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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., (© 2016 Wiley Periodicals, Inc.)

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., Competing Interests: none Competing Interests: The authors declare no competing interests., (© 2016 John Wiley & Sons, Ltd.)

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., (© 2016 by the Society for the Study of Reproduction, Inc.)

Objective: To determine if fertility treatment is associated with increased risk of severe maternal morbidity (SMM) compared with spontaneous pregnancies., Design: Retrospective cohort study., Setting: Academic medical center., Patient(s): In 2012, 6,543 women delivered live births >20 weeks gestation at our center. Women were categorized based on mode of conception: in vitro fertilization (IVF), non-IVF fertility treatment (NIFT), or spontaneous pregnancies., Intervention(s): None., Main Outcome Measure(s): The main outcome was presence of true SMM, such as eclampsia, respiratory failure, and peripartum hysterectomy. Deliveries were screened with the use of: 1) International Classification of Diseases 9 codes; 2) prolonged postpartum stay; 3) maternal intensive care unit admissions, and 4) blood transfusion. The charts of women meeting the screening criteria were reviewed to identify true SMM based on a previously validated method, recognizing that medical record review is the criterion standard., Result(s): Of the 6,543 deliveries, 246 (3.8%) were IVF conceptions and 109 (1.7%) NIFT conceptions. Sixty-nine cases of true SMM were identified (1.1%). In multivariate analyses, any fertility treatment (IVF + NIFT) was associated with increased risk of SMM compared with spontaneous conceptions. In a subset analysis of singletons only, the association between any fertility treatment (IVF + NIFT) and SMM was not statistically significant., Conclusion(s): Overall, fertility treatment increased risk for SMM events. Given the limited sample size, the negative finding with singleton gestations is inconclusive. Larger multicenter studies with accurate documentation of fertility treatment and SMM cases are needed to further clarify the risk associated with singletons., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Normal placentation during the first trimester sets the stage for the rest of pregnancy and involves a finely orchestrated cellular and molecular interplay of maternal and fetal tissues. The resulting intrauterine environment plays an important role in fetal programming and the future health of the fetus, and is impacted by multiple genetic and epigenetic factors. Abnormalities in placentation and spiral artery invasion can lead to ischemia, placental disease, and adverse obstetrical outcomes including preeclampsia, intrauterine growth restriction, and placental abruption. Although first trimester placentation is affected by multiple factors, preconception environmental influences such as mode of conception, including assisted reproductive technologies which result in fertilization in vitro and intrauterine influences due to sex differences, are emerging as potential significant factors impacting first trimester placentation., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women and the leading cause of anovulatory infertility. The prevalence of the syndrome ranges between 6 to 15% based on broader Rotterdam diagnostic criteria verses strict NIH diagnostic criteria.1 The condition is characterized by a combination of ovulatory dysfunction, hyperandrogenism and the presence of polycystic ovaries. PCOS has been associated with multiple metabolic alterations and consequences including impaired glucose tolerance, insulin resistance, hyperinsulinemia, type II diabetes, dyslipidemia, metabolic syndrome, obesity and subclinical cardiovascular disease. It remains unclear however if these associations lead to an increased risk of clinically significant long-term cardiovascular disease. Large prospective studies to date have not detected significant differences in overall cardiovascular morbidity and mortality in PCOS. The phenotypical variability in PCOS has made researching each of these associations challenging as different aspects of the syndrome may be contributing, opposing or confounding factors. The ability to detect significant differences in long-term cardiovascular outcomes may also be due to the variable nature of the syndrome. In this review, we attempt to describe a summary of the current literature concerning the metabolic alterations and cardiovascular consequences of polycystic ovary syndrome.

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., (© 2015 John Wiley & Sons Ltd.)

