Nomenclature of primary amenorrhea: A proposal document of the Japan Society of Obstetrics and Gynecology committee for the redefinition of primary amenorrhea.

AIM: The aim of this study was to provide medical terms to describe the condition of a girl who should be evaluated for primary amenorrhea in order to facilitate intervention at an appropriate time. METHODS: We performed a literature and clinical guidelines search for recent practices with regard to menarche and discussed relevant cases that had been experienced by committee members. Additionally, we theoretically reviewed medical terms defined in the Glossary Book of Obstetrics and Gynecology in Japan (Japan Society of Obstetrics and Gynecology, 3rd edition). RESULTS: The committee for the redefinition of primary amenorrhea proposed the introduction of two terms and the deletion of one term that had been defined by the Japan Society of Obstetrics and Gynecology, instead of changing the age definition of primary amenorrhea. 'Delayed menarche' was introduced to describe a condition in which a girl has never experienced cyclic menstruation (menarche) by 15-17 years of age. 'Late menarche' was also introduced to describe a condition in which a girl has experienced menarche at 15 years of age or older. 'Delayed menstruation,' which was defined as a condition in which a girl experiences menarche at 15-18 years of age, was deleted. CONCLUSION: The new terms 'delayed menarche' and 'late menarche' were introduced, and the term 'delayed menstruation' was deleted. The new system might help in the early detection and appropriate treatment of primary amenorrhea.

The education of traditional Japanese (Kampo) medicine: surveys of training hospitals and residents.

BACKGROUND: Japanese physicians prescribe Kampo medicine, but Kampo education is not standardized. We surveyed training hospitals and residents to identify problems and suggest solutions to promote Kampo education during and after residency. METHODS: This was a double questionnaire survey of 1011 training hospitals in Japan and 93 Tokai University School of Medicine graduates of 2011. RESULTS: There were 816 effective responses (81%) from the training hospitals. Most instructors (84%) thought physicians should have Kampo clinical skills; 67% thought positively about introducing Kampo education into clinical training; 23% of the hospitals provided Kampo education; 70% of instructors at hospitals without Kampo education indicated the lack of Kampo instructors, 16% lack of time, and 7% no necessity for Kampo education; hospitals permitted Kampo education through voluntary study (42%), lectures sponsored by Kampo manufacturers (35%), and study sessions with other hospitals (32%); independent study sessions (10%); smaller hospitals were less active in Kampo education than larger ones. The survey of residents had 72 effective responses (77%): 91% were interested in Kampo medicine; 96% thought it worth learning; 31% could learn it during residency; 52% were not satisfied with the training, 83% wanted to learn it; 73% thought it should be introduced into the curricula; 93% prescribed Kampo medicine, and residents who learned it prescribed it more; 48% were reluctant to prescribe it after residency; 89% thought Western and Kampo medicine should be integrated. CONCLUSIONS: Instructors knew Kampo education was needed, but little of it was taught, especially in small hospitals, because of the lack of Kampo instructors. Residents recognized the need for Kampo medicine and were motivated to learn it. Kampo medicine was mostly prescribed because instructors suggested it. Because of the limited opportunities to learn Kampo medicine, it should be taught during residency. In small hospitals, cooperation with other hospitals could be a solution to teach Kampo medicine.

Detection of human herpesviruses (HHVs) in semen of human male infertile patients.

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21-49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.

Accumulated HSV1-TK proteins interfere with spermatogenesis through a disruption of the integrity of Sertoli-germ cell junctions.

Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoli-germ cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed.

Current status of Kampo medicine curricula in all Japanese medical schools.

