A dose-response study on functional and transcriptomic effects of FSH on ex vivo mouse folliculogenesis.

Follicle-stimulating hormone (FSH) binds to its membrane receptor (FSHR) in granulosa cells to activate various signal transduction pathways and drive the gonadotropin-dependent phase of folliculogenesis. Poor female reproductive outcomes can result from both FSH insufficiency owing to genetic or non-genetic factors and FSH excess as encountered with ovarian stimulation in assisted reproductive technology (ART), but the underlying molecular mechanisms remain elusive. Herein, we conducted single-follicle and single-oocyte RNA sequencing analysis along with other approaches in an ex vivo mouse folliculogenesis and oogenesis system to investigate the effects of different concentrations of FSH on key follicular events. Our study revealed that a minimum FSH threshold is required for follicle maturation into the high estradiol-secreting preovulatory stage, and such threshold is moderately variable among individual follicles between 5-10 mIU/mL. FSH at 5, 10, 20, and 30 mIU/mL induced distinct expression patterns of follicle maturation-related genes, follicular transcriptomics, and follicular cAMP levels. RNA-seq analysis identified FSH-stimulated activation of G proteins and downstream canonical and novel signaling pathways that may critically regulate follicle maturation, including the cAMP/PKA/CREB, PI3K-AKT/FOXO1, and glycolysis pathways. High FSH at 20 and 30 mIU/mL resulted in non-canonical FSH responses including premature luteinization, high production of androgen and proinflammatory factors, and reduced expression of energy metabolism-related genes in oocytes. Together, this study improves our understanding of gonadotropin-dependent folliculogenesis and provides crucial insights into how high doses of FSH used in ART may impact follicular health, oocyte quality, pregnancy outcome, and systemic health.

Mechanisms of endometrial aging: lessons from natural conceptions and assisted reproductive technology cycles.

Until recently, the study of age-related decline in fertility has focused primarily on the ovary; depletion of the finite pool of oocytes and increases in meiotic errors leading to oocyte aneuploidy are well-established mechanisms by which fertility declines with advancing age. Comparatively little is known about the impact of age on endometrial function. The endometrium is a complex tissue comprised of many cell types, including epithelial, stromal, vascular, immune and stem cells. The capacity of this tissue for rapid, cyclic regeneration is unique to this tissue, undergoing repeated cycles of growth and shedding (in the absence of an embryo) in response to ovarian hormones. Furthermore, the endometrium has been shown to be capable of supporting pregnancies beyond the established boundaries of the reproductive lifespan. Despite its longevity, molecular studies have established age-related changes in individual cell populations within the endometrium. Human clinical studies have attempted to isolate the effect of aging on the endometrium by analyzing pregnancies conceived with euploid, high quality embryos. In this review, we explore the existing literature on endometrial aging and its impact on pregnancy outcomes. We begin with an overview of the principles of endometrial physiology and function. We then explore the mechanisms behind endometrial aging in its individual cellular compartments. Finally, we highlight lessons about endometrial aging gleaned from rodent and human clinical studies and propose opportunities for future study to better understand the contribution of the endometrium to age-related decline in fertility.

The impact of ovarian stimulation on the human endometrial microenvironment.