The process of implantation is highly complex and involves a delicate interplay between the embryo and the appropriate maternal environment. The failure to implant is thought to be due to maternal factors or embryonic factors. Inflammation can be a part of the normal physiologic process during implantation; however, there are also pathologic entities that adversely affect uterine receptivity. This review will focus on chronic endometritis and hydrosalpinges as two specific inflammatory processes that contribute to implantation failure. For both chronic endometritis and hydrosalpinges, we will review the diagnosis, pathophysiology, and effect on implantation following treatment. The existing literature conclusively demonstrates that hydrosalpinges should be addressed by either laparoscopic salpingectomy or proximal tubal occlusion prior to in vitro fertilization. The picture for chronic endometritis is less clear since the diagnosis and treatment of chronic endometritis are not standardized, and there are no available randomized controlled trials on this topic. Future studies may target gene expression arrays as a method for further elucidating the role of inflammatory markers in normal and abnormal implantation processes., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To evaluate the relationship between blastomere number and aneuploidy., Design: Historical cohort study., Setting: In vitro fertilization clinic., Patient(s): Two hundred fifty-nine patients undergoing in vitro fertilization (IVF) in combination with comprehensive chromosomal screening of embryos., Intervention(s): A total of 1,915 embryos were biopsied on day 3 and underwent comprehensive chromosomal screening with microarray-based comparative genomic hybridization., Main Outcome Measure(s): Relationship between day 3 blastomere number, aneuploidy rate, and progression to the blastocyst stage., Result(s): A number of day 3 blastomeres >9 was associated with significantly increased aneuploidy rates. Rapidly developing embryos were significantly more likely to blastulate regardless of their chromosomal status. Number of embryos per patient greater than 13 was independently associated with lower aneuploidy rates after controlling for maternal age. This trend was not significant with the use of a more clinically relevant threshold of greater than six embryos per patient., Conclusion(s): Embryos with 6-9 cells at the cleavage stage should be considered for transfer over embryos with >9 cells. Day 3 blastomere number may be used in conjunction with extended culture to improve selection of euploid embryos, especially when supernumerary embryos are available. Further studies are needed to show if these selection criteria improve clinical outcomes., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine whether there are differences in adverse pregnancy outcomes in very advanced maternal age (vAMA) women who conceived with assisted reproductive technologies (ART) compared with spontaneous conceptions., Design: Retrospective cohort study., Setting: Academic tertiary care medical center., Patient(s): A total of 472 women aged ≥45 years who delivered at one institution., Intervention(s): Mode of conception., Main Outcome Measure(s): Maternal and neonatal outcomes., Result(s): For singleton pregnancies, vAMA women who conceived with ART were significantly older (47.0 ± 2.3 vs. 45.6 ± 0.1 years), more likely to be white (88.1% vs. 75.6%), and less parous (0.4 ± 0.9 vs. 1.2 ± 1.8) than vAMA women who conceived spontaneously. They were at significantly increased risk for cesarean delivery (CD) (75.1% vs. 49.7%) and were more likely to undergo elective primary CD without labor (25.4% vs. 9.4%). Risk of retained placenta was also significantly higher (2.7% vs. 0%). Rates of other maternal complications and neonatal outcomes were similar. Subgroup analysis of ART singleton pregnancies did not demonstrate differences in women using autologous oocytes versus donor oocytes., Conclusion(s): Very advanced maternal age women who conceive after ART are more likely to be white, older, primiparous, and are more likely to proceed with an elective CD compared with vAMA women who conceive spontaneously. The increased risk of retained placenta in women who conceive with ART may indicate an underlying risk for placentation defects., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine whether BRCA carriers have a decreased ovarian reserve compared with women without BRCA mutations, because BRCA mutations may lead to accelerated oocyte apoptosis due to accumulation of damaged DNA., Design: Cross-sectional study., Setting: Academic tertiary care center., Patient(s): A total of 143 women, aged 18-45 years, who underwent clinical genetic testing for BRCA deleterious mutations because of a family history of cancer, were included. The cohort was classified into three groups: BRCA1 carriers, BRCA2 carriers, and women without BRCA mutations (controls). None had a personal history of breast or ovarian cancer., Intervention(s): None., Main Outcome Measure(s): The main outcome was serum antimüllerian hormone (AMH) level. Linear and logistic regression models adjusting for age and body mass index (BMI) were performed to determine the association between BRCA mutations and AMH., Result(s): BRCA1 mutation carriers had a significant decrease in AMH levels compared with controls after adjusting for age and BMI (0.53 ng/mL [95% confidence interval (CI) 0.33-0.77 ng/mL] vs. 1.05 ng/mL [95% CI 0.76-1.40 ng/mL]). Logistic regression confirmed that BRCA1 carriers had a fourfold greater odds of having AMH <1 ng/mL compared with controls (odds ratio 4.22, 95% CI 1.48-12.0). There was no difference in AMH levels between BRCA2 carriers and controls., Conclusion(s): BRCA1 carriers have lower age- and BMI-adjusted serum AMH levels compared with women without BRCA mutations. Our results contribute to the current body of literature regarding BRCA carriers and their reproductive outcomes. Larger prospective studies with clinical outcomes such as infertility and age at menopause in this population are needed to further substantiate our findings., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The forkhead transcription factor forkhead box L2 (FOXL2) is expressed in granulosa cells of small and medium follicles in the mouse ovary. Foxl2 female knockout mice exhibit primordial follicle depletion and primary ovarian failure, but evidence from adult female conditional Foxl2 knockout mice suggests that FOXL2 may also play a significant role in maintenance of ovarian differentiation at stages beyond the primordial follicle and initial wave of folliculogenesis. We previously showed that human FOXL2 functions as a transcriptional repressor of several key genes involved in granulosa cell proliferation and differentiation, including steroidogenic acute regulatory protein (STAR), P450aromatase (CYP19A1 (CYP19)), P450scc (CYP11A1 (CYP11A)), and cyclin D2 (CCND2). To elucidate the role of mouse FOXL2, we determined its role in transcriptional regulation in Chinese hamster ovary (CHO) cells and then confirmed our findings in mouse granulosa cells. We found that mouse FOXL2 represses the activities of the mouse Star, Cyp19a1, Cyp11a1 promoters in CHO cells, but may not repress the Ccnd2 promoter, and identified the minimal mouse Star, Cyp19a1, and Cyp11a1 promoter regions responsive to FOXL2 regulation. We then knocked down Foxl2 in mouse granulosa cells using siRNA, which resulted in significantly increased expression levels of mouse Star, Cyp19a1, and Cyp11a1 but not Ccnd2. To increase Foxl2 expression levels, we generated a mouse Foxl2 lentiviral construct and used it to infect mouse granulosa cells. Following lentiviral infection, the expression levels of mouse Star, Cyp19a1, and Cyp11a1, but not Ccnd2, decreased significantly. These data confirm that mouse FOXL2 functions as a transcriptional repressor of key granulosa cell genes that influence ovarian development.

