Clinical and Diagnostic Laboratory Immunology, September 2001, p. 955-958, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.955-958.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Detection of Y Chromosome DNA as Evidence of Semen
in Cervicovaginal Secretions of Sexually Active Women
Nicolas
Chomont,1
Gérard
Grésenguet,2
Michel
Lévy,3
Hakim
Hocini,1
Pierre
Becquart,1
Mathieu
Matta,4
Juliette
Tranchot-Diallo,1
Laurent
Andreoletti,4
Marie-Paule
Carreno,1
Michel D.
Kazatchkine,1 and
Laurent
Bélec1,4,*
INSERM U430, Hôpital
Broussais,1 Département de
Statistiques, Université René
Descartes,3 and Laboratoire de
Virologie, Hôpital Européen Georges
Pompidou,4 Paris, France, and Centre
National de Référence des Maladies Sexuellement
Transmissibles et du SIDA, Bangui, Central African
Republic2
Received 26 February 2001/Returned for modification 1 May
2001/Accepted 29 May 2001
 |
ABSTRACT |
The detection of traces of semen in cervicovaginal secretions (CVS)
from sexually active women practicing unprotected sex is a prerequisite
for the accurate study of cervicovaginal immunity. Two semen markers,
the prostatic-specific antigen (PSA) and the Y chromosome, were
detected in parallel in CVS obtained by a standardized vaginal washing
of consecutive women attending the principal medical center for
sexually transmitted diseases of Bangui, Central African Republic. PSA
was detected by immunoenzymatic capture assay in the cell-free fraction
of CVS, and the Y chromosome was detected by a single PCR assay of DNA
extracted by silica from the cell fraction (Y PCR). Fifty (19%)
cell-free fractions of the 264 
-globin-positive CVS samples were
positive for PSA, and 100 (38%) cell fractions of the CVS samples were
positive for the Y chromosome. All the 50 (19%) PSA-containing CVS
samples were also positive for the Y chromosome. Fifty (19%) CVS
samples were positive only for the Y chromosome, with no detectable
PSA. The remaining 164 (62%) CVS samples were both PSA and Y
chromosome negative. These findings demonstrate that CVS from sexually
active women may contain cell-associated semen residues unrecognized by
conventional immunoenzymatic assays used to detect semen
components. The detection of cell-associated male DNA with a highly
sensitive and specific procedure such as Y PCR constitutes a method of
choice to detect semen traces in female genital secretions.
| |
INTRODUCTION |
Mucosal immunity of the female
genital tract has recently gained special attention as an important
factor that could modulate the transmission of many sexually
transmitted infections (STIs), including human immunodeficiency
virus (HIV) infection. Furthermore, current concepts of designing
vaccines against viral infections acquired through sexual portals focus
on the potential interest in inducing specific mucosal immunity at the
sites of sexual exposure in association with systemic and cellular
immune responses.
Mucosal immunity is investigated by collecting cervicovaginal
secretions (CVS), either by vaginal washing (1) or by a
vaginal or cervical swab further treated with collecting buffer
(2). One potential methodological pitfall when sampling
CVS of sexually active women is the presence of contaminating semen in
the vaginal fluid that will bias the immunological characterization of
the collected genital fluid. Female participants in clinical studies are generally asked to avoid sexual intercourse and intravaginal medications for 3 (10, 11) to 5 (4) days
before sampling of CVS. However, semen residues may be detected in the
lower female genital tract up to 5 days after sexual intercourse
(12), and CVS collected from women at high risk for
sexually transmitted diseases have frequently been found to contain
traces of semen (13). Thus, ensuring that vaginal fluid is
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 analyzing
genital shedding of HIV in infected women. Finally, sensitive methods to detect traces of semen may be required in forensic medicine.
The presence of semen in CVS is assessed by microscopic observation of
motile spermatozoids (3), determination of acid phosphatase activity in CVS (9), and the detection of
semen components, including prostatic acid phosphatase,
prostatic-specific antigen (PSA) (6), and seminal
vesicle-specific antigen (5). The latter methods,
including those based on the immunochemical detection of semen-derived
molecules by immunocapture assays, may lack specificity and
sensitivity. The present study was undertaken to assess the validity of
using a highly sensitive PCR assay for the Y chromosome (designated Y
PCR) in the cellular fraction of CVS for detecting contaminating
semen in female genital fluids.
| |
MATERIALS AND METHODS |
Study population.
Two hundred seventy-four unselected
women attending the National Reference Center for Sexually
Transmissible Diseases and AIDS in Bangui, Central African Republic,
participated in the study. The Center offers multipurpose reproductive
health services, including STI services, and operates as the main
voluntary HIV testing and counselling site in Bangui. 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 genital and
pelvic examinations, 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 STI
syndrome or genital pathogen diagnosed.