STUDY QUESTION: How does ovarian stimulation (OS), which is used to mature multiple oocytes for ART procedures, impact the principal cellular compartments and transcriptome of the human endometrium in the periovulatory and mid-secretory phases? SUMMARY ANSWER: During the mid-secretory window of implantation, OS alters the abundance of endometrial immune cells, whereas during the periovulatory period, OS substantially changes the endometrial transcriptome and impacts both endometrial glandular and immune cells. WHAT IS KNOWN ALREADY: Pregnancies conceived in an OS cycle are at risk of complications reflective of abnormal placentation and placental function. OS can alter endometrial gene expression and immune cell populations. How OS impacts the glandular, stromal, immune, and vascular compartments of the endometrium, in the periovulatory period as compared to the window of implantation, is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study carried out between 2020 and 2022 included 25 subjects undergoing OS and 25 subjects in natural menstrual cycles. Endometrial biopsies were performed in the proliferative, periovulatory, and mid-secretory phases. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were processed to determine serum estradiol and progesterone levels. Both the endometrial transcriptome and the principal cellular compartments of the endometrium, including glands, stroma, immune, and vasculature, were evaluated by examining endometrial dating, differential gene expression, protein expression, cell populations, and the three-dimensional structure in endometrial tissue. Mann-Whitney U tests, unpaired t-tests or one-way ANOVA and pairwise multiple comparison tests were used to statistically evaluate differences. MAIN RESULTS AND THE ROLE OF CHANCE: In the periovulatory period, OS induced high levels of differential gene expression, glandular-stromal dyssynchrony, and an increase in both glandular epithelial volume and the frequency of endometrial monocytes/macrophages. In the window of implantation during the mid-secretory phase, OS induced changes in endometrial immune cells, with a greater frequency of B cells and a lower frequency of CD4 effector T cells. LARGE SCALE DATA: The data underlying this article have been uploaded to the Genome Expression Omnibus/National Center for Biotechnology Information with accession number GSE220044. LIMITATIONS, REASONS FOR CAUTION: A limited number of subjects were included in this study, although the subjects within each group, natural cycle or OS, were homogenous in their clinical characteristics. The number of subjects utilized was sufficient to identify significant differences; however, with a larger number of subjects and additional power, we may detect additional differences. Another limitation of the study is that proliferative phase biopsies were collected in natural cycles, but not in OS cycles. Given that the OS cycle subjects did not have known endometrial factor infertility, and the comparisons involved subjects who had a similar and robust response to stimulation, the findings are generalizable to women with a normal response to OS. WIDER IMPLICATIONS OF THE FINDINGS: OS substantially altered the periovulatory phase endometrium, with fewer transcriptomic and cell type-specific changes in the mid-secretory phase. Our findings show that after OS, the endometrial microenvironment in the window of implantation possesses many more similarities to that of a natural cycle than does the periovulatory endometrium. Further investigation of the immune compartment and the functional significance of this cellular compartment under OS conditions is warranted. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (R01AI148695 to A.M.B. and N.C.D.), Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD109152 to R.A.), and the March of Dimes (5-FY20-209 to R.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or March of Dimes. All authors declare no conflict of interest.

Vascular changes in the cycling and early pregnant uterus.

Uterine vascular remodeling is intrinsic to the cycling and early pregnant endometrium. Maternal regulatory factors such as ovarian hormones, VEGF, angiopoietins, Notch, and uterine natural killer cells significantly mediate these vascular changes. In the absence of pregnancy, changes in uterine vessel morphology and function correlate with different stages of the human menstrual cycle. During early pregnancy, vascular remodeling in rodents and humans results in decreased uterine vascular resistance and increased vascular permeability necessary for pregnancy success. Aberrations in these adaptive vascular processes contribute to increased risk of infertility, abnormal fetal growth, and/or preeclampsia. This Review comprehensively summarizes uterine vascular remodeling in the human menstrual cycle, and in the peri- and post-implantation stages in rodent species (mice and rats).

Mesenchymal stem cell secretome alters gene expression and upregulates motility of human endometrial stromal cells.

In brief: Endometrial stromal cell motility is fundamental to regeneration and repair of this tissue and crucial for successful reproduction. This paper shows a role for the mesenchymal stem cell (MSC) secretome in enhancing endometrial stromal cell motility. Abstract: Cyclic regeneration and repair of the endometrium are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) and umbilical cord (UC-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, mechanisms remain unclear. This study tested the hypothesis that the BM-MSC and UC-MSC secretomes upregulate human endometrial stromal cell (HESC) proliferation, migration, and invasion and activate pathways to increase HESC motility. BM-MSCs were purchased from ATCC and cultured from the BM aspirate of three healthy female donors. UC-MSCs were cultured from umbilical cords of two healthy male term infants. Using indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system, we demonstrated that co-culture of HESCs with BM-MSCs or UC-MSCs from all donors significantly increased HESC migration and invasion, whereas effects on HESC proliferation varied among BM-MSC and UC-MSC donors. Analysis of gene expression by mRNA sequencing and RT-qPCR showed that expression of CCL2 and HGF was upregulated in HESCs that had been cocultured with BM-MSCs or UC-MSCs. Validation studies revealed that exposure to recombinant CCL2 for 48 h significantly increased HESC migration and invasion. Increased HESC motility by the BM-MSC and UC-MSC secretome appears to be mediated in part by upregulated HESC CCL2 expression. Our data support the potential for leveraging MSC secretome as a novel cell-free therapy to treat disorders of endometrial regeneration.

