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Most cases of oral squamous cell carcinoma (OSCC) develop from visible oral potentially malignant disorders (OPMDs). The latter exhibit heterogeneous subtypes with different transformation potentials, complicating the early detection of OSCC during routine visual oral cancer screenings. To develop clinically applicable biomarkers, we collected saliva samples from 96 healthy controls, 103 low-risk OPMDs, 130 high-risk OPMDs, and 131 OSCC subjects. These individuals were enrolled in Taiwan's Oral Cancer Screening Program. We identified 302 protein biomarkers reported in the literature and/or through in-house studies and prioritized 49 proteins for quantification in the saliva samples using multiple reaction monitoring-MS. Twenty-eight proteins were successfully quantified with high confidence. The quantification data from non-OSCC subjects (healthy controls + low-risk OPMDs) and OSCC subjects in the training set were subjected to classification and regression tree analyses, through which we generated a four-protein panel consisting of MMP1, KNG1, ANXA2, and HSPA5. A risk-score scheme was established, and the panel showed high sensitivity (87.5%) and specificity (80.5%) in the test set to distinguish OSCC samples from non-OSCC samples. The risk score >0.4 detected 84% (42/50) of the stage I OSCCs and a significant portion (42%) of the high-risk OPMDs. Moreover, among 88 high-risk OPMD patients with available follow-up results, 18 developed OSCC within 5 y; of them, 77.8% (14/18) had risk scores >0.4. Our four-protein panel may therefore offer a clinically effective tool for detecting OSCC and monitoring high-risk OPMDs through a readily available biofluid.

The purpose of the study was to evaluate the specimen adequacy and diagnostic accuracy of loop conization in microinvasive carcinoma of the cervix. A retrospective study was conducted from 1997 to 2003 at the Colposcopic Clinic, Department of Gynecology and Obstetrics, Chang Gung Memorial Hospital, Taipei, Taiwan. Sixty-three consecutive patients with microinvasive carcinoma of the cervix receiving cold-knife conization (35 patients) or loop conization (28 patients) were included in the study. All patients underwent definitive hysterectomy. We reviewed the conization specimen together with the hysterectomied uterus to compare the two conization techniques with respect to the histopathologic interpretation and diagnostic accuracy. The mean depth of cone specimens was significantly less in the loop conization compared with cold-knife conization (1.65 versus 2.35 cm, P = 0.035). Regarding the application of conization, the loop conization was completed in a single slice in 27 patients (77.1%) and in multiple slices in 8 patients (22.9 %), in spite of encouragement to perform conization in a one-pass application when possible. However, the cold-knife specimens were invariably a single cone-shaped piece. As reviewed by microscopic examination, the rate of tissue transection was significantly higher in the loop group than in the cold-knife group (14.3% versus 0%, P = 0.04). Because of tissue transection and disorientation, pathologic evaluation of stromal status was inadequate in 11.4% (4/35) of the loop cones as opposed to none of the 28 cold-knife cones. After assessing the hysterectomy specimens, the clinical diagnoses in the loop group were downgraded in three patients compared with only one in the cold-knife group. Data from this investigation suggest that cervical cold-knife conization is superior to loop conization as a method to assess microinvasive cervical cancer.

The study was undertaken to test whether human telomerase reverse transcriptase (hTERT) transcripts in an individual blastomere could be used as an indicator for embryo development.Group A consisted of day 3 normal cleaving embryos at 4- to 8-cell stage, which were surplus and not allocated for uterine transfer. Group B consisted of arrested or fragmented embryos at the same stage, which were considered to be compromised. After blastomere dissociation, RNA purification, reverse transcription, and hTERT PCR amplification, the amplified product was separated by 3% gel electrophoresis.Eighty-six (90.5%) of the 95 intact blastomeres in group A and 78 (70.9%) of the 110 blastomeres in group B demonstrated hTERT mRNA expression. The difference was statistically significant (P0.05, chi-square). Eight (38.1%) of the 21 embryos in group A and 22 (62.9%) of the 35 embryos in group B had at least one blastomere that did not express hTERT mRNA under this procedure. The difference was not significant (P0.05, chi-square).General hTERT mRNA transcripts can be detected in most of the individual blastomeres but cannot be used as an indicator for early embryo development. Further investigations are necessary to elucidate its clinical application.

Objective: To evaluate the status of human papillomavirus (HPV) following radiotherapy, and its prognostic significance in cervical cancer. Materials and Methods: Multiple tissue sections obtained from 58 patients with cervical cancer before and after radiotherapy were analyzed for the presence of HPV types 16 and 18 DNA by using the polymerase chain reaction. Results: The overall frequencies of HPV-16/18 infection among cervical cancer patients were 93.1% (54/58) and 56.9% (33/58) before and after radiotherapy, respectively. Patients with HPV-16 DNA-containing tumors were significantly associated with a favorable tumor response following radiotherapy compared with patients who had HPV-18-positive tumors (92.9% vs 50%, p = 0.002). After a median follow-up of 5.1 years (range, 3.2–6.5 years), patients with HPV-18-positive tumors were found to have a significantly higher relapse rate than HPV-16-positive patients (50.0% vs 14.3%, p = 0.02). Conclusion: The results suggest that cervical cancer patients with HPV-18 DNA in their tumors have a significantly poorer response to radiotherapy. This implies that the identification of HPV genotypes in cervical carcinoma may play a valuable role in predicting tumor response to radiotherapy.

