Adolescents with varicocele have an impaired sperm–zona pellucida binding capacity

Abstract

Little is known about early changes of the semen profile (i.e., modifications of sperm concentration, progressive motility, or morphology) due to the presence of a varicocele in adolescence or about whether sperm functions become altered at such an early stage. Numerous in vitro bioassays have been proposed to assess the various sperm functions in adult infertile men. The ESHRE Andrology Special Interest Group concluded that that the sperm-zona pellucida binding assays are predictors of fertilization and in vitro fertilization (IVF) failure, providing powerful evidence for the clinical value of such tests (3). Furthermore, a recent meta-analysis demonstrated that the sperm-zona pellucida binding tests (particularly the hemizona assay, HZA), together with the induced-acrosome reaction assays, have the highest predictive power for fertilization outcome under in vitro conditions (4). In this prospectively designed, controlled, and blinded study we investigated the human sperm-zona pellucida binding capacity of ejaculates from adolescents with and without varicocele. This research protocol was approved by the institutional review board (protocol: 1361/98, Sao Paulo Federal University Ethical Committee). Signed consent forms were obtained from all adolescents and their parents. All adolescents had already initiated masturbation before being enrolled in the study. Authorization was also obtained from IVF patients who donated surplus oocytes (immature and unfertilized oocytes) for the HZA. Twenty adolescents with unilateral or bilateral varicocele grade II or III (study group), 20 adolescents without varicocele (control group), and a semen donor of recent known fertility (used as control in the HZA) were enrolled in this study, prospectively. These adolescents did not present any systemic illnesses, cryp- torchidism, orchitis, epididymitis, urethritis, or testicular atrophy in their history or physical examinations. All the adolescents were examined by the same urologist (MS) in a warm room (23° to 25°C), in the standing position. Varicoceles were classified as follows: Grade I, difficult to see but easily palpable during Valsalva maneuver; Grade II, visible and palpable without Valsalva maneuver; and Grade III, easily visible through the scrotal skin, great venous ectasy during Valsalva maneuver. Semen samples were collected in an area adjacent to the laboratory by masturbation, after 2 to 3 days of sexual abstinence. After semen liquefaction, a basic semen analysis was performed according to the World Health Organization criteria (5) and sperm morphology was evaluated by strict criteria after Diff-Quick staining. All ejaculates had a normal semen volume and presented 1 10 6 leukocytes/mL. Separation of the high motile sperm fractions was performed using a double wash swim-up procedure, and the purified populations of sperm with high motility were resuspended at 0.5 10 6 /mL in human tubal fluid (HTF, Irvine Scientific, Santa Ana, CA) supplemented with 0.3% human serum albumin (Irvine). The HZA was performed as published previously except that oocyte bisection into equal, matching hemizonae was performed manually using a micromanipulation knife (6, 7). In each experiment, the matching hemizonae from each pair were coincubated (in 5% CO2 at 37°C for 4 hours) with the semen of the adolescent (adjusted to 0.5 10 6 motile spermatozoa/mL) and with the donor sperm (also at 0.5 10 6 motile spermatozoa/mL), respectively. At the end of this period, the hemizonae were carefully rinsed in HTF and only spermatozoa tightly bound to the zona were counted under an inverted microscope (400 magnification). The hemizona index (HZI) was calculated as the number of adolescent's spermatozoa bound, divided by the number of donor's spermatozoa bound, multiplied by 100. The HZI expressed by each adolescent was calculated as the mean of the indices obtained with each bisected oocyte. Three oocytes were used per patient. The age of adolescents enrolled in the study and the sperm concentration and morphology were similar in