdrawal can occur through various mechanisms depending on species and physiological context. Parturition in most species involves inflammation within the uterine tissues and especially at the maternal-fetal interface. Local PGs and other inflammatory mediators may initiate parturition by inducing P4 withdrawal. Withdrawal of the P4 block is coordinated with increased E2 actions to enhance uterotonic signals mediated by OT and PGs to promote uterine contractions, cervix softening, and membrane rupture, i.e., labor. This review examines recent advances in research to understand the hormonal control of parturition, with focus on the roles of P4, E2, PGs, OT, inflammatory cytokines, and placental peptide hormones together with evolutionary biology of and implications for clinical management of human parturition.

Integrative analysis of noncoding mutations identifies the druggable genome in preterm birth.

Preterm birth affects ~10% of pregnancies in the US. Despite familial associations, identifying at-risk genetic loci has been challenging. We built deep learning and graphical models to score mutational effects at base resolution via integrating the pregnant myometrial epigenome and large-scale patient genomes with spontaneous preterm birth (sPTB) from European and African American cohorts. We uncovered previously unidentified sPTB genes that are involved in myometrial muscle relaxation and inflammatory responses and that are regulated by the progesterone receptor near labor onset. We studied genomic variants in these genes in our recruited pregnant women administered progestin prophylaxis. We observed that mutation burden in these genes was predictive of responses to progestin treatment for preterm birth. To advance therapeutic development, we screened ~4000 compounds, identified candidate molecules that affect our identified genes, and experimentally validated their therapeutic effects on regulating labor. Together, our integrative approach revealed the druggable genome in preterm birth and provided a generalizable framework for studying complex diseases.

A microbiota and dietary metabolite integrates DNA repair and cell death to regulate embryo viability and aneuploidy during aging.

During aging, environmental stressors and mutations along with reduced DNA repair cause germ cell aneuploidy and genome instability, which limits fertility and embryo development. Benevolent commensal microbiota and dietary plants secrete indoles, which improve healthspan and reproductive success, suggesting regulation of germ cell quality. We show that indoles prevent aneuploidy and promote DNA repair and embryo viability, which depends on age and genotoxic stress levels and affects embryo quality across generations. In young animals or with low doses of radiation, indoles promote DNA repair and embryo viability; however, in older animals or with high doses of radiation, indoles promote death of the embryo. These studies reveal a previously unknown quality control mechanism by which indole integrates DNA repair and cell death responses to preclude germ cell aneuploidy and ensure transgenerational genome integrity. Such regulation affects healthy aging, reproductive senescence, cancer, and the evolution of genetic diversity in invertebrates and vertebrates.

Tofacitinib Inhibits STAT Phosphorylation and Matrix Metalloproteinase-3, -9 and -13 Production by C28/I2 Human Juvenile Chondrocytes.

Purpose: This in vitro study was designed to determine the effect of the pan-Janus kinase inhibitor, Tofacitinib, on basal and interleukin-6 (IL-6)-induced signal transducers and activators of transcription (STAT) phosphorylation and matrix metalloproteinase (MMP) gene expression and MMP production by C28/I2 human chondrocytes. Methods: C28/I2 chondrocytes were grown to a confluent high-density and treated either with recombinant human IL-6 (rhIL-6; 10-20ng/mL) or maintained in the basal state for up to 60 min. MMP gene expression was determined using RT-PCR and MMP production by semi-quantitative immunohistochemistry. The effect of IL-6 with or without Tofacitinib on activation of STAT proteins was determined from quantitative Western blots. Results: C28/I2 chondrocytes produced STAT1, STAT3 and STAT5AB which were phosphorylated (p) following treatment with rhIL-6 for 30 min. Tofacitinib (2.5nM-100nM) decreased rhIL-6-induced activation of STAT1, STAT3, and STAT5AB as well as decreasing the expression of MMP3 and MMP13 but not MMP9, MMP1 or MMP2. In addition, Tofacitinib (50nM) reduced the number of rhIL-6-induced MMP3-, and MMP13- antibody-positive C28/I2 chondrocytes. However, Tofacitinib did decrease the number of MMP9-antibody-positive C28/I2 chondrocytes. Conclusion: Taken together, these data showed that Tofacitinib, a pan-JAK small molecule inhibitor employed for the medical therapy of rheumatoid arthritis was a potent inhibitor of rhIL-6-induced STAT phosphorylation that appeared to be coupled to the inhibition of MMP-3, -9 and -13 production by C28/I2 chondrocytes.

