Sexual Assault and Semen Persistence

Introduction

Sexual assault is usually a hidden crime where the only witnesses are the victim and the assailant. For a variety of reasons, even the victim may be unable to provide a detailed account of the assault or the identity of the perpetrator. Often the case history deteriorates into one person’s word against another. With such limited initial information, the physical and biological evidence collected from the victim, from the crime scene, and from the suspect will play a pivotal role in the objective and scientific reconstruction of the events in question. Rape is an excellent example of Locard’s Principle which states: Every contact leaves a trace. In addition to the hairs, fibers and debris that may be transferred in other types of violent crime, the rapist often leaves behind a personal biological signature that may include blood, saliva, and most importantly, semen. This semen evidence is frequently a cornerstone in the investigation and prosecution of the case.
In any sexual assault investigation, there are four main questions that semen evidence may help answer:
1. Did sexual contact occur? Positive recovery of any of the component parts of semen from the victim is generally considered conclusive proof that sexual contact took place. Recovery of semen from the vagina, however, does not necessarily prove intercourse since other modes of deposition may be responsible.
2. When (or in what time frame) did the sexual contact occur? The interval between semen deposition and evidence collection may be estimated by comparing the specific findings in the case with published norms and maximum reported recovery intervals.
3. Can a specific suspect be included or excluded as the potential source of the semen? Genetic profiles from the evidence material, the victim and the suspect can be developed using conventional se-rology (antigens and enzymes) and DNA. These profiles can then be compared and a confidence level established to include or exclude a specific suspect.
4. Was the sexual contact consensual or nonconsensual? If the victim is beyond the age of consent, semen findings are not helpful. If the victim is underage, then consent is moot and the recovery of semen is consistent with the commission of the crime.


Detection of Semen

The obvious first step in finding semen evidence is interviewing the victim. Initial evidence collection at the crime scene by the law enforcement officer or crime scene investigator should be guided by the victim’s description of the event. Items collected for forensic analysis may include clothing the victim wore during or after the assault, bedding, samples from furniture, vehicles, carpeting, or any item the victim may have used to clean up with after the assault (tissue, washcloth, etc.).
Additional victim-guided sampling will be obtained in the course of the forensic medical examination. If not already retained, the ‘assault’ clothing can be collected. Highest priority is directed to items the victim believes contain semen, areas of visible staining, and any clothing item that was nearest the genital area. The examiner will also obtain samples from the victim’s body. Again, any area the victim believes was in contact with semen will be carefully swabbed. Any material in the victim’s hair (pubic, head or body) will be clipped out and retained. Scanning the victim with an ultraviolet light source may reveal the fluorescence of dried semen, which was not visible in plain light. The examiner must collect samples from any body cavity or opening where sexual contact was attempted or completed. Unfortunately, the victims are rather inaccurate in their perceptions about semen deposition in or near body cavities. Possible explanations include the severe psychologic stress of the assault and the potential release of small, but detectable, amounts of semen in the pre-ejaculate emission. Semen has also been recovered in cases where the victim believed the assailant wore a condom, so samples should be obtained despite the history of condom use.
The process of collecting biological samples from the victim usually involves the application of moistened swabs to areas of possible dried stains and the insertion of dry swabs (under direct visualization) into body cavities to absorb any liquid secretions. Some jurisdictions prefer expectorated liquid saliva samples for oral cavity sampling or vaginal aspiration (or lavage) after vaginal penetration.
The proper handling and packaging of potential semen evidence cannot be overstressed. Any ‘moist’ or ‘wet’ evidence must be thoroughly air dried. In the case of swabs, this requires placement in a stream of cool air for at least 60min. Potential biological evidence must be packaged in containers that allow air circulation; never in plastic bags or sealed, nonporous tubes, jars, or boxes. Chain of custody must be meticulously documented and adhere to the prevailing local policies. Transportation, deposition, and storage of evidence must also strictly follow local protocol.

Evaluation of Potential Semen Evidence

Evidence material presented to the forensic laboratory for analysis of possible semen will usually consist of swabs taken from various locations on or in the victim and objects (clothing, fabric, matted hair, etc.) that may contain semen. Every laboratory will have its own specific protocol and sequence for analyzing the evidence but the general approach is similar (Table 1).

