Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ.

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.

TRIM71 mutations cause a neurodevelopmental syndrome featuring ventriculomegaly and hydrocephalus.

Congenital hydrocephalus (CH), characterized by cerebral ventriculomegaly, is one of the most common reasons for pediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate CH risk gene, however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated CH (totaling 2,697 parent-proband trios and 8,091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, CH, developmental delay, dysmorphic features, and other structural brain defects including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbor arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven additional patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome featuring ventriculomegaly and CH.

High-throughput mRNA-seq atlas of human placenta shows vast transcriptome remodeling from first to third trimester†.

The placenta, composed of chorionic villi, changes dramatically across gestation. Understanding differences in ongoing pregnancies are essential to identify the role of chorionic villi at specific times in gestation and develop biomarkers and prognostic indicators of maternal-fetal health. The normative mRNA profile is established using next-generation sequencing of 124 first trimester and 43 third trimester human placentas from ongoing healthy pregnancies. Stably expressed genes (SEGs) not different between trimesters and with low variability are identified. Differential expression analysis of first versus third trimester adjusted for fetal sex is performed, followed by a subanalysis with 23 matched pregnancies to control for subject variability using the same genetic and environmental background. Placenta expresses 14,979 polyadenylated genes above sequencing noise (transcripts per million > 0.66), with 10.7% SEGs across gestation. Differentially expressed genes (DEGs) account for 86.7% of genes in the full cohort [false discovery rate (FDR) < 0.05]. Fold changes highly correlate between the full cohort and subanalysis (Pearson = 0.98). At stricter thresholds (FDR < 0.001, fold change > 1.5), there remains 50.1% DEGs (3353 upregulated in first and 4155 upregulated in third trimester). This is the largest mRNA atlas of healthy human placenta across gestation, controlling for genetic and environmental factors, demonstrating substantial changes from first to third trimester in chorionic villi. Specific differences and SEGs may be used to understand the specific role of the chorionic villi throughout gestation and develop first trimester biomarkers of placental health that transpire across gestation, which can be used for future development of biomarkers for maternal-fetal health.

Sex differences in microRNA expression in first and third trimester human placenta†.

Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be due to sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing identified miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n = 113) and parturition (n = 47). Sequencing analysis identified 986 expressed mature miRNAs from female and male placentae at first and third trimester (baseMean>10). Of these, 11 sexually dimorphic (FDR < 0.05) miRNAs were identified in the first and 4 in the third trimester, all upregulated in females, including miR-361-5p, significant in both trimesters. Sex-specific analyses across gestation identified 677 differentially expressed (DE) miRNAs at FDR < 0.05 and baseMean>10, with 508 DE miRNAs in common between female-specific and male-specific analysis (269 upregulated in first trimester, 239 upregulated in third trimester). Of those, miR-4483 had the highest fold changes across gestation. There were 62.5% more female exclusive differences with fold change>2 across gestation than male exclusive (52 miRNAs vs 32 miRNAs), indicating miRNA expression across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex-independent and sex-specific placenta miRNA atlas across gestation, which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences.

Clinical utility of the endometrial receptivity analysis in women with prior failed transfers.

PURPOSE: To determine the utility of the endometrial receptivity analysis (ERA) in women with prior failed embryo transfers (ET). METHODS: This was a retrospective study of patients who underwent an ERA test with a subsequent frozen ET. Women were classified based on their indication for an ERA test: (1) ≥ 1 prior failed ET (cases), or (2) as a prophylactic measure (controls). A subset analysis of women with ≥ 3 prior failed transfers was performed. Pregnancy outcomes of the subsequent cycle were examined, including conception, clinical pregnancy, and ongoing pregnancy/live birth. RESULTS: A total of 222 women were included, 131 (59%) women with ≥ 1 prior failed ET and 91 (41%) controls. Among the 131 women with ≥ 1 prior failed ET, 20 women (9%) had ≥ 3 prior failed ETs. The proportion of non-receptive ERA tests in the three groups were the following: 45% (≥ 1 prior failed ET), 40% (≥ 3 prior failed ETs), and 52% (controls). The results did not differ between cases and controls. The pregnancy outcomes did not differ between women with ≥ 1 prior failed ET and controls. In women with ≥ 3 prior failed ETs, there was a lower ongoing pregnancy/live birth rate (28% vs 54%, P = 0.046). CONCLUSION: Women with ≥ 1 prior failed ET and ≥ 3 prior failed ETs had a similar prevalence of non-receptive endometrium compared to controls. Women with ≥ 3 prior failed ETs had a lower ongoing pregnancy/live birth rate despite a personalized FET, suggesting that there are additional factors in implantation failure beyond an adjustment in progesterone exposure.

