Christos Coutifaris

Effect of endometriosis on in vitro fertilization

OBJECTIVE To investigate the IVF outcome for patients with endometriosis. DESIGN Meta-analysis. SETTING Academic research center. PATIENT(S) A MEDLINE search and review of the literature were performed. Patients were classified by level of endometriosis, and controls were classified according to the indication for IVF. INTERVENTION(S) Bivariate analysis and multivariate logistic regression was used to estimate overall effect and control for confounding. MAIN OUTCOME MEASURE(S) Pregnancy rates, fertilization rate, implantation rates, and numbers of oocytes retrieved. RESULT(S) Twenty-two published studies were included in the overall analysis. The chance of achieving pregnancy was significantly lower for endometriosis patients (odds ratio, 0.56; 95% confidence interval, 0.44-0.70) when compared with tubal factor controls. Multivariate analysis also demonstrated a decrease in fertilization and implantation rates, and a significant decrease in the number of oocytes retrieved for endometriosis patients. Pregnancy rates for women with severe endometriosis were significantly lower than for women with mild disease (odds ratio, 0.60; 95% confidence interval, 0.42-0.87). CONCLUSION(S) Patients with endometriosis-associated infertility undergoing IVF respond with significantly decreased levels of all markers of reproductive process, resulting in a pregnancy rate that is almost one half that of women with other indications for IVF. These data suggest that the effect of endometriosis is not exclusively on the receptivity of the endometrium but also on the development of the oocyte and embryo.

DNA methylation and gene expression differences in children conceived in vitro or in vivo

Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth-weight, major and minor birth defects, and rare disorders involving imprinted genes, suggesting that epigenetic changes may be associated with assisted reproduction. We examined DNA methylation at more than 700 genes (1536 CpG sites) in placenta and cord blood and measured gene expression levels of a subset of genes that differed in methylation levels between children conceived in vitro versus in vivo. Our results suggest that in vitro conception is associated with lower mean methylation at CpG sites in placenta and higher mean methylation at CpG sites in cord blood. We also find that in vitro conception-associated DNA methylation differences are associated with gene expression differences at both imprinted and non-imprinted genes. The range of inter-individual variation in gene expression of the in vitro and in vivo groups overlaps substantially but some individuals from the in vitro group differ from the in vivo group mean by more than two standard deviations. Several of the genes whose expression differs between the two groups have been implicated in chronic metabolic disorders, such as obesity and type II diabetes. These findings suggest that there may be epigenetic differences in the gametes or early embryos derived from couples undergoing treatment for infertility. Alternatively, assisted reproduction technology may have an effect on global patterns of DNA methylation and gene expression. In either case, these differences or changes may affect long-term patterns of gene expression.

Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance

The semi-allograft embryo in the blastocyst stage implants itself in the endometrium, yet no immune rejection processes are activated. Embryonic trophoblast and maternal decidua produce corticotropin-releasing hormone (CRH) and express Fas ligand (FasL), a proapoptotic cytokine. We found that antalarmin, a CRH receptor type 1 antagonist, decreased FasL expression and promoted apoptosis of activated T lymphocytes, an effect which was potentiated by CRH and inhibited by antalarmin. Female rats treated with antalarmin showed a marked decrease in implantation sites and live embryos and diminished endometrial FasL expression. Embryos from mothers that lacked T cells or from syngeneic matings were not rejected when the mothers were given antalarmin. These findings suggested that locally produced CRH promotes implantation and maintenance of early pregnancy primarily by killing activated T cells.

Letrozole versus Clomiphene for Infertility in the Polycystic Ovary Syndrome

BACKGROUND Clomiphene is the current first-line infertility treatment in women with the polycystic ovary syndrome, but aromatase inhibitors, including letrozole, might result in better pregnancy outcomes. METHODS In this double-blind, multicenter trial, we randomly assigned 750 women, in a 1:1 ratio, to receive letrozole or clomiphene for up to five treatment cycles, with visits to determine ovulation and pregnancy, followed by tracking of pregnancies. The polycystic ovary syndrome was defined according to modified Rotterdam criteria (anovulation with either hyperandrogenism or polycystic ovaries). Participants were 18 to 40 years of age, had at least one patent fallopian tube and a normal uterine cavity, and had a male partner with a sperm concentration of at least 14 million per milliliter; the women and their partners agreed to have regular intercourse with the intent of conception during the study. The primary outcome was live birth during the treatment period. RESULTS Women who received letrozole had more cumulative live births than those who received clomiphene (103 of 374 [27.5%] vs. 72 of 376 [19.1%], P=0.007; rate ratio for live birth, 1.44; 95% confidence interval, 1.10 to 1.87) without significant differences in overall congenital anomalies, though there were four major congenital anomalies in the letrozole group versus one in the clomiphene group (P=0.65). The cumulative ovulation rate was higher with letrozole than with clomiphene (834 of 1352 treatment cycles [61.7%] vs. 688 of 1425 treatment cycles [48.3%], P<0.001). There were no significant between-group differences in pregnancy loss (49 of 154 pregnancies in the letrozole group [31.8%] and 30 of 103 pregnancies in the clomiphene group [29.1%]) or twin pregnancy (3.4% and 7.4%, respectively). Clomiphene was associated with a higher incidence of hot flushes, and letrozole was associated with higher incidences of fatigue and dizziness. Rates of other adverse events were similar in the two treatment groups. CONCLUSIONS As compared with clomiphene, letrozole was associated with higher live-birth and ovulation rates among infertile women with the polycystic ovary syndrome. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00719186.).

