The behavior of endometrial hyperplasia: a prospective study. Endometrial Hyperplasia Study Group.

OBJECTIVE: To clarify the behavior of endometrial hyperplasia in a prospective study. METHOD: Fifty-one patients with endometrial hyperplasia were followed up for 6 months. Samples of endometrial tissues were taken by uterine endometrial biopsy every 4 weeks during the first 3 months and at the end of follow-up. RESULTS: In 69% (35/51) of the patients histological picture of the endometrium became normal during the observation period. The lesions persisted in 17% (6/35) of the patients with simple hyperplasia, in 25% (1/4) of those with complex hyperplasia, in 14% (1/7) of those with simple atypical hyperplasia, and in 80% (4/5) of the patients with complex atypical hyperplasia. In the remaining 3 patients with simple hyperplasia, the lesions progressed to complex atypical hyperplasia by the end of follow-up, after showing a normal endometrium. CONCLUSION: Most cases of endometrial hyperplasia, except for complex atypical hyperplasia, disappeared spontaneously within a short period of time.

Detection of monocyte chemotactic and activating factor (MCAF) and interleukin (IL)-6 in human seminal plasma and effect of leukospermia on these cytokine levels.

PROBLEM: To demonstrate whether monocyte chemotactic and activating factor (MCAF) and interleukin-6 (IL-6) are present in the seminal plasma, and whether these presence is modulated by leukospermia. METHODS: Semen samples from 53 men were obtained by masturbation and examined for the presence of MCAF and IL-6 by enzyme immunoassay (EIA). Semen samples were obtained from 28 infertile men without leukospermia, 16 infertile men with leukospermia, and nine proven-fertile men. The correlation between the amount of MCAF in the seminal plasma with some spermiogram parameters and other cytokines such as IL-6 and IL-8 was statistically evaluated. RESULTS: Immunoreactive MCAF was detected in the seminal plasmas of all 53 subjects. The MCAF titer in the seminal plasma of patients with leukospermia (11.19 +/- 2.75 micrograms/l) was significantly higher than that in the seminal plasma of the patients without leukospermia (3.24 +/- 0.53 micrograms/l) and the fertile men (2.78 +/- 0.35 micrograms/l) (P < 0.001). The IL-6 titer in the seminal plasma of the patients with leukospermia (21.05 +/- 4.49 ng/l) was also significantly higher than that in the seminal plasma of the patients without leukospermia (8.77 +/- 1.92 ng/l) and the fertile men (6.94 +/- 1.27 ng/l) (P < 0.01). There was a high degree of correlation among the levels of MCAF, IL-6 and IL-8 in the seminal plasma. CONCLUSIONS: These findings demonstrated the presence of MCAF and IL-6 in the seminal plasma, and that the levels of these cytokines were elevated in the seminal plasma of the infertile patients with leukospermia.

Regulation of classical HLA class I genes in human choriocarcinoma cells by nuclear proteins binding to MHC class I regulatory elements.

PROBLEM: The regulation of classical HLA class I genes in choriocarcinoma have been reported. METHODS: We determined whether four choriocarcinoma cell lines expressed classical HLA class I or HLA-G by a reverse transcription-polymerase chain reaction (RT-PCR) and studied the regulatory mechanism of classical class I using a gel mobility shift assay. RESULTS: NUC1 and SCH expressed classical class I but not HLA-G. GCH1 and Jar did neither. Nuclear protein binding to the class I regulatory element (CRE) was detected in NUC1 and SCH. Interferon-gamma augmented both classical class I expression and the DNA-protein complex in NUC1. The DNA-protein complex was not observed in GCH1, and Jar showed a CRE-binding protein with different electrophoretic mobility and binding affinity from that of SCH and NUC1. CONCLUSION: The CRE is one of the regulatory elements of classical HLA class I genes in choriocarcinoma cells.

Long-term follow-up of patients with advanced ovarian cancers treated with intermittent administration of combination chemotherapy with cisplatin, doxorubicin and cyclophosphamide.

