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The aim of our study was to evaluate if the response to follicular GnRH agonist (GnRHa) trigger be used to predict intracycle ovarian response in GnRH antagonist cycles among women undergoing fertility preservation IVF. We conducted a prospective study of 146 GnRH antagonist oocyte pickup (OPU) cycles to evaluate GnRHa stimulation test (GAST). On day 2 of the cycle, basal E2 were measured, followed by injection of 0.2 mg GnRHa as part of the initial ovarian stimulation. 12 h later blood sampling was repeated (GAST E3). E2 response was used as test parameter. The major outcome was the number of mature cryopreserved oocytes. We found a linear correlation between both GAST E3 level and GAST E3/E2 ratio and number of M2 oocytes. ROC curve analysis of GAST E3, GAST E3/E2 ratio, AFC and day 3 FSH for > 15 M2 and < 5 M2 oocytes was calculated. For GAST E3 levels obtaining < 5 M2 oocytes, an AUC value of 0.79 was found. For GAST E3 levels obtaining > 15 M2 oocytes, AUC value of 0.8. Patients with GAST E3 ≤ 384 pmol/l has 58.6% risk to obtain < 5 oocytes. Patients younger than 35 with GAST E3 > 708 pmol/l have 66% chance for freezing > 15 oocytes. The response to single GnRHa administration during GnRH antagonist cycle can be used as biomarker of ovarian reserve. This simple, widely available marker, which reflect the estradiol response of small follicles, might predict the response of the specific cycle, and can potentially be used to adjust the treatment dose.Trial registration number: 0304-20-ASF., (© 2024. The Author(s).)

The role of prostaglandins (PGs) in the ovulatory process is known. However, the role of the ATP binding cassette subfamily C member 4 (ABCC4), transmembrane PG carrier protein, in ovulation remains unknown. We report herein that ABCC4 expression is significantly upregulated in preovulatory human granulosa cells (GCs). We found that PGE2 efflux in cultured human GCs is mediated by ABCC4 thus regulating its extracellular concentration. The ABCC4 inhibitor probenecid demonstrated effective blocking of ovulation and affects key ovulatory genes in female mice in vivo. We postulate that the reduction in PGE2 efflux caused by the inhibition of ABCC4 activity in GCs decreases the extracellular concentration of PGE2 and its ovulatory effect. Treatment of female mice with low dose of probenecid as well as with the PTGS inhibitor indomethacin or Meloxicam synergistically blocks ovulation. These results support the hypothesis that ABCC4 has an important role in ovulation and might be a potential target for non-hormonal contraception, especially in combination with PGE2 synthesis inhibitors. These findings may fill the gap in understanding the role of ABCC4 in PGE2 signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment and control of fertility., (© 2023 Federation of American Societies for Experimental Biology.)