Cervicovaginal sampling.
CVS were collected by a
standardized nontraumatic 60-s vaginal washing with 3.0 ml of
phosphate-buffered saline, as previously described (1).
The cellular fraction and the cell-free fraction of CVS were separated
by centrifugation at 1,000 × g for 10 min and kept
frozen at 
80°C until processing. The ratio of dilution of native
CVS introduced by the washing procedure has previously been calculated
as being 1:10 (1). Menstruating women and those with
genital bleeding were excluded from the study.
Detection of semen traces.
Detection of PSA and PCR
amplification of DNA of the Y chromosome were performed in parallel in
all collected CVS samples.
The detection and quantitation of PSA were performed with 150 µl of
the acellular fraction of CVS by an immunoenzymatic assay with a
threshold of positivity of 0.1 ng/ml (PSA IMX System; Abbott Laboratories, Chicago, Ill.). The cutoff for the presence of PSA antigen in cervicovaginal fluid was 0.4 ng/ml, determined as the mean
plus 2 standard deviations of the values obtained with this assay with
150-µl samples of CVS obtained from 30 healthy childbearing-aged HIV-seronegative Caucasian women claiming to be non-sexually active at
the time of sampling and recruited as controls. Positive and negative
controls for enzyme-linked immunosorbent assay (ELISA) were those
proposed by the manufacturer.
For PCR of the 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 assay with the primer set SRY3F (5'-CGC ATT CAT
CGT GTG GTC TCG-3') and SRY3R (5'-ATT CTT CGG CAG CAT CTT
CGC-3'), specific for a 229-bp region in the sex-determining
region (SRY), a gene located on the short arm of the Y chromosome
(7). The PCR consisted of an initial denaturation at
94°C for 4 min, followed by 38 cycles of amplification (94°C, 60 s; 66°C, 60 s; and 72°C, 120 s) and a
final elongation for 10 min at 72°C. The final PCR products were
visualized under UV transillumination by means of ethidium bromide
staining after electrophoresis with a 1.5% agarose gel. The positive
control for Y PCR was a 1:103 dilution (in distilled water)
of DNA extracted from 100,000 peripheral blood mononuclear cells
from a male donor; the negative control was undiluted DNA extracted
from 100,000 peripheral blood mononuclear cells from a female donor.
This Y PCR is able to detect five copies of Y DNA chromosome per µg
of total extracted DNA (7). Furthermore, we checked that
all CVS (cellular fraction) obtained from the 30 not-at-risk,
HIV-seronegative control women claiming to be non-sexually active at
time of sampling were Y DNA chromosome negative. To control the quality
of extracted DNA and the lack of PCR inhibitors, the ubiquitous
-globin gene was amplified by PCR, as previously described
(8).
| |
RESULTS |
Population characteristics and sample processing.
Two hundred
seventy-four women (mean age, 27 years; range, 15 to 48 years) 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, one to eight) reported lifetime partners. Seventy-one women
(26%) were found to be seropositive for HIV-1. DNA extracted from a
cellular pellet of CVS tested positive by PCR for the -globin gene
in 264 samples (96%). The 10 cervicovaginal samples testing negative
for the -globin gene, suggesting poor conservation or a low amount
of DNA in these samples, were excluded from the analysis.
Detection of PSA and Y chromosome in CVS.
When tested for the
presence of the PSA antigen, 50 of the 264 -globin-positive CVS
samples (19%) showed an optical density above the cutoff of
positivity (Fig. 1). The mean
concentration of PSA antigen ± standard deviation was 18.7 ± 19.9 ng/ml, with important differences among CVS samples. Thus, the
concentrations of PSA ranged from 0.4 to 2 ng/ml in 10 samples, from
2.1 to 10 ng/ml in 17 samples, and from 10.1 to 50 ng/ml in 12 samples
and were greater than 50.1 ng/ml in 11 samples (interquartile range, 2.8 to 39.3).
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|
FIG. 1.
Distribution of PSA concentrations among the 50 PSA-positive CVS samples. The box comprises the first to third
quartiles; the horizontal line shows the median.
|
|
The cellular fractions of the 264 -globin-positive CVS samples were
tested further for the SRY gene. One hundred samples (38%) gave an
amplicon as a unique and clearly distinguishable band of 129 bp and
were considered positive for the Y chromosome. All PSA-containing CVS
samples (n = 50; 19% of all CVS samples) were also
positive for SRY DNA. Fifty CVS samples (19%) were positive only for
the presence of the Y chromosome, with no detectable PSA. The number of
semen-containing CVS samples detected by Y PCR (n = 100) was significantly higher than the number of semen-containing CVS samples detected by PSA assay (n = 50). The
remaining 164 cervicovaginal samples (62%) were negative for both PSA
and the Y chromosome.
