Metformin reduces androgen receptor and upregulates homeobox A10 expression in uterine endometrium in women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) causes anovulation and is associated with a reduced clinical pregnancy rate. Metformin, which is widely used for treating PCOS, can lead to successful pregnancy by restoring the ovulation cycle and possibly improving endometrial abnormality during the implantation period. However, the mechanism by which metformin improves endometrial abnormality remains unknown. Women with PCOS have an aberrant expression of steroid hormone receptors and homeobox A10 (HOXA10), which is essential for embryo implantation in the endometrium. METHODS: In this study, we examined whether metformin affects androgen receptor (AR) and HOXA10 expression in PCOS endometrium in vivo and in human endometrial cell lines in vitro. Expression of AR and HOXA10 was evaluated by immunohistochemistry, fluorescent immunocytochemistry, and western blot analysis. RESULTS: AR expression was localized in both epithelial and stromal cells; however, HOXA10 expression was limited to only stromal cells in this study. In women with PCOS, 3 months after metformin treatment, the expression of AR was reduced in epithelial and stromal cells in comparison to their levels before treatment. In contrast, HOXA10 expression in the stromal cells with metformin treatment increased in comparison to its level before treatment. Further, we showed that metformin counteracted the testosterone-induced AR expression in both Ishikawa cells and human endometrial stromal cells (HESCs); whereas, metformin partly restored the testosterone-reduced HOXA10 expression in HESCs in vitro. CONCLUSIONS: Our results suggest that metformin may have a direct effect on the abnormal endometrial environment of androgen excess in women with PCOS. TRIAL REGISTRATION: The study was approved by the Ethical Committee of Fukushima Medical University (approval no. 504, approval date. July 6, 2006), and written informed consent was obtained from all patients. https://www.fmu.ac.jp/univ/sangaku/rinri.html.

Different mutation profiles between epithelium and stroma in endometriosis and normal endometrium.

STUDY QUESTION: Are there common mutation profiles between epithelial and stromal cells in ovarian endometriotic tissue and the normal endometrium? SUMMARY ANSWER: Our study revealed no common mutations between epithelial and stromal cells in ovarian endometriotic tissue and the normal endometrium. WHAT IS KNOWN ALREADY: Epithelial cells in both ovarian endometriotic tissue and the normal endometrium harbor somatic mutations in cancer-associated genes such as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and KRAS proto-oncogene, GTPase (KRAS). STUDY DESIGN, SIZE, DURATION: We performed a retrospective study to identify the mutation profiles of stromal cells in endometriotic tissue and the normal endometrium. We collected 11 endometriotic stroma samples and 10 normal endometrial stroma samples between 2013 and 2017 at a tertiary care center. PARTICIPANTS/MATERIALS, SETTING, METHODS: The laser microdissection method was used to obtain stromal cells in ovarian endometriotic and normal endometrial tissues from patients with ovarian endometriosis and/or other non-invasive gynecological diseases. Target gene sequencing was performed to assess and compare the mutation profiles of stromal cells with those of epithelial cells obtained in our previous study. For target gene sequencing, 76 genes were selected based on previous genomic analyses for ovarian endometriosis, normal endometrium, endometriosis-related ovarian cancer and endometrial cancer. MAIN RESULTS AND THE ROLE OF CHANCE: Stromal samples in ovarian endometrioma and normal endometrium harbor somatic mutations (18 mutations in 11 endometriosis samples and 16 mutations in 10 normal endometrial samples) but did not share any mutations with paired epithelial samples. The mutant allele frequency of stromal samples was significantly lower than that of epithelial samples in ovarian endometrioma (P = 6.0 × 10-11) and normal endometrium (P = 1.4 × 10-7). LIMITATIONS, REASONS FOR CAUTION: The number of genes evaluated in the mutational analysis was limited. Additionally, the functional roles of somatic mutations in stromal cells remain unclear. WIDER IMPLICATIONS OF THE FINDINGS: Different mutation profiles between paired epithelial and stromal cells in both ovarian endometrioma and normal endometrium suggest that origins of epithelial and stromal cells would be independent of each other in both normal endometrium and ovarian endometrioma; however, the theory of epithelial-mesenchymal transition is proposed in ovarian endometrioma. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the Japan Society for the Promotion of Science KAKENHI grant number JP15H02373 (Grant-in-Aid for Scientific Research A for I.I.), JP16H06267 (Grant-in-Aid for Young Scientists A for K.Y.), JP17K08688 (Grant-in-Aid for Scientific Research C for H.N.) and JP16H06279 (Grant-in-Aid for Scientific Research on Innovative Areas-Platforms for Advanced Technologies and Research Resources for H.N. and K.Y). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

