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Abstract Background The tetraspan protein epithelial membrane protein-2 (EMP2), which mediates surface display of diverse proteins, is required for endometrial competence in blastocyst implantation, and is uniquely correlated with poor survival from endometrial adenocarcinoma tumors. Because EMP2 is differentially expressed in the various stages of the murine and human estrous cycle, we tested the hypothesis that the steroid hormones progesterone and estrogen influence EMP2 expression and localization. Methods Frozen human proliferative and secretory endometrium were collected and analyzed for EMP2 expression using SDS-PAGE/Western blot analysis. The response of EMP2 to progesterone and estradiol was determined using a combination of real-time PCR, SDS-PAGE/Western blot analysis, and confocal immunofluorescence in the human endometrial carcinoma cell line RL95-2. To confirm the in vitro results, ovariectomized mice were treated with progesterone or estradiol, and EMP2 expression was analyzed using immunohistochemistry. Results Within normal human endometrium, EMP2 expression is upregulated in the secretory phase relative to the proliferative phase. To understand the role of steroid hormones on EMP2 expression, we utilized RL95-2 cells, which express both estrogen and progesterone receptors. In RL95-2 cells, both estradiol and progesterone induced EMP2 mRNA expression, but only progesterone induced EMP2 protein expression. To compare steroid hormone regulation of EMP2 between humans and mice, we analyzed EMP2 expression in ovarectomized mice. Similar to results observed in humans, progesterone upregulated endometrial EMP2 expression and induced EMP2 translocation to the plasma membrane. Estradiol did not promote translocation to the cell surface, but moderately induced EMP2 expression in cytoplasmic compartments in vivo. Conclusion These findings suggest that targeting of EMP2 to specific locations under the influence of these steroid hormones may be important for integrating the molecular responses required for implantation competence.

Introduction: Prior to pregnancy, hormonal changes lead to cellular adaptations in the endometrium allowing for embryo implantation. Critical for successful pregnancy establishment, innate immune cells constitute a significant proportion of uterine cells prior to arrival of the embryo and throughout the first trimester in humans and animal models. Abnormal uterine immune cell function during implantation is believed to play a role in multiple adverse pregnancy outcomes. Current work in humans has focused on uterine immune cells present after pregnancy establishment, and limited in vitro models exist to explore unique functions of these cells., Methods: With single-cell RNA-sequencing (scRNAseq), we comprehensively compared the human uterine immune landscape of the endometrium during the window of implantation and the decidua during the first trimester of pregnancy., Results: We uncovered global and cell-type-specific gene signatures for each timepoint. Immune cells in the endometrium prior to implantation expressed genes associated with immune metabolism, division, and activation. In contrast, we observed widespread interferon signaling during the first trimester of pregnancy. We also provide evidence of specific inflammatory pathways enriched in pre- and post-implantation macrophages and natural killer (NK) cells in the uterine lining. Using our novel implantation-on-a-chip (IOC) to model human implantation ex vivo, we demonstrate for the first time that uterine macrophages strongly promote invasion of extravillous trophoblasts (EVTs), a process essential for pregnancy establishment. Pre- and post-implantation uterine macrophages promoted EVT invasion to a similar degree as pre- and post-implantation NK cells on the IOC., Conclusions: This work provides a foundation for further investigation of the individual roles of uterine immune cell subtypes present prior to embryo implantation and during early pregnancy, which will be critical for our understanding of pregnancy complications associated with abnormal trophoblast invasion and placentation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mani, Garifallou, Kim, Simoni, Huh, Gordon and Mainigi.)

