研究者詳細 - 中村　智子

論文 - 中村　智子

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Susceptibility of Brca1<sup>(L63X/+)</sup> rat to ovarian reserve dissipation by chemotherapeutic agents to breast cancer Open Access

Kaseki S., Sonehara R., Motooka Y., Tanaka H., Nakamura T., Osuka S., Akatsuka S., Kajiyama H., Mashimo T., Imaoka T., Toyokuni S.

Cancer Science 2025年

出版者・発行元：Cancer Science

BRCA1 is one of the causative genes for hereditary breast and ovarian cancer syndrome with a high risk of early-onset breast cancer. Whereas olaparib (OLA), an inhibitor of poly-ADP-ribose polymerase, has been applied as adjuvant therapy to those cancer patients, its effect on ovarian reproductive function remains unelucidated. Recently, a rat model (MUT; Brca1(L63X/+) mutation) mimicking a human BRCA1 pathogenic variant has been established. Using this model, we evaluated the effects of OLA on ovarian reproductive function in comparison with the wild-type (WT) rats. MUT showed a significantly reduced number of primordial follicles and subfertility in accordance with aging. Oxidative stress was significantly elevated in the young MUT granulosa cells (GCs) accompanied by increased mTOR but decreased PTEN signals. OLA administration in MUT further decreased primordial follicles, with gene set enrichment analysis, indicating upregulated DNA repair pathways. Furthermore, a combination of OLA and cyclophosphamide (CPA) induced empty primordial follicles, recognized as CPA-induced severe ovarian toxicity. Whereas OLA + CPA caused greater reduction in primordial follicles both in MUT and WT in comparison with CPA alone, MUT ovaries were more susceptible to oxidative stress, potentially depleting primordial follicles via activation of GCs and inducing oocyte death due to accumulated DNA damage by OLA treatment. Our findings in this preclinical model underscore the importance of evaluating ovarian reserve prior to chemotherapy by performing reproductive consultation with female patients with BRCA1 pathogenic variants.

DOI： 10.1111/cas.16412

Small extracellular vesicles in follicular fluids for predicting reproductive outcomes in assisted reproductive technology Open Access

Muraoka A., Yokoi A., Yoshida K., Kitagawa M., Asano-Inami E., Murakami M., Bayasula , Miyake N., Nakanishi N., Nakamura T., Osuka S., Iwase A., Kajiyama H.

Communications Medicine 4 巻 ( 1 ) 2024年12月

出版者・発行元：Communications Medicine

Background: Assisted reproductive technology accounts for an increasing proportion of infertility treatments, and assessments to predict clinical pregnancy outcomes are desired. Extracellular vesicles exist in follicular fluid, and small non coding RNAs in extracellular vesicles underline the possibility of reflecting pregnancy potential. Methods: Follicular fluid samples are collected from 20 ovarian follicles of 15 infertile patients undergoing assisted reproductive technology. Extracellular vesicles are isolated by serial centrifugation and small RNA sequencing is performed to investigate the profiles of microRNAs and P-element-induced wimpy testis-interacting RNAs. Results: Small extracellular vesicles with a size range of approximately 100 nm are successfully isolated, and the small non coding RNA profiles of pregnant samples (n = 8) are different from those of non-pregnant samples (n = 12). Fourteen dysregulated small non coding RNAs are selected to identify the independent candidates [mean read count >100, area under the curve >0.8]. Among them, we find that a specific combination of small non coding RNAs (miR-16-2-3p, miR-378a-3p, and miR-483-5p) can predict the pregnant samples more precisely using a receiver operating characteristics curves analysis (area under the curve: 0.96). Furthermore, even in the same patients, the three microRNAs are differentially expressed between pregnant and non-pregnant samples. Conclusions: Our results demonstrate that small non coding RNAs derived from small extracellular vesicles in follicular fluid can be potential non-invasive biomarkers for predicting pregnancy, leading to their probable application in assisted reproductive technology. Further large-scale studies are required to validate the clinical usefulness of these small non coding RNAs.

DOI： 10.1038/s43856-024-00460-8

Serum miRNA as a predictive biomarker for ovarian reserve after endometrioma-cystectomy

Yabuki A., Muraoka A., Osuka S., Yokoi A., Yoshida K., Kitagawa M., Bayasura , Sonehara R., Miyake N., Nakanishi N., Nakamura T., Iwase A., Kajiyama H.

Reproductive Biology 24 巻 ( 1 ) 2024年3月

出版者・発行元：Reproductive Biology

Ovarian endometrioma (OE) is a common gynecological disease that is often treated with surgery and hormonal treatment. However, ovarian cystectomy can impair the ovarian reserve (OR). Previously, we showed that perioperative administration of dienogest (DNG) is an effective option for OR preservation. However, there were differences in the extent of OR preservation among patients following perioperative DNG treatment. In the current study, we performed a global examination of serum microRNAs (miRNAs) to identify accurate biomarkers that predict post-operative restoration of OR following perioperative DNG treatment. We also sought to identify specific miRNAs related to the anti-Müllerian hormone (AMH). miRNA sequencing was performed on serum samples obtained from twenty-seven patients who received perioperative DNG treatment. Candidate miRNAs were selected by comparing patients whose ORs were restored postoperatively (responder group, n = 7) with those whose ORs were not (non-responder group, n = 7). miR-370–3p and miR-1307–3p were significantly upregulated in the responder group, whereas miR-27b-3p was upregulated in the non-responder group. The pretreatment value of each miRNA could predict DNG responsiveness for OR following ovarian cystectomy (area under the curve [AUC] > 0.8). The quantitative polymerase chain reaction (qPCR) revealed only miR-1307–3p was found to be significantly upregulated in the responder group (P < 0.05). In addition, we identified miR-139–3p, miR-140–3p, and miR-629–5p as AMH-associated miRNAs. The transition of AMH showed a correlation with miR-139–3p (P < 0.05, r = −0.76). The miRNAs identified herein represent potential serum biomarkers of clinical value in predicting OR prior to DNG treatment.

DOI： 10.1016/j.repbio.2023.100821

The influence of radical trachelectomy on endometrial thickness in in vitro fertilization-embryo transfer

Yabuki A., Muraoka A., Tamauchi S., Seki T., Takeda T., Sonehara R., Miyake N., Nakamura T., Osuka S., Kajiyama H.

Journal of Obstetrics and Gynaecology Research 50 巻 ( 2 ) 頁： 218 - 224 2024年2月

出版者・発行元：Journal of Obstetrics and Gynaecology Research

Aim: Both morbidity and mortality rates of cervical cancer are increasing, especially in reproductive-aged women. Radical trachelectomy (RT) is an effective fertility-preserving surgery for early-stage cervical cancer. This study aimed to determine the influence of RT on endometrial thickness during in vitro fertilization-embryo transfer (IVF-ET). Methods: Forty-four patients had undergone RT, and 23 women undergoing IVF-ET treatment (105 ET cycles) were included. Endometrial thickness during hormone replacement therapy (HRT) was retrospectively evaluated and compared between patients with and without RT. Results: Eleven patients (50 ET cycles) in the RT group and 12 (52 ET cycles) in the control group were investigated. Compared with the control group, higher ET cancellation rates were observed in patients in the RT group (1 of 52 cycles [control group] vs. 8 of 50 cycles [RT group], p < 0.01). Endometrial thinning was not affected by patient age at first IVF-ET treatment, history of artificial abortion, preservation of uterine arteries during RT, or postoperative chemotherapy (p = 0.27, 1, 1, and 1, respectively). Conclusions: Our data revealed that RT influenced endometrial thickness in IVF-ET. This was not affected by the background of the patients or perioperative management in this study. We could not reveal the underlying mechanism, but it is postulated that the transient postoperative uterine blood flow status and postoperative infections may have some effect on the endometrium. To resolve these issues, accumulation of evidences are required. We recommend informing patients about the impact of RT on IVF-ET before starting assisted reproductive technology (ART).

DOI： 10.1111/jog.15841

Association between anti-Müllerian hormone levels and polycystic ovary syndrome in a general cohort of young women in Japan Open Access

Miyake N., Osuka S., Ohsawa I., Tonoike T., Uno T., Tsuzuki K., Bayasula , Sonehara R., Muraoka A., Nakamura T., Goto M., Iwase A., Kajiyama H.