MR appearance of normal uterine endometrium considering menstrual cycle: differentiation with benign and malignant endometrial lesions.

Background The thickness and signal intensity (SI) of normal uterine endometrium on T2-weighted (T2W) imaging changes depend on the menstrual cycle phase. Cases of normal endometrium that appear similar to endometrial lesions sometimes occur, and may result in misdiagnosis. Purpose To investigate normal endometrial appearance in luteal phase (LP) compared to that in follicular phase (FP), and to differentiate these appearances with those of endometrial lesions. Material and Methods Thirty-two normal volunteers prospectively underwent magnetic resonance (MR) examinations during LP and FP. Patients with pathologically confirmed endometrial polyps ( n = 9), hyperplasia ( n = 7), and cancer ( n = 15), who underwent MR examinations, were evaluated for comparison. Endometrial appearance was categorized into the following five types on sagittal T2W imaging and compared between LP, FP, and endometrial lesions: type 1, homogeneous higher SI; type 2, homogeneous iso SI; type 3, a bright midline and a peripheral iso SI layer; type 4, a lower/iso SI central line; and type 5, heterogeneous lower/iso SI. Endometrial thickness and SI were measured and also compared. Results Endometrial lesions were more frequently categorized as type 5 than normal endometrium ( P < 0.05). Endometrial thickness in LP (mean, 1.0 cm) was significantly greater than that in FP (0.6 cm), but not significantly different from polyps (1.1 cm), hyperplasia (1.0 cm), and cancer (0.9 cm). SI in FP was significantly higher than that in LP and that of all endometrial lesions. Conclusion Differentiation between normal endometrium in LP and endometrial lesions may be difficult based on thickness alone. Heterogeneous low SI may help to differentiate normal endometrium from endometrial lesions. Performing MR imaging during FP may also help due to higher SI of normal endometrium.

Clomiphene citrate affects the receptivity of the uterine endometrium.

Purpose: To investigate whether clomiphene citrate (CC) affects uterine receptivity or not, we evaluated pregnancy rates (PR) during the hormone replacement cycle (HRC) according to the period between the last day of CC administration and the day of embryo transfer (ET). Methods: From March 2008 through March 2010, a total of 378 treatment cycles among 378 patients who received CC and had to avoid fresh ET due to a thin uterine endometrium were recruited. All patients underwent thawed ET using HRC. PRs were evaluated according to the period between the last CC treatment and the day of ET. Results: PR for the groups in which the period between the last CC treatment and the day of ET increased to more than 91 days were significantly higher than that for group in which the period was less than 90 days (p < 0.05). Conclusions: A lower PR was shown by the patients who underwent thawed ET in the HRC within 90 days after their last CC treatment, which shows that CC affects the receptivity of the uterine endometrium.

Prostaglandin therapy during the proliferative phase improves pregnancy rates following frozen embryo transfer in a hormone replacement cycle.

