Denaturing gradient gel electrophoresis screening of the BRCA1 gene in cells from precancerous cervical lesions.

OBJECTIVE: To determine the integrity of the BRCA1 gene in archival, paraffin-embedded tissues from precancerous lesions of the uterine cervix. STUDY DESIGN: DNA was extracted from histologically documented precancerous cervical lesions (17 cases). Polymerase chain reactions were performed targeting exon 11 of BRCA1 (434-bp), the L1 consensus human papillomavirus (HPV) gene common to > 25 HPV types, as well as the beta-globin gene. The amplified products were analyzed using denaturing gradient gel electrophoresis. RESULTS: Mutation of the BRCA1 exon 11 gene was detected in > 76% of cases with precancerous lesions of the cervix. The mutations were either complete deletions or deletions of one or more nucleotides, leading to frame shifts. There were no significant differences in frequency of BRCA1 mutations among precancerous cervical tissues positive for the HPV L1 consensus gene (n = 9) when compared with HPV-negative tissues. CONCLUSION: The mutated BRCA1 gene was associated with 76% of 17 precancerous lesions of the cervix. The type of cervical intraepithelial neoplasia and the presence or absence of HPV were not related to the mutations. The role of BRCA1 mutation in the genesis of precancerous cervical lesions needs to be explored further.

Absence of human papillomavirus E6-E7 transforming genes from HPV 16 and 18 in malignant ovarian carcinoma.

Ovarian carcinoma is one of the frequent causes of death from malignancies in the United States. A report excited the scientific community when human papillomavirus were identified in advanced epithelial ovarian carcinoma tissues in 10 of 12 patients. A few studies also identified HPV DNA in ovarian carcinoma tissues. However, several researchers employing polymerase chain reaction techniques and using different oligonucleotide probes did not detect HPV DNA in ovarian carcinoma tissues. The objective was to determine the presence of the E6-E7 genes of HPV types 16 and 18 in archived paraffin-embedded malignant ovarian carcinoma using primers targeting. Archived human malignant ovarian cancer tissues (N = 20 cases) embedded in paraffin blocks were processed, and DNA was extracted and the presence of DNA verified by p53 amplifications. PCR analyses were performed on the extracted DNA together with appropriate controls. The results showed an absence of E6-E7 genes of HPV types 16 and 18 in ovarian carcinoma. However, the presence of other HPV types or gene regions is not ruled out and more studies are needed to resolve the question of HPV involvement in ovarian carcinogenesis.

Assessment of archived paraffin-embedded cervical condyloma tissues for mycoplasma-conserved DNA using sensitive PCR-ELISA.

Clinically, it is important to detect mycoplasmas because these organisms have been implicated in gastric and ovarian cancer, pneumonia, postabortal fever, pelvic inflammatory disease, pyelonephritis, endometritis, urethritis, perinatal mortality, arthritis, spontaneous abortion, infertility and interference with sperm development and they act as cofactors catalyzing the HIV disease state. Recently, the combined polymerase chain reaction and enzyme-linked immunosorbent assay method targeting the consensus DNA of over 15 species of mycoplasmas was shown to be superior for the detection of mycoplasmas. The objective was to determine if there was an association between mycoplasmas and cervical neoplasia. Cervical tissues, histopathologically categorized by cervical intraepithelial neoplasia (CIN) grade, flat or exophytic, and acanthosis or koilocytotic, were used. The results showed that mycoplasmas DNA were present in 21.4% of the condyloma tissues and in 33.3% of condyloma tissues with CIN. In contrast, mycoplasmas DNA were not detected when there were no CIN. The presence or absence of human papillomavirus (HPV) did not make a difference. Mycoplasmas DNA were present in 40.0 and 12.5% of the exophytic and flat condylomas, respectively. A higher percentage of cervical tissues graded with slight koilocytosis had (P = 0.05) mycoplasmas DNA compared with tissues graded with moderate koilocytosis. The detection of mycoplasmas DNA in archived cervical condyloma tissues with CIN corroborated previous reports of an association between mycoplasmas and CIN. However, the association between mycoplasmas and the presence of HPV could not be made in this study.

Polymerase chain reaction enzyme-linked immunosorbent assay detection of mycoplasma consensus gene in sperm with low oocyte penetration capacity.

