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Research question The recently developed in-vitro activation (IVA) approach provides a promising infertility treatment for patients with premature ovarian insufficiency. The IVA method promotes growth of residual ovarian follicles following ovarian tissue fragmentation leading to Hippo signalling disruption, together with in-vitro incubation with Akt stimulators. As poor ovarian response (POR) patients with decreased ovarian reserve (DOR) have multiple secondary follicles, this study tested whether Hippo signalling disruption alone using in-vitro ovarian cortical fragmentation, followed by autologous grafting, was sufficient to promote follicle growth. Design A case series study. Results In 9 out of 11 POR patients with DOR treated with a simplified IVA procedure, increases in antral follicle numbers in multiple growth waves were detected following FSH treatment. Subsequent injection with human chorionic gonadotrophin allowed retrieval of more mature oocytes for IVF (median antral follicle counts before and after IVA per ovarian stimulation: 1.0 versus 2.6) with 68.7% fertilization rates and 56.9% showing high-quality embryonic development. One natural conception and 16 embryo transfers in five patients resulted in one live birth, two ongoing pregnancies and one miscarriage. Three additional patients and the miscarriage patient have cryopreserved embryos for future transfer. Conclusions The present drug-free IVA approach may be suitable for POR patients with DOR, as it increased the number of antral follicles. The procedure also eliminated the need for 2-day incubation with drugs and required only one surgery. This approach could allow the retrieval of more oocytes in middle-aged women to achieve higher pregnancy rates and deserves proper evaluation in future randomized controlled trials.

Premature ovarian insufficiency (POI) occurs in at least 1% of all women and causes life-long health problems and psychological stress. Infertility caused by POI used to be considered absolute, with infertility treatment having little or no value. Generally, it has been thought that medicine can provide little service to these patients. The etiology of POI has been found to be genetic, chromosomal, and autoimmune. In addition, the increasing numbers of cancer survivors are candidates for iatrogenic POI, along with patients who have undergone ovarian surgery, especially laparoscopic surgery. Over 50 genes are known to be causally related to POI, and the disease course of some cases has been clarified, but in most cases, the genetic background remains unexplained, suggesting that more genes associated with the etiology of POI need to be discovered. Thus, in most cases, the genetic background of POI has not been clarified. Monosomy X is well known to manifest as Turner’s syndrome and is associated with primary amenorrhea, but recent studies have shown that some women with numerical abnormalities of the X chromosome can have spontaneous menstruation up to their twenties and thirties, and some even conceive. Hormone replacement therapy (HRT) is recommended for women with POI from many perspectives. It alleviates vasomotor and genitourinary symptoms and prevents bone loss and cardiovascular disease. POI has been reported to reduce quality of life and life expectancy, and HRT may help improve both. Most of the problems that may occur with HRT in postmenopausal women do not apply to women with POI; thus, in POI, HRT should be considered physiological replacement of estrogen (+progesterone). This review describes some new approaches to infertility treatment in POI patients that may lead to new treatments for POI, along with the development of more sensitive markers of secondary/preantral follicles and genetic diagnosis.

The thiazine dye toluidine blue (TB) is well known to stain mast cells and hyaline cartilage metachromatically, and thus is mostly often used for their identification. However, TB is not suitable for counterstaining in immunohistochemistry, because of its high-background staining in the cytoplasm of other cell species and in extracellular structures. To expand the knowledge about dyestuffs staining mast cells in consideration with their usage in immunohistochemistry, we determined the stainability of several thiazines and oxazines, which are structurally related compounds to TB, using sections of mast cell-containing tissues. We found that all azine dyes used metachromatically stained mast cells and cartilage. Among these dyes, an oxazines cresyl violet (CV) stained mast cells with lower background, suggesting that those are useful for detecting mast cells and for counterstaining in immunohistochemistry. To ascertain its utility, CV was used in immunostaining of bHSDs in sections from adult rat ovary. Immunopositive signals reflected by DAB development in brown were clearly detected even after CV staining. We conclude that, similar to thiazines, oxazines stain mast cells metachromatically, and that of these, CV is more useful as a counterstain in immunohistochemistry than TB.

Premature ovarian insufficiency (POI) is a clinical syndrome defined by the loss of ovarian activity before 40 years old. An autoimmune mechanism is suggested to be involved in the development of POI. Therefore, we examined the relationship between peripheral blood regulatory T (Treg) cells and autoantibodies in POI.Thirty POI patients and 23 control women were enrolled in the study. Using flow cytometry, we measured the abundance of CD4The number of CD4This is the first report of decreased numbers of effector Treg cells and increased CD4