AIM: To determine the efficacy of prostaglandin administration during the proliferative phase in order to improve pregnancy rates following frozen embryo transfer during a hormone replacement cycle (HRC). METHODS: From September 2010 through March 2012, patients (n = 135) were recruited who had undergone oocyte retrieval during a stimulation cycle with clomiphene and had deferred fresh embryo transfer (ET) due to a thin uterine endometrium. All patients were less than 40 years of age and underwent thawed ET following all embryo cryopreservation, and were randomly divided into two groups for thawed ET using a conventional hormone replacement cycle with or without prostaglandin derivatives (prostaglandin or conventional group). Prostaglandin derivatives were administrated during the proliferative phase. Pregnancy and implantation rates following frozen ET were compared between the two groups. RESULTS: Although the endometrial thickness on the day of ET was similar for the prostaglandin and conventional groups, the pregnancy and implantation rates for the prostaglandin group were 40.0% and 22.0%, respectively, which was significantly higher than the rates for the conventional group (P < 0.01). CONCLUSION: Among patients who avoided fresh ET due to a thin endometrium, the pregnancy rate following a thawed cycle was low. However, it was improved when prostaglandin derivatives were used during the proliferative phase.

Chronic Endometritis and Uterine Endometrium Microbiota in Recurrent Implantation Failure and Recurrent Pregnancy Loss.

The aim of this study was to evaluate whether chronic endometritis (CE) and uterine endometrium microbiota were associated with repeated implantation failures (RIFs) and recurrent pregnancy losses (RPLs). In this prospective study, uterine endometrial specimens were obtained from 24 women with RIF, 27 with RPL, and 29 fertile control women. Immunohistochemical staining of CD138 for CE and 16S ribosomal RNA (rRNA) sequencing analysis for uterine endometrium microbiota were performed simultaneously. To assess CE, Liu's method, McQueen scores and plasma cell count/10 mm2 were used. The frequency of CE (plasma cells > 5.15/10 mm2) was higher in women with RPL (29.6%) than in fertile controls (6.8%, p < 0.05). The plasma cell count/10 mm2 in women with RPL (median 1.53, range 0-252.6, p < 0.01) and women with RIF (median 0.6, range 0-6.98, p < 0.05) was higher than in fertile controls (median 0, range 0-29). The uterine endometrium microbiota in women with RPL or RIF was not significantly different from that in fertile controls. However, the relative dominance rate of Lactobacillus iners (median 4.7%, range 0-99.9 vs. median 0%, range 0-100, p < 0.001) and the positive rate of Ureaplasma species (36.3% vs. 8.6%, p < 0.05) were higher in 11 women with CE than in 69 women without CE. The results suggest that CE may be involved in the pathophysiology of RPL and RIF. Lactobacillus iners and Ureaplasma species may be associated with the etiology of CE.

A case of primary small cell neuroendocrine carcinoma of the uterus.

Neuroendocrine carcinoma of the uterine endometrium is extremely rare and found in <1% of all primary endometrial carcinomas. We report a case of neuroendocrine carcinoma of the endometrium detected in a 65-year-old woman and focus our attention on the main imaging features. The low apparent diffusion coefficient value and high maximum standardized uptake value for neuroendocrine cancer serve to distinguish this cancer from endometrial cancer.

Uterine endometrium microbiota and pregnancy outcome in women with recurrent pregnancy loss.

To evaluate whether uterine endometrium microbiota (UEM) is associated with pregnancy outcome in women with recurrent pregnancy loss (RPL). This prospective cohort study enrolled 67 women who had a history of two or more RPL. They underwent endometrial biopsy at midluteal phase for UEM analyses with 16 S ribosomal RNA sequence. Four women with inappropriate specimens were excluded. Therefore, 63 women were followed up for more than 14 months; 44 became pregnant, while 19 did not. Thirty of the 44 pregnancies ended in live births, including 24 full-term and six preterm deliveries. Three pregnancies were ongoing, and the remaining 11 ended in miscarriages, including eight miscarriages with normal chromosome karyotype and three miscarriages with abnormal karyotype. Clinical characteristics and UEM associated with risks for non-pregnancy, miscarriage with normal karyotype, and preterm delivery in subsequent pregnancies were evaluated. Multivariable logistic regression analyses revealed that the number of previous miscarriages (odds ratio 42.2, 95 % CI 1.19-1490, p = 0.040) and relative dominance rate of Ureaplasma species (odds ratio 24.2, 95 % CI 1.55-377, p = 0.023) in UEM were independent risk factors for subsequent miscarriage with normal karyotype; and relative dominance rate of Ureaplasma species in UEM was a risk factor for preterm delivery (odds ratio 109, 95 % CI 1.07-1110, p = 0.047). This study demonstrated for the first time that increases in Ureaplasma species in UEM of women with RPL were risks of miscarriage with normal chromosome karyotype and preterm delivery in subsequent pregnancies. UEM analysis for women with RPL before pregnancy may identify microbiota associated with adverse pregnancy outcomes.