Objective. The goal of this study was to determine/evaluate the negative predictive value of human papillomavirus (HPV) testing following conization of cervix uteri. Methods. A prospective analysis was undertaken on 79 cone biopsies of women with high-grade lesions (cervical intraepithelial neoplasia (CIN) III). HPV testing was performed on cervical smears before and after conization. We correlated the margin status (defined as positive cone margin or endocervical curettage status) and positive conization HPV status with the residual disease in a hysterectomy specimen. A Digene II kit was used to perform HPV testing. HPV detection was done by Hybrid Capture assay. Results. Of the 79 patients, 47(59.5%) had positive margins after conization. HPV testing was positive in 37 cases (78.7%) and negative in 10 cases (21.3%). Residual disease was found in 31 of 47 (66%) postconization hysterectomy specimens. No residual lesions were found in HPV-negative cases. Of the 32 cases with negative margins following conization, HPV testing was negative in 25 cases (78%) and was positive in 7 cases (22%). Among these 25 cases with negative HPV tests, no residual lesion was detected, and in 7 HPV-positive cases, only one residual lesion was found. Conclusion. HPV testing is potentially an effective tool in predicting residual dysplasia after conization and could potentially assist in the decision between hysterectomy and conservative follow-up in women with CIN III.

Objective. The aim of this study was to detect telomerase activity in peritoneal ascites and to assess whether it can be used as an assistant tool for the early detection of ovarian cancer. Methods. Telomerase activity was measured by TRAP assay in 47 patients with ovarian malignancies and 50 patients with benign uterine leiomyomas (control group). Results. All 26 peritoneal washing cytology positive cases were telomerase positive. Of the 21 peritoneal washing cytology negative cases, 3 were telomerase positive. When these 3 were reevaluated for peritoneal cytology, malignant ascitis was identified in 1. All telomerase negative cases were negative for peritoneal washing cytology. The sensitivity and specificity of peritoneal cytology and telomerase testing in correlation with true malignant cells were 96 (26/27) and 100% (20/20) versus 100 (27/27) and 90% (18/20), respectively. The false negative rate of peritoneal cytology was 4.7% (1/21). The false positive rate of the telomerase test in relation to malignant ascites was 6.9% (2/29). Conclusion. Our preliminary results reveal a high sensitivity and specificity of both telomerase testing and conventional cytology in peritoneal fluids. Our data suggest that the telomerase test in peritoneal fluids can be used as an adjuvant to cytopathological methods in the diagnosis of malignant peritoneal ascites, particularly in cases of negative cytology. In these cases, a review of peritoneal histocytology is advised.

The study was designed to investigate the expression of the inflammatory cytokines interleukin-1 beta and interleukin-6 in meconium-stained amniotic fluid and in fetal cord serum. Amniotic fluid and fetal cord serum specimens were collected from 10 and 9 women with meconium-stained and clear amniotic fluid, respectively, during Caesarean operation at labor The mean concentrations of interleukin-1 beta found in clear and meconium-stained amniotic fluid were 10.0 and 54.5 pg/ml, respectively, and the difference was not statistically significant. On the other hand, the concentrations of interleukin-6 in meconium-stained amniotic fluid (774 pg/ml) was significantly higher than that found in clear amniotic fluid (149 pg/ml) (P = 0.0036). The differences of levels of both interleukin-1 beta and interleukin-6 in fetal cord serum specimens were not significant between neonates born to mothers with either clear or meconium-stained amniotic fluid (P = 0.8702 and 0.2987, respectively). The results of this study suggest that the production of at least one of the inflammatory cytokines, interleukin-6, is associated with the meconium found in amniotic fluid.

In an attempt to seek out new factors that are related to colorectal carcinogenesis at the molecular level, subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues was performed. Subsequent screenings of the cDNA libraries, constructed from normal mucosal tissues, using the "subtractive probes" generated a total of 46 clones that were expressed in normal mucosa but were either expressed at a significantly reduced level or not expressed at all in cancer tissues. Partial nucleotide sequences of all of these cDNA clones were determined, and sequence homology analyses were performed with the Genbank database. Of the 46 cDNA samples, 44 contained substantial sequence homologies with 32 immunoglobulin gene fragments, a helix-loop-helix basic phosphoprotein gene, an acidic ribosomal phosphoprotein P2 gene, a BLR1 gene for Burkitt's lymphoma receptor 1 gene, D5S419 DNA segment containing (C-A) repeats, a glucokinase (GCK) gene, a Na+, K+-ATPase alpha-subunit gene, a histocompatibility system HLA-DR heavy-chain gene, a dystrophic gene, a mucin (MUC2) gene, a mu-glutathione S-transferase gene, a Menkes disease protein gene, and a 40-kDa keratin intermediate filament precursor gene. The remaining two cDNA clones (now registered under GenBank accession numbers U17714 and U20428) showed few (less than 60%) sequence homologies with any known sequences in the GenBank database and, therefore, may represent novel genes whose expression was down-regulated in human colorectal carcinomas. The possible clinical significance of these findings and the involvement of these two genes in the carcinogenesis of colorectal as well as other cancers are being investigated.

