Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism, and we previously found that androgens activate endoplasmic reticulum (ER) stress in granulosa cells from patients with PCOS. In addition, recent studies demonstrated the accumulation of advanced glycation end products (AGEs) in granulosa cells from PCOS patients, which contribute to the pathology. Therefore, we hypothesized that androgens upregulate the receptor for AGEs (RAGE) expression in granulosa cells by activating ER stress, thereby increasing the accumulation of AGEs in these cells and contributing to the pathology. In the present study, we show that testosterone increases RAGE expression and AGE accumulation in cultured human granulosa-lutein cells (GLCs), and this is reduced by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Knockdown of the transcription factor C/EBP homologous protein (CHOP), an unfolded protein response factor activated by ER stress, inhibits testosterone-induced RAGE expression and AGE accumulation. The expression of RAGE and the accumulation of AGEs are upregulated in granulosa cells from PCOS patients and dehydroepiandrosterone-induced PCOS mice. Administration of the RAGE inhibitor FPS-ZM1 or TUDCA to PCOS mice reduces RAGE expression and AGE accumulation in granulosa cells, improves their estrous cycle, and reduces the number of atretic antral follicles. In summary, our findings indicate that hyperandrogenism in PCOS increases the expression of RAGE and accumulation of AGEs in the ovary by activating ER stress, and that targeting the AGE-RAGE system, either by using a RAGE inhibitor or a clinically available ER stress inhibitor, may represent a novel approach to PCOS therapy.

Recent studies have uncovered the critical role of aryl hydrocarbon receptor (AHR) in various diseases, including obesity and cancer progression, independent of its previously identified role as a receptor for endocrine-disrupting chemicals (EDCs). We previously demonstrated that endoplasmic reticulum (ER) stress, a newly recognized local factor in the follicular microenvironment, is activated in granulosa cells from patients with polycystic ovary syndrome (PCOS) and a mouse model of the disease. By affecting diverse functions of granulosa cells, ER stress contributes to PCOS pathology. We hypothesized that expression of AHR and activation of its downstream signaling were upregulated by ER stress in granulosa cells, irrespective of the presence of EDCs, thereby promoting PCOS pathogenesis. In this study, we found that AHR, AHR nuclear translocator (ARNT), and AHR target gene cytochrome P450 1B1 (CYP1B1) were upregulated in the granulosa cells of PCOS patients and model mice. We examined CYP1B1 as a representative AHR target gene. AHR and ARNT were upregulated by ER stress in human granulosa-lutein cells (GLCs), resulting in an increase in the expression and activity of CYP1B1. Administration of the AHR antagonist CH223191 to PCOS mice restored estrous cycling and decreased the number of atretic antral follicles, concomitant with downregulation of AHR and CYP1B1 in granulosa cells. Taken together, our findings indicate that AHR activated by ER stress in the follicular microenvironment contributes to PCOS pathology, and that AHR represents a novel therapeutic target for PCOS.

Endometriosis exerts detrimental effects on ovarian physiology and compromises follicular health. Granulosa cells from patients with endometriosis are characterized by increased apoptosis, as well as high oxidative stress. Endoplasmic reticulum (ER) stress, a local factor closely associated with oxidative stress, has emerged as a critical regulator of ovarian function. We hypothesized that ER stress is activated by high oxidative stress in granulosa cells in ovaries with endometrioma and that this mediates oxidative stress–induced apoptosis. Human granulosa-lutein cells (GLCs) from patients with endometrioma expressed high levels of mRNAs associated with the unfolded protein response (UPR). In addition, the levels of phosphorylated ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), were elevated in granulosa cells from patients with endometrioma. Given that ER stress results in phosphorylation of ER stress sensor proteins and induces UPR factors, these findings indicate that these cells were under ER stress. H2O2, an inducer of oxidative stress, increased expression of UPR-associated mRNAs in cultured human GLCs, and this effect was abrogated by pretreatment with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor in clinical use. Treatment with H2O2 increased apoptosis and the activity of the pro-apoptotic factors caspase-8 and caspase-3, both of which were attenuated by TUDCA. Our findings suggest that activated ER stress induced by high oxidative stress in granulosa cells in ovaries with endometrioma mediates apoptosis of these cells, leading to ovarian dysfunction in patients with endometriosis.