Progesterone withdrawal and parturition.

The steroid hormone progesterone (P4), acting via the nuclear P4 receptors (PRs) in uterine cells, is essential for the establishment and maintenance of pregnancy. P4/PR signaling maintains pregnancy by promoting uterine quiescence and blocking the contractions of labor. Withdrawal of the P4/PR block to labor induces parturition. The success of pregnancy requires the timely birth of a mature neonate to a healthy mother, and to this end, the mechanism by which the P4/PR block is withdrawn, and how that process is physiologically controlled is critical. This review examines current understanding of cell and molecular biology of P4/PR withdrawal in the control of parturition.

Preterm birth update in Australasia: A report of the international symposium of Preterm Birth International Collaborative-Australasia branch.

Preterm birth (PTB) is one of the most important problems that pose dilemmas for both the obstetrician and neonatologist, placing a heavy burden psychologically and financially on the families involved, and triggering high socio-economic costs to the public healthcare. The rate of PTB in Asian countries has been ranked at top globally. To reduce the PTB rate, to promote the prevention and intervention for PTB, and to better understand the pathophysiology underlying PTB, the Preterm Birth International Collaborative Australia branch (PREBIC-AA) was launched in 2017. A series scientific activities including organizing annual research symposiums has been planned and organized among Australasian countries. Here we briefly updated the current progress in clinical management and translational research on PTB in Australasian countries that have been participated in PREBIC-AA.

Spatiotemporal regulation of human IFN-ε and innate immunity in the female reproductive tract.

Although published studies have demonstrated that IFN-ε has a crucial role in regulating protective immunity in the mouse female reproductive tract, expression and regulation of IFN-ε in the human female reproductive tract (hFRT) have not been characterized to our knowledge. We obtained hFRT samples from a well-characterized cohort of women to enable us to comprehensively assess ex vivo IFN-ε expression in the hFRT at various stages of the menstrual cycle. We found that among the various types of IFNs, IFN-ε was uniquely, selectively, and constitutively expressed in the hFRT epithelium. It had distinct expression patterns in the surface and glandular epithelia of the upper hFRT compared with basal layers of the stratified squamous epithelia of the lower hFRT. There was cyclical variation of IFN-ε expression in the endometrial epithelium of the upper hFRT and not in the distal FRT, consistent with selective endometrial expression of the progesterone receptor and regulation of the IFNE promoter by progesterone. Because we showed IFN-ε stimulated important protective IFN-regulated genes in FRT epithelium, this characterization is a key element in understanding the mechanisms of hormonal control of mucosal immunity.

Progesterone - Historical perspective.

Research leading to the discovery and characterization of progesterone (P4) began in the mid 1800 s and followed a path carved by key discoveries in the burgeoning field of endocrinology. The primary observations leading to the discovery of P4 was that the maternal corpus luteum (CL) is necessary for the establishment and maintenance of pregnancy. Experiments in animal models exploring the consequence of CL ablation and the effects of treatment with CL extract soon followed and formed the basis for the eventual isolation and characterization in 1930 s of the CL hormone, initially referred to as progestin, and subsequently named progesterone. In the following decades research into the physiology, biochemistry and molecular biology of P4 in the context of pregnancy provided fundamental insights into the hormonal control of pregnancy establishment, maintenance and termination. This review highlights the work of pioneering researchers and their seminal discoveries in the research history of P4.

Maternal-fetal conflict averted by progesterone- induced FOXP3+ regulatory T cells.