Maternal IL-33 critically regulates tissue remodeling and type 2 immune responses in the uterus during early pregnancy in mice.

The pregnant uterus is an immunologically rich organ, with dynamic changes in the inflammatory milieu and immune cell function underlying key stages of pregnancy. Recent studies have implicated dysregulated expression of the interleukin-1 (IL-1) family cytokine, IL-33, and its receptor, ST2, in poor pregnancy outcomes in women, including recurrent pregnancy loss, preeclampsia, and preterm labor. How IL-33 supports pregnancy progression in vivo is not well understood. Here, we demonstrate that maternal IL-33 signaling critically regulates uterine tissue remodeling and immune cell function during early pregnancy in mice. IL-33-deficient dams exhibit defects in implantation chamber formation and decidualization, and abnormal vascular remodeling during early pregnancy. These defects coincide with delays in early embryogenesis, increased resorptions, and impaired fetal and placental growth by late pregnancy. At a cellular level, myometrial fibroblasts, and decidual endothelial and stromal cells, are the main IL-33+ cell types in the uterus during decidualization and early placentation, whereas ST2 is expressed by uterine immune populations associated with type 2 immune responses, including ILC2s, Tregs, CD4+ T cells, M2- and cDC2-like myeloid cells, and mast cells. Early pregnancy defects in IL-33-deficient dams are associated with impaired type 2 cytokine responses by uterine lymphocytes and fewer Arginase-1+ macrophages in the uterine microenvironment. Collectively, our data highlight a regulatory network, involving crosstalk between IL-33-producing nonimmune cells and ST2+ immune cells at the maternal-fetal interface, that critically supports pregnancy progression in mice. This work has the potential to advance our understanding of how IL-33 signaling may support optimal pregnancy outcomes in women.

Gamma Secretase Inhibitors as Potential Therapeutic Targets for Notch Signaling in Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (uLMS) is a rare and aggressive cancer with few effective therapeutics. The Notch signaling pathway is evolutionarily conserved with oncogenic properties, but it has not been well studied in uLMS. The purpose of our study was to determine expression of Notch family genes and proteins and to investigate the therapeutic effect of γ-secretase inhibitors (GSIs), indirect inhibitors of Notch signaling, in uLMS. We determined expression of Notch genes and proteins in benign uterine smooth muscle tissue, fibroids, and uLMS samples by immunostaining and in two uLMS cell lines, SK-UT-1B (uterine primary) and SK-LMS-1 (vulvar metastasis) by RT-PCR, Western blot and immunostaining. We exposed our cell lines to GSIs, DAPT and MK-0752, and measured expression of HES1, a downstream effector of Notch. Notch proteins were differentially expressed in uLMS. Expression of NOTCH3 and NOTCH4 was higher in uLMS samples than in benign uterine smooth muscle and fibroids. Expression of NOTCH4 was higher in SK-LMS-1 compared to SK-UT-1B. Exposure of SK-UT-1B and SK-LMS-1 to DAPT and MK-0752 decreased expression of HES1 and decreased uLMS cell viability in a dose- and time-dependent manner that was unique to each GSI. Our findings suggest that GSIs are potential therapeutics for uLMS, albeit with limited efficacy.

Towards an Improved Understanding of the Effects of Elevated Progesterone Levels on Human Endometrial Receptivity and Oocyte/Embryo Quality during Assisted Reproductive Technologies.

Ovarian stimulation is an indispensable part of IVF and is employed to produce multiple ovarian follicles. In women who undergo ovarian stimulation with gonadotropins, supraphysiological levels of estradiol, as well as a premature rise in progesterone levels, can be seen on the day of hCG administration. These alterations in hormone levels are associated with reduced embryo implantation and pregnancy rates in IVF cycles with a fresh embryo transfer. This article aims to improve the reader's understanding of the effects of elevated progesterone levels on human endometrial receptivity and oocyte/embryo quality. Based on current clinical data, it appears that the premature rise in progesterone levels exerts minimal or no effects on oocyte/embryo quality, while advancing the histological development of the secretory endometrium and displacing the window of implantation. These clinical findings strongly suggest that reduced implantation and pregnancy rates are the result of a negatively affected endometrium rather than poor oocyte/embryo quality. Understanding the potential negative impact of elevated progesterone levels on the endometrium is crucial to improving implantation rates following a fresh embryo transfer. Clinical studies conducted over the past three decades, many of which have been reviewed here, have greatly advanced our knowledge in this important area.