Reproductive Medicine and Biology 23 巻 ( 1 ) 2024年1月

出版者・発行元：Reproductive Medicine and Biology

Purpose: Polycystic ovary syndrome (PCOS) significantly affects women. This study investigated the association between serum anti-Müllerian hormone (AMH) levels and menstrual cycle disorders, and AMH for PCOS in a general cohort of young Japanese women. Methods: We measured serum AMH levels in 528 healthy female students at two universities in Japan between 2014 and 2020. We investigated the association between serum AMH levels and hormone levels, menstrual cycle, and body mass index. Results: The mean (±standard deviation) AMH level was 4.78 ± 2.88 ng/mL. Correlations were observed between serum AMH and luteinizing hormone (LH) or LH/follicle-stimulating hormone (FSH) levels in women with irregular menstruation (LH: r = 0.542, p < 0.001; LH/FSH: r = 0.584, p < 0.001). The optimal serum AMH cutoff value that predicted LH ≥7.1 IU/L and LH/FSH ≥1.21 (PCOS diagnostic criteria revised by Japan Society of Obstetrics and Gynecology) in women with menstrual irregularities was 5.30 ng/mL (area under the curve: 0.815, sensitivity: 84.2%, specificity: 70.3%). Conclusions: Serum AMH can be measured during annual health checkups and may be a useful biomarker for early and arcuate diagnosis and intervention in women with PCOS.

DOI： 10.1002/rmb2.12615

Risk factors for non-previa placenta accreta spectrum in pregnancies conceived through frozen embryo transfer during a hormone replacement cycle in Japan Open Access

Matsuo S., Kotani T., Tano S., Ushida T., Imai K., Nakamura T., Osuka S., Goto M., Osawa M., Asada Y., Kajiyama H.

Reproductive Medicine and Biology 23 巻 ( 1 ) 2024年1月

出版者・発行元：Reproductive Medicine and Biology

Purpose: Non-previa placenta accreta spectrum (PAS) is associated with assisted reproductive technology (ART), particularly frozen embryo transfer during hormone replacement therapy (HRC-FET). We especially aimed to evaluate the prevalence and risk factors for non-previa PAS in HRC-FET pregnancies. Methods: Overall, 279 women who conceived through ART at three ART facilities and delivered at a single center were included in this retrospective study. Data regarding endometrial thickness at embryo transfer, previous histories, and type of embryo transfer—HRC-FET, frozen embryo transfer during a natural ovulatory cycle (NC-FET), and fresh embryo transfer (Fresh-ET)—were collected. Univariable logistic regression analyses were conducted. Results: The prevalence of non-previa PAS was 27/192 (14.1%) in the HRC-FET group and 0 (0.0%) in both the NC-FET and Fresh-ET groups. Significantly high odds ratio [95% confidence interval] of non-previa PAS was associated with a history of artificial abortion (6.45 [1.98–21.02]), endometrial thickness <8.0 mm (6.11 [1.06–35.12]), resolved low-lying placenta (5.73 [2.13–15.41]), multiparity (2.90 [1.26–6.69]), polycystic ovarian syndrome (2.62 [1.02–6.71]), and subchorionic hematoma (2.49 [1.03–6.04]). Conclusions: A history of artificial abortion, endometrial thickness <8.0 mm, and resolved low-lying placenta may help in antenatal detection of a high-risk population of non-previa PAS in HRC-FET pregnancies.

DOI： 10.1002/rmb2.12592

Serum-derived small extracellular vesicles as biomarkers for predicting pregnancy and delivery on assisted reproductive technology in patients with endometriosis Open Access

Muraoka A., Yokoi A., Yoshida K., Kitagawa M., Bayasula , Murakami M., Miyake N., Sonehara R., Nakamura T., Osuka S., Kajiyama H.

Frontiers in Endocrinology 15 巻 2024年

出版者・発行元：Frontiers in Endocrinology

Introduction: Endometriosis can cause of infertility, and evaluation methods for predicting clinical pregnancy outcomes are desired. Extracellular vesicles (EVs) exist in blood and it contains small non-coding RNAs (ncRNAs) that may reflect disease severity. In this study, we investigated small ncRNAs in serum EVs to identify specific biomarkers for predicting clinical pregnancy. Methods: Serum samples were collected from 48 patients who underwent assisted reproductive technology (ART). EVs were successfully isolated from serum samples and characterized using nanoparticle tracking assays, electron microscopy, and western blotting of EV’s markers. We performed small RNA sequencing and analyzed microRNA (miRNA) profiles in the infertility patients with and without endometriosis to detect pregnancy-predicting biomarkers. Results: Candidate miRNAs in serum EVs were selected by comparing patients without endometriosis who became pregnant (n = 13) with those who did not (n = 21). A total of 241 miRNAs were detected; however, no trends separated the two groups. Next, EVs from patients with endometriosis were analyzed and divided into pregnant (n = 4) and non-pregnant (n = 10) cases. Among the 224 candidate miRNAs, miRNA profiles of pregnant women with endometriosis were separated from those of non-pregnant women by receiver-operating characteristics (ROC) curve analysis (area under the curve [AUC] > 0.8). In patients with endometriosis, serum EVs may be useful for predicting possible pregnancy before infertility treatment. Finally, we used small RNA sequencing of the tissue to demonstrate that pregnancy-predicting miRNAs in serum EVs were produced from endometriosis lesions. Although no predictors were found from miRNAs in serum EVs without endometriosis, miRNAs in serum EVs of patients with endometriosis could provide novel noninvasive biomarkers to predict pregnancy and have potential clinical applicability in ART. Discussion: Further studies are required to examine the functional importance of these miRNAs to elucidate the pathological mechanisms of endometriosis and pregnancy.

DOI： 10.3389/fendo.2024.1442684

Brca2<sup>(p.T1942fs/+)</sup> dissipates ovarian reserve in rats through oxidative stress in follicular granulosa cells Open Access

Tanaka H., Motooka Y., Maeda Y., Sonehara R., Nakamura T., Kajiyama H., Mashimo T., Toyokuni S.

Free Radical Research 58 巻 ( 2 ) 頁： 130 - 143 2024年

出版者・発行元：Free Radical Research

Pathogenic variants of BRCA1/2 constitute hereditary breast and ovarian cancer (HBOC) syndrome, and BRCA1/2 mutant is a risk for various cancers. Whereas the clinical guideline for HBOC patients has been organized for the therapy and prevention of cancer, there is no recommendation on the female reproductive discipline. Indeed, the role of BRCA1/2 pathogenic variants in ovarian reserve has not been established due to the deficiency of appropriate animal models. Here, we used a rat model of Brca2(p.T1942fs/+) mutant of Sprague-Dawley strain with CRISPR-Cas9 editing to evaluate ovarian reserve in females. Fertility and ovarian follicles were evaluated and anti-Müllerian hormone (AMH) was measured at 8–32 weeks of age with a comparison between the wild-type and the mutant rats (MUT). MUT revealed a significantly smaller number of deliveries with fewer total pups. Furthermore, MUT showed a significant decrease in primordial follicles at 20 weeks and a low AMH level at 28 weeks. RNA-sequencing of the ovary at 10 weeks detected acceleration of the DNA damage repair pathway, which was accompanied by oxidative stress-induced DNA double-strand breaks, a decrease in PTEN, and an increase in mTOR in follicular granulosa cells. In conclusion, Brca2(p.T1942fs/+) dissipates primordial follicles via early activation of granulosa cells through oxidative stress, leading to earlier termination of fertility.

DOI： 10.1080/10715762.2024.2320405

Upregulated Ribosomal Pathway Impairs Follicle Development in a Polycystic Ovary Syndrome Mouse Model: Differential Gene Expression Analysis of Oocytes

Nakanishi N., Osuka S., Kono T., Kobayashi H., Ikeda S., Bayasula B., Sonehara R., Murakami M., Yoshita S., Miyake N., Muraoka A., Kasahara Y., Murase T., Nakamura T., Goto M., Iwase A., Kajiyama H.

Reproductive Sciences 30 巻 ( 4 ) 頁： 1306 - 1315 2023年4月

出版者・発行元：Reproductive Sciences

Polycystic ovary syndrome (PCOS), a common endocrine disorder, is associated with impaired oocyte development, leading to infertility. However, the pathogenesis of PCOS has not been completely elucidated. This study aimed to determine the differentially expressed genes (DEGs) and epigenetic changes in the oocytes from a PCOS mouse model to identify the etiological factors. RNA-sequencing analysis revealed that 90 DEGs were upregulated and 27 DEGs were downregulated in mice with PCOS compared with control mice. DNA methylation analysis revealed 30 hypomethylated and 10 hypermethylated regions in the PCOS group. However, the DNA methylation status did not correlate with differential gene expression. The pathway enrichment analysis revealed that five DEGs (Rps21, Rpl36, Rpl36a, Rpl37a, and Rpl22l1) were enriched in ribosome-related pathways in the oocytes of mice with PCOS, and the immunohistochemical analysis revealed significantly upregulated expression levels of Rps21 and Rpl36. These results suggest that differential gene expression in the oocytes of mice in PCOS is related to impaired folliculogenesis. These findings improve our understanding of PCOS pathogenesis.