BACKGROUND The presence of Epstein-Barr virus (EBV) has not been documented in previous reports of lymphoepithelioma-like carcinoma (LELC) of the uterine cervix by either polymerase chain reaction or in situ hybridization, and the histogenesis of the tumor remains unknown. Additionally, a relationship between human papillomavirus (HPV) and cervical LELC also has not been reported. METHODS In this article, the authors describe the clinical and histopathologic findings for 15 patients with cervical carcinoma that had a histologic pattern of LELC. The polymerase chain reaction detected the presence of EBV and HPV DNA sequences in cervical LELC. RESULTS All 15 tumors showed a typical syncytial growth pattern of undifferentiated cells with prominent lymphocytic infiltration. The detection rate of the EBV gene sequence in tissue samples from patients with LELC was more frequent than that in control patients with squamous cell carcinoma of the cervix (11 of 15 patients, 73.3%, vs. 4 of 15 patients, 26.7%; P = 0.01). However, the detection rate of HPV-16 and HPV-18 DNA was significantly lower in patients with LELC tumors than in patients with cervical squamous cell carcinoma (3 of 15 patients, 20.0%, vs. 12 of 15 patients, 80.0%; P = 0.001). After a median follow-up of 3.9 years (range, 1.8-5.3 years), the 15 patients showed no evidence of disease or metastasis after radical hysterectomy or radiotherapy. CONCLUSIONS The finding of EBV associations in cervical LELC supports the hypothesis that EBV may be involved in the pathogenesis of tumors that arise in the cervix. It is possible that cervical LELC may follow a different pathway in the pathogenesis of LELC in Asian women as compared with the more common forms of squamous cell carcinoma. Cancer 1997; 80:91-7. ©; 1997 American Cancer Society.

Objective: To investigate the presence of human papillomavirus (HPV) in human sperm cells and to evaluate potential effects of HPV on the sperm functions. Design: A descriptive clinical study. Patient(s): Specimens of semen were collected from 24 randomly selected patients who attended the fertility clinics at Chang Gung Memorial Hospital. Main Outcome Measure(s): The presence of HPV DNA and RNA were examined by polymerase chain reaction. Semen quality and sperm cell function were analyzed by computer-aided autoanalyzer. Result(s): Human papillomavirus type 16 DNA and RNA were found in 6 (25%) and 2 (8%) of the sperm cells specimens, respectively. Human papillomavirus type 18 DNA and RNA were present in 11 (46%) and 5 (21%) of the same sperm cells specimens, respectively. Incidence of asthenozoospermia among patients infected with either HPV was significantly higher than in those without HPV in their sperm cells (75% versus 8%). Although performance of curvilinear velocity, straight-line velocity, and mean amplitude of lateral head displacement was significantly lower in HPV-infected specimens, the differences of linearity, beat cross frequency, and straightness were not statistically significant. Conclusion(s): These results suggest that human papillomavirus can be found in human sperm cells and that certain HPV-specific genes are actively transcribed. Sperm motility parameters seem to be affected by the presence of HPV in the sperm cells, and also the incidence of asthenozoospermia may be associated with HPV infection.

PURPOSE Telomeres are tandem arrays of repeated DNA sequences located at the ends of eukaryotic chromosomes, and are synthesized by the enzyme telomerase. Loss of telomeric DNA may play an important role in the development of human cancers. However, very little is known about the status of telomerase during human cervical cancer development. PATIENTS AND METHODS Telomerase activity was measured by telomere repeat amplification protocol (TRAP) assay in 24 cervical cancers, one carcinoma in situ (CIS), and 20 cervical intraepithelial neoplasia (CIN) lesions. Adjacent nontumor cervical tissue from the same 24 cervical cancer patients and normal cervical tissues from 11 control individuals also were examined for the presence of telomerase activity. RESULTS Twenty two of the 24 (91.7%) cervical cancer specimens and the single CIS tissue were strongly positive for telomerase activity. Relatively weak but distinctive telomerase activity also was detectable in one of four CIN-I (25%), two of eight CIN-II (25%), and two of eight CIN-III (25%), respectively. However, telomerase activity was not found in the 24 corresponding nontumor cervical tissues from the same cervical cancer patients and the 11 normal cervical tissues from control individuals. CONCLUSION The majority of cervical cancers contain strong telomerase activity. Significant proportions of noncancerous CIN tissues also contain telomerase activity, although weaker than that in cervical cancer. It seems that there is a progressive increase of telomerase activity in association with an increased degree of cervical malignancy. These results seem to suggest that the expression of telomerase may play a crucial role in cervical cancer carcinogenesis.

Human papillomaviruses (HPV) contribute to the development of malignancies of the uterine cervix and the viral E6 and E7 oncogenes are invariably retained and expressed in cervical cancer tissues. Minor, but not major, structural aberrations have been found quite frequently in viral DNA recovered from cervical cancer tissues. We examined the presence of the DNA sequence of HPV type 18 in 33 cervical cancer tissues by polymerase chain reaction. HPV type 18 DNA sequences was found in 24 of these 33 cervical cancer tissue specimens, and at least 21 of these 24 specimens did not appear to retain all the region and open reading frames examined. Twelve of these 24 tissues seemed to harbor only the E6 and/or E7 genes. These results can be construed to suggest that the absence of viral genes other than E6 and E7 is quite frequent in HPV recovered from cervical cancer tissues and that the E6 and E7 genes are important in the carcinogenesis of cervical carcinoma. It is possible that the E6 and/or E7 alone may be sufficient to maintain the transformed phenotype of cervical cancer.

