A survey of the concentration of 19-OH F1α/F2α prostaglandins in the semen of fertile, infertile and vasectomized men and their stability in both liquid semen and semen stains

The levels of 19-hydroxy-prostaglandins F₁α/F₂α (PG F) in the semen of 19 vasectomized, 44 infertile and 8 fertile men were determined using a simple RIA technique. The mean concentrations observed in this survey were 45 μg/ml, 49.5 μg/ml and 59 μg/ml, respectively. No significant difference was recorded between the vasectomized and infertile groups; there were too few fertile samples available to undertake a meaningful statistical comparison. No reduction was observed in the levels of this PG in a liquid semen sample retained at room temperature over a 4 week period in the presence of a bacteriostat (sodium azide). However, a 30% reduction in the levels of 19-OH PG F occurred over the same time period when aliquots of the same semen sample were retained at either room temperature or at 4°C without azide. Finally, no reduction was observed in the concentration of 19-OH PG F in a series of 10-μl semen stains stored over a period of 6 weeks at room temperature.     

Radioimmunoassay detection limits for 19-OH F1 alpha/F2 alpha prostaglandin in normal, infertile and vasectomized semen stains. Analysis of saliva, sweat and urine for possible non-specific or matrix effects

The sensitivity of a simple radioimmunoassay (RIA) for the detection of 19-OH prostaglandin F1 alpha/F2 alpha (PG F) has been evaluated on a number of semen samples from vasectomized, infertile and fertile donors. The specificity of the test has been examined by testing saliva, sweat and urine from a number of male and female donors. The assay technique could readily detect the PG in semen stains prepared from 0.2 microliter of semen from normal, infertile and vasectomized donors. The detection limit of the assay system, based on the observed displacement, was calculated to be approximately 0.05 microliter semen. The assay could be conducted over a pH range of 7.5-10.5 even after the PG has been heated to 100 degrees C. The 19-OH series of PG were absent from sweat, saliva and female urine using the normal assay protocol; volumes in excess of 100 microliters of some urines particularly from women in labour and those with acute urinary tract infection showed some displacement. Low levels of PG were detected in 50% of the male urine analysed. However, urine samples from men who had engaged in recent sexual activity contained relatively high concentrations of PG which could be readily detected in 10 microliters of urine. These results emphasise the potential of these compounds as specific and sensitive markers for the presence of human semen.     

The evaluation of an ELISA for semen-specific 19-OH prostaglandin F1 alpha/F2 alpha using ex-casework swabs and stains

The application of an enzyme-linked immunosorbent assay (ELISA) for 19-OH Prostaglandin F1 alpha/F2 alpha (19-OH PG F) to casework analysis of seminal contamination of swabs and stains is reported. The results are compared to those where the identification of semen was based on the presence of acid phosphatase and spermatozoa. Five hundred and one samples were analysed and there was good agreement between the results of ELISA and conventional methods. The determination of 19-OH PG F confirmed both the presence of semen where spermatozoa were absent and indicated semen was present when acid phosphatase and spermatozoa were both absent. The results indicate that 19-OH PG F represents a useful marker for the casework identification of semen and is particularly valuable where spermatozoa are absent.     

The development of an enzyme-linked immunosorbent assay for 19-OH PG F1 alpha/F2 alpha

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of semen-specific 19-OH prostaglandin F1 alpha/F2 alpha (PGF). The assay can reliably and reproducibly detect as little as 2 pg 19-OH PGF/100 microliters and allows for the rapid analysis of large numbers of swab and stain extracts for the presence of semen.     

