AKT, p-AKT, ERK1/2 and p-ERK1/2 in Mural Granulosa Cells Are Not Correlated to Different Ovarian Stimulation Protocols in Patients Undergoing Assisted Reproductive Treatment

(1) Background: In this paper we aim to study the relationship between the expression levels of molecules involved in apoptotic/survival pathways, considered as molecular markers of oocyte competence (i.e., AKT, p-AKT, ERK1/2, and p-ERK1/2) in mural granulosa cells (MGCs) and the administration of r-FSH alone or combined with exogenous r-LH, in ovarian stimulation protocol. Moreover, we aim to evaluate oocyte competence by comparing normally cleaved embryos that were transferred in the uterus, with embryos that were arrested during in vitro culture. (2) Methods: The study included 34 normo-responder women undergoing ICSI procedures. All subjects were divided into two groups. Group A consisted of 18 women stimulated with r-FSH and used as a control group; Group B consisted of 14 women stimulated with r-FSH combined with r-LH. The MGCs were obtained from individual follicles. Immunoblot analyses were carried out to analyze the AKT, p-AKT, ERK1/2, and p-ERK1/2 levels in MGCs and to correlate them with the ovarian stimulation protocol. Furthermore, the oocyte competence was evaluated, for each follicle, according to the development of the embryo during in vitro culture and the pregnancy outcome. (3) Results: We found no significant difference in the levels of molecules in isolated MGCs between groups A and B. These results, in light of our previous research, suggest for the first time, to our knowledge, that cumulus cells and mural granulosa cells in the same follicle show different expression levels of molecules involved in the apoptotic mechanism. (4) Conclusions: Our results could clarify some controversial data in the literature where cumulative cell pools of cumulus and granulosa were analyzed, described as ovarian follicle cells, and used as markers of oocyte competence. In this paper, we found evidence that cumulus and granulosa cells need to be analyzed separately

APOPTOTIC ANALYSIS OF CUMULUS CELLS FOR THE SELECTION OF COMPETENT OOCYTES TO BE FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION (ICSI)

Oocyte quality is one of the main factors for the success of in vitro fertilization protocols. Apoptosis is known to affect oocyte quality and may impair subsequent embryonic development and implantation. The aim of this study was to investigate the apoptosis rate of single and pooled cumulus cells of cumulus cell\u2013oocyte complexes (COCs), as markers of oocyte quality, prior to intracytoplasmatic sperm injection (ICSI).We investigated the apoptosis rate by TUNEL assay (DNA fragmentation) and caspase-3 immunoassay of single and pooled cumulus cells of COCs. The results showed that DNA fragmentation in cumulus cells was remarkably lower in patients who achieved a pregnancy than in those who did not. Cumulus cell apoptosis rate could be a marker for the selection of the best oocytes to be fertilized by intracytoplasmatic sperm injection

APOPTOSIS RATE IN CUMULUS CELLS AS POSSIBLE MOLECULAR BIOMARKER FOR OOCYTE COMPETENCE.

Several lines of evidence showed that apoptosis rate of cumulus cells in oocytes derived by assisted reproductive technologies could be used as an indicator of fertilizing gamete quality. Aim of the study was to investigate the effects of three different ovarian stimulation protocols on the biological and clinical outcome in hyporesponder patients. Collected data showed a higher significant rate of DNA fragmentation index (DFI) in U group (patients treated with Highly Purified human Menopausal Gonadotrophin) than in P group (treated with recombinant human Follicle Stimulating Hormone (r-hFSH) combined with recombinant human Luteinizing Hormone (r-hLH)). Both groups R (treated with r-hFSH alone) and P showed a significant increase in collected and fertilized oocytes number, embryo quality number. This study showed that combined r-hFSH/r-hLH therapy could represent the best pharmacological strategy for controlled ovarian stimulation and suggests to use DFI as a biomarker of ovarian function in hyporesponder patients

