DNA fragmentation in morphologically normal human spermatozoa from teratozoospermic patients

CONRADO AVENDAÑO; ANAHÍ FRANCHI; STEVEN TAYLOR; MAHMOOD MORSHEDI; SILVINA BOCCA; SERGIO OEHNINGER

Barcelona, Spain.

Congreso; 24th Annual Meeting of the European Society of Human Reproduction and Embryology; 2008

European Society of Human Reproduction and Embryology

Introduction: The presence of a high percentage of spermatozoa with DNA damage may have a negative effect on the outcome of assisted reproductive technologies. ICSI bypasses natural sperm selection processes, as the embryologist subjectively chooses the spermatozoon to be injected into the oocyte based on its motility and morphology appearance. However, these selection criteria will not exclude, for example, the presence of a chromosomal abnormality. The exclusion of spermatozoa with nuclear defects can thus be expected to decrease the probability of accidental injection of a DNA-damaged spermatozoon into the oocyte. The objective of this study was to evaluate the presence of DNA fragmentation in spermatozoa with normal morphology obtained from the separated fractions of highly motile sperm, as these are the cells that with high probability will be chosen by the embryologist at the time of oocyte injection for ICSI. Materials and methods: Ejaculates from fertile donors (n ¼ 4) and infertile patients with moderate and severe teratozoospermia (n ¼ 10) were studied. Purified populations of highly motile sperm were obtained by swim-up. Sperm DNA fragmentation was evaluated by Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL), using In Situ Cell Death Detection Kit, Fluorescein. Simultaneous examination of normal sperm morphology (fixed wet preparation without staining) and DNA fragmentation in the same cell were performing using phase contrast and TUNEL respectively. A total of 400 cells were evaluated in two droplets per patient. During this examination, every time a spermatozoon with normal morphology was found, the light was immediately switched to the fluorescence to determine DNA integrity. Spermatozoa were considered normal when the head had a normal shape, a symmetrical and oval head configuration, vacuoles occupying less than 20% of the head area, an acrosomal region comprising 40?70% of the head area, a symmetrical insertion of the tail, and absence of midpiece or neck defects. Results: The proportion of TUNEL positive cells was 3.9%+2.9 (mean+SD) for the fertile group and 21.2%+13.4 for the infertile group. There was a significant difference between groups (p<0.05). The percentages of normal sperm morphology in the fixed wet preparations without staining examined under phase contrast were: fertile group, 7.5%+0.6 and infertile group 1.0%+0.3 (p<0.05). Next, the spermatozoa with normal morphology were examined with fluorescence for TUNEL analysis. No DNA fragmentation was found in spermatozoa with normal morphology in any of the samples from the fertile men. However, in all the samples from the patients in the infertile group between 20% and 66.6% of sperm with normal morphology presented DNA fragmentation (p<0.05). Conclusions: Spermatozoa from infertile men with moderate and severe teratozoospermia, with apparently normal morphology and recovered from the motile fractions after swim-up may have DNA fragmentation. Results pose a question on the use of ?normal morphology? alone as a reliable attribute for the selection of sperm for ICSI in this group of patients. Ideally, an evaluation of sperm morphology and DNA fragmentation in the same cell should be performed in all patients will be undergoing ICSI, in order to increase the fertility outcome and prevent or reduce the risk of inducing genetic alteration in the offspring.