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a β-adrenergic receptor-dependent (β-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the β-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Objective: To determine if ovarian responsiveness to gonadotropin stimulation differs by race/ethnicity and whether this predicts live birth rates (LBRs) in non-White patients undergoing in vitro fertilization (IVF)., Design: Retrospective cohort study., Setting: Academic infertility center., Patient(s): White, Asian, Black, and Hispanic patients undergoing ovarian stimulation for IVF., Intervention(s): Self-reported race and ethnicity., Main Outcome Measure(s): The primary outcome was ovarian sensitivity index (OSI), defined as (the number of oocytes retrieved ÷ total gonadotropin dose) × 1,000 as a measure of ovarian responsiveness, adjusting for age, body mass index, infertility diagnosis, and cycle number. Secondary outcomes included live birth and clinical pregnancy after first retrievals, adjusting for age, infertility diagnosis, and history of fibroids, as well as miscarriage rate per clinical pregnancy, adjusting for age, body mass index, infertility diagnosis, duration of infertility, history of fibroids, and use of preimplantation genetic testing for aneuploidy., Result(s): The primary analysis of OSI included 3,360 (70.2%) retrievals from White patients, 704 (14.7%) retrievals from Asian patients, 553 (11.6%) retrievals from Black patients, and 168 (3.5%) retrievals from Hispanic patients. Black and Hispanic patients had higher OSIs than White patients after accounting for those with multiple retrievals and adjusting for confounders (6.08 in Black and 6.27 in Hispanic, compared with 5.25 in White). There was no difference in OSI between Asian and White patients. The pregnancy outcomes analyses included 2,299 retrievals. Despite greater ovarian responsiveness, Black and Hispanic patients had lower LBRs compared with White patients, although these differences were not statistically significant after adjusting for confounders (adjusted odds ratio, 0.83; 95% confidence interval [CI], 0.63-1.09, for Black; adjusted odds ratio, 0.93; 95% CI, 0.61-1.43, for Hispanic). Ovarian sensitivity index was modestly predictive of live birth in White and Asian patients but not in Black (area under the curve, 0.51; 95% CI, 0.38-0.64) and Hispanic (area under the curve, 0.50; 95% CI, 0.37-0.63) patients., Conclusion(s): Black and Hispanic patients have higher ovarian responsiveness to stimulation during IVF but do not experience a consequent increase in LBR. Factors beyond differences in responsiveness to ovarian stimulation need to be explored to address the racial/ethnic disparity established in prior literature., Competing Interests: Declaration of interests I.T.L has nothing to disclose. D.S.B. has nothing to disclose. N.K. has nothing to disclose. S.S. reports funding from National Institutes of Health and AbbVie and consulting fees for participation on Ferring Oocyte Cryopreservation Advisory Board and is Co-Chair of the Society for Assisted Reproductive Technology Committee, outside the submitted work. M.M. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

In vitro fertilization (IVF) is associated with DNA methylation abnormalities and a higher incidence of adverse pregnancy outcomes. However, which exposure(s), among the many IVF interventions, contributes to these outcomes remains unknown. Frozen embryo transfer (ET) is increasingly utilized as an alternative to fresh ET, but reports suggest a higher incidence of pre-eclampsia and large for gestational age infants. This study examines DNA methylation in human placentas using the 850K Infinium MethylationEPIC BeadChip array obtained after 65 programmed frozen ET cycles, 82 fresh ET cycles and 45 unassisted conceptions. Nine patients provided placentas following frozen and fresh ET from consecutive pregnancies for a paired subgroup analysis. In parallel, eight mouse placentas from fresh and frozen ET were analyzed using the Infinium Mouse Methylation BeadChip array. Human and mouse placentas were significantly hypermethylated after frozen ET compared with fresh. Paired analysis showed similar trends. Sex-specific analysis revealed that these changes were driven by male placentas in humans and mice. Frozen and fresh ET placentas were significantly different from controls, with frozen samples hypermethylated compared with controls driven by males and fresh samples being hypomethylated compared with controls, driven by females. Sexually dimorphic epigenetic changes could indicate differential susceptibility to IVF-associated perturbations, which highlights the importance of sex-specific evaluation of adverse outcomes. Similarities between changes in mice and humans underscore the suitability of the mouse model in evaluating how IVF impacts the epigenetic landscape, which is valuable given limited access to human tissue and the ability to isolate specific interventions in mice., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Alterations in the epigenome are a risk factor in multiple disease states. We have demonstrated in the past that disruption of the epigenome during early pregnancy or periconception, as demonstrated by altered methylation, may be associated with both assisted reproductive technology and undesirable clinical outcomes at birth, such as low birth weight. We have previously defined this altered methylation, calculated based on statistical upper and lower limits of outlier CpGs compared to the population, as an 'outlier methylation phenotype' (OMP). Our aim in this study was to determine whether children thus identified as possessing an OMP at birth by DNA methylation in cord blood persist as outliers in early childhood based on salivary DNA methylation., Results: A total of 31 children were included in the analysis. Among 24 children for whom both cord blood DNA and salivary DNA were available, DNA methylation patterns, analyzed using the Illumina Infinium MethylationEPIC BeadChip (850 K), between cord blood at birth and saliva in childhood at age 6-12 years remain stable (R 2 range 0.89-0.97). At birth, three out of 28 children demonstrated an OMP in multiple cord blood datasets and hierarchical clustering. Overall DNA methylation among all three OMP children identified as outliers at birth was remarkably stable (individual R 2 0.908, 0.92, 0.915), even when only outlier CpG sites were considered (R 2 0.694, 0.738, 0.828)., Conclusions: DNA methylation signatures in cord blood remain stable over time as demonstrated by a strong correlation with epigenetic salivary signatures in childhood. Future work is planned to identify whether a clinical phenotype is associated with OMP and, if so, could undesirable clinical outcomes in childhood and adulthood be predicted at birth., (© 2022. The Author(s).)