Mouse Cre drivers: tools for studying disorders of the human female neuroendocrine-reproductive axis†.

Benign disorders of the human female reproductive system, such primary ovarian insufficiency and polycystic ovary syndrome are associated with infertility and recurrent miscarriage, as well as increased risk of adverse health outcomes, including cardiovascular disease and type 2 diabetes. For many of these conditions, the contributing molecular and cellular processes are poorly understood. The overarching similarities between mice and humans have rendered mouse models irreplaceable in understanding normal physiology and elucidating pathological processes that underlie disorders of the female reproductive system. The utilization of Cre-LoxP recombination technology, which allows for spatial and temporal control of gene expression, has identified the role of numerous genes in development of the female reproductive system and in processes, such as ovulation and endometrial decidualization, that are required for the establishment and maintenance of pregnancy in mammals. In this comprehensive review, we provide a detailed overview of Cre drivers with activity in the neuroendocrine-reproductive axis that have been used to study disruptions in key intracellular signaling pathways. We first summarize normal development of the hypothalamus, pituitary, ovary, and uterus, highlighting similarities and differences between mice and humans. We then describe human conditions resulting from abnormal development and/or function of the organ. Finally, we describe loss-of-function models for each Cre driver that elegantly recapitulate some key features of the human condition and are associated with impaired fertility. The examples we provide illustrate use of each Cre driver as a tool for elucidating genetic and molecular underpinnings of reproductive dysfunction.

Multisite Clinical Validation of Isothermal Amplification-Based SARS-CoV-2 Detection Assays Using Different Sampling Strategies.

Isothermal amplification-based tests have been introduced as rapid, low-cost, and simple alternatives to real-time reverse transcriptase PCR (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. The clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP coronavirus disease of 2019 [COVID-19] detection test and OptiGene COVID-19 direct plus RT-loop-mediated isothermal amplification [LAMP] test) was compared with that of clinical RT-PCR assays using different sampling strategies. A total of 1,378 participants were tested across 4 study sites. Compared with standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic site from which the sample was collected. Sensitivity for nasopharyngeal sampling was 65.4% (range across study sites, 52.8% to 79.8%), for midturbinate was 88.2%, for saliva was 55.1% (range across study sites, 42.9% to 77.8%), and for anterior nares was 66.7% (range across study sites, 63.6% to 76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall, 82.7%) and those with a higher viral load (overall, 92.4% for cycle threshold [CT] of ≤25). Sensitivity and specificity of the OptiGene direct plus RT-LAMP test, which was conducted at a single study site, were 25.5% and 100%, respectively. The Atila iAMP COVID test with midturbinate sampling is a rapid, low-cost assay for detecting SARS-CoV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-CoV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption. IMPORTANCE Numerous SARS-CoV-2 detection assays have been developed and introduced into the market under emergency use authorizations (EUAs). EUAs are granted primarily based on small studies of analytic sensitivity and specificity with limited clinical validations. A thorough clinical performance evaluation of SARS-CoV-2 assays is important to understand the strengths, limitations, and specific applications of these assays. In this first large-scale multicentric study, we evaluated the clinical performance and operational characteristics of two isothermal amplification-based SARS-CoV-2 tests, namely, (i) iAMP COVID-19 detection test (Atila BioSystems, USA) and (ii) COVID-19 direct plus RT-LAMP test (OptiGene Ltd., UK), compared with those of clinical RT-PCR tests using different sampling strategies (i.e., nasopharyngeal, self-sampled anterior nares, self-sampled midturbinate, and saliva). An important specific use for these isothermal amplification-based, rapid, low-cost, and easy-to-perform SARS-CoV-2 assays is to allow for a safer return to preventive clinical encounters, such as cancer screening, particularly in low- and middle-income countries that have low SARS-CoV-2 vaccination rates.