Chronic exposure to low concentrations of chlorpyrifos affects normal cyclicity and histology of the uterus in female rats.

Chlorpyrifos (CPF), the most used insecticide in Argentina, can act as an endocrine disruptor at low doses. We previously demonstrated that chronic exposure to CPF induces hormonal imbalance in vivo. The aim of this work was to study the effects of low concentrations of CPF (0.01 and 1 mg/kg/day) on the reproductive system of virgin adult rats. In the ovary, we studied the effects of CPF on steroidogenesis by determining steroid hormone content by RIA and CYP11 and CYP19 enzyme expression by qRT-PCR. The estrous cycle was evaluated by microscopic observation of vaginal smear, as well as by changes in uterine histology. In endometrium, we determined the fractal dimension and expression of PCNA, ERα and PR by IHC. Our results showed that chronic exposure to CPF affects ovarian steroid synthesis, causing alterations in the normal cyclicity of animals. In addition, CPF induced proliferative changes in the uterus, suggesting that it could affect reproduction or act as a risk factor in the development of uterine proliferative pathologies.

The Mutation of the Ap3b1 Gene Causes Uterine Hypoplasia in Pearl Mice.

The pearl (pe) mouse mutant has been identified as a model for Hermansky-Pudlak syndrome and bears a mutation in the beta3A subunit of the AP-3 complex, which has a core function in the biogenesis and function of various lysosomal-related organelles. Through large-scale mating, we found that female pearl mice also displayed reduced fertility with a smaller litter size. Abnormal uteri in both 1-month-old and 3-month-old mice were observed as having short and thin uterine horns, indicating abnormal development. Histological studies revealed that the endometrial epithelium and endometrial stoma of the uterus were both thinner than those in the normal controls. We examined some key factors in uterine development, including the Hoxa10, Hoxa11, and Wnt5a genes, and found that they all presented lower mRNA and protein levels. The pearl mouse could serve as a model for uterine hypoplasia, a common problem in female infertility.

Development of a novel dual reproductive organ on a chip: recapitulating bidirectional endocrine crosstalk between the uterine endometrium and the ovary.

Conventional 2D or even 3Din vitroculture models for human reproductive organs cannot properly recapitulate the bidirectional endocrine crosstalk between the uterine endometrium and the ovary. This crosstalk is essential for maintaining the various physiological features and functions of each tissue. Moreover, mostin vitromodels for the female reproductive tract also fail to mimic its multicellular structure. We therefore developed a novel 'dual reproductive organ on a chip' that reflects the bidirectional endocrine cross-talk and the complex multicellular structures by integrating various cellular components of both the human uterine endometrium and the ovary with several biodegradable natural polymers. Indeed, the bidirectional endocrine crosstalk between these two tissues is achieved through media sharing between channels, and it can markedly improve the viability of loaded cells within each chamber of the chip platform. In addition, we also identified a reliable reproductive toxicity marker, SERPINB2, which is significantly increased in response to various toxic exposures in both endometrial and ovarian follicular cells. Based on these findings, we next established a SERPINB2 luciferase reporter system that was specifically designed for detecting and quantifying the toxicity of certain substances. By introducing this SERPINB2 luciferase reporter system into the loaded cells within the chip platform, we ultimately developed an effective 'dual reproductive organ-on-chip' that was successfully used to predict the reproductive toxicity of various hazardous materials.

Platelet-Rich Plasma Therapy Enhances the Beneficial Effect of Bone Marrow Stem Cell Transplant on Endometrial Regeneration.