Despite high primary response rates with cisplatin-based combination chemotherapy, the overall survival rate for advanced ovarian cancers remains dismal. We designed a new systematic treatment approach with a combination chemotherapy consisting of cisplatin, doxorubicin and cyclophosphamide (cyclic PAC chemotherapy), with the aim of improving survival rates with minimal disturbance of quality of life. Cyclic PAC chemotherapy is a three-step chemotherapy with three courses of the PAC regimen in each step. A total of nine courses with a 3-month drug-free period between each step were administered over a 15-month period to patients with clinical stage IC-IV ovarian cancer who had undergone cytoreductive surgery. Forty-eight patients with stage IC-IV disease (34 patients with stage III and IV disease) were treated with cyclic PAC chemotherapy. Thirty-four patients with stage IC-IV disease (23 patients with stage III and IV disease) were treated by a brief course of PAC chemotherapy. Long-term survival and toxicity were evaluated for both treatment groups. Cyclic PAC chemotherapy improved the overall outcome of patients (66.6% 3-year and 56.5% 5-year survival rates) compared to brief PAC (41.2% 3-year and 23.5% 5-year survival rates) (P < 0.01). The outcome of patients with stage III-IV ovarian cancer of the cyclic PAC group (52.6% 3-year and 37.2% 5-year survival rates) was also superior to that of the brief PAC group (21.7% 3-year and 8.7% 5-year survival rates). Generally, the treatment was well tolerated. The toxicity was similar in both groups, although myelosuppresion and neurotoxicity were rather prominent in the cyclic PAC group. Cyclic PAC chemotherapy may lead to improved survival in advanced ovarian cancer, and merits further investigation in a randomized study.

Acrosome reaction induced in a limited population of human spermatozoa by progesterone (Ca(2+)-dependent) and ATP (Ca(2+)-independent).

We examined the kinetics of the acrosome reaction induced in human spermatozoa by progesterone (Ca(2+)-dependent) and adenosine 5'-triphosphate (ATP; Ca(2+)-independent). ATP and progesterone did not induce the acrosome reaction unless spermatozoa were incubated in a capacitation medium. ATP exhibited a constant induction of the acrosome reaction regardless of the incubation period, while progesterone began to induce the acrosome reaction after > or = 6 h of incubation. At 24 h of incubation, the percentages of spermatozoa in which the acrosome reaction had been induced by both progesterone and ATP were almost equal. To determine whether a limited population of human spermatozoa was reactive to both progesterone and ATP, we employed an MH61 bead binding method. When the acrosome-reacted spermatozoa were removed with MH61 beads, their percentage in the sperm suspension was decreased to < 3%. At 24 h of incubation, progesterone and ATP induced the acrosome reaction in 12.0 and 10.0% of spermatozoa, respectively. After MH61 bead binding was performed, the remaining spermatozoa did not react to the other activator (progesterone-->ATP, ATP-->progesterone). These findings indicate that only a limited population of human spermatozoa has the potential to undergo the acrosome reaction when stimulated by both progesterone and ATP.

Acrobeads test: a new diagnostic test for assessment of the fertilizing capacity of human spermatozoa.

OBJECTIVE: To determine the effectiveness of the Acrobeads test for predicting the outcome of IVF. DESIGN: Human spermatozoa express the CD46 molecule (membrane cofactor protein) on their heads after the acrosome reaction. CD46-positive spermatozoa formed a sperm-bead complex with immunobeads coated with anti-CD46 monoclonal antibody. In the Acrobeads test, fertilizing capacity was determined by assessing sperm-bead agglutination. SETTING: Department of Obstetrics and Gynecology, Osaka University Hospital. PARTICIPANTS: Thirty-seven donors of proven fertility and 88 male partners of infertile couples. MAIN OUTCOME MEASURES: We carried out the Acrobeads test and a sperm penetration assay (SPA) using zona-free hamster oocytes within 3 months before IVF and we then analyzed the results in relation to IVF outcome. RESULTS: The sensitivity of the Acrobeads test and SPA was 100% and 88%, respectively, whereas the specificity was 43% and 52%, respectively. The negative predictive value of the Acro-beads test was 100%, whereas that of the SPA was 73%. These results indicate that there was no significant difference between these two tests in terms of predicting IVF outcome. CONCLUSION: We suggested that the Acrobeads test be used to evaluate the fertilizing capacity of human spermatozoa because we should avoid using the SPA to prevent cruelty to animals.