Multi-site clinical validation of Isothermal Amplification based SARS-COV-2 detection assays using different sampling strategies.

BACKGROUND: Isothermal amplification-based tests were developed as rapid, low-cost, and simple alternatives to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) tests for SARS-COV-2 detection. METHODS: Clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP COVID-19 detection test and OptiGene COVID-19 Direct Plus RT-LAMP test) was compared to clinical RT-PCR assays using different sampling strategies. A total of 1378 participants were tested across four study sites. RESULTS: Compared to standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic collected site. Sensitivity for nasopharyngeal was 65.4% (range across study sites:52.8%-79.8%), mid-turbinate 88.2%, saliva 55.1% (range across study sites:42.9%-77.8%) and anterior nares 66.7% (range across study sites:63.6%-76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall 82.7%) and those with a higher viral load (overall 92.4% for ct≤25). Sensitivity and specificity of the OptiGene Direct Plus RT-LAMP test, conducted at a single study-site, were 25.5% and 100%, respectively. CONCLUSIONS: The Atila iAMP COVID test with mid-turbinate sampling is a rapid, low-cost assay for detecting SARS-COV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-COV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption.

Genotypic analysis of the female BPH/5 mouse, a model of superimposed preeclampsia.

Animal models that recapitulate human diseases and disorders are widely used to investigate etiology, diagnosis, and treatment of those conditions in people. Disorders during pregnancy are particularly difficult to explore as interventions in pregnant women are not easily performed. Therefore, models that allow for pre-conception investigations are advantageous for elucidating the mechanisms involved in adverse pregnancy outcomes that are responsible for both maternal and fetal morbidity, such as preeclampsia. The Blood Pressure High (BPH)/5 mouse model has been used extensively to study the pathogenesis of preeclampsia. The female BPH/5 mouse is obese with increased adiposity and borderline hypertension, both of which are exacerbated with pregnancy making it a model of superimposed preeclampsia. Thus, the BPH/5 model shares traits with a large majority of women with pre-existing conditions that predisposes them to preeclampsia. We sought to explore the genome of the BPH/5 female mouse and determine the genetic underpinnings that may contribute to preeclampsia-associated phenotypes in this model. Using a whole genome sequencing approach, we are the first to characterize the genetic mutations in BPH/5 female mice that make it unique from the closely related BPH/2 model and the normotensive background strain, C57Bl/6. We found the BPH/5 female mouse to be uniquely different from BPH/2 and C57Bl/6 mice with a genetically complex landscape. The majority of non-synonymous consequences within the coding region of BPH/5 females were missense mutations found most abundant on chromosome X when comparing BPH/5 and BPH/2, and on chromosome 8 when comparing BPH/5 to C57Bl/6. Genetic mutations in BPH/5 females largely belong to immune system-related processes, with overlap between BPH/5 and BPH/2 models. Further studies examining each gene mutation during pregnancy are warranted to determine key contributors to the BPH/5 preeclamptic-like phenotype and to identify genetic similarities to women that develop preeclampsia.

Anti-Müllerian hormone levels among contraceptive users: evidence from a cross-sectional cohort of 27,125 individuals.

