STIX Assay Reveals Significantly Reduced Levels of SPTRX3 Protein in Spermatozoa of Men Who Conceived Twins via Assisted Fertilization and Embryo Transfer. (2009)

Thioredoxins (TRX) are a class of redox proteins that function as general protein oxidoreductases involved in antioxidant defense, modulation of cell growth and differentiation, regulation of apoptosis and activation of membrane ion channels. Mammals are equipped with an additional set of thioredoxins with an expression pattern restricted to male germ cells. One of such thioredoxins, the Spermatid-Specific Thioredoxin 3 (SPTRX3 or TXNDC8), is retained postmeiotically in round spermatids in close association with the developing acrosomal granule. Once the sperm acrosome is formed, SPTXR3 accumulates in the cytoplasmic lobe of the elongating spermatids ready to be discharged in the residual bodies and phagocytosed by the Sertoli cells. The SPTRX3 protein remains undetectable in most fully differentiated normal spermatozoa of humans and various animal species. In contrast, it is retained in the superfluous cytoplasm filling the nuclear vacuoles and wrapping around the sperm tail midpieces in fully differentiated but morphologically defective human spermatozoa. Based on the above observations, a sperm quality biomarker assay termed Sperm Thioredoxin Immunoassay (STIX) has been developed. As a part of the STIX biomarker validation, a set of sperm samples donated by men from 45 infertile couples was evaluated. Each semen sample was subjected to a two-step purification procedure using PureSperm gradient (Step 1) and sperm swim-up procedure (Step 2). Raw semen, as well as Step 1 and Step 2 purified spermatozoa were subjected to flow cytometric STIX analysis, generating a total of 135 samples. Two important observations were made: 1) In some sperm samples, the proportion of STIX-positive spermatozoa increased five fold or more once the samples passed through gradient and swim-up purification. As the cellular debris was removed by PureSperm and Swim-up, the proportion of STIX-positive spermatozoa was increased dramatically. Sperm purification is routinely used for sperm preparation prior to ICSI procedure. Consequently, this observation implies that even the two step sperm purification may not be an efficient procedure to avoid the use of defective spermatozoa for ICSI treatment. 2) Men who conceived twins (i.e. those who presumably produced most viable embryos in vitro) had the percentage of STIX-positive spermatozoa reduced after PureSperm gradient separation in a statistically significant manner, compared to men who conceived a singleton pregnancy (p=0.01) and those who produced no pregnancy (p=0.02). No more than three embryos were transferred in any of the treated couples. Most importantly, none of the available conventional semen parameters showed statistically significant differences. These findings have significant value for clinical decision making regarding the number of embryos to be transferred. As illustrated by a recent highly publicized case of an octuplet mom, there are major ethical and medical concerns regarding multiple embryo transfer after assisted fertilization. The STIX test can identify a superior sperm sample most likely to produce multiple implantations and pregnancies, providing infertility clinicians with a useful tool for deciding on single vs. multiple embryo transfer. Removing the STIX-positive spermatozoa from the ICSI pool could lead to more confidence in transferring fewer embryos and ultimately lowering the multiple birth rates while maintaining high pregnancy rates. (poster)