Pregnancy stimulates an intricately coordinated assortment of physiological changes to accommodate growth of the developing fetus, while simultaneously averting rejection of genetically foreign fetal cells and tissues. Despite increasing evidence that expansion of immune-suppressive maternal regulatory T cells enforces fetal tolerance and protects against pregnancy complications, the pregnancy-associated signals driving this essential adaptation remain poorly understood. Here we show that the female reproductive hormone, progesterone, coordinates immune tolerance by stimulating expansion of FOXP3+ regulatory T cells. Conditional loss of the canonical nuclear progesterone receptor in maternal FOXP3+ regulatory T cells blunts their proliferation and accumulation, which is associated with fetal wastage and decidual infiltration of activated CD8+ T cells. Reciprocally, the synthetic progestin 17α-hydroxyprogesterone caproate (17-OHPC) administered to pregnant mice reinforces fetal tolerance and protects against fetal wastage. These immune modulatory effects of progesterone that promote fetal tolerance establish a molecular link between immunological and other physiological adaptions during pregnancy.

The selective progesterone receptor modulator-promegestone-delays term parturition and prevents systemic inflammation-mediated preterm birth in mice.

BACKGROUND: Progesterone, acting via its nuclear receptors called progesterone receptors, promotes myometrial relaxation during pregnancy, and suspension of this activity triggers labor. We previously found that 20α-hydroxysteroid dehydrogenase causes a local withdrawal of progesterone in the term and preterm myometrium by converting the progesterone into an inactive form before it accesses the progesterone receptors. OBJECTIVE: We hypothesized that a selective progesterone receptor modulator called promegestone, which is not metabolized by 20α-hydroxysteroid dehydrogenase, would sustain progesterone receptor signaling and prevent/delay term labor and preterm labor in mice. STUDY DESIGN: In the term labor mouse model, promegestone (0.2 mg/dam) or a vehicle were administered subcutaneously in timed-pregnant CD-1 mice at gestational days 15, 16, and 17 (term gestational days, 19.5). In the inflammation preterm labor model, pregnant mice received promegestone or a vehicle on gestational days 15, 16, and 17, which was 24 hours before, immediately before, and 24 hours after systemic bacterial endotoxin (50 µg intraperitoneal; lipopolysaccharide group) or vehicle (saline) administration. The maternal and fetal tissues were collected on gestational day 16 6 hours after lipopolysaccharide±promegestone injection and at term gestational day 18.75. The protein levels of 10 cytokines were measured by multiplex immunoassay in maternal plasma and amniotic fluid. Myometrial, decidual, and placental messenger RNA levels of multiple cytokines and procontractile proteins were evaluated by real-time polymerase chain reaction and confirmed by immunoblotting. RESULTS: Promegestone prevented term labor and maintained mice pregnancy postterm >24 hours. The litter size and fetal weights were not different from the controls. Promegestone prevented systemic bacterial-endotoxin-induced preterm labor in 100% of the mice, blocked uterine contractions, significantly inhibited all systemic inflammation-induced myometrial cytokines, and partially inhibited decidual and placental inflammation. Promegestone did not prevent bacterial-endotoxin-induced fetal toxicity. CONCLUSION: Promegestone a selective progesterone receptor modulator that binds progesterone receptors with high affinity and is not metabolized by 20α-hydroxysteroid dehydrogenase could completely suppress term parturition and systemic bacterial-endotoxin-induced preterm birth in mice. We suggest that such selective progesterone receptor modulators may represent a potential therapeutic approach to the prevention of preterm labor in women at high risk of preterm birth.

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.

Pro-inflammatory signals induce 20α-HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal.

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α-HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection-induced) and associated activation of transcription factors: NF-кB and AP-1, common to term and preterm labour. We found that 20α-HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro-inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12-O-tetradecanoylphorbol-13-acetate (TPA, mimicking inflammation), induced 20α-HSD gene expression and increased 20α-HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up-regulation of GJA1 in the hTERT-HM cells. The NF-кB /AP-1 transcription factors mediated effects of LPS and TPA on 20α-HSD gene transcription. Both pro-inflammatory stimuli induced 20α-HSD promoter activity in LPS/TPA-treated cells which was significantly attenuated by inhibition of NF-кB (JSH: 20 µM) or AP-1 signalling (T5224: 10 µM). Deletion of NF-кB consensus sites abrogated LPS-mediated promoter induction, while removal of AP-1 sites reversed the TPA-mediated induction of 20α-HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF-кB or AP-1 induce 20α-HSD transcription and subsequent local P4 withdrawal resulting in up-regulation of GJA1 and activation of myometrium that precedes labour.