DOI： 10.1007/s43032-022-01095-7

Follicle development and its prediction in patients with primary ovarian insufficiency: Possible treatments and markers to maximize the ability to conceive with residual follicles Open Access

Osuka S., Kasahara Y., Iyoshi S., Sonehara R., Myake N., Muraoka A., Nakamura T., Iwase A., Kajiyama H.

Reproductive Medicine and Biology 22 巻 ( 1 ) 2023年1月

出版者・発行元：Reproductive Medicine and Biology

Background: Primary ovarian insufficiency (POI) is characterized by the development of hypergonadotropic hypogonadism before 40 years of age and leads to intractable infertility. Although in vitro fertilization and embryo transfer with donated eggs enables pregnancy, not a few patients desire pregnancy using their oocytes. However, follicular development is rare and unpredictable in patients with POI. Thus, there is a need for treatments that promote the development of residual follicles and methods to accurately predict infrequent ovulation. Methods: This review discusses the effects of various treatments for obtaining eggs from POI patients. Furthermore, this study focused a potential marker for predicting follicular growth in patients with POI. Main Findings: Different treatments such as hormone-replacement therapy, dehydroepiandrosterone supplementation, platelet-rich plasma injection, and in vitro activation have shown varying degrees of effectiveness in retrieving oocytes from patients with POI. To predict follicle development in the cycle, elevated serum estradiol and reduced follicle-stimulating hormone (FSH) levels are important. However, these markers are not always reliable under continuous estradiol-replacement therapy. As a novel marker for predicting follicle growth, serum anti-Müllerian hormone (AMH) levels, measured using the picoAMH enzyme-linked immunosorbent assay, were found to predict follicle growth in patients and the cycle. Conclusion: This review highlights the challenges and available interventions for achieving pregnancy using a patient's oocytes in cases of POI. We believe that a combination of currently available treatments and prediction methods is the best strategy to enable patients with POI to conceive using their own eggs. Although AMH levels may predict follicle growth, further research is necessary to improve the chances of successful follicular development and conception in patients with POI.

DOI： 10.1002/rmb2.12556

Unkeito promotes follicle development by restoring reduced follicle-stimulating hormone responsiveness in rats with polycystic ovary syndrome Open Access

Yoshita S., Osuka S., Shimizu T., Fujitsuka N., Matsumoto C., Bayasula , Miyake N., Muraoka A., Nakanishi N., Nakamura T., Goto M., Kajiyama H.

Frontiers in Endocrinology 14 巻 2023年

出版者・発行元：Frontiers in Endocrinology

Background: Polycystic ovary syndrome (PCOS) is a common disorder resulting in irregular menstruation and infertility due to improper follicular development and ovulation. PCOS pathogenesis is mediated by downregulated follicle-stimulating hormone receptor (FSHR) expression in granulosa cells (GCs); however, the underlying mechanism remains elusive. Unkeito (UKT) is a traditional Japanese medicine used to treat irregular menstruation in patients with PCOS. In this study, we aimed to confirm the effectiveness of UKT in PCOS by focusing on follicle-stimulating hormone (FSH) responsiveness. Methods: A rat model of PCOS was generated by prenatal treatment with 5α-dihydrotestosterone. Female offspring (3-week-old) rats were fed a UKT mixed diet or a normal diet daily. To compare the PCOS phenotype in rats, the estrous cycle, hormone profiles, and ovarian morphology were evaluated. To further examine the role of FSH, molecular, genetic, and immunohistological analyses were performed using ovarian tissues and primary cultured GCs from normal and PCOS model rats. Results: UKT increased the number of antral and preovulatory follicles and restored the irregular estrous cycle in PCOS rats. The gene expression levels of FSHR and bone morphogenetic protein (BMP)-2 and BMP-6 were significantly decreased in the ovarian GCs of PCOS rats compared to those in normal rats. UKT treatment increased FSHR staining in the small antral follicles and upregulated Fshr and Bmps expression in the ovary and GCs of PCOS rats. There was no change in serum gonadotropin levels. In primary cultured GCs stimulated by FSH, UKT enhanced estradiol production, accompanied by increased intracellular cyclic adenosine monophosphate levels, and upregulated the expression of genes encoding the enzymes involved in local estradiol synthesis, namely Cyp19a1 and Hsd17b. Furthermore, UKT elevated the expression of Star and Cyp11a1, involved in progesterone production in cultured GCs in the presence of FSH. Conclusions: UKT stimulates ovarian follicle development by potentiating FSH responsiveness by upregulating BMP-2 and BMP-6 expression, resulting in the recovery of estrous cycle abnormalities in PCOS rats. Restoring the FSHR dysfunction in the small antral follicles may alleviate the PCOS phenotype.

DOI： 10.3389/fendo.2023.1228088

Tamoxifen Activates Dormant Primordial Follicles in Mouse Ovaries Open Access

Wei W., Komatsu K., Osuka S., Murase T., Bayasula B., Nakanishi N., Nakamura T., Goto M., Iwase A., Masubuchi S., Kajiyama H.

Reproductive Sciences 29 巻 ( 12 ) 頁： 3404 - 3412 2022年12月

記述言語：日本語出版者・発行元：Reproductive Sciences

Our previous study found that 17β-estradiol (E2) suppresses primordial follicle activation and growth in cultured mouse ovaries. In this study, we administered tamoxifen, an estrogen receptor antagonist, into the abdominal cavity of mice to clarify the relationship between primordial follicle activation and the physiological concentration of E2 in mouse ovaries. The results showed that tamoxifen promoted primordial follicle activation. Administration of tamoxifen promoted degradation of the extracellular matrix surrounding primordial follicles in the ovaries. Furthermore, tamoxifen decreased the expression of stefin A, an inhibitor of cathepsins that digest some proteins and extracellular matrix, in the ovaries. Mechanical stress produced by the extracellular matrix reportedly suppresses the activation of primordial follicles. The collective results show that tamoxifen can promote primordial follicle activation through the degradation of the extracellular matrix surrounding primordial follicles. Our results indicate that E2 suppresses primordial follicle activation in vivo and that tamoxifen may be useful as a therapeutic agent against infertility. Graphical abstract: [Figure not available: see fulltext.]

DOI： 10.1007/s43032-022-00896-0

Effectiveness of NLRP3 Inhibitor as a Non-Hormonal Treatment for ovarian endometriosis Open Access

Murakami M., Osuka S., Muraoka A., Hayashi S., Bayasula , Kasahara Y., Sonehara R., Hariyama Y., Shinjo K., Tanaka H., Miyake N., Yoshita S., Nakanishi N., Nakamura T., Goto M., Kajiyama H.

Reproductive Biology and Endocrinology 20 巻 ( 1 ) 2022年12月

出版者・発行元：Reproductive Biology and Endocrinology

Background: Endometriosis is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process that affects 10% of women of reproductive age. Ovarian endometriosis (OE) is the most common lesion in endometriosis and may cause infertility, in addition to dysmenorrhea. Hormonal treatments, which are the conventional treatment methods for endometriosis, suppress ovulation and hence are not compatible with fertility. The inflammasome is a complex that includes Nod-like receptor (NLR) family proteins, which sense pathogen-associated molecular patterns and homeostasis-altering molecular processes. It has been reported that the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing (NLRP) 3 inflammasome, which contributes to the activation of interleukin-1 beta (IL-1β), might be related to the progression of endometriosis. Therefore, the aim of the present study was to evaluate non-hormonal therapies for OE, such as inhibitors of the NLRP3 inflammasome. Methods: The expression of NLRP3 was measured in the eutopic endometrium (EM) of patients with and without endometriosis and OE samples, as well as stromal cells derived from the endometrium of patients with and without endometriosis and OE samples (endometrial stromal cells with endometriosis [ESCs] and cyst-derived stromal cells [CSCs]). The effects of an NLRP3 inhibitor (MCC950) on ESCs and CSCs survival and IL-1β production were evaluated. We then administered MCC950 to a murine model of OE to evaluate its effects on OE lesions and ovarian function. Results: NLRP3 gene and protein expression levels were higher in OE and CSCs than in EM and ESCs, respectively. MCC950 treatment significantly reduced the survival of CSCs, but not that of ESCs. Moreover, MCC950 treatment reduced the co-localization of NLRP3 and IL-1β in CSCs, as well as IL-1β concentrations in CSCs supernatants. In the murine model, MCC950 treatment reduced OE lesion size compared to phosphate-buffered saline treatment (89 ± 15 vs. 49 ± 9.3 mm3 per ovary; P < 0.05). In the MCC950-treated group, IL-1β and Ki67 levels in the OE-associated epithelia were reduced along with the oxidative stress markers of granulosa cells. Conclusions: These results indicated that NLRP3/IL-1β is involved in the pathogenesis of endometriosis and that NLRP3 inhibitors may be useful for suppressing OE and improving the function of ovaries with endometriosis.