This study aimed to investigate the potential effect of platelet-rich plasma (PRP) therapy on treatment using bone marrow stem cell (BMSC) transplant for uterine horn damage, and to reveal the potential underlying molecular mechanism. Uterine horn damage was established in a rat model, which can be repaired by transplant using BMSCs receiving control or PRP treatment. Immunohistochemistry was conducted to evaluate thickness and expression of α-SMA and vWF in the regenerated endometrium tissues. mRNA and proteins of insulin-like growth factor-1 (IGF-1) and interleukin-10 (IL-10) were measured both in the regenerated endometrium tissues and in cultured BMSCs to evaluate the effect of PRP treatment on their expression. Enzyme-linked immunosorbent assay was employed to measure the secretory levels of IL-10 in cultured BMSCs. Multi-differentiation assays were performed to address the effect of PRP treatment on tri-lineage potential of cultured BMSCs. Chromatin immunoprecipitation and luciferase reporter assays were applied to analyze NF-κB subunit p50 binding on IL-10 promoter and the resulted regulatory effect. PRP treatment significantly improved the efficacy of BMSC transplant in repairing uterine horn damage of rats, and elevated IGF-1 and IL-10 expression in regenerated endometrium tissues and cultured BMSCs, as well as enhanced tri-lineage differentiation potential of BMSCs. On the other hand, p50 inhibition and silencing suppressed the PRP-induced expression and secretion of IL-10 without affecting IGF-1 in the BMSCs. Furthermore, p50 was able to directly bind to IL-10 promoter to promote its expression. Data in the current study propose a working model, where PRP therapy improves endometrial regeneration of uterine horn damage in rats after BMSC transplant therapy, likely mediated through the NF-κB signaling pathway subunit p50 to directly induce the expression and production of IL-10.

Marijuana use differentially affects cannabinoid receptor expression in early gestational human endometrium and placenta.

Marijuana is one of the most abused drugs among pregnant women leading to maternal and fetal abnormalities. Cannabinoids are the active ingredients of marijuana, which interact with cannabinoid receptors such as CNR1 and CNR2 to activate cellular signaling pathways. Human endometrium and placenta are known to express CNR1 and CNR2 and can respond to cannabinoid signaling. In this study, we show that marijuana use significantly increases mRNA or protein expression of CNR1 and CNR2 in human endometrium from the first and early second trimester pregnancies, with minor effects on placental expression of CNRs.

Uterine Function: From Normal to Polycystic Ovarian Syndrome Alterations.

BACKGROUND: The endometrium is one of the most important female reproductive organs. Polycystic ovarian syndrome (PCOS) is a reproductive and endocrine pathology that affect women of reproductive age. PCOS negatively affects the endometrium, leading to implantation failure and proliferative aberrations. METHODS: We conducted a search at the http://www.ncbi.nlm.nhi.gov/pubmed/electronic database using the following key words: endometrial steroid receptors, endometrium, uterine function, endometrium and PCOS, implantation window, implantation and PCOS, implantation markers, inflammation, oxidative stress. We selected the articles based on their titles and abstracts, then we analyzed the full text and classified the articles depending on the information provided according to the sections of the present review. RESULTS: The endocrine and metabolic abnormalities displayed in women with PCOS promote complex effects on the endometrium, leading to a low rate of implantation and even infertility. Women with PCOS show alterations in the Hypothalamic-Pituitary- Ovarian axis, which results in constant circulating levels of estrogen, similar to those at the early follicular phase, and a deficiency in the withdrawal of estrogen and progesterone. Besides this deficiency in the withdrawal of estrogen and progesterone, the insulin/ glucose pathway, adhesion molecules, cytokines and the inflammatory cascade, together with the establishment of a pro-oxidative status, lead to an imbalance in the uterine function, which in turn leads to implantation failure or even endometrial cancer. CONCLUSION: Women with PCOS display a dysregulation of the Hypothalamic-Pituitary- Ovarian axis, which alters the steroid pathway. In addition, the deficiency in the withdrawal of estrogen and progesterone in the endometrium results in abnormal endometrial cellular proliferation. The imbalance in adipose tissue observed in PCOS patients reinforces the increase in circulating hormones. The present review describes the role of hormones, metabolites, cytokines, adhesion molecules and the insulin/glucose pathway related to the uterine endometrium in women with PCOS and their role in implantation failure and development of endometrial cancer.

