Expression of epidermal growth factor receptor and HER-2/neu in normal and neoplastic cervix, vulva, and vagina.

Monoclonal antibodies were used to localize immunohistochemically epidermal growth factor receptor and HER-2/neu in normal and neoplastic frozen tissue samples from the lower genital tract of women. In squamous epithelia of the cervix, vulva, and vagina, epidermal growth factor receptor and HER-2/neu both were expressed most strongly by basal keratinocytes. Expression of both of these cell surface molecules decreased as cells underwent differentiation toward the mucosal surface. In contrast, both epidermal growth factor receptor and HER-2/neu were expressed throughout the entire thickness of the epithelium by undifferentiated squamous cells in squamous metaplasia, raised condyloma, and carcinoma in situ. In 34 squamous cancers of the cervix, vulva, and vagina, all malignant cells were found to have moderate to heavy staining for epidermal growth factor receptor. Staining of 33 of these cancers for HER-2/neu was light, although one patient who presented with distant metastases had heavy staining for HER-2/neu. These data suggest that although overexpression of HER-2/neu in squamous cancers of the lower genital tract is a rare event, it may be associated with aggressive biologic behavior.

Diagnosis of preterm rupture of membranes: is testing for alpha-fetoprotein better than ferning or nitrazine?

This article evaluates the role of alpha-fetoprotein (AFP) testing in the diagnosis of ruptured membranes in the preterm patient. AFP is normally found in amniotic fluid and is not present in vaginal secretions. Testing for AFP in vaginal fluid was compared with nitrazine evaluation and ferning in two groups of preterm patients, those with intact membranes and those with ruptured membranes. There was no difference in the sensitivities of AFP, nitrazine, or ferning in diagnosing ruptured membranes. The specificities of AFP and ferning were similar and both were significantly better than nitrazine. Duration of membrane rupture for greater than 24 hours negatively affected the accuracy of all three tests. Blood-tinged amniotic fluid interfered with AFP and nitrazine evaluation. AFP does not appear to offer a significant advantage over nitrazine testing and ferning in the evaluation of patients with possible ruptured membranes.

Localization of immunoglobulins in the chicken oviduct.

Studies were made on the distribution of lymphoid tissues and immunoglobulin (Ig: IgA, IgG and IgM)-containing cells (cIg: cIgA, cIgG and cIgM) and the localization of immunoglobulins (Igs) in the oviducal walls of laying hens. Lymphocyte accumulations were occasionally observed, located mainly in the middle infundibulum and in the regions from the isthmus to the vagina. The number of cIgG significantly predominated over that of cIgA or cIgM in the mucosal connective tissue of the magnum and the isthmus. In contrast, in the regions other than the magnum and the isthmus, these three types of cIg were fewer in number. Igs were localized in some superficial epithelial cells (SECs) and glandular cells (GlCs) of the oviduct. Many IgG-containing SECs were found in the infundibulum, the isthmus, and the cranial and major uterus. IgA- or IgM-containing SECs were rare throughout the oviduct. Three types of Ig-containing GlCs were numerously found in the magnum, though lymphocyte accumulations were scarce there. In the isthmus, many IgG-containing GlCs were found, while IgA- or IgM-containing GlCs were rarely observed. Ig-containing GlCs in the magnum were considerably decreased in number after the egg passage. The results suggest that the maternal Igs are transferred to the egg mainly through GlCs in the magnum of the chicken oviduct, and that the oviducal lymphoid tissues have little relationship to the passive immunity.

An evaluation of the polymerase chain reaction method for forensic applications.

This paper describes the use of polymerase chain reaction (PCR) for amplifying small amounts of DNA obtained from samples of interest to the forensic scientist. A region of the HLA DQalpha (DQa) locus was amplified in DNA prepared from the following: hair roots, liquid blood, blood-stains, semen and vaginal swabs (semen free and semen contaminated). A population study was conducted using DNA from 78 unrelated individuals. The observed distribution of HLA DQa alleles varied from that reported for an American population but obeyed the Hardy-Weinberg equilibrium. Interpretation problems associated with the PCR technique are discussed.

Inflammatory epithelial changes and nonviral cervicovaginal pathogens.

