Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.

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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 ...

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