Objective To investigate the possible presence and expression of human papillomavirus viruses (HPV) in human seminal plasma and sperm cells. Design Controlled clinical study. Setting A major medical center affiliated with a medical college. Patients Twenty-four randomly selected patients who attended Fertility Clinics at the Chang Gung Memorial Hospital. Interventions Specimens of semen were collected from volunteered patients. Main Outcome Measure The presence of HPV types 16 and 18 DNA and RNA sequences were examined by polymerase chain reaction. Results Human papillomavirus type 16 E6 and E7 DNA and RNA sequences were found in two and zero seminal plasma specimens, respectively, and in six and two sperm cells specimens, respectively. Deoxyribonucleic acid and RNA sequences of HPV type 18 were found in eight and two seminal specimens and in 11 and 5 sperm cells specimens, respectively. Conclusion These results seem to suggest that HPV cannot only infect human sperm cells, certain HPV genes are expressed actively in infected sperm cells. The virus-infected sperm cells conceivably can behave as vectors or carriers for the transmission of HPV, to sexual partner during sexual contact, to fetuses through fertilized eggs, or both.

The expression of genes coding for inflammatory cytokine interleukin-1-alpha (IL-1 alpha), IL-6, interferon-gamma and tumor necrosis factor-alpha from 15 normal cervix, 11 cervical intraepithelial neoplasia and 13 cervical cancer tissues was investigated. The cytokine messenger ribonucleic acids were reverse transcribed and amplified in the presence of biotinylated and dinitrophenylated primers. Amplified DNA was then captured onto streptavidin-coated microwell plate and quantitatively measured in a colorimetric reaction using ant-DNP antibodies conjugated to horse radish peroxidase. There is no change of IL-1 alpha, IL-6 and tumor necrosis factor-alpha gene expression in either cervical intraepithelial neoplasia or cervical cancer tissues. But the transcription of interferon-gamma gene is significantly reduced in both cervical intraepithelial neoplasia and cervical cancer tissue as compared to normal cervix. This study demonstrated that reverse transcription and quantitative polymerase chain reaction coupling to colorimetric microwell plate assay is a sensitive and useful method to quantitate multiple cytokine gene expression. Our results also suggest that cervical epithelial cells are capable to express cytokines and that interferon-gamma may play a role in the pathogenesis of cervical cancer since its reduced expression may influence inflammation and immunity of the cervical tissues.

Genetic aberrations were examined to assess the possible roles that p53 and retinoblastoma susceptibility genes might have played in the development of small cell cervical carcinomas. Cervical cancer tissues from 12 patients with small cell cervical carcinoma that were free of human papillomavirus were analyzed. The presence of mutational alterations were examined by polymerase chain reaction-single-strand conformation polymorphism and by direct DNA sequencing. None of 12 small cell cervical carcinomas were found to contain mutations in regions of p53 and retinoblastoma susceptibility genes that were functionally important and where most mutations in human tumors have been found. Furthermore, there was no evidence indicative of loss of heterozygosity of chromosome region 17p13 (in which p53 is located) in these tumors. These data seem to suggest that whereas mutant type of p53 and retinoblastoma susceptibility genes may exhibit “oncogenic” function in many human tumors, mutational inactivation of these genes may not be an important feature in the carcinogenic development of human papillomavirus-negative small cell cervical carcinomas. © 1994 Wiley-Liss, Inc.

Fetal cells can be identified by using the polymerase chain reaction to test for the presence of human Y-chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood of women who bear a male fetus. Thirty-one pregnant women were studied in the first trimester to determine when fetal cells become detectable in the maternal circulation. Among the 19 women whose peripheral blood samples were positive for Y-chromosome-specific DNA sequences, the presence of fetal cells was quite case-variable from the 6th to 12th gestational weeks. Twenty-eight women who had given birth to their first male babies were studied postpartum to determine when fetal cells disappear from the maternal circulation. Fetal cells can still be detected in maternal blood 10 months postpartum in some cases. These results suggest that identification of fetal cells in the maternal circulation is possible. Nevertheless, interpretation of fetal cells in maternal circulation should be handled very carefully with respect to when these fetal cells first became detectable and potential interference from previous pregnancies.

DNA was prepared from peripheral blood mononuclear cells of 114 Chinese with low erythrocyte mean corpuscular volume and analyzed by allele-specific DNA amplification for the presence of mutant alleles in the beta-globin gene that account for about 90% of beta-thalassemia in Chinese. A total of 9 mutations of the five most frequent mutant alleles were detected in 8 individuals. All mutant sequences were confirmed later by DNA sequencing. However, no mutation of these mutant alleles was detected in the remaining 106 individuals with low erythrocyte mean corpuscular volume including 22 who also had Hb A2 content of 6.0% or more. Our results seem to suggest that the presence of beta-thalassemia allele does not correlate very well with red blood cell indices and that direct DNA analysis by allele-specific DNA amplification is an accurate method to identify beta-thalassemia heterozygotes.

To further our understanding of the mechanism of action of regulatory and structural genes involved in human sex determination, we have examined three sex-reversed 46,XY females and two of their normal fathers for any mutational alteration in ZFY and SRY genes, using polymerase chain reaction and single-strand conformation polymorphism and by subsequent DNA sequencing. We could not find any mutation in the ZFY and SRY genes of these patients and their fathers. The results seem to suggest that although ZFY and SRY may be required for initiation of testis differentiation and male sex determination, sex-reversed females may predominantly result from alterations in genes either downstream or secondary to ZFY or SRY.