DOI： 10.1186/s12958-022-00924-3

Predictive factors for massive hemorrhage in women with retained products of conception: a prospective study Open Access

Sonehara R., Nakamura T., Iwase A., Nishida K., Takikawa S., Murakami M., Yoshita S., Muraoka A., Miyake N., Nakanishi N., Osuka S., Goto M., Kajiyama H.

Scientific Reports 12 巻 ( 1 ) 2022年12月

出版者・発行元：Scientific Reports

Retained products of conception (RPOC) is a common cause of postpartum bleeding, which may be life-threatening; however, no evidence-based guidelines exist to assist in evaluating the risk of massive hemorrhage in women with RPOC. In this prospective study, we aimed to evaluate the predictive factors for massive hemorrhage in women with RPOC. The primary and secondary endpoints were to validate the usefulness of power Doppler color scoring (PDCS) in evaluating hypervascularity and to identify other predictive factors (such as maximum RPOC diameter and serum βhCG and Hb level at first visit), respectively. Among the 51 women with RPOC included in this study, 16 (31.5%) experienced massive hemorrhage during follow-up. None of the women with PDCS 1 or 2 (18) experienced massive hemorrhage, whereas 16 (48.5%) women with PDCS 3 or 4 (33) did. Multiple logistic regression analysis showed that the odds ratio [95% confidence interval] (P value) for PDCS, assisted reproductive technology (ART), and low serum hemoglobin (Hb) levels were 22.39 [2.25 − 3087.92] (P = 0.004), 5.72 [1.28 − 33.29] (P = 0.022), and 4.24 [0.97 − 22.99] (P = 0.056), respectively. Further, the decision tree method identified PDCS, ART, and low serum Hb levels as potential predictive factors for massive hemorrhage. This study identified PDCS as useful predictor of massive hemorrhage in women with RPOC. With additional inclusion of factors such as ART and low serum Hb levels, the risk of massive hemorrhage may be effectively evaluated, leading to better management of women of reproductive age.

DOI： 10.1038/s41598-022-15564-1

Mexiletine in spinal and bulbar muscular atrophy: a randomized controlled trial

Yamada S., Hashizume A., Hijikata Y., Inagaki T., Ito D., Kishimoto Y., Kinoshita F., Hirakawa A., Shimizu S., Nakamura T., Katsuno M.

Annals of Clinical and Translational Neurology 9 巻 ( 11 ) 頁： 1702 - 1714 2022年11月

出版者・発行元：Annals of Clinical and Translational Neurology

Objective: Patients with spinal and bulbar muscular atrophy (SBMA) often experience muscular weakness under cold exposure. Methods: In our previously conducted observational study, we assessed nerve conduction and grip strength to examine the effect of cold exposure on motor function, based on which we conducted a randomized controlled trial to evaluate the efficacy and safety of mexiletine hydrochloride in SBMA (MEXPRESS). Results: In the observational study, 51 consecutive patients with SBMA and 18 healthy controls (HCs) were enrolled. Of the patients with SBMA, 88.0% experienced cold paresis. Patients with SBMA exhibited greater prolongation of ulnar nerve distal latency under cold (SBMA, 5.6 ± 1.1 msec; HC, 4.3 ± 0.6 msec; p <0.001); the change in the distal latencies between room temperature and cold exposure conditions correlated with the change in grip power. In the MEXPRESS trial, 20 participants took mexiletine or lactose, three times a day for 4 weeks with a crossover design. There was no difference in distal latencies at room temperature and under cold exposure between mexiletine and placebo groups as the primary endpoint. However, tongue pressure and 10-sec grip and release test under cold exposure were improved in the mexiletine group. There were no serious adverse events throughout the study period. Interpretation: Cold paresis is common and associated with prolongation of distal latency in SBMA. The results of the phase II clinical trial revealed that mexiletine showed short-term safety, but it did not restore cold exposure-induced prolongation of distal latency.

DOI： 10.1002/acn3.51667

Functional Lactotrophs in Induced Adenohypophysis Differentiated from Human iPS Cells Open Access

Miyake N., Nagai T., Suga H., Osuka S., Kasai T., Sakakibara M., Soen M., Ozaki H., Miwata T., Asano T., Kano M., Muraoka A., Nakanishi N., Nakamura T., Goto M., Yasuda Y., Kawaguchi Y., Miyata T., Kobayashi T., Sugiyama M., Onoue T., Hagiwara D., Iwama S., Iwase A., Inoshita N., Arima H., Kajiyama H.

Endocrinology (United States) 163 巻 ( 3 ) 2022年3月

記述言語：日本語出版者・発行元：Endocrinology (United States)

Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.

DOI： 10.1210/endocr/bqac004

Primate-specific POTE-actin gene could play a role in human folliculogenesis by controlling the proliferation of granulosa cells Open Access

Kasahara Y., Osuka S., Takasaki N., Bayasula , Koya Y., Nakanishi N., Murase T., Nakamura T., Goto M., Iwase A., Kajiyama H.

Cell Death Discovery 7 巻 ( 1 ) 頁： 186 2021年12月

記述言語：日本語出版者・発行元：Cell Death Discovery

Patients with primary ovarian insufficiency (POI) often have a high prevalence of autoimmune disorders. To identify antigenic molecules associated with ovarian autoimmunity, we performed immunoprecipitation (IP) screening using serum from patients with POI and the established human granulosa cell line (HGrC1). POTE ankyrin domain family member E (POTEE) and POTE ankyrin domain family member F (POTEF), proteins specific to primates, were identified as candidate antigens. Using immunohistochemistry (IHC) with human ovarian tissue, POTEE or POTEF was weakly seen in the granulosa cells (GCs) of primordial follicles and primary follicles, and strongly in large antral follicles and luteal cells. Interestingly, no signals were detected in growing GCs in secondary, preantral, and small antral follicles. Thus, to explore the function of POTEE and POTEF in human folliculogenesis, we established HGrC1 cell lines with drug-inducible expression of POTEF. Expression of POTEF significantly suppressed cell proliferation in HGrC1 cells. Furthermore, chaperonin containing TCP-1 complex (CCT) components, which affect folding proteins required for cell proliferation, was bound to the actin domain of POTEF protein. Although CCT is normally localized only around the Golgi apparatus, TCP-1α, a component of CCT, co-migrated closer to the cell membrane when POTEF expression was induced. These data suggest that the interaction between POTEF and CCT components impairs the usual function of CCT during cell growth. In addition, over-accumulation of POTEF in HGrC1 cells leads to autophagic failure. It was recently reported that knockout of an autophagic gene in mice leads to a phenotype similar to human POI. These results suggested that a proper amount of POTEF is required for the maintenance of GCs in follicle pools, whereas POTEF overaccumulation might be involved in follicle atresia and the development of POI. We also showed the possibility that POTEF could be an antigen involved in ovarian autoimmunity.

DOI： 10.1038/s41420-021-00566-1

Impact of perioperative use of GnRH agonist or dienogest on ovarian reserve after cystectomy for endometriomas: a randomized controlled trial Open Access

Muraoka A., Osuka S., Yabuki A., Bayasula , Yoshihara M., Tanaka H., Sonehara R., Miyake N., Murakami M., Yoshita S., Nakanishi N., Nakamura T., Goto M., Iwase A., Kajiyama H.

