New molecular markers for the evaluation of gamete quality

Purpose: Only 30 % of IVF cycles result in a pregnancy, so that multiple embryos need to be replaced, per treatment cycle, to increase pregnancy rates, resulting in a multiple gestation rate of 25 %. The use of new markers in the gamete selection, could reduce the number of the oocytes to be fertilized and embryos to be produced, but the tools to evidence the gamete competence remain unavailable and more studies are needed to identify bio-markers to select the best oocyte and sperm to produce embryos with higher implantation potentiality. Methods: To define oocyte competence, the apoptosis of the surrounding cumulus cells and the oxygen consumption rates for individual oocytes before fertilization seems to provide a non-invasive marker of oocyte competence and hence a quantitative assessment of the reproductive potential for the oocyte. The chromatin integrity seems to be used also as biological marker of sperm competence, together with the morphological evaluation of large vacuoles in the head. Results: The apoptosis rate of cumulus cells lower than 25 % and an higher oxygen consumption could be an evidence of an overall metabolic activity, related to a better fertilization ability and embryo cleavage quality. The apoptosis rate of the sperm chromatin, evaluated by direct Tunel in situ analysis, seems to be, also for the male gamete, a marker of competence and implantation potentiality, in particular when it is lower than 20 %. The evaluation of the presence of large vacuoles in the sperm head prior to perform ICSI seems to increase the implantation rate, but it is not associated to chromatin integrity. Conclusions: The biological concept of competence appears unrelated to any morphological parameters, so that it is necessary to investigate new molecular markers in the gamete selection. Apoptosis of cumulus cells in the oocytes and spermatozoa, revealing the presence of large vacuoles, could help to determine the competence of the gamete to be fertilize. © 2013 Springer Science+Business Media New York

Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian stimulation for in vitro fertilization procedures.

Objective To investigate the effects of recombinant (r-) LH supplementation in “low responder” patients undergoing ovarian stimulation with r-FSH for an IVF program. The apoptosis rate in cumulus cells was used as an indicator of oocyte quality. Design Comparison of the rate of DNA fragmentation and caspase-3 activity in cumulus cells in women stimulated with r-LH and r-FSH, versus patients treated with r-FSH alone (control). Setting In vitro fertilization (IVF) laboratory. Patient(s) Forty patients undergoing assisted fertilization programs treated with a GnRH agonist, or r-FSH treatment begun on day 3 of the cycle (control). In the r-LH group, from day 8 of gonadotropin stimulation, 150 IU per day of r-LH were administered. Intervention(s) Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine-triphosphate (dUTP) nick-end labeling (TUNEL) assay, and anti-caspase-3 cleaved immunoassay, to detect apoptosis in human cumulus cells. Main Outcome Measure(s) Difference in DNA fragmentation rate between cumulus cells derived from r-LH treatment and cumulus cells derived from control patients. Result(s) No differences were observed between the two groups in the total amount of r-FSH administered and in the number of retrieved oocytes per patient. A statistically significant increase in the number of immature oocytes and in the E2 serum peak was observed in the control group. The number of transferred embryos was significantly higher in the r-LH group. Pregnancy and implantation rates were higher in the r-LH group, but without statistical significance. The apoptosis rate in cumulus cells was higher in the control group than in the r-LH group. Conclusion(s) This study suggests that supplementation with r-LH improves the chromatin quality of cumulus cells involved in the control of oocyte maturation

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