Evaluation of the 190H analogues of prostaglandins E1, E2, F1 alpha and F2 alpha as specific markers for the identification of human semen in body fluid mixtures

The specificity of antisera raised against each of the prostaglandin series 190H E1/E2 and 190H F1 alpha/F2 alpha, produced in males, was evaluated by radioimmunoassay. Further, the ability of these antisera to detect semen specific prostaglandins in mixtures of body fluids was examined. Antisera directed against the 190H E1/E2 series cross-reacted with prostaglandin E1 and marginally with E2. Antisera raised to the 190H F1 alpha/F2 alpha series were, however, highly specific to the semen specific prostaglandins 190H F1 alpha/F2 alpha and 190H E1/E2. It was possible to detect picogramme quantities of contaminating 190H F1 alpha/F2 alpha on vaginal swabs taken up to 72 h after intercourse and on vaginal swabs stored at room temperature for up to 2 years. These prostaglandins were not detected on semen free vaginal swabs, in faecal material, saliva, urine or in a sample of human milk (stain). A limited study of casework material is also described. Detection of the 190H F series, as a group, has considerable potential in the identification of human semen at picogramme levels, eliminating the need for alternative chemical tests and extensive microscopic examination.     

The application of a simple RIA technique for the detection of 19-OH F1 alpha/F2 alpha prostaglandin, a specific semen marker, in semen contaminated vaginal swabs: time since intercourse studies

The specificity of the 19-OH F1 alpha/F2 alpha prostaglandin antisera for the detection of semen in seminal/vaginal mixtures, has been evaluated. Using a parallel curve test we found that the antibody showed a high specificity for these seminal prostaglandins in seminal/vaginal mixtures at concentrations of between 2 pg and 40 pg/100 microliter. The precision of the assay has been improved by the use of a donkey-anti-rabbit ferritin-bound second antibody. The application of this detection system makes it possible to complete an assay within 4.5 h. A survey of 50 semen-free vaginal swabs obtained from 3 donors, taken throughout the menstrual cycle, showed no trace of these prostaglandins. They were also not detected in the vaginal secretions of two further donors who were undergoing medication. Using only 10-microliter aliquots of a seminal/vaginal swab extract, prepared in 500 microliter, it was possible to detect semen up to approx. 80 h after one sexual act.     

Application of an immunological method to detect trace amounts of dried human semen

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.     

DNA profiling of azoospermic semen samples from vasectomized males by using Y-PLEX 6 amplification kit

Post-vasectomized azoospermic semen samples (N = 6) were analyzed for short tandem repeats (STR) on the Y-chromosome by using Y-PLEX 6 and the 310 Genetic Analyzer. We have observed a wide variation in the yield of extracted DNA from 12.5-1,000 ng. This variation was attributed to the number of epithelial and/or white blood cells that are present in these azoospermic samples. DNA profiles of these vasectomized males were obtained for all six Y-STR loci, namely DYS393, DYS 19, DYS389II, DYS390, DYS391, and DYS385 amplified by using the Y-PLEX 6.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

The polymorphism of DIA3 was investigated by isoelectric focusing in semen samples from 235 unrelated Japanese volunteers and patients. Besides the three common phenotypes seven samples of the type 3-1 were observed. However, readable isoenzyme patterns were not demonstrated in semen samples of oligospermia under about 10 X 10(6)/ml sperm cells. The allele frequencies were DIA3*1 = 0.821, DIA3*2 = 0.164, and DIA3*3 = 0.015. The DIA3*1 frequency in oligospermia (0.765) was lower than that in normospermia (0.836). The isoelectric focusing method was successfully applied to phenotyping DIA3 in seminal stains; each phenotype was demonstrated at 37 degrees C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4 degrees C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.     

Use of the "SMITEST" PSA card to identify the presence of prostate-specific antigen in semen and male urine

To determine whether the prostate-specific antigen (PSA) could be identified in semen using the "SMITEST" PSA immunochromatographic membrane test card, we examined semen and other body fluids, including urine. Although PSA activity was detected in semen with high sensitivity using the "SMITEST" PSA card, it was also detected in adult male urine. However, the lower detectable limit in the urine was 1000-fold lower than that in semen. The concentration of PSA in adult male urine was found to be 800 ng/ml using the card. PSA activity usually can be detected in urine of individuals over 14 years old and it has been detected in urine from children as young as 11 years old. Therefore, the appearance of PSA in urine may occur anytime between the age of 12 and 14 years. To determine the stability of PSA activity in urine, dried samples of urine on filter paper were kept at room temperature for up to 3 years. Although the immunoreactive line showing PSA activity became weak after storage, it was still detectable, but faint, after 3 years. In addition, PSA activity was not detected in male serum or saliva and in the urine from human females, male cats or male dogs using the PSA card. We conclude that the PSA card is useful for identification of PSA in both semen and adult male urine.     