Mode of conception does not affect fetal or placental growth parameters or ratios in early gestation or at delivery.

PURPOSE: Ratio of fetal weight to placenta size varies by mode of conception (fertility treatments utilized) in animals. Our objective was to assess whether fertility treatments also affect these ratios in humans. METHODS: In this retrospective study, we assessed two cohorts: (a) early gestation cohort, women with singleton pregnancies who underwent first trimester vaginal ultrasound and (b) delivered cohort, women who delivered a live-born, singleton infant with placenta disposition to pathology. Crown rump length (CRL) and estimated placental volume (EPV) were calculated from first trimester ultrasound images using a validated computation. Infant birth weight (BW), pregnancy data, placental weight (PW), and placental histopathology were collected. Fetal growth-to-placental weight ratios (CRL/EPV; BW/PW) and placentas were compared by mode of conception. Linear regression was used to adjust for confounding variables. RESULTS: Two thousand one hundred seventy patients were included in the early gestation cohort and 1443 in the delivered cohort. Of the early gestation cohort (a), 85.4% were spontaneous conceptions, 5.9% Non-IVF Fertility (NIFT), and 8.7% IVF. In the delivered cohort (b), 92.4% were spontaneous, 2.1% NIFT, and 80 5.5% IVF. There were no significant differences between fetal growth-to-placental weight parameters, ratios, and neonatal birth measurements based on mode of conception. Placenta accreta was significantly higher in the patients receiving fertility treatments (1.2 versus 3.6%, p < 0.05). CONCLUSIONS: Mode of conception does not appear to influence fetal growth-to-placental weight ratios throughout gestation. In addition, findings in animal models may not always translate into human studies of infertility treatment outcomes.

Fertility Treatment Is Associated with Stay in the Neonatal Intensive Care Unit and Respiratory Support in Late Preterm Infants.

Late preterm infants are at risk for short-term morbidities. We report that late preterm singletons conceived with fertility treatment have increased risk for admission to the neonatal intensive care unit and respiratory support compared with spontaneously conceived infants. Fertility treatment may be a risk factor to consider in managing late preterm infants.

Childbirth Education Class and Birth Plans Are Associated with a Vaginal Delivery.

BACKGROUND: To determine whether the mode of delivery was different between women who attended childbirth education (CBE) class, had a birth plan, or both compared with those who did not attend CBE class or have a birth plan. METHODS: This is a retrospective cross-sectional study of women who delivered singleton gestations > 24 weeks at our institution between August 2011 and June 2014. Based on a self-report at the time of admission for labor, women were stratified into four categories: those who attended a CBE class, those with a birth plan, both, and those with neither CBE or birth plan. The primary outcome was the mode of delivery. Multivariate logistic regression analyses adjusting for clinical covariates were performed. RESULTS: In this study, 14,630 deliveries met the inclusion criteria: 31.9 percent of the women attended CBE class, 12.0 percent had a birth plan, and 8.8 percent had both. Women who attended CBE or had a birth plan were older (p < 0.001), more likely to be nulliparous (p < 0.001), had a lower body mass index (p < 0.001), and were less likely to be African-American (p < 0.001). After adjusting for significant covariates, women who participated in either option or both had higher odds of a vaginal delivery (CBE: OR 1.26 [95% CI 1.15-1.39]; birth plan: OR 1.98 [95% CI 1.56-2.51]; and both: OR 1.69 [95% CI 1.46-1.95]) compared with controls. CONCLUSION: Attending CBE class and/or having a birth plan were associated with a vaginal delivery. These findings suggest that patient education and birth preparation may influence the mode of delivery. CBE and birth plans could be used as quality improvement tools to potentially decrease cesarean rates.

Function and Hormonal Regulation of GATA3 in Human First Trimester Placentation.