Screening tests

Ultraviolet light To help identify occult semen stains, the evidence may be scanned with an ultraviolet light (or alternative light source). Dried semen may fluoresce under UV illumination and highlight stains that were previously invisible thus directing the criminalist to sample areas of higher potential yield.
Presumptive chemical tests for semen Substances within the evidence swabs or stains that allow the identification and/or the individualization of semen must be solubilized and extracted for analysis. Presumptive testing for semen helps to confirm the sampling location as a potentially positive site. The two commonly used presumptive tests for semen are both colorometrics. The Florence test identifies the presence of choline and the Brentamine test detects acid phosphatase. Both of these assays are qualitative.

Table 1 Evaluation of potential semen evidence in sexual assault investigation

Screening tests Ultraviolet light scanning Presumptive chemical tests for semen Choline (Florence test) Acid phosphatase (Brentamine test)
Confirmatory tests Spermatozoa Motile Non-motile
Proteinase K enhancement Fluorescence in situ hybridization (FISH) Noncellular semen markers Acid phosphatase p30
Individualization of semen evidence Blood group typing
ABO(H) system
Lewis antigens Enzyme typing
Phosphoglucomutase (PGM)
Peptidase A (Pep A) DNA

Confirmatory testing

Confirmatory testing for semen involves solubilization of evidence material followed by appropriate vortexing and centrifugation, which yields a supernatant and a cell pellet. The cell pellet is used primarily to detect spermatozoa and for DNA analysis. The supernatant portion is most helpful to detect noncellular markers in semen when sperm are not detected and to develop genetic profiling or grouping.
Spermatozoa Identification of one or more intact spermatozoa is conclusive proof of the presence of semen and hence affirms sexual contact. The condition and collection site of the sperm may be very helpful in defining the time interval between deposition and collection (also known as the postcoital interval or PCI).
Motile sperm The forensic medical examiner has the unique opportunity to observe motile sperm collected from the victim during the evidentiary exam. Recovery of motile sperm has only been reported from samples taken from the vagina or cervix. The technique requires the preparation of a ‘wet mount’ slide (vaginal or cervical swab sample placed on a slide with a drop of saline plus a cover slip) and examined with a phase-contrast microscope. The maximum reported recovery times are shown in Table 2.
Nonmotile sperm Although nonmotile but morphologically intact spermatozoa may be seen on the wet mount slide performed during the forensic medical examination, the identification of nonmotile sperm is usually based on the stained smears prepared by the forensic laboratory from the evidence swabs. Commonly employed staining methods include Oppitz (Christmas-tree-stain), hematoxylin and eosin (H & E) and Papanicolaou (pap smear). The slides are usually evaluated by plain light microscopy at 400 x . Some laboratories score the concentration of spermatozoa seen. This is usually reported as a range from ‘few’ (less than five sperm per field) to ’4+’ (many in every field). If the sample has been contaminated by other bodily fluids (saliva, vaginal secretions, etc.), epithelial cells and cellular debris may make the identification of spermatozoa more problematic. Selective degradation of epithelial cells and debris may be accomplished by treating the cell extract with a mixture of proteinase K and sodium dodecyl sulfate before staining and microscopic examination.
Table 2 summarizes the maximum reported recovery times for motile and nonmotile sperm collected from various anatomic locations. These ‘record’ times were compiled from multiple published studies and represent a variety of methodologies. Recent investigators have been able to compare sperm concentrations (few to 4+) found on the stained smears to the time since offense to refine estimates of probable postcoital interval.
A number of factors may influence the recovery of spermatozoa. Because the number of sperm collected per swab may be very low, most authorities suggest obtaining multiple swabs from the same site to maximize recovery and thus provide more forensic material for analysis. Victim position following the assault plays an important role in the amount of vaginal spermatozoa available for sampling. Gravity drainage creates significant loss of vaginal evidence if the victim is upright after deposition. Conversely, if the victim remains recumbent following the act, recovery is likely to be greater. This has been a legitimate criticism against comparing volunteer studies (more likely recumbent) to casework studies (more often upright). If only a small number of sperm are recovered from the vagina, the possibility of sperm deposition from a contaminated object (e.g. finger) must be considered as an alternative explanation to penile penetration.
The detection of sperm on anal swabs must be interpreted cautiously. Recovery of anal sperm from a male sodomy victim or from a female sodomy victim without any detectable vaginal sperm is consistent with anal sexual contact. Difficulty arises when the female victim has both vaginal and anal sperm. Drainage of vaginal material may contaminate the anal area. Sampling technique, specific labeling of ‘anal’ and ‘rectal’ samples and sperm concentrations all bear on the interpretation of the individual case.
Table 2 Maximum reported recovery times for spermatozoa collected from body cavities in living sexual assault victims

