The Earlier Apoptosis in Human Sperm: Its Correlation with Semen Parameters and Assisted Reproduction Outcome

Abstract

The combination of Syto16/7-AAD allows the detection of apoptosis at an earlier stage than that of annexin V/7-AAD. The effects of the earlier apoptosis of spermatozoa on fertilization rate and post-embryonic development have not been investigated systematically. The objective of this study is to assess the relationship between the earlier apoptosis of spermatozoa in IVF patients and seminal parameters (concentration and morphology), to assess the effect of the earlier apoptosis of spermatozoa on ART outcomes, especially with regard to clinical pregnancy. The earlier apoptosis of spermatozoa was detected by syto16/7AAD using flow cytometry (FCM) in 98 couples (98 IVF) before and after density gradient centrifugation (DGC), and its relationships with the fertilization rate, the normal fertilization rate, the cleavage rate, the good embryo rate and the cumulative clinical pregnancy rate were assessed. Meanwhile, the correlation with seminal parameters (concentration, motility and morphology) was also analyzed. The rate of the earlier apoptosis was significantly negatively correlated to sperm concentration(r = -.302, p = .003) and membrane integrity rate(r = -.862; p < .01) after DGC. There was also a positive correlation between the rate of the earlier apoptosis and sperm mortality rate (r = .225, p = .027) after DGC. A highly significantly negative correlation was found between the rate of the earlier apoptosis and outcomes in IVF patients: the fertilization rate and the normal fertilization rate decreased as the rate of the earlier apoptosis increased (r = -.271, -.288; p = .008,.004). The cumulative clinical pregnancy rate also significantly differed between patients with the earlier apoptotic rate ≥15% and <15% (57.89% vs. 81.69%, p < .01) after DGC. The earlier apoptosis of spermatozoa seems to affect fertilization, however, it does not affect the early embryo development proficiency in IVF procedures. Because of the decreased fertilization rate, the earlier apoptosis of spermatozoa can compromise the number of available embryos and good embryos, resulting in cumulative clinical pregnancy loss.

Keywords: fertility rate; good embryo rate; membrane integrity; sperm; the cumulative clinical pregnancy rate; the earlier apoptosis.

Figure 1.

Flow Cytometry Analysis of the Earlier Apoptosis of Spermatozoa Staining with Syto16/7-AAD. Flow cytometry analysis of a representative sperm sample exhibits the percentages of the earlier apoptotic spermatozoa shown in the gated area are 64.36% (left, Q3), 25.77% (middle, Q3), and 6.21% (right, Q3), the percentages of the spermatozoa with membrane integrity are 7.91% (left, Q4), 33.86% (middle, Q4) and 64.12% (right, Q4), respectively.

Figure 2.

The Comparison of the Percentage of Sperm Apoptosis Before DGC with That After DGC. Flow cytometry analysis of a representative sperm sample exhibits that the percentage of the earlier apoptotic sperm is 23.09 %(left, Q3) before DGC, and the percentage of the earlier apoptotic sperm is 3.28%(right, Q3) after DGC. DGC = density gradient centrifugation.