Objective: To study the expression and function of adiponectin and its receptors in mouse and human follicle cells and in early embryo development., Design: Whole ovaries, granulosa cells, and cumulus-oocyte complexes isolated from immature mice before and during hormone-induced ovulation were used to analyze the expression of adiponectin, its receptors, and ovulation-related genes; human cumulus cells and granulosa cells were isolated from patients undergoing in vitro fertilization (IVF) procedures., Setting: Multicenter., Patient(s): Women in IVF programs in Japan and the United States., Intervention(s): None., Main Outcome Measure(s): Expression of adiponectin receptors and fertility., Result(s): Adiponectin expression is absent/low in mouse and human granulosa cells and cumulus cells. Adiponectin receptors are hormonally regulated in mouse granulosa and cumulus cells in vivo and in culture. Adiponectin differentially alters the expression of Adipor1/Adipor2 as well as genes related to steroidogenesis, ovulation, and apoptosis in cumulus cells versus granulosa cells. Adiponectin enhances oocyte maturation and early embryo development in mouse and human IVF procedures., Conclusion(s): Adiponectin can modulate not only follicle growth but also embryo development in mice and humans., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal -57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding.

Purpose: To determine the expression of SMAD transcripts in human granulosa cells., Methods: Luteinized mural granulosa cells were harvested from forty women undergoing oocyte retrieval, and RNAs were isolated. SMAD expression levels were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (q-RTPCR)., Results: SMAD1-7 and 9 are expressed in human granulosa cells, with SMAD2, 3 and 4 showing the highest expression levels. Peak estradiol (E2) levels correlated with the number of oocytes retrieved during IVF. Oocyte number showed no correlation with SMAD expression levels or ratios. Fertilization rates also did not correlate with the expression levels of individual SMADs, but did correlate with higher SMAD4:SMAD3 ratios (p = 0.0062) and trended with SMAD4:SMAD2 (p = 0.0698)., Conclusions: SMAD transcripts are differently expressed in human granulosa cells, where they may mediate TGF-beta superfamily signaling during folliculogenesis and ovulation. Further, the relative expression ratios of SMAD2, 3 and 4 may differentially affect fertilization rate.