To examine the role of hepatitis C virus (HCV) infection in spontaneous hepatitis B surface antigen (HBsAg) clearance during the course of chronic hepatitis B virus (HBV) infection, serum specimens from 32 asymptomatic HBsAg carriers and 22 patients with chronic hepatitis type B who underwent spontaneous HBsAg clearance were studied for antibody to HCV (anti-HCV) using commercial EIAs. The results were compared with those of control groups matched for age, sex, hepatitis B e antigen, antibody to hepatitis delta virus, and cirrhosis. Eight (25%) of the asymptomatic carriers and 9 (41%) of the patients with chronic hepatitis were seropositive for anti-HCV in contrast to 1.6% and 9.1% of their respective control groups (P less than .01). Serum alanine aminotransferase level was persistently abnormal after HBsAg clearance in one asymptomatic carrier and in four patients with chronic hepatitis. These patients were seropositive for anti-HCV and at least one of them was negative for HBV-DNA by polymerase chain reaction. The data suggest that HCV superinfection may not only suppress HBV or terminate the HBsAg carrier state but may also assume the role of HBV as the cause of persistent hepatitis or transaminase elevation.

Deoxyribonucleic acid sequences of human ZFY (zinc-finger-Y) gene, a Y-chromosome-specific gene and candidate for the testis-determining factor, has been identified by an in vitro enzymatic deoxyribonucleic acid amplification method in peripheral blood specimens of women pregnant with male fetuses. This technique permits detection of ZFY gene deoxyribonucleic acid sequences in as few as a single male cell among 1,000,000 female cells. Maternal blood results were confirmed by amplification of ZFY gene deoxyribonucleic acid sequences in chorionic villus cells and by karyotyping in 33 of 36 pregnant women. There was no false-positive male result, and two of the three blood specimens with false-negative results were obtained from pregnant women at a very early gestational age. With properly designed guidelines, this deoxyribonucleic acid amplification method may be an alternative to determine the fetal sex for those pregnancies at risk for X-linked genetic disorders.

Objective: The objective of this study was to examine the possibility of intrauterine human papillomavirus infection of fetuses by transplacental transmission of human papillomavirus before delivery. Study Design: Specimens of cervicovaginal cells and peripheral blood mononuclear cells were obtained from 52 consecutive pregnant women in the third trimester of pregnancy. Cord blood specimens were also obtained from the neonates born to these mothers. Presence of human papillomavirus types 16 and 18 deoxyribonucleic acid was analyzed by an in vitro enzymatic deoxyribonucleic acid amplification method. Results: Human papillomavirus type 16 deoxyribonucleic acid was found in 6 (11.5%) cervicovaginal and in 9 (17.3%) peripheral blood mononuclear cell specimens. Seven cord blood specimens from neonates born to mothers who were positive for peripheral blood mononuclear cell human papillomavirus type 16 deoxyribonucleic acid were found to contain human papillomavirus type 16 deoxyribonucleic acid. One cervicovaginal and two peripheral blood mononuclear cell specimens contained human papillomavirus type 18 deoxyribonucleic acid, but none of the cord blood specimens contained human papillomavirus type 18 deoxyribonucleic acid. Conclusion: These results seem to suggest possible transplacental transmission of the virus and the potential association of such transmission with the status of human papillomavirus in peripheral blood mononuclear cells.

The in vitro DNA amplification technique of polymerase chain reaction was used to evaluate the possible presence of human papillomavirus (HPV) in small cell carcinoma of the uterine cervix. None of the 12 cases examined contain detectable amounts of either HPV type 16, 18, 31, or 33 DNA. On the other hand, HPV types 16 and 18 DNA were found in 14 (93.3%) and 9 (60.0%) of 25 invasive cervical squamous carcinoma tissues. The results seem to suggest that these types of HPV are not present or are present in extremely small quantities in cervical small cell carcinoma. Such an absence of HPV DNA makes it unlikely that these types of HPV play any etiological role in the pathogenesis of cervical small cell carcinoma.

The presence of Chlamydia trachomatis in cervicovaginal cells and amniotic fluid of pregnant women was examined by culture and by polymerase chain reaction deoxyribonucleic acid amplification methods. Chlamydial deoxyribonucleic acid sequences were found in nine of 31 (29.0%) cervicovaginal cell specimens, five of which also were positive by culture method. Amniotic fluid specimens from two of the nine (22.2%) cervicovaginal chlamydial deoxyribonucleic acid-positive women and none from the remaining 22 cervicovaginal chlamydial deoxyribonucleic acid-negative women were found to contain chlamydial deoxyribonucleic acid. The results underscore the significance and frequency of intraamniotic chlamydial infection in women with urogenital chlamydial infections.

The presence of hepatitis type B virus (HBV) DNA in serum specimens from 926 apparently healthy people with normal liver functions was determined by polymerase chain reaction; 41.2% of people with positive results for HBV surface antigen (HBsAg) (94 of 228) and 95.2% of people with positive results for HBV e antigen (HBeAg) (60 of 63) were found to have positive results for serum HBV DNA. On the other hand, serum HBV DNA was found in 11.0% (77 of 698) of HBsAg-negative people and in 13% (69 of 530) of those who had positive results for serum antibodies directed against HBsAg. The results seem to suggest that HBV DNA can be found in a significant portion of apparently healthy people with normal liver function who are either seronegative for HBsAg or seropositive for antibodies directed against HBsAg.