BACKGROUND: There have been a few but not precise surveys of the current status of traditional Japanese Kampo education at medical schools in Japan. Our aim was to identify problems and suggest solutions for a standardized Kampo educational model for all medical schools throughout Japan. METHODS: We surveyed all 80 medical schools in Japan regarding eight items related to teaching or studying Kampo medicine: (1) the number of class meetings, target school year(s), and type of classes; (2) presence or absence of full-time instructors; (3) curricula contents; (4) textbooks in use; (5) desire for standardized textbooks; (6) faculty development programmes; (7) course contents; and (8) problems to be solved to promote Kampo education. We conducted descriptive analyses without statistics. RESULTS: Eighty questionnaires were collected (100%). (1) There were 0 to 25 Kampo class meetings during the 6 years of medical school. At least one Kampo class was conducted at 98% of the schools, ≥4 at 84%, ≥8 at 44%, and ≥16 at 5%. Distribution of classes was 19% and 57% for third- and fourth-year students, respectively. (2) Only 29% of schools employed full-time Kampo medicine instructors. (3) Medicine was taught on the basis of traditional Japanese Kampo medicine by 81% of the schools, Chinese medicine by 19%, and Western medicine by 20%. (4) Textbooks were used by 24%. (5) Seventy-four percent considered using standardized textbooks. (6) Thirty-three percent provided faculty development programmes. (7) Regarding course contents, "characteristics" was selected by 94%, "basic concepts" by 84%, and evidence-based medicine by 64%. (8) Among the problems to be solved promptly, curriculum standardization was selected by 63%, preparation of simple textbooks by 51%, and fostering instructors responsible for Kampo education by 65%. CONCLUSIONS: Japanese medical schools only offer students a short time to study Kampo medicine, and the impetus to include Kampo medicine in their curricula varies among schools. Future Kampo education at medical schools requires solving several problems, including curriculum standardization.

Failure to detect significant association between estrogen receptor-alpha gene polymorphisms and endometriosis in Japanese women.

OBJECTIVES: The aim of the study was to test whether estrogen receptor 1 (ESR1) gene polymorphisms are correlated with the risk of the development of endometriosis in Japanese women, as a preliminary study. METHODS: To compare allelic frequencies and genotype distributions, a case-control study of 100 affected women and 143 women with no evidence of disease was performed using 10 microsatellite repeat markers and 66 single-nucleotide polymorphisms (SNPs) in the ESR1 gene region. RESULTS: Although our results might be insufficient to detect genetic susceptibility, owing to the small sample size and low genetic power, statistical analysis of the differences in allelic frequency between the cases and controls at each microsatellite locus demonstrated that no microsatellite locus in the ESR1 gene displayed a significant association with the disease when multiple testing was taken into account. Also, there were no statistically significant differences in the SNP allele frequencies and genotypes between the cases and controls when multiple testing was taken into account. CONCLUSION: The findings in our pilot study suggest that ESR1 polymorphisms do not contribute to endometriosis susceptibility.

Lack of an association human dioxin detoxification gene polymorphisms with endometriosis in Japanese women: results of a pilot study.

OBJECTIVES: Endometriosis is a chronic disease caused by the presence of endometrial tissue in ectopic locations outside the uterus. Chronic exposure to the environmental pollutant dioxin has been correlated with an increased incidence in the development of endometriosis in non-human primates. We have therefore examined whether there is an association between the polymorphisms of ten dioxin detoxification genes and endometriosis in Japanese women. METHODS: This was a pilot study in which 100 patients with endometriosis and 143 controls were enrolled. The prevalence of five microsatellite and 28 single nucleotide polymorphism markers within ten dioxin detoxification genes (AhR, AHRR, ARNT, CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, GSTT1, NAT2) was examined. RESULTS: Taking into account that this analysis was a preliminary study due to its small sample size and genetic power, the results did not show any statistically significant difference between the cases and controls for any of the allele and genotype frequency distributions examined. In addition, no significant associations between the allele/genotype of all polymorphisms and the stage (I-II or III-IV) of endometriosis were observed. CONCLUSION: Based on the findings of this pilot study, we conclude the polymorphisms of the ten dioxin detoxification genes analyzed did not contribute to the etiology of endometriosis among our patients.

