Establishment Of Pregnancy Research Articles

Single-cell sequencing analysis of the maternal–fetal interface

The microenvironment at the maternal–fetal interface is optimized to facilitate the development and survival of the fetus during pregnancy. It involves a balance between cell populations and interactions of the fetal placenta with various cell types (ie, stromal cells, endothelial cells, immune cells, and fibroblasts) that are embedded in the maternal endometrium/decidua. Aberrant shifts in cell populations and deranged cell–cell interactions are closely related to pregnancy disorders. Thus, analysis of the dynamic changes in cell populations and their interactions at the maternal–fetal interface in normal and complicated pregnancies is essential to provide insights into the fundamental processes involved in the establishment and maintenance of normal pregnancy, and how these processes are dysregulated. Thus, informing novel pathways for therapeutic targets of pregnancy complications. Single-cell sequencing (SCS) is a powerful tool for transcriptome analysis at single-cell resolution. Combined with information on the developmental trajectory and function of different cell populations, SCS can provide an unparalleled opportunity for refining the spatiotemporal cell atlas to elaborate dynamic changes in cell populations and their interactions in tissues that consist of highly heterogeneous cell populations such as the maternal–fetal interface. This minireview briefly summarizes traditional methods and their limitations for analyzing maternal–fetal interface cell–cell interactions, and introduces the current applications, advantages, limitations, and prospective applications of SCS in research on maternal–fetal interactions.

221 Putative Gene Regulators in the Equine Endometrium, Corpus Luteum and Embryo During Early Pregnancy in the Horse

Abstract It is estimated that 17.3% of all pregnancies in the horse are lost from the time of detection to day 40, which has significant economic impact. But still, equine reproductive research has limited funding opportunities. Understanding the dynamics between the embryo, endometrium, and corpus luteum (CL) is essential to ameliorate those losses. Pig embryonic interferon (IFN) gamma is essential for the establishment of pregnancy. In ruminants, IFN tau is the maternal recognition signal. The equine embryo seems to express IFN alpha, gamma, delta, and epsilon but higher expression was only identified on day(D) 16 embryos, after the maternal recognition of pregnancy (MRP), so the IFN role during pregnancy is unknown. Oxytocin and prostaglandins have been demonstrated to have a role during luteolysis and luteostasis in the horse, and therefore investigation the embryonic, endometrium, and CL putative gene regulators during the equine MRP is warranted. Oxytocinase (LNPEP), an aminopeptidase that metabolizes oxytocin (OXT) has been recently investigated with other putative gene regulators in the horse during the expected time of MRP. OXT and LNPEPmRNA were identified in the equine embryo, endometrium, and CL, but OXT/neurophysin-1 protein was not detected in the embryo, and present in the CL. Most of the endometrial and luteal mRNA differences were identified around D12 post-ovulation, suggesting being a critical time for MRP. Luteal OXT was not different between pregnant and non-pregnant mares. LNPEP mRNA was upregulated in pregnant and non-pregnant mares D10 and 12 luteal tissues along with prostaglandin E synthase (PTGES). Embryonic trophoblast LNPEP and PTGES have higher relative abundance on D15 conceptus compared earlier embryos, suggesting a potential role during embryonic fixation. The increased expression of LNPEP is suggested to have a role in decreasing oxytocin availability and shifting the ratio PGE:PGF to a PGE that will support luteal function during early pregnancy.

PSVIII-B-13 Machine Learning-Based co-Expression Network Analysis Unravels Fertility-Related Genes in Beef Cattle