To investigate the incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection, a prospective follow-up study was conducted in two consecutive groups of patients. Group I consisted of 984 patients (859 men and 125 women) with biopsy-proven chronic type B hepatitis, whereas group II consisted of 1,598 asymptomatic chronic carriers (998 men and 600 women) with normal serum aminotransferase activity. During a mean follow-up period of 4.0 ± 2.3 yr, 19 patients (1.9%) of group I cleared HBsAg from their serum, whereas 35 patients (2.2%) in group II did so in a mean follow-up period of 2.7 ± 1.4 yr. The annual incidence of delayed serum HBsAg clearance was 0.5% in group I and 0.8% in group II (p < 0.02). The cumulative probability of HBsAg clearance was also higher in group II than in group I (p

The presence of human cytomegalovirus (HCMV) was tested in 388 cervicovaginal cells specimens obtained from the same number of pregnant women. HCMV was detected in 5·41%, 11·6% and 13·9% of these specimens by conventional culture, in situ DNA hybridization and polymerase chain reaction (PCR) methods, respectively. The sensitivities of detecting HCMV by in situ hybridization and PCR methods were 76·2% and 90·5% and the specificities were 92·1% and 90·5%, respectively, when compared with conventional culture method. The PCR compared favourably with both conventional culture and in situ hybridization methods and it may become a valuable and useful tool for the early and rapid detection of HCMV in clinical specimens.

The presence of chlamydial deoxyribonucleic acid (DNA) was evaluated by DNA hybridization in endocervical cells of infertile and normal fertile women. Chlamydial DNA was detected in 49 of 186 (26.3%) infertile patients, which is significantly more common than in fertile control individuals (12.5%, or 8 of 64 individuals). Among infertile patients, 49.3% (33 of 67) of those with tubal factors as cause of infertility and 13.4% (16 of 119) of those with nontubal factors were found to contain chlamydial DNA in their endocervical cells. The results show that chlamydial DNA could be found significantly more frequently in endocervical cells of infertile patients with tubal factor than those without tubal factors or in normal controls.

We designed a self-sampling method to collect exfoliated genital cells for human papilloma virus (HPV) detection. The aim was to assess whether it was suitable as an assistant tool for the early detection of cervical pre-cancer and cancer in a special category of the women who are not frequently screened for cervical cancer.We compared the results of HPV detection that were self-obtained and physician-obtained cervical swabs from the same patient that were analyzed using hybrid capture II assay. The diagnostic rate of cervical pre-cancer and cancer between self-obtained method and physician-obtained method were analyzed.A total of 1194 women were prospectively registered from September 1997 through September 1999. Among them, 144 (12.1%) of self-test samples and 155 (13%) of physician-obtained samples were oncogenetic associated-HPV positive. Statistically, no significant differences existed in the screening rate for cervical cancer using either the self-collected samples or the physician-obtained samples (p.05). The sensitivity of cervical precancer or cancer detection using self-obtained HPV testing was higher (96.3%) as compared with the Pap smear (79.2%) (p.02).The detection correlation of the HPV test between the self-obtained method and physician-obtained method was 93%. Our results indicated that self-sampling was a reliable method for testing for HPV. The identification of HPV infection through the self-obtained method can be used in early identification of high-risk women with cervical precancer and cancer especially in underserved populations.

Human papillomaviruses were detected by an in vitro enzymatic DNA amplification method in cells obtained from vulvar swabs of 9 of 61 (14.8 %) young women without prior experience of sexual intercourse and in 7 of 57 (12.3 %) young women with prior experience. The prevalence of human papillomavirus DNA in these two groups of women was not significantly different (x2=0.16, p>0.5; 95 % confidence interval −0.165 to 0.215). These results suggest that genital human papillomavirus is not sexually transmitted in all cases and that it may be acquired by modes other than sexual contact.

Objective To determine the transmission rate of human papillomavirus (HPV) in newborn infants of HPV-positive women and to assess the relationship between perinatal HPV transmission and mode of delivery. Methods Three hundred one pregnant women were selected: vaginal delivery (n = 160) or cesarean delivery (n =141). We assessed the presence of the HPV types 16 and 18 DNA sequences in buccal and genital swabs of neonates born to HPV-positive mothers, using the polymerase chain reaction. Result The overall frequency of HPV 16/18 infection among the pregnant women was 22.6% (68/301). At birth, the overall frequency of HPV transmission from HPV 16/18positive mothers to newborns was 39.7% (27/68). A significantly higher rate of HPV 16/18 infection was found at birth when infants were delivered vaginally than when infants were delivered by cesarean (18/35 or 51.4% versus 9/33 or 27.3%, P = .042). However, there was no significant difference in the incidence of perinatal HPV infection between the HPV types 16 and 18 in either vaginal delivery group or in the cesarean delivery group (all P > .100). No significant difference was found between the buccal and genital sites (27/68 versus 21/68, P = .234) or between male and female infants overall (12/36 versus 15/32, P = .255). Conclusion The findings suggest that neonates are at higher risk for exposure to HPV after vaginal delivery than after cesarean delivery.

OBJECTIVE: The objectives of this study were to evaluate the effect of chemotherapy on the continual presence of human papillomavirus deoxyribonucleic acid sequences in bulky cervical cancer tissues and the relationship between the presence of human papillomavirus and the response of these patients to chemotherapy. STUDY DESIGN: Multiple tissue sections obtained from 33 patients with bulky cervical cancer both before and after chemotherapy were analyzed for the presence of human papillomavirus types 16 and 18 by deoxyribonucleic acid amplification. RESULTS: The cytotoxic effects of chemotherapy did not significantly alter the continual presence of human papillomavirus deoxyribonucleic acid sequences in these tissues ( p = 0.8048). The presence of human papillomavirus type 16 deoxyribonucleic acid in tumors treated with neoadjuvant chemotherapy was significantly associated with favorable tumor response compared with type 18-positive patients and type 16/18-negative patients (94.7% vs 42.9%, p = 0.0059 and 94.7% vs 44.4%, p = 0.0004, respectively). Additionally, patients with type 18 deoxyribonucleic acid had a significantly higher risk of recurrence than did type 16-positive patients ( p = 0.0123). CONCLUSIONS: These results seem to suggest that the presence of human papillomavirus deoxyribonucleic acid sequences may serve as a marker to predict the response of bulky cervical cancer to chemotherapy and may be useful in reassessing neoadjuvant treatment for those patients who are free of human papillomavirus or those with type 18 deoxyribonucleic acid. (Am J Obstet Gynecol 1997;176:865-9.)