Aim The aim of this study was to investigate whether the consecutive administration of recombinant thrombomodulin (r-TM) for 4 days improves maternal and fetal conditions and physiological outcomes in an N′-nitro-L-arginine-methyl ester hydrochloride-induced and low-dose endotoxin-induced pre-eclampsia (PE). Methods r-TM or saline was administrated i.v. to normal pregnant and experimental PE rats for 4 days. The maternal condition, vascular endothelial growth factor receptor-1 (VEGFR-1), fetal conditions, uteroplacental blood flow (UPBF), and oxygenation in the placenta and fetal brain was evaluated on gestational day 21. Results Significant increases in the mean arterial blood pressure, VEGFR-1 values and fetal death rate were observed in PE rats compared with control rats, while maternal and fetal bodyweight and fetal brain weight were substantially lower. Hypoperfusion and hypo-oxygenation in both the placenta and fetal brain tissues occurred in PE rats. Although r-TM failed to improve hypertension and affect the differences in maternal bodyweight between the groups, r-TM significantly improved hypoperfusion and fetal and maternal conditions, including VEGFR-1 values (6.5 ± 4.0 vs 2.2 ± 2.7 ng/mL, PE vs PE with r-TM, respectively; P

Successful development of preimplantation embryos is essential for reproduction. Growth factors secreted by reproductive tracts are important for the development of preimplantation embryos. FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase receptor related to colony-stimulating factor-1 receptor and c-KIT, promoting preimplantation embryo development following their ligand binding. We found expression of FLT3 ligand transcripts in the oviducts and uteri of pregnant mice. The transcripts for its receptor, FLT3, were detectable throughout the early embryonic stages with an increase after the eight-cell stage. In contrast, the expression of FLT3 ligand was negligible after the morula stage, suggesting potential paracrine actions of FLT3 ligand produced by the reproductive tracts. Treatment with FLT3 ligand promoted the development of two-cell embryos to the morula and blastocyst stages in a dose-dependent manner with increases in cell proliferation, but not inhibition of cell survival. The eff...

Exposure to 3,3'-iminodipropionitrile (IDPN) causes persistent neurotoxicity, while its reproductive toxicity in female rats is transient, indicating that gonadotropin-releasing hormone (GnRH) neurons and gonadotrophs receive little or no damage from IDPN and that the transient gonadal toxicity may be also observed in males. To clarify these points, the acute toxic effects of IDPN on hypothalamic-pituitary-gonadal axis of male rats were examined histologically, biochemically and serologically. A single intraperitoneal injection of IDPN (1000 mg/kg body weight) induced signs of neurotoxicity within a day; nevertheless, GnRH neurons were not affected throughout the experimental period. Four days after IDPN treatment, the plasma level of testosterone but not gonadotropins decreased and active caspase 3-immunopositive spermatids increased; both parameters returned to normal levels afterwards. Data from our studies revealed that while IDPN had little or no toxic effect on GnRH neurons or gonadotrophs it was transiently toxic to gonads in both sexes.

Primary ovarian insufficiency (POI) and polycystic ovarian syndrome are ovarian diseases causing infertility. Although there is no effective treatment for POI, therapies for polycystic ovarian syndrome include ovarian wedge resection or laser drilling to induce follicle growth. Underlying mechanisms for these disruptive procedures are unclear. Here, we explored the role of the conserved Hippo signaling pathway that serves to maintain optimal size across organs and species. We found that fragmentation of murine ovaries promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth, and the generation of mature oocytes. In addition to elucidating mechanisms underlying follicle growth elicited by ovarian damage, we further demonstrated additive follicle growth when ovarian fragmentation was combined with Akt stimulator treatments. We then extended results to treatment of infertility in POI patients via disruption of Hippo signaling by fragmenting ovaries followed by Akt stimulator treatment and autografting. We successfully promoted follicle growth, retrieved mature oocytes, and performed in vitro fertilization. Following embryo transfer, a healthy baby was delivered. The ovarian fragmentation-in vitro activation approach is not only valuable for treating infertility of POI patients but could also be useful for middle-aged infertile women, cancer patients undergoing sterilizing treatments, and other conditions of diminished ovarian reserve.

Sirtuins, a family of NAD+-dependent protein deacetylases, regulate important physiological events including aging and cell metabolism, mainly by protecting cells/tissues from oxidative damage. Ovarian aging decreases the quality of oocytes through induction of mitochondrial dysfunction and increases in DNA strand breaks by accumulation of reactive oxygen species. However, involvement of sirtuins in regulating oocyte quality with aging has not been determined. Here, we found the expression of sirtuin genes (Sirt1–7) in mouse ovaries and isolated oocytes and cumulus cells in a cell-specific manner. Based on real-time RT-PCR, all seven sirtuin genes were detected in the ovary with Sirt2 transcript levels showing the highest abundance. Oocyte expressed high levels of Sirt6, whereas the expressions of Sirt1, Sirt2, Sirt4, and Sirt6 were high in cumulus cells. When comparing samples from young and aged mice, oocyte levels of Sirt1–7 mRNA were not different. However, Sirt2 and Sirt6 transcript levels ...