Pregnancies resulting from fresh in vitro fertilization (IVF) cycles exposed to supraphysiologic estrogen levels have been associated with higher rates of low birth weight and small for gestational age babies. We identified GATA3, a transcription factor selectively expressed in the trophectoderm during the blastocyst stage of embryo development, in an upstream analysis of genes that were differentially methylated in chorionic villus samples between IVF and non-IVF infertility treatment pregnancies. In this study, we investigate the hypothesis that GATA3 is hormonally regulated and plays an important functional role in trophoblast migration, invasion, and placentation. We found that GATA3 expression was hormonally regulated by estradiol in HTR8/SVneo first trimester trophoblast cells; however, no change in expression was seen with progesterone treatment. Furthermore, GATA3 knockdown resulted in decreased HTR8/SVneo cell migration and invasion compared with controls. RNA sequencing of GATA3 knockdown cells demonstrated 96 differentially regulated genes compared with controls. Genes known to play an important role in cell-cell and cell-extracellular matrix interactions, cell invasion, and placentation were identified, including CTGF, CYR61, ADAMTS12, and TIMP3 Our results demonstrate estradiol down-regulates GATA3, and decreased GATA3 expression leads to impaired trophoblast cell migration and invasion, likely through regulation of downstream genes important in placentation. These results are consistent with clinical data suggesting that supraphysiologic estrogen levels seen in IVF pregnancies may play an important role in attenuated trophoblast migration, invasion, and impaired placentation. GATA3 appears to be an important regulator of placentation and may play a role in impaired outcomes associated with fresh IVF cycles.

Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

BACKGROUND: Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. RESULTS: Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CONCLUSIONS: CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd.

Basal metabolic rate in women with PCOS compared to eumenorrheic controls.

OBJECTIVE: PCOS is associated with obesity and insulin resistance. Efforts have focused on whether an abnormal energy homeostasis contributes to the development of obesity in these patients. There are conflicting results in the literature regarding whether women with PCOS have an altered basal metabolic rate (BMR), thereby leading to difficulties in weight loss. The objective of this study is to compare basal metabolic rate (BMR) in women with PCOS and controls. DESIGN: Cross-sectional study. PATIENTS: One hundred and twenty-eight PCOS patients diagnosed by original NIH consensus criteria and 72 eumenorrheic, non-hirsute controls were recruited from an academic medical centre. MEASUREMENTS: Assessment of BMR using the InBody portable bioelectrical impedance analysis (BIA) device and insulin resistance by HOMA-IR indices. RESULTS: PCOS women were younger than controls. As expected, PCOS subjects had higher body mass index (BMI), serum androgens and estimated insulin resistance. After adjusting for age and BMI, there was no significant difference in BMR between PCOS subjects (adjusted mean 5807 kJ/day, 95% CI 5715-5899) and controls (adjusted mean 5916 kJ/day, 95% CI 5786-6046) (P = 0·193). BMR was also comparable in a secondary analysis comparing PCOS women with and without insulin resistance. CONCLUSIONS: After adjusting for age and BMI, there was no difference in BMR between PCOS women and controls.

Utilization of noninvasive prenatal testing: impact on referrals for diagnostic testing.

OBJECTIVE: Since the introduction of noninvasive prenatal testing (NIPT), a marked decrease in prenatal diagnostic testing (chorionic villus sampling [CVS] and amniocentesis) has been observed with unknown potential effects on genetic diagnosis of these pregnancies. The purpose of this study was to understand the impact of NIPT on genetics counseling referrals, diagnostic testing with CVS/amniocentesis, and appropriate use of NIPT. STUDY DESIGN: A retrospective cohort study was performed on all women referred for genetic counseling and prenatal testing during the 2 years preceding the introduction of NIPT (pre-NIPT) and 2 years following (post-NIPT). The primary outcome was the difference in the number of women referred for genetic counseling and prenatal diagnosis during the pre-NIPT period compared with the post-NIPT period. The secondary outcome was the difference in the number of women referred who were not considered candidates for NIPT between the 2 study periods. RESULTS: There was a statistically significant reduction in the number of referrals for genetic counseling and diagnostic testing in the post-NIPT compared with the pre-NIPT period (2824 vs 3944, P = .001), a reduction of 28.4%. During the post-NIPT period there was a significant reduction in referrals of women who would not be candidates for NIPT (467 pre-NIPT vs 285 post-NIPT, P = .043). In women who had diagnostic testing with CVS during the study period, 32.4% of the aneuploidies identified would not have been detected by NIPT. CONCLUSION: There was a significant reduction in the number of patients referred for genetic counseling and prenatal diagnosis following the introduction of NIPT. In addition, there was a significant reduction in the number of patients referred for counseling and testing who would not be candidates for NIPT. This suggests that an increasing number of potential patients are being offered NIPT screening instead of diagnostic testing, including those at risk for fetal single gene disorders and aneuploidies not detectable by NIPT, potentially leading to misdiagnosis.