BACKGROUND: Anti-Müllerian hormone has become the clinical biomarker-based standard to assess ovarian reserve. As anti-Müllerian hormone testing becomes more common, more individuals are seeking to interpret the values obtained while using contraceptives. To appropriately counsel women, a better understanding of anti-Müllerian hormone levels in women using different contraceptives is needed. OBJECTIVE: To study the association between different forms of contraceptives and anti-Müllerian levels in women of reproductive age. STUDY DESIGN: This is a cross-sectional study including 27,125 US-based women aged 20 to 46 years, accessing reproductive hormone results through Modern Fertility and who provided informed consent to participate in the research. Anti-Müllerian hormone levels were collected through dried blood spot card (95.9%) or venipuncture (4.1%), and previous work has shown high correlation between hormone levels collected by these 2 methods. Multiple linear regressions were run to compare anti-Müllerian hormone levels in women using contraceptives with women not on any contraceptive, controlling for age, age of menarche, body mass index, smoking, sample collection method, cycle day, and self-reported polycystic ovary syndrome diagnosis. We also analyzed whether duration of contraceptive use predicted anti-Müllerian hormone levels in users of the hormonal intrauterine device and combined oral contraceptive pill, given the size of these contraceptive groups. RESULTS: Mean anti-Müllerian hormone levels were statistically significantly lower in women using the combined oral contraceptive pill (23.68% lower; coefficient, 0.76; 95% confidence interval, 0.72-0.81; P<.001), vaginal ring (22.07% lower; coefficient, 0.78; 95% confidence interval, 0.71-0.86; P<.001), hormonal intrauterine device (6.73% lower; coefficient, 0.93; 95% confidence interval, 0.88-0.99; P=.014), implant (23.44% lower; coefficient, 0.77; 95% confidence interval, 0.69-0.85; P<.001), or progestin-only pill (14.80% lower; coefficient, 0.85; 95% confidence interval, 0.76-0.96; P=.007) than women not on any contraceptive when controlling for covariates. Anti-Müllerian hormone levels were not significantly different when comparing women not using any contraceptives to those using the copper intrauterine device (1.57% lower; coefficient, 0.98; 95% confidence interval, 0.92-1.05, P=.600). Associations between contraceptive use and anti-Müllerian hormone levels did not differ based on self-reported polycystic ovary syndrome diagnosis. Duration of hormonal intrauterine device use, but not of combined oral contraceptive pill use, was slightly positively associated with anti-Müllerian hormone levels, although this small magnitude effect is likely not clinically meaningful (coefficient, 1.002; 95% confidence interval, 1.0005-1.003; P=.007). CONCLUSION: Current hormonal contraceptive use is associated with a lower mean anti-Müllerian hormone level than that of women who are not on contraceptives, with variability in the percent difference across contraceptive methods. These data provide guidance for clinicians on how to interpret anti-Müllerian hormone levels assessed while on contraceptives and may facilitate more patients to continue contraceptive use while being evaluated for their ovarian reserve.

Implications for preeclampsia: hypoxia-induced Notch promotes trophoblast migration.

In the first trimester of human pregnancy, low oxygen tension or hypoxia is essential for proper placentation and placenta function. Low oxygen levels and activation of signaling pathways have been implicated as critical mediators in the promotion of trophoblast differentiation, migration, and invasion with inappropriate changes in oxygen tension and aberrant Notch signaling both individually reported as causative to abnormal placentation. Despite crosstalk between hypoxia and Notch signaling in multiple cell types, the relationship between hypoxia and Notch in first trimester trophoblast function is not understood. To determine how a low oxygen environment impacts Notch signaling and cellular motility, we utilized the human first trimester trophoblast cell line, HTR-8/SVneo. Gene set enrichment and ontology analyses identified pathways involved in angiogenesis, Notch and cellular migration as upregulated in HTR-8/SVneo cells exposed to hypoxic conditions. DAPT, a γ-secretase inhibitor that inhibits Notch activation, was used to interrogate the crosstalk between Notch and hypoxia pathways in HTR-8/SVneo cells. We found that hypoxia requires Notch activation to mediate HTR-8/SVneo cell migration, but not invasion. To determine if our in vitro findings were associated with preeclampsia, we analyzed the second trimester chorionic villous sampling (CVS) samples and third trimester placentas. We found a significant decrease in expression of migration and invasion genes in CVS from preeclamptic pregnancies and significantly lower levels of JAG1 in placentas from pregnancies with early-onset preeclampsia with severe features. Our data support a role for Notch in mediating hypoxia-induced trophoblast migration, which may contribute to preeclampsia development.

Complement in Reproductive White Adipose Tissue Characterizes the Obese Preeclamptic-Like BPH/5 Mouse Prior to and During Pregnancy.