Clonal Expansion and Diversification of Cancer-Associated Mutations in Endometriosis and Normal Endometrium.

Endometriosis is characterized by ectopic endometrial-like epithelium and stroma, of which molecular characteristics remain to be fully elucidated. We sequenced 107 ovarian endometriotic and 82 normal uterine endometrial epithelium samples isolated by laser microdissection. In both endometriotic and normal epithelium samples, numerous somatic mutations were identified within genes frequently mutated in endometriosis-associated ovarian cancers. KRAS is frequently mutated in endometriotic epithelium, with a higher mutant allele frequency (MAF) accompanied by arm-level allelic imbalances. Analyses of MAF, combined with multiregional sequencing, illuminated spatiotemporal evolution of the endometriosis and uterine endometrium genomes. We sequenced 109 single endometrial glands and found that each gland carried distinct cancer-associated mutations, demonstrating the heterogeneity of the genomic architecture of endometrial epithelium. Remarkable increases in MAF of mutations in cancer-associated genes in endometriotic epithelium suggest retrograde flow of endometrial cells already harboring cancer-associated mutations, with selective advantages at ectopic sites, leading to the development of endometriosis.

Developmental exposures to bisphenol S, a BPA replacement, alter estrogen-responsiveness of the female reproductive tract: a pilot study.

Developmental exposures to bisphenol A (BPA), an estrogen receptor agonist, can disrupt development of the female reproductive tract in rodents and non-human primates. Due to an increased public knowledge of negative health effects associated with BPA exposure, BPA has begun to be phased out of many consumer products and in some cases it has been replaced with structurally similar compounds including bisphenol S (BPS). This study examined CD-1 mice exposed to a low dose of BPS during early development (200 µg/kg/day from gestational day 8 until postnatal day 19). BPS altered expression of estrogen-responsive genes in both the uterus and ovary, and induced increases in ovarian follicular development in pre-pubertal females evaluated at postnatal day 22. Prior studies have revealed that developmental exposures to environmental chemicals including BPA alter the response of animals to hormonal or carcinogen challenges experienced later in life. To evaluate whether early life exposures to BPS alter responses of females to an estrogen challenge, additional females were exposed to ethinyl estradiol from postnatal day 19 through postnatal day 21. BPS-treated females responded abnormally to this estrogen challenge, displaying heightened responses in the uterus and diminished responses in the ovary. Although additional studies are needed to characterize the mechanisms by which BPS alters the female reproductive tract, this pilot study provides evidence that a common BPA replacement chemical may have endocrine disrupting properties.

NK22 Cells in the Uterine Mid-Secretory Endometrium and Peripheral Blood of Women with Recurrent Pregnancy Loss and Unexplained Infertility.

PROBLEM: We aimed to investigate natural killer 22 (NK22) cells in the peripheral blood and the uterine endometrium of women with unexplained recurrent pregnancy loss (URPL) and unexplained infertility (UI). METHOD OF STUDY: Peripheral blood and endometrial samples were collected from women with URPL (n = 43) and UI (n = 38). Intracellular cytokine production, such as IL-22, IFN-γ and TNF-α, and the expression of NKp46 on NK cells were analyzed by three-color flow cytometry. RESULTS: The percentages of endometrial CD56(+) /IL-22(+) and CD56(dim) /IL-22(+) cells in women with URPL were significantly higher than those of UI (P < 0.05, respectively). In addition, the percentage of CD56(bright) /IL-22(+) cells in women with RPL was negatively correlated with those of CD56(bright) /IFN-γ(+) and CD56(bright) /TNF-α(+) in both peripheral blood and endometrial NK cells. This was not seen in women with UI. The percentage of CD56(bright) /IL-22(+) cells was negatively correlated with CD56(bright) /NKp46 expressing NK cells in peripheral blood. CONCLUSION: Endometrial NK22 cells are differently regulated in women with URPL and UI. Women with URPL have higher level of NK22 cells with a potential to induce NK2 shift than women with UI.