Abstract Reproductive efficiency is a critical component of a sustainable cow-calf system. While several studies have identified factors underlying fertility, the genetic mechanisms contributing to this complex trait are still unclear. Our goal was to identify a set of predictive biomarker signatures from transcriptomics data associated with female cattle fertility. We implemented a multi-tiered approach using machine learning (ML) feature selection, gene co-expression network, and functional analysis. To this end, we retrieved public data from the Gene Expression Omnibus database (GEO GSE171577). The RNA-Seq data was generated from uterine luminal epithelial cells of recipient cows sampled on day four before embryo transfer. The data (n = 18 non-pregnant – NP and n = 25 pregnant – P) were analyzed using a standard pipeline based on FastQC, MultiQC, STAR, and DESeq2. Genes with expression values > 0.5 counts per million in 50% of the samples were filtered out. Feature and model selection was implemented through BioDiscML. Further, the PCIT algorithm was used to create gene co-expression networks from 15,039 genes kept after quality control. Our ML approach identified nine genes as predictors of pregnancy status. The genes included: SERPINE3, MRTFA, MEF2B, NAA16, ARHGEF7, PDCD1, FNDC1, ENSBTAG00000054585, and ENSBTAG00000019474. The networks from P and NP cows resulted in five and four thousand significantly co-expressed gene pairs, respectively. We then kept 1,837 pairs with a |r| > 0.7 and were co-expressed with the gene predictors from the ML analysis. Biological processes, such as vasculature development, oxidative phosphorylation, and focal adhesion were over-represented by genes from the P network. We identified immune system development, negative regulation of the biological process, and protein modification over-represented processes in the NP gene network. We have demonstrated the potential of combining different methods to identify fertility-related biomarkers and have provided insights into the complex genomic basis underlying pregnancy establishment in cattle.

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species. However, the temporal changes in maternal vitamin D status throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated. The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (n = 5–11 gilts/GD). Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (P < 0.05). The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight, ovulation rate, mean litter weight, number of live foetuses, percentage prenatal survival, and sex ratio of the litter was assessed. Maternal 25(OH)D (P = 0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (P < 0.05) concentrations were inversely associated with gilt weight. Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90 (P < 0.05). This study has provided novel insights into temporal changes in maternal vitamin D status throughout gestation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight, percentage prenatal survival and percentage of male foetuses in the litter. Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.

Expression of Key Steroidogenic Enzymes in Human Placenta and Associated Adverse Pregnancy Outcomes

Abstract Steroid hormones, including progestagens, estrogens, androgens, corticosteroids, and their precursor cholesterol, perform essential functions in the successful establishment and maintenance of pregnancy and normal fetal development. As the core endocrine organ at the prenatal stage, the human placenta is involved in the biosynthesis, metabolism, and delivery of steroid hormones. Steroidogenic pathways are tightly regulated by placenta-intrinsic cytochrome P450 and hydroxysteroid dehydrogenase. However, the relationship between placental steroidogenic enzyme expression and adverse pregnancy outcomes is controversial. In this review, we summarize the possible upstream regulatory mechanisms of placental steroidogenic enzymes in physiologic and pathophysiologic states. We also describe the human placental barrier model and examine the potential of single-cell sequencing for evaluating the primary functions and cellular origin of steroidogenic enzymes. Finally, we examine the existing evidence for the association between placental steroidogenic enzyme dysregulation and adverse pregnancy outcomes.

Insulin-like growth factor system components expressed at the conceptus-maternal interface during the establishment of equine pregnancy.

In many species, the insulin-like growth factors (IGF1 and IGF2), their receptors and IGF binding proteins play important roles in preparing the endometrium for implantation, and regulating conceptus growth and development. To determine whether the IGF system may contribute to conceptus-maternal interaction during equine pre-implantation development, we evaluated mRNA expression for IGF system components in conceptuses, and endometrium recovered from pregnant and cycling mares, on days 7, 14, 21 and 28 after ovulation. We also investigated expression of IGF1, IGF2 and their receptors 6 and 11 days after transfer of day 8 embryos to synchronous (day 8) or asynchronous (day 3) recipient mares. Expression of IGF1 and IGF2, IGF1R, IGF2R, INSR and IGFBPs 1, 2, 4 and 5 was evident in endometrium and conceptus membranes during days 7–28. Endometrial IGF2, INSR, IGFBP1 and IGFBP2 expression increased between days 7 and 28 of pregnancy. In conceptus membranes, expression of all IGF system components increased with developmental stage. Immunohistochemistry revealed strong expression of IGF1, IGF2 and IGF1R in both endometrium and conceptus membranes, whereas INSR was highly expressed in endometrium but barely detectable in the conceptus. Finally, a negatively asynchronous uterine environment retarded IGF1, IGF2 and INSR expression in the conceptus, whereas in the endometrium only INSR expression was altered by asynchrony. The presence of IGFs, their receptors and IGFBPs in the endometrium and conceptus during early equine pregnancy, and down-regulation in the conceptus following asynchronous embryo transfer, suggest a role in conceptus-maternal communication during the preparation for implantation.