Correlation of Polycystic Ovarian Syndrome Phenotypes With Pregnancy and Neonatal Outcomes.

OBJECTIVE: To compare pregnancy and neonatal outcomes in women with hyperandrogenic polycystic ovarian syndrome (PCOS) phenotypes compared with nonhyperandrogenic PCOS phenotypes. METHODS: We conducted a retrospective cohort study of participants in the PPCOS (Pregnancy in Polycystic Ovary Syndrome) I and II randomized controlled trials; all of the participants met the National Institutes of Health diagnostic criteria for PCOS and were then sorted into three of the four Rotterdam criteria categories based on medical interview, demographics, physical examination, and laboratory data. The two hyperandrogenic (A and B) Rotterdam categories were compared with the nonhyperandrogenic phenotype of PCOS (phenotype D). Our outcomes of interest were clinical pregnancy, pregnancy loss, live birth, obstetric complications (including preterm labor, preeclampsia, gestational diabetes, intrauterine growth restriction, and premature rupture of membranes), and neonatal outcomes (including jaundice, respiratory distress syndrome, neonatal hospitalization, and neonatal infection). RESULTS: Of the 1,376 participants included in the study, 1,249 (90.8%) had hyperandrogenic PCOS phenotypes compared with 127 (9.2%) nonhyperandrogenic PCOS (nonhyperandrogenic PCOS). Compared with participants with nonhyperandrogenic PCOS, those with hyperandrogenic PCOS had higher body mass index (BMI) (35.5±8.9 vs 31.9±9.3 kg/m 2 , P <.001), fasting insulin (21.6±27.7 vs 14.7±15.0 micro-international units/mL, P <.001), and homeostatic model assessment for insulin resistance score (5.01±9.1 vs 3.4±4.1, P =.0002). Age and race were similar between groups. Months attempting pregnancy were greater in participants with hyperandrogenic PCOS compared with nonhyperandrogenic PCOS (41.8±37.3 vs 33.9±32.0). The proportion of participants who achieved pregnancy (29.9% vs 40.2%, P =.02) and live birth rates (20.1% vs 33.1%, P =.001) were lower among those with hyperandrogenic PCOS compared with nonhyperandrogenic PCOS, although pregnancy loss rates did not differ significantly (23.9% vs 32.3%, P =.06). The hyperandrogenic PCOS group had lower odds of live birth compared with the nonhyperandrogenic PCOS group (odds ratio [OR] 0.51, CI, 0.34-0.76), even after adjusting for BMI (adjusted odds ratio [aOR] 0.59, CI, 0.40-0.89). The hyperandrogenic PCOS group also had lower odds of achieving pregnancy compared with the nonhyperandrogenic PCOS group (OR 0.63, CI, 0.44-0.92); however, this association was no longer significant after adjusting for BMI (aOR 0.74, CI, 0.50-1.10). The overall low prevalence of prenatal complications and neonatal outcomes precluded a meaningful comparison between the two groups. CONCLUSION: Participants with hyperandrogenic PCOS achieved lower rates of pregnancy and live birth compared with those with nonhyperandrogenic PCOS. Evaluating distinct PCOS phenotypes may allow for individualized guidance regarding the probability of pregnancy and live birth. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov , NCT00068861 and NCT00718186.

Alterations in nonesterified free fatty acid trafficking rather than hyperandrogenism contribute to metabolic health in obese women with polycystic ovary syndrome.