Objective: To assess the influence of coronavirus disease 2019 (COVID-19) messenger ribonucleic acid vaccine on ovarian response and in vitro fertilization (IVF) treatment outcomes., Design: A retrospective cohort study., Setting: A tertiary university-affiliated medical center and a private medical center., Patient(s): The study included a total of 400 patients, 200 vaccinated women and 200 age-matched unvaccinated women, who underwent IVF in January-April 2021., Intervention(s): None., Main Outcome Measure(s): The mean number of oocytes retrieved and clinical pregnancy rates in vaccinated vs. unvaccinated patients., Result(s): A total of 200 patients underwent oocyte retrieval 14-68 days after receiving COVID-19 vaccination. No difference was found in the mean number of oocytes retrieved per cycle (10.63 vs. 10.72) between vaccinated and unvaccinated patients. Among 128 vaccinated and 133 unvaccinated patients who underwent fresh embryos transfers, no difference was demonstrated in the clinical pregnancy rates (32.8% vs. 33.1%), with 42 and 44 clinical pregnancies, respectively. The fertilization rates and mean number of cryopreserved embryos were similar between the 2 groups in freeze-all cycles (55.43% vs. 54.29% and 3.59 vs. 3.28, respectively). Among vaccinated and unvaccinated patients who underwent fresh embryo transfers, no difference was noted in the fertilization rate (64.81% vs. 61.98%) and transferred embryos' quality. Regression models applied demonstrated no effect of the vaccine on oocyte yields and pregnancy rates., Conclusion(s): The COVID-19 messenger ribonucleic acid vaccine did not affect the ovarian response or pregnancy rates in IVF treatment. Women should be vaccinated for COVID-19 before attempting to conceive via IVF treatments, given the higher risk of severe illness in pregnant women., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The superiority of day 5 blastocysts compared to day 6 blastocysts in fresh cycle transfers was previously demonstrated and attributed mainly to endometrial asynchrony. Data from frozen blastocysts transfers showed conflicting results, possibly due to heterogeneous patient population and embryo quality. The aim of this study was to compare clinical pregnancy rate (CPR) and live birth rate (LBR) between transfers of vitrified day 5 blastocysts and day 6 blastocysts in oocyte donation, blastocyst-only cycles. In a retrospective, multi-center study, with a single oocyte donation program, a total of 1840 frozen embryo transfers (FET's) were analyzed, including 1180 day 5 blastocysts and 660 day 6 blastocysts transfers. Day 5 blastocyst transfers had better embryonic development and significantly higher CPRs (34.24% vs. 20.15%, P < 0.0001), higher LBRs (26.89% vs. 14.77%, P < 0.0001), less cycles to LBR (1.83 ± 0.08 vs. 2.39 ± 0.18, P = 0.003) and shorter time to LBRs (76.32 ± 8.7 vs. 123.24 ± 19.1 days, P = 0.01), compared to day 6 transfers, respectively. A multivariate stepwise logistic regression indicated, that day 5 transfer was an independent factor for CPRs (OR 1.91; 95% CI 1.43-2.54, P < 0.001) and LBRs (OR 2.26; 95% CI 1.19-4.28, P = 0.01), regardless of embryo quality, compared to day 6. In conclusion, day 5 blastocysts in oocyte donation program have significantly higher CPRs and LBRs, and present shorter time to delivery, compared to day 6 blastocysts, regardless of embryo quality.

Background: DCN (decorin) is a proteoglycan known to be involved in regulating cell proliferation, collagen fibril organization and migration. In our global transcriptome RNA-sequencing approach to systematically identify new ovulation-associated genes, DCN was identified as one of the highly regulated genes. We therefore hypothesize that DCN may have a role in ovulatory processes such as cell migration and proliferation., Aim: To characterize the expression, regulation and function of the proteoglycan DCN in the human ovarian follicles during the preovulatory period., Methods: The in-vivo expression of DCN mRNA in mural (MGCs) and cumulus (CGCs) granulosa cells was characterized using quantitative RT-PCR and western blot. A signaling study was performed by treating human MGCs cultures with gonadotropins and different stimulators and inhibitors to determine their effect on DCN expression by qRT- PCR and elucidate the pathways regulating these proteins. In a functional study, KGN granulosa cell line was used to study cell migration with a scratch assay., Results: DCN mRNA expression was significantly higher in MGCs compared to CGCs. DCN mRNA was significantly higher in CGCs surrounding mature metaphase II (MII) oocytes compared to CGCs of germinal vesicle (GV) and metaphase I (MI) oocytes. hCG significantly increased DCN mRNA and protein expression levels in cultured MGCs. Using signal transduction activators and inhibitors, we demonstrated that DCN induction by LH/hCG is carried out via PKA, PKC, ERK/MEK, and PI3K pathways. We showed that DCN expression is also induced in high-density cell cultures, in a dose-dependent pattern. In addition, progesterone induced a significant increase in DCN secretion to the media. MGCs from follicles of endometriosis patients exhibited reduced (about 20% of) mRNA transcriptions levels compared to MGCs follicles of control patients. More significantly, we found that DCN has an inhibiting effect on KGN cell migration., Conclusions: Our study indicates that DCN is a unique ovulatory gene. Our findings support the hypothesis that DCN plays an important new role during the preovulatory period and ovulation, and stress its involvement in endometriosis infertility. A better understanding of DCN role in ovulation and endometriosis may provide treatment for some types of infertility.

Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown. The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs. hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion. Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6-8 h after hCG, while 24 h of hCG stimulation was needed for late induced genes. Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24 h later., (Copyright © 2019 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.)

