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It is well established that mouse ovarian granulosa cells secrete urokinase plasminogen activator (uPA) under gonadotropin stimulation. The synthesis and secretion of the enzyme correlate well with the time of follicular rupture in vivo. Moreover, uPA is secreted by the trophoblast at the time of implantation. In the present study, we have analyzed whether the absence of uPA could influence follicular growth, ovulation, and embryo implantation. Our data show fewer preantral follicles in uPA -/- ovaries but no decrease in hormonally induced ovulation. However, we observed a significant decrease in the number of implanted embryos in uPA -/- animals and, therefore, a lower number of pups per family. Adding uPA to the epithelial and stromal uterine cell culture medium strongly upregulates the expression of prostaglandin-endoperoxide synthase 2 (Ptgs2), the enzyme required for prostaglandin production and embryo implantation. The uPA inhibitor amiloride abrogated this increase., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Omentin (ITLN1) is a novel adipokine mainly expressed in the white adipose tissue. It plays a crucial role in the metabolic homeostasis and insulin sensitivity. Our last study documented that ITLN1 levels in the adipose tissue and plasma are lower in fat Meishan (MS) compared to normal weight Large White (LW) pigs. The aim of this study was to investigate transcript and protein concentrations of ITLN1 as well as its immunolocalisation in the ovarian follicles and examine the molecular mechanism involved in the regulation of its expression in response to gonadotropins (FSH, LH) and steroids (P4, T, E2). Ovarian follicles were collected from LW and MS sows on days 2-3, 10-12, and 14-16 of the oestrous. We found the elevated ITLN1 expression in the ovarian follicles and the increase of concentrations in follicular fluid (FF) of LW pigs vs MS pigs; in both breeds of pigs, the levels of ITLN1 increased with the oestrous progression. We noted ITLN1 signals in oocyte, granulosa and theca cells. Gonadotropins and steroids increased ITLN1 levels in the ovarian follicle cells of LW pigs, while in MS pigs, we observed only the stimulatory effect of LH and T. Both extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3'-kinase (PI3K) were involved in the regulation of ITLN1. Our study demonstrated the levels and regulation of ITLN1 in the porcine ovarian follicles through ERK1/2 and PI3K signaling pathways., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ovarian response to stimulation mainly determines the length of stimulation. However, there is no clarity in the literature regarding the optimal duration required to achieve oocyte maturity in patients with the poor ovarian response (POR) defined by Bologna criteria. Therefore, a total of 267 cycles that fulfilled the inclusion criteria were selected retrospectively. Group A constitute of patients with a stimulation period < 9 d ( n  = 70); and group B included patients with a stimulation period ≥ 9 d ( n  = 133). The results showed that antral follicle count (5.72 ± 1.82 vs. 5.10 ± 1.78, p  = 0.023), serum oestradiol level on hCG day (1286.88 ± 778.18 pg/mL vs. 820.14 ± 479.04 pg/mL, p  = 0.001), and total gonadotropin dose used (2949.53 ± 727.92 IU vs 2020.94 ± 415.17 IU, p  = 0.0001) were higher in group B when compared to group A. Although the number of total (5.47 ± 3.32 vs 3.86 ± 2.15, p  = 0.0001) and mature oocytes retrieved (4.34 ± 2.88 vs 2.84 ± 1.67, p  = 0.0001) were higher in group B, no significant difference was observed in the pregnancy rates between groups (25.6 vs 15.7%, p  > 0.05). In conclusion, no deleterious effect of a shorter duration of stimulation on cycle outcome was seen in patients with POR.

In Brief: The SCN regulates ovulation by stimulating the preovulatory surge of gonadotropins. This study revealed an additional role in the sensitization of the hypothalamus to estradiol that changes along the estrous cycle and the side of the nucleus., Abstract: Ovulation is timed by neural signals originating at the suprachiasmatic nucleus (SCN) that trigger ovulation when converge with high estradiol levels, which indicates the maturation of ovarian follicles. We have shown that the hypothalamic regulation of ovulation is asymmetrical and we hypothesized that the paired SCN could contribute to such symmetries. We unilaterally lesioned the SCN of rats at each stage of the estrous cycle and evaluated the acute effects on the progression of their estrous cycle, follicular development and ovulation. Lesions prevented progression of the estrous cycle when performed in estrus/metestrus but not in diestrus/proestrus. Abnormalities in follicular development were observed in the nonovulating lesioned rats and this was independent of the side of the SCN destroyed and the stage of the cycle when surgery was performed. Groups of lesioned rats were then hormonally primed with GnRH or estradiol to assess the neuroendocrine pathway altered by the treatment. GnRH restored ovulation, suggesting that both SCN are needed for proper triggering of the preovulatory surge of GnRH and that unilateral lesion does not impair the sensitivity of the pituitary or the ovary to GnRH and gonadotropins, respectively. With regard to restoring ovulation, estradiol was asymmetrically effective in rats lesioned in estrous, partially effective in rats operated at diestrus and ineffective in rats at metestrus. Our results indicate that the SCN regulates the activity of the hypothalamic-pituitary-ovarian axis not only by modulating the preovulatory surge of GnRH/gonadotropins but also by promoting the hypothalamic integration of estrogenic signals from the ovaries in an asymmetric and stage-dependent fashion.

Omentin-1 (OMNT1) is an adipokine involved in the regulation of energy metabolism, insulin sensitivity, and reproduction. The present study was the first to investigate the plasma levels and expression of OMNT1 in the anterior pituitary (AP) gland on days 2-3, 10-12, 14-16, and 17-19 of the estrous cycle of normal-weight Large White (LW) and fat Meishan (MS) pigs. Next, we determined the effect of GnRH, LH, and FSH on the OMNT1 levels in cultured AP cells. The gene and protein expression of OMNT1 in AP fluctuated during the estrous cycle, with a higher expression in MS than in LW (except on days 10-12). However, plasma levels of OMNT1 were higher in LW than in MS. OMNT1 was localized in somatotrophs, lactotrophs, thyrotrophs, and gonadotrophs. In LW pituitary cells, GnRH and gonadotropins stimulated OMNT1 protein expression (except FSH on days 14-16) and had no effect on OMNT1 levels in the culture medium. In MS pituitary cells, we observed that GnRH and LH increased while FSH decreased OMNT1 protein expression. These findings showed OMNT1 expression and regulation in the porcine AP and suggested that OMNT1 could be a new player modifying the pituitary functions., (© 2023. The Author(s).)

Recent studies have demonstrated that visfatin participates in the regulation of female reproduction. Due to the lack of data concerning the level of visfatin in the ovarian follicles of pigs, one of the most economically important livestock species, the aim of this study was to investigate the expression and localisation of visfatin and the follicular fluid concentration in the ovarian follicles of prepubertal and mature gilts. We also aimed to examine the in vitro effects of gonadotropins (LH, FSH), insulin, progesterone (P 4 ), oestradiol (E 2 ), prostaglandin E 2 (PGE 2 ) and F 2α (PGF 2α ) on visfatin levels. In the present study, we have demonstrated that visfatin expression is dependent on the maturity of the animals and the stage of ovarian follicle development. Visfatin signal was detected in individual follicular compartments from primordial to antral follicles and even in atretic follicles. We have shown that the expression of visfatin in granulosa cells was higher than in theca cells. The level of visfatin is upregulated by LH, FSH, E 2, and P 4 and downregulated by insulin, while prostaglandins have modulatory effects, dependent on the dose and type of ovarian follicular cells. To summarise, our research has shown that visfatin is widely expressed in the ovarian follicles of prepubertal and mature pigs, and its expression is regulated by gonadotropins, insulin, steroids, and prostaglandins, suggesting that visfatin appears to be an important intra-ovarian factor that could regulate porcine ovarian follicular function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles., Aims: The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries., Methods: One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight., Key Results: In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05)., Conclusions: Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development., Implications: This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHβ, FSHβ, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The enkephalins are known to regulate many physiological functions, including reproduction in vertebrates. However, the role of leucine-enkephalin (L-ENK) in the ovarian recrudescence activity of reptiles is not known. In the present study, we studied the influence of L-ENK on seasonal and FSH-induced ovarian recrudescence during the breeding and non-breeding phases of the cycle in the tropical and subtropical gecko Hemidactylus frenatus. In the first experiment, treatment with 5 and 25 μg L-ENK resulted in a dose-dependent inhibitory effect on the hypothalamic gonadotropin-releasing hormone (GnRH) neurons and ovary, as indicated by a significantly decreased percent area of GnRH-immunoreactive (GnRH-ir) fibres in the median eminence and pars distalis of the pituitary gland, concomitant with complete absence of stage V (late vitellogenic) follicles in the ovary compared to those of experimental controls. In the second experiment, administration of FSH to lizards in the regression phase stimulated the recruitment of stage IV and V (vitellogenic) follicles in contrast to their absence in initial controls or treatment controls. However, similar treatment of FSH in combination with 25 μg L-ENK did not result in the development of stage IV or V follicles. Together, these results suggest for the first time that treatment with 5 and 25 μg L-ENK exerts a dose-dependent inhibitory effect on the hypothalamic GnRH release into the median eminence and pituitary gland, leading to the blockade of ovarian recrudescence. These results also suggest a possible direct inhibitory effect of L-ENK at the level of the ovary in the gecko., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