OBJECTIVE: To determine whether alterations in nonesterified fatty acid (NEFA) dynamics or degree of hyperandrogenism (HA) contribute to the difference in insulin sensitivity between women with metabolically healthy obese polycystic ovary syndrome (PCOS) (MHO-PCOS) and women with metabolically unhealthy obese PCOS (MUO-PCOS). DESIGN: Prospective cross-sectional study. SETTING: Tertiary-care academic center. PATIENTS: One hundred twenty-five obese women with PCOS. INTERVENTION: Consecutive obese (body mass index [BMI] ≥ 30 kg/m2) oligo-ovulatory women (n = 125) with PCOS underwent an oral glucose tolerance test and a subgroup of 16 participants underwent a modified frequently sampled intravenous glucose tolerance test to determine insulin-glucose and -NEFA dynamics. MAIN OUTCOME MEASURES: Degree of insulin resistance (IR) in adipose tissue (AT) basally (Adipo-IR) and dynamically (the nadir in NEFA levels observed [NEFAnadir], the time it took for NEFA levels to reach nadir [TIMEnadir], and the percent suppression in plasma NEFA levels from baseline to nadir [%NEFAsupp]); peak lipolysis rate (SNEFA) and peak rate of NEFA disposal from plasma pool (KNEFA); whole-body insulin-glucose interaction (acute response of insulin to glucose [AIRg], insulin sensitivity index [Si], glucose effectiveness [Sg], and disposition index [Di]); and HA (hirsutism score, total and free testosterone levels, and dehydroepiandrosterone sulfate levels). RESULTS: A total of 85 (68%) women were MUO-PCOS and 40 (32%) were MHO-PCOS using the homeostasis model of assessment of IR. Subjects with MUO-PCOS and MHO-PCOS did not differ in mean age, BMI, waist-to-hip ratio, HA, and lipoprotein levels. By a modified frequently sampled intravenous glucose tolerance test, eight women with MUO-PCOS had lesser Si, KNEFA, and the percent suppression in plasma NEFA levels from baseline to nadir (%NEFAsupp) and greater TIMEnadir, NEFAnadir, and baseline adipose tissue IR index (Adipo-IR) than eight subjects with MHO-PCOS, but similar fasting NEFA levels and SNEFA. Women with MUO-PCOS had a higher homeostasis model of assessment-ß% and fasting insulin levels than women with MHO-PCOS. In bivalent analysis, Si correlated strongly and negatively with Adipo-IR and NEFAnadir, weakly and negatively with TIMEnadir, and positively with KNEFA and %NEFAsupp, in women with MUO-PCOS only. CONCLUSION: Independent of age and BMI, women with MUO-PCOS have reduced NEFA uptake and altered insulin-mediated NEFA suppression, but no difference in HA, compared with women with MHO-PCOS. Altered insulin-mediated NEFA suppression, rather than HA or lipolysis rate, contributes to variations in insulin sensitivity among obese women with PCOS.

A systematic review of genome-wide analyses of methylation changes associated with assisted reproductive technologies in various tissues.

IMPORTANCE: Because analytic technologies improve, increasing amounts of data on methylation differences between assisted reproductive technology (ART) and unassisted conceptions are available. However, various studies use different tissue types and different populations in their analyses, making data comparison and integration difficult. OBJECTIVE: To compare and integrate data on genome-wide analyses of methylation differences due to ART, allowing exposure of overarching themes. EVIDENCE REVIEW: All studies undertaking genome-wide analysis of human methylation differences due to ART or infertility in any tissue type across the lifespan were assessed for inclusion. FINDINGS: Seventeen studies were identified that met the inclusion criteria. One study assessed trophectoderm biopsies, 2 first-trimester placenta, 1 first-trimester fetal tissue, 2 term placenta, 7 cord blood, 3 newborn dried blood spots, 1 childhood buccal smears, 1 childhood peripheral blood, and 2 adult peripheral blood. Eleven studies compared tissues from in vitro fertilization (IVF) conceptions with those of unassisted conceptions, 4 compared intracytoplasmic sperm injection with unassisted conceptions, 4 compared non-IVF fertility treatment (NIFT) with unassisted conceptions, 4 compared NIFT with IVF, and 5 compared an infertile population (conceiving via various methods) with an unassisted presumably fertile population. In studies assessing placental tissue, 1 gene with potential methylation changes due to IVF when compared with unassisted conceptions was identified by 2 studies. In blood, 11 potential genes with methylation changes due to IVF compared with unassisted conceptions were identified by 2 studies, 1 of which was identified by 3 studies. Three potentially affected genes were identified by 2 studies involving blood between intracytoplasmic sperm injection and unassisted populations. There were no overlapping genes identified in any tissue type between NIFT and unassisted populations, between NIFT and IVF, or the infertility combined population when compared with the unassisted fertile population. CONCLUSIONS: Comparing studies is challenging due to differing variables between analyses. However, even in similar tissue types and populations, overlapping methylation changes are limited, suggesting that differences due to ART are minimal. RELEVANCE: Information from this systematic review is significant for providers and patients who provide and use ART to understand methylation risks that may be associated with the technology.

