Tiziana Marsella

Anti-Müllerian hormone-based prediction model for a live birth in assisted reproduction

Prediction of assisted reproduction treatment outcome has been the focus of clinical research for many years, with a variety of prognostic models describing the probability of an ongoing pregnancy or a live birth. This study assessed whether serum anti-Müllerian hormone (AMH) concentrations may be incorporated into a model to enhance the prediction of a live birth in women undergoing their first IVF cycle, by analysing a database containing clinical and laboratory information on IVF cycles carried out between 2005 and 2008 at the Mother-Infant Department of University Hospital, Modena. Logistic regression was used to examine the association of live birth with baseline patient characteristics. Only AMH and age were demonstrated in regression analysis to predict live birth, so a model solely based on these two criteria was generated. The model permitted the identification of live birth with a sensitivity of 79.2% and a specificity of only 44.2%. In the prediction of a live birth following IVF, a distinction, however moderate, can be made between couples with a good and a poor prognosis. The success of IVF was found to mainly depend on maternal age and serum AMH concentrations, one of the most relevant and valuable markers of ovarian reserve.

Technical aspects of oocyte cryopreservation

Since the successful development in the mouse, the oocyte cryopreservation has been applied with varying success to a number of different species including the human. The recently reported successes in terms of pregnancies obtained by human oocyte cryopreservation are encouraging. Several studies typically reported different rates of survival (20-80%), fertilization (30-60%) and cleavage (32-100%). This variability of results throws some doubts on the usefulness of oocyte cryopreservation in IVF treatment cycles. It remains to be determined whether the relatively different success rates reported in literature, mainly in terms of survival rate, are due to methodological differences. We tried to investigate the effect of some factors on the oocyte survival rate after thawing: the presence or absence of cumulus oophorus and the exposure time of the oocytes to cryoprotectant. We suggest that a combination of several factors including both morphological and biophisical ones can affect the oocyte survival rate.

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.

Different concentrations of interleukins in the peritoneal fluid of women with endometriosis: relationships with lymphocyte subsets.

The present study explored the possible relationships between immune cell subsets and interleukin (IL)-12 or IL-13 levels in the peritoneal fluid of patients with and without endometriosis. Peritoneal fluid samples were obtained from 80 women while they were undergoing laparoscopy for pain, infertility, tubal ligation or reanastomosis. The American Fertility Society scoring system was used to determine the extension of endometriosis. The peritoneal fluid mononuclear cells were analyzed for immunophenotyping using cytometry, whereas peritoneal fluid concentrations of interleukins were measured using two ultrasensitive commercially available enzyme-linked immunosorbent assay kits. Significantly higher peritoneal fluid IL-12 levels were found in women with moderate or severe endometriosis (stages III and IV) than in healthy controls (p<0.01). Conversely, subjects with endometriosis showed remarkably lower peritoneal fluid IL-13 concentrations than controls, independent of the severity of the disease (p<0.05). Considering immune system effectors, patients with endometriosis presented a significantly higher peritoneal fluid CD8+/CD4+ratio when compared with healthy controls. Moreover, the number of peritoneal fluid CD8+and CD4+activated T cells was significantly lower in the former than in the latter group, independent of the endometriosis stage. Connections were observed between peritoneal fluid interleukins and peritoneal fluid T cells: both patients with endometriosis and controls presented an inverse correlation between peritoneal fluid activated T cells and IL-13 levels, and a direct correlation between peritoneal fluid T cells and IL-12 concentrations. These data seem to suggest that a reciprocal modulation exists between peritoneal fluid cytokines and T lymphocyte subsets in patients with endometriosis.

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.

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.

Human embryo development and pregnancies in an homologous granulosa cell coculture system.

