Summary. An evaluation of a method utilizing zona-free hamster ova to test the fertility of human spermatozoa has shown that (i) the induction of superovulation in immature animals provides the most convenient method of obtaining mature ova for study; (ii) motile spermatozoa are best prepared by the technique of layering; (iii) an 18 h incubation at 37°C (which is associated with capacitation) in an atmosphere of air (pH of medium 8·2) is preferable to one of 5% CO2 (pH of medium 7·2); (iv) the incubation and insemination densities of spermatozoa should be >1 × 106 and <10 × 106/ml; (v) spermatozoa do not remain motile, or capable of binding to or penetrating ova, after about 30 h in culture; and (vi) intra- and inter-assay variations are acceptable. The spermatozoa from 15 healthy men of proven fertility and 15 subfertile patients with normal spermiograms were evaluated for their ability to bind to and penetrate zona-free hamster ova. Of the 476 ova inseminated with spermatozoa from the fertile men > 5 spermatozoa/ovum consistently bound to the vitelline membrane and 284 ova 59·7%) had swollen sperm heads or pronuclei (still with tails attached) in their ooplasm. The range of individual penetration rates was 23·5–88·9%. Of the 586 ova tested with spermatozoa from the infertile subjects only 11 (1·9%) showed any evidence of penetration (range of individual penetration rates 0–8·7%) and binding to the vitelline membrane was poor (0 or <5 spermatozoa/ ovum). Spermatozoa from a further 9 infertile men who had abnormal spermiograms also gave poor penetration rates (4/300 ova, 1·3%). It is concluded that this bioassay has a useful role as an additional test to the classic spermiogram, but that its routine use is best reserved for selected cases of unexplained infertility.
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