Leveraging chorionic villus biopsies for the derivation of patient-specific trophoblast stem cells.

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TS CV ) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TS CT ) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development.

Sexually dimorphic DNA methylation and gene expression patterns in human first trimester placenta.

BACKGROUND: Fetal sex and placental development impact pregnancy outcomes and fetal-maternal health, but the critical timepoint of placenta establishment in first trimester is understudied in human pregnancies. METHODS: Pregnant subjects were recruited in late first trimester (weeks 10-14) at time of chorionic villus sampling, a prenatal diagnostic test. Leftover placenta tissue was collected and stored until birth outcomes were known, then DNA and RNA were isolated from singleton, normal karyotype pregnancies resulting in live births. DNA methylation was measured with the Illumina Infinium MethylationEPIC BeadChip array (n = 56). Differential methylation analysis compared 25 females versus 31 males using a generalized linear model on 743,461 autosomal probes. Gene expression sex differences were analyzed with RNA-sequencing (n = 74). An integrated analysis was performed using linear regression to correlate gene expression and DNA methylation in 51 overlapping placentas. RESULTS: Methylation analysis identified 151 differentially methylated probes (DMPs) significant at false discovery rate < 0.05, including 89 (59%) hypermethylated in females. Probe cg17612569 (GABPA, ATP5J) was the most significant CpG site, hypermethylated in males. There were 11 differentially methylated regions affected by fetal sex, with transcription factors ZNF300 and ZNF311 most significantly hypermethylated in males and females, respectively. RNA-sequencing identified 152 genes significantly sexually dimorphic at false discovery rate < 0.05. The 151 DMPs were associated with 18 genes with gene downregulation (P < 0.05) in the direction of hypermethylation, including 2 genes significant at false discovery rate < 0.05 (ZNF300 and CUB and Sushi multiple domains 1, CSMD1). Both genes, as well as Family With Sequence Similarity 228 Member A (FAM228A), showed significant correlation between DNA methylation and sexually dimorphic gene expression, though FAM228A DNA methylation was less sexually dimorphic. Comparison with other sex differences studies found that cg17612569 is male-hypermethylated across gestation in placenta and in human blood up to adulthood. CONCLUSIONS: Overall, sex dimorphic differential methylation with associated differential gene expression in the first trimester placenta is small, but there remain significant genes that may be regulated through methylation leading to differences in the first trimester placenta.

Fetal sex and placenta development affect pregnancy outcomes for both the fetus and mother throughout pregnancy, including risk of miscarriages, preterm birth, preeclampsia, and other outcomes. Epigenetics, the "overlay" of regulatory signals on DNA which affects how DNA is read, is not well understood in early pregnancy when critical placenta developments are happening that affect the rest of pregnancy. Here, we use leftover placenta biopsy samples (n = 56) donated by Cedars-Sinai patients with informed consent to learn about first trimester human placenta DNA methylation differences due to fetal sex. Out of the total 743,461 sites analyzed, we identified 151 sites significantly affected by fetal sex after correcting p-values to reduce false positives (false discovery rate < 0.05). We also performed an analysis to look at multiple sites and identified 11 regions across the genome with significant DNA methylation changes due to fetal sex. Furthermore, because DNA methylation is a regulatory mark on DNA which typically dampens gene expression, we also compared the DNA methylation sex differences to placental RNA-sequencing gene expression analysis using the same tissue from a mostly overlapping patient group (n = 74 total sequenced, n = 51 overlap). We identify 18 genes which show both significant DNA methylation differences and gene expression changes. The most significant gene was transcription factor ZNF300 with higher DNA methylation in males and reduced gene expression in males (and thus higher gene expression in females). This study identifies some sex differences that continue until later pregnancy and others that are unique to first trimester.

High-throughput mRNA sequencing of human placenta shows sex differences across gestation.

