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High follicle-stimulating hormone (FSH) doses during ovarian stimulation are detrimental to ovulatory follicle function and decrease live birth rate in cattle and women. However, the mechanism whereby excessive FSH causes ovarian dysfunction is unknown. This study tested the hypothesis that excessive FSH during ovarian stimulation induces premature luteinization of ovulatory-size follicles. Small ovarian reserve heifers were injected twice daily for 4 days with 70 IU (N = 7 heifers) or 210 IU (N = 6 heifers) Folltropin-V [commercial FSH-enriched preparation of porcine pituitary glands with minor (<1%) luteinizing hormone (LH) contamination, cpFSH]. Ovulatory-size (≥10 mm) follicles were excised from ovaries after the last cpFSH injection and hormone concentrations in follicular fluid (FF) were determined using ELISA. Luteinization was monitored by assessing cumulus cell–oocyte complex (COC) morphology and measuring concentrations of estradiol (E), progesterone (P), and oxytocin (O) in FF. COCs were classified as having compact (cCOC) or expanded (eCOC) cumulus cell layers, and as estrogen-active (E:P in FF ≥1), estrogen-inactive (EI, E:P in FF ≤1 > 0.1), or extreme-estrogen-inactive (EEI, E:P in FF ≤0.1). A high proportion (72%) of ovulatory-size follicles in 210 IU, but not 70 IU, dose heifers displayed eCOCs. The high doses also produced higher proportions of EI or EEI follicles which had lower E:P ratio and/or E but higher P and/or O concentrations compared with the 70 IU dose heifers. In conclusion, excessive cpFSH doses during ovarian stimulation may induce premature luteinization of most ovulatory-size follicles in heifers with small ovarian reserves. Summary Sentence Excessive doses of Folltropin-V (commercial FSH-enriched preparation from porcine pituitary glands with minor LH contamination) during ovarian stimulation of heifers with small ovarian reserves reduce intrafollicular estradiol while inducing premature luteinization of most ovulatory-size follicles.

Background The production of sex steroids by follicular cells is proposed to be influenced by the maturity of the incumbent oocyte. Thus steroid levels may reflect suitability of an oocyte for IVF. We examined follicular fluids and granulosa cell production of steroid from IVF patients in order to test the relationship between steroid levels and fertilization. Methods Follicular fluid and granulosa cells were extracted from 206 follicles of 35 women undergoing controlled ovarian stimulation. Follicular fluid was assayed for estradiol, progesterone and testosterone. Granulosa cells were cultured from individual follicles and their culture media assayed for production of these hormones after 24 hrs in vitro. Levels of steroids were correlated with follicular diameter, oocyte recovery and subsequent fertilization. Results Follicular fluid levels of progesterone were 6100 times higher than that of estradiol, and 16,900 times higher that of testosterone. Despite the size of follicle triggered after controlled luteinisation, the levels of progesterone and testosterone were maintained at relatively constant levels (median 98.1 micromoles/L for progesterone, and 5.8 nanomoles/L for testosterone). However, estradiol levels were slightly lower in the larger follicles (follicular diameter 10-15 mm, median 25.3 nanomoles/L; follicles > = 15 mm, median 15.1 nanomoles/L; linear correlation r = -0.47, p < 0.0001). With respect to oocyte recovery, no steroid showed a significant association in follicular fluid levels. Similarly no difference in follicular fluid steroid levels was found for those oocytes that did or did not fertilize. Significant quantities of progesterone were produced by the granulosa cells but production was constant regardless of the size of follicle from which the cells originated. Estradiol levels were only detectable in 10 of 121 cultures examined, and testosterone in none. Interestingly, when an oocyte was present follicular estradiol levels correlated with progesterone levels. However, when absent, follicular estradiol levels correlated with testosterone levels but not with progesterone. Conclusions The principle steroid product of luteinized pre-ovulatory granulosa is progesterone, a differentiation triggered by the gonadotropin surge. However, absolute steroid levels are associated with follicular size, not oocyte maturation/ability to fertilize.