Apoptosis in human unfertilized oocytes after Intracytoplasmic Sperm Injection

Objective To investigate the presence of programmed cell death in unfertilized oocytes after intracytoplasmic sperm injection (ICSI), assuming that previous apoptotic events could be correlated with the fertilization failure. Design Comparison of the rate of DNA fragmentation in human oocytes at different stages of maturation soon after pick-up (control) and in unfertilized oocytes after ICSI treatment. Setting In vitro fertilization (IVF) laboratory with extensive ICSI experience. Patient(s) Sixty-three patients undergoing assisted fertilization by ICSI. Intervention(s) Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay and anticaspase-3 cleaved immunoassay to detect apoptosis in control and ICSI-treated oocytes. Main Outcome Measure(s) Differences in the percentage of oocytes demonstrating DNA fragmentation between control oocytes and unfertilized ICSI treated oocytes at different stages of maturation. Result(s) The DNA fragmentation, by TUNEL assay, appeared in all the immature control oocytes, but only 37% of mature oocytes showed DNA fragmentation. This DNA fragmentation was observed in 88.8% of the oocytes unfertilized after ICSI; furthermore, DNA fragmentation appeared as well in the sperm injected into the cytoplasm. Conclusion(s) The study has shown DNA fragmentation in human oocytes unfertilized after ICSI. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. Caspase-3 immunoassay suggests the presence of apoptosis. The high percentage of oocytes demonstrating DNA fragmentation in the unfertilized oocytes could be correlated with fertilization failure

FSH administration reduces significantly sperm apoptosis only in the case of high DFI value: a study in idiopathic dispermic patients

Introduction: In the last decades sperm DNA quality has been recognized as one of the most important markers of male reproductive potential (Lewis and Aitken, 2005; Ozmen, 2007; Tarozzi, 2007), in contrast to standard semen parameters as sperm density, motility and morphology, which do not act as powerful discriminators between fertile and infertile men. DNA damage in the male germ line is a major contributor to infertility, miscarriage and birth defects in the offspring. In animal models, it has been unequivocally demonstrated that the genetic integrity of the male germ line plays a major role in determining the normality of embryonic development. In humans, many studies showed that sperm DNA damage is associated with impaired embryo cleavage (8), higher miscarriage rates (9) and also with a significantly increased risk of pregnancy loss after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) (10). Specifically, above a threshold of 30% of sperms with fragmented DNA, chances for pregnancy are close to zero, either by means of natural conception or intrauterine insemination (Spano M, 2000; Bungum M, 2007). Since there is a clear relationship between sperm DNA damage and poor assisted reproduction technology (ART) outcomes, efforts should be directed in developing treatments to improve sperm DNA quality to be introduced into clinical use. The aim of this observational study was to investigate the effects of r-FSH administration on sperm DNA fragmentation of iOAT patients undergoing ICSI, comparing the DNA fragmentation index (DFI) before and after 90 days of FSH therapy. Matherial and Methods: Fifty-three iOAT men, with a median age of 33,6 ± 7,6 years, referred to our clinics because of fertility problems after at least two years of natural attempts, were selected for the study. In all patients DNA fragmentation was evaluated sperm prior to treatment with 150 IU of recombinant human FSH (GONAL-f®, Merck Serono) three times at week for at least three months. Patients were re-evaluated after a 3-month period with semen analysis and DNA fragmentation. Sperm DNA fragmentation index (DFI) was investigated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end labelling (TUNEL) assay. Data were analysed using the paired t-test and chi-square as appropriate. A p-value <0.05 was considered statistically significant. Results: After 3 months of r-FSH treatment, no significant differences was observed between baseline and post therapy semen sample parameters including sperm count, motility, and the percentage of normal sperm forms. IThe percentage of sperm DNA fragmentation in the total of patients dropped from 20.8 ± 9.1 to 15.1 ± 8.9 (P < 0.05) (see Tab I). Interestingly, no statistical difference was found in sperm DFI when patients showed a baseline DFI ≤15% (10.5 ± 4.2 vs 11.4 ± 4.5). We found an evident and statistically significant DFI reduction in patients with sperm baseline DFI value ≥15% (24.37 ± 9.6 vs 15.4 ± 4.6). Conclusion: Our data seems to demonstrate that FSH acts as a strong anti-apoptotic agent in reducing DNA fragmentation in iOAT patients. The therapy may be a specific pretreatment for infertile male partners of couples undergoing ICSI, specifically in the case that basal DFI is higher than 15%, reducing the percentage of spermatozoa with DNA integrity anomalies suggesting a positive effect on the reproductive outcome

Treating Mitroflow dysfunction by means of an open valve-in-valve Perceval implantation

We described a case report about treating Mitroflow dysfunction with a Perceval implantation : a valve in valve open inplantatio