Adenosarcomas originating from sites other than uterine endometrium.

We report three cases of adenosarcomas arising from extraendometrium of the uterus: one arising from the ovary, one from the paracolpium and one from the endocervix of the uterus. Microscopically, they consisted of an admixture of benign-appearing epithelial and mesenchymal components with hypercellularity and minimal atypia. Two of the tumors were initially misdiagnosed as endometriosis and one was diagnosed as adenofibroma. One patient had several recurrences and died 7 years after the initial laparotomy and another patient had sarcomatous overgrowth which invaded the muscular tissues of the large intestine. Thus it appears that adenosarcoma occasionally shows grave clinical behavior, despite the benign or low-grade appearance of its microscopic features. Problems of diagnosis and management of this tumor are discussed. An aggressive therapeutic approach including wide surgical excision is recommended even in questionable cases.

Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-alpha and interferon-gamma.

BACKGROUND: Macrophages and T lymphocytes have been identified in the regressing corpus luteum, and they are thought to participate in structural luteolysis (destruction and removal of luteal cells). Since these cells produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), we investigated the effects of these two cytokines on death of luteal cells in vitro. METHODS: Mouse luteal cells were cultured in serum-free medium with TNF-alpha at 0, 500, 1,000, 3,000, or 5,000 U/ml in the presence or absence of IFN-gamma at 1,000 U/ml for 3 or 6 days. Then, for estimation of the actions of these cytokines on induction of luteal cell death, we determined the number of viable cells, the percentage of fragmented DNA in total DNA extracted from cultured cells, and the percentage of cells with fragmented DNA in their nuclei by the trypan blue exclusion test, the sensitive micromethod for DNA assay, and the in situ DNA 3' end labeling method, respectively. DNA fragmentation was also analysed by agarose gel electrophoresis, and cultured cells were examined by electron microscopy. RESULTS: On day 3 of culture, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500-5,000 U/ml did not decrease the number of viable cells, but a combination of IFN-gamma (1,000 U/ml) and TNF-alpha (5,000 U/ml) did. On day 6, IFN-gamma alone at 1,000 U/ml or TNF-alpha alone at 500, 1,000 and 3,000 U/ml did not decrease the number of viable cells, whereas TNF-alpha alone at 5,000 U/ml did, and combinations of IFN-gamma and TNF-alpha at 1,000, 3,000, and 5,000 U/ml decreased the number of viable cells in proportion to the concentration of TNF-alpha. On days 3-6 of culture, combinations of IFN-gamma and TNF-alpha that decreased the number of viable cells also increased the percentages of fragmented DNA in total DNA of cultured luteal cells and the percentages of luteal cells with fragmented DNA in their nuclei. Agarose gel electrophoresis of fragmented DNA showed a ladder-like pattern, and electron microscopic examination showed luteal cells with the characteristics of apoptosis. CONCLUSIONS: The presence of IFN-gamma modulates the ability of TNF-alpha to induce a reduction in the number of viable cells, although TNF-alpha alone at high concentrations can induce a reduction in the number of viable cells.

Analysis of clonality by amplification of short tandem repeats. Carcinomas of the female reproductive tract.