The forkhead transcription factor (FOXL2) is an essential transcription factor in the ovary. It is important in ovarian development and a key factor in female sex determination. In addition, FOXL2 plays a significant role in the postnatal ovary and follicle maintenance. The diverse transcriptional activities of FOXL2 are likely attributable to posttranslational modifications and binding to other key proteins involved in granulosa cell function. Mutations of FOXL2 lead to disorders of ovarian function ranging from premature follicle depletion and ovarian failure to unregulated granulosa cell proliferation leading to tumor formation. Thus, FOXL2 is a key regulator of granulosa cell function and a master transcription factor in these cells.

Our investigation of differences in first-trimester placentation and/or fetal biometry in pregnancies conceived spontaneously compared with those conceived through fertility treatment, and comparing less invasive fertility treatment with in vitro fertilization found no statistically significant difference in first-trimester abnormal placentation or fetal growth between pregnancies conceived spontaneously and those conceived through fertility treatment, or between the in vivo and in vitro subgroups., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine if pregnancies conceived through infertility treatment are at increased risk of cytogenetic abnormalities in the late first trimester compared with spontaneously conceived pregnancies, or if there is increased risk when comparing less invasive infertility treatment (in vivo group) to in vitro fertilization (in vitro group)., Design: Retrospective case-controlled study., Setting: University hospital., Patient(s): A total of 1,606 women who spontaneously conceived and 559 women who conceived through infertility treatment undergoing chorionic villus sampling (CVS)., Intervention(s): None., Main Outcome Measure(s): Cytogenetic abnormalities diagnosed by CVS., Result(s): No difference in cytogenetic abnormalities was found when comparing spontaneously conceived pregnancies to those conceived through infertility treatment (7.0% versus 5.4%). We also found no difference in the prevalence of cytogenetic abnormalities when comparing in vivo and in vitro fertilization subgroups (4.7% versus 5.8%). Finally, no difference was found when comparing the prevalence of different types of cytogenetic abnormalities between groups., Conclusion(s): Infertility treatment does not increase the risk of carrying a cytogenetically abnormal fetus in the late first trimester, nor does it increase the preponderance for any specific type of abnormality., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Forkhead L2 (FOXL2) is expressed in the ovary and acts as a transcriptional repressor of the steroidogenic acute regulatory (StAR) gene, a marker of granulosa cell differentiation. Human FOXL2 mutations that produce truncated proteins lacking the COOH terminus result in blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome type I, which is associated with premature ovarian failure (POF). In this study, we investigated whether FOXL2's activity as a transcriptional repressor is regulated by phosphorylation. We found that FOXL2 is phosphorylated at a serine residue and, using yeast two-hybrid screening, identified LATS1 as a potential FOXL2-interacting protein. LATS1 is a serine/threonine kinase whose deletion in mice results in an ovarian phenotype similar to POF. Using coimmunoprecipitation and kinase assays, we confirmed that LATS1 binds to FOXL2 and demonstrated that LATS1 phosphorylates FOXL2 at a serine residue. Moreover, we found that FOXL2 and LATS1 are coexpressed in developing mouse gonads and in granulosa cells of small and medium follicles in the mouse ovary. Last, we demonstrated that coexpression with LATS1 enhances FOXL2's activity as a repressor of the StAR promoter, and this results from the kinase activity of LATS1. These results provide novel evidence that FOXL2 is phosphorylated by LATS1 and that this phosphorylation enhances the transcriptional repression of the StAR gene, a marker of granulosa cell differentiation. These data support our hypothesis that phosphorylation of FOXL2 may be a control mechanism regulating the rate of granulosa cell differentiation and hence, follicle maturation, and its dysregulation may contribute to accelerated follicular development and POF in BPES type I.

