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

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.

Assisted reproductive technologies (ART) are medical procedures used to address infertility. Follicular fluid (FF)is a liquid that surrounds the immature egg cells (oocytes) and provides hormones and nutrients necessary for their maturation and eventual development into embryos. We analyzed genetic components within the FF as a potential predictor of reproductive outcomes following ART. Here, we show that a specific combination of genetic elements produced by the FF in successful pregnancies did not occur in unsuccessful pregnancies. Our findings may help to provide a non-invasive approach to determining reproductive outcomes during ART procedures.

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

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.

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

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).

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

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.

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

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.

Differentiation of human induced pluripotent stem cells into hypothalamic vasopressin neurons with minimal exogenous signals and partial conversion to the naive state.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

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

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 ßhCG 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.

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

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-1ß), 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-1ß 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-1ß in CSCs, as well as IL-1ß 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-1ß 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-1ß 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.

Functional Lactotrophs in Induced Adenohypophysis Differentiated From Human iPS Cells.

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.

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

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.

Adrenergic stimulation-released 5-HT stored in adrenergic nerves inhibits CGRPergic nerve-mediated vasodilatation in rat mesenteric resistance arteries.

BACKGROUND AND PURPOSE: 5-HT is taken up by and stored in adrenergic nerves and periarterial nerve stimulation (PNS) releases 5-HT to cause vasoconstriction in rat mesenteric arteries. The present study investigated whether PNS-released 5-HT stored in adrenergic nerves affects the function of perivascular calcitonin gene-related peptide-containing (CGRPergic) nerves. EXPERIMENTAL APPROACH: Rat mesenteric vascular beds without endothelium and with active tone were perfused with Krebs solution. Changes in perfusion pressure in response to PNS and CGRP injection were measured before (control) and after perfusion of Krebs solution containing 5-HT (10 µM) for 20 min. Distributions of 5-HT- and TH-immunopositive fibres in mesenteric arteries were studied using immunohistochemical methods. KEY RESULTS: PNS (1-4 Hz) frequency dependently caused adrenergic nerve-mediated vasoconstriction followed by CGRPergic nerve-mediated vasodilatation. 5-HT treatment inhibited PNS-induced vasodilatation without affecting exogenous CGRP-induced vasodilatation, while it augmented PNS-induced vasoconstriction. Guanethidine (adrenergic neuron blocker), methysergide (non-selective 5-HT receptor antagonist) and BRL15572 (selective 5-HT1D receptor antagonist) abolished inhibition of PNS-induced vasodilatation in 5-HT-treated preparations. Combined treatment with 5-HT and desipramine (catecholamine transporter inhibitor), but not fluoxetine (selective 5-HT reuptake inhibitor), did not inhibit PNS-induced vasodilatation. Exogenous 5-HT inhibited PNS-induced vasodilatation, which was antagonized by methysergide. In immunohistochemical experiments, 5-HT-immunopositive nerves, colocalized with adrenergic TH-immunopositive nerves, were observed only in 5-HT-treated mesenteric arteries, but not in control preparations or arteries co-treated with desipramine. CONCLUSIONS AND IMPLICATIONS: These results suggest that 5-HT can be taken up by and released from adrenergic nerves in vitro by PNS to inhibit CGRPergic nerve transmission in rat mesenteric arteries.