Choice of the best strategy.
Taken together, our experimental
observations indicate that the detection of semen by means of an
immunocapture assay for PSA is easy (automatic) and relatively
inexpensive, but it lacks sensitivity. The detection of Y DNA in the
cellular fraction of CVS by PCR is both very specific (no
cross-amplification is possible in the female genital tract) and
sensitive (high sensitivity of the gene amplification procedure used),
but it is time consuming and relatively expensive.
A mathematical approach for the choice of the best strategy to be used
is proposed in the Appendix. One can suppose that all PSA-containing
CVS samples are TPPSA (where
TPPSA is the number of true-positive
samples), since all PSA-positive samples were confirmed to be Y
PCR positive; then, 1 
1 = 50/(50 + 50), and 1 = 0.50; one can also assume that
1 is very close to 0 (1 
0),
since all PSA-positive samples were defined according to a threshold of
positivity used in the PSA assay established with cervicovaginal
samples from a control group of women who were not sexually active at
the time of sampling; q is approximately equal to 3, since
an ELISA is approximately three times less expensive than a simple PCR
(14). Thus, equation A8 (see Appendix) may be written as
follows: Pc = 1/1.50 0.66. Strategy A
(i.e., the use of PSA for all cervicovaginal samples and Y PCR with
PSA-negative samples) should be considered if the value of
Pe reaches or exceeds 66%; below the critical
value Pc, strategy B (Y PCR in all samples) appears less expensive and should be preferred (Fig.
2).
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|
FIG. 2.
Estimated costs of strategy A (detection of PSA in all
cervicovaginal samples and use of Y PCR for PSA-negative samples)
(CA) and strategy B (Y PCR in all samples),
CB, as a function of the expected
prevalence of semen residue in the study population
(Pe), according to equations A2 and A3, assuming
that factor 1 is very close to 0 (1 0) and that the cost of Y PCR is q-fold that of PSA
immunochemical detection (q > 1). When the
Pe reaches the critical value
Pc in the study population (0.66, according
to equation A8, where 1 = 0.50 and q = 3), CA and CB are
equivalent. Strategy A is preferable if Pe is
>66% in the study population; below Pc,
strategy B is less expensive and should be preferred.
|
|
If one supposes that the initial screening ELISA (SELISA) used in
strategy A has a sensitivity, Se, such that
SeSELISA = 1 3 and a specificity Sp,
such that SpSELISA = 1 3
that is very close to 1(3 0),
Pc may be considered a function of
3, as follows:
|
(1)
|
or
|
(2)
|
Under these experimental conditions, Pc is
inversely proportional to the sensitivity of the screening
immunoenzymatic assay. In other terms, the higher the sensitivity of
the ELISA (SeSELISA), the lower is the critical
value Pc and the greater are the chances that
strategy A is preferred for a population of sexually active women in
which the Pe is high.
| |
DISCUSSION |
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 CVS collected from
women practicing unprotected sexual intercourse, did not show 50 (50%) of 100 Y PCR-positive CVS. Our findings show that CVS from
sexually active women may contain semen unrecognized by conventional
immunoenzymatic assays used to detect semen components. The detection
of semen components in the female genital secretions after penovaginal
intercourse depends on the clearance of the semen components and on the
sensitivity of methods used. Although the rate of 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 female lower genital tract. One
may hypothesize that the rate of 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 by a highly
sensitive and specific procedure such as Y PCR constitutes a method of
choice to detect semen traces in female genital secretions.
Because PCR is time consuming and relatively expensive, the possibility
of screening CVS for soluble semen components by means of an
immunoenzymatic assay and reserving Y PCR for confirmation of the
negative samples is interesting. In the study population of women
living in Central Africa, the prevalence of semen retention in
cervicovaginal fluid was as high as 38%. Using a mathematical approach
taking into account the expected prevalence of semen-containing CVS in
a given population, the strategy of screening CVS samples for PSA and
to further confirming only the PSA-negative samples by Y PCR was
estimated to be more expensive than systematically searching for Y DNA
material in the cellular fraction of each genital secretion. In studies
requiring genital secretion samples from voluntary participants
claiming to have avoided sexual intercourse prior to genital sampling,
as well as in pathophysiological studies including women having
unprotected sex, the prevalence of women with semen-containing CVS may
be expected to be low or at least below the critical value of
Pc. Under these conditions, our model shows that
the best strategy consists of directly detecting the Y chromosome
without prescreening by the detection of soluble semen components.