It has been recently recognized that prenatal androgen exposure is involved in the development of polycystic ovary syndrome (PCOS) in adulthood. In addition, the gut microbiome in adult patients and rodents with PCOS differs from that of healthy individuals. Moreover, recent studies have suggested that the gut microbiome may play a causative role in the pathogenesis of PCOS. We wondered whether prenatal androgen exposure induces gut microbial dysbiosis early in life and is associated with the development of PCOS in later life. To test this hypothesis, we studied the development of PCOS-like phenotypes in prenatally androgenized (PNA) female mice and compared the gut microbiome of PNA and control offspring from 4 to 16 weeks of age. PNA offspring showed a reproductive phenotype from 6 weeks and a metabolic phenotype from 12 weeks of age. The αdiversity of the gut microbiome of the PNA group was higher at 8 weeks and lower at 12 and 16 weeks of age, and the βdiversity differed from control at 8 weeks.However, a significant difference in the composition of gut microbiome between the PNA and control groups was already apparent at 4 weeks. Allobaculum and Roseburia were less abundant in PNA offspring, and may therefore be targets for future interventional studies. In conclusion, abnormalities in the gut microbiome appear as early as or even before PCOS-like phenotypes develop in PNA mice.Thus, the gut microbiome in early life is a potential target for the prevention of PCOS in later life. K E Y W O R D S androgens, delayed effects of prenatal exposure, gastrointestinal microbiome, polycystic ovary syndrome How to cite this article: Kusamoto A, Harada M, Azhary JMK, et al. Temporal relationship between alterations in the gut microbiome and the development of polycystic ovary syndrome-like phenotypes in prenatally androgenized female mice.

Purpose:In 2017, the first guidelines for fertility preservation in cancer patients were published in Japan. However, the impact of the guidelines remains unknown.Therefore, the authors conducted a nationwide survey on cryopreservation procedures in the period from shortly before to after publication of the guidelines (2016-2019) and compared the results with our previous survey (2011)(2012)(2013)(2014)(2015). The authors also surveyed reproductive specialists' awareness of the guidelines and implementation problems. Methods:The authors sent a questionnaire to 618 assisted reproductive technology facilities certified by the Japanese Society of Obstetrics and Gynecology. How to cite this article: Kunitomi C, Harada M, Sanada Y, et al. The possible effects of the Japan Society of Clinical Oncology Clinical Practice Guidelines 2017 on the practice of fertility preservation in female cancer patients in Japan.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-age women, affecting up to 15% of women in this group, and the most common cause of anovulatory infertility. Although its etiology remains unclear, recent research has revealed the critical role of endoplasmic reticulum (ER) stress in the pathophysiology of PCOS. ER stress is defined as a condition in which unfolded or misfolded proteins accumulate in the ER because of an imbalance in the demand for protein folding and the protein-folding capacity of the ER. ER stress results in the activation of several signal transduction cascades, collectively termed the unfolded protein response (UPR), which regulates various cellular activities. In principle, the UPR restores homeostasis and keeps the cell alive. However, if the ER stress cannot be resolved, it induces programmed cell death. ER stress has recently been recognized to play diverse roles in both physiological and pathological conditions of the ovary. In this review, we summarize current knowledge of the roles of ER stress in the pathogenesis of PCOS. ER stress pathways are activated in the ovaries of both a mouse model of PCOS and in humans, and local hyperandrogenism in the follicular microenvironment associated with PCOS is responsible for activating these. The activation of ER stress contributes to the pathophysiology of PCOS through multiple effects in granulosa cells. Finally, we discuss the potential for ER stress to serve as a novel therapeutic target for PCOS.

Endoplasmic reticulum (ER) stress activated in granulosa cells contributes to the pathophysiology of polycystic ovary syndrome (PCOS). In addition, recent studies have demonstrated that Notch signaling plays multiple roles in the ovary via cell-to-cell interactions. We hypothesized that ER stress activated in granulosa cells of antral follicles in PCOS induces Notch signaling in these cells, and that activated Notch signaling induces aberrant cumulus-oocyte complex (COC) expansion. Expression of Notch2 and Notch-target transcription factors was increased in granulosa cells of PCOS patients and model mice. ER stress increased expression of Notch2 and Notch-target transcription factors in cultured human granulosa-lutein cells (GLCs). Inhibition of Notch signaling abrogated ER stress-induced expression of genes associated with COC expansion in cultured human GLCs, as well as ER stress-enhanced expansion of cumulus cells in cultured murine COCs. Furthermore, inhibition of Notch signaling reduced the areas of COCs in PCOS model mice with activated ER stress in the ovary, indicating that Notch signaling regulates COC expansion in vivo. Our findings suggest that Notch2 signaling is activated in granulosa cells in PCOS and regulates COC expansion. It remains to be elucidated whether aberrant COC expansion induced by the ER stress-Notch pathway is associated with ovulatory dysfunction in PCOS patients.