To assess the relationship between inflammatory epithelial changes (IECs) and cervicovaginal infection, 249 women with cytologic diagnoses of IEC were studied. The findings showed a Chlamydia infection in 15.7% of the cases, Monilia in 38.1%, Trichomonas in 1.6% and a lateral vaginal wall pH of greater than 5.5 in 29.7%. These results were compared with those for 211 controls drawn from the same population; women with abnormal cytologic reports were excluded from the control group. The controls had a prevalence of yeast and trichomoniasis no different from that of the IEC group. In contrast, the prevalence of chlamydial infection (7.6%) and pH greater than 5.5 (10.7%) were significantly lower in the controls than in the IEC cases. Chlamydia was found in 30.2% of 43 women with an atypical transformation zone, equally distributed between women whose biopsies were normal and those whose biopsies showed cervical intraepithelial neoplasia. The traditional guidelines of treating cases with cytologic diagnoses of IEC prior to obtaining a repeat smear may need reevaluation in the light of these findings.

Comparison of Cytobrush and cervicovaginal lavage sampling methods for the detection of genital human papillomavirus.

The development of an accurate method for the detection and typing of genital human papillomavirus is of substantial clinical importance. This virus has been implicated as an etiologic agent in the development of cervical neoplasia. To detect human papillomavirus infection with maximum sensitivity, cells must be collected and assayed for human papillomavirus deoxyribonucleic acid. We compared two noninvasive methods of sampling exfoliated cervical cells--cervicovaginal lavage and scrape-Cytobrush. Seventy-four patients newly referred to the colposcopy clinic were divided randomly for cell sampling by either cervicovaginal lavage followed by scrape-Cytobrush or, conversely, scrape-Cytobrush followed by cervicovaginal lavage. Restriction analysis and Southern blot hybridization were used to test all the samples thus obtained for human papillomavirus. Overall, test results from 42 patients (56.8%) were positive for human papillomavirus deoxyribonucleic acid. Twenty-six (31.1%) tested positive for human papillomavirus by both sampling methods, and 32 (43.2%) tested negative for human papillomavirus by both methods. One (1.4%) tested positive with scrape-Cytobrush sampling but negative with cervicovaginal lavage, while 15 (20.3%) tested negative with scrape-Cytobrush but positive with cervicovaginal lavage (p less than 0.001, McNemar's test). These data, combined with previous work from our group, suggest that, of the available methods, cervicovaginal lavage, coupled with human papillomavirus deoxyribonucleic acid hybridization, is the most sensitive noninvasive method for harvesting cells for molecular identification of human papillomavirus in the female lower genital tract.

ABO and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique.

A new method for ABO and Lewis typing of body fluids is described. It combines the advantages of a good antigen binding to nitrocellulose membranes, the need of only very small amounts of stain material and the high sensitivity of an enzyme-linked immunosorbent assay for antigen detection. This is of special interest because conventional ABO and Lewis typing of secretion stains need relatively large stain dimensions. The method is very easy to handle, does not need any expensive equipment and gives a permanent record. Furthermore the high sensitivity offers the possibility of analyzing even sweat and urine stains without the need of concentrating these extracts.

Information from penile swabs in sexual assault cases.

A survey has been made to assess the evidential value of tests carried out on 660 casework penile swabs. Most were from suspects in sexual assaults and were examined to see if the donor had had recent anal, oral or vaginal intercourse. The swabs were tested for one or more of the following: blood, faeces, saliva, vaginal secretions, semen. Blood was seldom found, it was usually weak and insufficient for grouping. Faeces were only identified on a pair of swabs from a dead homosexual showing that proof of buggery by this means is rare. Amylase, suggestive of saliva and oral intercourse, was occasionally detected. Glycogen-rich epithelial cells were sometimes present indicating vaginal intercourse. Semen was frequently found but its presence may not result from a recent sexual act. An ABO group different from the donor was obtained from a fifth of the swabs typed. Grouping in other blood group systems was rarely attempted or successful. Penile swabs provided a means of detecting a victim's ABO blood group on a suspect when it would not have been possible to demonstrate the suspect's group on samples from the victim. They also had value in assaults involving more than one offender. The main limitation of penile swabs was the paucity of material on them and the sampling site affected the interpretation of the results.

Association between bacterial vaginosis and acute cystitis in women using diaphragms.

We hypothesized that the increased vaginal fluid pH and altered vaginal microflora characteristic of bacterial vaginosis might predispose young women to introital colonization with Escherichia coli and to acute cystitis. To evaluate this hypothesis, we studied 291 women who presented with acute urinary symptoms for association of clinically defined bacterial vaginosis and vaginal conditions associated with this syndrome (increased vaginal fluid pH, absence of lactobacilli, and abnormal vaginal fluid gas-liquid chromatographic patterns) with E coli introital colonization and urinary tract infection. Escherichia coli introital colonization and urinary tract infection were both significantly more frequent among women with a high vaginal fluid pH, an absence of vaginal lactobacilli, or an abnormal vaginal fluid gas-liquid chromatographic pattern characteristic of bacterial vaginosis. Escherichia coli introital colonization was also more frequent in women with bacterial vaginosis. These associations and an association of bacterial vaginosis and E coli urinary tract infection were strong only among the 144 women who were diaphragm users. We conclude that bacterial vaginosis, or an altered vaginal microflora as reflected by an abnormal gas-liquid chromatographic pattern characteristic of bacterial vaginosis, is associated with E coli introital colonization and acute symptomatic urinary tract infection in women who use diaphragms.