Background: The cumulus expansion process is one of the LH mediated ovulatory processes. Hyaluronan synthase 2 (HAS2) regulates the synthesis of hyaluronic acid, the main component of the cumulus expansion process. Recently, the lncRNA HAS2 antisense RNA 1 (HAS2-AS1) was identified in our global transcriptome RNA-sequencing of novel ovulation associated genes. The role of HAS2-AS1 in HAS2 regulation w.as studied previously with contradictive results in different models but not in the ovary. Taken together the induction of HAS2-AS1 and the important role of HAS2 in the cumulus expansion process, we hypothesize that HAS2-AS1 regulate HAS2 expression and function in the ovary. Therefore we undertook to study the expression, regulation, and possible functional role of HAS2-AS1 in the human ovary., Results: HAS2-AS1, located within the HAS2 gene that was highly regulated in our library. We found that HAS2-AS1 express mainly in cumulus cells (CCs). Furthermore, HAS2-AS1 showed low expression in immature CCs and a significant increase expression in mature CCs. Functional studies reveal that inhibition of HAS2-AS1 by siRNA caused decrease expression of HAS2. Furthermore, inhibition of HAS2-AS1 by siRNA results in decrease migration of granulosa cells., Conclusions: Our results suggest that HAS2-AS1 is an LH/hCG target gene that plays a positive role in HAS2 expression and thus might play a role in regulating cumulus expansion and migration.

Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.

Purpose: Few clinical options for fertility preservation are available to females with cancer, and data about clinical outcomes is limited. Potential supplementary approaches to fertility preservation include retrieval of immature oocytes followed by in vitro maturation (IVM) and storage. The aim of this study was to evaluate post-thawing outcomes of immature oocytes collected both by transvaginal aspiration and from excised ovarian tissue., Methods: We conducted a retrospective cohort study of patients treated in a single tertiary center. We reviewed the records of 119 cancer patients who underwent ovarian tissue cryopreservation and immature oocyte harvesting for fertility preservation. All embryos and oocytes that were frozen and thawed were included in the study. Post-thawing outcomes were evaluated., Results: Thirty-five stored embryos from eight patients were thawed. Twenty-nine embryos survived (82% survival rate) and were transferred. Six oocytes were thawed, two oocytes survived, and no oocytes were fertilized. Only one PCOS patient became pregnant, resulting in the normal delivery of a healthy baby., Conclusions: Although a relatively high number of mature oocytes and embryos can be stored with the combined procedure, the limited rate of pregnancies represents a poor reproductive outcome. Therefore, this approach should be reserved for special groups with limited options.

Purpose: Partial thawing of a vial of cryopreserved sperm (shaving) is sometimes applied as a measure to preserve sperm for further use, particularly in cases of very restricted sperm quantity. However, mechanical violence may disrupt the sperm-wall and lead to impaired in-vitro fertilization (IVF) outcomes., Material and Methods: In a retrospective case-control study at a tertiary, university-affiliated medical center, we compared the IVF/intracytoplasmic sperm injection (ICSI) outcomes of patients who used donor sperm following partial thawing (shaving) of the vial of cryopreserved sperm (n = 99) to a control group consisting of patients for whom the vial of sperm was completely thawed before use (n = 99)., Results: While no differences were observed in the rates of oocyte fertilization, the mean number of top-quality embryos (TQE) was significantly lower in the shaving group than in the complete thawing group (1.33 ± 0.17 vs. 1.87 ± 0.17, p < 0.02). Experimental analysis of aliquots from the same donors revealed significantly reduced motility in sperm samples that were shaved vs. fully thawed (6.5 vs. 37.1%, p < 0.001)., Conclusions: In cases in which available cryopreserved sperm samples are limited, shaving of the vial without thawing can be used but with caution and only when absolutely necessary. Further, large prospective studies are needed to better clarify whether there is post-thawing sperm damage and to compare IVF outcomes after these two thawing methods.

Prostaglandins (PGs) play an important role in the ovulatory process. However, the role of the PG transporter (PGT) in this context remains unknown. We report that the expression of PGT, a transmembrane PG carrier protein, is markedly up-regulated in preovulatory human granulosa cells (GCs). Treatment with human chorionic gonadotropin (hCG), an ovulatory trigger, significantly increases the expression of PGT mRNA and protein in human GCs both in vivo and in vitro. The hCG-induced increase in the expression of PGT in cultured human GCs is mediated via protein kinase A and protein kinase C by way of the extracellular signal-regulated kinase pathway. PGT in cultured human GCs mediates the uptake of PGE2, thereby regulating its extracellular concentration. In vivo treatment of mice with PGT inhibitors effectively blocks ovulation and markedly attenuates the expression of key ovulatory genes. We hypothesize that the inhibition of PGT activity in GCs increases the extracellular concentration of PGE2, the ability of which to exert its ovulatory effect is compromised by desensitization of its cognate receptors. Together, these findings support the idea that PGT is an important mediator of ovulation and that its inhibitors may be viewed as potential candidates for nonhormonal contraception. These findings may also fill the gap in the understanding of PGT signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment of infertility or subfertility in women by using nonsteroidal PG-based therapeutic approaches., (Copyright © 2016, American Association for the Advancement of Science.)