High-progesterone environment preserves T cell competency by evading glucocorticoid effects on immune regulation.

Progesterone (P4) and glucocorticoid (GC) play crucial roles in the immunoregulation of a mother to accept and maintain a semi-allogenic fetus. P4 concentration increases during pregnancy and becomes much higher in the placenta than in the other peripheral tissues, wherein the concentration of cortisol (COR), the most abundant GC and a strong immunosuppressor, remains uniform throughout the rest of the body. Here, we evaluated the effect of a high-P4 environment on pregnant immunity by comparing it with COR. Naïve T cell proportion increased transiently in peripheral blood of pregnant women just after delivery and decreased after one month. T cells stimulated with superantigen toxic-shock-syndrome-1 (TSST-1) in the presence of P4 stayed in the naïve state and did not increase, irrespective of the presence of COR, and reactive T cells could not survive. Treatment of T cells with P4 without T cell receptor (TCR) stimulation transiently suppressed T cell activation and proliferation, whereas the levels remain unaltered if P4 was not given before stimulation. Comparison of the engraftment and response against specific antigens using hu-PBL-NOG-hIL-4-Tg mice showed that P4-pretreated lymphocytes preserved CD62L expression and engrafted effectively in the spleen. Moreover, they produced antigen-specific antibodies, whereas COR-pretreated lymphocytes did not. These results suggest that a high-P4 environment suppresses T cell activation and induces T cell migration into lymphoid tissues, where they maintain the ability to produce anti-pathogen antibodies, whereas COR does not preserve T cell function. The mechanism may be pivotal in maintaining non-fetus-specific T cell function in pregnancy.

Epigenetic assessment of environmental chemicals detected in maternal peripheral and cord blood samples.

Epigenetic alteration is an emerging paradigm underlying the long-term effects of chemicals on gene functions. Various chemicals, including organophosphate insecticides and heavy metals, have been detected in the human fetal environment. Epigenetics by DNA methylation and histone modifications, through dynamic chromatin remodeling, is a mechanism for genome stability and gene functions. To investigate whether such environmental chemicals may cause epigenetic alterations, we studied the effects of selected chemicals on morphological changes in heterochromatin and DNA methylation status in mouse ES cells (ESCs). Twenty-five chemicals, including organophosphate insecticides, heavy metals and their metabolites, were assessed for their effect on the epigenetic status of mouse ESCs by monitoring heterochromatin stained with 4¢,6-diamino-2-phenylindole (DAPI). The cells were surveyed after 48 or 96 h of exposure to the chemicals at the serum concentrations of cord blood. The candidates for epigenetic mutagens were examined for the effect on DNA methylation at genic regions. Of the 25 chemicals, five chemicals (diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se) and octachlorodipropyl ether (S-421)) caused alterations in nuclear staining, suggesting that they affected heterochromatin conditions. Hg and Se caused aberrant DNA methylation at gene loci. Furthermore, DEP at 0.1 ppb caused irreversible heterochromatin changes in ESCs, and DEP-, Hg- and S-421-exposed cells also exhibited impaired formation of the embryoid body (EB), which is an in vitro model for early embryos. We established a system for assessment of epigenetic mutagens. We identified environmental chemicals that could have effects on the human fetus epigenetic status.

An immunohistochemical study of metroplastic surgical specimens from patients with a septate uterus.