Vagina Cervix Mouth Rectum Anus
Motile sperm 6-28 h 3-7.5 days
Nonmotile sperm 14 h to 10 days 7.5-19 days 2-31 h 4-113h 2-44h

The recovery of spermatozoa from, in or around the mouth is generally low. Some authorities now advocate oral sampling by expectorated liquid saliva to improve the yield over the standard swab technique.
Fluorescence in situ hybridization A new molecular cytogenic analysis method offers an innovative approach to improve the identification of male cells from sexual assault evidence materials. Fluorescence in situ hybridization (FISH) uses a Y chromosome specific DNA probe to identify Y-bearing (male) cells. This technique sensitively identifies not only spermatozoa, but also nonsperm cells (epithelial and segmented neutrophils) of male origin. Although forensic experience with this technique in sexual assault cases is limited, it offers a sensitive and specific alternative to conventional methods for the primary or confirmatory detection of spermatozoa. It also offers the additional potential for confirming male-female contact when sperm and other semen markers are absent.
Noncellular semen markers When evaluation of the potential semen evidence yields a positive presumptive test or a negative presumptive test but high clinical suspicion and the analysis fails to identify spermatozoa, further testing is required. The objective is to detect the presence of markers that are specific and unique to seminal plasma but independent of sperm cell presence. The two most commonly employed semen constituents are acid phosphatase and the glycoprotein, p30.
Acid phosphatase Acid phosphatase is not a single enzyme but an array of related isoenzymes from a variety of sources. The forensic interest in acid phosphatase in the evaluation of sexual assault evidence is based on the fact that acid phosphatase activity in human semen is 500-1000 times greater than in any other normal bodily fluid. Unfortunately, the use of acid phosphatase as a marker for semen is compromised because the vagina is also a source of the same type of acid phosphatase. Since seminal and vaginal acid phosphatases cannot be reliably discriminated qualitatively, the only approach to differentiating azoospermic semen from vaginal secretions is by quantitative analysis. Finding a ‘significantly’ elevated acid phosphatase level is consistent with the presence of semen.
A number of factors, however, complicate the interpretation of any given sample. Considerable variation in baseline endogenous vaginal acid phos-phatase levels occurs not only between individuals, but also within a single individual. Pregnancy, phase of the menstrual cycle, bacterial vaginosis, use of certain feminine hygiene products or other contaminants may produce elevated endogenous acid phos-phatase values. Measurable acid phosphatase activity in vaginal samples declines after semen deposition because of drainage and dilution from vaginal secretions. The rate of decline is quite valuable. There is little consensus in the literature regarding a reliable threshold level to consistently separate endogenous vaginal acid phophatase from seminal. There is also no agreement on the time frame in which acid phos-phatase analysis is useful for forensic purposes following deposition. Reports vary from as short as 3 h to as long as 72 h. Because physiologic, temporal, quantitative and methodologic variables all potentially affect measured values, the interpretation of a specific specimen should be in the context of the database and experience of the forensic laboratory performing the test.
p30 The glycoprotein, p30 (also known as prostate specific antigen or PSA), is derived from prostate epithelial cells and is found in seminal plasma, male urine, and blood. p30 has not been found in any female body tissue or fluid. There are no significant differences in p30 recovery from vasectomized men compared to nonvasectomized controls. The finding of any p30 in evidence material confirms the presence of semen. A positive p30 analysis reliably identifies semen regardless of whether acid phosphatase is elevated or spermatozoa are detected.
As with other markers, the level of p30 declines after deposition in the vagina. The disappearance is log-linear and fairly predictable. The mean time to reach the lower limits of vaginal detection is 27 h with a range of 13-47 h. p30 thus provides an additional point of reference with which to estimate the post-coital interval. p30 is quite durable in dried stains and has been detected up to 10 years later in material stored at room temperature.
Monoclonal antibody mouse antihuman semen-5 (MHS-5) Another semen-specific antigenic protein has been detected but is not widely used in the forensic evaluation of rape. MHS-5 is produced in the seminal vesicle epithelium and is not found in any other bodily fluid besides semen and has no cross-reactivity with other body fluids. The mean vaginal decay time has not been established. Although forensic experience with the technique is limited, it may potentially be a viable adjunct in the arsenal of confirmatory semen tests.

