Original research article| Volume 88, ISSUE 3, P387-395, September 2013

TSPY4 is a novel sperm-specific biomarker of semen exposure in human cervicovaginal fluids; potential use in HIV prevention and contraception studies



      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.


      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.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Contraception
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Mauck C.K.
        • Doncel G.F.
        Biomarkers of semen in the vagina: applications in clinical trials of contraception and prevention of sexually transmitted pathogens including HIV.
        Contraception. 2007; 75: 407-419
        • Roewer L.
        Y chromosome STR typing in crime casework.
        Forensic Sci Med Pathol. 2009; 5: 77-84
        • Bahamondes L.
        • Diaz J.
        • Marchi N.M.
        • Castro S.
        • Villarroel M.
        • Macaluso M.
        Prostate-specific antigen in vaginal fluid after exposure to known amounts of semen and after condom use: comparison of self-collected and nurse-collected samples.
        Hum Reprod. 2008; 23: 2444-2451
        • Minnis A.M.
        • Steiner M.J.
        • Gallo M.F.
        • et al.
        Biomarker validation of reports of recent sexual activity: results of a randomized controlled study in Zimbabwe.
        Am J Epidemiol. 2009; 170: 918-924
        • Pang B.C.
        • Cheung B.K.
        Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen.
        Forensic Sci Int. 2007; 169: 27-31
        • Hobbs M.M.
        • Steiner M.J.
        • Rich K.D.
        • Gallo M.F.
        • Warner L.
        • Macaluso M.
        Vaginal swab specimen processing methods influence performance of rapid semen detection tests: a cautionary tale.
        Contraception. 2010; 82: 291-295
        • Gallo M.F.
        • Behets F.M.
        • Steiner M.J.
        • et al.
        Validity of self-reported 'safe sex' among females sex workers in Mombasa, Kenya–PSA analysis.
        Int J STD AIDS. 2007; 18: 33-38
        • Thomsen S.C.
        • Gallo M.F.
        • Ombidi W.
        • et al.
        Randomised controlled trial on whether advance knowledge of prostate-specific antigen testing improves participant reporting of unprotected sex.
        Sex Transm Infect. 2007; 83: 419-420
        • Walsh T.L.
        • Frezieres R.G.
        • Peacock K.
        • et al.
        Use of prostate-specific antigen (PSA) to measure semen exposure resulting from male condom failures: implications for contraceptive efficacy and the prevention of sexually transmitted disease.
        Contraception. 2003; 67: 139-150
        • Chen M.P.
        • Macaluso M.
        • Blackwell R.
        • et al.
        Self-reported mechanical problems during condom use and semen exposure. Comparison of two randomized trials in the United States and Brazil.
        Sex Transm Dis. 2007; 34: 557-562
        • Mauck C.K.
        • Weaver M.A.
        • Schwartz J.L.
        • Walsh T.
        • Joanis C.
        Critical next steps for female condom research—report from a workshop.
        Contraception. 2009; 79: 339-344
        • McCluggage W.G.
        • Ganesan R.
        • Hirschowitz L.
        • Miller K.
        • Rollason T.P.
        Ectopic prostatic tissue in the uterine cervix and vagina: report of a series with a detailed immunohistochemical analysis.
        Am J Surg Pathol. 2006; 30: 209-215
        • Macaluso M.
        • Lawson L.
        • Akers R.
        • et al.
        Prostate-specific antigen in vaginal fluid as a biologic marker of condom failure.
        Contraception. 1999; 59: 195-201
        • Negri C.
        • Tosi F.
        • Dorizzi R.
        • et al.
        Antiandrogen drugs lower serum prostate-specific antigen (PSA) levels in hirsute subjects: evidence that serum PSA is a marker of androgen action in women.
        J Clin Endocrinol Metab. 2000; 85: 81-84
        • Obiezu C.V.
        • Scorilas A.
        • Magklara A.
        • et al.
        Prostate-specific antigen and human glandular kallikrein 2 are markedly elevated in urine of patients with polycystic ovary syndrome.
        J Clin Endocrinol Metab. 2001; 86: 1558-1561
        • Chomont N.
        • Gresenguet G.
        • Levy M.
        • et al.
        Detection of Y chromosome DNA as evidence of semen in cervicovaginal secretions of sexually active women.
        Clin Diagn Lab Immunol. 2001; 8: 955-958
        • Kastelic V.
        • Budowle B.
        • Drobnic K.
        Validation of SRY marker for forensic casework analysis.
        J Forensic Sci. 2009; 54: 551-555
        • Vogel T.
        • Schmidtke J.
        Structure and function of TSPY, the Y-chromosome gene coding for the "testis-specific protein".
        Cytogenet Cell Genet. 1998; 80: 209-213
        • Lau Y.F.