Ovulation is induced by the luteinizing hormone (LH) surge and accompanied by granulosa cell luteinization and ovarian angiogenesis. Semaphorin 3E (Sema3E)‐Plexin‐D1 pathway regulates angiogenesis in other tissues, but its role in the ovary is unknown. Evidence indicates that Sema3E‐Plexin‐D1 pathway plays an important role in the mouse ovary. The expression of Sema3E and its receptor, Plexin‐D1, is dynamically regulated in the mouse ovary downstream of the LH surge. This regulation requires the modulation of chromatin accessibility by CCAAT/enhancer‐binding proteins α and β. Intraovarian injection of recombinant Sema3E results in reduced ovulation, impaired corpus luteum formation, and aberrant ovarian angiogenesis. These in vivo physiological abnormalities are consistent with altered expression of genes regulating these processes, and with data from in vitro cultured granulosa cells and ovarian stromal tissues treated with Sema3E or neutralizing antibody of Plexin‐D1. The findings pinpoint Sema3E‐Plexin‐D1 pathway as a potential therapeutic target for fertility and infertility management. The Semaphorin 3E (Sema3E)‐Plexin‐D1 pathway mediated by C/EBPα and C/EBPβ downstream of the luteinizing hormone (LH) surge plays important roles in the mouse preovulatory ovary. Timely activation and suppression of this pathway during the preovulatory stage are crucial for ovulation, corpus luteum formation, and proper angiogenesis. The Sema3E‐Plexin‐D1 pathway may serve as a potential new therapeutic target for managing ovulation and luteal function.

Krüppel-like factor 4 (Klf4) plays an important role in the transition from proliferation to differentiation in a wide variety of cells. Previous studies demonstrated its critical role in the luteal transition of preovulatory granulosa cells (GCs). This study used cultured rat preovulatory GCs to investigate the mechanism by which luteinizing hormone (LH) regulates Klf4 gene expression. Klf4 mRNA and protein were rapidly and transiently induced by LH treatment, reaching peak levels after 45 min and declining to basal levels by 3 h. Pretreatment with the protein synthesis inhibitor cycloheximide had no effect on LH-stimulated Klf4 expression, indicating that Klf4 is an immediate early gene in response to LH. To investigate the signaling pathway involved in LH-induced Klf4 regulation, the protein kinase A (PKA) and protein kinase C (PKC) pathways were evaluated. A-kinase agonists, but not a C-kinase agonist, mimicked LH in inducing Klf4 transcription. In addition, specific inhibitors of A-kinase abolished the stimulatory effect of LH on Klf4 expression. Truncation of a Klf4 expression construct to −715 bp (pKlf4-715/luc) had no effect on transcriptional activity, whereas deletion to −402 bp (pKlf4-402/luc) dramatically reduced it. ChIP analysis revealed in vivo binding of endogenous Sp1 to the −715/−500 bp region and maximal transcriptional responsiveness to LH required the Sp1 binding element at −698/−688 bp, which is highly conserved in mice, rats, and humans. These findings demonstrate that Klf4 is activated by LH via the cAMP/PKA pathway and a putative Sp1 binding element at −698/−688 bp is indispensable for activation and suggest that Klf4 could be a target for strategies for treating luteal phase insufficiency induced by an aberrant response to the LH surge.

MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3' untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.

The in vitro granulosa cell (GC) model presents a valuable tool to explore antral follicle development. A full understanding of the reasons and blocking methods that occur during in vitro luteinization of sheep GCs, stimulated by serum culture, is a complex goal that has not been completely achieved. Herein, the phenomenon and causes of GC differentiation, as well as the methods for inhibiting luteinization in an in vitro culture system, were investigated by immunofluorescence, Western blot, RT-qPCR, and ELISA techniques. The results reveal that, when compared to fresh GCs, FSHR protein levels in primary GCs significantly decreased in serum-containing media, while STAR protein levels significantly increased, implying that sheep GCs can differentiate in serum-containing media. LH concentrations were significantly greater in serum-containing media compared to serum-free media. The LH receptor (LHR) mRNA expression in primary-generation GCs steadily increased with longer culture times, indicating that LH-LHR signaling leads to GC luteinization in vitro. In primary and second-generation GCs, 180 nmol/L BAY-899, an LHR-specific antagonist, significantly increased FSHR protein expression, reduced STAR protein synthesis, and inhibited P4 secretion within 48 h of in vitro culture compared to controls. BAY-899 showed no adverse effects on fifth-generation GCs growth, implying that BAY-899 can inhibit GC luteinization while not affecting cell proliferation. In conclusion, this study found that the LHR antagonist BAY-899 can preserve the features of sheep GCs in vitro by suppressing the spontaneous luteinization process caused by LH-LHR signaling, which has a key methodological implication for studying the mechanics of antral follicle formation in vivo.