Background Ovarian tissue cryopreservation by rapid cooling (vitrification) is a convenient fertility preservation option. However, the progress of vitrified ovarian tissue after transplantation is not well understood in primates. Methods For tissues from cynomolgus monkeys, we used closed straw vitrification and open cryosupport vitrification in which tissues are immersed directly into liquid nitrogen. Following warming, ovarian cortical pieces were autotransplanted and their function was monitored by computed tomography (CT), hormone assays and oocyte recovery, ICSI and embryo transfers (ETs). Results Hormone cycles were restored in 6 of 7 animals in a mean of 126 days with no significant difference between the two vitrification regimens. The presence of new blood vessels supplying the grafted ovarian tissue was confirmed by contrast-enhanced CT. Oocyte retrieval from two monkeys after transplantation of the ovarian cortex vitrified by cryosupport vitrification yielded a total of nine oocytes of which six fertilized after ICSI, but ETs did not lead to any pregnancies. Conclusions This work shows that CT can give insight into ovarian function after heterotopic transplantation, and that heterotopic autografts of vitrified ovarian cortex can give rise to long-term ovarian function and embryos in a primate model. It remains to be established how outcomes following rapid vitrification compared with outcomes following conventional slow cooling procedures.

Heat shock protein 27 (hsp27) is expressed by squamous cell carcinoma of the uterine cervix. Results from an earlier study by our group indicted that hsp27 may be a diagnostic marker for cervical intraepithelial neoplasia (CIN) and carcinoma. p16 expression is known to be elevated in intraepithelial uterine cervical cancer and grades 2 and 3 lesions (CIN2, CIN3), but has also been reported to be negative in 5-20% of cervical cancer and CIN lesions. The aim of our study was to confirm immunohistochemically the expression of hsp27 and p16 in cervical lesions. Formalin-fixed, paraffin-embedded cervical tissue specimens obtained between 2002 and 2010 were investigated for hsp27 and p16 expression. Positive staining was detected for hsp27 in 63% of normal cervical tissues, 47% of CIN1 lesions, 75% of CIN2 lesions, 92% of CIN3 lesions, and 100% of squamous cell carcinomas (SCC); the corresponding rates for p16 positivity were 29, 47, 67, 92, and 75%, respectively. Positive staining for both hsp27 and p16 was observed in 6% of normal cervical tissues and in 19% of CIN1, 18% of CIN2, 85% of CIN3, and 75% of SCC specimens. Hsp27 or p16 positivity had a sensitivity of 95.6 or 84.7% and a specificity of 37.2 or 70.5%, respectively, for the identification of CIN3 or SCC lesions; when both hsp27 and p16 were assessed, both the sensitivity and specificity were improved. In conclusion, both hsp27 and p16 immunohistochemistry is a useful tool for the diagnosis of CIN3 lesions or cervical SCC.

Objective To obtain insight into the effects of androstenedione on ovarian folliculogenesis and oogenesis. Design Experimental study. Setting St. Marianna University School of Medicine. Animal(s) Prepubertal (14-day-old) BDF1 female mice. Intervention(s) Early secondary follicles were isolated from the ovaries and were cultured individually in vitro with or without androstenedione (10 −11 to 10 −5 M) for 12 days. Thereafter, the follicles were treated with hCG and epidermal growth factor (EGF). Main Outcome Measure(s) Diameters and morphology of follicles and oocytes; E 2 and P secretion; and chromatin configuration and expression of growth differentiation factor 9 (GDF9) in oocytes were examined. Result(s) Early secondary follicles developed to the preovulatory stage. Androstenedione treatments increased the follicle diameters, reduced survival rates of follicles, and promoted the formation of follicles with abnormal morphology, including misshapen oocyte. The secretion of E 2 and P was significantly higher in androstenedione-exposed follicles. Androstenedione prevented the alteration in chromatin configuration and reduced oocyte GDF9 expression. When follicles cultured with androstenedione were treated with hCG and EGF, the first polar body exclusion, chromosome alignment on metaphase plate, and spindle assembly were inhibited in the oocytes. Conclusion(s) These results demonstrate that excess androgen induces abnormalities in the morphology and function of developing oocytes, which impairs oocyte meiotic competence.

To preserve the fertility of patients who undergo chemotherapy and/or radiotherapy, procedures for cryopreservation of female germ cells have been investigated. Cyropreservation methods differ according to follicle stage because the mammalian ovary contains a large number of oocytes at different growth stages. Follicles at very early stages, for example the primordial and primary stages, are usually cryopreserved within ovarian cortical tissue because they need surrounding somatic cells for subsequent development. In contrast, fully‐grown oocytes in Graafian follicles are cryopreserved without any other cells at the metaphase II stage. Recently, ultra‐rapid cooling was incorporated into cryopreservation procedures for human ovaries. In this review, we describe oocyte freezing, the development of ultra‐rapid cooling systems for ovarian tissues, freezing of human ovaries, and ovarian transplantation.

Conjoined twinning is a unique complication of monochorionic pregnancy. This report describes the clinical findings in two cases of conjoined twins, and discusses their management. One case involved thoracopagus complicating a triplet pregnancy, and the other involved cephalothoracopagus, in which the outcome was intrauterine fetal death due to abruptio placentae after amniocentesis. Recent improvements in ultrasound imaging have facilitated the diagnosis of conjoined twins as early as the first trimester. Although many mothers opt to terminate pregnancy when conjoined twins are diagnosed, a few do not, as in the cases described. In such cases, pregnancy management, including accurate determination of the degree of organ fusion and psychological follow up, are important. On the basis of the two present cases, we present a systematic flow diagram for management of conjoined twin pregnancy from the time of diagnosis until delivery.

