Gene expression profiles of human granulosa cells treated with bioequivalent doses of corifollitropin alfa (CFA) or recombinant human follicle-stimulating hormone (recFSH).

Using recombinant DNA technologies, a chimeric gene containing the coding sequences of follicle stimulating hormone (FSH) ß-subunit and C-terminal peptide of the human chorionic gonadotrophin (hCG) ß-subunit have been designed to generate a new gonadotrophin named corifollitropin alfa (CFA). CFA has longer elimination half-life and slower rate of absorption compared with FSH, which makes CFA a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of CFA or recFSH in primary cultures of human granulosa cells (hGCs). Gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that CFA is more effective than recFSH in inducing aromatase gene expression after 6 and 24 h from the initial stimulation in agreement with its long-acting characteristic.

The anti-Müllerian hormone (AMH) induces forkhead box L2 (FOXL2) expression in primary culture of human granulosa cells in vitro.

PURPOSE: Anti-Müllerian hormone (AMH) and forkhead box L2 (FOXL2) are two pivotal genes expressed in human granulosa cells (hGCs) where both genes share similar inhibitory functions on activation and follicular growth in order to preserve the ovarian follicle reserve. Furthermore, AMH and FOXL2 contribute to inhibit steroidogenesis, decreasing or preventing the activation of gonadotrophin-dependent aromatase CYP19A1 cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The purpose of this study is to evaluate the role of AMH in regulating the expression of FOXL2. METHODS: Primary cultures of hGCs were treated with increasing concentrations of recombinant human AMH (rhAMH; range 10-100 ng/ml) for 3 h. Negative controls were performed using corresponding amounts of AMH vehicle. Total RNA or proteins were purified and quantified by spectrophotometry. FOXL2 and CYP19A1 gene expression, normalized by reference gene ribosomal protein S7 (RpS7), was evaluated by RT-qPCR. Each reaction was repeated in triplicate. Statistical analysis was performed. Extracted proteins were analyzed by immunoblot using anti-FOXL2 and anti-ß-actin as primary antibodies. RESULTS: rhAMH treatments tested did not modulate the basal expression of aromatase CYP19A1 gene. rhAMH (50 ng/ml) was able to increase FOXL2 gene expression and its intracellular content. CONCLUSIONS: This study demonstrated the existence of an AMH-FOXL2 relationship in hGCs. AMH is capable of increasing both gene and protein expression of FOXL2. Because FOXL2 induces AMH transcription, these ovarian factors could be finely regulated by a positive feedback loop mechanism to preserve the ovarian follicle reserve.

Modulation of gonadotrophin induced steroidogenic enzymes in granulosa cells by d-chiroinositol.

BACKGROUND: d-chiroinositol (DCI) is a inositolphosphoglycan (IPG) involved in several cellular functions that control the glucose metabolism. DCI functions as second messenger in the insulin signaling pathway and it is considered an insulin sensitizer since deficiency in tissue availability of DCI were shown to cause insulin resistance (IR). Polycystic ovary syndrome (PCOS) is a pathological condition that is often accompanied with insulin resistance. DCI can positively affects several aspect of PCOS etiology decreasing the total and free testosterone, lowering blood pressure, improving the glucose metabolism and increasing the ovulation frequency. The purpose of this study was to evaluate the effects of DCI and insulin combined with gonadotrophins namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on key steroidogenic enzymes genes regulation, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and cytochrome P450 side-chain cleavage (P450scc) in primary cultures of human granulosa cells (hGCs). We also investigated whether DCI, being an insulin-sensitizer would be able to counteract the expected stimulator activity of insulin on human granulosa cells (hGCs). METHODS: The study was conducted on primary cultures of hGCs. Gene expression was evaluated by RT-qPCR method. Statistical analysis was performed applying student t-test, as appropriate (P < 0.05) set for statistical significance. RESULTS: DCI is able to reduce the gene expression of CYP19A1, P450scc and insulin-like growth factor 1 receptor (IGF-1R) in dose-response manner. The presence of DCI impaired the increased expression of steroidogenic enzyme genes generated by the insulin treatment in gonadotrophin-stimulated hGCs. CONCLUSIONS: Insulin acts as co-gonadotrophin increasing the expression of steroidogenic enzymes genes in gonadotrophin-stimulated granulosa cells. DCI is an insulin-sensitizer that counteracts this action by reducing the expression of the genes CYP19A1, P450scc and IGF-1R. The ability of DCI to modulate in vitro ovarian activity of insulin could in part explain its beneficial effect when used as treatment for conditions associated to insulin resistance.