Individualization of semen stains

Once evidence material has been shown to contain semen, the next step is to individualize the semen by developing a genetic profile of the donor. This genetic profile can then be compared with the genetic profiles of the victim and the suspect. The suspect can thus be included as the possible assailant or excluded from consideration. The more definitive the genetic typing, the greater the probability of excluding innocent suspects and the more restricted the guilty suspect group.
Semen contains many polymorphic protein markers but only a few are amenable to forensic analysis because most are only recovered in amounts too small for reliable detection or evaluation. Conventional forensic serology is usually limited to soluble blood group antigens (ABO (H) system and Lewis antigens), phosphoglucomutase (PGM) and peptidase A (Pep A).
It would not be an overstatement to say that the recent explosion in DNA technology is revolutionizing forensic science. Given the fact that DNA is both highly polymorphic and extremely stable, the forensic laboratory now has the capability to employ individualizing techniques on minute evidence samples with extraordinarily high sensitivity and discriminatory potential.
Bloodgroup typing The human red cell membrane contains hundreds of genetically determined antigens. The best studied and most important forensically is the ABO group. Closely associated with the ABO system is the ‘H’ antigen, which is a precursor of the A and B antigens. Although the H and ABO loci are not genetically linked, their functional relationship has prompted the convention of treating these antigens as a single group.
Although most red cell antigens are bound to the cell membrane, the ABO(H) antigens are soluble and can be secreted into bodily fluids (including saliva, semen and vaginal secretions). This ability to secrete soluble antigens is under the control of a pair of genes, Se and se. With Se being dominant, homozygous (Se Se) and heterozygous (Se se) individuals are ‘secretors’ and homozygous (se se) individuals are ‘nonsecre-tors”. About 80% of the population are secretors. Clinically, the secretor status of an individual can be checked by comparing that individual’s ABO blood type with the presence or absence of the same ABO(H) antigens analyzed from a saliva sample.
The detection of Lewis antigens in the blood is another method of establishing secretor status. Lewis (a— b+) phenotypes are secretors and Lewis (a+ b —) are not. The secretor status of Lewis (a — b— ) individuals are variable. In addition to secretor information, Lewis antigen phenotypes have different distributions in various racial groups.
The application of blood group typing in sexual assault cases requires the comparison of blood group substances recovered in the evidence material with those of the victim and the suspect (Table 3). The secretor status of both is also important for the proper interpretation of results. A rough quantitative assessment of the amount of antigen deposited by the assailant may be inferred by the concentration of spermatozoa found on the vaginal smear (few to 4+). The estimation of foreign antigen amounts may be used with the measured amounts of recovered antigens to further refine the interpretation of blood group results. As with the other marker assays, temporal, quantitative, methodological and physiologic variables may limit the usefulness of the blood group contribution to the genetic profile.
Despite these limitations and the substantial discriminatory power of DNA, conventional blood group typing retains a significant role in the evaluation of sexual assault evidence. Traditional grouping is cheap, fast and universally available. ABO blood grouping may be superior to DNA analysis for typing saliva or semen that contains few or no sperm. Seminal blood groups have been detected in the vagina up to 21 h after deposition. Seminal blood groups are rarely recovered from the mouth or anus/rectum.
Enzyme typing Phosphoglucomutase (PGM) and peptidase A (Pep A) are the two enzyme markers commonly used in the genetic profiling of semen evidence in sexual assault cases. These enzymes are found in semen and vaginal secretions regardless of ABO type or secretor status. Phosphoglucomutase is polymorphic in all populations and can be subdivided into ten distinct subgroups. Although peptidase A is polymorphic in many racial groups, it is most commonly used as a discriminator in cases in which the perpetrator is thought to be Black.
The measurable activity of both PGM and Pep A decline rapidly in the postcoital vagina with PGM usually dropping below threshold by 6h and Pep A not usually recovered after 3 h. An additional problem with PGM is that semen contaminated with saliva can exhibit aberrant PGM isoenzyme patterns.

