Establishment of a Human Nonluteinized Granulosa Cell Line that Transitions from the Gonadotropin-Independent to the Gonadotropin-Dependent Status

名古屋大学Nagoya University博士(医学)doctoral thesi

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

BackgroundPolycystic 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.MethodsA 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.ResultsUKT 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.ConclusionsUKT 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

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

IntroductionEndometriosis 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.MethodsSerum 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.ResultsCandidate 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.DiscussionFurther studies are required to examine the functional importance of these miRNAs to elucidate the pathological mechanisms of endometriosis and pregnancy

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

Abstract Background: Patients with primary ovarian insufficiency (POI) occasionally present with follicle growth; however, it is difficult to predict exactly which cycles will show follicle growth. Serum anti-Müllerian hormone (AMH), a useful marker for ovarian reserve, is produced in early-stage follicles. Therefore, AMH levels should reflect the presence of small follicles, which are difficult to detect using ultrasonography, and may be useful as a marker for predicting follicle growth in patients with POI. Methods: Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit, we found very low serum AMH levels in patients with POI. We assessed the follicle growth of each patient during each cycle to clarify the usefulness of measuring serum AMH levels for predicting follicle growth in patients with POI under hormone replacement therapy (HRT). Results: A total of 91 cycles of 19 patients with POI were analyzed in this study. Five patients presented with positive serum AMH levels during the observational periods, and all five experienced follicle growth. Only two of the 14 patients with negative serum AMH levels had follicle growth. Serum AMH and follicle-stimulating hormone (FSH) levels in cycles with follicle growth were significantly higher than in cycles without follicle growth. The median serum AMH level (2.77 pg/mL; 25th, 75th percentiles: 0.0, 9.64) in cycles with follicle growth 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 FSH (&lt; 10 mIU/mL).Conclusions: Measurement of very low levels of serum AMH using picoAMH assays is useful for prediction of follicle growth in patients with POI under HRT conditions. Trial registration: UMIN-CTR UMIN000029464. Retrospectively registered on April 4, 2018. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000033550</jats:p

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

Abstract Background Patients with primary ovarian insufficiency (POI) occasionally present with follicle growth; however, it is difficult to predict exactly which cycles will show follicle growth. Serum anti-Müllerian hormone (AMH), a useful marker for ovarian reserve, is produced in early-stage follicles. Therefore, AMH levels should reflect the presence of small follicles, which are difficult to detect using ultrasonography, and may be useful as a marker for predicting follicle growth in patients with POI. Methods Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit, we found very low serum AMH levels in patients with POI. We assessed the follicle growth of each patient during each cycle to clarify the usefulness of measuring serum AMH levels for predicting follicle growth in patients with POI under hormone replacement therapy (HRT). Results A total of 91 cycles of 19 patients with POI were analyzed in this study. Five patients presented with positive serum AMH levels during the observational periods, and all five experienced follicle growth. Only two of the 14 patients with negative serum AMH levels had follicle growth. Serum AMH and follicle-stimulating hormone (FSH) levels in cycles with follicle growth were significantly higher than in cycles without follicle growth. The median serum AMH level (2.77 pg/mL; 25 th , 75 th percentiles: 0.0, 9.64) in cycles with follicle growth 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 FSH (&lt; 10 mIU/mL). Conclusions Measurement of very low levels of serum AMH using picoAMH assays is useful for prediction of follicle growth in patients with POI under HRT conditions.</jats:p

Usefulness of ultrasensitive anti-Müllerian hormone assay to predict follicle growth in patients with primary ovarian insufficiency

Abstract Background: Patients with primary ovarian insufficiency (POI) present with follicle growth occasionally, however, it is difficult to predict the exact cycles with follicle growth. Serum anti-Müllerian hormone (AMH), a useful marker for ovarian reserve, is produced in early-stage follicles. Therefore, AMH levels should reflect the existence of small follicles which are difficult to detect using ultrasonography and may be useful for predicting follicle growth in patients with POI. Methods: Using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) kit, we found very low serum AMH levels of patients with POI; we further assessed the follicle growth of each patient and their cycles to clarify the usefulness of measuring serum AMH levels for predicting the follicle growth in patients with POI. Results: A total of 91 cycles of 19 patients with POI were analyzed in this study. Five patients presented with positive serum AMH levels during the observational periods and all five experienced follicle growth. Only two of the 14 patients with negative serum AMH levels had follicle growth. Serum AMH and follicle-stimulating hormone (FSH) levels in cycles with follicle growth were significantly higher than in cycles without follicle growth. The median serum AMH level (2.77 pg/mL; 25th, 75th percentiles: 0.0, 9.64) in cycles with follicle growth were 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 FSH (&lt;10 mIU/mL).Conclusions: Measuring very low levels of serum AMH using picoAMH assays is useful for the prediction of follicle growth in patients with POI.</jats:p

Tamoxifen Activates Dormant Primordial Follicles in Mouse Ovaries

AbstractOur 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</jats:p

Effect of the neuropeptide phoenixin and its receptor GPR173 during folliculogenesis

Folliculogenesis is a complex process, defined by the growth and development of follicles from the primordial population. Granulosa cells (GCs) play a vital role in every stage of follicular growth through proliferation, acquisition of gonadotropic responsiveness, steroidogenesis and production of autocrine/paracrine factors. A recently discovered hypothalamic neuropeptide phoenixin is involved in the regulation of the reproductive system. Phoenixin acts through its receptor, G protein-coupled receptor 173 (GPR173), to activate the cAMP/PKA pathway leading to the phosphorylation of CREB (pCREB). Here, we demonstrated the expression patterns of phoenixin and GPR173 in human ovary and explored its role in folliculogenesis. Phoenixin and GPR173 were both expressed in the human ovarian follicle, with increased expression in GCs as the follicle grows. Phoenixin treatment at 100 nM for 24 h induced the proliferation of human non-luteinized granulosa cell line, HGrC1 and significantly increased the expression levels of CYP19A1, FSHR, LHR and KITL, but decreased NPPC expression levels. These effects were suppressed by GPR173 siRNA. The expression level of CREB1, pCREB and estradiol (E2) production in the culture medium was significantly enhanced by phoenixin treatment in a concentration-dependent manner. Phoenixin also significantly increased the follicular area in a murine ovarian tissue culture model, leading to an increased number of ovulated oocytes with a higher level of maturation. Taken together, our data demonstrate that phoenixin is an intraovarian factor that promotes follicular growth through its receptor GPR173 by accelerating proliferation of GCs, inducing E2 production and increasing the expression of genes related to follicle development.</jats:p

Effectiveness of NLRP3 Inhibitor as a Non-Hormonal Treatment for Ovarian Endometriosis

Abstract 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 for endometriosis suppress ovulation; hence, they are not compatible with fertility. The inflammasome is a complex that includes Nod-like receptor (NLR) family proteins that sense pathogen-/danger‐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 the inhibitors of the NLRP3 inflammasome. Methods The expression of NLRP3 was measured in the eutopic endometrium (EM) of patients with/without endometriosis and OE and stromal cells derived from the endometrium of patients with endometriosis and OE (endometrial stromal cells [ESCs] and cyst-derived stromal cells [CSCs]). The effect of an NLRP3 inhibitor (MCC950) on ESC and CSC survival and IL-1β production was 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 and IL-1β concentrations in CSC 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 &lt; 0.05). In addition, IL-1β and Ki67 levels in the OE-associated epithelia and oxidative stress markers of granulosa cells were reduced in the MCC950-treated group. Conclusions These results indicate 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.</jats:p