Adnexal Torsion during Pregnancy after Oocyte In Vitro Maturation and Intracytoplasmic Sperm Injection Cycle.

We report a case of right adnexal torsion during pregnancy after an oocyte in vitro maturation and intracitoplasmic sperm injection cycle in patient with polycystic ovary syndrome. A 31-year-old woman with a typical clinical disorder of polycystic ovarian syndrome was included in an oocyte in vitro maturation program. Right adnexal torsion occurred two days after embryo transfer, and laparoscopy detorsion was successfully performed with preservation of adnexa. The patient had a full-term pregnancy and delivered a healthy infant at 40 weeks of gestation. To our knowledge this is the first report of adnexal torsion after an oocyte in vitro maturation and intracitoplasmic sperm injection program.

Embryo quality and implantation rate in two different culture media: ISM1 versus Universal IVF Medium.

OBJECTIVE: To compare the outcome of two different culture media marketed by the MediCult AS Company (Jyllinge, Denmark)-Universal IVF Medium and ISM1 Medium culture-which, in addition to glucose, pyruvate, and energy-providing components, also contain amino acids, nucleotides, vitamins, and cholesterol. DESIGN: Laboratory and retrospective clinical study. SETTING: University teaching hospital. PATIENT(S): A total of 726 patients, undergoing IVF-intracytoplasmic sperm injection procedure, comparable in mean age range, oocyte retrieval, and infertility indication, were included in the study. Laboratory quality and standard procedures were maintained unaffected. INTERVENTION(S): Oocyte retrieval, different embryo culture media. MAIN OUTCOME MEASURE(S): Embryo quality, ongoing pregnancy, and implantation rate. RESULT(S): The frequency of good-quality embryos (79% vs. 74%) and the percentages of ongoing pregnancy (27.5% vs. 18%) and implantation rate (15% vs. 10%) were significantly higher in the group treated with ISM1 Medium rather than Universal IVF Medium. CONCLUSION(S): ISM1 Medium culture seems to improve the performance of embryonic growth and development, as well as increasing the percentage of pregnancy.

Seminal plasma total antioxidant capacity and semen parameters in patients with varicocele.

Total antioxidant capacity (TAC) was evaluated in the seminal plasma of infertile patients with varicocele in relation to their semen parameters. The study recruited 60 patients affected by varicocele and 10 fertile non-varicocele subjects as controls. Controls had normal semen parameters and proven fertility. On the basis of semen parameters, patients with varicocele were grouped into normozoospermic (n = 12), asthenozoospermic (n = 8), oligoasthenozoospermic (n = 40). The group with oligosthenozoospermia was divided into mild (<20 x 10(6)/ml; > or =15 x 10(6)/ml), moderate (<15 x 10(6)/ml; > or =5 x 10(6)/ml), and severe (<5 x 10(6)/ml), based on sperm count. Antioxidant activity was measured in seminal plasma and peripheral blood using the free oxygen radicals defence test. No significant differences were observed in peripheral blood TAC concentrations between controls and groups. In patients with varicocele and moderate oligoasthenozoospermia or severe oligoasthenozoospermia, seminal plasma TAC concentrations were significantly lower (P < 0.05) than in controls and normozoospermic patients with varicocele. Moreover, in patients with severe oligosthenozoospermia, seminal plasma TAC concentrations were also significantly lower (P < 0.05) than in asthenozoozpermic patients with varicocele. In all subjects, concentrations of TAC showed a positive correlation with sperm concentration (r = 0.93, P < 0.05) and motility (r = 0.92, P < 0.05).

Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue.