We investigated the role of protein kinase c (PKC) -α and -β during the ovarian follicular dynamics using estrous cycle, gonadotropin-induced ovulation, and antral follicle culture, 4-vinylcyclohexene diepoxide (VCD)-induced premature ovarian failure (POF) in the SD rat models. We found the higher activity of PKC during the proestrus stage along with expression of PKC-α during the estrus and metestrus stages of the estrous cycle while PKC-β expression was increased during the diestrus, proestrus, and estrus stages. In response to pregnant mare gonadotropin (PMSG)-induced follicular recruitment and ovulation, the phosphorylated (Thr-642) PKC-β was increased. PKC activity inhibition by hispidin during the proestrus stage resulted in decreased antral follicles and corpus luteum. Treatment with hispidin resulted in the downregulation of granulosa cell (GC) biomarker, follicle stimulating hormone receptor (FSHR) expression in the cultured pre-antral follicle. During the forskolin-induced luteinization of human granulosa cells, the expression level of PKC-α and β (I and II) was decreased. In the POF condition, the activity of total PKC and the expression levels of PKC-α and β (I and II) were increased. Immunostaining depicted ubiquitous expression of PKC-α in the ovary during the estrous cycle and POF conditions. Taken together, we conclude the association of PKC-α and -β (I and II) during ovarian follicular dynamics where the expression level of PKC-α is increased, but the expression level of PKC-β (I and II) is suppressed in the POF condition in the SD rat model., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest in the context of the ‘Employment and consultancies, Grants, fees and honoraria, Ownership of stock or shares, Royalties, Patents (pending and actual), and Board membership., (Copyright © 2023 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Purpose: An impact of different gonadotrophins selection for ovarian stimulation (OS) on oocyte competence has yet to be defined. In this study, we asked whether an association exists between OS protocol and euploid blastocyst rate (EBR) per metaphase-II (MII) oocytes., Methods: Cycles of first preimplantation genetic testing for aneuploidies conducted by women ≥ 35 years old with their own metaphase-II oocytes inseminated in the absence of severe male factor (years 2014-2018) were clustered based on whether recombinant FSH (rec-FSH) or human menopausal gonadotrophin (HMG) was used for OS, then matched for the number of fresh inseminated eggs. Four groups were outlined: rec-FSH (N = 57), rec-FSH plus rec-LH (N = 55), rec-FSH plus HMG (N = 112), and HMG-only (N = 127). Intracytoplasmic sperm injection, continuous blastocyst culture, comprehensive chromosome testing to assess full-chromosome non-mosaic aneuploidies and vitrified-warmed euploid single embryo transfers (SETs) were performed. The primary outcome was the EBR per cohort of MII oocytes. The secondary outcome was the live birth rate (LBR) per first SETs., Results: Rec-FSH protocol was shorter and characterized by lower total gonadotrophin (Gn) dose. The linear regression model adjusted for maternal age showed no association between the Gn adopted for OS and EBR per cohort of MII oocytes. Similarly, no association was reported with the LBR per first SETs, even when adjusting for blastocyst quality and day of full blastulation., Conclusion: In view of enhanced personalization in OS, clinicians shall focus on different endpoints or quantitative effects related to Gn action towards follicle recruitment, development, and atresia. Here, LH and/or hCG was administered exclusively to women with expected sub/poor response; therefore, we cannot exclude that specific Gn formulations may impact patient prognosis in other populations., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ovarian function in teleosts, like in other vertebrates, is regulated by two distinct gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Gonadotropin effects are mediated by membrane-bound G protein-coupled receptors localized on the surface of follicle cells. Gonadotropin receptor activation results in increased intracellular cAMP, the most important second cellular signaling molecule. FSH stimulation induces the production of 17β-estradiol in the cells of growing follicles to promote vitellogenesis in oocytes. In contrast, in response to LH, fully grown post-vitellogenic follicles gain the ability to synthesize maturation-inducing steroids, which induce meiotic resumption and ovulation. All these events were induced downstream of cAMP. In this review, we summarize studies addressing the role of the cAMP pathway in gonadotropin-induced processes in teleost ovarian follicles. Furthermore, we discuss future problems concerning cAMP signaling in relation to teleost ovarian function and the differences and similarities in the gonadotropin-induced cAMP signaling pathways between mammals and teleosts., Competing Interests: Declaration of Competing Interest Authors declare no competing interest., (Copyright © 2022. Published by Elsevier Inc.)

Objective: To examine the effect of letrozole on oocyte quality and pregnancy outcome in assisted reproductive technology (ART)., Methods: This double blind placebo controlled clinical trial was conducted in Vali-Asr Infertility Center. Infertile women candidate for IVF that underwent antagonist protocol were selected. Eligible women randomly allocated into treatment (letrozole/Let group) and control (placebo) group. Participants received letrozole 5 mg/day or placebo at the time of gonadotropin start until trigger day in the same manner. Number of oocyte retrieved, metaphase II oocyte number, high grade oocyte number (G1), high quality embryo, Chemical and clinical pregnancy rate and OHSS (ovarian hyperstimulation syndrome) rate was recorded. 216 infertile women (104 in letrozole and 112 in the control group) were evaluated., Results: In the Let group estradiol level was significantly lower ( p&#95; value < .001) and testosterone significantly higher than in the control group ( p&#95; value = .02). The number of retrieved oocytes, MII oocytes, G1 oocytes, and 2PN was significantly lower in the Let group ( p  < .05). No significant difference was found in the day of stimulation, total gonadotropin dose, OHSS rate, and clinical pregnancy rate between the two groups ( p  > 0.05)., Conclusions: According to the results, letrozole may reduce oocyte quality and cause poor IVF outcomes as well.

Two isoforms of the gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are expressed in mammals, and the presence of one or more GnRH-like peptides has been demonstrated in the male reproductive tract. GnRH and its receptors (GnRHR) are present in human and non-human primate testis, prostate, epididymis, seminal vesicle, spermatozoa and seminal human plasma. GnRH-II is site-specific and acts directly in an inhibitory or stimulatory fashion. Previous studies speculated that GnRH-II could disrupt specific sperm processes, such as sperm motility or capacitation and could be utilized as an effective contraceptive agent. Our study aimed to investigate the in-vitro effects of GnRH-I and GnRH-II on Vervet monkey sperm function. Electro-ejaculated semen samples from 10 Vervet monkeys (Chlorocebus aethiops) were used to select motile sperm populations. Sperm aliquots were incubated with GnRH-I and GnRH-II at different concentrations for 1 h, where after sperm motility and kinematic parameters were assessed using the automated Sperm Class Analyser. Additional sperm aliquots were incubated with two 10-amino acid control peptides, a non-related peptide and an inactive peptide to exclude the possible influence on sperm motility from other peptides with a structure similar to GnRH. Additionally, a GnRHR-I antagonist (GnRHR-A), Cetrorelix, was tested to establish its antagonistic capability on GnRH. The effect of selected concentrations of GnRH-I and GnRH-II on sperm vitality and acrosome intactness was also evaluated after 10- and 60 min exposure. Analysis of the percentage total sperm motility revealed that different concentrations for GnRH-I and GnRH-II inhibited sperm motility significantly. While sperm progressiveness was also notably affected and a trend of decreased sperm kinematics were evident, no effect was found on sperm vitality or acrosome intactness. The non-related and inactive peptides had no impact on sperm motility. The GnRHR-A demonstrated no effect on sperm motility and effectively blocked the inhibitory outcome on the motility of both GnRH isoforms. While GnRH-I or GnRH-II at low-dose concentrations resulted in in-vitro inhibition of sperm motility, it appears to have no adverse effects on other sperm functional parameters evaluated. These collective observations possibly indicate an essential role for GnRH in the in-vivo process of sperm selection in the female reproductive tract., (© 2022 Wiley-VCH GmbH.)