Reproductive Biology and Endocrinology 19 巻 ( 1 ) 頁： 179 2021年12月

記述言語：日本語出版者・発行元：Reproductive Biology and Endocrinology

Background: Ovarian endometrioma is a common gynecological disease that is often treated with surgery or hormonal treatment. Ovarian cystectomy, a surgical procedure for ovarian endometrioma, can result in impaired ovarian reserve. Methods: We conducted a randomized controlled trial to evaluate the efficacy of hormonal treatment [gonadotropin-releasing hormone agonist (GnRHa) or dienogest (DNG)] for preserving ovarian reserve after cystectomy for ovarian endometrioma. The primary endpoint was the level of serum Anti-Müllerian hormone (AMH) as a marker of ovarian reserve. Results: Before and after laparoscopic surgery, 22 patients in the GnRHa group and 27 patients in the DNG group were administered hormonal treatment for a total of 4 months. After 1-year follow-up, >60% of the patients in the DNG group retained over 70% of their pretreatment AMH levels, whereas no patient in the GnRHa group retained their AMH levels after cystectomy (P < 0.01). Interleukin-6 (IL-6) is a key cytokine involved in inflammation. Compared with the GnRHa group, patients in the DNG group had lower IL-6 levels at the end of treatment. Conclusions: Our data revealed that DNG is more effective than GnRHa in preserving ovarian reserve after cystectomy of ovarian endometrioma. This is achieved through the reduction of the inflammatory response during the perioperative period and other endometriosis-related inflammatory reactions. Trial registration: The registration number of this trial is UMIN-CTR, UMIN000018569, registered 6 August 2015, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000021492, and Japan Registry of Clinical Trials, jRCTs041180140, registered 29 March 2019, https://jrct.niph.go.jp/en-latest-detail/jRCTs041180140. This randomized controlled trial was conducted in accordance with the CONSORT guidelines.

DOI： 10.1186/s12958-021-00866-2

Clinical Aspects of Adolescent Endometriosis Open Access

Nakamura T.

Endocrines 2 巻 ( 3 ) 頁： 301 - 310 2021年9月

出版者・発行元：Endocrines

Early diagnosis and long-term management of endometriosis is important in adolescent girls considering their potential for future pregnancy and need for preventing disease progression. However, symptoms and clinical findings of adolescent endometriosis may differ from those of typical adult endometriosis, making diagnosis difficult. In adolescents, menstrual pain may present as acyclic and unresponsive to commonly used medication. Typical imaging findings in adult endometriosis, such as ovarian endometriotic cysts and fibrotic scars, are less common in adolescents. Peritoneal lesions, characteristic of early-stage endometriosis, are commonly found in this age group. It should be noted that endometriosis may also be found in adolescents before menarche, because of premenarcheal endometriosis or congenital uterine anomaly and outflow obstruction; the latter requiring surgical correction. Although surgery is reported to be effective for pain, postsurgical recurrence rate is high, and the effect of hormonal treatment is controversial. The optimal timing for surgical intervention also remains to be determined. Here, we aim to identify the unique characteristics of endometriosis in adolescents to achieve early diagnosis and optimal management for this group of patients.

DOI： 10.3390/endocrines2030028

Very Low Levels of Serum Anti-Müllerian Hormone as a Possible Marker for Follicle Growth in Patients with Primary Ovarian Insufficiency Under Hormone Replacement Therapy Open Access

Kasahara Y., Osuka S., Bayasula , Nakanishi N., Murase T., Nakamura T., Goto M., Kotani T., Iwase A., Kikkawa F.

Reproductive Sciences 28 巻 ( 1 ) 頁： 31 - 36 2021年1月

出版者・発行元：Reproductive Sciences

Patients with primary ovarian insufficiency (POI) occasionally present with follicle growth; however, accurately predicting cycles accompanied by follicle growth is challenging. Early-stage follicles produce serum anti-Müllerian hormone (AMH), a useful marker of ovarian reserve. Therefore, serum AMH levels indicate growth of small follicles (which are difficult to detect ultrasonographically) and may predict follicle growth in patients with POI. Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit, we observed very low serum AMH levels in patients with POI. We further evaluated follicle growth in each patient during each cycle to determine the usefulness of measuring serum AMH levels as a predictor of follicle growth in patients with POI who receive hormone replacement therapy (HRT). We investigated 19 patients with POI in whom we analyzed 91 cycles; 14 cycles showed positive and 77 cycles showed negative results on serum AMH testing. The rate of cycles showing follicle growth in AMH-positive cycles was higher than that in AMH-negative cycles (64.3% vs. 6.5%, p = 0.0001). The median serum AMH level (7.7 pg/mL [25th and 75th percentiles 4.6 pg/mL and 22.3 pg/mL, respectively]) in AMH-positive cycles was lower than the lower limit of detection of conventional AMH ELISA kits. The positive predictive value of positive serum AMH levels for follicle growth was higher than that of follicle-stimulating hormone (< 10 mIU/mL). These results indicate that a very low level of serum AMH detected using picoAMH assays is a useful predictor of follicle growth in patients with POI receiving HRT.

DOI： 10.1007/s43032-020-00278-4

Establishment and characterization of cell lines from human endometrial epithelial and mesenchymal cells from patients with endometriosis Open Access

Muraoka A., Osuka S., Kiyono T., Suzuki M., Yokoi A., Murase T., Nishino K., Niimi K., Nakamura T., Goto M., Kajiyama H., Kondo Y., Kikkawa F.

F and S Science 1 巻 ( 2 ) 頁： 195 - 205 2020年11月

記述言語：日本語出版者・発行元：F and S Science

Objective: To establish and characterize cell lines derived from human endometrial epithelial cells (ECs) and mesenchymal cells (MCs) from patients with and without endometriosis. Design: In vitro experimental study. Setting: University and national cancer center research institute. Patient(s): Two women with endometriosis and two women without endometriosis. Intervention(s): Sampling of endometrial ECs and MCs. Main Outcome Measure(s): Establishing immortalized endometrial ECs and MCs with quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunocytochemical analysis, and RNA sequence profiling performed to characterize the immortalized cells and a cell proliferation assay, three-dimensional culture, and assays for hormone responses performed to characterize the features of ECs. Result(s): The qRT-PCR, immunocytochemical analysis, and Western blot analysis revealed that the ECs and MCs maintained their original features. Moreover, the immortalized cells were found to retain responsiveness to sex steroid hormones. The ECs formed a gland-like structure in three-dimensional culture, indicating the maintenance of normal EC phenotypes. The RNA sequence profiling, principal component analysis, and clustering analysis showed that the gene expression patterns of the immortalized cells were different from those of cancer cells. Several signaling pathways that were statistically significantly enriched in ECs and MCs with endometriosis were revealed. Conclusion(s): We successfully obtained four paired immortalized endometrial ECs and MCs from patients with and without endometriosis. Using these cells could help identify diagnostic and therapeutic targets for endometriosis. The cell lines established in this study will thus serve as powerful experimental tools in the study of endometriosis.

DOI： 10.1016/j.xfss.2020.09.001

Novel ovarian endometriosis model causes infertility via iron-mediated oxidative stress in mice Open Access

Hayashi S., Nakamura T., Motooka Y., Ito F., Jiang L., Akatsuka S., Iwase A., Kajiyama H., Kikkawa F., Toyokuni S.

Redox Biology 37 巻頁： 101726 2020年10月

出版者・発行元：Redox Biology

Ovarian endometriosis (OE) provides women of reproductive age with not only severe menstrual pain but also infertility and an increased risk for ovarian carcinogenesis. Whereas peritoneal endometriosis models have been developed with syngeneic implantation of minced uterine tissue and oncogenic K-ras allele with conditional Pten deletion within ovarian surface epithelium generated preneoplastic endometrial glandular morphology, followed by endometrioid adenocarcinoma, there has been no mouse model of OE similar to human counterparts, applicable to preclinical studies. Here we for the first time established a murine OE model that reveals infertility, and evaluated the involvement of iron catalyzed oxidative stress in the pathogenesis. Minced uterine tissue from female mice was implanted on ovarian surface of syngeneic mice after bursectomy to induce OE. Ectopic growth of endometrium was observed in association with ovary 4 weeks after implantation in 85.7% (12/14) of the operated mice with our protocol. Endometriotic lesions involved intestine, pancreas and peritoneal wall. Fibrosis around the ovary was prominent and increased time-dependently in the OE group. Iron accumulation was significantly increased in the OE group, leading to oxidative stress in each stage of the follicles as evaluated by 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2′-deoxyguanosine. Expression of follicle stimulating hormone receptor in the follicles revealed a significant decrease during pre-antral, antral and pre-ovulatory phases in the OE group. Finally, the number of pups was significantly reduced in the OE group in comparison to the controls. This model affords an opportunity to evaluate agents or procedures to counteract ovarian endometriosis in the preclinical settings.

