Peter Fedorcsak

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

Obesity is a risk factor for early pregnancy loss after IVF or ICSI

Background. Experience with polycystic ovary syndrome shows that insulin resistance is related to early pregnancy loss. This association was examined by comparing pregnancy outcome in obese and lean women.

Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition.

Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct measurements of chromatin states have been hindered by technical difficulties in profiling histone modifications from small quantities of cells. Recent improvements allow for 500 cell-equivalents of chromatin per reaction, but require 10,000 cells for initial steps or require a highly specialized microfluidics device that is not readily available. We developed a micro-scale chromatin immunoprecipitation and sequencing (μChIP–seq) method, which we used to profile genome-wide histone H3 lysine methylation (H3K4me3) and acetylation (H3K27ac) in mouse immature and metaphase II oocytes and in 2-cell and 8-cell embryos. Notably, we show that ~22% of the oocyte genome is associated with broad H3K4me3 domains that are anti-correlated with DNA methylation. The H3K4me3 signal becomes confined to transcriptional-start-site regions in 2-cell embryos, concomitant with the onset of major zygotic genome activation. Active removal of broad H3K4me3 domains by the lysine demethylases KDM5A and KDM5B is required for normal zygotic genome activation and is essential for early embryo development. Our results provide insight into the onset of the developmental program in mouse embryos and demonstrate a role for broad H3K4me3 domains in MZT.

In vitro fertilization is a successful treatment in endometriosis-associated infertility

OBJECTIVE To assess success rates of IVF and intracytoplasmic sperm injection in women with various stages of endometriosis. DESIGN Retrospective cohort study. SETTING Reproductive medicine unit in a university hospital. PATIENT(S) Infertile women (n = 2,245) with various stages of endometriosis or tubal factor infertility. INTERVENTION(S) IVF or intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S) Dose of FSH, number of oocytes retrieved, fertilization rate, implantation rate, pregnancy rate (PR), live birth/ongoing PR. RESULT(S) Women with endometriosis had similar pregnancy and live birth/ongoing PR as did women with tubal factor infertility, but the American Society for Reproductive Medicine (ASRM) stage I and II endometriosis patients had a lower fertilization rate, and stage III and IV patients required more FSH and had fewer oocytes retrieved. Splitting the stage III and IV groups into patients with and without endometriomas showed that the endometrioma group required more FSH and had a significantly lower pregnancy and live birth/ongoing PR. CONCLUSION(S) With the exception of patients with endometrioma, infertile women with various stages of endometriosis have the same success rates with IVF and intracytoplasmic sperm injection as patients with tubal factor. This contrasts with the systematic review on which the European Society of Human Reproduction and Embryology bases its recommendations.

Effects of Leptin and Leukemia Inhibitory Factor on Preimplantation Development and STAT3 Signaling of Mouse Embryos In Vitro

Abstract Preimplantation mouse embryos simultaneously express receptors for leptin and leukemia inhibitory factor (LIF), both of which trigger activation of STAT3 (Signal Transducer and Activator of Transcription) protein. To examine the joint effects of leptin and LIF on embryonic development, we studied preimplantation development and activation of STAT3 signaling of mouse embryos after exposure to leptin and/or LIF in vitro. Two-cell mouse embryos (Day 2) were cultured in the presence of leptin and/or LIF. Significantly fewer leptin-exposed than control embryos hatched by Day 5 and by Day 6 of development. In addition, cells of leptin-exposed Day 5 blastocysts showed a higher rate of DNA fragmentation, which is a sign of apoptosis. Leukemia inhibitory factor alone had no effect on the rates of embryonic development or DNA fragmentation. Simultaneous exposure of embryos to leptin and LIF increased the proportion of hatching embryos and decreased the rate of apoptosis compared to embryos exposed to leptin only. Leptin treatment was associated with an increased phospho-STAT3-specific immunofluorescence in the cell membrane of blastocysts, which was not observed in LIF-exposed embryos. In conclusion, LIF modifies the effect of leptin during preimplantation embryo development in mice, presumably by interfering with activation of STAT3 signaling.

