Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

Seiji Watanabe 0 0 Department of Anatomy, Hirosaki University School of Medicine , 5 Zaifucho, Hirosaki 036-8562 , Japan BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media. Introduction ICSI is the only means of treatment for infertile patients showing severe asthenozoospermia. Numerous pregnancies and healthy births have been reported using intracytoplasmic injection of immotile sperm (Kahraman et al., 1996; Nijs et al., 1996; Barros et al., 1997; Vandervorst et al., 1997; Ved et al., 1997; Wang et al., 1997; Nagy et al., 1998; Sallam et al., 2001), although cases have also been reported that failed to obtain fertilized oocytes despite repeated attempts. Moreover, it is known that the fertilization and pregnancy rates are lower than for ICSI using motile sperm (Nagy et al., 1998; Shulman et al., 1999). Frequent structural chromosomal abnormality is one of the explanations for the problems of ICSI using immotile sperm. Rybouchkin et al. (1997) conducted chromosome analysis of immotile sperm from infertile patients and demonstrated that the incidences of structural chromosome aberrations were in proportion to the incidences of dead immotile sperm. In contrast, when our group attempted a preliminary cytogenetic analysis of immotile human sperm obtained from a healthy man, there was no significant increase in structural chromosome aberrations as compared with motile sperm (S.Watanabe, unpublished data). This observation may have resulted from the fact that the immotile sperm were obtained from a fertile donor, but we hypothesized that the difference in the results between the previous report and this observation could be attributed to whether immotile sperm were exposed to culture media before microinjection. To confirm this, in this study immotile sperm were cytogenetically analysed using injection into mouse oocytes before and after exposure to several kinds of culture media. Materials and methods Collection of mouse oocytes B6D2F1 female mice, 611 weeks old, were induced to superovulate by i.p. injection of 7 IU pregnant mares serum gonadotrophin followed by i.p. injection of 7 IU HCG 48 h later. Oocytecumulus cell complexes were collected from oviducts 16 h after the HCG injection. The oocytes were freed from cumulus cells by 5 min treatment of 0.1% hyarulonidase dissolved in HEPES-CZB medium (Kimura and Yanagimachi, 1995) containing 0.3% bovine serum albumin (BSA; Sigma, Carlsbad, CA, USA). The oocytes were washed twice with fresh HEPES-CZB medium and were then incubated up to 3 h in CZB medium containing 0.3% BSA (Chatot et al., 1989; 1990) at 37 C, under 5% CO2 in air before micromanipulation. Collection of human sperm Human semen samples were obtained from two fertile donors (donor I and II) showing normozoospermia according to WHO criteria (WHO, 1999). All the semen samples from donor I were liquefied for 30 min at 37 C in air, and were immediately used for ICSI. All the samples from donor II were used after cryopreservation according to a previously described method (Watanabe and Kamiguchi, 2001a). During the experiment, the fresh or frozen sperm samples were pretreated according to the manner shown in Table I. Preparation of injection chamber For ICSI, an injection chamber was prepared by placing four kinds of medium droplets on a cover of 10-cm plastic dish and covering them with mineral oil. The first was a sperm droplet (5 ml) for sperm selection. The second droplet (5 ml) was seminal plasma or one of four kinds of culture media [HEPES-BWW, HEPES-BWW modified with 20 mmol/l NaCl, Dulbeccos phosphate-buffered saline (PBS), modified-human tubal fluid (HTF)] for sperm storage. HEPESBWW modified with 20 mmol/l NaCl, modified-HTF and Dulbeccos PBS will hereafter be abbreviated as mBWW, mHTF and PBS, respectively. Compositions of the four kinds of media are shown in Table II. The third droplet (5 ml) was 10% polyvinylpyrrolidone (PVP) dissolved in PBS for sperm immobilization. The fourth droplet (20 ml) was HEPES-CZB containing 0.3% BSA for mouse oocytes. The injection chamber was placed on the microcool plate (Kitazato supply, Tokyo, Japan) of the inverted microscope with Hoffmans modulation contrast optics. The temperature of the microcool plate was maintained at 1718 C (Watanabe, 2003). Experiment 1: injection of initially immotile human sperm Injection of immotile human sperm into mouse oocytes was performed as described previously (Watanabe, 2003). To avoid contamination of the secondary immotile sperm, which probably resulted from centrifugation or dilution with culture media, liquefied fresh semen was used for the sperm droplet (the first droplet). The immotile human sperm were sucked into an injection pipette, and were then transferred into the 10% PVP droplet (the second droplet). As soon as the immotile sperm were freed from sticky seminal plasma as much as possible by repetitive gentle pipetting, a few Piezo-pulses were Experiment Group Semen Motility Medium Centrifugation 1 2 3 F-T = frozenthawed. Seminal plasma Seminal plasma Immotile HEPES-BWW Immotile mBWW Immotile PBS Immotile mHTF Immotile Seminal plasma Seminal plasma + Seminal plasma + HEPES-BWW + applied to the cells, taking into consideration that this process was indispensab (...truncated)