DOI： 10.1016/j.redox.2020.101726

Mutant FOXL2<sup>C134W</sup> hijacks SMAD4 and SMAD2/3 to drive adult granulosa cell tumors Open Access

Weis-Banke S.E., Lerdrup M., Kleine-Kohlbrecher D., Mohammad F., Sidoli S., Jensen O.N., Yanase T., Nakamura T., Iwase A., Stylianou A., Abu-Rustum N.R., Aghajanian C., Soslow R., da Cruz Paula A., Koche R.P., Weigelt B., Christensen J., Helin K., Cloos P.A.C.

Cancer Research 80 巻 ( 17 ) 頁： 3466 - 3479 2020年9月

出版者・発行元：Cancer Research

The mutant protein FOXL2C134W is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2C134W also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2C134W binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFb mitigated the transcriptional effect of FOXL2C134W. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2C134W and its interaction with SMAD4 as potential therapeutic targets to this condition.

DOI： 10.1158/0008-5472.CAN-20-0259

Focal Adhesion Kinase-Mediated Sequences, Including Cell Adhesion, Inflammatory Response, and Fibrosis, as a Therapeutic Target in Endometriosis

Nagai T., Ishida C., Nakamura T., Iwase A., Mori M., Murase T., Bayasula , Osuka S., Takikawa S., Goto M., Kotani T., Kikkawa F.

Reproductive Sciences 27 巻 ( 7 ) 頁： 1400 - 1410 2020年7月

出版者・発行元：Reproductive Sciences

Endometriosis has several distinguishing features in the ectopic endometrium, including chronic inflammation and fibrosis. According to the retrograde menstruation theory, endometriotic cells are derived from eutopic endometrial cells, and adhesion of endometrial cells to the extracellular matrix can be the initial step in the development of endometriosis. Therefore, we hypothesized that cell adhesion, which mediates a sequence of events in the development of endometriosis triggering inflammatory responses and tissue fibrosis could be a possible therapeutic target for endometriosis. We found co-upregulation of focal adhesion kinase (FAK) and monocyte chemoattractant protein-1 (MCP-1) in the endometriotic tissues compared with that in the normal endometrium. MCP-1 secretion was significantly higher in the endometriotic stromal cells than in the eutopic endometrial stromal cells. Furthermore, co-culture of U937 cells and endometriotic stromal cells upregulated secretion of transforming growth factor-β1 (TGF-β1). A FAK inhibitor significantly inhibited the secretion of MCP-1 in the endometriotic stromal cells and TGF-β1 in the co-culture with macrophages. FAK inhibitor treatment in the murine endometriosis model demonstrated a decrease in the formation of endometriotic lesions as well as the expression of MCP-1 and TGF-β1. Our results suggest that the FAK-mediated sequential development of endometriosis, including inflammatory response and tissue fibrosis, can be a new therapeutic target in endometriosis.

DOI： 10.1007/s43032-019-00044-1

Primary Follicles Suppress the Growth of Primordial Follicles.

Wei Wei, Komatsu Kouji, Murase Tomohiko, Sonehara Reina, Miyake Natsuki, Murakami Mayuko, Ganieva Umida, Bayasula Bayasula, Yoshita Sayako, Muraoka Ayako, Hayashi Shotaro, Nakanishi Natsuki, Kasahara Yukiyo, Takasaki Nobuyoshi, Nakamura Tomoko, Osuka Satoko, Goto Maki, Kikkawa Fumitaka

REPRODUCTIVE SCIENCES 27 巻 ( SUPPL 1 ) 頁： 283A - 283A 2020年3月

The Function of POTEF in Human Granulosa Cells, an Antigen for Ovarian Autoimmunity, in POI Patients.

Kasahara Yukiyo, Osuka Satoko, Takasaki Nobuyoshi, Wei Wei, Nakanishi Natsuki, Bayasula Bayasula, Nakamura Tomoko, Murase Tomohiko, Goto Maki, Kikkawa Fumitaka

REPRODUCTIVE SCIENCES 27 巻 ( SUPPL 1 ) 頁： 199A - 199A 2020年3月

Involvement of Transcription Factor 21 in the Pathogenesis of Fibrosis in Endometriosis Open Access

Ganieva U., Nakamura T., Osuka S., Bayasula , Nakanishi N., Kasahara Y., Takasaki N., Muraoka A., Hayashi S., Nagai T., Murase T., Goto M., Iwase A., Kikkawa F.

American Journal of Pathology 190 巻 ( 1 ) 頁： 145 - 157 2020年1月

出版者・発行元：American Journal of Pathology

Repeated tissue injury and repair and fibrosis play a pivotal role in endometriosis. Fibrotic tissue consists of extracellular matrix proteins, regulated by transcriptional factors promoting cell proliferation and survival. Periostin is one of the putative key extracellular matrix proteins. This study aimed to determine whether transcription factor 21 (TCF21) is involved in the development of endometriosis as an upstream regulatory gene of periostin. Formalin-fixed, paraffin-embedded tissue samples [normal endometrium of women without endometriosis; eutopic endometrium of women with endometriosis; ovarian endometriosis (OE); and deep infiltrating endometriosis (DIE)] and respective cells were analyzed. Basal, transiently stimulated, and knocked down periostin and TCF21 concentrations in stromal cells of women with or without endometriosis were examined. Periostin and TCF21 expressions were undetected in normal endometrium of women without endometriosis, weakly positive in eutopic endometrium of women with endometriosis, moderately positive in OE, and strongly positive in DIE. Type 2 helper T-cell cytokines (IL-4, IL-13, and transforming growth factor-β1) increased the mRNA expression of periostin and TCF21. These cytokines, periostin, and TCF21 colocalized in the stroma of OE and DIE. siRNA against human TCF21 gene suppressed periostin expression. Transfection of TCF21 plasmid vector into stromal cells of women without endometriosis, which originally expressed neither periostin nor TCF21, resulted in TCF21 and periostin expression. TCF21 and periostin are involved in the regulation of fibrosis in endometriosis. TCF21 may be a promising therapeutic target and biomarker in endometriosis.

DOI： 10.1016/j.ajpath.2019.09.008

若年者における子宮内膜症査読有り

中村智子

東海産婦人科内視鏡手術研究会雑誌 8 巻頁： 3 - 9 2020年

虫垂子宮内膜症により完全型虫垂重積を生じた一例

中村智子, 林祥太郎, 清水顕, 村岡彩子, 仲西菜月, 新美薫, 大須賀智子, 後藤真紀

日本産科婦人科内視鏡学会雑誌 36 巻 ( 1 ) 頁： 185 - 188 2020年

記述言語：日本語出版者・発行元：日本産科婦人科内視鏡学会

<p> Appendiceal intussusception secondary to endometriosis is extremely rare. We report a case of left ovarian endometriosis with endometriosis-induced appendiceal intussusception in a 42-year-old woman presenting with rectal bleeding and left-sided abdominal pain. Colonoscopy revealed a cecal mass measuring 30 mm in diameter. Magnetic resonance imaging revealed extensive pelvic adhesions and a left-sided endometrioma measuring 20 mm in diameter. Laparoscopy revealed isolated appendiceal intussusception and deep infiltrating pelvic endometriosis. Laparoscopic ileocecal resection and left ovarian cystectomy were performed. Histopathological examination confirmed the diagnosis of ovarian endometriosis with endometriosis-induced appendiceal intussusception. Although rare, endometriosis-induced appendiceal intussusception should be considered in the differential diagnosis in women with endometriosis presenting with an appendiceal mass. Coordination between gynecologists and gastrointestinal surgeons is essential for an effective surgical approach to ensure optimal management.</p><p></p>

DOI： 10.5180/jsgoe.36.1_185

Upregulation of Fibroblast Growth Factors Caused by Heart and Neural Crest Derivatives Expressed 2 Suppression in Endometriotic Cells: A Possible Therapeutic Target in Endometriosis

Kato N., Iwase A., Ishida C., Nagai T., Mori M., Bayasula , Nakamura T., Osuka S., Ganiyeva U., Qin Y., Miki R., Kikkawa F.