Objective: To assess the impact of the COVID-19 pandemic on attitudes toward planned oocyte cryopreservation (OC)., Design: Cross-sectional study., Setting: Internet-based survey questionnaire distributed nationally., Patients: One thousand women aged 21-45 years, stratified by age ≤35 or >35 years., Interventions: None., Main Outcome Measures: Change in the likelihood of considering OC because of the pandemic., Results: Of the participants who reported that the pandemic altered their likelihood of considering OC (15.2%, n = 152), 52.6% (n = 80) reported an increased and 47.3% (n = 72) reported a decreased likelihood of considering OC. Vaccination status did not affect the likelihood of considering OC. In multivariable analysis, history of COVID-19 infection (odds ratio [OR] 1.57; 95% confidence interval [CI] 1.00-2.45), government-subsidized insurance (OR 1.47; 95% CI 0.97-2.21), loss of health insurance because of the pandemic (OR 2.32; 95% CI 1.15-4.66), working more (OR 2.99; 95% CI 1.62-5.51) or less (OR 2.54; 95% CI 1.65-3.90) because of the pandemic, and relationship status (divorced, separated, or widowed [OR 0.44; 95% CI 0.20-0.99]) were significantly associated with a change in the likelihood of considering OC because of the pandemic. Of those who believed that the COVID-19 pandemic influenced their childbearing plans (28.3%, n = 283), 64.0% (n = 181) deferred childbearing and 29.7% (n = 84) expedited childbearing. The pandemic's economic impact, concerns regarding safety of pregnancy/childbirth, and safety of childrearing were cited as most influential on childbearing (67%, 70%, 58%, respectively) and on the likelihood of considering OC (47%, 45%, and 34%, respectively)., Conclusions: Through its negative impact on financial security and perceived safety, the COVID-19 pandemic has altered the likelihood of considering OC in >15% of reproductive-aged women and reproductive timelines in 30%. Vaccination has not significantly modified these changes., (© 2022 The Authors.)

Successful establishment of pregnancy requires adhesion of an embryo to the endometrium and subsequent invasion into the maternal tissue. Abnormalities in this critical process of implantation and placentation lead to many pregnancy complications. Here we present a microenigneered system to model a complex sequence of orchestrated multicellular events that plays an essential role in early pregnancy. Our implantation-on-a-chip is capable of reconstructing the three-dimensional structural organization of the maternal-fetal interface to model the invasion of specialized fetal extravillous trophoblasts into the maternal uterus. Using primary human cells isolated from clinical specimens, we demonstrate in vivo-like directional migration of extravillous trophoblasts towards a microengineered maternal vessel and their interactions with the endothelium necessary for vascular remodeling. Through parametric variation of the cellular microenvironment and proteomic analysis of microengineered tissues, we show the important role of decidualized stromal cells as a regulator of extravillous trophoblast migration. Furthermore, our study reveals previously unknown effects of pre-implantation maternal immune cells on extravillous trophoblast invasion. This work represents a significant advance in our ability to model early human pregnancy, and may enable the development of advanced in vitro platforms for basic and clinical research of human reproduction., (© 2022. The Author(s).)

Objective: To assess whether the mode of conception and embryo biopsy impact first-trimester human chorionic gonadotropin (hCG) dynamics and subsequent risk of small for gestational age (SGA) or large for gestational age (LGA)., Design: Retrospective cohort study., Setting: University fertility center., Patients: Six hundred-two pregnant patients with singleton live births., Interventions: Serial serum hCG measurements were obtained between 10 and 28 days postconception to determine the within-woman rate of change in hCG (slope) by mode of conception (unassisted pregnancy, fresh embryo transfer (ET), frozen ET, and frozen ET following preimplantation genetic testing for aneuploidy (PGT-A)., Main Outcome Measures: Primary outcomes included birth weight, SGA, and LGA., Results: Mode of conception is not independently associated with birth weight, SGA, or LGA. Mediation analysis revealed an expected one-day increase in log-transformed hCG varied by mode of conception: unassisted (0.41), fresh ET (0.39), frozen ET (0.42), PGT-A (0.44). Human chorionic gonadotropin rise has a positive effect on birth weight (55 g per SD increase in hCG slope) and is associated with SGA (odds ratio, 0.65), but not with LGA (odds ratio, 1.18)., Conclusions: Human chorionic gonadotropin rise is an important mediator of the mode of conception/birth weight relationship. Preimplantation genetic testing for aneuploidy has the highest rate of hCG rise, followed by frozen ET, unassisted, and fresh ET. Faster rise is associated with higher birth weight and lower risk of SGA but does not impact LGA risk. Importantly, PGT-A does not increase the risk of extreme birth weight relative to other modes of conception evaluated.