Vaginal fluid prolactin: a reliable marker for the diagnosis of prematurely ruptured membranes. Comparison with vaginal fluid alpha-fetoprotein and placental lactogen.

The aim of this study is the evaluation of the reliability of vaginal fluid (VF) prolactin (PRL) for detecting prematurely ruptured membranes (PROM) and the comparison of this marker with vaginal fluid alpha-fetoprotein (AFP) and placental lactogen (HPL). In 21 pregnant women with recent or prolonged PROM from 20 to 41 weeks' gestation, in whom intact membranes were never found subsequently VF- and MS-PRL, -AFP and -HPL were measured by enzyme immunoassays, which are sensitive and very rapid. The same markers were also measured in MS, VF and urine samples (U) in 12 pregnant women of the same gestational age, without PROM, in whom the membranes were ruptured later during labor. In PROM, independently of prematurity and duration of PROM VF-PRL levels were significantly higher (2-10-fold) than the paired MS-PRL (p less than 0.0001) and ranged from 130 to 2315 ng/ml. In contrast, VF-PRL and urine PRL concentrations in pregnancies without PROM were very low or undetectable (range: 0-5 ng/ml and 0.15-1 ng/ml, respectively). Vaginal fluid AFP values in PROM from 20th to the 33rd week of pregnancy were significantly higher (5-50-fold) than the paired MS-AFP (p less than 0.01) and ranged from 103 to 5500 ng/ml. In PROM after the 33rd week of pregnancy, VF-AFP values were either lower (1/3), or equal to, or even higher (up to 2-fold) than MS-PRL. On the contrary in pregnancies with intact membranes, VF-AFP were always less than 9 ng/ml and urine AFP was undetectable (range: 0.2-1.1 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Vaginal swabs: endogenous and postcoital components.

The relationship between components found on vaginal swabs was examined to determine whether the presence and quantity of a particular component could be used to predict the presence of others or to help interpret grouping results. Vaginal swabs were tested for the presence of blood, cellular material, spermatozoa, acid phosphatase, p30, ABH, Lewis, EsD, GLO I, PGM and PGM subtype. Methods included rocket electrophoresis and p-nitrophenyl phosphate assay for acid phosphatase; rocket and cross-over electrophoresis for p30. Results from 323 semen-free and postcoital vaginal swabs from known donors are presented. Comparison of methods showed that seminal acid phosphatase and p30 were detected more often by the rocket techniques. Attributing the grouping results to semen, based on the reactivity of any single component, could lead to erroneous conclusions. Activation of vaginal components in the presence of semen and endogenous vaginal levels are discussed.

Menstrual variation of estrogen receptor content in vaginal tissue.

Estrogen receptor content in vaginal tissue was quantified in 12 healthy female volunteers during the menstrual cycle. Epithelial biopsies were obtained at cycle days 10 and 21 and investigated by a monoclonal enzyme immunoassay. Measurable amounts of estrogen receptors were found in all women at both sampling occasions. In women with an ovulatory menstrual cycle, a significant fall in V-receptor content was recorded during the luteal phase. Structural and functional changes in the vaginal epithelium during the menstrual cycle may be important for the diagnosis and susceptibility to certain viral infections.

Detection of human papillomavirus deoxyribonucleic acid in exfoliated cervicovaginal cells as a predictor of cervical neoplasia in a high-risk population.