Aim: Anxiety and depression are common in cancer patients, because diagnosis of cancer raises the fear of death. Although mental problems are often overlooked in cancer patients, it is important to control psychological distress, improve the quality of life, encourage patients to express requests about cancer therapy appropriately, and reduce the burden on family members. Material & Methods: There were 214 patients admitted to the Department of Obstetrics and Gynecology of St. Marianna University Hospital for treatment of cancer between January 2007 and December 2008. At 2 weeks after learning the diagnosis of cancer, these patients completed a Hospital Anxiety and Depression Scale (HADS) questionnaire, and their psychological characteristics were investigated in relation to age, tumor type and time after learning the diagnosis. The cut-off value for intervention to manage maladjustment and major depression was set at a HADS score of 11. Results: The HADS score was 11 in 118 of the 214 patients (55.1%). The HADS score for anxiety was higher in younger patients, while the HADS score for depression was higher in older patients. There were no significant correlations between the HADS score and the type of gynecologic cancer (cervical cancer, endometrial cancer and ovarian cancer) or the time after learning the diagnosis. Conclusion: Assessment based on the HADS score revealed a high prevalence of psychological problems after announcement of the diagnosis of gynecologic cancer. This emphasizes the importance of psychiatric intervention when patients are informed of their condition.

Formation of a fistula to a digestive organ is an extremely rare phenomenon in cases of ovarian carcinoma. We report a case of ovarian clear-cell carcinoma complicated by formation of a sigmoid colon fistula, and review the related literature. A 61-year-old woman, who had undergone hysterectomy and right salpingo-oophorectomy due to myoma and an ovarian tumor, developed bloody bowel discharge and abdominal distention. Computed tomography revealed a huge pelvic tumor with a thickened wall and internal gas. As the patient also had severe anemia and peritonitis, emergency laparotomy was performed, and intraoperatively it was noted that the tumor was tightly attached to the sigmoid colon, and contained bloody pus. Left salpingo-oophorectomy was performed and pathological examination of the specimen revealed fistula formation between the ovarian tumor and the sigmoid colon. The tumor was diagnosed as left ovarian clear-cell carcinoma, but no diverticulum or direct tumor invasion was evident around the fistula. The patient was given chemotherapy with paclitaxel and carboplatin, and she is now doing well after 9 months with no evidence of tumor recurrence. Although fistulation to the digestive tract is very rare in cases of ovarian cancer, it must be diagnosed and treated promptly because severe inflammation can make it potentially life-threatening.

Leiomyosarcoma (LMS) of the fallopian tube is exceedingly uncommon. So far as we investigated, only eighteen cases of LMS of the fallopian tube have been reported. Here we report a nineteenth case which was International Federation of Gynecology and Obstetrics stage IIIc LMS of the fallopian tube successfully treated with intraperitoneal cisplatin followed by prolonged oral etoposide. A 70-year-old female was introduced to our institute due to intrapelvic tumor and ascites. Because of elevated serum lactate dehydrogenase and CA125 as well as the findings of pelvic magnetic resonance imaging and computerized tomography, the patient was suspected to have ovarian cancer. In laparotomy, the large pelvic tumor was seemed to originate from the right fallopian tube. Pathologically, the patient was diagnosed as stage IIIc fallopian tube LMS. At the end of the operation, cisplatin was given intraperitoneally followed by prolonged oral etoposide. Although a lot of dissemination was noted throughout the peritoneal cavity, the patient is alive without any evidence of recurrence for more than 6 years since the initial operation. In this uncommon entity, a cisplatin- and etoposide-based regimen could be considered.

The purpose of this study was to investigate whether slow-rate freezing or vitrification is better for cryopreservation of ovary tissues pretreated with gonadotropin-releasing hormone agonist. In this nonclinical study performed in rats, leuprorelin acetate was administered to female Wistar rats, aged 6–8 weeks. After confirming arrest of the estrous cycle by examination of vaginal smears, ovarian tissue was cryopreserved by vitrification and slow-rate freezing prior to thawing and autotransplantation. The time required for estrous cycle recovery was assessed from vaginal smears in each group starting from day 1 of transplantation. Estradiol levels were also monitored after transplantation. The estrous cycle recovered after transplantation of ovarian tissue frozen by either method, but recovery was significantly faster after transplantation of vitrified tissue. The estradiol level also recovered by 10 days after transplantation. Ovarian function was restored after transplantation of tissue preserved by either vitrification or slow-rate freezing after pretreatment with leuprorelin acetate. This method may be applicable for patients scheduled to undergo cryopreservation of ovarian tissue before chemotherapy.

