Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Contraception. 2013 Sep;88(3):387-95. doi: 10.1016/j.contraception.2012.11.022. Epub 2012 Dec 7.
Developing an objective, reliable method to determine semen exposure in cervicovaginal fluids is important for accurately studying the efficacy of vaginal microbicides and contraceptives. Y-chromosome biomarkers offer better stability, sensitivity, and specificity than protein biomarkers. TSPY4 belongs to the TSPY (testis-specific protein Y-encoded) family of homologous genes on the Y-chromosome. Using a multiplex PCR amplifying TSPY4, amelogenin, and Sex-determining region in the Y chromosome (SRY), our objective was to determine whether a gene in the TSPY family was a more sensitive marker of semen exposure in cervicovaginal fluids than SRY.
The multiplex polymerase chain reaction (PCR) was developed using sperm and vaginal epithelial (female) DNA. Diluted sperm DNA and mixed male/female DNA was used to determine the sensitivity of the multiplex PCR. Potential interference of TSPY4 amplification by components in cervicovaginal and seminal fluids was determined. TSPY4 and SRY amplification was also investigated in women participating in a separate IRB-approved clinical study in which cervicovaginal swab DNA was collected before semen exposure and at various time points after exposure.
TSPY4, SRY, and amelogenin were amplified in sperm DNA, but only amelogenin in female DNA. The limit of sperm DNA from which TSPY4 could be amplified was lower than SRY (4 pg vs 80 pg). TSPY4 could also be amplified from mixed male/female DNA. Amplification was not affected by cervicovaginal and seminal components. Using cervicovaginal swab DNA from three women before and after semen exposure, TSPY4 was detected up to 72 h post exposure while SRY detection was observed up to 24-48 h. TSPY4 was detected up to 7 days post exposure in one out of three women.
We have demonstrated that TSPY4 is a new sensitive, and sperm-specific biomarker. The multiplex PCR incorporating this new biomarker has potential to be an objective measure for determining semen exposure in clinical trials of vaginal products such as contraceptives and HIV pre/post-exposure prophylaxis agents.
开发一种客观、可靠的方法来确定宫颈阴道液中的精液暴露情况对于准确研究阴道杀微生物剂和避孕药的效果非常重要。Y 染色体生物标志物比蛋白质生物标志物具有更好的稳定性、敏感性和特异性。TSPY4 属于 Y 染色体上的 TSPY（睾丸特异性蛋白 Y 编码）家族同源基因。本研究采用多重 PCR 扩增 TSPY4、amelogenin 和 Y 染色体性别决定区（SRY），旨在确定 TSPY 家族中的一个基因是否比 SRY 更敏感地标记宫颈阴道液中的精液暴露情况。
采用精子和阴道上皮（女性）DNA 开发多重聚合酶链反应（PCR）。使用稀释的精子 DNA 和混合的男性/女性 DNA 来确定多重 PCR 的灵敏度。还确定了 TSPY4 扩增是否受到宫颈阴道和精液成分的干扰。还研究了 TSPY4 和 SRY 在参与另一项经 IRB 批准的临床研究的女性中的扩增情况，该研究在精液暴露前和暴露后不同时间点收集宫颈阴道拭子 DNA。
TSPY4、SRY 和 amelogenin 在精子 DNA 中扩增，但仅在女性 DNA 中扩增 amelogenin。可以扩增 TSPY4 的精子 DNA 下限低于 SRY（4pg 对 80pg）。TSPY4 也可以从混合的男性/女性 DNA 中扩增。扩增不受宫颈阴道和精液成分的影响。使用 3 名女性在精液暴露前后的宫颈阴道拭子 DNA，在精液暴露后 72 小时内检测到 TSPY4，而在 24-48 小时内观察到 SRY 检测。在 3 名女性中的 1 名中，在精液暴露后 7 天检测到 TSPY4。
我们已经证明 TSPY4 是一种新的敏感、精子特异性生物标志物。该多重 PCR 结合了这种新的生物标志物，有可能成为临床试验中客观测量阴道产品（如避孕药和 HIV 暴露前/后预防剂）中精液暴露的一种方法。