A medicolegal study on enzymic fluorometry of choline in human semen

Studies have been conducted on an enzymic fluorometric method based on an initial rate of reaction for the determination of choline. The reaction system consists of choline oxidase coupled to peroxidase and homovanillic acid. Concentrations of choline as low as 0.1 nmol could be detected by this procedure. The concentration of free choline in normal semen was 18.7 to 29.5 mumol/mL. Free choline in other body fluids was negligible. The choline concentrations in seminal stains maintained at room temperature were not changed during a 30-day period. Those concentrations in seminal fluids kept at room temperature were detected until at least the fifth day.     

Validation of the use of a commercially available kit for the identification of prostate specific antigen (PSA) in semen stains

PSA is currently being used to detect and monitor quantitatively the development of prostate cancer by serum levels of PSA and has also been found to be present in high concentrations in semen. Elegantly simple, sensitive, and reproducible methods have been developed for analysis of the presence of PSA, including the Tandem-E PSA Immunoenzymetric Assay. The most common procedures for the forensic identification of semen have focused on the microscopic detection of sperm, acid phosphatase activity, and immunoelectrophoretic methods for the detection of PSA. Although these methods have been used for many years, there are problems associated with each method. The Tandem-E PSA Immunoenzymetric Assay detected PSA in 100% of the forensic casework fabric samples, 80% of the forensic casework vaginal swabs and 100% of the vasectomized individuals tested. The cut-off value was determined to be 1.77 ng/mL. These results indicate that this method can be used to identify the presence of semen in forensically significant specimens.     

Typing study of human semen DIA3 by isoelectric focusing--distribution in the Wuhan population, China

Human semen DIA3 typing was studied by isoelectric focusing on ultra-thin-layer polyacrylamide gel which resulted in a simpler and more definite separation of the products of DIA3 alleles than hitherto. In 198 semen samples collected from unrelated Chinese males four different phenotypes were observed. The DIA3 allele frequencies were calculated: DIA 3(1) = 0.7727, DIA 3(2) = 0.2172, DIA 3(3) = 0.0101. The results of the stability study of 12 laboratory-prepared semen stains stored at room temperature suggested that DIA3 in seminal strains is a relatively stable genetic marker. Our gene frequencies have been compared to those reported in other populations.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.     

Secreted blood group substances: distributions in semen and stabilities in dried semen stains

A sensitive microplate hemagglutination-inhibition technique has been used to ascertain the distributions of secreted blood group substances (BGS) in a population of 176 semen specimens and to characterize the stability of these substances in dried semen stains. The BGS concentrations in semen were found to vary throughout a wide range of titer. Despite this latitude of variation, the titers for the component BGS within the blood groups could be described by a log-normal distribution function. Studies of a number of sequential semen specimens obtained from the same donors revealed that the intraindividual variation in BGS titers was much more limited than the interindividual BGS titers. Attempts to correlate variations in titers between A and H in Group A semen or B and H in Group B semen indicated that the levels of these component substances vary independently. Studies of the stability of BGS in Groups A and O semen suggested that these substances were stable when the semen stains were stored at -20 degrees C, 4 degrees C, or at ambient laboratory temperature in a dry state. In contrast, stains stored at 37 degrees C under humid conditions suffered a dramatic loss in BGS titer, with the half-life of the BGS being on the order of 30 days.     