Objective To investigate the effects of androstenedione on ovarian follicle development. Design Experimental study. Setting University research laboratory. Animal(s) Female Wistar-Imamichi rats and BDF1 mice. Intervention(s) Rats were injected with androstenedione. Ovarian follicles of mice were cultured in the presence of androstenedione. Main Outcome Measure(s) Ovarian morphology; ovarian cell types undergoing apoptosis; ovarian expression of cytochrome P450 aromatase (P450arom), cytochrome P450 side-chain cleavage (P450scc), and cyclin-dependent kinase inhibitor p27 kip1 ; serum levels of T, E 2 , and P in rats; and ultrastructure of granulosa cells from cultured follicles of mice. Result(s) In androstenedione-treated rat ovaries, follicular cysts were formed, and apoptotic cells were found in the inner part of granulosa cell layers of antral follicles. Androstenedione administration down-regulated expression of P450arom but up-regulated expression of P450scc and p27 Kip1 in the granulosa cells of antral follicles. Serum T levels were significantly increased in androstenedione-treated rats. In mouse follicles exposed to androstenedione, the granulosa cells contained abundant lipid droplets and mitochondria with complex tubular cristae. Conclusion(s) Excess androgen enhances apoptosis in the inner part of granulosa cell layers of antral follicles, resulting in the formation of follicular cysts. It is also demonstrated that androgen stimulates premature luteinization of granulosa cells.

Water intoxication may be induced by the use of intrauterine fluid overload in hysteroscopic surgery. We report a patient with a uterine myoma and anemia (Hb, 9.3 g/dl), who developed severe hyponatremia during a hysteroscopic myomectomy. The submucosal myoma was resected using a monopolar resectoscope following 6 courses of GnRH agonist therapy. The procedure was performed by a combination of lumbar and intravenous anesthesia. The total amount of 3% solbitol was 27,000 ml and the duration of the procedure was 75 minutes. Severe hyponatremia (104 mEq/l) was diagnosed during the procedure, although there were no abnormal symptoms and vital signs. The serum Na level recovered to a normal level following the administration of diuretics. This case suggests that hyponatremia may occur during extensive resection of non-protruding submucosal myomas. Therefore, frequent measurements of intake and output of fluids and measurement of the serum Na level are required to prevent severe hyponatremia during hysteroscopic procedures for such cases.

Objective It has been reported that an ovarian tumor is diagnosed during pregnancy in 0.21-1.24% of cases. Patients with ovarian tumor who underwent surgery in pregnancy from 2004 to 2008 were reviewed.Design During these 5-years, there were 3,837 deliveries in our hospital, and 16 of which underwent surgery due to an ovarian tumor. A retrospective analysis of these cases was performed.Results Laparoscopic surgery was performed in 9 patients, while the remaining 7 patients underwent open abdominal surgery. The tumor was determined to be malignant in 2 patients. The mean patient age at the time of surgery was 31.4 years, and the median number of gestational age at surgery was 16 weeks. The median value for the maximal tumor diameter was 9 cm. Overall, among the patients with ovarian tumors complicating their pregnancy, the pathological diagnosis was a mature cystic teratoma in 9 patients, which was the predominant type, followed by an endometriotic cyst in 3 patients and a malignant tumor in 2 patients. These malignant tumors consisted of an immature teratoma (G2) and a yolk sac tumor, and these patients are currently undergoing chemotherapy.Conclusion Patients should be examined for the possible presence of an ovarian tumor early in gestation. Once a tumor is present and indication of operation is judged to be appropriate, it is necessary to carefully determine the best time and method for the surgery in each individual patient.

CapsuleSuccessful pregnancy and delivery after endoscopic revision of a uterine bicornis bicollis and septate vagina.AbstractInternal genital abnormalities which occur in fetuses are often diagnosed after menarche with symptoms of dysmenorrhea. We report a patient who was diagnosed with uterine bicornis bicollis and a septate vagina. She achieved a successful pregnancy and delivery after undergoing endoscopic surgery in childhood.

The CAG repeat lengths weighted by X-inactivation ratios were significantly shorter in 58 Japanese patients with premature ovarian failure (POF) than in 42 age-matched control females with normal menses. The results suggest that short CAG repeats with a relatively high androgen receptor function may constitute a susceptibility factor for the development of POF.

TGP (thermo-reversible gelation polymer) is a high molecular compound that has so-gel transmitting temperature of 221C Since solid cancer tissue grows in this polymer three-dimensionally, and fibroblasts scarcely grow in it, TGP is suitable for chemosensitivity assays for solid tumors. In this study, a chemosensitivity test using TGP was applied to recurrent gynecologic cancer patients in order to evaluate its utility and efficacy. In some ovarian cancer cases, expression of anticancer drug resistance-related proteins was also analyzed.Recurrent tumor tissues were surgically obtained with informed consent. After these tissues were minced and incubated for 4 days with CDDP, mitomycin C, 5-fluorouracil, paclitaxel, and CPT-11, the sensitivity against these drugs was estimated. Western blotting was performed in 8 recurrent ovarian cancer tissues in order to analyze the expression of Bcl-2, MRP2, BCRP, and GST-pi.The total evaluability rate of this assay was 90.6% (29/32). Sensitive drugs could be determined in 5 of 7 ascites samples (71.4%) and in 2 of 3 intra-tumoral fluid samples (66.7%). The overall clinical response rate of chemotherapy determined by these results was 50.0%. There were significant correlations between the IC50 of CDDP and Bcl-2, BCRP, GST-pi, and between that of 5-FU and MRP 2.Although this was a preliminary study, the chemosensitivity test using this new material appears to be useful for designing 'made-to order' salvage chemotherapy for pretreated recurrent gynecologic patients. In order to overcome multidrug resistance, the mechanisms of multidrug resistance should be further investigated.