Study question From when do abnormality in gut microbiome and phenotypes of PCOS appear during the process of growth? Summary answer Reproductive phenotypes of PCOS appear from 6 weeks and metabolic phenotypes from 12 weeks onward. Alteration in gut microbiome appears as early as 4 weeks. What is known already The etiology of PCOS remains largely unknown, however PCOS is considered as a complex multigenic disorder with strong epigenetic and environmental influence. Previous studies have suggested that fetal over-exposure to androgens could be the main factor of the development of PCOS after birth. On the other hands, recent studies on both human and PCOS rodent models have demonstrated the association between PCOS and alteration of gut microbiome in adulthood. Furthermore, it was recently reported that gut microbiome in obese adolescent with PCOS is different from obese adolescent without PCOS. Study design, size, duration A rodent PCOS model induced by prenatal dehydroepiandrosterone (DHT) exposure was applied to this study. Phenotypes and gut microbiome were compared between PCOS model mice (n = 12/group) and control mice (n = 10/group) at each stage of growth; 4 weeks (prepuberty), 6 weeks (puberty), 8 weeks (adolescent), 12 weeks (young adult), and 16 weeks (adult). The determinants for PCOS phenotypes are onset of puberty, estrous cycle, morphology of ovaries, serum testosterone level, body weight, and insulin resistance. Participants/materials, setting, methods Pregnant dams were subcutaneously injected on days of 16, 17, and 18 of gestation with either sesame oil for control groups or sesame oil containing 250µg of DHT for prenatal DHT groups. The evaluation of PCOS phenotypes and gut microbiome in female offspring were performed at each stage of growth. For examination of gut microbiota, next generation sequencing and bioinformatics analysis of 16S rRNA genes were performed on DNA extracted from mouse fecal samples. Main results and the role of chance Prenatal DHT mice exhibited delayed puberty onset, disrupted estrous cycle, and significantly increased testosterone levels from 6 weeks onward. Significantly increased atretic antral follicles were observed in prenatal DHT mice at 6, 12, and 16 weeks. Prenatal DHT mice showed significantly decreased body weight at 4, 6, 8 weeks and increased body weight from 12 weeks onward. As for gut microbiome, alpha-diversity was significantly different between control and prenatal DHT mice from 8 weeks onward and beta-diversity was significantly different at 6 and 8 weeks. Altered composition of gut microbiota was observed as early as 4 weeks. At phylum level, Firmicutes are significantly increased in prenatal DHT mice at 4 and 8 weeks and decreased at 16 weeks. Actinobacteria phylum showed significant decrease at 6 and 8 weeks in prenatal DHT mice. At genus level, relative abundance of several bacterial taxa significantly differed between control and prenatal DHT mice; some taxa, such as Allobaculum, Adlercreutzia, Bilophila, Clostridium, Gemella, Gemmiger, Roseburia, Ruminococcus, Staphylococcus, and Sutterella, exhibited constant increase or decrease in prenatal DHT mice during the process of growth. Interestingly, Roseburia was never detected in prenatal DHT mice, while approximately half of control mice harbored Roseburia at 12 and 16 weeks. Limitations, reasons for caution It is not clearly determined whether alteration in gut microbiome is cause or result of PCOS development, although the changes in gut microbiome seemed to precede the appearance of typical PCOS phenotypes in the present study. Mouse model does not completely recapitulate human PCOS. Wider implications of the findings: Our findings suggest that prenatal androgen exposure causes alteration of gut microbiome from pre-puberty onward, even before PCOS phenotypes become apparent. Intervention for girls at risk of PCOS with pre/pro-biotics may prevent them from developing PCOS in future. Trial registration number Not applicable