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

Epoetin alfa increases frataxin production in Friedreich's ataxia without affecting hematocrit.

Objective of the study was to test the efficacy, safety, and tolerability of two single doses of Epoetin alfa in patients with Friedreich's ataxia. Ten patients were treated subcutaneously with 600 IU/kg for the first dose, and 3 months later with 1200 IU/kg. Epoetin alfa had no acute effect on frataxin, whereas a delayed and sustained increase in frataxin was evident at 3 months after the first dose (+35%; P < 0.05), and up to 6 months after the second dose (+54%; P < 0.001). The treatment was well tolerated and did not affect hematocrit, cardiac function, and neurological scale. Single high dose of Epoetin alfa can produce a considerably larger and sustained effect when compared with low doses and repeated administration schemes previously adopted. In addition, no hemoglobin increase was observed, and none of our patients required phlebotomy, indicating lack of erythropoietic effect of single high dose of erythropoietin. © 2010 Movement Disorder Society

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

Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism

Abstract PURPOSE: An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. METHODS: In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. RESULTS: After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p 15 %), while no significant variation occurred in the patients with DFI values ≤15 %. CONCLUSIONS: Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 %