Background: Despite significant advances in our understanding of the pathophysiology of preeclampsia (PE), there are still many unknowns and controversies in the field. Women undergoing frozen-thawed embryo transfer (FET) to a hormonally prepared endometrium have been found to have an unexpected increased risk of PE compared to women who receive embryos in a natural FET cycle. The differences in risk have been hypothesized to be related to the absence or presence of a functioning corpus luteum (CL)., Objective and Rationale: To evaluate the literature on secretory products of the CL that could be essential for a healthy pregnancy and could reduce the risk of PE in the setting of FET., Search Methods: For this review, pertinent studies were searched in PubMed/Medline (updated June 2020) using common keywords applied in the field of assisted reproductive technologies, CL physiology and preeclampsia. We also screened the complete list of references in recent publications in English (both animal and human studies) on the topics investigated. Given the design of this work as a narrative review, no formal criteria for study selection or appraisal were utilized., Outcomes: The CL is a major source of multiple factors regulating reproduction. Progesterone, estradiol, relaxin and vasoactive and angiogenic substances produced by the CL have important roles in regulating its functional lifespan and are also secreted into the circulation to act remotely during early stages of pregnancy. Beyond the known actions of progesterone and estradiol on the uterus in early pregnancy, their metabolites have angiogenic properties that may optimize implantation and placentation. Serum levels of relaxin are almost undetectable in pregnant women without a CL, which precludes some maternal cardiovascular and renal adaptations to early pregnancy. We suggest that an imbalance in steroid hormones and their metabolites and polypeptides influencing early physiologic processes such as decidualization, implantation, angiogenesis and maternal haemodynamics could contribute to the increased PE risk among women undergoing programmed FET cycles., Wider Implications: A better understanding of the critical roles of the secretory products of the CL during early pregnancy holds the promise of improving the efficacy and safety of ART based on programmed FET cycles., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The use of quantitative PCR (qPCR) and other polymerase chain reaction (PCR)-based methods in the field of human in vitro fertilization blastocoel fluid analysis can potentially be utilized for assisting clinicians in embryo selection based on specific gene expression patterns. Since typical Comparative cycle threshold ( C t ) analysis utilizes one threshold for runs per gene target and requires an inherent control group, this method is inadequate for analysis of small stochastic systems, such as embryonic-derived fluid. We mathematically demonstrate analytical modifications upon the Comparative C t qPCR workflow to incorporate a variable fluorescence threshold (utilizing only the parameters defined in the Comparative C t method), and subsequently demonstrate the typical workflow in which this modified method can successfully quantifiably analyze embryonic blastocoel fluid qPCR analysis., (© The Author(s) 2021. Published by Oxford University Press.)