This study evaluates the effect of GnRH agonist (GnRHa) trigger for ovulation induction among women with advanced maternal age (AMA). This is a retrospective study performed at a single assisted reproductive technology centre, 2012 to 2020. A total of 306 patients with 515 IVF cycles who were triggered with GnRHa for Ovum Pick Up (OPU), were divided into two groups according to maternal age: age ≥ 40 and age < 40. The groups were compared for demographics, stimulation parameters of IVF treatment and IVF treatment outcomes. The patients in the age < 40 group were approximately 10 years younger than the patients in the age ≥ 40 group (31 ± 5.4 vs. 41.5 ± 1.3 years, p < 0.001). The age ≥ 40 group had significantly higher mean E2/retrieved oocytes ratio, compared to the age < 40 group (310.3 ± 200.6 pg/ml vs. 239 ± 168.2 pg/ml, p = 0.003), and a lower mean MII/retrieved oocyte (35 ± 37.8 vs. 43.4 ± 35.9, p = 0.05, respectively). Multivariable logistic regression analysis for E2/retrieved oocytes demonstrated that age < 40 and total dose of gonadotropins were significant variables. In conclusion, GnRHa for ovulation triggering in high responder patients prior to OPU appears to be a good option for AMA. However, this population is characterized by different parameters of ovarian response that require further evaluation., (© 2022. The Author(s).)

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Granulosa cells (GCs) are multilayered somatic cells within the follicle that provide physical support and microenvironment for the developing oocyte. In recent years, the role of Neuregulin-1 (NRG1), a member of the EGF-like factor family, has received considerable attention due to its neurodevelopmental and cardiac function. However, the exact physiological role of NRG1 in GC is mainly unknown. In order to confirm that NRG1 plays a regulatory role in rat GC functions, endogenous NRG1-knockdown studies were carried out in GCs using RNA interference methodology., Results: Knockdown of NRG1 in GCs resulted in the enhanced expression and secretion of the cytokines and chemokines. In addition, the phosphorylation of PI3K/Akt/ERK1/2 was significantly low in GCs under these experimental conditions. Moreover, in vitro experimental studies suggest that tumor necrosis factor-α (TNFα) treatment causes the physical destruction of GCs by activating caspase-3/7 activity. In contrast, exogenous NRG1 co-treatment of GCs delayed the onset of TNFα-induced apoptosis and inhibited the activation of caspase-3/7 activity. Furthermore, current experimental studies suggest that gonadotropins promote differential expression of NRG1 and ErbB3 receptors in GCs of the antral follicle. Interestingly, NRG1 and ErbB3 were intensely co-localized in the mural and cumulus GCs and cumulus-oocyte complex of pre-ovulatory follicles in the estrus stage., Conclusions: The present studies suggest that gonadotropins-dependent NRG1-signaling in GCs may require the balance of the cytokines and chemokines expression and secretion, ultimately which may be supporting the follicular maturation and oocyte competence for ovulation and preventing follicular atresia., (© 2022. The Author(s).)

Objectives: Diabetes mellitus (DM) is a chronic disease associated with serious complications, including male infertility. Umbelliferone (UMB) is a coumarin with promising antioxidant, anti-inflammatory and other beneficial effects. This study investigated the ameliorative effect of UMB against testicular injury, oxidative stress and altered steroidogenesis in rats with type 2 DM., Methods: Rats received a high fat diet for 4 weeks followed by a single injection of streptozotocin. Diabetic rats were treated with UMB or pioglitazone (PIO) for 6 weeks and samples were collected for analysis., Key Findings: Diabetic rats exhibited hyperglycemia, insulin resistance and dyslipidemia associated with increased serum pro-inflammatory cytokines, and decreased gonadotropins and testosterone. UMB significantly ameliorated metabolic alterations, decreased pro-inflammatory cytokines, and increased gonadotropins and testosterone levels. UMB prevented testicular injury, suppressed lipid peroxidation and nitric oxide and increased antioxidants in diabetic rats. In addition, UMB upregulated testicular gonadotropins receptors, steroidogenesis markers (steroidogenic acute regulatory protein, cytochrome P450 family 17 subfamily A member 1 [CYP17A1], 3β-hydroxysteroid dehydrogenase [3ß-HSD] and 17ß-hydroxysteroid dehydrogenase [17ß-HSD]), and peroxisome proliferator-activated receptor gamma (PPARγ) expression., Conclusions: UMB prevents testicular injury by preventing metabolic alterations, suppressing oxidative damage and inflammation, and boosting antioxidant defenses in diabetic rats. UMB enhanced pituitary-gonadal axis and steroidogenesis and upregulated testicular PPARγ in diabetic rats. Thus, UMB may represent a protective agent against testicular injury and sexual dysfunction associated with chronic hyperglycemia., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Precocious male maturation causes reduced welfare and increased production costs in Atlantic salmon ( Salmo salar ) aquaculture. The pituitary produces and releases follicle-stimulating hormone (Fsh), the gonadotropin triggering puberty in male salmonids. However, little is known about how Fsh production is regulated in Atlantic salmon. We examined, in vivo and ex vivo , transcriptional changes of gonadotropin-related genes accompanying the initial steps of testis maturation, in pituitaries of males exposed to photoperiod and temperature conditions promoting maturation (constant light and 16°C). Pituitary fshb , lhb and gnrhr2bba transcripts increased in vivo in maturing males (gonado-somatic index > 0.1%). RNA sequencing (RNAseq) analysis using pituitaries from genetically similar males carrying the same genetic predisposition to mature, but differing by responding or not responding to stimulatory environmental conditions, revealed 144 differentially expressed genes, ~2/3rds being up-regulated in responders, including fshb and other pituitary hormones, steroid-related and other puberty-associated transcripts. Functional enrichment analyses confirmed gene involvement in hormone/steroid production and gonad development. In ex vivo studies, whole pituitaries were exposed to a selection of hormones and growth factors. Gonadotropin-releasing hormone (Gnrh), 17β-estradiol (E 2 ) and 11-ketotestosterone (11-KT) up-regulated gnrhr2bba and lhb , while fshb was up-regulated by Gnrh but down-regulated by 11-KT in pituitaries from immature males. Also pituitaries from maturing males responded to Gnrh and sex steroids by increased gnrhr2bba and lhb transcript levels, but fshb expression remained unchanged. Growth factors (inhibin A, activin A and insulin-like growth factor 1) did not change gnrhr2bba , lhb or fshb transcript levels in pituitaries either from immature or maturing males. Additional pituitary ex vivo studies on candidates identified by RNAseq showed that these transcripts were preferentially regulated by Gnrh and sex steroids, but not by growth factors, and that Gnrh/sex steroids were less effective when incubating pituitaries from maturing males. Our results suggest that a yet to be characterized mechanism up-regulating fshb expression in the salmon pituitary is activated in response to stimulatory environmental conditions prior to morphological signs of testis maturation, and that the transcriptional program associated with this mechanism becomes unresponsive or less responsive to most stimulators ex vivo once males had entered pubertal developmental in vivo ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Crespo, Skaftnesmo, Kjærner-Semb, Yilmaz, Norberg, Olausson, Vogelsang, Bogerd, Kleppe, Edvardsen, Andersson, Wargelius, Hansen, Fjelldal and Schulz.)

Purpose: of the research: To achieve male fertility preservation and restoration, experimental strategies for in vitro germ cell differentiation are required. The effects of two different culture conditions on in vitro maintenance and differentiation of non-human primate germ cells was studied. Three testes from three 6-month-old marmosets were cultured using a gas-liquid interphase system for 12 days. Testicular maturation in pre-culture control and samples cultured in gonadotropin and serum supplemented and non-supplemented culture samples was evaluated using Periodic Acid-Schiff (PAS) and immunohistochemical stainings., Principle Results: Gonadotropins and serum-supplemented tissues demonstrate up to meiotic differentiation (BOULE + Pachytene spermatocyte) and advanced localization of germ cells (MAGEA4+). Moreover, complex (with gonadotropin and marmoset monkey serum) conditions induced progression in somatic cell maturation with advanced seminiferous epithelial organization, maintenance of encapsulation of cultured fragments with peritubular-myoid cells, preservation of tubular structural integrity and architecture., Major Conclusions: We report stimulation-dependent in vitro meiotic transition in non-human primate testes. This model represents a novel ex vivo approach to obtain crucial developmental progression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