Reproductive Sciences 26 巻 ( 7 ) 頁： 979 - 987 2019年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Reproductive Sciences

Several features exist that distinguish endometriotic cells from eutopic endometrial cells. Progesterone resistance is one of the main distinguishing features, although how progesterone resistance affects the phenotype of endometriotic cells is not fully elucidated. Heart and neural crest derivatives expressed 2 (HAND2) is a transcriptional factor that plays an important role in maintaining endometrial function in a progesterone-dependent manner. Therefore, we explored whether progesterone-dependent HAND2 is implicated in the progression of endometriosis. HAND2 was less expressed by endometriotic tissues compared to endometrial tissues. Suppression of HAND2 expression induced fibroblast growth factor 1 (FGF1), FGF2, and FGF9 in endometriotic stromal cells and consequently enhanced migration and invasion capacity. AZD4547, a FGF receptor inhibitor, diminished the migration and invasion of endometriotic cells in vitro. In the murine model of endometriosis, AZD4547 showed suppressive effects on the development of endometriotic lesions at a relatively low concentration. In conclusion, we demonstrated that FGF1, FGF2, and FGF9 are downstream effectors of HAND2 in endometriotic cells. Since HAND2-dependent FGFs play roles in enhancing invasive capacity of endometriotic cells, our results suggest that FGF receptor inhibitors, such as AZD4547, can be promising therapeutic targets for endometriosis.

DOI： 10.1177/1933719118802053

Molecular mechanism of FSHR expression induced by BMP15 in human granulosa cells Open Access

Shimizu, K; Nakamura, T; Bayasula; Nakanishi, N; Kasahara, Y; Nagai, T; Murase, T; Osuka, S; Goto, M; Iwase, A; Kikkawa, F

JOURNAL OF ASSISTED REPRODUCTION AND GENETICS 36 巻 ( 6 ) 頁： 1185 - 1194 2019年6月

出版者・発行元：Journal of Assisted Reproduction and Genetics

Purpose: Follicle-stimulating hormone receptor (FSHR) expression in granulosa cells is critical in enabling follicles to achieve accelerated growth. Although FSHR expression has been reported to be epigenetically regulated, the mechanism is unclear. Cooperation between oocytes and granulosa cells is also essential for normal follicular growth. Among oocyte-derived factors, bone morphogenetic protein 15 (BMP15) promotes follicular growth and is suggested to have epigenetic effects. We examined the role of BMP15 in the acquirement of FSHR in human granulosa cells. Methods: Immortalized non-luteinized human granulosa (HGrC1) cells were stimulated with trichostatin A (TSA) or BMP15 to analyze FSHR expression, histone modifications, and USF1/2 binding at the FSHR promoter region. Histone acetyl transferase (HAT) activity and phosphorylation of Smad 1/5/8 and p38 MAPK were examined with or without BMP15, SB203580, and LDN193189. CYP19A1 expression and estradiol production were also studied. Results: TSA and BMP15 induced FSHR mRNA expression in a dose-dependent manner and histone modifications were observed with increased binding of USF1/2. BMP15 increased FSHR protein expression, which was suppressed by LDN193189. BMP15 increased phosphorylation of Smad 1/5/8 and significantly increased HAT activity, which was inhibited by LDN193189, but not by SB203580. BMP15 increased phosphorylation of p38 MAPK and USF1. LDN193189 suppressed BMP15-induced phosphorylation of both p38 MAPK and USF1, whereas SB203580 suppressed the phosphorylation of USF1. BMP15 increased CYP19A1 mRNA expression and estradiol production. Conclusion: BMP15 induced FSHR expression in human granulosa cells through Smad and non-Smad pathways. This mechanism of FSHR induction by BMP15 may be utilized for controlling follicular growth.

DOI： 10.1007/s10815-019-01469-y

Animal models of polycystic ovary syndrome: A review of hormone-induced rodent models focused on hypothalamus-pituitary-ovary axis and neuropeptides Open Access

Osuka S., Nakanishi N., Murase T., Nakamura T., Goto M., Iwase A., Kikkawa F.

Reproductive Medicine and Biology 18 巻 ( 2 ) 頁： 151 - 160 2019年4月

出版者・発行元：Reproductive Medicine and Biology

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder among women of reproductive age and a major cause of infertility; however, the pathophysiology of this syndrome is not fully understood. This can be addressed using appropriate animal models of PCOS. In this review, we describe rodent models of hormone-induced PCOS that focus on the perturbation of the hypothalamic-pituitary-ovary (HPO) axis and abnormalities in neuropeptide levels. Methods: Comparison of rodent models of hormone-induced PCOS. Main findings: The main method used to generate rodent models of PCOS was subcutaneous injection or implantation of androgens, estrogens, antiprogestin, or aromatase inhibitor. Androgens were administered to animals pre- or postnatally. Alterations in the levels of kisspeptin and related molecules have been reported in these models. Conclusion: The most appropriate model for the research objective and hypothesis should be established. Dysregulation of the HPO axis followed by elevated serum luteinizing hormone levels, hyperandrogenism, and metabolic disturbance contribute to the complex etiology of PCOS. These phenotypes of the human disease are recapitulated in hormone-induced PCOS models. Thus, evidence from animal models can help to clarify the pathophysiology of PCOS.

DOI： 10.1002/rmb2.12262

Protective effects of mangafodipir against chemotherapy-induced ovarian damage in mice Open Access

Qin, Y; Iwase, A; Murase, T; Bayasula; Ishida, C; Kato, N; Nakamura, T; Osuka, S; Takikawa, S; Goto, M; Kotani, T; Kikkawa, F

REPRODUCTIVE BIOLOGY AND ENDOCRINOLOGY 16 巻 ( 1 ) 頁： 106 2018年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Reproductive Biology and Endocrinology

Background: Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs. Methods: Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H2O2), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment. Results: Mangafodipir attenuated apoptosis induced by H2O2 and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects. Conclusions: Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.

DOI： 10.1186/s12958-018-0426-y

TCF21 REGULATION OF PERIOSTIN IN THE PROGRESSION OF ENDOMETRIOTIC FIBROSIS.

Ganieva, U; Nakamura, T; Nguyen, PX; Wei, W; Murakami, M; Miyake, N; Nakanishi, N; Kasahara, Y; Takasaki, N; Muraoka, A; Hayashi, S; Nagai, T; Murase, T; Osuka, S; Goto, M; Iwase, A

FERTILITY AND STERILITY 110 巻 ( 4 ) 頁： E393 - E393 2018年9月

Thyroid Autoantibodies do not Impair the Ovarian Reserve in Euthyroid Infertile Women: A Cross-Sectional Study

Osuka, S; Iwase, A; Goto, M; Takikawa, S; Nakamura, T; Murase, T; Kato, N; Bayasula; Kotani, T; Kikkawa, F

HORMONE AND METABOLIC RESEARCH 50 巻 ( 7 ) 頁： 537 - 542 2018年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Hormone and Metabolic Research

Patients with primary ovarian insufficiency (POI) have a high prevalence of thyroid autoimmune disorders. However, the extent of the contribution of thyroid autoantibodies or elevated thyroid-stimulating hormone (TSH) levels to decreased ovarian reserve is unclear. Therefore, we evaluated the serum levels of anti-Müllerian hormone (AMH) and thyroid autoantibodies [antithyroperoxidase antibody (TPOAb), and antithyroglobulin antibody (TgAb)] in euthyroid infertile women. One hundred and fifty-three women with normal menstrual cycles were recruited for this retrospective study. Serum levels of AMH were compared between patients with positive and negative thyroid autoantibodies. The correlation between serum levels of AMH and each thyroid autoantibody was also evaluated. Participants were observed to be either TPOAb or TgAb positive (n=27), only TPOAb positive (n=8), only TgAb positive (n=7), TPOAb and TgAb positive (double positive; n=12), and TPOAb and TgAb negative (double negative; n=126). No significant differences were found in serum AMH levels between the TPOAb- or TgAb-positive women and the antibody-double negative women. Serum AMH levels did not show a significant correlation with the concentration of TgAb or TPOAb. On the other hand, serum AMH levels negatively correlated with TSH levels in patients who were either positive for TPOAb or TgAb. Thyroid autoantibodies are not likely to influence ovarian reserve in euthyroid women whose TSH levels fall within the normal range although elevated TSH levels may be involved in the decline of serum AMH levels.

DOI： 10.1055/a-0637-9430

Neuropeptide phoenixin (PNX) and its novel receptor GPR173 induce COX-1/COX-2 expression and PGE2 production through the CREB signaling pathway

Nguyen, XP; Iwase, A; Murase, T; Ganieva, U; Qin, Y; Bayasula, B; Shimizu, K; Osuka, S; Nakamura, T; Goto, M; Kikkawa, F

HUMAN REPRODUCTION 33 巻頁： 287 - 287 2018年7月

The regulation of ovarian follicular growth by Anti-Müllerian Hormone

Nakamura T., Murase T., Osuka S., Goto M., Iwase A.