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.

OP-6 - NLRP3 regulates inflammatory response in the uterus by seminal plasma and induces establishment of pregnancy in mice

An appropriate immune response by seminal plasma (SP) is essential for a successful pregnancy and is regarded as a sterile inflammatory response. In contrast, the overactivation of NLRP3 inflammasome is involved in pregnancy complications. We investigated whether NLRP3 inflammasome regulates SP-induced immune response for establishment of pregnancy. The pregnancy continuation rate of wild-type (WT) female mice mated with NLRP3-knockout (KO) male mice was lower, and immune response in their uterus were higher than mated with WT male mice. SP of WT or NLRP3-KO mice was injected into the uterus of mice. The implantation numbers were found to decrease in NLRP3-KO-SP group compared with the WT-SP group. Also, cytokine array analysis showed that most cytokine levels of NLRP3-KO-SP were lower than those of WT-SP. In murine macrophages, treatment with WT-SP increased mRNA expression of inflammatory cytokines and protein expression of macrophage differentiation markers, but not in treatment with NLRP3-KO-SP, suggesting that regulatory capacities of SP differed between WT and NLRP3-KO mice. Moreover, NLRP3 protein was detected in WT-SP, but not in NLRP3-KO-SP. In murine macrophages, treatment with NLRP3 protein increased protein expression of M2 type marker. These results suggest that NLRP3 plays an important role in regulating postcoitus immune response for establishment of pregnancy.

Post-mating diclofenac vs. carprofen treatment on serum progesterone levels and reproductive outcomes in Hungarian-Merino ewes during the non-breeding season.

The maternal recognition process is crucial for the establishment of healthy pregnancy. In this process, anti-luteolytic applications are one of the main reproductive strategies to manage the embryonic losses and maximize reproductive profitability in farm animals. The aim of this study was to investigate the efficacy of post-mating NSAID treatments on reproductive parameters (pregnancy rate, lambing rate, multiple birth rate, litter size) and serum progesterone levels in ewes stimulated with progesterone non-breeding season. For this purpose, two different experiments (diclofenac and carprofen) were conducted in the same ewe flock induced with short-term progestogen-based protocol in the non-breeding season for two consecutive years. In experiment 1 (n=85), 42 ewes were injected with 2.5mg/kg diclofenac on the 9th and 10th days post-mating, and the rest were not treated and served as control. In experiment 2 (n=82), 40 ewes were injected with 1.4mg/kg carprofen on the 9th days post-mating, and the rest were not treated as control. In both experiments, blood samples were collected from all ewes on days 9, 12 and 13 post-mating to measure serum progesterone levels. In both experiments, there were no differences both reproductive parameters and serum progesterone levels when compared to the control groups. It was concluded that post-mating diclofenac and carprofen treatments in the critical period have no significant effects on both reproductive parameters and serum progesterone levels in ewes in the non-breeding season.

Infertility and the Endometrium

Background: A couple’s infertility can originate from the male and/or the female. In women, the uterus provides the site where the maternal-fetal interface is established and maintained. Final blastocyst development occurs within the uterine cavity, then the blastocyst must attach to and implant into the endometrium (the inner uterine surface), via its outermost trophectodermal cells. Beneath the epithelium, these differentiate into syncytial trophoblast and invasive trophoblast — the latter progress through the endometrium to invade the spiral arteries converting them to the flaccid blood sacs of the placenta. Therefore, the endometrium plays a critical role in establishment of pregnancy. Objectives: To critically examine current knowledge of endometrial preparation for blastocyst implantation and placental development at the cellular and molecular level and to evaluate measures to improve implantation success. Mechanism: Literature searching by leading experts in the field. Findings: A wealth of new knowledge resulting from ‘omics’ technologies and new functional models has greatly enhanced our knowledge, but this information is yet to be translated into enhanced outcomes. Conclusions: The endometrium remains the ‘black box’ of infertility. Extensive trials do not support current adjuvant therapies as being better than placebo while effectively timed testing for endometrial preparedness for implantation is still urgently needed.

Inflammation-Related Molecules at the Maternal-Fetal Interface during Pregnancy and in Pathologically Altered Endometrium.