New Findings: What is the central question of this study? Giot1, the gene for gonadotropin inducible ovarian transcription factor 1 (GIOT1), is upregulated in osmotically challenged rats: does Giot1 gene expression in the paraventricular nucleus have a role in controlling fluid intake following dehydration and what is the role of ovarian hormones in the modulation of GIOT1 actions? What is the main finding and its importance? GIOT1 acts to regulate water and salt intake as well as hormone secretion after dehydration. The identification of genes that participate in the hormone and behavioural responses involved with hydromineral homeostasis is essential for future exploration of novel drug targets for the treatment of metabolic disease., Abstract: In order to maintain body fluid balance after dehydration, hypothalamic neurons of the paraventricular nucleus (PVN) are activated to promote secretion of vasopressin (AVP) and oxytocin (OXT) from the neurohypophysis, and to modulate the behavioural allostatic responses of thirst and salt appetite. Gonadotropin inducible transcription factor (GIOT1) is a Krüppel-type zinc finger protein induced by gonadotropins and oestradiol (E2). This transcription factor is expressed in the hypothalamus, specifically in the PVN where expression of Giot1 mRNA increases following hydromineral challenges such as water deprivation or salt loading, although its physiological role is not clear. We hypothesize that GIOT1 has a central role in the integrated homeostatic and allostatic responses to disturbances in hydromineral balance, especially in the presence of female gonadal hormones. Female rats with intact ovaries or ovariectomized rats were subjected to specific microinjection of a lentiviral vector mediating Giot1 knockdown in the PVN. Three weeks after injection, rats were subjected to 48 h water deprivation, and thereafter water and salt intake were evaluated. Giot1 knockdown in PVN reduced water and saline intake as well as AVP and OXT secretion. Furthermore, Giot1 knockdown had profound effects on gene expression in the PVN, reducing the abundance of transcripts encoded by the Avp, Oxt, Nr4a1 and Crh genes. In conclusion, the present study shows for the first time that GIOT1 in the PVN regulates both transcription and fluid intake, although any connection to ovarian hormones remains to be established., (© 2021 The Authors. Experimental Physiology © 2021 The Physiological Society.)

Bone morphogenetic proteins (BMPs) are expressed in different cell types of the human ovarian follicle and play important roles in the regulation of ovarian function. BMP-9, also known as growth differentiation factor-2 (GDF-2), belongs to the transforming growth factor-beta (TGF-β) superfamily. BMP-9 is mainly synthesized in the liver and secreted into the blood which allows it to regulate various physiological and pathological functions. To date, the expression of BMP-9 in the human ovary and its function in human granulosa cells remains unknown. In the present study, we detect the protein expression of BMP-9 in the human follicular fluid. Using the primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization as a cell model, we show that treatment with BMP-9 downregulates steroidogenic acute regulatory protein (StAR) expression and suppresses progesterone (P4) production. The expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are not affected by BMP-9 treatment. Mechanistically, treatment of hGL cells with BMP-9 activates both SMAD1/5/8 and SMAD2/3 signaling pathways. Blocking the activations of SMAD1/5/8 and SMAD2/3 by pharmacological inhibitors or knockdown of SMAD4 attenuates the inhibitory effects of BMP-9 on StAR expression and P4 production. This study reveals a novel function of BMP-9 in the regulation of ovarian steroidogenesis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Unpredictability in acquiring an adequate number of high-quality oocytes following ovarian stimulation is one of the major complications in controlled ovarian hyperstimulation (COH). Genetic predispositions of variations could alter the immunological profiles and consequently be implicated in the variability of ovarian response to the stimulation., Design: Uncovering the influence of variations in AMHR2, LHCGR, MTHFR, PGR, and SERPINE1 genes with ovarian response to gonadotrophin stimulation in COH of infertile women., Methods: Blood samples of the women with a good ovarian response (GOR) or with a poor ovarian response (POR) were collected. Genomic DNA was extracted, and gene variations were genotyped by TaqMan SNP Genotyping Assays using primer-probe sets or real-time PCR Kit., Results: Except for PGR (rs10895068), allele distributions demonstrate that the majority of POR patients carried minor alleles of AMHR2 (rs2002555, G-allele), LHCGR (rs2293275, G-allele), MTHFR (rs1801131, C-allele, and rs1801133, T-allele), and SERPINE1 (rs1799889, 4G allele) genes compared to the GOR. Similarly, genotypes with a minor allele in AMHR2, LHCGR, MTHFR, and SERPINE1 genes had a higher prevalence among POR patients with the polymorphic genotypes. However, further genotype stratification indicated that the minor alleles of these genes are not associated with poor response. Multivariate logistic analysis of clinical-demographic factors and polymorphic genotypes demonstrated a correlation between FSH levels and polymorphic genotypes of SERPINE1 in poor response status., Conclusions: Despite a higher prevalence of AMHR2, LHCGR, MTHFR, and SERPINE1 variations in the patients with poor ovarian response, it seems that these variations are not associated with the ovarian response., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To evaluate the association, if any, between serum antimüllerian hormone (AMH) levels and probability of clinical pregnancy and spontaneous abortion (SAB) in the infertility setting., Design: Retrospective cohort study., Setting: Academic fertility center., Patient(s): A total of 1,861 gonadotropin stimulation/intrauterine insemination cycles stratified by AMH levels into 3 groups: Low, <25th percentile (<0.7 ng/mL); Middle, ≥25th and <75th percentile (0.7-4.4 ng/mL); and High, ≥75th percentile (≥4.5 ng/mL)., Intervention(s): Intrauterine insemination after stimulation with gonadotropins., Main Outcome Measure(s): Cumulative probability of clinical pregnancy over a maximum of 3 and/or 6 cycles and SAB incidence risk rate (IRR). The Kaplan-Meier failure function (log rank test), Cox proportional hazards models, and multilevel mixed-effects Poisson regression models were performed to compare outcomes among the AMH groups., Result(s): Overall, in both unadjusted and adjusted models, the probability of achieving a clinical pregnancy was higher in the Middle and High AMH groups compared with that in the Low AMH group, both over 3 (hazard ratios [95% confidence interval], 1.55 [1.05-2.29] and 1.85 [1.22-2.81], respectively) and 6 (1.71 [1.17-2.48] and 2.12 [1.42-3.16], respectively) cycles. In the unadjusted models, the SAB IRR was higher among the Low AMH group (IRR [95% confidence interval], 2.17 (1.11-4.24]), with the relationship persisting after adjusting for age (1.83 [0.93-3.60]). When the SAB IRR were calculated separately for the subpopulations with and without polycystic ovary syndrome, a similar relationship was noted among the latter in the unadjusted (1.94 [0.97-3.88]) and adjusted (1.74 [0.86-3.49]) analyses., Conclusion(s): In women undergoing gonadotropin stimulation/intrauterine insemination, AMH appears to affect the probability of achieving a clinical pregnancy. A possible negative impact, independent of age, on the risk of SAB was also suggested., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

One of the most widely used types of assisted reproduction technology is the in vitro fertilization (IVF), in which women undergo controlled ovarian stimulation through the administration of the appropriate hormones to produce as many mature follicles, as possible. The most common hormone combination is the co-administration of gonadotropin-releasing hormone (GnRH) analogues with recombinant or urinary-derived follicle-stimulating hormone (FSH). In the last few years, scientists have begun to explore the effect that different gonadotropin preparations have on granulosa cells' maturation and apoptosis, aiming to identify new predictive markers of oocyte quality and successful fertilization. Two major pathways that control the ovarian development, as well as the oocyte-granulosa cell communication and the follicular growth, are the PI3K/Akt/mTOR and the Hippo signaling. The purpose of this article is to briefly review the current knowledge about the effects that the different gonadotropins, used for ovulation induction, may exert in the biology of granulosa cells, focusing on the importance of these two pathways, which are crucial for follicular maturation. We believe that a better understanding of the influence that the various ovarian stimulation protocols have on these critical molecular cascades will be invaluable in choosing the best approach for a given patient, thereby avoiding cancelled cycles, reducing frustration and potential treatment-related complications, and increasing the pregnancy rate. Moreover, individualizing the treatment plan will help clinicians to better coordinate assisted reproductive technology (ART) programs, discuss the specific options with the couples undergoing IVF, and alleviate stress, thus making the IVF experience easier., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Papageorgiou, Mastora, Zikopoulos, Grigoriou, Georgiou and Michaelidis.)

