Ensuring that cervicovaginal secretions obtained from sexually active women are free of semen is essential to avoid misinterpretation of the data and accurately assess the immune response in the female genital tract. Similar precautionary measures should be undertaken when analysing genital shedding of HIV in infected women. The presence of semen in cervicovaginal secretions (CVS) may be assessed by the immunochemical detection of semen-derived components by immunocapture assays, including prostatic acid phosphatase, prostatic-specific antigen (PSA)  and seminal vesicle-specific antigen . However, the latter methods may lack specificity and sensitivity. The present study was carried out to validate a highly sensitive polymerase chain reaction (PCR) for the Y chromosome in the cellular fraction of CVS for detecting contaminating semen in female genital fluids.
Two hundred and thirteen unselected women attending the National Réference Center for Sexually Transmissible Diseases and AIDS in Bangui, Central African Republic participated in the study. We followed the ethical recommendations of the Ministry of Health of the Central African Republic, and verbal informed consent was obtained from all participants. Women entering the study underwent general, genital and pelvic examination, during which CVS were collected as described below. A 7 day follow-up appointment was arranged for all women, and appropriate treatment was provided free of charge for any treatable sexually transmitted infection syndrome or genital pathogen diagnosed. CVS were collected by a standardized non-traumatic 60 s vaginal washing with 3.0 ml of phosphate-buffered saline (PBS), as previously described . The cellular fraction and the cell-free fraction of CVS were separated by centrifugation at 1000 g for 10 min and were kept frozen at −80°C until processing.
The detection of PSA and PCR amplification of DNA of the Y chromosome were performed in parallel in all CVS samples collected. The detection and quantitation of PSA were performed in 150 μl of the acellular fraction of CVS using an immunoenzymatic assay with a threshold of positivity of 0.1 ng/ml (PSA IMX System, Abbott Laboratories, Abbott Park, Chicago, IL, USA). The cutoff for the presence of PSA antigen in cervicovaginal fluid was 0.4 ng/ml, determined as the mean + 2 standard deviations (SD) of the values obtained with this assay in 150 μl of CVS obtained from 30 healthy childbearing-aged HIV-seronegative Caucasian women claiming to be not sexually active at the time of sampling and recruited as controls. For the PCR of Y chromosome, DNA was extracted from the cellular pellet of CVS using the QIAamp DNA kit, according to the manufacturer's recommendations (Qiagen AG, Basel, Switzerland). One microgram of extracted DNA was processed for Y chromosome DNA amplification, by means of a single PCR using as primer set, SRY3F and SRY3R, specific for a 229 bp region in the sex-determining region (SRY), a gene located on the short arm of the Y chromosome, as described . In order to control the quality of extracted DNA and the lack of PCR inhibitors, the ubiquitous β-globin gene was amplified by PCR.
A total of 213 women (mean age 27 years; range 15–48) were eligible for enrollment. None refused to participate in the study. The median age of first sexual intercourse was 16 years, with a median of two (range 1–8) reported lifetime partners. Forty-four women were found to be seropositive for HIV-1 (20.6%). DNA extracted from the cellular pellet of CVS was tested positive by PCR for the β-globin gene in 204 samples (96%). The nine cervicovaginal samples tested negative for the β-globin gene, suggesting poor conservation or a low amount of DNA in these samples, were excluded from the analysis. When tested for the presence of the PSA antigen, 41 of the 204 β-globin-positive CVS samples (20%) showed an optical density above the cutoff of positivity. The mean concentration of PSA antigen ± SD was 19.9 ± 20.0 ng/ml, with important differences among CVS samples. The concentrations of PSA thus ranged from 0.4 to 2 ng/ml in nine samples; from 2.1 to 10 ng/ml in 11 samples; from 10.1 to 50 ng/ml in 11 samples; and were above 50.1 ng/ml in 10 samples (interquartile range 2.9–39.9). The cellular fractions of the 204 β-globin-positive CVS were further tested for the SRY gene. Seventy-three (36%) samples gave an amplicon as a unique and clearly distinguishable band of 129 base pairs, and were considered to be positive for the Y chromosome. All PSA-containing CVS (n = 41, 20% of CVS) were also positive for SRY DNA. Thirty-two CVS samples (16%) were only positive for the presence of the Y chromosome, with no detectable PSA. The number of semen-containing CVS detected by the Y PCR was significantly higher than the number of semen-containing CVS detected by PSA detection (P < 0.001). The remaining 131 (64%) cervicovaginal samples were both PSA and Y chromosome negative.
In the present study, we demonstrate that the PSA immunocapture assay, one of the most sensitive, specific and commonly used immunoenzymatic assays available to detect semen in the CVS collected from women practising unprotected sexual intercourse , did not identify 32 out of 73 (44%) Y PCR-positive CVS. Our findings show that cervicovaginal secretions from sexually active women may contain semen unrecognized by conventional immunoenzymatic assay used to detect semen components. The detection of semen components in female genital secretions after peniovaginal intercourse depends on the clearance of the semen components and on the sensitivity of the methods used. Although the clearance of semen-associated DNA deposited in the vagina is unknown, it is likely that the DNA protected in the nucleus of spermatozoids or male gamete precursors is relatively stable in the lower female genital tract. One may hypothesize that the clearance of semen-associated DNA in the female genital tract is lower than that of soluble molecules such as PSA, in agreement with our observation that the Y chromosome was always amplified when PSA was detected in the CVS of sexually active women. The data suggest that the detection of male DNA with a higly sensitive and specific procedure such as Y PCR constitutes a method of choice to detect semen traces in female genital secretions.
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