The clonal composition of cancers of the female reproductive tract was evaluated by analysis of patterns of X-chromosome inactivation. Using DNA extracted from frozen tissues or paraffin-embedded archival specimens as template, polymerase chain reaction (PCR) was performed to generate amplified DNA fragments of exon 1 of the X-linked androgen receptor gene, which contains a highly polymorphic trinucleotide repeat. Predigestion of tumor DNA with methylation-sensitive restriction endonuclease Hha I or Hpa II permitted selective PCR amplification from the methylated (uncleaved) allele. Of a total of 54 tumors analyzed, 50 cases showed heterozygosity (93%) and were therefore informative for clonal analysis. Monoclonal composition of the tumors was suggested in a total of 49 of 50 cases, including 12 adenocarcinomas of the uterine endometrium, 13 squamous cell carcinomas of the uterine cervix, 6 adenocarcinomas of the uterine endocervix, and 18 epithelial tumors of the ovary. However, polyclonal composition was observed in one mucinous carcinoma of the ovary, in which we previously showed that both GGT-->GAT and GGT-->GTT mutations are present in > 20% of total K-ras copies in the tissue. Our studies demonstrate the utility of PCR amplification of highly polymorphic repetitive sequences for analysis of patterns of X-chromosome inactivation. This approach is practical for the analysis of clonal cell composition in a high proportion of both formalin-fixed and frozen archival tissues.

Alteration of the p53 tumor suppressor gene occurs independently of K-ras activation and more frequently in serous adenocarcinomas than in other common epithelial tumors of the human ovary.

To clarify the role of the p53 tumor suppressor gene in the development of human ovarian epithelial tumors and to study the association of p53 alterations with K-ras activation, a series of 70 common epithelial ovarian tumors from Japanese patients was studied. These included 31 serous adenocarcinomas, 12 mucinous adenocarcinomas, 5 mucinous tumors of borderline malignancy, 13 endometrioid adenocarcinomas, and 9 clear cell carcinomas. Allelic loss, recognized at the polymorphic site in codon 72 of the p53 gene, was detected in 14 of 36 (39%) informative cases by restriction fragment length polymorphism analysis and by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR)-amplified DNA fragments. Mutations in the highly conserved regions of the p53 gene were detected by SSCP analysis of PCR-amplified fragments. Mutations were found in 22 of 70 (31%) ovarian tumors, including 1 of 5 mucinous tumors of borderline malignancy. Mutations were subsequently characterized by direct sequencing. Single missense base substitutions were detected in 13 ovarian carcinomas and in one case of mucinous tumor of borderline malignancy. Short (1-8 bp) deletions and insertions were found in 8 cases. Mutations in the p53 gene occurred more frequently in serous adenocarcinomas (14/31, 45%) than in all nonserous types of malignant epithelial tumors combined (7/34, 21%; P = 0.032). Point mutations in K-ras were identified by dot blot hybridization analysis of PCR-amplified fragments with mutation-specific oligonucleotides and by direct sequencing. The overall frequency of K-ras mutations was 19/70 (27%). K-ras mutations were found in 12 of 17 (71%) mucinous tumors (8/12 mucinous carcinomas [67%] and 4/5 mucinous tumors of borderline malignancy [80%]), and occurred more frequently than in serous carcinomas (4/31, 13%; P = 0.00009) or in all nonmucinous types of ovarian epithelial tumors combined (7/53, 13%; P = 0.00002). These data suggest that different combinations of oncogenes and/or tumor suppressor genes may be involved in the genesis and development of histologically distinct categories of common epithelial tumors of the human ovary.

Immunohistochemical analysis of p53 in gynecologic tumors.

Immunohistochemical staining for the p53 protein was performed in microwave-fixed, paraffin-embedded sections of normal, premalignant and malignant tissues of the female genital tract using a monoclonal antibody, PAb 1801. No staining was detected in normal and premalignant tissues, whereas nuclear staining of cancer cells was observed in 12 (22%) of 55 cervical squamous cell carcinomas, 4 (25%) of 16 cervical adenocarcinomas, 37 (42%) of 88 endometrial carcinomas, 23 (38%) of 60 ovarian adenocarcinomas, and 6 (100%) of 6 squamous cell carcinomas arising in dermoid cysts. Of interest, 1 of 7 endometrial cancers with concomitant atypical hyperplasia showed weak nuclear staining in a few atypical hyperplastic glands in addition to the cancerous lesions. Although staining was associated with cancers having a high histologic grade and serous papillary adenocarcinomas of the endometrium, it did not correlate with invasion, metastasis, or clinical stage. Comparison of the staining patterns with molecular analysis of mutations in the p53 gene showed the expected correlation of nuclear staining with missense mutations but not with nonsense mutations, which consistuted one third of all mutations found in this series. In addition, cytoplasmic staining did not predict mutation.