PURPOSE To determine the presence of cervical cancer cells in circulating peripheral blood of stage IVb cervical cancer patients with metastasis to distant organs. PATIENTS AND METHODS Cervical cancer tissue from 15 stage IVb cervical cancer patients with metastasis were analyzed for the presence of human papillomavirus (HPV) type 16 DNA by nested polymerase chain reaction (PCR). The presence of transcriptional products of the HPV type 16 E6-transforming gene in the peripheral blood of the same 15 cancer patients was analyzed by reverse transcription and PCR. Cervical tissues and peripheral-blood specimens from 12 normal healthy individuals served as controls. RESULTS Thirteen of 15 (86.7%) cervical cancer tissues from same number of patients were found to contain HPV type 16 DNA. Peripheral-blood specimens from 12 of 13 (92.3%) cervical HPV DNA-positive patients were found to contain HPV-specific mRNA detectable by reverse transcription (RT) and PCR. Cervical tissues from all 12 normal controls were HPV-free. None of the peripheral-blood specimens from two cervical HPV-negative cancer patients and 12 normal controls contained detectable amounts of mRNA of HPV type 16 E6-transforming gene. CONCLUSION The most likely source of the HPV-specific mRNA detected in the peripheral blood of cervical cancer patients with metastasis is the cervical cancer cells derived from or shed from the cervix. The presence of HPV E6 mRNAs in peripheral blood may be a sensitive indicator of circulating cervical cancer cells. If PCR positivity is proven to be able to predict disease progression reliably, these findings may have clinical applications in the treatment of cervical and many other cancers.

Treatment results of 26 patients with squamous cell carcinoma (SCC) arising in mature cystic teratoma of the ovary were analyzed. Four nulliparous patients with stage Ia tumors underwent conservative salpingo-oophorectomy. Following surgery, 2 patients had successful pregnancies. The remaining 7 patients with stage Ia tumors were observed after hysterectomy and bilateral salpingo-oophorectomy. Fifteen patients with stage Ic–IV tumors underwent cytoreductive surgery followed by cis -platinum-based chemotherapy with or without sequential radiotherapy. The mean survival was 63.9 months. The overall actuarial disease-free survival at 2 years was 69%, and by stage was as follows: stage I, 100% (13/13); stage II, 100% (2/2); stage III, 30% (3/10); and stage IV, 0% (0/1). A significant difference in disease-free survival was noted in stage ( P = 0.0001). Optimal versus suboptimal operation was associated with a median Kaplan–Meier survival of 65 months versus 34.8 months, with actuarial disease-free survival at 2 years of 60 and 0%, respectively ( P = 0.0210). Our study shows that 67% (16/24) of the patients had SCC antigen levels exceeding 2 ng/ml, which by stage was as follows: stage I, 5/11 (45%); stage II, 1/2 (50%); stage III, 9/10 (90%); and stage IV, 1/1 (100%). After completion of treatment, all 8 patients with recurrent lesions had reelevated SCC antigen levels in series SCC antigen monitoring. In conclusion, positive prognostic factors of disease-free survival were optimal cytoreduction and lower FIGO stage. We suggest that multimodality therapy, including aggressive cytoreduction followed by cis -platinum-based chemotherapy with or without sequential radiotherapy, is recommended. In addition, we suggest that serum SCC antigen monitoring may be helpful in early detection of cancer recurrence.

Objective To determine if sperm cells differentially take up or retain different regions of human papilloma virus (HPV) type 18 genome. Design A descriptive clinical study. Setting A major medical center affiliated with a medical college. Patients Twenty-three randomly selected patients who attended Fertility Clinics at the Chang Gung Memorial Hospital. Semen specimens were obtained from volunteered patients who attended fertility clinics at the Chang Gung Memorial Hospital. Main Outcome Measure The presence of various regions of HPV type 18 genome in sperm cells was determined by polymerase chain reaction. Results Among 23 sperm specimens that were positive for HPV type 18 DNA by polymerase chain reaction, the upstream regulatory region, E6, E7, E1, and L1 regions or open region frames were detected in 4 (17%), 7 (30%), 19 (83%), 5 (22%), and 1 (4%) specimens, respectively. The differential display of the presence of various regions of the HPV type 18 genome was not the result of different amplification and detection efficiencies of these DNA fragments. Conclusion These results suggest that the oncogenic portion of HPV DNA is present in spermatozoa. Furthermore, the E6 and E7 regions of the viral genome preferentially were taken up and/or retained by the human sperm cells.