This study assesses the relatively high incidence of the expression of paralogs of several pseudogenes within the cascade of expression of functional genes in the rat ovary in response to an ovulation-stimulating dose of gonadotropin. Immature Wistar rats were primed with 10 IU equine chorionic gonadotropin subcutaneously, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG subcutaneously. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for RT-PCR differential display to detect gene expression in the ovarian tissue. Sequence analyses of differentially expressed cDNAs revealed that ∼27% (i.e. 22/82 clones) of the transcripts were fragments of paralogs of known pseudogenes. Out of the 22 clones reported here, 12 have high sequence similarity to the cytochrome P450 pseudogene Cyp21a1-ps, and 5 have high sequence similarity to both the Cyp21a1-ps and the aldo-keto reductase gene Akr1c6. The remaining five clones were paralogs of the endogenous retrovirus SC1 that has heavily infested the rat genome. Northern analyses reveal that peak expression of all the 22 paralogs occurs at 4–8 h into the ovulatory process. In situ hybridization shows that expression of these pseudogenes is primarily in the granulosa layer of ovulatory follicles. In summary, the results reveal that ovarian expression of Cyp21a1-ps- and SC1-like pseudogenes occurs concurrently with the ovulatory process.

Objectives This study proposed a method for assessing menopause-specific health literacy (knowledge and beliefs about menopausal symptoms which aid their recognition, assessment, and management) using a vignette methodology. Study design A cross-sectional web-based survey was conducted in September 2015 among Japanese women aged 30⿿59 years. Of 1236 women surveyed, 1196 eligible participants who were not under treatment for menopausal symptoms were included. Main outcome measures: Participants were presented with a vignette describing a woman with menopausal symptoms and were then asked a series of questions to assess their recognition of menopausal symptoms, attitude, subjective norm, perceived behavior control, availability, and intention to seek medical care if they themselves had the problems described in the vignette. Results The majority (87%) of participants correctly labelled the vignette as menopausal symptoms and 60% expressed an intention to seek medical care if they had the symptoms presented. Logistic regression showed that attitude, subjective norm, and perceived behavior control were significant predictors of the intention to seek medical care. A structural equation model depicting these relationships with intention to seek medical care revealed acceptable fit indices: goodness of fit index (GFI) = 0.948, adjusted goodness of fit index (AGFI) = 0.913, comparative fit index (CFI) = 0.883, and root mean square error of approximation (RMSEA) = 0.089. Subjective norm had the greatest direct effect on intention to seek medical care. Conclusions The assessment of menopause-specific health literacy may be useful for understanding why women hesitate to seek medical care for menopausal symptoms and for developing interventions to improve the coping behaviors of women with menopausal symptoms.

In this study, the preliminary analyses were conducted of enzymatic activities of uridine phosphorylase (UP) and thymidine phosphorylase (TP) in normal tissues and cancer tissues of the uterine cervix. The study was performed on 27 patients of cervical cancer, treated first in our hospital. Normal cervical tissues obtained from 15 patients undergoing hysterectomy for benign diseases were used as controls. The supernatant of the homogenated cervical tissues and the stroma (5-FU and ribose-1-P or deoxyribose-1-P) were analyzed by high performance liquid chromatography, and then the UP and TP activities calculated. TP activity was significantly greater than UP activity (P < 0.0001). Both UP and TP showed significantly greater activity in cancer tissues than in normal tissues (P < 0.0001). In the TP activity of the cancer tissues, there was no significant difference among the histological types, while the TP activity tended to be significantly higher in the cases with lymph node metastasis. These results showed that the TP-mediated route seemed important as the 5FU metabolic pathway in the uterine cervical tissues, and TP enzymatic activity might be associated with lymph node metastasis.

The intermediate filament protein nestin was originally found to be expressed in neuronal progenitor cells, but recent studies have shown that other cell types, including endocrine and vascular endothelial cells, express nestin. In the present study, we examined the expression and localization of nestin in the ovaries of developing, peripubertal, and adult rats. RT-PCR and Western blot analyses revealed that nestin mRNA and proteins were expressed in adult rat ovaries. Immunohistochemical analyses using adult rat ovaries showed that nestin was mainly localized to capillary endothelial cells of theca interna in follicles with more than two layers of granulosa cells and that its expression increased with follicle growth. Ontogenetically, ovarian nestin expression started at the peripubertal period when the first gonadotropin surge occurs. To test the possibility that gonadotropins induce nestin expression, prepubertal (postnatal d 21) rats were sc injected with equine chorionic gonadotropin (eCG) and/or human chorionic gonadotropin (hCG). A single injection of hCG, but not eCG, was sufficient to induce nestin expression in follicles, mainly in capillary endothelial cells of theca interna. Furthermore, pretreatment with an inhibitor of vascular endothelial growth factor receptor prevented the induction of the nestin expression by hCG. These findings demonstrate that the endogenous LH surge induces nestin expression in capillary endothelial cells of theca interna via the vascular endothelial growth factor signaling pathway. Nestin may be involved in angiogenesis in growing follicles, which is followed by follicle maturation and subsequent ovulation.