Type-IV collagenase and tissue inhibitor of metalloproteinase in ovarian cancer tissues.

OBJECTIVE: We examined the specific expression of gelatinase/type-IV collagenase and tissue inhibitor of metalloproteinase (TIMP) in clinical ovarian cancer tissue. METHODS: Molecular weight-specific gelatinase/type-IV collagenase activity was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis in which substrate was included (zymography). The expression of TIMP mRNA was examined by Northern blot analysis. RESULTS: Zymography revealed that in ovarian cancer the activity of a 92-kDa gelatinase/type-IV collagenase was always greater than that of a 64-kDa gelatinase/type-IV collagenase in contrast to the situation in the normal ovary. Northern blot analysis revealed no remarkable difference of TIMP mRNA expression between cancer and normal ovarian tissues. CONCLUSIONS: These results indicate that the higher activity of the 92-kDa gelatinase/type-IV collagenase enzyme, relative to that of the 64-kDa enzyme, is involved in the malignant phenotype of ovarian cancer, while the inhibitor of these enzymes, TIMP, is distributed in a widespread fashion in the tissue, and its levels are not correlated with the malignancy.

Detection of high-risk human papillomavirus in the cervix and semen of sex partners.

To investigate the possibility of sexual transmission of human papillomavirus (HPV), 53 married couples were examined for the presence of HPV-16 and -18 DNAs in the uterine cervix and semen using the polymerase chain reaction method. Twenty-three of the 53 women and 12 of the 53 male partners were positive for HPV-16 DNA. No HPV-18 DNA was detected in samples from any of the partners. In 27 pairs, both partners were negative for HPV DNA in cervix or semen; in the remaining 26 pairs, at least 1 of the partners was HPV-16-positive. In 9 (35%) of these 26 pairs, both partners were infected. Furthermore, 9 (75%) of the 12 women with HPV-positive partners were HPV-positive, while 9 (39%) of the 23 men with HPV-positive female partners were HPV-positive. These findings suggest an increased risk of HPV transmission via sexual intercourse, thereby underscoring the importance of preventive care against HPV infection during intercourse.

Clonal analysis of human gynecologic cancers by means of the polymerase chain reaction.

Clonality of human gynecologic cancers was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of 1 of 2 X-chromosomes by methylation in females. Among 52 gynecologic cancers tested, 25 were found to be heterozygous for the BstXI polymorphism of the PGK gene. All the 25 gynecologic cancers (4 cervix, 11 endometrium, 7 ovary and 3 fallopian tube) analyzed by the PCR-based method were monoclonal in origin while adjacent normal tissues were polyclonal. When DNA samples were prepared from widely separated sites of tumors and/or metastatic lesions, every sample was found to be monoclonal, and the same allele of the PGK gene was inactivated in each case. These results demonstrate that clonal analysis by PCR offers a good method for studying clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign and malignant gynecologic lesions.

Clinicopathological characteristics of p53 overexpression in endometrial cancers.

Using immunohistochemical methods, we analyzed the association between nuclear p53 overexpression and various clinicopathological parameters in patients with endometrial cancers. Formalin-fixed and paraffin-embedded tissue sections from 139 cases of endometrial cancer (endometrioid type, 126; serous papillary type, 12; and clear-cell type, 1) were stained with anti-p53 monoclonal antibody (MAb) DO7. Overexpression of p53 was associated with high malignant potential, including extensive muscular invasion, advanced surgical stage, high histological grade, serous papillary type and a personal history of cancer. Lymph-node metastasis was also related to p53 overexpression with marginal significance. Survival curves determined by the Kaplan-Meier method and univariate analysis showed p53 overexpression to be associated with a poor outcome in endometrial cancer patients. However, multivariate analysis using the stepwise Cox proportional-hazard model showed that whereas lymph-node metastasis, a personal history of cancer and muscular invasion were related to poor survival rates, p53 overexpression was not. Consequently, p53 overexpression itself does not appear to be an independent prognostic factor in endometrial cancer, although a still larger sample of patient material would be required to assess this issue definitively.