Forkhead L2 (FOXL2) is a member of the forkhead/hepatocyte nuclear factor 3 (FKH/HNF3) gene family of transcription factors and acts as a transcriptional repressor of the Steroidogenic Acute Regulatory (StAR) gene, a marker of granulosa cell differentiation. FOXL2 may play a role in ovarian follicle maturation and prevent premature follicle depletion leading to premature ovarian failure. In this study, we found that FOXL2 interacts with Ubc9, an E2-conjugating enzyme that mediates sumoylation, a key mechanism in transcriptional regulation. FOXL2 and Ubc9 are co-expressed in granulosa cells of small and medium ovarian follicles. FOXL2 is sumoylated by Ubc9, and this Ubc9-mediated sumoylation is essential to the transcriptional activity of FOXL2 on the StAR promoter. As FOXL2 is endogenous to granulosa cells, we generated a stable cell line expressing FOXL2 and found that activity of the StAR promoter in this cell line is greatly decreased in the presence of Ubc9. The sumoylation site was identified at lysine 25 of FOXL2. Mutation of lysine 25 to arginine leads to loss of transcriptional repressor activity of FOXL2. Taken together, we propose that Ubc9-mediated sumoylation at lysine 25 of FOXL2 is required for transcriptional repression of the StAR gene and may be responsible for controlling the development of ovarian follicles.

Objective: To determine if mosaicism that occurs in infertility and assisted reproductive technologies continues in the late first trimester and if this is unique to infertility or occurs in all pregnancies., Design: Retrospective case-controlled study., Setting: University hospital., Patient(s): 5337 consecutive chorionic villus samplings (CVS)., Intervention(s): None., Main Outcome Measure(s): Mosaic karyotypes at CVS., Result(s): We confirmed 69 mosaic karyotypes, a rate of 1.29%. Comparing spontaneous pregnancies with pregnancies from infertility treatment, no difference was found in the prevalence of mosaicism: 1.22% versus 1.32%, respectively. Subgroup analysis of infertile couples, comparing in vitro (assisted reproduction) with in vivo fertilization (other treatments) revealed a mosaicism rate of 1.84% and 0.41%, respectively. Confined placental mosaic (CPM) rates for infertility treated pregnancies and spontaneously conceived pregnancies were 0.88% and 0.92%, respectively. Subgroup analysis of infertile patients revealed a CPM rate of 1.15% for in vitro fertilization treatment and 0.41% for in vivo fertilization treatment. These results were not statistically significant., Conclusion(s): There was no difference in the prevalence of mosaicism at the end of the first trimester in pregnancies conceived spontaneously compared with those with infertility. There was no difference in the prevalence of mosaicism when in vitro and in vivo treatment were compared.

Members of the forkhead box O1 (FOXO) family of transcription factors are expressed in granulosa cells during various stages of follicle development, and evidence from rodent and other model systems suggests that they may be involved in regulating follicular activation and oocyte maturation. In this report, we show that FOXO1, FOXO3, and FOXO4 are expressed in human luteinized mural granulosa cells, which may suggest that these transcription factors are also involved in human folliculogenesis and luteinization.

Blepharophimosis-ptosis-epicanthus inversus syndrome type I is an autosomal disorder caused by mutations in FOXL2 gene and associated with premature ovarian failure in women by a dominant inheritance. FOXL2 is a recently identified protein that belongs to forkhead family transcription factor, of which signaling pathways are still unknown. Here, we show that FOXL2 induces apoptosis in both Chinese hamster ovary cells and rat granulosa cells, and it interacts with DP103, a DEAD box-containing protein. Overexpression of DP103 itself did not affect cell viability while its coexpression with FOXL2 led to the potentiation of cell death. Our results present previously undiscovered functions of these proteins, an apoptotic activity of FOXL2 in the ovary and a modulating activity of DP103 by interacting with FOXL2.