Simultaneous identification of alpha-L-fucosidase and phosphoglucomutase (PGM) subtyping in semen stains

Seminal fluid and stains were analyzed by isoelectric focusing to determine the donor phenotype in the alpha-L-fucosidase (AlFuc) polymorphic system. The enzyme is found in both seminal fluid and spermatazoa. Three common phenotypes exist and can be identified in fluid specimens stored at 4 degrees C for more than a year. Untreated semen specimens display more than eight distinct bands of alpha-L-fucosidase activity with isoelectric points of pH 6.6 and below. Neuraminidase-treated specimens have enhanced banding patterns cathodally with a loss of activity in anodal bands making it easier to phenotype specimens. Semen stains maintained in dehumidified chambers at 25 or 37 degrees C retained activity for at least one month and could be accurately phenotyped. Activity was observed in semen specimens maintained at -20 degrees C in the dried state for a period of one year, whereas a complete loss of activity was observed after two weeks in similar specimens maintained at 25 or 37 degrees C under humid conditions. Of seventy-four semen stains analyzed, two had no apparent activity. Of the remaining seventy-two specimens 56, 32, and 12% were phenotyped as FUC 1-1, FUC 2-1, and FUC 2-2, respectively. Calculated gene frequencies are FUC1 = 0.72 and FUC2 = 0.28. Following analysis of alpha-L-fucosidase, the agarose gel can be chemically developed to reveal the PGM1 subtyping pattern. The ability to phenotype both systems in semen stains significantly improves the ability of the analyst to individualize this type of physical evidence. The probability of discrimination for these two combined systems is approximately 0.89.     

Changes in the amount of prostaglandin F2 alpha in incision wounds of the skin with different time intervals after injury

A radioimmunological method is presented for the determination of the quantity of prostaglandin F2 alpha in cut wounds on the skin of guinea pigs. Rapidly increasing quantities of prostaglandin F2 alpha can be found in skin cuts, and the level reaches 71 ng/g within the 1st hour after the injury. In the postmortem period, the quantities calculated in the cuts while the animals were still alive gradually decreased and reached a value of 17 ng/g in the 6th h after death. In postmortem cuts, inflicted in the 8th h after death, the prostaglandin was 14-18 ng/g. In later postmortem cuts the quantity was about 9-10 ng/g. Establishing the dynamics of the quantitative changes permits investigation of the prostaglandin to be used to certify whether the victim was alive or not, as well as when the skin damage was inflicted.     

Transferrin (TF) typing from semen stains using isoelectric focusing and immunoblotting: correlation of TF types among blood, semen, urine, and vaginal secretion.

We describe a method for obtaining nondistorted and reproducible transferrin (TF) typing from liquid semen and semen stains. Isoelectric focusing of TF isoproteins on polyacrylamide gel (IEF-PAGE, pH 4 to 6.5) was accomplished using a 0.5 mm thick gel. The separated isoproteins were then visualized by immunoblotting with TF-specific antibody. Pretreatment of semen samples with neuraminidase enhanced the TF band resolution. The method was reliable, sensitive and simple, with a high resolution. When maintained at room temperature, laboratory-prepared semen stains were TF-typable for up to at least 50 weeks. The TF types in semen stains were correlated with the types found in the corresponding blood and urine samples. TF typing could therefore provide an additional discriminant characteristic in the forensic examination of semen stains. An evaluation of TF typing by IEF-PAGE and immunoblotting was also performed on casework samples submitted to our laboratory.     

alpha-L-fucosidase phenotyping in human placentae, semen and seminal stains

The polymorphism of alpha-L-fucosidase (Fu) was investigated in a Japanese population from samples of placentae and semen, using isoelectric focusing. The gene frequencies of placental types were Fu1 = 0.748 and Fu2 = 0.252, and those of seminal types were Fu1 = 0.739 and Fu2 = 0.261. The coincidence in the distribution between the placental and seminal types suggests that the Fu types occurring in placentae and in semen are controlled by the same Fu alleles. The Fu typing was possible in seminal stains stored at 4 degrees C for up to 9 weeks, at room temperature for up to 7 weeks and at 37 degrees C for up to 4 weeks. The Fu types were still detectable at semen dilutions of up to 1:4. This polymorphism would provide a useful genetic marker for the medicolegal grouping of seminal stains.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.