The PCR detection of Y DNA in the cellular fraction of CVS appears more
sensitive and is likely to be more specific than the immunochemical
detection of soluble semen components, such as PSA, in establishing the
presence of semen. Obviously, the use of Y PCR might not be recommended
as the best tool to assess recent sexual intercourse, for example, in
forensic medicine. We conclude that the detection of semen in
cervicovaginal fluid is an essential prerequisite to accurately assess
HIV-specific cervicovaginal immunity or genital shedding of HIV in
HIV-infected women, as well as in HIV-negative women exposed to the
virus through sexual activity.
| |
APPENDIX |
Two strategies may be considered in assessing the presence of
semen in CVS. In strategy A, all samples are tested for the presence of
PSA, and then PSA-negative samples are tested by PCR. In strategy B,
all CVS samples are screened for the SRY gene. We have developed a
mathematical approach that takes into account the expected prevalence
of semen residues in the study population (Pc)
and the relative cost of each procedure used to detect semen residues
(C).
The PSA immunocapture assay is characterized by its sensitivity
(SePSA) and specificity
(SpPSA), where TPPSA
represents the number of true-positive samples in a population of
n women, TNPSA is the number of
true-negative samples, FPPSA is the number of false-positive samples, and FNPSA is the number
of false-negative samples, as follows:
where 1 is a constant and
SePSA corresponds to the percentage of
semen-containing cervicovaginal samples that have been correctly
detected by PSA detection. In the following equation
1 is a constant and SpPSA
corresponds to the percentage of semen-free cervicovaginal samples that
have been found to be truly negative for the presence of PSA.
Similarly, Y PCR is characterized by its sensitivity
(SeY) and specificity
(SpY), as follows, where
TPY represents the number of true-positive
samples in the tested population of women, TNY is the number of true-negative samples, FPY is
the number of false-positive samples, and FNY is
the number of false-negative samples:
and
where 2 and 2 are constants.
According to the high specificity and sensitivity of PCR procedure, one
can assume that the specificity of the Y PCR is close to 1, giving
2 0, and that its sensitivity is also very
close to 1, giving 2 0.
The total cost of the strategy A, CA, may be
expressed as follows:
![C<SUB>A</SUB>=n(C<SUB>PSA</SUB>)+[n P<SUB>e</SUB><UP> · &agr;<SUB>1</SUB></UP>+n(1−P<SUB>e</SUB>) · (1−<UP>&bgr;</UP><SUB>1</SUB>)] C<SUB>Y</SUB>](/content/vol8/issue5/fulltext/955/img007.gif)
|
(A1)
|
where CPSA represents the cost per sample
of the PSA immunocapture assay, and CY is the
cost per sample of the Y PCR. If one considers that
CY is q-fold
CPSA (q > 1), equation A1
becomes:
![C<SUB>A</SUB>=n(C<SUB>PSA</SUB>)+[n P<SUB>e</SUB><UP> · &agr;</UP><SUB>1</SUB>+n(1−P<SUB>e</SUB>) · (1−<UP>&bgr;</UP><SUB>1</SUB>)]qC<SUB>PSA</SUB>](/content/vol8/issue5/fulltext/955/img008.gif)
|
(A2)
|
![C<SUB>A</SUB>=n(C<SUB>PSA</SUB>) [1+q+q(<UP>&agr;<SUB>1</SUB> + &bgr;<SUB>1</SUB></UP>−1)P<SUB>e</SUB>−q<UP>&bgr;<SUB>1</SUB></UP>]](/content/vol8/issue5/fulltext/955/img009.gif)
|
(A3)
|
The total cost of strategy B, CB, may be
expressed as follows:
|
(A4)
|
![C<SUB>B</SUB>=n(q[C<SUB>PSA</SUB>])](/content/vol8/issue5/fulltext/955/img011.gif)
|
(A5)
|
The costs of strategies A and B are equivalent, when
Pe reaches the critical value,
Pc, deduced from equation A6 and A7:
|
(A6)
|
|
(A7)
|
![P<SUB>c</SUB>=[1−<UP>&bgr;<SUB>1</SUB></UP>]<UP>/</UP>q (1−<UP>&agr;<SUB>1</SUB> − &bgr;<SUB>1</SUB></UP>)](/content/vol8/issue5/fulltext/955/img014.gif)
|
(A8)
|
| |
ACKNOWLEDGMENTS |
This study was supported by the Institut National de la
Santé et de la Recherche Médicale, the Université
Pierre et Marie-Curie (Paris VI), and the Agence Nationale de
Recherches sur le SIDA. N.C. is recipient of a scholarship of the
Ministère de l'Education Nationale, de la Recherche et de la
Technologie, Paris, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, Hôpital Européen Georges Pompidou, 20 rue
Leblanc, 75 908 Paris Cedex 15, France. Phone: 331 56 09 39 59. Fax:
331 56 09 24 47. E-mail:
laurent.belec{at}egp.ap-hop-paris.fr.
| |
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Clinical and Diagnostic Laboratory Immunology, September 2001, p. 955-958, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.955-958.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
