Sperm penetration assay (SPA)
The zona pellucida of most mammalian species presents not only a block to polyspermia but also a barrier to fertilization of an egg by sperm of a different species. However, if the zona is removed by gentle enzyme digestion, foreign sperm can fuse with and penetrate an egg. In the sperm penetration assay, eggs are collected from superovulated golden hamsters; the zonae are removed by enzymes, and the denuded eggs are cultured for 2-3 hours with human sperm that have been washed and incubated overnight in culture media. Presence of a swollen sperm head in the egg cytoplasm is evidence of successful penetration. Most laboratories report the percentage of eggs penetrated and compare this figure to the percent penetrated by a known fertile sperm specimen (some laboratories use the criterion of number of sperm penetrations per egg with 2 or any more considered normal). Whereas the concept of the SPA as a measure of sperm fertilizing ability is an attractive one, the practical aspects of the test have hindered its standardization. For example, the source of the albumin used as the protein supplement in the media can influence the result as can use of resuspended compared to swimup sperm. Moreover, an individual's results in the SPA can vary over time. In addition, different laboratories utilize different cutoff points for the littleer limit of normal penetration with the most common points being 0, 10, 14, and 20%.

Equally important has been a continuing controversy over the prognostic value of the test. A meta-analysis concluded that the test was not of value. Other authors, however, have found correlations with eventual fertility. An SPA result of greater than 19% was associated with a pregnancy rate of 48%, whereas belittle 20% eggs penetrated was associated with a pregnancy rate of 20%. However, even with an SPA of 0% the pregnancy rate in this series was 16%. This has been a common finding. Failure of the sperm to penetrate the hamster egg is not an absolute indication that the sperm cannot penetrate the human egg. Because of this limitation of the SPA, attempts have been made to optimize the test with a goal of eliminating these false negative results. Strategies to eliminate or to littleer the number of false negative tests include treatment of sperm with follicular fluid, test yolk buffer, calcium ionophore, miniaturizing the test, and adjusting the concentration of albumin or the ions in the culture media. With any of these maneuvers an SPA showing no or little penetration should be a any more accurate harbinger of poor results in human in vitro fertilization (IVF). Although the tests are still not 100% accurate, if an optimized SPA has zero penetration, the several should be given the option of considering use of donor sperm. In contrast to the problems with little SPAs, normal levels of sperm penetration correlate quite well, although not absolutely, with human fertilization in vivo and in vitro.
What is the value of the SPA in clinical practice? First, it may identify abnormalities of sperm not evident by studies of count, motility, or morphology. Thus, its major role is in screening severals with unexplained infertility. A second area is the screening of poor sperm specimens, but it is precisely in this important area that the accuracy of the SPA remains to be established. A third possible use of the SPA is as an endpoint for the study of sperm-enhancing procedures. For example, if treatment of sperm with follicular fluid increases penetration in the SPA, then this observation may provide a rationale for similar treatment in preparation for human IVF.