Table 3 ABO blood types and antigens in the forensic evaluation of sexual assault.

Victim’s ABO phenotype Expected antigens from the victim Foreign antigens from the assailant
O H A and/or B
A A and H B
B B and H A
AB A and B and H None

DNA profiling The primary advantage of DNA profiling in sexual assault investigations is its ability to accurately individualize semen that contains only minimal numbers of spermatozoa. DNA can also be used to differentiate multiple donors in mixed stains whether there are mixed sources of the same bodily fluid or different bodily fluids contributed by different individuals. DNA technology also enhances other techniques like polymerase chain reaction, restriction fragment length polymorphism, ABO genotyping and fluorescence in situ hybridization of Y-bearing male cells. The already long list of DNA advancements is growing rapidly. As the use of DNA techniques becomes cheaper, more widely available and more uniformly accepted by the courts, the greater will be the role of the forensic laboratory in the investigation and prosecution of sexual assault cases.

Postmortem Detection of Semen

Very little has been published about the postmortem recovery of semen from rape-homicide victims. One report found morphologically intact spermatozoa in the vagina of a rape-homicide victim who had been dead 16 days. A more recent report describes the recovery of intact spermatozoa from the vagina of a homicide victim at 34 days postmortem. This victim also had detectable p30 on the vaginal swab. No information was available in either report as to the time interval between deposition of semen and death.
A few anecdotal reports have been published describing the recovery of sperm from dead bodies. In these cases there was no history of sexual assault, but swabs were obtained at autopsy as a ‘precaution’. Little or no information was available about the time of deposition relative to the time of death. The longest reported postmortem vaginal recovery was 3-4 months. One body with vaginal sperm was immersed in water up to 2.5 weeks. One mummified corpse yielded vaginal sperm 5-6 weeks postmortem. A mouth swab was positive 4 days after death.

Failure to Recover Semen

When the history anticipates the recovery of semen from a rape victim but the forensic evaluation fails to find any semen evidence, there are a number of potential explanations besides the obvious suggestion of a false complaint (Table 4). No semen will be recovered if none was deposited (sexual dysfunction, condom use). Mechanical elimination (drainage, hygiene activities), biological elimination (degradation) or

Table 4 Factors that may limit the detection or recovery of semen from the sexual assault victim

No seminal constituents recovered Sexual dysfunction in the assailant Condom use
Time delay between the assault and the exam:
Drainage of vaginal contents
Degradation of seminal secretions
Dilution by endogenous components Victim hygiene (douching, bathing, gargling, etc.) Physiologic activity (urinating, defecating, menses)
No spermatozoa recovered Impaired spermatogenesis: Germinal aplasia/hypoplasia Hormonal deficiencies (pituitary, thyroid, gonadal) Testicular irradiation Drugs/toxins Neoplasm Varicocele
Infection (mumps, tuberculosis, gonorrhea)
Elevated scrotal temperature
Chronic alcoholism Depleted stores (frequent ejaculation may create transient
azoospermia) Impaired delivery
Vasectomy
Trauma
Congenital abnormalities
physiologic dilution (or any combination) may yield negative results.
Seminal plasma markers may be present, but many factors may preclude the recovery of spermatozoa. Impaired spermatogenesis, depleted stores, or impaired delivery may all play a role in the production of azoospermic semen.

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