OBJECTIVE: To determine the prevalence of mycoplasmas in washed sperm and to compare the penetration of zona-free hamster oocytes by sperm with and without mycoplasmas. DESIGN: Prospective comparative study. SETTING: Clinical and academic research environment. PATIENT(S): Semen from 34 male patients. INTERVENTION(S): Specimens were divided, Percoll washed, and scanned for differences in kinematic parameters. Sperm DNA was extracted and assayed for mycoplasma DNA using the polymerase chain reaction-ELISA method targeting the consensus gene of 15 mycoplasma species. Remaining sperm were processed by centrifuge, Percoll, or TEST (TES and Tris) Yolk Buffer (TYB) and assessed for penetration capacity. MAIN OUTCOME MEASURE(S): Detection of mycoplasma DNA. RESULT(S): Mycoplasma DNA was detected in 29.4% of the Percoll-washed sperm. The penetration of oocytes by mycoplasma-positive sperm (59.5% +/- 17.3%; mean +/- SEM) was less than mycoplasma-negative sperm (86.8% +/- 5.4%) in the TYB-processed group. CONCLUSION(S): Mycoplasma DNA is demonstrated in almost a third of the Percoll-washed sperm. Because there were no other cell types except sperm, the results suggest that the mycoplasmas were either internalized or attached to the membranes. The reduced penetration by mycoplasma-positive sperm after 48-hour TYB suggest mycoplasmas required time to affect sperm function. Similarities between hypo-osmotic swelling and between kinematic parameters suggest that the mechanism does not involve differences in membrane integrity and in motility patterns.

Prevalence of mycoplasma conserved DNA in malignant ovarian cancer detected using sensitive PCR-ELISA.

Mycoplasmas are tiny polymorphic prokaryotic organisms (0.2-0.3 microm) that lack a cell wall and reside ubiquitously at the cell membrane or internalized into the cell. The organisms have been implicated in many diseases including functioning as cofactors catalyzing the HIV disease state. The oncogenic potential of mycoplasmas was only recently realized when they were shown to cause chromosomal changes and in vitro cell transformations through gradual progressive chromosomal loss and translocations. While a recent study linked mycoplasmas with gastric cancer, the association between mycoplasmas and ovarian cancer has not been established. Recently, a commercial assay which combined polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods was developed for the detection of mycoplasmas. The present objective was to determine the prevalence of mycoplasmas in archived paraffin-embedded malignant ovarian cancer tissue. The combined PCR-ELISA procedure was used with consensus primers targeting for 15 species of mycoplasmas and acholeplasmas. Archived human malignant ovarian cancer tissues (N = 27 cases) embedded in paraffin blocks were processed, and DNA was extracted and the presence of DNA verified. The extracted DNA specimens were randomly divided into three groups for analyses. PCR-ELISA assays were performed on extracted DNA together with appropriate negative and positive controls. The results showed mycoplasmas were present in 59.3% of the malignant ovarian cancer specimens. PCR-ELISA analysis of Neisseria gonorrhea and Chlamydia trachomatis controls did not produce cross-reacting false-positive results. The results suggest an association between mycoplasmas and malignant ovarian cancer. A 59.3% prevalence rate was demonstrated for mycoplasmas in paraffin-embedded ovarian cancer tissues. The mechanism involved in oncogenesis by mycoplasmas remains to be elucidated.

Blastocysts exhibit preferential uptake of DNA fragments from the E6-E7 conserved region of the human papillomavirus.

The objective of the study was to determine if embryos at the blastocyst stage differentially took up exogenous human papillomavirus (HPV) DNA derived from the different HPV types and to determine whether the capture process was active or passive. In vivo fertilized mouse embryos were cultured to the blastocyst stage in vitro. The mouse blastocysts were incubated for 2 hr in the presence of a mixture of exogenous DNA fragments derived from HPV types 6b, 11, 16, and 18. The blastocysts were rigorously washed and analyzed for differential uptake of HPV gene sequences using the polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis. PCR analysis detected HPV type 18 in only 40% of the blastocyst groups while detecting the other HPV types in 100% of the blastocysts. The negative control group did not show HPV DNA after PCR ruling out possible PCR artifacts. Formalin-fixed blastocysts also showed uptake of HPV DNA. In conclusion, the data suggest a role for embryos as passive vectors for foreign DNA and that the degree of DNA uptake varies with different types of HPV DNA.

A new concept for anastomosis of the fallopian tube: tissue reconstruction with nonpenetrating, arcuate, legged clips in the rat model.