Background: Targeted drug delivery is a novel method to specifically deliver anticancer therapeutics to tumor sites. Gonadotropin-releasing hormone (GnRH) is a decapeptide, and its target binding property has attracted attention as a means of targeted drug delivery. Human pancreatic ribonuclease 1 (hpRNase1) has been shown to exert anticancer properties, when fused to a targeting moiety. The goal of the present study was to add a GnRH targeting peptide to the N-terminus of hpRNase1 to specifically target GnRH receptor (GnRH-R) expressing cells., Methods: This in vitro study was conducted at Shiraz Institute for Cancer Research (Shiraz, Iran) in 2019. The coding sequence of GnRH and hpRNase1 were fused, and the chimeric protein together with non-fused hpRNase1 were produced in E. coli (BL21). The recombinant proteins were purified, and their biological activity was evaluated using MTT and apoptosis assays. Non-parametric Kruskal-Wallis tests with Dunn's post hoc tests were performed to determine the significant differences between the study groups., Results: GnRH-hpRNase1 chimeric protein specifically inhibited the proliferation of PC-3 (P=0.021), LNCaP (P=0.034), and AD-Gn (P=0.041) cells, while the growth of negative cells (AD-293) was not significantly affected (P=0.081). GnRH-hpRNase1 decreased the IC50 values more than non-fused hpRNase1, by approximately 26.5-fold (P=0.036) for PC-3 cells, and exerted its growth inhibitory effects through apoptosis induction., Conclusion: Fusion of GnRH to hpRNase1 structure produced an enzyme, which could specifically target tumor cells. This approach can be used to eliminate tumors that harbor GnRH-R., (Copyright: © Iranian Journal of Medical Sciences.)

Aim: To evaluate the overall performance and oocyte quality of follicular phase stimulation (FPS) vs. luteal phase stimulation (LPS) among patients undergoing double ovarian stimulation (DuoStim)., Materials and Methods: Observational retrospective two-center cohort study including 79 infertile women who underwent a total of 87 DuoStim cycles between January 2017 and May 2019. Besides assessing baseline characteristics in order to determine the patients' clinical profile, we analyzed the FPS and LPS regarding the total dose of gonadotropin received, the duration of stimulation, the number and maturity of oocytes, fertilization and blastocyst formation rates, and the number of blastocysts obtained., Results: The patients' baseline characteristics were compatible with a diminished ovarian reserve and poor reproductive prognosis. While the luteal phase needed longer stimulation (12 days (5-19) vs. 11 (7-16), p  < .001) and slightly higher gonadotropin doses (2946 ± 890 IU vs. 2550 ± 970 IU, p  < .001), no significant differences were detected in the oocyte maturity, fertilization, and blastocyst formation rates. However, the number of oocytes retrieved (5 (0-16) vs. 4 (0-15), p  = .006), mature oocytes (4 (0-15) vs. 3 (0-11), p  = .032), and blastocysts obtained (70 vs. 53) were substantially greater after LPS., Conclusions: The DuoStim strategy in poor prognosis patients increases the number of oocytes and blastocysts available. Moreover, the number of oocytes and blastocysts obtained are higher after LPS when compared to FPS. Thus, it should be considered for selected patients in order to not only improve reproductive outcomes but also shorten the time to pregnancy.

The unpredictable variability in patients' responses to gonadotropins represents one of the most intractable IVF treatment problems. Identifying the genetic variants associated with ovarian responses to gonadotropins is an important step towards developing individualised pharmacogenetics protocols for ovarian stimulation. The purpose of the current study was to evaluate correlations between FSHR rs6165, FSHR rs616, and ESR1 rs2234693 gene variants and the degree of ovarian response to gonadotropin in Egyptian women undergoing ICSI treatment. Two hundred and eighty Egyptian women (mean age of 20-35) undergoing ICSI treatment were enrolled in a cross-sectional study conducted between January 2017 and May 2019. The women were classified into three groups based on ovarian response: normal responders (retrieved oocytes = 4-15) (n = 80), poor responders (retrieved oocytes < 4) (n = 92), and high responders (retrieved oocytes> 15) (n = 108). Genomic DNA was extracted from blood samples, and PCR and DNA sequencing were performed to identify genetic variations in the different study groups. FSHR and ESR1 genetic variants were then compared in normal, poor, and high responders. DNA sequencing results showed significant differences in the frequencies of FSHR rs6166 and ESR1 rs2234693 genotypes in poor responders compared with normal responders (P ≤ 0.001 and P ≤ 0.001, respectively). In contrast, no significant differences in the frequencies of FSHR rs6166, FSHR rs6165, or ESR1 rs2234693 genotypes were observed in high responders compared with normal responders (P ≤ 0.074, P ≤ 0.353, and P ≤ 0.060, respectively). These results suggest that FSHR and ESR1 gene variants could predict the degree of ovarian response to Controlled ovarian hyperstimulation in Egyptian women., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Nearly a century ago, studies by Lancelot Hogben and others demonstrated that ovulation in female Xenopus laevis can be induced via injection of mammalian gonadotropins into the dorsal lymph sac, allowing for egg production throughout the year independent of the normal reproductive cycles. Hormonally induced females are capable of producing thousands of eggs in a single spawning, which can then be fertilized to generate embryos or used as a substrate for generation of egg extracts. The protocol for induction of ovulation and subsequent egg collection is straightforward and robust, yet some of its details may vary among laboratories based on prior training, availability of necessary reagents, or the experimental objectives. As the goal of this protocol is not to describe every single variation possible for acquiring eggs but to provide a simple and clear description that can be easily applied by researchers with no prior working experience with X. laevis , we focus on describing the method we use at the National Xenopus Resource-that is, inducing ovulation in X. laevis via dorsal lymph sac injection of gonadotropic hormones and the stimulation of egg laying through application of gentle pressure to the females., (© 2021 Cold Spring Harbor Laboratory Press.)

Polycystic ovary syndrome (PCOS) is characterized by an oligo-anovulation, hyperandrogenism and polycystic ovarian morphology combined with major metabolic disturbances. However, despite the high prevalence and the human and economic consequences of this syndrome, its etiology remains unknown. In this study, we show that female Goto-Kakizaki (GK) rats, a type 2 diabetes mellitus model, encapsulate naturally all the reproductive and metabolic hallmarks of lean women with PCOS at puberty and in adulthood. The analysis of their gestation and of their fetuses demonstrates that this PCOS-like phenotype is developmentally programmed. GK rats also develop features of ovarian hyperstimulation syndrome. Lastly, a comparison between GK rats and a cohort of women with PCOS reveals a similar reproductive signature. Thus, this spontaneous rodent model of PCOS represents an original tool for the identification of the mechanisms involved in its pathogenesis and for the development of novel strategies for its treatment.

A relaxin-like gonad-stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B-chain (21 aa), C-peptide (47 aa), and A-chain (24 aa). There are three putative processing sites (Lys-Arg) between the B-chain and C-peptide, between the C-peptide and A-chain, and within the C-peptide. This structural organization revealed that the mature AscRGP is composed of A- and B-chains with two interchain disulfide bonds and one intrachain disulfide bond. The C-terminal residues of the B-chain are Gln-Gly-Arg, which is a potential substrate for formation of an amidated C-terminal Gln residue. Non-amidated (AscRGP-GR) and amidated (AscRGP-NH 2 ) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP-GR and AscRGP-NH 2 induced oocyte maturation and ovulation in similar dose-dependent manners. This is the first report on a C-terminally amidated functional RGP. Collectively, these results suggest that AscRGP-GR and AscRGP-NH 2 act as a natural gonadotropic hormone in A. scoparius., (© 2020 Wiley Periodicals LLC.)

Objective: To study the association of endometrial thickness (EMT) with live birth rates (LBR) in ovarian stimulation with intrauterine insemination (OS-IUI) treatments for unexplained infertility., Design: Prospective cohort analysis of the Reproductive Medicine Network's Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) randomized controlled trial., Setting: Multicenter randomized controlled trial., Patients: A total of 868 couples with unexplained infertility (n=2,459 cycles)., Interventions: OS-IUI treatment cycles (n = 2,459) as part of the AMIGOS clinical trial., Main Outcome Measures: Live birth rates; unadjusted and adjusted risk ratios (RR) for live birth by EMT category, calculated using generalized estimating equations., Results: The overall mean EMT on day of human chorionic gonadotropin administration in cycles with a live birth was significantly greater than in those without. Compared to the referent EMT group of 9 to 12 mm, the unadjusted RR for live birth for the EMT groups of ≤5 and 6-8 were 0.48 and 0.92, respectively. The test for trend indicated evidence of decreasing LBR with decreasing EMT. After adjustment for ovarian stimulation medication, a linear trend was no longer supported. Stratified analyses revealed no differences in associations by treatment group., Conclusions: In OS-IUI for unexplained infertility, higher LBR are observed with increasing EMT; however, EMT is not significantly associated with LBR when adjusted for OS treatment type. Appreciable LBR are seen at all EMT, even those of ≤5 mm, suggesting that OS-IUI cycles should not be canceled for thin endometrium., Clinical Trial Registration Number: NCT01044862., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess the effects PCOS on live birth rates when transferring a single fresh ideal blastocyst., Methods: A retrospective cohort study performed at the university-affiliated reproductive center. Women with PCOS and a control group of normal ovulatory women who underwent their first fresh embryo transfer with single ideal grade blastocyst were included in the study. Demographic, stimulation information and pregnancy outcomes were collected and analysed. The primary outcome was live birth rates, and secondary outcomes included pregnancy and clinical pregnancy rates., Results: 71 Women with PCOS and 272 normal ovulatory controls underwent their first embryo transfer and met the inclusion and exclusion criteria. PCOS patient were younger (31.0 ± 3.7 vs. 33.1 ± 3.2, p = 0.0001), with higher AFC (40.0 ± 9.3 vs. 13.3 ± 4.6, p = 0.0001), required lower dose of gonadotropins to stimulate (1198 ± 786 vs. 1891 ± 1224, p = 0.0001), and had higher serum testosterone levels (2.3 ± 0.7 vs. 1.1 ± 0.3, p = 0.0001). No significant difference was found between the two groups regarding the number of previous pregnancies, the number of previous full-term pregnancies, the level of basal serum FSH, estradiol level at triggering and the BMI. When compared by Chi squared testing pregnancy rates, clinical pregnancy rates and live birth rates did not differ. However, when controlling (with multivariate stepwise logistic regression) for confounders, live birth rates were lower among the women with PCOS (p = 0.035, CI: 0.18-0.92)., Conclusion: After controlling for confounders, when transferring a fresh single ideal blastocyst, live birth rates were lower among the women with PCOS than normal ovulatory controls.