        Gonadoblastoma, testicular and prostate cancers, and the TSPY gene.
        Am J Hum Genet. 1999; 64: 921-927
        • Skaletsky H.
        • Kuroda-Kawaguchi T.
        • Minx P.J.
        • et al.
        The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
        Nature. 2003; 423: 825-837
        • Lemos D.C.
        • Rios A.F.L.
        • Caetano L.C.
        • et al.
        Use of the TSPY gene for sexing cattle.
        Genet Mol Biol. 2005; 28: 117-119
        • Zimmermann B.
        • El-Sheikhah A.
        • Nicolaides K.
        • Holzgreve W.
        • Hahn S.
        Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.
        Clin Chem. 2005; 51: 1598-1604
        • Fichorova R.N.
        • Rheinwald J.G.
        • Anderson D.J.
        Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins.
        Biol Reprod. 1997; 57: 847-855
        • Santos F.R.
        • Pandya A.
        • Tyler-Smith C.
        Reliability of DNA-based sex tests.
        Nat Genet. 1998; 18: 103
        • Morikawa T.
        • Yamamoto Y.
        • Miyaishi S.
        A new method for sex determination based on detection of SRY, STS and amelogenin gene regions with simultaneous amplification of their homologous sequences by a multiplex PCR.
        Acta Med Okayama. 2011; 65: 113-122
        • Mauck C.K.
        • van der Straten A.
        Using objective markers to assess participant behavior in HIV prevention trials of vaginal microbicides.
        J Acquir Immune Defic Syndr. 2008; 49: 64-69
        • Skoler-Karpoff S.
        • Ramjee G.
        • Ahmed K.
        • et al.
        Efficacy of Carraguard for prevention of HIV infection in women in South Africa: a randomised, double-blind, placebo-controlled trial.
        Lancet. 2008; 372: 1977-1987
        • Abdool Karim Q.
        • Abdool Karim S.S.
        • Frohlich J.A.
        • et al.
        Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women.
        Science. 2010; 329: 1168-1174
        • Michael N.L.
        Oral preexposure prophylaxis for HIV-Another arrow in the Quiver?.
        N Eng J Med. 2010; 363: 2663-2665
        • van der Straten A.
        • Van Damme L.
        • Haberer J.E.
        • Bangsberg D.R.
        Unraveling the divergent results of pre-exposure prophylaxis trials for HIV prevention.
        AIDS. 2012; 26: F13-F19
        • Graves H.C.
        • Sensabaugh G.F.
        • Blake E.T.
        Postcoital detection of a male-specific semen protein. Application to the investigation of rape.
        N Eng J Med. 1985; 312: 338-343
        • Reynolds R.
        • Varlaro J.
        Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene sequences.
        J Forensic Sci. 1996; 41: 2479-2486
        • Sullivan K.M.
        • Mannucci A.
        • Kimpton C.P.
        • Gill P.
        A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin.
        Biotechniques. 1993; 1993: 636-641
        • Jadack R.A.
        • Yuenger J.
        • Ghanem K.G.
        • Zenilman J.
        Polymerase chain reaction detection of Y-chromosome sequences in vaginal fluid of women accessing a sexually transmitted disease clinic.
        Sex Transm Dis. 2006; 33: 22-25
        • Zenilman J.M.
        • Yuenger J.
        • Galai N.
        • Turner C.F.
        • Rogers S.
        Polymerase chain reaction detection of Y chromosome sequences in vaginal fluid: preliminary studies of a potential biomarker for sexual behavior.
        Sex Transm Dis. 2005; 32: 90-94
        • Brotman R.M.
        • Melendez J.H.
        • Smith T.D.
        • Galai N.
        • Zenilman J.M.
        Effect of menses on clearance of Y-chromosome in vaginal fluid: implications for a biomarker of recent sexual activity.
        Sex Transm Dis. 2010; 37: 1-4
        • Mayntz-Press K.A.
        • Sims L.M.
        • Hall A.
        • Ballantyne J.
        Y-STR profiling in extended interval (>3days) postcoital cervicovaginal samples.
        J Forensic Sci. 2008; 53: 342-348
        • Lau Y.F.
        • Kido T.
        • Li Y.M.
        The TSPY gene family. World Scientific Press, New Jersey2007
        • Devany S.A.
        • Palomaki G.E.
        • Scott J.A.
        • Bianchi D.W.
        Noninvasive fetal sex determination using cell-free fetal DNA.
        J Am Med Assoc. 2011; 306: 627-636
        • Giardina E.
        • Pietrangeli I.
        • Martone C.
        • et al.
        Whole genome amplification and real-time PCR in forensic casework.
        BMC Genomics. 2009; 10: 159-167
        • Finning K.M.
        • Chitty L.S.
        Non-invasive fetal sex determination: impact on clinical practice.
        Semin Fetal Neonatal Med. 2008; 13: 69-75
        • Melendez J.H.
        • Giles J.A.
        • Yuenger J.D.
        • et al.
        Detection and quantification of Y-chromosomal sequences by real-time PCR using the LightCycler System.
        Sex Transm Dis. 2007; 34: 617-619