Stanniocalcin is a glycoprotein hormone important in the maintenance of calcium and phosphate homeostasis in fish. Two related mammalian stanniocalcin genes, STC1 and STC2, were found to be expressed in various tissues as paracrine regulators. We have demonstrated the existence of a second stanniocalcin gene in fish, designated fish STC2, with only 30% identity to fish STC1. However, phylogenetic analysis and comparison of the genomic structure of STC genes in vertebrates indicated that STC1 and STC2 genes were probably derived from a common ancestor gene. Based on the prominent expression of mammalian STC1 in the ovary, we tested STC2 expression in rat ovary and the regulation of STC2 expression by gonadotropins. Treatment of immature rats with pregnant mare serum gonadotropin increased STC2 transcripts, whereas subsequent treatment with human chorionic gonadotropin suppressed STC2 expression. Real-time PCR analyses also demonstrated that STC2 is expressed mainly in thecal layers. In situ hybridization studies also revealed that STC2 is expressed in thecal cell layers of antral and preovulatory follicles after gonadotropin stimulation. To elucidate the physiological functions of STC2, recombinant human and fish STC2 proteins were generated and found to be N-glycosylated homodimers. In cultured granulosa cells, treatment with human or fish STC2 suppressed FSH-induced progesterone, but not estradiol or cAMP, production. The STC2 suppression of progesterone production was associated with the inhibition of FSH-induced CYP11A and 3beta-hydroxysteroid dehydrogenase expression. Thus, STC2 is a functional homodimeric glycoprotein, and thecal cell-derived STC2 could play a paracrine role during follicular development.

The American Society for Reproductive Medicine (ASRM) conducted a survey of its domestic members to develop a strategic plan and policies for the Society. This survey was very valuable in assessing the needs of its members and demonstrated that the ASRM is addressing their needs.

Premature ovarian failure in a subgroup of women with blepharophimosis-ptosis-epicanthus inversus type 1 syndrome has been associated with nonsense mutations in the gene encoding a Forkhead transcription factor, Forkhead L2 (FOXL2). However, the exact function of FOXL2 in the ovary is unclear. We investigated the expression of FOXL2 in the mouse ovary during follicular development and maturation by RT-PCR and in situ hybridization. The FOXL2 mRNA is expressed in ovaries throughout development and adulthood and is localized to the undifferentiated granulosa cells in small and medium follicles as well as cumulus cells of preovulatory follicles. FOXL2 belongs to a group of transcription factors capable of interacting with specific DNA sequences in diverse gene promoters. With the presence of multiple putative forkhead DNA consensus sites, the promoter of the human steroidogenic acute regulatory (StAR) gene was used to test for regulation by FOXL2. Cotransfection studies revealed that wild-type FOXL2 represses the activity of the StAR promoter, and the first 95 bp upstream of the transcriptional start site of the StAR gene is sufficient for FOXL2 repression. EMSAs confirmed that FOXL2 interacts directly with this region. Analyses using FOXL2 mutants also demonstrated the importance of the entire alanine/proline-rich carboxyl terminus of FOXL2 for transcriptional repression. Furthermore, these mutations produce a protein with a dominant-negative effect that disables the transcriptional repressor activity of wild-type FOXL2. Dominant-negative mutations of FOXL2 could increase expression of StAR and other follicle differentiation genes in small and medium follicles to accelerate follicle development, resulting in increased initial recruitment of dormant follicles and thus the premature ovarian failure phenotype.

During the periovulatory period, the mammalian ovary is the site of dramatic functional and structural changes, leading to oocyte maturation, follicle rupture, and corpus luteum formation. To a large extent, these processes result from changes in the transcriptome of various ovarian cell types. To develop a broader view of periovulatory changes in gene expression in the ovary and to identify further genes involved in periovulatory events, we used the recently developed DNA array technology. Immature female eCG-primed rats were killed either immediately before or 6 h after ovulation induction with hCG. Total ovarian RNA was isolated and used to prepare radiolabeled cDNA probes, which were hybridized to DNA arrays representing approximately 600 rat genes. Quantitative analysis identified a multitude of regulated gene messages, including several genes involved in extracellular matrix degradation and lipid/steroid metabolism previously reported to be induced by hCG. This screening also identified a group of candidate genes whose ovarian expression and gonadotropin regulation was hitherto unknown. The induction of three of these genes, encoding cutaneous fatty acid-binding protein, the interleukin-4 receptor alpha chain, and prepronociceptin, was confirmed and further characterized by Northern blot analysis. In addition, in situ hybridization analysis showed that hCG administration resulted in exclusive or predominant expression of all three genes in theca cells. These results demonstrate that DNA arrays can be used to identify genes regulated during the periovulatory period, thus contributing to a more detailed understanding of the molecular mechanisms of ovulation.