The development and maturity of follicles are regulated by sex hormones and growth factors. It has been proven that peri-ovarian adipose tissue (POAT) plays an important role in folliculogenesis and fertility in the female ICR and KM mice. The aim of the present study was to further investigate whether the removal of bilateral POAT affected follicular development and lipid metabolism in the female C57BL/6 J mice. Female C57BL/6 J mice at 6-week old were sham-operated (Sham) or removed bilateral POAT (Surgery). After 2 weeks, the mice were subjected to the body composition analysis and indirect calorimetry measurement. Our results show that the Surgery mice exhibited abnormal follicular development, including increased follicular dysplasia and atresia, decreased serum sex hormone levels, and abnormal expression of follicular development-related genes. Correspondingly, the endometrial thickness of the Surgery mice was less than the Sham mice. In addition, the Surgery mice had abnormal lipid metabolism, including reduced fat mass, increased energy expenditure, and up-regulated gene and protein expression involved in lipolysis. These data confirmed the importance of POAT in the follicular development in the female reproduction and suggested the contribution of POAT to the whole-body lipid metabolism., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Oocyte pick up (OPU) coupled with IVP produce over 1 million cattle embryos per year and has been most successful in Bos indicus derived breeds that contain large numbers of antral follicles on their ovaries. More recently, this technology has been applied on a large scale to Bos taurus cattle, where hormone manipulation is generally employed to improve the developmental competence of the COCs. Hormone manipulation, and specifically the use of FSH priming before OPU, has been strategically used in the intensively managed dairy cow, where genomic evaluation and juvenile IVP can produce additional significant genetic gains., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of ovarian superstimulatory treatments in cattle is to obtain the maximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes. While factors inherent to the donor animal are critical, an increased knowledge of ovarian physiology, gonadotropin biochemistry and the ability to manipulate ovarian function have provided alternatives for the design of simple and successful protocols for superovulation in cattle. Recent protocols have also been made more user-friendly and allowed for the grouping of donors for successful superovulation. Although the number of reports associating FSH profiles with superovulatory response is limited, studies designed to reduce the number of FSH treatments necessary to induce superstimulation may provide guidance for the development of optimized gonadotropin treatment protocols. Although high peak levels of circulating FSH following a single administration of Folltropin-V have been shown to be associated with a reduced superstimulatory response, the ideal treatment protocol would seem to be to increase circulating FSH levels to values comparable to those required for the induction of follicle wave emergence, and to maintain these levels for at least 72 h (or 36 h for superstimulation prior to ovum pick-up) to allow follicles to reach an ovulatory size and acquire the capacity to ovulate., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nutrition is an important source of exogenous AGEs and thermally processed foods present in western-style diets contain a large amount of these pro-inflammatory AGEs. Additionally, the intake of dietary AGEs could upregulate ovarian gene expression of inflammatory macrophage markers. The objective of this study was to investigate the effect of diet rich in AGEs on estrous cyclicity and ovarian function in a mouse model. Six-week old C57BL/6 J female mice were randomly subjected to either a diet low in AGEs (L-AGE) or a diet high in AGEs (H-AGE) for a total of 13 weeks. Experiments performed included daily vaginal smears to assess estrous cyclicity, ovarian superovulation with gonadotropins to assess the number of oocytes released, whole ovarian tissue mRNA quantification by RT-PCR to quantify genes involved in folliculogenesis, steroidogenesis, and macrophage markers, and ovarian morphology for follicle count. Outcome measures included estrous cyclicity, number of oocytes following superovulation, expression of genes involved in folliculogenesis, steroidogenesis, and macrophage infiltration as well as the number of primordial, primary, secondary, antral follicles and corpora lutea. Compared to mice on L-AGE diet, mice on H-AGE spent significantly longer time in the diestrus phase, had similar number of oocytes released following ovarian superovulation, and showed significant alterations in genes involved in steroidogenesis (increase in Star mRNA expression levels) and folliculogenesis (increase in Gdf-9 and Fshr mRNA expression levels). Mouse macrophage marker F4/80 mRNA expression was upregulated in mice on H-AGE diet compared to mice on L-AGE diet. Finally, mice on H-AGE diet had significantly fewer corpora lutea in their ovaries. These results indicate that the ingestion of high amounts of dietary AGEs could disrupt folliculogenesis and steroidogenesis that might lead to abnormal estrous cyclicity. Intake of dietary AGEs could also upregulate ovarian gene expression of inflammatory macrophage markers., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Co-administration of letrozole during the first 5 days of ovarian stimulation was suggested to improve IVF outcomes in poor responders. We aimed to determine whether poor/sub-optimal responders might benefit from Letrozole co-treatment throughout the entire stimulation course., Methods: We retrospectively reviewed the medical files of women who demonstrated poor (oocyte yield ≤3) and sub-optimal (4 ≤ oocyte yield ≤9) ovarian response during conventional multiple-dose antagonist stimulation protocols and were co-treated in a subsequent cycle with 5 mg Letrozole from the first day of stimulation until trigger day. A self-paired comparison between gonadotropins-only and gonadotropins-letrozole cycles was performed., Results: Twenty-four patients were included. Mean patients' age was 39.83 ± 4.60 and mean day-3-FSH was 12.77 ± 4.49 IU/m. Duration of stimulation and total gonadotropins dose were comparable between the two cycle groups. Peak estradiol levels were significantly lower in gonadotropins-letrozole cycles (2786.74 ± 2118.53 vs 1200.13 ± 535.98, p < 0.05). Number of retrieved oocytes (3.29 ± 2.15 vs 6.46 ± 3.20, p < 0.05), MII-oocytes (2.47 ± 1.65 vs 5.59 ± 3.20, p < 0.05), 2PN-embryos (1.78 ± 1.50, 4.04 ± 2.74, p < 0.05) and top-quality embryos (0.91 ± 0.97 vs. 2.35 ± 1.66, p < 0.05) were significantly higher in the gonadotropins-letrozole cycles. Clinical pregnancy rate in gonadotropins-letrozole cycles was 31.5%., Conclusion: Letrozole co-treatment during the entire stimulation course improves ovarian response and IVF outcomes in poor/sub-optimal responders.

PIWI-interacting RNAs (piRNAs) play an important role in gametogenesis, fertility and embryonic development. The current study investigated the effect of different doses of pregnant mare serum gonadotrophin/human chorionic gonadotrophin (PMSG/hCG) and repeated ovarian stimulation (OS) on the expression of the Mili, Miwi, Mael, Tdrd1, Tdrd9, qnd Mitopld genes, which have crucial roles in the biogenesis and function of piRNAs. Here, we found that after treatment with 7.5 I.U. PMSG/hCG and two repeated rounds of OS, both the mRNA and protein levels of Tdrd9, Tdrd1 and Mael showed the greatest decrease in the ovarian tissue, but the plasma E2 levels showed the strongest increases (p<0.05). However, we found that the Mitopld, Miwi and Mili gene levels were decreased significantly after treatment with 12.5 I.U. PMSG/hCG. Our results suggested that exogenous gonadotropin administration leads to a significant decrease in the expression of the Mili, Miwi, Mael, Tdrd1, Tdrd9 and Mitopld genes, which are critically important in the piRNA pathway, and the changes in the expression levels of Tdrd9, Tdrd1 and Mael may be associated with plasma E2 levels. New comprehensive studies are needed to reduce the potential effects of OS on the piRNA pathway, which silences transposable elements and maintains genome integrity, and to contribute to the safety of OS., Competing Interests: The authors declare that there is no conflict of interest.