Superovulation with gonadotropins alters the hormonal milieu during early embryo development and placentation, and may be responsible for fetal and placental changes observed after in vitro fertilization (IVF). We hypothesized that superovulation has differential effects depending on timing of exposure. To test our hypothesis, we isolated the effect of superovulation on pre- and peri-implantation mouse embryos. Blastocysts were obtained from either natural mating or following superovulation and mating, and were transferred into naturally mated or superovulated pseudopregnant recipient mice. Fetal weight was significantly lower after peri-implantation exposure to superovulation, regardless of preimplantation exposure (p = 0.006). Placentas derived from blastocysts exposed to superovulation pre- and peri-implantation were larger than placentas derived from natural blastocysts that are transferred into a natural or superovulated environment (p < 0.05). Fetal-to-placental weight ratio decreased following superovulation during the pre- or peri-implantation period (p = 0.05, 0.01, respectively) and these effects were additive. Peg3 DNA methylation levels were decreased in placentas derived from exposure to superovulation both pre- and peri-implantation compared with unexposed embryos and exposure of the preimplantation embryo only. Through RNA sequencing on placental tissue, changes were identified in genes involved in immune system regulation, specifically interferon signaling, which has been previously implicated in implantation and maintenance of early pregnancy in mice. Overall, we found that the timing of exposure to gonadotropin stimulation can have differential effects on fetal and placental growth. These findings could impact clinical practice and underscores the importance of dissecting the role of procedures utilized during IVF on pregnancy complications., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Children conceived by Assisted Reproductive Technologies (ART) are at moderately increased risk for a number of undesirable outcomes, including low birth weight. Whether the additional risk is associated with specific procedures used in ART or biological factors that are intrinsic to infertility has been the subject of much debate, as has the mechanism by which ART or infertility might influence this risk. The potential effect of ART clinical and laboratory procedures on the gamete and embryo epigenomes heads the list of mechanistic candidates that might explain the association between ART and undesirable clinical outcomes. The reason for this focus is that the developmental time points at which ART clinical and laboratory procedures are implemented are precisely the time points at which large-scale reorganization of the epigenome takes place during normal development. In this manuscript, we review the many human studies comparing the epigenomes of ART children with children conceived in vivo, as well as assess the potential of individual ART clinical and laboratory procedures to alter the epigenome.

Preterm birth (PTB) affects approximately 1 in 10 pregnancies and contributes to approximately 50% of neonatal mortality. However, despite decades of research, little is understood about the etiology of PTB, likely due to the multifactorial nature of the disease. In this study, we examined preterm and term placentas, from unassisted conceptions and those conceived using in vitro fertilization (IVF). IVF increases the risk of PTB and causes epigenetic change in the placenta and fetus; therefore, we utilized these patients as a unique population with a potential common etiology. We investigated genome-wide DNA methylation in placentas from term IVF, preterm IVF, term control (unassisted conception) and preterm control pregnancies and discovered epigenetic dysregulation of multiple genes involved in cell migration, including members of the ADAMTS family, ADAMTS12 and ADAMTS16. These genes function in extracellular matrix regulation and tumor cell invasion, processes replicated by invasive trophoblasts (extravillous trophoblasts (EVTs)) during early placentation. Though expression was similar between term and preterm placentas, we found that both genes demonstrate high expression in first- and second-trimester placenta, specifically in EVTs and syncytiotrophoblasts. When we knocked down ADAMTS12 or ADAMTS16in vitro, there was poor EVT invasion and reduced matrix metalloproteinase activity, reinforcing their critical role in placentation. In conclusion, utilizing a population at high risk for PTB, we have identified a role for ADAMTS gene methylation in regulating early placentation and susceptibility to PTB.

Purpose: Epidemiologic data suggest that in vitro fertilization (IVF) is associated with an increased risk of disorders of placentation including preeclampsia and fetal growth restriction. Specifically, studies have demonstrated that singleton pregnancies conceived following a fresh embryo transfer are at an increased risk of delivering an infant with low birth weight compared to those conceived following a frozen embryo transfer. The mechanism responsible for this association remains unclear. Procedures utilized in IVF have also been linked with epigenetic changes and gene expression changes in both fetal and maternal tissues. Data suggest that modifications in the maternal endometrium can lead to disordered trophoblast invasion and placentation. This study examines the effect of ovarian stimulation on endometrial gene expression and DNA methylation during the window of implantation to examine potential pathways playing a role in the adverse outcomes associated with IVF., Methods: Endometrial biopsies were obtained from oocyte donors and age-matched naturally cycling women 11 days following oocyte retrieval in donors or 12 days following luteinizing hormone (LH) surge in naturally cycling women. Global gene expression was analyzed via Affymetrix Human Gene 1.1 ST array and confirmed with RT-qPCR. DNA methylation was assessed with the Infinium DNA methylation 450 K BeadChip., Results: Analysis of endometrial gene expression from 23 women (11 oocyte donors and 12 controls) demonstrated 165 genes with a greater than twofold change in expression between donors and controls. While there were 785 genes with significant differential methylation in the endometrium of donors when compared with control subjects, none of the genes with altered expression showed significant changes in DNA methylation. Analysis of the differentially expressed genes showed enrichment for genes involved in endometrial remodeling including PLAT, HSPE2, MMP2, and TIMP1. Validation studies using RT-qPCR found a 73% reduction in expression of heparanase 2 (HSPE2) an enzyme associated with both angiogenesis and cell invasion, a greater than twofold increase in tissue-type plasminogen activator (PLAT), a serine protease participating in matrix degradation, and a 70% increase in MMP2, a gelatinase involved in collagen and fibronectin breakdown., Conclusions: Superovulation alters expression of genes critical to endometrial remodeling during early implantation. Such changes could lead to altered trophoblast migration and impaired endovascular invasion. These findings offer a potential mechanism for the adverse perinatal outcomes observed following embryo transfer during fresh IVF cycles.