Specific types of human papillomavirus are currently implicated as etiologic agents of precancerous and cancerous lesions of the cervix. We have previously described the use of cervicovaginal lavage and molecular hybridization to detect human papillomavirus infections of the cervix. We report here the predictive value of this method of human papillomavirus detection to identify women with biopsy proved dysplastic and cancerous lesions of the cervix. One hundred ninety-one women from a city hospital colposcopy clinic underwent concurrent Papanicolaou smear, cervicovaginal lavage, and coloposcopically directed cervical biopsy. Human papillomavirus deoxyribonucleic acid was detected in 114 (59.7%) of these women. Of the positive results, human papillomavirus type 16 accounted for 23.7%, human papillomavirus type 18 for 10.5%, human papillomavirus type six or 11 for 6.2%, related human papillomavirus types for 52.6%, and 7.0% contained more than one type. The distribution of human papillomavirus types was similar in both women younger than 40 years of age and in older women. Eighty-nine of 128 (69.5%) women less than 40 years old with cervical lesions had positive findings of human papillomavirus, and 18 of 29 (62.1%) older women with cervical lesions had positive findings of human papillomavirus. Detection of human papillomavirus types 16 and 18 identified only 35 of 157 (22.3%) women with cervical lesions. The sensitivity of detecting all types of human papillomavirus as a predictor of a biopsy proved lesion (68.0%) was comparable with the sensitivity of cytologic examination alone (74.0%). However, human papillomavirus detection combined with the Papanicolaou smear provided an increased overall sensitivity of 89.3% (p less than 0.01). In fact, women either positive for human papillomavirus or having abnormal cytologic findings were 11.8 times more likely to have a biopsy proved cervical lesion than human papillomavirus-negative women with negative cytologic results (95% confidence interval for odds ratio: 5.3 to 26.6). We conclude that the sensitivity of cytologic examination plus human papillomavirus detection is superior to the use of either cytologic studies or human papillomavirus detection alone in identifying patients with cervical lesions.

Correlation of histology with human papillomavirus DNA detection in the female genital tract.

Genital condyloma and intraepithelial neoplasia secondary to Human Papillomavirus (HPV) infection are characterized by perinuclear halos and marked nuclear atypia (koilocytotic atypia) on cytologic and histologic examination. However, at times the histologic findings, including the degree of nuclear atypia, may be suggestive but not absolutely diagnostic of an HPV related neoplasm. HPV DNA sequences were detected in 63 and 56% of colposcopically visible vaginal and cervical lesions, respectively, that were diagnosed as condyloma or intraepithelial neoplasia. HPV DNA sequences were detected in 14 and 47% of vaginal and cervical lesions, respectively, that did not fulfill the histologic criteria of condyloma or intraepithelial neoplasia (i.e., "nondiagnostic"). When examining cervices from patients with no visible lesion and no recent history of an abnormal pap smear, 5.5% had detectable HPV DNA sequences. The histologic findings in this group were equivalent to the virus-negative cases and similar to the "nondiagnostic" cervical lesions. These findings suggest that the detection rate of HPV DNA in "nondiagnostic" tissues is dependent on the site and presence or absence of a visible lesion. The rate is similar in cervical lesions regardless of the histologic findings whereas it is less in vaginal lesions when the histologic criteria of condyloma or intraepithelial neoplasia are not detected.

The endogenous concentration and subcellular distribution of androgens in normal human premenopausal endometrium, myometrium and vagina.

The endogenous concentrations of testosterone (Testo), androstenedione (Adion), 5-androstene-3 beta, 17 beta-diol (Adiol), dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) have been measured in endometrium, myometrium and vagina from 23 premenopausal women at different stages of the cycle. The tissue to plasma gradient was positive for all androgens except DHEAS; a significant correlation existed between plasma and tissue levels of Adiol and DHEAS. In all tissues significant correlations were calculated between Adiol and DHEA, Adion and Testo, and Adiol and DHEAS. Except for Testo, androgen concentrations were different in the tissues with a significant correlation between the tissues, each tissue seeming to be able to create its own specific intratissue androgen pattern. During the cycle Testo was highest in myometrium and vagina and Adion in myometrium and endometrium during the secretory phase. Except for DHEA, androgens were preferentially localized in the cytosol fraction of the cells. From this localization and the known inhibitory effects of androgens on the activity of estrogen metabolizing enzymes, it is postulated that androgens play a role in enzymatic rather than receptor-mediated processes in uterine cells.

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ.

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.

Vaginal colonization by bacteria and yeast.

A number of different factors have been found to influence the microflora of the vagina. Studies showing an increased rate of colonization associated with pregnancy, menses, oral contraceptive use, and estrogen replacement therapy illustrate the influence of hormones, glycogen content, and pH on vaginal colonization. The microorganisms found in the vagina may also be dependent on the existence of specific attachment sites, which may differ in the various areas of the vagina. Vaginal colonization appears to be altered in patients with diabetes mellitus, those receiving steroid therapy, and immunosuppressed individuals. Surgical manipulation of the vagina and the use of broad-spectrum antibiotics may also contribute to the growth of organisms in the vaginal tract.

The ABH reactions of seminal stains.

The ABO grouping results from approx. 1000 seminal stains have been collected and analysed. Most of the stains came from rape cases where the ABO and secretor status of both complainant and suspect were known. The results of the survey provided information concerning the usefulness of elution and inhibition as methods of body fluid grouping, the relative strengths of reaction of the A, B and H antigens in body fluids and the interpretation of the ABH reactions of body fluid stains.