Journal of Mammalian Ova Research 35 巻 ( 1 ) 頁： 13 - 19 2018年4月

出版者・発行元：Journal of Mammalian Ova Research

Anti-Müllerian hormone (AMH) was originally discovered as the factor responsible for the regression of the Müllerian duct during male sexual differentiation. Through studies of AMH knockout mice, AMH has also been found to regulate primordial follicle recruitment and FSH-dependent cyclic recruitment. However, the details of how AMH influences follicular growth have not been elucidated. Since the early 2000s, when serum AMH concentration was found to be a reliable biochemical marker of ovarian reserve, AMH has been in the spotlight in reproductive medicine. Several studies of AMH have led to new insights on the mechanism of AMH-regulated follicular growth. Here, we review from the earliest studies to the latest findings, AMH regulation of follicle growth with reference to the potential clinical uses of AMH and AMH inhibitors.

DOI： 10.1274/jmor.35.13

Clinical application of serum anti-Müllerian hormone as an ovarian reserve marker: A review of recent studies.

2018年3月

Directive Action of Neuropeptide Phoenixin and Its Receptor GPR173 during Folliculogenesis.

Nguyen, PX; Iwase, A; Murase, T; Ganieva, U; Qin, Y; Bayasula, B; Shimizu, K; Osuka, S; Nakamura, T; Goto, M; Kikkawa, F

REPRODUCTIVE SCIENCES 25 巻頁： 321A - 322A 2018年3月

FOXL2C134W-induced CYP19 expression via cooperation with SMAD3 in HGrC1 cells.

Belli M, Iwata N, Nakamura T, Iwase A, Stupack D, Shimasaki S

Endocrinology 2018年2月

Follicle dynamics: visualization and analysis of follicle growth and maturation using murine ovarian tissue culture.

Murase T, Iwase A, Komatsu K, Bayasula, Nakamura T, Osuka S, Takikawa S, Goto M, Kotani T, Kikkawa F

Journal of assisted reproduction and genetics 35 巻 ( 2 ) 頁： 339-343 2018年2月

膀胱鏡を併用し腹腔鏡下に膀胱部分切除術を施行した膀胱子宮内膜症の2例

石田千晴, 村岡彩子, 邨瀬智彦, 中村智子, 大須賀智子, 後藤真紀, 野元正崇, 岩瀬明

日本産科婦人科内視鏡学会雑誌 34 巻 ( 2 ) 頁： 204 - 210 2018年

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：日本産科婦人科内視鏡学会

<p> Bladder endometriosis is a rare pelvic dysfunction with painful urination and hematuria.</p><p> We successfully managed two cases of bladder endometriosis by cystoscopy-assisted laparoscopic partial cystectomy. </p><p><b>Case 1:</b> A 40-year-old woman presented with urodynia during menstruation for four years. Cystoscopy revealed a dark-red polypoid lesion on the posterior bladder wall. She underwent hormonal therapy with GnRH-agonist for six months followed by dienogest for a few months. She finally decided to undergo an operation because of poor improvement of symptoms. Incision line was determined using cystoscopy with a margin from both ureteral orifices and then laparoscopic partial cystectomy was performed. She was discharged after confirmation of no leakage by cystography at post-operative day 7. </p><p><b>Case 2:</b> A 44-year-old woman was diagnosed with endometriosis at the age of 30 and had been taking low dose estrogen and progestin (LEP). She discontinued LEP owing to breast cancer and then started to feel pain during menstruation, urination, and defecation. Cystoscopy revealed a polypoid lesion on the posterior bladder wall. She underwent laparoscopic hysterectomy, bilateral salpingo-oophorectomy, and cystoscopy-assisted partial cystectomy. Her postoperative course was uneventful, and she was discharged at post-operative day 8. Altogether, this case report suggests that using cystoscopy to avoid unnecessary ureterovesicostomy and maintaining adequate distance from both ureteral orifices is useful to determine the incision line. Cooperation with urologists is necessary when performing surgery in patients with bladder endometriosis.</p>

DOI： 10.5180/jsgoe.34.2_204

Retrospective analysis of magnetic resonance imaging for differentiating intraligamentous leiomyomas from subserosal leiomyomas

Shimizu, K; Iwase, A; Sakurai, Y; Nakamura, T; Osuka, S; Takikawa, S; Goto, M; Kikkawa, F

EUROPEAN JOURNAL OF OBSTETRICS & GYNECOLOGY AND REPRODUCTIVE BIOLOGY 215 巻頁： 256 - 257 2017年8月

PAI-1 in granulosa cells is suppressed directly by statin and indirectly by suppressing TGF- and TNF- in mononuclear cells by insulin-sensitizing drugs

Yamada-Nomoto, K; Yoshino, O; Akiyama, I; Iwase, A; Ono, Y; Nakamura, T; Harada, M; Nakashima, A; Shima, T; Ushijima, A; Osuga, Y; Chang, RJ; Shimasaki, S; Saito, S

AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 78 巻 ( 1 ) 2017年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Journal of Reproductive Immunology

Problem: Plasminogen activator inhibitor-1 (PAI-1) is elevated in women with polycystic ovary syndrome (PCOS), but the regulation in granulosa cells (GCs) is unclear. Method of study: PAI-1 expression in PCOS ovaries was investigated immunohistologically. PAI-1 expressions in HGrC1, a human GC cell line, were investigated at mRNA and activity levels. The expressions of TGF-β and TNF-α in peritoneal fluid mononuclear cells (PFMCs) were measured with quantitative PCR. Results: Little PAI-1 expression is observed in healthy GCs, whereas GCs of PCOS and atretic follicle exhibit distinct expression in vivo. In vitro study using HGrC1 shows that TGF-β and TNF-α increase PAI-1 mRNA and its activity, and both together exhibit a synergistic effect. The expression of PAI-1 mRNA is suppressed by simvastatin. Moreover, insulin-sensitizing drugs (metformin, pioglitazone, and rosiglitazone) suppress LPS-induced TGF-β and TNF-α mRNA expression in PFMC. Conclusion: Statin and insulin-sensitizing drugs may provide a potential therapy for PCOS via down-regulation of PAI-1 expression in GCs and down-regulation of TGF-β and TNF-α expression in PFMC, respectively.

DOI： 10.1111/aji.12669

Growth Differentiation Factor 9 Induces Granulosa Cell Proliferation via Inhibition of the Expression of AMH Type II Receptor.

Iwase Akira, Osuka Satoko, Bayasula Bayasula, Takikawa Sachiko, Murase Tomohiko, Nakamura Tomoko, Goto Maki, Kikkawa Fumitaka

REPRODUCTIVE SCIENCES 24 巻頁： 163A-163A 2017年3月

Analysis of Follicular Growth and Oocyte Maturation in Cultured Murine Ovarian Tissue.

Murase Tomohiko, Iwase Akira, Nguyen Phuoc Xuan, Ganieva Umida, Qin Ying, Nakanishi Natsuki, Kasahara Yukiyo, Nagai Takashi, Shimizu Ken, Ishida Chiharu, Kato Nao, Osuka Satoko, Nakamura Tomoko, Takikawa Sachiko, Goto Maki, Kikkawa Fumitaka

REPRODUCTIVE SCIENCES 24 巻頁： 292A-292A 2017年3月

Kisspeptin in the Hypothalamus of 2 Rat Models of Polycystic Ovary Syndrome

Osuka, S; Iwase, A; Nakahara, T; Kondo, M; Saito, A; Bayasula; Nakamura, T; Takikawa, S; Goto, M; Kotani, T; Kikkawa, F

ENDOCRINOLOGY 158 巻 ( 2 ) 頁： 367 - 377 2017年2月

CYP51A1 induced by growth differentiation factor 9 and follicle-stimulating hormone in granulosa cells is a possible predictor for unfertilization 査読有り

Nakamura T, Iwase A, Bayasula B, Nagatomo Y, Kondo M, Nakahara T, Takikawa S, Goto M, Kotani T, Kiyono T, Kikkawa F

Reproductive sciences 22 巻 ( 3 ) 頁： 377-84 2015年3月

A Placental Site Trophoblastic Tumor Complicated with Arteriovenous Malformation: A Case Report

Tomoko Nakamura, Akira Iwase, Chiharu Ishida, Sachiko Takikawa, Maki Goto, Fumitaka Kikkawa

Clinical Case Reports 5 巻 ( 9 ) 2015年

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