BACKGROUND: Controlled-rate freezing of ovarian cortical tissue for preservation of fertility among young women facing chemo- or radio-therapy is a widely accepted procedure. To improve the method for cryopreservation of ovarian tissue, particularly the stroma, we carried out a systematic comparison of vitrification versus slow programmed freezing. METHODS: Ovarian tissue from 20 women, donated during Caesarean section, was used for parallel comparison of survival and detailed light and electron microscopic (EM) morphology of oocytes, granulosa cells and ovarian stroma after freezing (slow freezing and vitrification), thawing and 24-h culture. Using tissue obtained from the same patient, we compared four cryopreservation protocols and fresh tissue. The cryoprotectants used in slow freezing were 1,2-propanediol (PrOH)-sucrose and ethylene glycol (EG)-sucrose. For vitrification, tissues were incubated for 5 or 10 min in three solutions containing a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP). RESULTS: Cryopreservation using controlled-rate freezing and vitrification preserved the morphological characteristics of ovarian tissue generally well. As revealed by morphological analysis, particularly EM, the ovarian stroma was significantly better preserved after vitrification than after slow freezing (P < 0.001). The follicles were similarly preserved after all freezing methods. CONCLUSIONS: Vitrification using a combination of PrOH, EG, DMSO and PVP was comparable to slow freezing in terms of preserving follicles in human ovarian tissue. Ovarian stroma had significantly better morphological integrity after vitrification than after controlled-rate freezing.

Human ovarian tissue cryopreservation: effect of sucrose concentration on morphological features after thawing.

Recent improvements in techniques in clinical assisted reproduction have led to an increased interest in the cryopreservation of human ovarian tissue as a way of preserving fertility and ovarian steroidogenic activity in young cancer patients. Acceptable follicular survival in frozen-thawed human ovarian tissue has generally been reported. Since a 0.3 mol/l sucrose concentration in cryopreservation solutions evidently increases human oocyte survival after cryopreservation, the aim of this study was to observe the effect of sucrose concentrations of 0.2 mol/l and 0.3 mol/l on human ovarian tissue survival after thawing. Ovarian cortical slices from 10 patients, 22-36 years of age, were cryopreserved slowly using 0.2 mol/l or 0.3 mol/l sucrose with 1,2-propanediol (1.5 mol/l) as the cryoprotectants. Light and electron microscopy were used for the histological analyses. Results showed that both treatments produced an increase in damaged cells; however, the use of 0.3 mol/l sucrose showed a smaller percentage of damaged germ cells than 0.2 mol/l sucrose, and therefore was less detrimental to the thawed ovarian tissue. However as the damage occurred principally in the stroma and follicular cells rather than in the oocytes, the suitability of these cryopreservation protocols must be further evaluated prior to considering the use of stored ovarian cortex for autografting after thawing.

Comparison of a gonadotropin-releasing hormone (GnRH) antagonist and GnRH agonist flare-up regimen in poor responders undergoing ovarian stimulation.

OBJECTIVE: To compare the efficacy of flare-up and GnRH-antagonist treatment in poor-responder patients. DESIGN: Randomized prospective study. SETTING: Assisted reproduction center. PATIENT(S): Fifty-five poor-responder patients undergoing intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Thirty patients received GnRH agonist on the 1st day of menstruation, followed by exogenous gonadotropins from the 2nd day. Twenty-five patients received exogenous gonadotropins starting on the second day of menstruation, followed by GnRH antagonist when the leading follicle reached 14 mm in diameter. MAIN OUTCOME MEASURE(S): The total dose of FSH administered during the ovarian stimulation, as well as the number of mature oocytes retrieved, embryo quality, fertilization, implantation, and pregnancy rates were evaluated. RESULT(S): The number of ampules and units of FSH administered were significantly less in the flare-up than in the antagonistic group. The numbers of mature oocytes retrieved and of top-quality embryos transferred were significantly greater in the flare-up than in the GnRH-antagonist group. The fertilization rate (84% vs. 63%) was significantly higher in the flare-up than in the GnRH-antagonist group. The implantation and pregnancy rate were similar in the two groups. CONCLUSION(S): The flare-up protocol appears to be more effective than the GnRH-antagonist protocol in terms of mature oocytes retrieved, fertilization rate, and top-quality embryos transferred in poor-responder patients.