Complete surgical removal of minimal and mild endometriosis improves outcome of subsequent IVF/ICSI treatment

Surgical eradication of minimal and mild endometriosis has been shown to increase the birth rate both spontaneously and after intrauterine insemination. This study from a reproductive medicine unit at a referral university hospital examined whether surgical eradication of minimal and mild endometriosis prior to IVF improved the treatment outcome. Records of infertile patients with minimal and mild endometriosis (American Society for Reproductive Medicine stages I and II) with no prior IVF/intracytoplasmic sperm injection (ICSI) treatments were analysed. During the first treatment cycle, women who had undergone complete removal (n=399) of endometriotic lesions experienced, compared with women with diagnostic laparoscopy only (n=262), a significantly improved implantation rate (30.9% versus 23.9%, P=0.02), pregnancy rate (40.1% versus 29.4%, P=0.004) and live-birth rate per ovum retrieval (27.7% versus 20.6%, P=0.04). Surgical removal of minimal and mild endometriotic lesions also gave shorter time to first pregnancy and a higher cumulative pregnancy rate. The study shows that women with stages I and II endometriosis undergoing IVF/ICSI have significantly shorter time to pregnancy and higher live-birth rate if all visible endometriosis is completely eliminated at the time of diagnostic surgery. Surgical elimination of minimal and mild endometriosis has been shown to increase the birth rate both spontaneously and after intrauterine insemination. In this study from a reproductive medicine unit at a referral university hospital, we examined whether surgical elimination of minimal and mild endometriosis prior to IVF improved the outcome of this treatment as well. During the first IVF treatment cycle, women who had undergone complete surgical removal of endometriosis experienced, compared with women who still had their endometriosis, an improved rate of embryo implantation, pregnancy rate and live birth rate per ovum retrieval. Surgical removal of minimal and mild endometriotic lesions also gave shorter time to first pregnancy and a higher cumulative pregnancy rate. In summary, our study shows that women with minimal and mild endometriosis undergoing IVF have shorter time to pregnancy and higher live-birth rate if all visible endometriosis is completely eliminated before the start of treatment.

The effect of metformin on ovarian stimulation and in vitro fertilization in insulin-resistant women with polycystic ovary syndrome: an open-label randomized cross-over trial

Metformin effectively restores insulin sensitivity in insulin-resistant women with polycystic ovary syndrome (PCOS). We examined whether metformin ,given prior to and during ovarian stimulation for in vitro fertilization (IVF) ,altered follicle stimulating hormone (FSH) requirement and increased the number of collected oocytes in these women. Seventeen insulin-resistant women with PCOS were recruited to our IVF unit to receive two consecutive cycles of ovarian stimulation with or without metformin co-treatment ,the order of treatments being randomized using a table of random numbers. Metformin treatment (1500 mg/day) started 3 weeks before downregulation with buserelin acetate and was continued throughout ovarian stimulation with human recombinant FSH. Nine women completed both cycles ,the results of eight women being excluded because of pregnancy after the first cycle (n = 4) or because the protocol of the study was not followed (n = 4). Mean total FSH dose was 2301 IU (range 1500-6563 IU) in metformin cycles and 2174 IU (range 1200-3900 IU) in parallel control cycles ,while the mean number of collected oocytes was 8.6 (range 2-28) and 4.6 (range 1-16) ,respectively. Bayesian analysis showed probabilities of 0.05 that metformin reduces FSH requirement by at least 10% ,and of 0.61 that at least 10% more oocytes are collected after metformin co-treatment. Co-administration of metformin is therefore likely to increase the number of oocytes collected after ovarian stimulation in insulin-resistant women with PCOS but is unlikely to reduce the requirement for FSH.

Impaired insulin action on granulosa-lutein cells in women with polycystic ovary syndrome and insulin resistance

We studied the in vitro response to insulin of granulosalutein cells derived from patients with polycystic ovary syndrome (PCOS) and clinically defined insulin resistance. Insulin sensitivity was assessed by continuous infusion of glucose with model assessment test (CIGMA). Insulin resistant (PCOS-IR; n = 8), non-insulin resistant (PCOS-NIR; n = 9) patients with PCOS, and women with tubal factor infertility (TF; n = 8) underwent controlled ovarian stimulation with long-term gonadotropin-releasing hormone (GnRH) agonist, recombinant follicle stimulating hormone (FSH), and in vitro fertilization. Primary cultures of granulosa-lutein cells were incubated with insulin (10, 100, 500 ng/ml) and/or luteinzing hormone (LH) (10, 100 ng/ml) in the presence of low density lipoprotein (100 μg/ml). The progesterone and lactate accumulation were measured in the culture medium. LH potently stimulated the progesterone secretion in all groups. Insulin alone had no effect on progesterone release in any of the groups, but stimulated lactate formation in the PCOS-NIR and TF groups. Insulin augmented the effect of LH on progesterone secretion selectively in the PCOS-NIR group. The expression of the insulin receptor was determined by Western blotting in separate cultures of granulosa-lutein cells, and showed receptor down-regulation in the PCOS-IR patients. We infer that the in vitro effect of insulin on progesterone and lactate release by granulosa-lutein cells is impaired in insulin resistant PCOS patients.