Blastocyst Vitrification and Trophectoderm Biopsy Cumulatively Alter Embryonic Gene Expression in a Mouse Model.

Although embryo vitrification has been used extensively in human assisted reproductive technology (ART) and animal models, epidemiologic evidence and randomized controlled trials suggest differences in pregnancy/perinatal outcomes (birthweight, risk for preterm birth, and pre-eclampsia) between babies born from fresh versus frozen embryo transfers. To address the uncertainty surrounding the effects of laboratory manipulations of embryos on clinical outcomes, we subjected mouse blastocysts to increasing levels of manipulation for transcriptome analysis. Blastocysts were randomly divided into four groups: no manipulation (control), single vitrification/thaw (1 vit), double vitrification/thaw (2 vit), and single vitrification/thaw plus trophectoderm biopsy and again vitrified/thawed (2 vit + bx). Three sets of 15 blastocysts in each group were pooled for RNA sequencing, and differentially expressed genes (DEGs) and pathways were determined by statistical analysis. Blastocysts were also stained for ZO-1 and F-actin to assess cytoskeletal integrity. Freeze/thaw and biopsy manipulation affected multiple biological pathways. The most significant differences were detected in genes related to innate immunity, apoptosis, and mitochondrial function, with the magnitude of change proportional to the extent to manipulation. Significant disruptions were also seen in cytoskeletal staining, with greater disruptions seen with greater of manipulation. Our data suggests that embryo vitrification and biopsy affect embryo gene transcription, with several identified DEGs that may have plausible mechanisms for the clinical outcomes seen in human offspring following ART. Further study is required to determine whether these alterations in gene expression are associated with clinical differences seen in children born from fresh or frozen embryo transfer.

Enhanced drug delivery to the reproductive tract using nanomedicine reveals therapeutic options for prevention of preterm birth.

Inflammation contributes to nearly 4 million global premature births annually. Here, we used a mouse model of intrauterine inflammation to test clinically used formulations, as well as engineered nanoformulations, for the prevention of preterm birth (PTB). We observed that neither systemic 17a-hydroxyprogesterone caproate (Makena) nor vaginal progesterone gel (Crinone) was sufficient to prevent inflammation-induced PTB, consistent with recent clinical trial failures. However, we found that vaginal delivery of mucoinert nanosuspensions of histone deacetylase (HDAC) inhibitors, in some cases with the addition of progesterone, prevented PTB and resulted in delivery of live pups exhibiting neurotypical development. In human myometrial cells in vitro, the P4/HDAC inhibitor combination both inhibited cell contractility and promoted the anti-inflammatory action of P4 by increasing progesterone receptor B stability. Here, we demonstrate the use of vaginally delivered drugs to prevent intrauterine inflammation-induced PTB resulting in the birth of live offspring in a preclinical animal model.

Superovulation with gonadotropin-releasing hormone agonist or chorionic gonadotropin for ovulation trigger differentially affects leukocyte populations in the peri-implantation mouse uterus.

