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.

The anti-Müllerian hormone (AMH) acts as a gatekeeper of ovarian steroidogenesis inhibiting the granulosa cell response to both FSH and LH.

PURPOSE: Anti Müllerian Hormone (AMH) has a negative and inhibitory role in many functions of human granulosa-lutein cells (hGCs) including notoriously the reduction of the aromatase CYP19A1 expression induced by follicle-stimulating hormone (FSH). No data have been provided on the possible role of AMH in modulating the response to luteinizing hormone (LH) (alone or combined with FSH) as well as its effect on other enzymes involved in steroidogenesis including aromatase P450scc. The aim of this study was to investigate the role of AMH as regulator of the basal and stimulated steroids production by hGCs. METHODS: Primary culture of hGCs were incubated with hormones AMH, LH, and FSH, alone or in combination. The CYP19A1 and P450scc messenger RNA (mRNA) expression, normalized by housekeeping ribosomal protein S7 (RpS7) gene, was evaluated by reverse transcriptase quantitative PCR (RT-qPCR). Each reaction was repeated in triplicate. Negative controls using corresponding amount of vehicle control for each hormone treatment were performed. RESULT: AMH did not modulate the basal mRNA expression of both aromatase genes at any of the concentrations tested. Meanwhile, the strong mRNA induction of CYP19A1 and P450scc generated by a 24-h gonadotropin treatment (alone and combined) was suppressed by 20 ng/ml AMH added to culture medium. CONCLUSIONS: These findings contribute in clarifying the relationship between hormones regulating the early phase of steroidogenesis confirming that AMH is playing a suppressive role on CYP19A1 expression stimulated by gonadotropin in hGCs. Furthermore, a similar inhibitory effect for AMH was observed on P450scc gene expression when activated by gonadotropin treatment.

Live birth rates in the different combinations of the Bologna criteria poor ovarian responders: a validation study.

PURPOSE: to compare the baseline characteristics and chance of live birth in the different categories of poor responders identified by the combinations of the Bologna criteria and establish whether these groups comprise a homogenous population. METHODS: database containing clinical and laboratory information on IVF treatment cycles carried out at the Mother-Infant Department of the University Hospital of Modena between year 2007 and 2011 was analysed. This data was collected prospectively and recorded in the registered database of the fertility centre. Eight hundred and thirty women fulfilled the inclusion/ exclusion criteria of the study and 210 women fulfilled the Bologna criteria definition for poor ovarian response (POR). Five categories of poor responders were identified by different combinations of the Bologna criteria. RESULTS: There were no significant differences in female age, AFC, AMH, cycle cancellation rate and number of retrieved oocytes between the five groups. The live birth rate ranged between 5.5 and 7.4 % and was not statistically different in the five different categories of women defined as poor responders according to the Bologna criteria. CONCLUSION: The study demonstrates that the different groups of poor responders based on the Bologna criteria have similar IVF outcomes. This information validates the Bologna criteria definition as women having a uniform poor prognosis and also demonstrates that the Bologna criteria poor responders in the various subgroups represent a homogenous population with similar pre-clinical and clinical outcomes.

Human MATER localization in specific cell domains of oocytes and follicular cells.

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

Influence of multiple transrectal electroejaculations on semen parameters and intracytoplasmic sperm injection outcome.

OBJECTIVE: To compare semen parameters and intracytoplasmic sperm injection (ICSI) outcome in spinal cord-injured subjects who underwent single (group 1) or multiple (group 2) electroejaculations before ICSI. DESIGN: Prospective, randomized, controlled study. SETTING: Department of gynecology, obstetrics, and pediatric science in a reproductive medicine unit at a major Italian university. PATIENT(S): Thirty-four healthy women with a male partner with SCI who were seeking assisted reproduction services. INTERVENTION(S): Transrectal electroejaculation, controlled ovarian hyperstimulation, and ICSI. MAIN OUTCOME MEASURE(S): Sperm concentration, morphology, and motility and fertilization and pregnancy rates after ICSI. RESULT(S): Sperm was successfully retrieved in 94.1% of cases. In male subjects who underwent multiple electroejaculations, statistically significant improvements in sperm concentration and total sperm motility rate were observed. The overall fertilization rate was 63.6%. The number of oocytes retrieved and injected was comparable between the two groups. A total of nine clinical pregnancies were achieved. The pregnancy rate was statistically significantly higher in group 2 (n = 6/16; 37.5%) than in group 1 (n = 3/16; 18.75%). CONCLUSION(S): These data suggest that multiple electroejaculation has a positive effect on semen parameters and ICSI outcome.