The blastocyst expresses paternally derived alloantigens and induces inflammation during implantation. However, it is necessary for the onset of pregnancy. An abnormal response might result in a pathological course of pregnancy or pregnancy failure. On the other hand, a state of maternal immune tolerance is necessary to ensure the normal development of pregnancy by suppressing inflammatory processes. This article discusses recognized mechanisms and the significance of inflammatory processes for embryo implantation and pregnancy establishment. We would also like to present disorders involving excessive inflammatory response and their influence on events occurring during embryo implantation. The chain of correlation between the processes responsible for embryo implantation and the subsequent physiological course of pregnancy is complicated. Many of those interrelationships are still yet to be discovered. Undoubtedly, their recognition will give hope to infertile couples for the emergence of new treatments that will increase the chance of giving birth to a healthy child.

Bedeutung natürlicher Killerzellen in der Frühschwangerschaft

BackgroundNatural killer (NK) cells play a pivotal role in the establishment of a successful pregnancy. They are found in peripheral blood (peripheral NK cells) as well as in the endometrium and decidua (uterine NK cells).ObjectiveThis article aims to provide an overview of the origin, function, and diagnostics of NK cells, as well as to highlight possible therapeutic options.ResultsDifferent types of NK cells differ significantly in their cytotoxicity. About 90% of uterine NK cells show low cytotoxicity with high cytokine production, whereas peripheral NK cells show high cytotoxicity and low cytokine production. Elevated uterine and peripheral NK cells are associated with miscarriage, hypertension, preeclampsia, and fetal growth restriction.ConclusionA detailed understanding of the various mechanisms of implantation and (early) pregnancy is paramount to improving the success rates of reproductive interventions, as well as to prevent pregnancy complications. There is a growing number of studies on immunomodulatory therapies for NK cell changes investigating, e.g., corticosteroids, lipid infusions, and immunoglobulins. However, the studies are heterogeneous in design, methodology, intervention, and study population, which makes not only comparison and interpretation difficult, but also the development of evidence-based therapeutic strategies.

Prospectively assessed perceived stress associated with early pregnancy losses among women with history of pregnancy loss.

What is the association between perceived stress during peri-conception and early pregnancy and pregnancy loss among women who have experienced a prior pregnancy loss? Daily perceived stress above the median is associated with over a 2-fold risk of early pregnancy loss among women who have experienced a prior loss. Women who have experienced a pregnancy loss may be more vulnerable to stress while trying to become pregnant again. While prior research has indicated a link between psychological stress and clinically confirmed miscarriages, research is lacking among a pre-conceptional cohort followed prospectively for the effects of perceived stress during early critical windows of pregnancy establishment on risk of both hCG-detected pregnancy losses and confirmed losses, while considering important time-varying confounders. Secondary data analysis of the EAGeR trial (2007-2011) among women with an hCG-detected pregnancy (n = 797 women). Women from four US clinical centers enrolled pre-conceptionally and were followed ≤6 cycles while attempting pregnancy and, as applicable, throughout pregnancy. Perceived stress was captured via daily diaries and end-of-month questionnaires. Main outcome measures include hCG-detected and clinically recognized pregnancy losses. Among women who had an hCG-confirmed pregnancy, 188 pregnancies (23.6%) ended in loss. Women with high (>50th percentile) versus low (≤50th percentile) peri-implantation or early pregnancy weekly perceived stress had an elevated risk of experiencing any pregnancy loss (hazard ratio (HR): 1.69, 95% CI: 1.13, 2.54) or clinical loss (HR: 1.58, 95% CI: 0.96, 2.60), with higher risks observed for women experiencing an hCG-detected loss (HR: 2.16, 95% CI: 1.04, 4.46). Models accounted for women's age, BMI, employment, marital status, income, education, race, parity, prior losses, exercise and time-varying nausea/vomiting, caffeine, alcohol and smoking. We were limited in our ability to clearly identify the mechanisms of stress on pregnancy loss due to our sole reliance on self-reported perceived stress, and the lack of biomarkers of different pathways of stress. This study provides new insight on early pregnancy perceived stress and risk of pregnancy loss, most notably hCG-detected losses, among women with a history of a prior loss. Our study is an improvement over past studies in its ability to account for time-varying early pregnancy symptoms, such as nausea/vomiting, and lifestyle factors, such as caffeine, alcohol and smoking, which are also risk factors for psychological stress and pregnancy loss. This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (Contract numbers: HHSN267200603423, HHSN267200603424, HHSN267200603426, HHSN275201300023I). Additionally, K.C.S. was supported by the National Institute on Aging of the National Institutes of Health under Award Number K01AG058781. The authors have no conflicts of interest to disclose. #NCT00467363.