Purpose: To elucidate the etiology of recurrent pregnancy loss in patients with congenital uterine anomalies, an immunohistochemical technique was used to quantitatively evaluate the vascular arrangement of septate uteri with respect to vascular density and morphology. Methods: Nine specimens obtained from patients who had undergone metroplastic surgery for the treatment of a septate uterus and 10 control specimens from patients who had undergone a hysterectomy because of cervical carcinoma were used in this study. Formalin-fixed paraffin-embedded uterine specimens were then immunostained for CD34, which is specifically expressed in vascular endothelial cells. Results: The mean blood vessel count (mean ± SD) for the myometrium was 149.7 ± 22.7/field in the septate uteri and 162.2 ± 36.4/field in the control uteri; these values were not significantly different. However, the total vessel cross-sectional areas, as evaluated quantitatively using the KS400 image analysis system, were 10350.4 ± 1024.3 µm2/field for the septate uteri and 12002.9 ± 2232.3 µm2/field for the control uteri; these values were significantly different (p < 0.05). The vessel morphology expressed by vessel irregularity and deformity showed a characteristic change in the septate uterus. Conclusions: A significant difference in the distribution of the blood vessels existed between the septate and control uteri, presumably impairing blood flow in the myometrium and the adverse pregnancy outcome.

Tips and Techniques for Laparoscopic Tubal Reanastomosis: A Case Report.

Tubal reanastomosis or tubal reversal, a surgical method used to reverse tubal sterilization, may be an option for women who for various reasons wish to reestablish their fertility. A 38-year-old Chinese woman, gravida 2, para 2 (both delivered through cesarean section) presented to our outpatient gynecology clinic requesting bilateral tubal recanalization. After other causes of infertility were excluded, laparoscopic tubal reanastomosis was performed. Here, we present our tips and techniques for laparoscopic tubal reanastomosis that rapidly resulted in an intrauterine pregnancy, which delivered at term. Laparoscopic tubal reanastomosis is a well-established procedure with good prognosis, as reported in the literature. For women who wish to become pregnant after tubal sterilization, it is necessary to present the option of surgery as well as in vitro fertilization.

Determination of phthalates in diet and bedding for experimental animals using gas chromatography-mass spectrometry.

We have developed a gas chromatography-mass spectrometry method to measure five phthalates (dibutyl phthalate, butylbenzyl phthalate, di-2-ethylhexyl phthalate, diisooctyl phthalate, and diisononyl phthalate) in diets and beddings for experimental animals. The recoveries from diets and beddings spiked with five phthalates were 98.8%-148% with coefficients of variation of 0.4%-7.8% for diets and 94.7%-146% with coefficients of variation of 1.0%-5.0% for beddings. We analyzed commercial animal diets and beddings, and found that the levels of phthalates varied from sample to sample; the concentrations of five phthalates were 141-1,410 ng/g for diets and 20.5-7,560 ng/g for beddings.

Determination of five phthalate monoesters in human urine using gas chromatography-mass spectrometry.

We have developed a gas chromatography-mass spectrometry (GC-MS) method to determine five phthalate monoesters (monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-(2-ethylhexyl) phthalate (MEHP), monoisononyl phthalate (MINP) and monobenzyl phthalate (MBz)) in human urine. Human urine samples were subjected to enzymatic deconjugation of the glucuronides followed by extraction with hexane. The extracted phthalate monoesters were methylated with diazomethane, purified on a Florisil column and then subjected to GC-MS analysis. The recoveries from urine spiked with five phthalate monoesters were 86.3%-119% with coefficients of variation of 0.6%-6.1%. We measured phthalate monoester levels in human urine by analyzing 36 samples from volunteers. MBP and MEP were detected in all samples, and their median concentrations were 60.0 and 10.7 ng/mL, respectively. MBzP and MEHP were found in 75% and 56% of samples, and their median concentrations were 10.9 and 5.75 ng/mL, respectively. MINPs were not detected in most samples (6% detectable). Women had significantly (p < 0.05) higher mean concentrations of MBP and MEP than men. The estimated daily exposure levels for the four parent phthalates excluding diisononyl phthalate ranged from 0.27 to 5.69 mug/kg/day (median).

HSV type 1 thymidine kinase protein accumulation in round spermatids induces male infertility by spermatogenesis disruption and apoptotic loss of germ cells.