Leptin and leptin binding activity in the preovulatory follicle of polycystic ovary syndrome patients.

To investigate the clinical importance of leptins intraovarian effects, we studied the concentration of leptin and leptin binding activity in the plasma and in the follicular fluid of PCOS patients (n=20; median BMI: 27.1 kg/m2, range 19.7-36.3) undergoing controlled ovarian stimulation with long-term GnRH agonist, recombinant FSH, and in vitro fertilization. Follicular fluid and blood samples were collected during follicle aspiration for IVF. Total leptin concentration was measured by radioimmunoassay, and specific leptin binding activity was accessed by a gel filtration column assay. Follicular fluid and plasma leptin levels were similar (median 1135 pmol/l vs. 1409 pmol/l; p=0.81). Follicular fluid to plasma leptin ratio was independently associated with cumulative FSH dose (r=0.63; p=0.006) and insulin resistance index (r=-0.45; p=0.04). Specific leptin binding activity was higher in the plasma than in the follicular fluid [median 7.94% vs. 3.49%; p<0.001]. When multivariate analysis was used to predict FSH consumption, only follicular fluid leptin levels were significantly associated with cumulative FSH dose (r=0.46; p=0.04). We infer that at least in part by increased intrafollicular leptin levels, obesity directly affects ovarian function in PCOS, and may induce a relative resistance to gonadotropin stimulation. This intraovarian effect of leptin can be even more profound because of low leptin binding activity in the preovulatory follicle of obese patients.To investigate the clinical importance of leptins intraovarian effects, we studied the concentration of leptin and leptin binding activity in the plasma and in the follicular fluid of PCOS patients (n=20; median BMI: 27.1 kg/m2, range 19.7- 36.3) undergoing controlled ovarian stimulation with long-term GnRH agonist, recombinant FSH, and in vitro fertilization. Follicular fluid and blood samples were collected during follicle aspiration for IVF. Total leptin concentration was measured by radioimmunoassay, and specific leptin binding activity was accessed by a gel filtration column assay. Follicular fluid and plasma leptin levels were similar (median 1135 pmol/l vs. 1409 pmol/l; p=0.81). Follicular fluid to plasma leptin ratio was independently associated with cumulative FSH dose (r=0.63; p=0.006) and insulin resistance index (r=- 0.45; p=0.04). Specific leptin binding activity was higher in the plasma than in the follicular fluid [median 7.94% vs. 3.49%; p<0.001]. When multivariate analysis was used to predict FSH consumption, only follicular fluid leptin levels were significantly associated with cumulative FSH dose (r=0.46; p=0.04). We infer that at least in part by increased intrafollicular leptin levels, obesity directly affects ovarian function in PCOS, and may induce a relative resistance to gonadotropin stimulation. This intraovarian effect of leptin can be even more profound because of low leptin binding activity in the preovulatory follicle of obese patients.

Sustained fertility from 22 to 41 years of age in women with polycystic ovarian syndrome

BACKGROUND Subfertility due to chronic anovulation is common in women with polycystic ovary syndrome (PCOS) and is often treated with IVF. Women with PCOS have an increased ovarian follicle and oocyte count, increased ovarian reserve and/or a slower rate of follicle atresia. If so, one would expect women with PCOS to display a delayed reduction in fertility with advancing age as compared with eumenorrheic women. METHODS To test this hypothesis, we compared oocyte count and live birth rates among two groups undergoing IVF, 500 women with PCOS and 500 eumenorrheic women with infertility due to tubal factor only. RESULTS Across the age range of 22-41 years, oocyte count and live birth rates remained stable in women with PCOS. In the eumenorrheic comparison group, these parameters decreased significantly with age. CONCLUSIONS Women with PCOS display sustained fertility with advancing age as compared with infertile eumenorrheic women.