Correlations between concentrations of interleukin-12 and interleukin-13 and lymphocyte subsets in the follicular fluid of women with and without polycystic ovary syndrome.

OBJECTIVE: To investigate a possible correlation between interleukin-12 (IL-12) and IL-13 levels and lymphocyte subsets in the preovulatory follicles of patients with and without polycystic ovarian syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: University hospital. PATIENT(S): Seventy-eight infertile women undergoing IVF-embryo transfer. INTERVENTION(S): The subjects underwent blood sampling, ovum retrieval, and embryo transfer. MAIN OUTCOME MEASURES: Follicular fluid levels of T, androstenedione (A); IL-12, IL-13, activated T cells, T helper, and T-suppressor lymphocytes. RESULT(S): The level of IL-12 detected in follicular fluid (FF) was significantly lower in patients with PCOS than in normally ovulating women (mean: 1.47 +/- 0.3 pg/mL vs. 2.25 +/- 0.7 pg/mL, respectively); in contrast, FF IL-13 concentrations were significantly higher in the patients with PCOS than in the normally ovulating women (mean: 32.5 +/- 3.7 pg/mL vs. 19.6 +/- 2.5 pg/mL, respectively), as was the total number of activated T lymphocytes (11.5% +/- 1.5% vs. 4.8% +/- 0.4%). A significant correlation was observed between FF activated T-cell concentrations and FF IL-12, IL-13, T, and A levels. No significant differences were observed when these data were compared with embryological parameters. CONCLUSION(S): The present study shows significant differences in the correlation between FF IL-12 and IL-13 levels and T lymphocyte numbers in the subset of patients with PCOS as compared to normally ovulating women.

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.

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.

Adjuvant L-arginine treatment in controlled ovarian hyperstimulation: a double-blind, randomized study.

BACKGROUND: Enhanced vascularization appears to be important for follicular selection and maturation in both spontaneous and stimulated IVF cycles. Nitric oxide, formed in vivo from L-arginine, may play a key role in follicular maturation and ovulation. METHODS: To evaluate the role of L-arginine supplementation in controlled ovarian hyperstimulation, 37 IVF patients were divided into two groups according to ovarian stimulation protocols: group I, GnRH agonist plus pure (p)FSH plus oral L-arginine (n = 18); and group II, GnRH agonist plus pFSH plus placebo (n = 19). Hormonal, ultrasonographic and Doppler evaluations were performed, and plasma and follicular fluid nitrite/nitrate concentrations were monitored. RESULTS: Thirty-two patients completed the study. In group I (n = 16), plasma L-arginine concentrations increased from (basal) 87 +/- 12 micromol to 279 +/- 31 micromol (P = 0.002) on the day of beta-HCG administration. In this group, pFSH treatment was shorter (P = 0.039) than in group II (n = 16). The number of the follicles > or =17mm was lower (P = 0.038) in group I than group II. The "good quality" embryos were fewer in number (P = 0.034) and pregnancy rate, both per patient (P = 0.024) and per embryo transfer (P = 0.019), was lower in group I. In the L-arginine group, an increased follicular fluid concentration of nitrite/nitrate was observed. On day 8 of the cycle, elevated plasma estradiol levels were associated with decreased blood flow resistances of perifollicular arteries. Follicular fluid concentrations of nitrite/nitrate were inversely correlated with embryo quality (r = -0.613; P = 0.005) and perifollicular artery pulsatility index (r = -0.609; P = 0.021). CONCLUSIONS: L-Arginine supplementation may be detrimental to embryo quality and pregnancy rate during controlled ovarian hyperstimulation cycles.