Premutations of the fragile X mental retardation 1 (FMR1) gene are associated with increased risk of primary ovarian insufficiency. Here we examined the localization of the Fmr1 gene protein product, fragile X mental retardation protein (FMRP), in rat ovaries at different stages, including fetus, neonate, and old age. In ovaries dissected from 19 days postcoitum embryos, the germ cells were divided into 2 types: one with decondensed chromatin in the nucleus was FMRP positive in the cytoplasm, but the other with strongly condensed chromatin in the nucleus was FMRP negative in the cytoplasm. The FMRP was predominantly localized to the cytoplasm of oocytes in growing ovarian follicles. Levels of FMRP in oocytes from elderly (9 or 14 months of age) ovaries were lower than in those from younger ovaries. These results suggest that FMRP is associated with the activation of oogenesis and oocyte function. Especially, FMRP is likely to be implicated in germline development during oogenesis.

The transporter protein genes and lipids in human ovarian carcinoma-derived KF28 cells with anticancer-drug-sensitive properties were compared with those in resistant cells, taxol-resistant KF28TX, cisplatin-resistant KFr13, and taxol- and cisplatin-resistant KFr13TX, to identify the molecules required for anticancer-drug resistance. In accordance with previous reports, taxol and cisplatin resistance was closely correlated with expression of the multidrug resistance 1 and bile acid export pump, and multidrug resistance-associated protein 2 genes, respectively. In addition, we found a distinct difference in glycosphingolipids between the sensitive and resistant cells. Although GlcCer was the major glycolipid (83.0%) in sensitive cells, GalCer, LacCer and, particularly, Gb3Cer were characteristically increased in all resistant cells, irrespective of whether the resistance was to taxol or cisplatin, and comprised 65–84% of total glycosphingolipids. GM3, which was present at 0.04 µg/mg dry weight in the sensitive cells, showed a twofold increase in the taxol-resistant cells, but was absent in the cisplatin-resistant cells. The altered glycolipid composition was proven to be due to enhanced or suppressed expression of the respective sugar transferase genes. In addition, the ceramide moiety of ceramide monohexoside in the sensitive cells constituted 83% of non-hydroxy fatty acids, but that in the resistant cells comprised 67–74% of α-hydroxy fatty acids. Thus, cells containing Gb3Cer with α-hydroxy fatty acids were found to survive selectively in the presence of taxol and cisplatin, and modification of the glycolipid structure was revealed to occur in association with anticancer-drug resistance. (Cancer Sci 2006; 97: 1321–1326)

The significance of cyclooxygenase-2 (COX-2) expression in ovarian cancer has been discussed. In this study, we found increased expression of COX-1 mRNA and protein in three out of 10 ovarian cancer cell lines. Prostaglandin E 2 (PGE 2 ) production was elevated in these three cell lines, but not in other seven cell lines. COX-2 protein was not detected in any of the cell lines. Cytosolic prostaglandin E synthase (cPGES) mRNA and protein were detected in all 10 cell lines. Membrane-associated PGES-1 (mPGES-1) was detected in some of the ovarian cell lines, but its presence did not correspond with PGE 2 production. In contrast, mPGES-2 mRNA and protein were detected in all 10 cell lines. A nonselective COX inhibitor (indometacin) and a selective COX-1 inhibitor (SC-560) strongly inhibited PGE 2 production by the three cell lines, while selective COX-2 inhibitors (NS-398 and rofecoxib) did not inhibit PGE 2 production. In addition, increased expression of COX-1, not COX-2 protein was observed in the mass of ovarian cancer tissues from 22 patients when compared with that in normal tissue. These findings suggest that COX-1 might be a major enzyme regulating PGE 2 production in ovarian cancer cells.

To introduce Complementary and Alternative Medicine (CAM) into a hospital department of obstetrics and gynecology with the goal of achieving integrative medicine, the authors investigated the effectiveness of aromatherapy for outpatients with menopausal symptoms.The department of obstetrics and gynecology, St. Marianna University Hospital, Kawasaki, Japan.Participants in the aromatherapy trial received a 30 minute aromatherapy session performed by an aromatherapist that included a consultation, massage, and home care guidance. After they carried out approximately 1 month of home care, they received a second aromatherapy session. The patients' physical and mental health status was measured at the start and endpoint of the trial by use of the Kupperman index (KI), the self-rating depression scale, and consultation.Fifteen (15) outpatients (mean age, 54.3 +/- 7.4 years) with menopausal symptoms in the department of obstetrics and gynecology of St. Marianna University Hospital.The mean value of the KI score was significantly lowered after the aromatherapy trial from 31.4 +/- 6.8 to 22.9 +/- 6.1 (p = 0.001).This investigation shows that aromatherapy could be effective as a CAM method for menopausal symptoms in the setting of a hospital obstetrics and gynecology department.

We successfully established a novel ovarian granulosa tumor cell line (HSOGT). The tumor tissue of the ovary was derived from a 25 year-old Japanese woman under her consent. The cell line was maintained for over 14 months, subcultured more than 73 times, and had a population doubling time of 18.9 hours. Phase contrast microscopy displayed a pavement-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy and the mode was 83; many marker chromosomes were observed. The HSOGT was also successfully xenotransplanted into nude mice. The cell line produced estradiol and has preserved some characters of granulosa cells with stable growth in vitro. We firmly believe that this cell line will be a most useful tool for endocrinological investigation of human granulosa cells.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin (N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 - 1 2 , 10 - 1 0 , or 10 - 8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 - 1 0 and 10 - 8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 - 1 2 to 10 - 8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole-O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.