OBJECTIVE: To evaluate the titanium Kirsch-Zhu microclip microsurgical reanastomoses of the fallopian tubes. DESIGN: Compare the reanastomoses of the rat uterine horn between Kirsch-Zhu clips (Cushman Engineering, Albuquerque, NM) (group A) and conventional suture microsurgical techniques (group B). SETTING: Microsurgery Research Laboratory, Loma Linda University Medical Center, Loma Linda, California. PATIENTS, PARTICIPANTS: Fifteen SD rats Harlan (Harlan Sprague-Dawley Corp., Indianapolis, IN) were done in groups A and B and six in control group (C). MAIN OUTCOME MEASURE: Pregnancy rate, litter size, tissue of procedure, and histologic results. RESULTS: The clip technique was shorter in procedure time and resulted in equal fertility rate and litter size. Histologically, there were less granulomatous formation and histiocytic inflammation, but muscularis thinning and fibrosis were noted with the clip. CONCLUSIONS: The Kirsch-Zhu clip has potential for application to human fallopian tube reanastomoses.

Nonpenetrating, arcuate-legged clip reconstruction of the rat uterine horn.

A new method for reconstructing rat uterine horn was developed in which nonpenetrating, arcuate-legged clips are applied in interrupted fashion to everted seromuscular edges, forming an elastomeric flanged joint. This anastomosis has unusual physical and morphologic properties, with improved tissue healing and luminal restitution. Clipping is easier than suturing, and resulted in equivalent fertility rate (50-60%) and litter size. It also is associated with less granuloma formation and hystiocytic infiltration than suture. The new technique has the potential of endoscopic translation for human tubal reconstruction.

Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid.

The present study demonstrated the presence of HPV gene sequences in Percoll-washed sperm cells using polymerase chain reaction primers targeting smaller gene regions. Up to 64% of the sperm specimens were shown to contain gene sequences indicative of the presence of HPV. Human papillomavirus type 16 was detected about twice as often as HPV type 18. The results suggest the possible role of sperm as a vector for HPV.

Pregnancy outcome in Swiss-Webster mice infected with Chlamydia trachomatis.

OBJECTIVE: The objective of this study was to observe pregnancy outcomes in mice infected transvaginally with Chlamydia trachomatis. METHODS: Pregnant mice were inoculated transvaginally with either C. trachomatis (CT) or sterile calf serum (CON) on pregnancy day 4. Pregnancy outcomes as well as genital tract histology and culture were compared. Statistical analysis was performed using Fisher's exact test and Student's t-test. RESULTS: Twenty-four of 26 CT mice had positive uterine cultures for C. trachomatis. Inflammation occurred in 9 (34.6%) (P = 0.002, 95% confidence interval = 1.7-3.5) and intrauterine fetal demise occurred in 5 (19.2%) (P = 0.05, 95% confidence interval = 1.6-2.9) of CT mice. No mice in the CON group (0/24) had positive uterine cultures, developed inflammation, or experienced intrauterine fetal demise. CONCLUSIONS: Lower genital tract chlamydial infection is associated with intrauterine fetal demise in Swiss-Webster mice.

Malignant teratoma arising in a dysgenetic gonad.

A case of malignant teratoma arising within a dysgenetic gonad in a 21-year-old phenotypic female with a 46 XY karyotype is presented. Admixtures of dysgerminoma, yolk sac tumor in close juxtaposition to embryoid bodies and elements of choriocarcinoma were also present. The contralateral gonad was an unidentifiable fibrovascular streak. Neither gonadoblastoma nor coarse calcifications (such as commonly found in gonadoblastoma) could be identified. We believe that the present case arose de novo in a dysgenetic gonad and, uncharacteristically, was not associated with a gonadoblastoma.

Uptake of exogenous human papilloma virus L1 DNA by oocytes and detection by the polymerase chain reaction.

OBJECTIVE: The purpose of this study was to determine if oocytes were capable of taking up exogenous DNA such as human papillomaviral (HPV) DNA and evaluate the zona pellucida as a barrier to the entry of foreign DNA into the oocyte. METHODS: The experiment consisted of four groups of hamster oocytes exposed to HPV DNA fragments: Group A, zona-free oocytes (n = 5); Group B, oocytes with an intact zona pellucida (n = 5); Group C, oocytes fixed in 4% buffered formalin solution for 20 min (n = 5); and Group D, zona-free oocytes (n = 4). Group C oocytes served as an internal control to ensure adequate washing of the oocytes after incubation. RESULTS: The zona pellucida was not a barrier to foreign DNA molecules. The PCR did not detect L1-HPV and beta-globin gene sequences in the untreated hamster oocyte. Uptake of the smaller DNA fragments such as that amplified from the beta-globin region was independent of active oocyte cell processes. CONCLUSION: Oocytes cultured in vitro can passively take up exogenous DNA fragments. The results suggest a possible role of oocytes as vectors for foreign DNA.

Endometrioid carcinoma of the oviduct.