Epidemiological studies suggest that babies born following in vitro fertilization (IVF) and fresh embryo transfer are of lower birthweight than babies born following frozen embryo transfer, although the mechanism responsible for this phenotype is not known. We developed a novel mouse model that isolates the independent effects of embryo freezing and the superovulated environment, which cannot be performed in humans. We transferred blastocysts that had been vitrified and warmed, mixed with with fresh blastocysts, into individual pseudopregnant recipients produced by either natural mating or mating following injection with equine chorionic gonadotropin and human chorionic gonadotropin and hCG (superovulation). We found that superovulation of the recipient dams led to significantly lower fetal weight at term while blastocyst vitrification had no significant effect on fetal weight (1.43 ± 0.24 g fresh-natural, 1.30 ± 0.28 g vitrified-natural vs. 1.09 ± 0.20 fresh-superovulated, 0.93 ± 0.23 g vitrified-superovulated, P < 0.0001). Doppler ultrasound revealed increased median umbilical artery resistance in the placentae of near-term dams exposed to superovulation compared to naturally mated dams (0.927 vs 0.904, P = 0.02). Additionally, placental microvascular density was lower in superovulated compared to naturally mated dams (1.24 × 10-3 vessel/micron vs 1.46 × 10-3 vessels/micron, P = 0.046). Gene expression profiling suggested alterations in fetal genes involved in glucorticoid regulation. These results suggest a potential mechanism for altered birthweight following superovulation in our model and may have implications for human IVF., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: We analyzed placental DNA methylation levels at repeated sequences ( LINE1 elements) and all CCGG sites (the LUMA assay) to study the effect of modifiable clinical or laboratory procedures involved in in vitro fertilization. We included four potential modifiable factors: oxygen tension during embryo culture, fresh embryo transfer vs frozen embryo transfer, intracytoplasmic sperm injection (ICSI) vs conventional insemination or day 3 embryo transfer vs day 5 embryo transfer., Results: Global methylation levels differed between placentas from natural conceptions compared to placentas conceived by IVF. Placentas from embryos cultured at 20% oxygen showed significant differences in LINE1 methylation compared to in vivo conceptions, while those from embryos cultured at 5% oxygen, did not have significant differences. In addition, placentas from fresh embryo transfer had significantly different LINE1 methylation compared to placentas from in vivo conceptions, while embryos resulting from frozen embryos were not significantly different from controls. On sex-stratified analysis, only males had significant methylation differences at LINE1 elements stratified for the modifiable factors. As expected, LINE1 methylation was significantly different between males and females in the control population. However, we did not observe sex-specific differences in the IVF group. We validated this sex-specific observation in an additional cohort and in opposite sex IVF twins., Conclusion: We show that two clinically modifiable factors (embryo culture in 5 vs 20% oxygen tension and fresh vs frozen embryo transfer) are associated with global placental methylation differences. Interestingly, males appear more vulnerable to such treatment-related global changes in DNA methylation than do females.

Although time-lapse analysis of early embryo cleavage parameters (morphokinetics) predicts blastocyst development, it has not been definitively linked to establishing pregnancy and live birth. For example, a direct comparison of the developmental potential of embryos with optimal kinetic parameters compared to suboptimal kinetics has not been performed with human embryos. To ascertain whether such a linkage exists, we developed a mouse model of morphokinetic analysis of early embryo cleavage using time-lapse microscopy to predict blastocyst formation and tested whether cleavage parameters predict pregnancy outcome by transferring morphokinetically optimal and suboptimal embryos into a single host. Using classification and regression trees, we established that the timing of the second and third mitotic divisions (division from two to three and three to four cells, respectively) predicts blastocyst development in the mouse. Using this prediction model, we found that the incidence of sustained implantation at mid-gestation was significantly higher for the optimal compared to suboptimal embryos. In addition, the incidence of resorption among implanted embryos was significantly higher in the suboptimal compared to the optimal group. Transcript profiling of optimal and suboptimal embryos revealed minimal differences between the two groups, suggesting that time-lapse imaging of early embryo cleavage events provides additional information regarding developmental competence apart from gene expression., (© 2016 by the Society for the Study of Reproduction, Inc.)