Establishment and characterization of human vaginal malignant melanoma xenotransplants.

Human vaginal malignant melanoma represents rare gynecological malignancies of poor prognosis. We have established a melanoma tumor line in nude mice, designated Mela-1, and have examined the histological and biological characteristics of this tumor. The Mela-1 tumor has preserved the histological, histochemical and biological characteristics of malignant melanoma even after 20 passages. Tumor cells are of epitheloid shape varying in size. An ultrastructural study revealed that the tumor cells were characterized by the presence of cells with deeply indented nuclei, and both types of melanosomes, eumelanosomes and pheomelanosomes, in various stages of maturation with vesiculo-globular bodies in the cytoplasm. Melanin analysis of the tumor indicated the Mela-1 tumor to be pheomelanic. Immunohistochemical examinations revealed that the Mela-1 cells were stained positively by melanoma-associated antibody (NKI/C3) and by antibodies for S-100 protein and vimentin, and negatively for keratin and CEA. The levels of AFP, CA125 and CEA in sera of tumor-bearing mice were within normal range. The 5-S-cysteinyldopa level in sera of tumor-bearing mice correlated well with the size of the tumor. Chromosomal analysis showed the human karyotype with great heterogeneity and a modal number of 102 chromosomes. Thus the Mela-1 tumor will be useful in establishing the biological characteristics in the search for an effective treatment of human malignant melanoma of the vagina.

Loss of biological activity of human chorionic gonadotropin (hCG) by the amino acid substitution on the "CMGCC" region of the alpha-subunit.

In order to study the bioactive sites of the glycoprotein hormones, we have prepared five point mutants on the CMGCC (Cys28-Met29-Gly30-Cys31-Cys32) region of the human alpha-subunit by using site-directed mutagenesis. Each mutant human chorionic gonadotropin (hCG) agr; cDNA and a wild-type hCG beta cDNA were transcribed by T3 RNA polymerase, and the mixture of the hCG alpha mRNA and hCG beta mRNA was microinjected into Xenopus laevis oocytes. All five mutant hCGs produced in oocyte culture supernatants were detected as immunoreactive forms by enzyme immunoassay. In contrast, four mutants (Cys28-->Tyr28, Gly30-->Arg30, Ala30, Asp30) were devoid of biological activity in vitro bioassay using the production of testosterone with mouse Leydig cells. These results indicate that the CMGCC region in the alpha-subunit, particularly the cysteine residue at position 28 and the glycine residue at position 30, plays an important role in the biosynthesis of glycoprotein hormones.

Regulation of early gene expression of human papillomavirus type 16 by inflammatory cytokines.

The host's immune reaction against human papillomavirus (HPV) infection remains poorly understood. Inflammatory cytokines undoubtedly play a key role through activating and coordinating the immune response. However, their direct interactions with the HPV genes remain unclear. In the present study, the effects of various inflammatory cytokines on HPV16 gene expression were investigated. In a CAT assay, tumor necrosis factor (TNF) alpha and interleukin-1 (IL-1) alpha were shown to repress HPV16 early gene expression at the transcriptional level through the noncoding region (NCR), whereas IL-6 and interferon-gamma did not. In Northern blot analysis, TNF and IL-1 were also shown to repress HPV16 E6/E7 mRNA expression in the HPV16-immortalized human keratinocyte cell line. The TNF- and IL-1-responsive elements in the HPV16 NCR were determined to lie within the cell-type-specific enhancer, where there are several binding sites for nuclear factors involved in HPV16 early gene regulation, suggesting the participation of these factors in TNF and IL-1 regulations. Thus, TNF and IL-1 were shown to have antiviral effects on HPV through down-regulation of its gene transcription. This is the first demonstration that TNF and IL-1 are involved in HPV gene regulation. These functions of inflammatory cytokines are presumed to contribute to the host's defense against HPV infection.