HSV type 1 thymidine kinase (HSV1-TK)-introduced transgenic rodents and HSV-infected humans were reported to suffer male infertility. The present study aimed to find novel clues to clarify the cause of HSV1-TK-induced male infertility using an HSV1-tk transgenic rat line. Two truncated HSV1-TK proteins, 37 and 39kDa, were produced and accumulated in the round spermatids, and their transcription initiation site was identified for the first time at the 65 base downstream of the translation start point of the full-length 43kDa HSV1-TK. Spermatozoa from those young transgenic rats showed malformed heads, looped tails, and missing cell membrane in heads and tails. Furthermore, age-dependent germ cell loss was observed. TUNEL assay suggested that this germ cell loss is caused by increased apoptotic germ cell death. These results suggest that the expression of HSV1-TK in testes brings about not only abnormal spermiogenesis but also a loss of germ cells due to apoptosis. These findings could provide a novel clue to elucidate the molecular mechanism underlying male infertility in transgenic animals and HSV-infected patients.

The antigenic binding sites of autoantibodies to factor XII in patients with recurrent pregnancy losses.

Recently, numerous studies have suggested an association between factor XII (FXII) deficiency and recurrent pregnancy losses, and between autoantibodies to FXII and recurrent pregnancy losses. Autoantibodies to FXII rather than FXII deficiency may be a risk factor for recurrent pregnancy losses. To know the pathogenesis of autoantibodies to FXII, epitope mapping study was done. Seventeen anti-FXII antibody positive recurrent pregnancy loss patients were chosen for this study. We used synthetic peptides in inhibition and direct binding studies to identify the antigenic binding site of autoantibodies to FXII. Among plasmas from 17 recurrent pregnancy loss patients who were positive for autoantibodies to FXII, 13 patients (76.5%) recognized amino acids 1-30, the amino-terminal heavy chain region that is known as factor XII binding site to platelet glycoprotein Ibalpha.

Spontaneous platelet aggregation in normal subject assessed by a laser light scattering method: an attempt at standardization.

Platelet aggregometry by the laser light scattering (LS) method is sufficiently sensitive to detect small platelet aggregates that form spontaneously in vitro in the absence of agonists. Platelet aggregation without agonists is named spontaneous platelet aggregation (SPA). Since SPA has been suggested to be associated with various thrombotic diseases, it is essential to measure SPA and to establish a standard range of SPA values. In this study, we measured SPA in 167 healthy subjects by the LS method and attempted to clarify various factors influencing SPA, including the blood collection procedure. We also attempted to establish a tentative standard range of SPA values. SPA was quantitatively measured in terms of the maximum total LS intensity, which reflects small aggregates formed over 10 minutes (SMAX) and the area under the total LS intensity curve of small aggregates (SAUC). Since both the values of SMAX and SAUC were skewed and the log SMAX and log AUC values showed a normal distribution, the statistical analyses were performed using log SMAX and log SAUC. The log SMAX and log SAUC were significantly higher in the samples collected using a tourniquet and/or a 21 G needle, than in those collected without a tourniquet and/or with an 18 G needle. The log SAUC values were significantly lower in samples obtained with a syringe and/or 3.8% sodium citrate than in those obtained in vacuum sampling tubes and/or 3.13% or 3.14% sodium citrate. The Ht and plasma glucose concentration influenced the log SMAX values. We propose that to standardize SPA measurements, the measurements should be completed within two hours of blood sample collection and collected using the regular concentration of citrate. The standard range of SMAX values measured in samples obtained using a tourniquet and a 21 G needle was 2.0-23.99 (*10(3) mV*count). The standard range of SAUC values measured under same conditions was 0.58-9.12 (*10(6) mV*count*min). The standard range of SMAX values measured in samples obtained using a tourniquet, 21 G needle and a vacuum tube was 1.7-29.51 (*10(3) mV*count). The standard range of SAUC values measured under same conditions was 0.59-9.33 (*10(6) mV*count*min).