We have identified a novel molecular phenotype that defines a subgroup of newborns who have highly disrupted epigenomes. We profiled DNA methylation in cord blood of 114 children selected from the lowest and highest quintiles of the birth weight distribution (irrespective of their mode of conception) at 96 CpG sites in genes we have found previously to be related to birth weight or growth and metabolism. We identified those individuals in each group who differed from the mean of the distribution by the greatest magnitude at each site and for the largest number of sites. Such 'outlier' individuals differ substantially from the rest of the group in having highly disrupted methylation levels at many CpG sites. We find that children from the lowest quintile of the birth weight distribution have a significantly greater number of disrupted CpGs than children from the highest quintile of the birth weight distribution. Among children from the lowest quintile of the birth weight distribution, 'outlier' individuals are significantly more common among children conceived in vitro than children conceived in vivo. These observations are novel and potentially important because they associate a molecular phenotype (multiple and large DNA methylation differences) in normal somatic tissues (cord blood) with both a prenatal exposure (conception in vitro) and a clinically important outcome (low birth weight). These observations suggest that some individuals are more susceptible to environmentally mediated epigenetic alterations than others., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The oocyte-to-embryo transition entails genome activation and a dramatic reprogramming of gene expression that is required for continued development. Superimposed on genome activation and reprogramming is development of a transcriptionally repressive state at the level of chromatin structure. Inducing global histone hyperacetylation relieves this repression and histone deacetylases 1 and 2 (HDAC1 and HDAC2) are involved in establishing the repressive state. Because SIN3A is an HDAC1/2-containing complex, we investigated whether it is involved in reprogramming gene expression during the course of genome activation. We find that Sin3a mRNA is recruited during maturation and that inhibiting its recruitment not only inhibits development beyond the 2-cell stage but also compromises the fidelity of reprogramming gene expression. The SIN3A that is synthesized during oocyte maturation reaches a maximum level in the mid-1-cell embryo and is essentially absent by the mid-2-cell stage. Overexpressing SIN3A in 1-cell embryos has no obvious effect on pre- and postimplantation development. These results provide a mechanism by which reprogramming can occur using a maternally inherited transcription machinery, namely, recruitment of mRNAs encoding transcription factors and chromatin remodelers, such as SIN3A., (© 2015 by the Society for the Study of Reproduction, Inc.)

Background: We, and others, have demonstrated previously that there are differences in DNA methylation and transcript levels of a number of genes in cord blood and placenta between children conceived using assisted reproductive technologies (ART) and children conceived in vivo. The source of these differences (the effect of ART versus the underlying infertility) has never been determined in humans. In this study, we have attempted to resolve this issue by comparing placental DNA methylation levels at 37 CpG sites in 16 previously identified candidate genes in independent populations of children conceived in vivo ('fertile control' group) with ART children conceived from two groups: either autologous oocytes with infertility in one or both parents ('infertile ART' group) or donor oocytes (obtained from young fertile donors) without male infertility ('donor oocyte ART' group)., Results: Of the 37 CpG sites analyzed, significant differences between the three groups were found in 11 CpGs (29.73 %), using ANOVA. Tukey's post hoc test on the significant results indicated that seven (63.63 %) of these differences were significant between the donor oocyte ART and fertile control groups. In addition, 20 of the 37 CpGs analyzed had been identified as differentially methylated between ART and fertile control groups in an independent population in a prior study. Of these 20 CpG sites, 9 also showed significant differences in the present population. An additional 9 CpGs were found to be significantly different between the two groups. Of these 18 candidate CpGs, 12 CpGs (in seven candidate genes) also showed significant differences in placental DNA methylation levels between the donor oocyte ART and fertile control groups., Conclusions: These data suggest strongly that the DNA methylation differences observed between ART and in vivo conceptions are associated with some aspect of ART protocols, not simply the underlying infertility.