Objective: To investigate the effect of gonadotropin (Gn) on embryo aneuploidy rate and pregnancy outcome during preimplanptation genetic testing for aneuploidy (PGT-A) cycles. Methods: The clinical data of patients undergoing PGT-A cycle at the First Medical Center of the PLA General Hospital from January 1, 2013 to May 31, 2019 were retrospectively analyzed. Patients were divided into younger patient group (<35 years old) and elder patient group (≥35 years old) by maternal age, then divided into two groups in line with Gn dosage (≤2 250 U, >2 250 U), and into four groups by number of oocytes retrieved (1-5, 6-10, 11-15 and ≥16 oocytes). The embryo aneuploidy rate and pregnancy outcome between the groups were compared. Logistic regression was used to analyze the relationship between the cumulative amount of Gn, embryo aneuploidy rate and live-birth rate. Results: A total of 402 cycles (338 patients) and 1 883 embryos were included in the study. (1) In the younger patients, the aneuploidy rate was 52.5% (304/579) in the group of Gn≤2 250 U and 48.6% (188/387) in the group of Gn > 2 250 U, with no significant difference between them ( P= 0.232). In the elderly patients, the difference in embryo aneuploidy rate between the two Gn group [57.9% (208/359) versus 60.6% (319/526)] was not statistically significant ( P= 0.420). (2) The embryonic aneuploidy rate in different protocol of ovary stimulation was analyzed,in the younger group, the embryonic aneuploidy rate in patients using antagonist long protocol was 50.3% (158/314), it was 50.0% (121/242) in agonist long protocol, 52.1% (207/397) in agonist short protocol and 6/13 in luteal phase protocol, no statistical difference was found in above groups ( P= 0.923); in the elder group, embryonic aneuploidy rate was 60.8% (191/314) in antagonist protocol, 58.4% (132/226) in agonist long protocol, 59.2%(199/336) in agonist short protocol, 5/9 in luteal phase protocol, respectively,no significant difference was found ( P= 0.938). (3) In the younger patients, the aneuploidy rate in 1-5 oocytes group, 6-10 oocytes group, 11-15 oocytes group and ≥16 oocytes group was 37.9% (11/29), 54.0% (94/174), 52.5% (104/198) and 50.1% (283/565) respectively, no significant difference was found between the groups ( P= 0.652); while in the elder patients, the difference between aneuploidy rate in each retrieved oocytes group [73.6% (89/121), 57.5% (119/207), 56.3% (108/192), 57.8% (211/365)] was statistically significant ( P= 0.046). (4) Logistic regression analysis of age, cumulative dosage of Gn, number of oocytes obtained, and embryo aneuploidy rate showed that there was no association between the amount of Gn and embryo aneuploidy rate ( P> 0.05); the increase in maternal age would increase the risk of aneuploidy rate of embryos, which was statistically significant ( OR= 1.031, 95 %CI : 1.010-1.054, P= 0.004); the increase in oocytes retrived would significantly decrease the risk of aneuploidy ( OR= 0.981, 95 %CI : 0.971-0.991, P< 0.01). (5) There was no significant difference in biochemical pregnancy rate [55.6% (80/144) versus 52.1% (63/121)], clinical pregnancy rate [50.0% (72/144) versus 47.9% (58/121)] and live-birth rate [46.5% (67/144) versus 40.5% (49/121)] between different Gn dosage groups ( P= 0.613, P= 0.738, P= 0.324). The logistic regression analysis showed that the maternal age, the cumulative dosage of Gn, the number of oocytes obtained, and the ovarian stimulation protocol had no effect on the live-birth rate (all P> 0.05). Conclusions: In PGT-A cycle, the dosage of Gn has no association with the embryo aneuploidy rate and pregnancy outcome. In the patients ≥35 years old, the increase in number of oocytes obtained may decrease the risk of aneuploidy. Age is an important factor affecting the embryo aneuploidy in PGT-A cycle.

In human, the use of freshly recovered granulosa cells for experiments remains difficult. Because of the single use of human cells, the experiments cannot be repeated, and no additional conditions can be tested afterwards with the cells of the same patient. Therefore, granulosa cell cryopreservation could be a good alternative to keep part of these cells for later controls or experiments. The aim of this study is to compare the responsiveness to FSH of fresh and frozen-thawed human primary granulosa-lutein cells (hGLC) and determine if cryopreserved granulosa cells can be used in place of fresh cells. Two cryopreservation methods were also compared: a conventional versus a simplified freezing method. This experimental study was undertaken at Igyxos S.A., Nouzilly, France. Seventy women undergoing oocyte retrieval at the IVF Unit from Bretonneau University Hospital (Tours, France) were recruited in 2016. Fresh and frozen-thawed hGLC were cultured for 7 days and then stimulated by r-FSH for 48 h. To assess r-FSH efficacy and potency, extracellular cAMP accumulated in the supernatant for each stimulation point was measured. We demonstrated that hGLC remain responsive to FSH stimulation after freezing-thawing and 7 days of pre-culture. They are able to secrete cAMP with a similar EC50 value as fresh hGLC, but FSH efficacy is lowered. As our study did not show any significant difference between the two freezing methods concerning the sensitivity of hGLC to FSH, hGLC could be cryopreserved with the simplified freezing method without taking up too much time for IVF laboratories.

In this study, we investigated in an androgenized rat model the involvement of autophagy and mitochondrial dynamics in granulosa cells in the pathogenesis of polycystic ovarian syndrome (PCOS) and its modulation by exogenous gonadotropin (eCG). We found 5α-dihydrotestosterone (DHT) treatment reduces ovarian length and weight with predominantly late antral and/or preovulatory stage follicles and no corpora lutea. DHT increased the population of large lysosomes (>50 micron) and macroautophagy, an event associated with granulosa cell apoptosis. Increased granulosa cell Dynamin Related Protein 1 (Drp1) content in the DHT group was accompanied by increased circular and constricted, but reduced rod-shaped, mitochondria. eCG eliminated all atypical follicles and increased the number of late antral and preovulatory follicles with less granulosa cell apoptosis. eCG-treated rats had a higher proportion of connected mitochondria, and in combination with DHT had a lower proportion of circular and constricted mitochondria than rats treated with DHT alone, suggesting that eCG induces mitochondrial fusion and attenuates fission in granulosa cells. In summary, we observed that DHT-induced up-regulation of Drp1 is associated with excessive mitochondrial fission, macroautophagy and apoptosis in granulosa cells at the antral stage of development in an androgenized rat model for PCOS, a response partially attenuated by exogenous gonadotropin.

We studied the influence of the estrous cycle on the morphology of preovulatory (germinal vesicle, GV) oocytes in mice and their capacity to meiotic maturation in vitro. After standard injections of eCG gonadotropin (PMSG, Follimag) to females at different stages of the estrous cycle, the maximum levels of GV oocytes (26±1/mouse) were isolated from the ovaries of animals injected with the hormone during estrus. The capacity of isolated GV oocytes to meiotic maturation in vitro decreased in the following order: estrus (75.5±2.3%), metestrus (67.9±3.4%), proestrus (57.8±4.4%), and diestrus (50.6±5.6%); the differences between estrus and diestrus/proestrus were significant (p<0.05). After eCG injections during estrus, GV oocytes differed from other oocytes by lesser total diameter, lesser diameter of cytoplasm, lesser thickness of zona pellucida, and moderately dilated perivitelline space. These signs reflected higher competence of the "estrous" GV oocytes for meiotic maturation in vitro. Hormone stimulation of females with eCG, with consideration for the stage of the estrous cycle, seems to be an effective method for improving the quality of GV oocytes isolated from mouse ovaries.