In patients with poor response to ovarian stimulation with gonadotrophins, growth hormone (GH) is sometimes used to increase paracrine insulin-like growth factor-1 (IGF-1) effect. We postulated that dehydroepiandrosterone (DHEA) administration to poor responders would augment gonado-trophin effect via a similar mechanism. Baseline ovarian stimulation response to a cycle with DHEA in five healthy non-smoking women <41 years old was compared with day 3 FSH <20 mIU/ml. All had documented poor response to vigorous gonadotrophin administration. After day 2 ultrasounds, DHEA-sulphate (DHEA-S), FSH, human chorionic gonadotrophin (HCG), and testosterone were measured, and the women were given 80 mg/day of oral micronized DHEA for 2 months. While still on DHEA, they underwent ovarian stimulation with FSH given i.m. twice a day, and HCG (10 000 IU) at follicular maturity, followed by intrauterine insemination. Cycle parameters assessed were peak oestradiol, and peak oestradiol/ampoule. The DHEA/ovarian stimulation cycles occurred between 4 and 24 months after the control cycles. After 2 months DHEA treatment, DHEA-S increased to 544 +/- 55 microg/dl, and testosterone increased to 67.3 +/- 6.1 ng/dl. All five subjects (six cycles; one subject had two DHEA cycles) had increased responsiveness; peak oestradiol concentrations increased from 266.3 +/- 69.4 pg/ml to 939.8 +/- 418.9 pg/ml. The oestradiol/ampoule ratio increased in all six cycles, by a mean of 2.94 +/- 0.50 fold (P = 0.012). One of the cycles resulted in a delivered twin pregnancy. In this small series, DHEA improved response to ovarian stimulation even after controlling for gonadotrophin dose. Supplemental DHEA treatment during ovarian stimulation may represent a novel way to maximize ovarian response.

The expression and function of estrogen receptor ERalpha/beta subtypes and ERbeta variants in granulosa cells have been determined using several integrated approaches:, Western blotting, indirect immunofluorescence, RT-PCR, and transient transfection assays. Each of these approaches has provided specific details concerning the dynamics of ER expression, ER functional activity, and estradiol (E) regulation of target genes in granulosa cells. Specifically, the studies presented herein document that messenger RNAs (mRNAs) encoding ERbeta and its splice variants, as well as mRNA encoding ERalpha, are expressed in granulosa cells of immature rats before and during culture in serum-free medium. The results also provide the first documentation that functional (DNA binding and transcriptionally active) ER is present in cultured granulosa cells and that its ability to bind consensus estrogen response element (ERE) oligonucleotide and to transactivate an ERE promoter-reporter construct is associated with the level (type?) of receptor protein as well as the stage of granulosa cell differentiation. Using a labeled ERE consensus oligonucleotide and antibodies specific for ERbeta and ERalpha, we show that ERbeta but not ERalpha was detected (supershifted in electrophoretic mobility shift assays) in extracts of granulosa cells cultured overnight (0 h) in defined medium alone. When the cells were cultured with FSH and testosterone (T) to stimulate their differentiation, ERbeta binding activity, as well as immunoreactive ERbeta as determined by Western blot analyses, decreased progressively from 24 to 48 h and was undetectable by 72 h. ERbeta mRNA was low, and ERbeta binding activity was not observed in luteinized granulosa cells. ERalpha DNA binding activity was not observed in any of the granulosa cell cultures, although low levels of immunoreactive ERalpha were detected by Western blot analyses. Immunofluorescent analyses documented that ERbeta, as well as ERalpha, were localized to granulosa cell nuclei and that the intensity of nuclear staining was related to agonist stimulation and differentiation: forskolin increased, whereas E decreased immunostaining for ERbeta and ERalpha at 48 h. When an ERE-E1b-luciferase vector was transfected into granulosa cells of unprimed rats, basal luciferase activity was low but increased by forskolin (3-4x) and by E (2x), responses to both agonists being blocked by the ER antagonist, ICI. When the same vector was transfected into differentiated granulosa cells (cultured for 48 h with FSH/T), forskolin alone increased activity. Collectively, these results show that ERbeta protein is preferentially expressed in immature granulosa cells, is functionally active (binds DNA), can transactivate (either as a homodimer or heterodimer with ERalpha) ERE-containing promoter constructs, and might be associated with increased expression of the endogenous gene encoding c-Jun.

