Abstract
Background
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.
Study Design
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.
Results
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.
Conclusions
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.
Keywords
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Article info
Publication history
Published online: December 10, 2012
Accepted:
November 30,
2012
Received in revised form:
November 29,
2012
Received:
October 16,
2012
Identification
Copyright
© 2013 Elsevier Inc. Published by Elsevier Inc. All rights reserved.