OBJECTIVE: To investigate the effect of superovulation with human chorionic gonadotropin (hCG) or gonadotropin-releasing hormone agonist (GnRHa) trigger on leukocyte density and expression of leukocyte-specific genes in the peri-implantation period in the mouse uterus. DESIGN: Laboratory research. SETTING: University laboratory facility. INTERVENTIONS: Female mice were mated to fertile male mice in one of three protocols: (1) natural mating or mating following injection with pregnant mare serum gonadotropin followed by trigger with (2) GnRHa or (3) hCG. Female mice were killed prior to implantation, 3 days after ovulation (E3.5), and the ovaries and uterine tissue were collected. Total RNA was isolated and assayed using quantitative reverse transcription polymerase chain reaction, and the uterine tissue was stained for histologic analysis of immune cell markers. MAIN OUTCOME MEASURES: Endometrial leukocyte (CD45) and vessel density (CD31) by immunohistochemical staining; expression of leukocyte markers CD11b, CD335, and CD22, by quantitative reverse transcription polymerase chain reaction in the whole uterine tissue. RESULTS: Superovulation decreased (compared with controls) the endometrial leukocyte density, based on the number of cells staining for CD45, and endometrial vessel density, based on the number of cells staining for CD31. Leukocyte density was additionally decreased in the GnRHa trigger group compared with that in the hCG trigger group. Superovulation with hCG and GnRHa triggers decreased the uterine expression of the B-cell marker CD22 compared with controls. The expression of the natural killer cell marker CD11b was decreased by the hCG trigger but not by the GnRHa. Abundance of mRNA encoding the CD335 natural killer cell marker was not affected by superovulation or trigger agent. CONCLUSIONS: In mice, superovulation with the GnRHa trigger compared with that with the hCG trigger differentially alters key immunologic factors in the uterine peri-implantation. These altered immunologic factors have roles in angiogenesis that may assist in elucidating the effects of assisted reproductive technologies on implantation efficiency and fetal growth and development.

Developing a theoretical evolutionary framework to solve the mystery of parturition initiation.

Eutherian mammals have characteristic lengths of gestation that are key for reproductive success, but relatively little is known about the processes that determine the timing of parturition, the process of birth, and how they are coordinated with fetal developmental programs. This issue remains one of biology's great unsolved mysteries and has significant clinical relevance because preterm birth is the leading cause of infant and under 5 year old child mortality worldwide. Here, we consider the evolutionary influences and potential signaling mechanisms that maintain or end pregnancy in eutherian mammals and use this knowledge to formulate general theoretical evolutionary models. These models can be tested through evolutionary species comparisons, studies of experimental manipulation of gestation period and birth timing, and human clinical studies. Understanding how gestation time and parturition are determined will shed light on this fundamental biological process and improve human health through the development of therapies to prevent preterm birth.

Advancing human health in the decade ahead: pregnancy as a key window for discovery: A Burroughs Wellcome Fund Pregnancy Think Tank.

Recent revolutionary advances at the intersection of medicine, omics, data sciences, computing, epidemiology, and related technologies inspire us to ponder their impact on health. Their potential impact is particularly germane to the biology of pregnancy and perinatal medicine, where limited improvement in health outcomes for women and children has remained a global challenge. We assembled a group of experts to establish a Pregnancy Think Tank to discuss a broad spectrum of major gestational disorders and adverse pregnancy outcomes that affect maternal-infant lifelong health and should serve as targets for leveraging the many recent advances. This report reflects avenues for future effects that hold great potential in 3 major areas: developmental genomics, including the application of methodologies designed to bridge genotypes, physiology, and diseases, addressing vexing questions in early human development; gestational physiology, from immune tolerance to growth and the timing of parturition; and personalized and population medicine, focusing on amalgamating health record data and deep phenotypes to create broad knowledge that can be integrated into healthcare systems and drive discovery to address pregnancy-related disease and promote general health. We propose a series of questions reflecting development, systems biology, diseases, clinical approaches and tools, and population health, and a call for scientific action. Clearly, transdisciplinary science must advance and accelerate to address adverse pregnancy outcomes. Disciplines not traditionally involved in the reproductive sciences, such as computer science, engineering, mathematics, and pharmacology, should be engaged at the study design phase to optimize the information gathered and to identify and further evaluate potentially actionable therapeutic targets. Information sources should include noninvasive personalized sensors and monitors, alongside instructive "liquid biopsies" for noninvasive pregnancy assessment. Future research should also address the diversity of human cohorts in terms of geography, racial and ethnic distributions, and social and health disparities. Modern technologies, for both data-gathering and data-analyzing, make this possible at a scale that was previously unachievable. Finally, the psychosocial and economic environment in which pregnancy takes place must be considered to promote the health and wellness of communities worldwide.