Objective: To compare conventional IVF with ICSI in the subfertile male population using sibling oocytes. Results from males with isolated severe teratozoospermia also are analyzed., Design: Prospective experimental study., Setting: University based IVF clinic., Patient(s): Group A: 18 patients with one or more abnormalities in count, motility, or morphology. Group B: 20 patients with isolated severe teratozoospermia (< or = 4% Kruger Strict Criteria)., Intervention(s): Ovulation induction, random allocation of sibling oocytes, and IVF or ICSI., Main Outcome Measure(s): Fertilization rates (fertilization per cycle, fertilization per oocytes, and fertilization per couple) and embryo quality., Result(s): In group A, fertilization occurred in 13 of 18 (72%) of IVF cycles and 17 of 18 (94%) of ICSI cycles. Overall, 69 of 120 (58%) oocytes fertilized after IVF, whereas 80 of 131 (61%) fertilized after ICSI. The mean (+/-SEM) percent of oocytes fertilized per couple was 44.6%+/-9.0% with IVF and 62.7%+/-5.6% with ICSI (not statistically significant). In group B, fertilization occurred in 18 of 20 (90%) cycles after IVF and 20 of 20 (100%) cycles with ICSI. Overall, 54 of 113 (48%) of the oocytes fertilized after IVF, whereas 82 of 124 (66%) fertilized with ICSI. The mean (+/-SEM) percent of oocytes fertilized per couple was 50.9%+/-7.1 % with IVF and 66.6%+/-4.7% with ICSI. No statistically significant difference in embryo quality after IVF versus ICSI was demonstrated., Conclusion(s): With severe teratozoospermia, ICSI results in higher fertilization rates than conventional IVF, without altering embryo quality. In our subfertile male population, there is a trend toward improved fertilization with ICSI, with less failed fertilization.

Objective: To report a case of laparoscopic treatment of a heterotopic primary abdominal pregnancy after IVF with preservation of the concurrent intrauterine pregnancy., Design: Case report., Setting: University-based IVF program., Patient(s): A woman with a heterotopic abdominal pregnancy after IVF-ET., Intervention(s): Pituitary down-regulation with luteal leuprolide acetate, ovulation induction with menotropins, IVF-ET, progesterone in oil for luteal support, laparoscopy, and resection of the abdominal gestation., Main Outcome Measure(s): Human chorionic gonadotropin levels, pelvic ultrasound examinations, and laparoscopic and pathologic findings., Result(s): A heterotopic abdominal pregnancy occurred after IVF-ET and was treated successfully with laparoscopy. The concurrent intrauterine pregnancy was delivered at term., Conclusion(s): Early diagnosis of an ectopic abdominal pregnancy allowed successful laparoscopic treatment, without sequelae to the intrauterine gestation.

Congenital hypopituitarism may be due to hypothalamic failure. The case presented below belonged to this category. In addition, the demonstration of absent septum pellucidum placed this case in the category of suprasellar dysplasia. The patient was 21 years old, with primary amenorrhea and lack of development of secondary sex characteristics. The laboratory findings confirmed the diagnosis of hypothyroidism, hypocortisolism, hypogonadotropism, hyperprolactinemia and normal growth hormone. Stimulation studies revealed a subnormal response of cortisol to adrenocorticotrophic hormone stimulation, subnormal response of follicle stimulating hormone and luteinizing hormone to gonadotropin releasing hormone stimulation, normal response of prolactin to thyrotropin releasing hormone stimulation and exaggerated response of thyroid stimulating hormone to thyrotropin releasing hormone stimulation. The patient was treated with thyroid supplementation. Magnetic resonance imaging showed a hypoplastic infundibulum, ectopic neurohypophysis, small anterior pituitary gland and absent septum pellucidum. Congenital hypopituitarism may be part of a large spectrum of midline brain abnormalities.