Downregulation of krüppel-like factor 6 expression modulates extravillous trophoblast cell behavior by increasing reactive oxygen species

IntroductionPlacental extravillous trophoblasts play a crucial role in the establishment of a healthy pregnancy. Reactive oxygen species (ROS) may contribute to their differentiation and function as mediators in signaling processes or might cause oxidative stress resulting in trophoblast dysfunction. The krüppel-like transcription factor 6 (KLF6) regulates many genes involved in essential cell processes where ROS are also involved. However, whether KLF6 regulates ROS levels has not been previously investigated. Materials and MethodsKLF6 was silenced by siRNAs in HTR8-SV/neo cells, an extravillous trophoblast model. Total and mitochondrial ROS levels, as well as mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. The expression of genes and proteins of interest were analyzed by qRT-PCR and Western blot, respectively. Cell response to oxidative stress, proliferation, viability, morphology, and migration were evaluated. ResultsKLF6 downregulation led to an increase in ROS and NOX4 mRNA levels, accompanied by reduced cell proliferation and increased p21 protein expression. Catalase activity, 2-Cys peroxiredoxin protein levels, Nrf2 cytoplasmic localization and hemoxygenase 1 expression, as well as mitochondrial membrane potential and cell apoptosis were not altered suggesting that ROS increase is not associated with cellular damage. Instead, KLF6 silencing induced cytoskeleton modifications and increased cell migration in a ROS-dependent manner. DiscussionPresent data reveal a novel role of KLF6 on ROS balance and signaling demonstrating that KLF6 downregulation induces an increase in ROS levels that contribute to extravillous trophoblast cell migration.

Study of progesterone level during first trimester of pregnancy in cows.

Measurement of progesterone is an indirect method for pregnancy diagnosis in many livestock species including cattle, buffaloes, sheep, and goats. Conception extends the life of the corpus luteum (CL) by preventing the luteolytic mechanism from being triggered, thus prolonging and maintaining its functional characteristics, ensuring continued high progesterone levels (Spencer, et.al., 1995). (Shemesh et al., 1973) proposed that the difference in peripheral plasma progesterone levels between pregnant and non pregnant cows, 19 days after insemination, can form the basis for a very early pregnancy test. (Laing and Heap 1972) first documented this in milk to diagnose cows in early pregnancy Progesterone maintains the uterine endometrium in a state which supports embryonic development, implantation, and foetoplacental development. Progesterone concentrations vary with the stage of the estrous cycle which makes it one of the most commonly studied reproductive hormones in bovine ruminants for pregnancy detection and ovarian activity (Kaneko,et.al.,1994). Studies in the bovine estrous cycle indicate that the milk or serum progesterone concentrations reach a maximum value 13-14 days after estrus, and if the animal is pregnant, these continue to remain elevated up to day 21 after fertilization (Parkinson, et al.,1994) . These high levels of progesterone in serum or milk between days 18 and 24 after insemination form the basis of establishment of pregnancy in cattle (Sasser, and Ruder,1987; Shemesh,1973). Interferon-???? exerts its antiluteolytic effect by inhibiting the endometrial expression of oxytocin receptors, through which oxytocin stimulates pulsatile PGF2???? release (Wolf,2003). Although low progesterone concentrations at 18 to 24 days after breeding can accurately predict non pregnancy, high progesterone concentrations during this period are not the specific indicators of pregnancy due to variations among cows in duration of the estrous cycle as well as the incidence of early or late embryonic mortality. The advantages of progesterone assay for pregnancy diagnosis include noninvasive collection of milk sample and the feasibility to conduct the test on the farm using commercial cow-side milk progesterone test kits [Pennington,1985 ; Gowan,1982 ; Wimpy,et.al.,1986), though the sensitivity gets compromised to some extent with these assay kits.&#x0D; Key Word: progesterone, pregnancy, cows

Low-Dose Tacrolimus Promotes the Migration and Invasion and Nitric Oxide Production in the Human-Derived First Trimester Extravillous Trophoblast Cells In Vitro.