Simulated neonatal exposure to DEHP and MEHP from PVC enteral nutrition products.

The leaching of di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) from medical products made of polyvinyl-chloride (PVC) to enteral nutrition (EN) for neonatal patients was determined in a simulated study. The study simulated a typical case of EN administration to a neonatal patient (body weight, 3 kg) in a neonatal care unit (temperature, 25 degrees C); the medical products used were an irrigator and catheter containing DEHP (9.1-31.8%, w/w) as a plasticizer. The worst-case daily exposures of the neonatal patient to DEHP and MEHP by the administration of EN were estimated to be 148 and 3.72 microg/(kg day), respectively, as assessed from the levels of these compounds leaching from the medical products to the EN. The use of DEHP-free medical products reduced the exposure of DEHP and MEHP to the minimum levels contained in the EN at preparation. A transition to DEHP-free medical products for neonatal patients would be effective in reducing the exposure of neonatal patients to DEHP via EN administration.

Exome and copy number variation analyses of Mayer-Rokitansky-Küster- Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is characterized by congenital absence of the vagina and uterus. We conducted genome-wide SNP analyses and exome sequencing to detect the causes of MRKH syndrome. We identified de novo variants of MYCBP2, NAV3, and PTPN3 in three families and a variant of MYCBP2 in a sporadic case. Here, we demonstrated the partial genetic makeup of Japanese MRKH syndrome.

Presence of human herpes virus 1-thymidine kinase in testis of azoospermic infertile herpes-infected patients.

Infection with human herpes virus 1 (HHV1) is a suspected cause of human male infertility. However, the correlation between HHV1 infection and infertility is still unclear. We have previously generated transgenic rats that ectopically express the HHV1 thymidine kinase gene (HHV1-TK) in post-meiotic spermatids and found they had aberrant spermatogenesis and infertility. Therefore, we hypothesized that human infertility might be caused by HHV1 infection. Here, we examined whether HHV1-TK is expressed in human testis by analyzing the presence of its transcript and protein. Specimens were collected by biopsy from 30 azoospermic infertile male patients. RT-PCR and immunohistochemistry showed that 23 patients were positive for HHV1-TK expression, while seven patients were negative. Thus, we demonstrated HHV1-TK expression, indicating HHV1 infection, in the testis of human azoospermic infertile males for the first time; our findings represent a great advancement toward the verification of our hypothesis that HHV1-TK expression might cause human infertility.

Identification of PRTFDC1 silencing and aberrant promoter methylation of GPR150, ITGA8 and HOXD11 in ovarian cancers.

Methylated promoter CpG islands (CGIs) can be used to find novel tumor-suppressor genes and disease markers. In this study, to identify promoter CGIs aberrantly methylated in human ovarian cancers, we performed a genome-wide screening for differentially methylated DNA fragments using methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 185 DNA fragments specifically methylated in an ovarian cancer cell line (ES-2), compared with a normal human ovarian surface epithelial cell line (HOSE6-3), and 33 of them were derived from putative promoter CGIs. Ten ovarian cancer cell lines were analyzed by methylation-specific PCR, and seven (GPR150, LOC222171, PRTFDC1, LOC339210, ITGA8, C9orf64 and HOXD11) of the 33 CGIs were methylated in one or more of the cell lines. Their downstream genes were barely expressed in cell lines without unmethylated DNA molecules by quantitative reverse-transcription-PCR. Demethylation of methylated cell lines with 5-aza-2'-deoxycytidine restored expression of two genes (PRTFDC1 and C9orf64). In primary ovarian cancers, CGIs of GPR150 (in 4 of 15 cancers), ITGA8 (2/15), PRTFDC1 (1/15), and HOXD11 (1/15) were methylated. Silencing of PRTFDC1 was revealed here for the first time, and aberrant methylation of GPR150, ITGA8 and HOXD11 could be candidate tumor markers.