The impact of the prematurely elevated serum progesterone on the late follicular phase, commonly known as premature luteinization (PL), is a matter of continuing debate. Available evidence supports that serum progesterone ≥ 1.5 ng/ml on the day of ovulation triggering could reduce the pregnancy potential in fresh in vitro fertilization (IVF) cycles by jeopardizing endometrial receptivity. Causes of PL during ovarian stimulation are unclear. Recent studies point toward the daily follicle-stimulating hormone dosage, duration of controlled ovarian stimulation, number of oocytes retrieved, and peak estradiol level as factors affecting the incidence of PL. Emerging data show additional influence on embryo quality. The prevention of PL has been challenging. The key elements in preventing PL include individualization of ovarian stimulation according to patient’s ovarian reserve, proper ovulation trigger timing, and use of medications such as corticosteroids and metformin. Embryo cryopreservation with deferred embryo transfer is the established strategy to overcome PL, yet it is an extra burden to the IVF laboratory and increased cost for patients. Herein, we review the up-to-date knowledge of this frequent IVF problem including causes, proposed diagnostic criteria, and its impact on endometrial receptivity, embryo quality, and pregnancy outcomes. The preventive measures and rescue strategies are also discussed.

Various cells are localized to appropriate positions in tissues and induced a unique gene expression and functions. In this study, we show for the first time that age-related changes in ovarian hardness and elasticity are linked to abnormal signal transduction affects in secondary follicles. Immature ovaries had lower hardness and elasticity indices, whereas aged ovaries had lower elasticity and higher hardness indices than mature ovaries. Importantly, an optimal hardness and elasticity is important for normal gene expression profile of secondary follicles. We reproduced the ovarian hardness and elasticity using alginate beads with different concentration and viscosities, for three-dimensional culture of secondary follicles. In the aged-ovarian hardness group, follicular diameter decreased significantly and the expression of inflammation-related markers increased significantly. In the immature-ovarian hardness group, follicular diameter increased significantly and the expression of differentiation and proliferation markers increased significantly. The mechanism transmitted the hardness and elasticity to the change of gene expression was that YAP, transmit the physical conditions to nucleus via actin network, was significantly increased in both condition of immature or aged ovary. The abnormal signaling associated with the age-related changes in ovarian hardness and elasticity may aid in the development of treatments for abnormal follicular development in infertility.

Background: Oral progesterone is recommended as an alternative to gonadotropin-releasing hormone (GnRH) agonists and antagonists to prevent luteinizing hormone (LH) surge in assisted reproductive technology (ART) cycles. However, there are little data regarding its use. Objective: We aimed to compare the effect of oral Utrogestan and Cetrotide (a GnRH antagonist) on preventing LH surge in ART cycles. Materials and Methods: In this randomized clinical trial, 100 infertile women undergoing ART who received recombinant follicle-stimulating hormone (FSH) at 150- 225 IU/day were randomly assigned to receive either Utrogestan 100 mg twice a day (case group) or GnRH antagonist protocol (control group) from cycle day 3 until the trigger day. Triggering was performed with 10,000 IU hCG) when there were at least three mature follicles. Viable embryos were cryopreserved for transfer in the next cycle for both groups. The number of oocytes retrieved and transferred embryos were compared between groups. Results: The case group had significantly higher progesterone levels on triggering day, more follicles of >14 mm with higher maturity, and more oocytes retrieved with a higher rate of embryos transferred. A small increase in the pregnancy rate was observed in the case group, with no significant between-group differences. The most important result was the lack of premature LH surge in either group upon serum LH assessment on the triggering day. Conclusion: Utrogestan is an alternative treatment that could reduce the LH surge rate and increase the ART outcomes including the number of oocytes retrieved and transferred embryos compared with GnRH agonists and antagonists. Key words: In vitro fertilization, Premature luteinization, Utrogestan.

PurposePremature luteinization (PL) affects 12.3–46.7% of fresh in vitro fertilization cycles, and there is accumulating evidence confirming its negative effect on success rates. However, despite its clinical significance, PL is poorly understood and defined. This narrative review aims to provide a fresh look at the phenomenon of PL by summarizing the existing evidence and re-evaluating fundamental issues.MethodsA thorough electronic search was conducted covering the period from 1978 until January 2018 in PubMed, Embase, and Medline databases, and references of relevant studies were cross-checked. Meeting proceedings of the European Society of Human Reproduction and Embryology and the American Society for Reproductive Medicine were also hand searched.ResultsIn the curious case of PL, one should go back to the beginning and re-consider every step of the way. The pathogenesis, definition, measurement methods, clinical implications, and management strategies are discussed in detail, highlighting controversies and offering “food for thought” for future directions.ConclusionsAuthors need to speak the same language when studying PL in order to facilitate comparisons. The terminology, progesterone cut-off, measurement methods and days of measurement should be standardized and globally accepted; otherwise, there can be no scientific dialog. Future research should focus on specific patient profiles that may require a tailored approach. Progesterone measurements throughout the follicular phase possibly depict the progesterone exposure better than an isolated measurement on the day of hCG. Adequately powered randomized controlled trials should confirm which the best prevention and management plan of PL is, before introducing any strategy into clinical practice.