Preeclampsia (PE) is a serious hypertensive disorder of pregnancy characterized by abnormal placental development with an unknown etiology. To better understand which women will develop PE, a number of maternal risk factors have been identified, including obesity. Visceral white adipose tissue (WAT) contains inflammatory mediators that may contribute to PE. To explore this, we utilized the blood pressure high (BPH)/5 mouse model of superimposed PE that spontaneously recapitulates the maternal PE syndrome. We hypothesized that BPH/5 visceral WAT adjacent to the female reproductive tract (reproductive WAT) is a source of complement factors that contribute to the inflammatory milieu and angiogenic imbalance at the maternal-fetal interface in this model and in preeclamptic women. To test our hypothesis, we calorie-restricted BPH/5 females for two weeks prior to pregnancy and the first seven days of pregnancy, which attenuated complement component 3 (C3) but not complement factor B, nor complement factor D, (adipsin) in the reproductive WAT or the implantation site in BPH/5. Furthermore, calorie restriction during pregnancy restored vascular endothelial and placental growth factor mRNA levels in the BPH/5 implantation site. These data show maternal reproductive WAT may be a source of increased C3 during pregnancy, which is increased at the maternal-fetal interface in preeclamptic BPH/5 mice. It also suggests that calorie restriction could regulate inflammatory mediators thought to contribute to placental dysfunction in PE. Future studies are necessary to examine the effect of calorie restriction on C3 throughout pregnancy and the role of maternal obesity in PE.

Endothelial Jagged1 Antagonizes Dll4/Notch Signaling in Decidual Angiogenesis during Early Mouse Pregnancy.

Maternal spiral arteries and newly formed decidual capillaries support embryonic development prior to placentation. Previous studies demonstrated that Notch signaling is active in endothelial cells of both decidual capillaries and spiral arteries, however the role of Notch signaling in physiologic decidual angiogenesis and maintenance of the decidual vasculature in early mouse pregnancy has not yet been fully elucidated. We used the Cdh5-CreERT2;Jagged1(Jag1)flox/flox (Jag1∆EC) mouse model to delete Notch ligand, Jag1, in maternal endothelial cells during post-implantation, pre-placentation mouse pregnancy. Loss of endothelial Jag1 leads to increased expression of Notch effectors, Hey2 and Nrarp, and increased endothelial Notch signaling activity in areas of the decidua with remodeling angiogenesis. This correlated with an increase in Dll4 expression in capillary endothelial cells, but not spiral artery endothelial cells. Consistent with increased Dll4/Notch signaling, we observed decreased VEGFR2 expression and endothelial cell proliferation in angiogenic decidual capillaries. Despite aberrant Dll4 expression and Notch activation in Jag1∆EC mutants, pregnancies were maintained and the decidual vasculature was not altered up to embryonic day 7.5. Thus, Jag1 functions in the newly formed decidual capillaries as an antagonist of endothelial Dll4/Notch signaling during angiogenesis, but Jag1 signaling is not necessary for early uterine angiogenesis.

Dynamic Expression of Interleukin-33 and ST2 in the Mouse Reproductive Tract Is Influenced by Superovulation.

Interleukin-33 (IL-33) is an IL-1 family cytokine with pleiotropic effects on diverse cell types. Dysregulated IL-33 signaling has been implicated in pregnancy-related disorders, including preeclampsia and recurrent pregnancy loss, and in ovarian function in women undergoing controlled ovarian stimulation for in vitro fertilization. To date, expression of IL-33 and its receptor subunit, ST2, in the female reproductive tract remains poorly characterized. We identify IL-33-expressing oocytes surrounded by ST2-expressing granulosa cells at all stages of follicular development, in addition to IL-33+ and ST2+ non-endothelial cells in the ovarian stroma and theca layer in ovaries from adult mice. These expression patterns are similar in estrus- and diestrus-stage adults and in pubescent mice, suggesting a role for IL-33 signaling in ovarian function throughout development and in the estrous cycle. In the uterus, we find expression of IL-33 and ST2 in glandular and luminal epithelia during estrus and at the initiation of pregnancy. Uterine IL-33 expression was modulated by the estrous cycle and was reduced in pubescent females. Last, superovulation increases transcripts for IL-33 and the soluble form of ST2 (sST2) in ovaries, and for IL-33 in uteri. Collectively, our findings lay the foundation for studies identifying cell type-specific requirements for IL-33/ST2 signaling in the establishment and maintenance of mouse pregnancy.

Dominant-negative inhibition of canonical Notch signaling in trophoblast cells does not disrupt placenta formation.