Placentation is one of the most important determinants for a successful pregnancy, and this is dependent on the process of trophoblast migration and invasion. Progesterone receptors (PGR) are critical effectors of progesterone (P4) signaling that is required for trophoblast migration and invasion conducive to a successful gestation. In immune complicated pregnancies, evidence has shown that abnormal placentation occurs because of aberrant expression of PGR. Therapeutic intervention with tacrolimus (FK506) was able to restore PGR expression and improve pregnancy outcomes in immune-complicated gestations; however, the exact mode of action of tacrolimus in assisting placentation is not clear. Here, we attempt to uncover the mode of action of tacrolimus by examining its effects on trophoblast invasion and migration in the human-derived extravillous trophoblast (EVT) cell line, the HTR-8/SVneo cells. Using a variety of functional assays, we demonstrated that low-dose tacrolimus (10 ng/mL) was sufficient to significantly (p < 0.001) stimulate the migration and invasion of the HTR-8/SVneo cells, inducing their cytosolic/nuclear progesterone receptor expression and activation, and modulating their Nitric Oxide (NO) production. Moreover, tacrolimus abrogated the suppressive effect of the NOS inhibitor Nω- Nitro-L-Arginine Methyl Ester (L-NAME) on these vital processes critically involved in the establishment of human pregnancy. Collectively, our data suggest an immune-independent mode of action of tacrolimus in positively influencing placentation in complicated gestations, at least in part, through promoting the migration and invasion of the first trimester extravillous trophoblast cells by modulating their NO production and activating their cytosolic/nuclear progesterone-receptors. To our knowledge, this is the first report to show that the mode of action of tacrolimus as a monotherapy for implantation failure is plausibly PGR-dependent.

Reference gene selection in bovine caruncular epithelial cells under pregnancy-associated hormones exposure

Examination of transcriptional regulation occurring during pregnancy establishment and maintenance requires the identification of endogenous reference genes characterized by high expression stability. Since the expression of some reference genes may be modulated by pregnancy-associated hormones, the goal of our study was to identify suitable reference genes unaffected by hormonal treatment. In our study bovine caruncular epithelial cells were subjected to progesterone, estrogen and prostaglandin F2α treatment. Ten candidate reference genes (ACTR1A, CNOT11, HDAC1, HPRT1, RPL19, RPS9, SDHA, SUZ12, UXT and ZNF131) were evaluated with the use of four approaches (geNorm, NormFinder, BestKeeper, delta Ct). We found that RPS9 and SUZ12 displayed the highest expression stability in the tested material. Moreover, HPRT1 and SDHA were found inappropriate for RT-qPCR data normalization as they demonstrated the highest expression variability out of all candidates analysed. Hence geNorm calculations shown that the use of just two best-performing genes would be sufficient for obtaining reliable results, we propose that RPS9 and SUZ12 be used as suitable endogenous controls in future studies investigating gene expression in normal and compromised pregnancies.

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.

Prss29 Cre recombinase mice are useful to study adult uterine gland function.

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

Impact of endometrial claudin-3 deletion on murine implantation, decidualization, and embryo development†.

The composition of cell contacts in the endometrium plays an important role in the process of embryo implantation and the establishment of pregnancy. In previous studies, we showed an induction of the tight junction protein claudin-3 in the developing decidua from day 6.5 of pregnancy onward. To evaluate the role of this specific claudin-3 distribution, we here evaluated the effect of an endometrial claudin-3 deletion in implantation and embryo development in claudin-3 knockout mice. Claudin-3 knockout mice were fertile but revealed a slightly reduced amount of implantation sites as well as of litter size. Though implantation sites showed morphologically regularly developed embryos and deciduas, depth of ectoplacental cone invasion was reduced in tendency compared to controls. The weight of the implantation sites on day 6.5 and 8.5 of pregnancy as well as the weight of the embryos on day 17.5 of pregnancy, but not of the placentas, was significantly reduced in claudin-3 knockout mice due to a maternal effect. This could be due to an impairment of decidualization as substantiated by a downregulation of the transcription of various decidua-associated genes in the early implantation sites of claudin-3 knockout mice. The fact that claudin-3 knockout mice are nevertheless fertile possibly may be compensated by the presence of other claudins like claudin-4 and claudin-10.