Differential binding of X- and Y-chromosome-bearing human spermatozoa to zona pellucida in vitro

The sex of human offspring has been associated with the day in the mother's menstrual cycle on which insemination occurs, with male zygotes being form ed earlier in the fertile period than female zygotes. Using an In vitro env ironment designed to mimic the in vivo milieu, we tested the hypothesis tha t Y-chromosome-bearing spermatozoa survive functionally longer than X-chrom osome-bearing spermatozoa, and that this differential functional survival i s a contributing factor to the in vivo phenomenon. Donor semen was processe d by swim-up and incubated at 37 degreesC in culture me medium for 0, 24 an d 48 h, with human zona pellucida (hemizona, HZ) being used to select funct ional spermatozoa. A second set of in vitro storage conditions, 4 degreesC in test-yolk refrigeration buffer, was used to determine whether changing t he incubation conditions alters the process. The sex chromosome of the sper matozoa was determined using fluorescent in situ hybridization (FISH). For spermatozoa incubated at 37 degreesC, the percentage of functional (HZ boun d) Y-bearing spermatozoa was significantly increased (P < 0.05) at 48 h (55 .4 + 2.9%) but not at 0 h (50.5 + 0.7 %) or 24 h (52.8 + 3.1 %) compared to swim-up spermatozoa (50.6 + 0.3 %). No difference in the percentage of fun ctional Y-spermatozoa was observed at any time-point with storage at 4 degr eesC in refrigeration buffer. Thus, we demonstrated that significantly more Y-bearing spermatozoa were capable of zona binding than X-bearing spermato zoa at 48 h at 37 degreesC incubation, with an observed Y:X ratio of 1.15 f or these zona-bound spermatozoa compared to 1.02 for post-swim-up spermatoz oa. We conclude that a differential functional survival appears to exist be tween X-bearing and Y-bearing spermatozoa, with the latter exhibiting a lon ger functional survival under in vitro conditions.