Endometrioid carcinoma is a malignant tumor which microscopically resembles endometrial adenocarcinoma, but arises from extrauterine sites, most commonly the ovary. Although its association with endometriosis has received considerable discussion, it appears that most of the malignant endometrioid tumors arise de novo. We present a case of endometrioid carcinoma which is outstanding for its primary site in the oviduct and for its histologic appearance, which incorporated a close interplay between both stromal and epithelial elements. The neoplasm was found incidentally, when the woman presented with postmenopausal bleeding due to adenomatous hyperplasia of the endometrium. The patient presented with a stage I, grade I tumor which did not invade the wall of the oviduct. Twelve years following a TAH-BSO and postoperative radiation, the tumor recurred, requiring additional surgery and chemotherapy. The patient is currently disease free, 2 years from her recurrence.

Malignant mixed müllerian tumor of the oviduct.

Malignant mixed müllerian tumor is an uncommon gynecologic neoplasm, especially in the oviduct, where fewer than 30 have been described. We report two additional cases which clinically mimicked carcinoma of the oviduct. Both patients were postmenopausal and had the chief complaint of abdominal pain associated with increased abdominal girth. Presurgical vaginal cytologies were negative and both diagnoses were made only with histologic examination of tissues removed at surgery. As most of the cases from the literature involved low-parity women, the high parity of these women is unusual. Unfortunately both presented with stage III disease and died of recurrent disease 18 and 12 months following surgery.

Fallopian tube carcinoma: a clinicopathological study of 17 cases.

From 1969 through 1986, 17 patients with primary adenocarcinoma of the fallopian tube were treated at the Loma Linda University Medical Center. Stages I, II, and III of the disease were present in 6, 5, and 6 patients, respectively. The mean age of the patients was 59.9 years. Vaginal bleeding or discharge (57%), followed by abdominal pain or discomfort (29%), was the most common symptom in our patients. A palpable pelvic mass was detected in two-thirds of the patients. One case of carcinosarcoma, one case of mixed mesodermal tumor, and one case of endometrioid carcinoma are included. No patient in this series had a correct preoperative diagnosis. Therapy consisted of surgical resection, usually followed by various combinations of adjuvant radiation therapy and/or chemotherapy. The overall 5-year survival rate was 31%. Five patients (29%) are alive without evidence of disease. This study supports the need for collaboration among large centers to define the optimal adjuvant therapy of this disease. In the absence of the desired treatment protocols, such lesions should be approached in a manner similar to that used for ovarian cancers.

An analysis of endometrial biopsies performed for infertility.

The authors evaluated 774 endometrial biopsies that were performed for infertility. Complications arose in 3.6%. Lag of more than 2 days was found in 19%; luteal phase defect (LPD) was diagnosed in 5.7%. Most of the incidence of LPD can be predicted from chance occurrence. There was no association between abnormal biopsies and basal body temperature patterns, or between pathology, pregnancy outcome, and treatment. Exceptions included women with multiple spontaneous abortions and patients treated with clomiphene citrate (CC). An endometrial biopsy was performed in a pregnancy cycle in 4.0%, with an abortion rate not significantly different from the total study group. The authors conclude that an endometrial biopsy is relatively safe; however, the diagnostic and therapeutic consequences are limited. Endometrial biopsies may be useful only if performed in cases of habitual abortion or ovulation induction with CC.

Stromal invasion in endometrial adenocarcinoma.

The ability to predict myometrial invasion by endometrial adenocarcinoma is useful for preoperative treatment purposes. The presence of stromal invasion is one possible method of predicting myometrial invasion, but criteria for the presence of stromal invasion have been lacking. Recently, criteria for its detection were proposed. We evaluated the validity of the proposed criteria in two conditions: (1) atypical hyperplasia (119 cases) and (2) endometrial adenocarcinoma Stage I, grade 1 (83 cases). Adenocarcinoma without stromal invasion according to the criteria of Kurman and Norris was present in 43 cases and adenocarcinoma with stromal invasion was present in 40 cases. This series demonstrated the ability of prehysterectomy endometrial sampling to predict the probability of myometrial penetration. In addition, none of our patients without stromal invasion in the resected uterus had myometrial penetration and no metastases have occurred. In those showing stromal invasion in the excised uterus, the myometrium was invaded in 34 of 51 cases (67%). Since myometrial penetration was not present when there was no stromal invasion in the resected uterus, stromal invasion deserves further evaluation and should be considered in future classification of endometrial adenocarcinoma.

Microinvasive squamous carcinoma of the vulva: present status and reassessment.

A retrospective analysis of 24 patients with early invasive squamous carcinoma was performed. No nodal metastases were noted in these patients. Based on a review of the literature, no absolute definition of microinvasive carcinoma could be formulated, but a treatment outline has been formulated based on depth of invasion for Stage I lesions.