Penetration of human spermatozoa through the zona pellucida of nonviable human oocytes.

Abstract

The purpose of this study was to evaluate the penetrating capacity of human sperm through the intact zonae of nonviable human oocytes and the influence of oocyte storage conditions and gamete coincubation times on penetration rates. Immature oocytes were obtained from surgically removed ovarian tissue and from in vitro fertilization (IVF) and subjected to four storage conditions: 1) storage at 4 degrees C for up to 48 hours in culture medium (refrigeration, n = 53), 2) cryopreservation in 1.5 mol/L propanediol with storage at -196 degrees C (PrOH; n = 49), 3) cryopreservation in 2.0 mol/L dimethylsulfoxide with storage at -70 degrees C (DMSO; n = 20), and 4) storage in a hypertonic salt solution at 4 degrees C (salt; n = 30). Semen was obtained from fertile donors (n = 7) and after a wash and swim-up, samples were adjusted to 500,000 motile sperm/mL. Zonae and sperm were coincubated in 100-microL drops under oil, and penetration was assessed at 5 and 20 hours. Penetration was evidenced by visualization of at least one sperm head in the perivitelline space at 200x magnification. Zona penetration rates were 50.9, 32.7, 23.3, and 10.0% for refrigeration, PrOH, salt, and DMSO storage, respectively (P < .004). There was a significantly higher number of sperm in the perivitelline space after 20 hours' coincubation (3.8 +/- 0.2) compared to 5 hours (0.8 +/- 0.1) (P < .0006). This represented an increase in the penetration rate from 31.2% (34 of 109) at 5 hours to 37.6% (41 of 109) at 20 hours, although the difference was not significant. These results demonstrate that the zona pellucida of nonviable human oocytes can be used in a zona penetration assay and that refrigerated and PrOH-frozen intact zonae have the highest rates of penetration. The decrease in penetrability seen in DMSO and salt-stored oocytes may be due to alterations occurring in the zona pellucida.