INTRODUCTION: Fetal sex affects fetal and maternal health outcomes in pregnancy, but this connection remains poorly understood. As the placenta is the route of fetomaternal communication and derives from the fetal genome, placental gene expression sex differences may explain these outcomes. OBJECTIVES: We utilized next generation sequencing to study the normal human placenta in both sexes in first and third trimester to generate a normative transcriptome based on sex and gestation. STUDY DESIGN: We analyzed 124 first trimester (T1, 59 female and 65 male) and 43 third trimester (T3, 18 female and 25 male) samples for sex differences within each trimester and sex-specific gestational differences. RESULTS: Placenta shows more significant sexual dimorphism in T1, with 94 T1 and 26 T3 differentially expressed genes (DEGs). The sex chromosomes contributed 60.6% of DEGs in T1 and 80.8% of DEGs in T3, excluding X/Y pseudoautosomal regions. There were 6 DEGs from the pseudoautosomal regions, only significant in T1 and all upregulated in males. The distribution of DEGs on the X chromosome suggests genes on Xp (the short arm) may be particularly important in placental sex differences. Dosage compensation analysis of X/Y homolog genes shows expression is primarily contributed by the X chromosome. In sex-specific analyses of first versus third trimester, there were 2815 DEGs common to both sexes upregulated in T1, and 3263 common DEGs upregulated in T3. There were 7 female-exclusive DEGs upregulated in T1, 15 female-exclusive DEGs upregulated in T3, 10 male-exclusive DEGs upregulated in T1, and 20 male-exclusive DEGs upregulated in T3. DISCUSSION: This is the largest cohort of placentas across gestation from healthy pregnancies defining the normative sex dimorphic gene expression and sex common, sex specific and sex exclusive gene expression across gestation. The first trimester has the most sexually dimorphic transcripts, and the majority were upregulated in females compared to males in both trimesters. The short arm of the X chromosome and the pseudoautosomal region is particularly critical in defining sex differences in the first trimester placenta. As pregnancy is a dynamic state, sex specific DEGs across gestation may contribute to sex dimorphic changes in overall outcomes.

Racial and ethnic disparities in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that impacts women worldwide. There are several racial and ethnic differences in PCOS phenotypes and in PCOS- associated metabolic dysfunction. In this review, we summarize the current literature on disparities in the diagnosis and outcomes associated with PCOS in the United States. Future studies are needed to address gaps in knowledge for racial and ethnic-specific differences in PCOS, and include a large number of non-White and/or Hispanic participants in PCOS studies.

Regional Variation in Hormonal and Metabolic Parameters of White and Black Women With PCOS in the United States.

CONTEXT: Ongoing research is needed to determine geo-epidemiologic differences of polycystic ovary syndrome (PCOS). OBJECTIVE: Determine hormonal and metabolic parameters of women with PCOS in 2 environments. METHODS: Prospective cohort study. SETTING: Tertiary-care based specialty clinics in Alabama and California. PATIENTS OR OTHER PARTICIPANTS: A total of 1610 women with PCOS by National Institutes of Health Criteria from 1987 to 2010. INTERVENTIONS: Interview, physical examination, laboratory studies. MAIN OUTCOMES MEASURES: Demographic data, menstrual cycle history, and hormonal and metabolic parameters were collected. Hirsutism was defined as modified Ferriman-Gallwey scores ≥4. Androgen values greater than laboratory reference ranges or >95th percentile of all values were considered elevated (hyperandrogenemia). Metabolic parameters included body mass index (BMI), waist-hip-ratio (WHR), glucose tolerance test, and homeostatic model assessment for insulin resistance (HOMA-IR) scores. RESULTS: Alabama women with PCOS were younger with a higher BMI. After adjustment for age and BMI, Alabama women with PCOS were more likely hirsute (adjusted odds ratio [aOR], 1.8; 95% CI, 1.4-2.4; P < 0.001), with elevated HOMA-IR scores (adjusted beta coefficient 3.6; 95% CI, 1.61-5.5; P < 0.001). California women with PCOS were more likely to have hyperandrogenemia (free testosterone aOR, 0.14; 95% CI, 0.11-0.18; P < 0.001; total testosterone aOR, 0.41; 95% CI, 0.33-0.51). Results were similar when stratified by White race. In Black women with PCOS, BMI and WHR did not differ between locations, yet differences in androgen profiles and metabolic dysfunction remained. CONCLUSION: Alabama women with PCOS, regardless of Black or White race, were more likely hirsute with metabolic dysfunction, whereas California women with PCOS were more likely to demonstrate hyperandrogenemia, highlighting potential environmental impacts on PCOS.