Anxiolytic Metabolites of Progesterone: Correlation with Mood and Performance Measures following Oral Progesterone Administration to Healthy Female Volunteers

Progesterone is readily reduced in humans to its A-ring metabolites, allopregnanolone (3 alpha-hydroxy-5 alpha-pregnan-20-one) and pregnanolone (3 alpha-hydroxy-5 beta-pregnan-20-one). The latter have been reported to have anxiolytic, hypnotic and anesthetic actions when administered to laboratory animals and (or) humans. Consequently, we measured allopregnanolone and pregnanolone in 18 healthy females, ages 18-25, at the time of peak plasma progesterone following an oral dose of micronized progesterone (1,200 mg) in a double-blind, placebo-controlled study. The plasma levels of the parent steroid and metabolites were compared with changes in mood, cognition, and motor performance following progesterone administration. We observed good correlations between plasma progesterone and plasma allopregnanolone (r = 0.85), plasma pregnanolone (r = 0.81) and the combined metabolites (r = 0.92). Plasma allopregnanolone was significantly correlated with measures of fatigue, confusion and immediate recall, and these correlation coefficients were somewhat greater than those for plasma progesterone and these same behavioral measures. Significant changes in fatigue, delayed verbal recall and symbol copying were experienced by subjects who achieved high levels (> or = 95.55 nmol/l) of these anxiolytic metabolites, while those with lower metabolite levels reported no negative effects. These data suggest that allopregnanolone and pregnanolone may contribute to or mediate the observed behavioral effects of progesterone.

Total testosterone assays in women with polycystic ovary syndrome: Precision and correlation with hirsutism

CONTEXT There is no standardized assay of testosterone in women. Liquid chromatography mass spectrometry (LC/MS) has been proposed as the preferable assay by an Endocrine Society Position Statement. OBJECTIVE The aim was to compare assay results from a direct RIA with two LC/MS. DESIGN AND SETTING We conducted a blinded laboratory study including masked duplicate samples at three laboratories--two academic (University of Virginia, RIA; and Mayo Clinic, LC/MS) and one commercial (Quest, LC/MS). PARTICIPANTS AND INTERVENTIONS Baseline testosterone levels from 596 women with PCOS who participated in a large, multicenter, randomized controlled infertility trial performed at academic health centers in the United States were run by varying assays, and results were compared. MAIN OUTCOME MEASURE We measured assay precision and correlation and baseline Ferriman-Gallwey hirsutism scores. RESULTS Median testosterone levels were highest with RIA. The correlations between the blinded samples that were run in duplicate were comparable. The correlation coefficient (CC) between LC/MS at Quest and Mayo was 0.83 [95% confidence interval (CI), 0.80-0.85], between RIA and LC/MS at Mayo was 0.79 (95% CI, 0.76-0.82), and between RIA and LC/MS at Quest was 0.67 (95% CI, 0.63-0.72). Interassay variation was highest at the lower levels of total testosterone (≤50 ng/dl). The CC for Quest LC/MS was significantly different from those derived from the other assays. We found similar correlations between total testosterone levels and hirsutism score with the RIA (CC=0.24), LC/MS at Mayo (CC=0.15), or Quest (CC=0.17). CONCLUSIONS A testosterone RIA is comparable to LC/MS assays. There is significant variability between LC/MS assays and poor precision with all assays at low testosterone levels.

Inter- and intra-individual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology.

Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

Ovarian stimulation and low birth weight in newborns conceived through in vitro fertilization.