The presence of human papillomavirus (HPV) type 16 and 18 transcripts was determined by reverse transcription/polymerase chain reaction (PCR) in ovarian and endometrial carcinoma tissue. HPV-16 DNA sequences were detected in 50.0% (9/18) of the ovarian carcinomas and in 44.4% (8/18) of endometrial carcinomas. HPV-18 DNA sequences were found in 16.7% (3/18) of both the ovarian and endometrial carcinomas. Using RNA-PCR analysis, we found 3 out of 9 (33.3%) of the HPV-16 DNA-positive and 1 in 3 (33.3%) of the HPV-18 DNA-positive ovarian carcinomas were transcriptionally active, contrary to the HPV-16 DNA-positive or HPV-18 DNA-positive endometrial carcinomas. The results suggest that HPV RNA may be detected in ovarian carcinomas though its biological significance remains to be elucidated. HPV RNA is not demonstrable in endometrial carcinoma using the current primer sets. Further investigations are necessary for a final conclusion.

Ultra-violet light irradiation of containers and components used in in vitro DNA amplification reactions catalyse by Taq DNA polymerase is a simple and effective method to reduce carry-over contamination and can reduce false-positive results. However, we found that prolonged exposure of water in polypropylene microcentrifuge tubes to u.v. light can result in reduction of amplification efficiency by at least two orders of magnitude when these water specimens are used in amplification reaction mixtures. Although the mechanism that causes this inhibition of DNA amplification is unclear now, the results seem to suggest that u.v. irradiation for routine anti-contamination purposes should be used with caution.

The human papillomavirus was detected in cervicovaginal cells by the polymerase chain reaction in 14 of 37 (37.8 %) patients attending a sexually transmitted disease (STD) clinic and in 6 of 43 healthy young women (14.0 %) undergoing routine gynecologic examinations who served as controls. The results indicated that even the more malignant types of human papillomaviruses were not uncommon among the control group, and that the prevalence of human papillomavirus infection was significantly higher in STD clinic patients than in the control group. These findings confirm the suggestion that factors other than human papillomavirus infections may be involved in the pathogenesis of cervical cancer.

Hepatitis B virus (HBV) DNA was detected by in vitro enzymatic DNA amplification techniques in 66.7% (six of nine) of hepatitis B virus surface antigen (HBsAg)-positive and in 21.1% (7 of 33) of HBsAg-negative pregnant women. Five of the HBV DNA and HBsAg-positive women and one HBV DNA-positive but HBsAg-negative woman gave birth to infants positive for serum HBV DNA at time of birth. These results suggest that HBsAg-negative pregnant women are potentially capable of transmitting HBV DNA to their infants.

In vitro DNA amplification by means of the polymerase chain reaction (PCR) was used to amplify dengue types 1 and 2 viral genomes in cultured cells and in the serum of persons infected with dengue virus. Results of the present investigation suggest that the PCR method is type-specific in detecting dengue virus and has a detection sensitivity of less than 100 plaque-forming units (pfu) for both serotypes of the virus. The PCR method may be useful for detecting and typing dengue virus in clinical and epidemiological specimens.

Polymerase chain reaction (PCR) was used to amplify and identify the presence of the DNA of human papillomavirus (HPV) types 6, 11, 16, and 18 in peripheral blood mononuclear cells (PBMCs) of women with and without urogenital HPV infections. HPV DNA of various types was found in PBMCs of 13 of 25 (52.0%) patients with urogenital HPV infections and in none of the 19 control subjects who are free of urogenital HPV infections. The presence of HPV DNA in PBMCs may impair the immunologic functions of the lymphocytes and play a role in the epidemiology of HPV infections and the pathogenesis of HPV-induced diseases.

Cloned Gardnerella vaginalis DNA were selected from a plasmid DNA library constructed with partially restriction endonuclease Hind III-digested genomic DNA of G. vaginalis to serve as DNA probe in detecting G. vaginalis. The level of detection was determined to be approximately 10,000 cells by slot-blot DNA hybridization. This probe DNA will not cross-hybridize with DNA of a number of non-Gardnerella micro-organisms commonly found in female genital tract. The DNA probe-based hybridization test may become a useful tool for the identification of G. vaginalis in uncultured clinical specimens.

The polymerase chain reaction (PCR) was used to identify mycobacterial DNA sequences in uncultured clinical specimens. Two oligonucleotide primers derived from the sequence of a gene that codes for the 65-kilodalton antigen of Mycobacterium tuberculosis amplified DNA from all 11 species of mycobacteria tested. Amplified DNAs of nontuberculosis mycobacteria were found to be approximately 20 to 40 bases shorter than those from M. tuberculosis and Mycobacterium bovis BCG. DNA equivalent to that present in as few as 40 M. tuberculosis cells either alone or in the presence of DNA equivalent to that in 10(6) human cells could be detected. Results from analysis of cultured bacteria and clinical specimens showed PCR was sensitive and specific both in detecting mycobacteria and in differentiating M. tuberculosis and BCG from other species of mycobacteria. The PCR method with the primers reported here may become a useful tool in the early and rapid detection of mycobacterial infections in uncultured clinical specimens.

The polymerase chain reaction (PCR) was used to identify human papillomavirus (HPV) in cervicovaginal cells in normal individuals and in patients with cervical intraepithelial neoplasia (CIN). By use of a set of primers encoding the E6 region of the HPV genome, the presence of HPV DNA was demonstrated in the cervicovaginal cells of 43 (42.2%) of 102 normal individuals and in all 12 CIN patients. High sensitivity of the PCR method produced an additional 9 positive results on second sampling from 48 individuals who were initially HPV-negative. On the other hand, 26 (24.3%) of 107 HPV-positive individuals were HPV-negative when sampled a second time 5-7 days later. The data suggested that retest probably should be considered for patients clinically suspected of having HPV infection whose initial test results are negative for HPV DNA. Also, single HPV DNA-positive results should be accepted with caution.