背景:便秘症のカルチノイド症候群を呈した卵巣甲状腺腫性カルチノイドの1例を経験し, その腫瘍発生の検索のために二重染色による免疫組織化学的な検索を行った.症例:48歳, 女性. 画像診断で右卵巣に手拳大の腫瘤を認め, 成熟型奇形腫が疑われ摘出手術を施行し, 甲状腺腫性カルチノイドと診断された. 捺印細胞像では, コロイド様の無構造物質を背景に, 腫瘍細胞はシート状集塊または散在性, 一部濾胞構造, 腺管様構造, 索状配列を示していた. 細胞は比較的小型の類円形から円柱形で, 細胞質はレース状ないし顆粒状でライトグリーンに淡染し, 核は類円形, 核縁は薄く, クロマチンは軽度増量し, やや粗顆粒状で均等分布していた. 組織学的に腫瘍はカルチノイド, 甲状腺腫およびそれらの移行部分から構成されていた. 免疫組織化学的に移行部分はthyroglobulin, chromogranin A両者に陽性を示した.結論:本例の診断に大切なことは, カルチノイドに特徴的な細胞所見と, 甲状腺腫としてのコロイドを含む濾胞構造に着目したことであった. また免疫組織化学的染色結果から, 本腫瘍発生は, 腫瘍細胞が多分化能を持っていることと密接な関係があると考えた.

We report on fluorescence in situ hybridization (FISH) analysis in 30 mosaic or nonmosaic females diagnosed as having apparently simple terminal X deletions by standard G-banding analysis. FISH studies for DXZ1, the Xp and Xq telomere regions, and the whole X chromosome painting were carried out for the 30 females, indicating rearranged X chromosomes with signal patterns discordant with terminal deletions in 6 cases: one dic(X)(DXZ1++) chromosome, two der(X)(qtel++) chromosomes, one Xq− (qtel+) chromosome, and two der(X)(ptel++) chromosomes. Additional FISH studies were performed for the 6 cases using probes defining 12 loci on the X chromosome, showing large Xp deletion and small Xp duplication in the dic(X)(DXZ1++) chromosome, partial Xp deletions and partial Xq duplications in the two der(X)(qtel++) chromosomes, an interstitial Xq deletion in the Xq− (qtel+) chromosome, and partial Xq deletions and partial Xp duplications in the two der(X)(ptel++) chromosomes. Clinical assessment of the 6 cases revealed tall and normal stature in the two mosaic cases with the der(X)(ptel++) chromosomes that were shown to be associated with SHOX duplication. The results suggest that unusual X chromosome rearrangements are often misinterpreted as simple terminal X deletions, and that FISH analysis is useful for precise structural determination and better genotype-phenotype correlation of the X chromosome aberrations. © 2001 Wiley-Liss, Inc.

The effect of melatonin on endometrial cancer cell growth was investigated using two cell lines, SNG-II and Ishikawa, which are different in their estrogen receptor status. A physiological concentration of melatonin (10(-9) M) showed no growth inhibitory effect on SNG-II cells, which are estrogen receptor-negative at all cell densities and incubation times. In contrast, melatonin significantly inhibited Ishikawa cells, which are estrogen receptor-positive at all cell densities tested after 96 hr incubation. The greatest inhibition of Ishikawa cell growth was observed at 10(-9) M melatonin, compared with other supra (10(-6), 10(-8) M) or subphysiological concentrations (10(-10), 10(-12) M). This growth inhibitory effect of melatonin on Ishikawa cells was completely blocked by 10(-10) to 10(-8) M concentrations of 17-beta estradiol administration. Pretreatment with luzindole, which is a selective melatonin receptor antagonist, prior to the addition of melatonin also blocked the inhibitory effect of melatonin on Ishikawa cells. This is the first study to demonstrate an anti-proliferative effect of physiological melatonin on endometrial cancer cells in vitro. The present study revealed that melatonin also inhibits the growth of estrogen receptor positive endometrial cancer cells and that this effect of the pineal indole may be mediated by both steroid and melatonin receptors.

To examine the effect of melatonin on in vitro fertilization and embryonic development, mouse embryos after insemination in vitro were cultured in a physiological medium with or without melatonin. Melatonin increased the fertilization rate significantly at a concentration between 10(-6) and 10(-4) M (27.6 vs. 43.9 or 40.4%, P < 0.01). Furthermore, a significant increase in the rate of embryos reaching the four-cell stage (16.0 vs. 26.7%, P < 0.01), the eight-cell stage (12.1 vs. 25.8 or 23.5%, P < 0.01), and blastulation (8.9 vs. 23.5 or 17.5%, P < 0.01) was observed when the embryos were cultured in a medium containing 10(-8) or 10(-6) M melatonin. These results demonstrate that melatonin supports fertilization and early embryo development after in vitro fertilization.