Endogenous estrogens play major roles in many aspects of female reproductive development in fish. In order to develop a relatively high-throughput assay to determine the potential impact on reproductive development, vitellogenic rainbow trout ovarian follicles were exposed to a suite of contaminants in vitro and then assessed for the ability to produce estradiol-17β (E2) after a 500 ng/ml salmon gonadotropin (sGTH) challenge. There was a positive correlation between ovarian follicle size and E2 production, but an inverse correlation between size and responsiveness to sGTH. Significant impacts on E2 levels were observed following treatment with different endocrine disrupting chemicals, such as 17α-ethinylestradiol (EE2), prochloraz, or trenbolone. EE2 was remarkably potent and significantly reduced ovarian follicle responsiveness to sGTH at concentrations as low as 0.1 nM. Of the other contaminants tested, only tamoxifen impacted E2 levels, and only at concentrations near the limits of solubility. Flutamide, fluoxetine, 4-hydroxy tamoxifen, hydroxyflutamide, and norfluoxetine had little or no impact. Quantitative PCR analyses of steroidogenesis-related genes were carried out on EE2 treated ovarian follicles, but significant transcriptional responses to EE2 were not observed. Overall, this study suggests that xenoestrogens and anti-estrogens are more likely to interfere with ovarian E2 synthesis than other classes of EDCs. This also provides a template for further testing of the effects of EDCs on ovarian function., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To explore the changes and correlations of anti-Müllerian hormone (AMH) and stem-cell factors (SCF) in different ovarian reserve patients during controlled ovarian hyperstimulation (COH) and the effects on COH outcomes., Methods: Serum at six different timepoints during GnRH-antagonist protocol and follicular fluid (FF) on oocyte retrieval day of 52 patients with polycystic ovary syndrome (PCOS), 61 patients with normal ovarian reserve (NOR) and 42 patients with diminished ovarian reserve (DOR) were collected. AMH and SCF were assessed using enzyme-linked immunosorbent assay., Results: During COH, AMH in the PCOS group was the highest, but SCF did the opposite, and serum AMH gradually decreased, while SCF inversely increased. In the PCOS group, SCF on the first and fourth days of gonadotropin (Gn) administration was negative with Gn dosage (r = - 0.362, P < 0.05; r = - 0.344, P < 0.05). In the NOR group, the basal AMH was also negative with Gn dosage (r = - 0.297, P < 0.05) and positive with COH outcomes (number of retrieved oocytes, MII oocytes, and 2PN fertilization) as well as serum SCF after Gn administration. In the DOR group, both AMH and SCF were significantly associated with COH outcomes. Serum AMH in the DOR group after Gn administration and FF AMH showed a negative correlation with SCF., Conclusions: Serum AMH decreased, while SCF increased during COH. AMH and SCF are effective for Gn time and dosage adjustment and predicting COH outcomes for NOR and DOR patients. In addition, serum AMH in DOR patients after Gn administration and FF AMH has a negative effect on SCF.

Controlled ovarian hyperstimulation cycle with exogenous gonadotropins (COH) is associated with clinical complications. The aim of this work was to determine whether COH alters the physiological antioxidant status of follicular fluid in women with no reproductive dysfunction, compared to the natural cycle (NC). In this longitudinal study, forty-one women (oocyte donors) consecutively underwent NC and COH. Follicular fluid was collected at oocyte retrieval and different redox biomarkers were determined: total antioxidant activity (TAA), oxygen radical absorbance capacity (ORAC), nitric oxide, α- and γ-tocopherol, the fatty acid composition, activities of superoxide dismutase, catalase, total and Se-dependent glutathione peroxidases, and the antioxidant paraoxonase (PON) family. Results showed that TAA (1.70 ± 0.03 mM versus 1.86 ± 0.03 mM, p < 0.05), α-tocopherol (4.37 ± 0.26 μM versus 5.74 ± 0.30 μM, p < 0.05), PON1 paraoxonase (245 ± 24 nmol/min/ml versus 272 ± 27 nmol/min/ml, p < 0.05), PON1 arylesterase (87.2 ± 4.6 μmol/min/ml versus 99.3 ± 4.8 μmol/min/ml, p < 0.05), and PON3 simvastatinase (13.48 ± 0.52 nmol/min/ml versus 16.29 ± 0.72 nmol/min/ml, p < 0.001) were significantly lower in COH versus NC. Fatty acids from COH were more saturated, increasing palmitate and decreasing the n-6 and total polyunsaturated fatty acids (PUFAs). Docosahexaenoic acid also increased (p < 0.05). Results suggest that COH could lead to premature ovarian aging and provide new insights into the possible prevention of the adverse effects of ovarian hyperstimulation by directing therapeutic applications to the maintenance of the redox balance and fatty acid status, with special attention to paraoxonase proteins and docosahexaenoic acid., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n = 154) and from perimenopausal and postmenopausal women (n = 28). Serum concentrations of BMP15 and GDF9 were analyzed in women relative to age, anti-Müllerian hormone, number of oocytes retrieved, and polycystic ovary syndrome (PCOS) after superovulation for in vitro fertilization. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/mL, respectively), and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64-fold and 15-fold, respectively, between women, but they did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15 concentration, was associated with oocyte number retrieved in patients without PCOS (P = 0.018). GDF9 and BMP15 associations with oocyte number differed significantly (P < 0.05) with PCOS status. GDF9 concentrations were lower in poor responders (women with fewer than four oocytes retrieved or with cancelled cycles; P = 0.020). Serum BMP15, but not GDF9, was lower in women >55 years of age, compared with women of reproductive age (P < 0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine., (Copyright © 2019 Endocrine Society.)

The aim of this study was to investigate if there is an adverse effect of multiple controlled ovarian stimulation (COS) on the maturity of oocytes (MI and MII), fertilization rate, embryo developmental qualities and clinical pregnancy rates in donation cycles. In total, 65 patients undergoing oocyte donation cycles multiple times were included in this study. Patients were grouped as group A that consisted of donors with ≤2 stimulation cycles while B consisted of donors with ≥3 stimulation cycles; and group C included donors who had ≤15 oocytes, while group D had donors with ≥16 oocytes. Numbers of oocytes obtained, MI and MII oocytes, fertilization, embryo quality and clinical pregnancy outcomes were compared. Significant statistical differences were observed in total number of oocytes obtained, maturity of oocytes (MI and MII), fertilization rate, embryo qualities and clinical pregnancy outcomes of donors in groups A-D. Donors with ≤2 ovarian stimulation cycles had lower numbers of immature oocytes than donors with three or more stimulation cycles. However, donors with ≥3 stimulation cycles had higher numbers of mature oocytes, zygotes, with better day 3 embryo qualities and higher clinical pregnancy rates than donors with ≤2 stimulation cycles. Repeated COS does not seem to have any adverse effect on ovarian response to higher dose of artificial gonadotropin, as quality of oocytes collected and their embryological developmental potential were not affected by the number of successive stimulation cycles. The effect of multiple COS on the health of the oocyte donor needs to be assessed for future purpose.

Objective: To determine if natural selection and follicular stimulation produces a lower risk for embryonic aneuploidy than that attained following superovulation with exogenous gonadotropins., Design: Prospective observational with historical control group., Setting: Large academically affiliated private practice., Patient(s): All patients presenting for their evaluation for infertility were offered participation in the study., Intervention(s): All participants in the natural cycle group underwent an unstimulated in vitro fertilization (IVF) cycle. A subsequent frozen embryo transfer was performed if a euploid blastocyst was attained., Main Outcome Measure(s): Rates of embryonic aneuploidy attained in unstimulated IVF cycles were compared to those observed in age-controlled historical cohort undergoing conventional stimulated IVF cycles with exogenous gonadotropins., Result(s): Aneuploidy rates were equivalent in unstimulated and stimulated IVF cycles. The prevalence of aneuploidy in natural cycles increased with the age of the female partner in a manner identical to that seen in stimulated IVF cycles. Finally, sustained implantation rates of euploid blastocysts were equivalent in natural and stimulated IVF cycles., Conclusion(s): Rates of embryonic aneuploidy are not impacted by follicular stimulation with exogenous gonadotropins. Prior concerns of inducing a higher risk of embryonic aneuploidy are not supported by this data., Clinical Trial Registration Number: NCT01866618., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication in women undergoing induction of ovulation with human chorionic gonadotropin (hCG) for assisted reproductive techniques. Amphiregulin (AREG) is the most abundant epidermal growth factor receptor (EGFR) ligand expressed in human granulosa cells and follicular fluid and can be upregulated by luteinizing hormone (LH)/hCG. However, whether the expression levels of AREG, EGFR, and HER2 change in the granulosa cells of OHSS patients remains unknown. If it does, whether these molecules are involved in the development of OHSS requires investigation. In the present study, we showed that AREG, EGFR, and HER2 transcripts in granulosa cells as well as follicular fluid AREG proteins were elevated in OHSS patients. Increased AREG levels were associated with transcript levels and follicular content of vascular endothelial growth factor (VEGF), the marker for OHSS pathology. Treatment of cultured granulosa cells with AREG stimulated VEGF expression and secretion, with granulosa cells from OHSS patients showing more rapid and pronounced increases than the non-OHSS group. In addition, siRNA-mediated knockdown of EGFR and AREG attenuated the hCG-induced upregulation of VEGF. This study demonstrated that granulosa cell-secreted AREG plays an important role in the development of OHSS, suggesting that the EGFR/HER2-mediated signaling could be a novel drug target for the prevention and treatment of OHSS., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)