Proper development and function of the mammalian placenta requires interactions between embryo-derived trophoblasts and uterine endothelial cells to form mosaic vessels that facilitate blood flow to a developing conceptus. Notch signaling utilizes a cell-cell contact dependent mechanism to drive cell behaviors, such as differentiation and invasion. In mice, Notch2 is needed for proper placentation and embryo survival. We used transgenic mice with a dominant-negative form of Mastermind-like1 and Cyp19-Cre and Tpbpa-Cre drivers to inhibit canonical Notch signaling in trophoblasts. Both Cre drivers resulted in robust placental expression of dominant-negative Mastermind-like1. All pregnancies progressed beyond mid-gestation and morphological analyses of placentas revealed no differences between mutants and controls. Our data suggest that mouse placentation occurs normally despite dominant negative inhibition of trophoblast canonical Notch signaling and that Notch2 signaling via the canonical pathway is not necessary for placentation.

Concordance of Fingerstick and Venipuncture Sampling for Fertility Hormones.

BACKGROUND: Minimally invasive fingerstick sampling allows testing of reproductive hormone levels at home, providing women with increased access to tests that can screen for conditions such as polycystic ovarian syndrome, primary ovarian insufficiency, and pituitary and thyroid dysfunction. METHOD: We present a measurement procedure comparison study of matched venipuncture and fingerstick samples from 130 women aged 18-40 years, tested on menstrual cycle day 3. Samples were measured for anti-müllerian hormone, estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), testosterone, thyroid-stimulating hormone (TSH), and free thyroxine (T4) levels. Samples were tested using U.S. Food and Drug Administration-cleared immunoassays, with a modified reconstitution step for fingerstick samples. EXPERIENCE: Venipuncture and fingerstick hormone values were concordant and linear across all assay ranges. There was no evidence of systematic bias across the assay ranges, and bias measures were below recommended guidelines. The correlation between venipuncture and fingerstick was between 0.99 and 1.0 for each hormone. Each assay displayed a high degree of precision (less than 13% coefficient of variation) and a high level of accuracy (average recovery equaled 95.5-102.3%). CONCLUSION: Venipuncture and fingerstick samples can be used interchangeably to measure anti-müllerian hormone, E2, FSH, LH, PRL, testosterone, TSH, and free T4 levels. Fingerstick sampling provides doctors and women more convenient testing options. FUNDING SOURCE: The study was sponsored by Modern Fertility.

Celecoxib restores angiogenic factor expression at the maternal-fetal interface in the BPH/5 mouse model of preeclampsia.

Preeclampsia (PE), a hypertensive disease of pregnancy, is a leading cause of fetal and maternal morbidity/mortality. Early angiogenic and inflammatory disturbances within the placenta are thought to underlie the development of the maternal PE syndrome and poor pregnancy outcomes. However, the exact etiology remains largely unknown. Here, we use the BPH/5 mouse model of PE to elucidate the way in which inflammation early in pregnancy contributes to abnormal expression of angiogenic factors at the maternal-fetal interface. We have previously described improvement in maternal hypertension and fetal growth restriction in this model after treatment with the anti-inflammatory cyclooxygenase-2 (Cox2) specific inhibitor celecoxib. To further characterize the mechanisms by which celecoxib improves poor pregnancy outcomes in BPH/5 mice, we determined expression of angiogenic factors and complement pathway components after celecoxib. In BPH/5 implantation sites there was increased hypoxia inducible factor-1α ( Hif1α), heme oxygenase-1 ( Ho-1), and stem cell factor ( Scf) mRNA concomitant with elevated prostaglandin synthase 2 ( Ptgs2), encoding Cox2, and elevated VEGF protein. Angiopoietin 1 ( Ang1), tunica interna endothelial cell kinase-2 receptor ( Tie2), complement factor 3 ( C3), and complement factor B ( CfB) were increased in midgestation BPH/5 placentae. Whereas BPH/5 expression levels of VEGF, Ang1, and Tie2 normalized after celecoxib, placental C3 and CfB mRNA remained unchanged. However, celecoxib did reduce the pregnancy-specific circulating soluble fms-like tyrosine kinase-1 (sFlt-1) rise in BPH/5 mice at midgestation. These data show that elevated Cox2 during implantation contributes to placental angiogenic factor imbalances in the BPH/5 mouse model of PE.