AbstractPurpose: Our purpose was to determine the effects of thecoculture of embryos on human granulosa cells (GCs) inpatients in the first cycle of IVF-ET treatment and in patientswith repeated implantation failures and to investigate thepresence of specific proteins in a 48-hr GC conditionedmedium and the GC ultrastructural characteristics. Methods: Eighteen patients with tubal or idiopathicinfertility were enrolled in this study: 7 patients (Trial 1) were inthe first cycle of IVF-ET treatment and 11 patients (Trial 2)had repeated implantation failures (one to five). Embryosfrom each patient were cocultured randomly either onhomologous granulosa cells or on a conventional culturemedium. Results: At the end of the coculture period (day 5 or 6),50% of the embryos (Trial 1) reached the blastocyst stage,with respect to 35% in Trial 2. The pregnancy rate perretrieval was 14.2 and 9%, respectively, in Trial 1 and inTrial 2. Many conditioned media showed proteins of 24–29kDa. and some of them showed additional proteins of 90kDa. The ultrastructural analysis of GCs showed healthy,metabolically active, protein-synthesizing, and mostlysteroidogenic cells. Conclusions: GC cultures improve embryo development butnot pregnancy rates both in Trial 1 and in Trial 2.

Follicular fluid and human granulosa cell cultures: Influence on sperm kinetic parameters, hyperactivation, and acrosome reaction

OBJECTIVE To evaluate the influence of human granulosa cell (GC) cultures and follicular fluid (FF) on sperm kinetic parameters, hyperactivation, and the acrosome reaction compared with the influence of human tubal fluid (HTF) and Hams F-10 medium. DESIGN Sperm kinetic parameters, hyperactivation, and the acrosome reaction were evaluated after 6 hours of incubation in HTF, Hams F-10 medium, FF, and GC cultures. SETTING Infertility and In Vitro Fertilization Centre, Reproductive Endocrinology Unit, Institute of Obstetrics and Gynaecology. PATIENT(S) Sixteen normal semen samples. INTERVENTION(S) Sperm kinetic parameters and hyperactivation were analyzed using an automated videomicrography system, the acrosome reaction was performed using a triple-stain technique, and progesterone and 17OH-progesterone levels were measured with the use of commercially available kits. MAIN OUTCOME MEASURE(S) Sperm kinetic parameters, hyperactivation, acrosome reaction. RESULT(S) The percentage of motile sperm, the mean curvilinear velocity, and the mean of the maximum amplitude of lateral head movement were increased significantly after 6 hours of incubation in FF or GC cultures compared with incubation in HTF or Hams F-10 medium, whereas the mean linearity was decreased significantly. Follicular fluid and GC cultures significantly increased hyperactivation and the acrosome reaction compared with the values obtained using HTF and Hams F-10 medium. Progesterone and 17OH-progesterone levels were increased significantly after incubation in FF and GC cultures compared with HTF and Hams F-10 medium. CONCLUSION(S) Follicular fluid and GC cultures increase sperm motility parameters, hyperactivation, and the acrosome reaction. This effect may be related to GC detoxification of the microenvironement or GC secretion of peptides, glycoproteins, growth factors (insulin-like growth factors 1 and 2), or steroids (progesterone and 17OH-progesterone).

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.

The effect of leuprorelin on steroidogenesis of human preovulatory granulosa cells in vitro

AbstractPurpose: LHRH analogues are commonly used in in vitro fertilization protocols to induce hypogonadotropic hypogonadism. The aim of our study was to evaluate the action of the LHRH agonist leuprorelin on the E2 steroidogenesis of human preovulatory granulosa cells. Results: FSH causes a significant increase in E2 production which is double that of the basal condition (P<0.01). At concentrations of 1, 10, and 100 ng/ml, leuprorelin does not produce any modification with respect to the basal condition during the 24- or 72-hr culture period. The FSH, added at different analogue concentrations, produces a significant increase in E2 production as compared to the basal condition (P<0.05) and the E2 production percentage is similar to the values obtained with FSH alone during the 24- or 72-hr culture period. Conclusions: Leuprorelin has no effect on the in vitro E2 production at any concentration. The treatment with different doses of leuprorelin does not suppress FSH-stimulated E2 production. Our findings suggest that human granulosa cells are not acutely sensitive to a direct action on E2 steroidogenesis by LHRH analogues.