OBJECTIVE: Singleton neonates born after in vitro fertilization (IVF) are at increased risk for low birth weight, preterm delivery, or both. We sought to assess whether the alteration of the peri-implantation maternal environment resulting from ovarian stimulation may contribute to increased risk of low birth weight in IVF births. METHODS: The Society for Assisted Reproductive Technologies database was used to identify IVF-conceived neonates born in the United States between 2004 and 2006. Associations were assessed in neonates born after fresh compared with frozen and thawed embryo transfer in women of similar ovarian responsiveness, in paired analysis of neonates born to the same woman after both types of embryo transfer, and in neonates born after oocyte donation. RESULTS: Of 56,792 neonates identified, 38,626 and 18,166 were conceived after transfer of fresh and frozen embryos, respectively. In singletons, there was no difference in preterm delivery. However, the odds of overall low birth weight (10% compared with 7.2%; adjusted odds ratio [OR] 1.35; 95% confidence interval [CI] 1.20–1.51), low birth weight at term (2.5% compared with 1.2%, adjusted OR 1.73, 95% CI 1.31–2.29), and preterm low birth weight (34.1% compared with 23.8%, adjusted OR 1.49, 95% CI 1.24–1.78) were all significantly higher after fresh embryo transfer. In singletons, after either fresh or frozen embryo transfer in the same patient, this association was even stronger (low birth weight: 11.5% compared with 5.6%, adjusted OR 4.66, 95% CI 1.18–18.38). In oocyte donor recipients who do not undergo any ovarian hormonal stimulation for either a fresh or a frozen embryo transfer, no difference in low birth weight was demonstrated (11.5% compared with 11.3% adjusted OR 0.99, 95% CI 0.82–1.18). CONCLUSION: The ovarian stimulation-induced maternal environment appears to represent an independent mediator contributing to the risk of low birth weight, but not preterm delivery, in neonates conceived after IVF. LEVEL OF EVIDENCE: II

N-Cadherin-Mediated Human Granulosa Cell Adhesion Prevents Apoptosis : A Role in Follicular Atresia and Luteolysis?

Studies suggest that cell-cell interactions may regulate apoptosis, and in particular, the calcium-dependent cell adhesion molecule N-cadherin has been shown to be capable of modulating this process. Rat granulosa cells (GCs) are known to express N-cadherin whereas cAMP is known to induce apoptosis in human and rat GCs. Based on these observations, we hypothesized that N-cadherin regulates human GC apoptosis via a cAMP-dependent mechanism. N-cadherin expression was evaluated in ovarian follicles and corpora lutea utilizing immunohistochemical techniques and in luteinized GCs in culture using immunoblotting, flow cytometric analysis, immunohistochemistry, and indirect immunofluorescence techniques utilizing anti-N-cadherin antibodies directed against both the extracellular and cytoplasmic domains of the molecule. Apoptosis was assessed by TUNEL and DNA fragmentation analysis and confirmed by flow cytometric cell cycle analysis and electron microscopy. The rate of GC apoptosis was found to be two- to three-fold lower among aggregated cells, as compared with single cells. N-cadherin was found to be expressed by aggregating GCs in vitro and GCs cultured in the presence of either N-cadherin function disrupting antibodies or peptides exhibiting enhanced rates of apoptosis. GCs in situ stained intensely for N-cadherin in preantral and normal growing preovulatory follicles as well as early corpora lutea. N-cadherin was weak in atretic follicles and regressing corpora lutea. Exposure of GCs to cAMP increased apoptosis while decreasing N-cadherin protein expression in a dose-dependent manner. Cell culture under serum-free conditions increased apoptosis and decreased N-cadherin expression, in part through cleavage of the extracellular domain of the molecule. The metalloproteinase inhibitor 1-10-phenanthroline inhibited the cleavage of the extracellular domain of N-cadherin and concomitantly inhibited the serum-deprivation-induced apoptosis of aggregated GCs. Collectively, these observations suggest that down-regulation of N-cadherin or the absence of a functional extracellular domain of the molecule prevents cell aggregation and is associated with GC apoptosis. In addition, cAMP induces apoptosis in a dose-dependent manner, and this process is dependent, at least in part, on regulation of the N-cadherin molecule at the surface of the cells. We conclude that N-cadherin-mediated GC signaling plays a central role in follicular and luteal cell survival.

Regulated expression of cadherin‐11 in human epithelial cells: A role for cadherin‐11 in trophoblast‐endometrium interactions?

Cadherin‐11 is a novel member of the cadherin supergene family. Cadherin‐11 expression is localized to mesenchymal tissue and specific regions of the neural tube during mouse embryogenesis. Here we report that cadherin‐11 is spatiotemporally expressed in the epithelial cells of the human placenta. Cadherin‐11 mRNA levels were low in freshly isolated cytotrophoblast cells but increased as the cytotrophoblast cells aggregated and fused to form syncytiotrophoblast cells in vitro. The increase in cadherin‐11 mRNA levels was concomitant with a decrease in E‐cadherin expression. Cadherin‐11 was localized to the syncytial trophoblast and extravillous cytotrophoblasts, but not the villous cytotrophoblasts of the human placenta by immunohistochemistry. As both of the former cell types have intimate interactions with the endometrium, we examined cadherin‐11 expression in the human endometrium. Cadherin‐11 was detected in the glandular and surface epithelium of the endometrium at all stages of the menstrual cycle. However, cadherin‐11 was abundant only in the stroma in the late secretory stage of the menstrual cycle. The accumulation of cadherin‐11 in the stroma correlated with decidualization. Taken together, our observations demonstrate that cadherin‐11 is expressed in certain epithelial cell lineages and suggest the possibility that cadherin‐11 plays an important role in mediating trophoblast‐endometrium interactions.