Objective: To investigate the impact of intracytoplasmic sperm injection (ICSI) and assisted hatching (AH) on assisted reproductive technology (ART) outcomes in initial cycles with diminished ovarian reserve (DOR) as the primary diagnosis., Design: Retrospective cohort study of cycles from the Society for Assisted Reproductive Technology (SART) Clinic Outcome Reporting System database., Setting: Not applicable., Patient(s): A total of 422,949 fresh, nondonor, initial ART cycles of which 8,597 were diagnosed with only elevated FSH and 38,926 were diagnosed with only DOR according to the SART DOR categorization., Intervention(s): None., Main Outcome Measure(s): Live birth and clinical pregnancy rates., Result(s): ICSI and AH were associated with diminished odds of live birth in SART DOR-only cycles (adjusted odds ratio [AOR] 0.88, 95% confidence interval [CI] 0.81-0.96 for ICSI; AOR 0.77, 95% CI 0.71-0.84 for AH). No association between odds of live birth and either ICSI or AH in elevated FSH-only cycles was observed. The combination of ICSI and AH was associated with significantly lower odds of live birth in SART DOR-only cycles but not in elevated FSH-only cycles., Conclusion(s): In initial ART cycles for which the only indication relates to a diagnosis of DOR, AH and ICSI are not associated with improved live birth rates., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Epidemiologic studies have shown an increased rate of adverse perinatal outcomes, including small for gestational age (SGA) births, in fresh in vitro fertilization (IVF) cycles compared with frozen embryo transfer cycles. This increase is not seen in the donor oocyte population, suggesting that it is the peri-implantation environment created after superovulation that is responsible for these changes. During a fresh IVF cycle, multiple corpora lutea secrete high levels of hormones and other factors that can affect the endometrium and the implanting embryo. In this review, we discuss both animal and human data demonstrating that superovulation has significant effects on the endometrium and embryo. Additionally, potential mechanisms for the adverse effects of gonadotropin stimulation on implantation and placental development are proposed. We think that these data, along with the growing body of epidemiologic evidence, support the proposal that frozen embryo transfer should be considered preferentially, particularly in high responders, as a means to potentially decrease at least some of the adverse perinatal outcomes associated with IVF., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: The tetraspan protein epithelial membrane protein-2 (EMP2), which mediates surface display of diverse proteins, is required for endometrial competence in blastocyst implantation, and is uniquely correlated with poor survival from endometrial adenocarcinoma tumors. Because EMP2 is differentially expressed in the various stages of the murine and human estrous cycle, we tested the hypothesis that the steroid hormones progesterone and estrogen influence EMP2 expression and localization., Methods: Frozen human proliferative and secretory endometrium were collected and analyzed for EMP2 expression using SDS-PAGE/Western blot analysis. The response of EMP2 to progesterone and estradiol was determined using a combination of real-time PCR, SDS-PAGE/Western blot analysis, and confocal immunofluorescence in the human endometrial carcinoma cell line RL95-2. To confirm the in vitro results, ovariectomized mice were treated with progesterone or estradiol, and EMP2 expression was analyzed using immunohistochemistry., Results: Within normal human endometrium, EMP2 expression is upregulated in the secretory phase relative to the proliferative phase. To understand the role of steroid hormones on EMP2 expression, we utilized RL95-2 cells, which express both estrogen and progesterone receptors. In RL95-2 cells, both estradiol and progesterone induced EMP2 mRNA expression, but only progesterone induced EMP2 protein expression. To compare steroid hormone regulation of EMP2 between humans and mice, we analyzed EMP2 expression in ovarectomized mice. Similar to results observed in humans, progesterone upregulated endometrial EMP2 expression and induced EMP2 translocation to the plasma membrane. Estradiol did not promote translocation to the cell surface, but moderately induced EMP2 expression in cytoplasmic compartments in vivo., Conclusion: These findings suggest that targeting of EMP2 to specific locations under the influence of these steroid hormones may be important for integrating the molecular responses required for implantation competence.

The establishment of pregnancy requires a successful molecular interaction between the trophectoderm cells of the blastocyst stage embryo and the endometrial cells of the uterus. These interactions are complex and require synchronous development and coordinated endocrine, paracrine, and autocrine communication. In this study, we demonstrate that the tetraspan protein epithelial membrane protein-2 (EMP2) is involved in these molecular interactions during implantation. EMP2, which is highly expressed in the uterus, translocates from an intracellular location to the apical surface of the endometrial epithelium during the window of implantation and is expressed in decidualized stromal cells. We developed plasmid constructs that utilized either ribozyme-mediated or short hairpin RNA-mediated mechanisms to target endometrial EMP2 mRNA for destruction. These constructs were transfected into the mouse uterus on day 1 of pregnancy using the technique of in vivo reproductive tract gene transfer. Reduction in EMP2 expression by either method resulted in a significant decrease in the number of implantation sites in the treated uterine horns as compared to control horns. These studies indicate a previously unknown function of tetraspan proteins in implantation and could provide a molecular framework for the development of therapeutic modalities for both contraception and fertility.