The aim of this study was to evaluate the safety and efficacy of combined ovarian tissue cryopreservation and oocyte aspiration just before ovarian tissue cryobanking. A retrospective cohort study of fertility preservation patients treated in 2007-2013 in one tertiary centre was performed. A total of 255 cancer patients were admitted for fertility preservation: 142 patients underwent ovarian tissue cryopreservation only (OTC), 56 underwent OTC plus oocyte retrieval from ovarian tissue (OTIVM), nine underwent oocyte aspiration and in-vitro maturation (AIVM) and 48 underwent all three procedures. The total number of oocytes, total number of metaphase II (MII) oocytes, maturation rate, fertilization rate and total number of cryopreserved oocytes between groups were compared. The study found significantly more oocytes (P < 0.001), more MII oocytes (P < 0.001), better maturation rate (P < 0.01) and more cryopreserved oocytes (P < 0.05) with all three compared with OTIVM or OTC. No adverse outcome was observed by performing oocyte retrieval before ovarian resection for cryopreservation. In conclusion, oocyte aspiration just before ovarian tissue cryobanking is safe and gains more oocytes with a better maturation rate than ovarian tissue oocyte cryobanking alone. Better results were obtained with 3 days of stimulation before oocyte retrieval., (Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: Serum AMH is declining with age and is highly associated with ovarian follicular reserve and disordered folliculogenesis. However, the precise role of AMH in the process of human follicular aging has still to be determined., Aim: This study investigates AMH level in the follicular fluid (FF) and mRNA expression pattern in cumulus and mural granulosa cells of human ovarian follicles in relation to age., Methods: We conducted a prospective study. Sixty-eight women undergoing In vitro fertilization (IVF) treatment were enrolled in the study. We obtained FF, mural and cumulus granulosa cells from large preovulatory follicles (17-20 mm) of 21-35 years old women (n = 40) and 40-45 years old women (n = 28) during oocyte pickup., Results: Higher level of AMH mRNA expression in cumulus cells was observed in the older age group compared to the younger (P <0.01). In accordance with AMH mRNA expression results, FF AMH protein levels were significantly higher in the older group than in the younger group (4.7 ± 1.1 ng\ml and 2.3 ± 0.2 ng\ml respectively, p < 0.002)., Conclusions: AMH is highly expressed and secreted from cumulus GCs of advanced age patients. This remarkable correlation between AMH mRNA levels in cumulus cells in respect to age suggests that AMH may be involved in follicular aging process.

Objective: To determine the prevalence of placental complications in patients conceived through donor versus autologous oocytes., Study Design: A retrospective cohort study including 2 groups of patients who conceived through in vitro fertilization using: (1) donor oocyte (n = 139) and (2) autologous oocyte (n = 126). Only singleton gestations were included. The rate of placental complications including preeclampsia, gestational hypertension, and intrauterine growth restriction was compared between these 2 groups., Results: The women who conceived using donor oocytes were older compared with women who conceived using autologous oocytes (median maternal age 45 vs 41, P < .01). The rate of hypertensive diseases of pregnancy including gestational hypertension and preeclampsia was significantly higher in ovum donor recipients compared with women conceived with autologous oocytes (25% vs 10%, P < .01). Similarly, the rate of intrauterine growth restriction was also higher among patients conceived through oocyte donation although it did not reach statistical significance (9.3% vs 4%, P = .08). When maternal age was restricted to ≤45 years, the rate of hypertensive diseases of pregnancy remained significantly higher among ovum donor compared with autologous oocyte recipients (22% vs 10%, P = .02). Adjustment for maternal age, gravidity, parity, and chronic hypertension revealed that oocyte donation was independently associated with higher rate of hypertensive diseases of pregnancy (P = .01)., Conclusion: Patients conceived through oocyte donation have an increased risk for placental complications of pregnancy. These findings support the 'immunologic theory' suggesting that immunologic intolerance between the mother and the fetus may play an important role in the pathogenesis of preeclampsia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Poliovirus receptor (PVR), regulator of G-protein signaling-11 (RGS11), and erythrocyte protein band-4.1-like 3 (EPB41L3) have been proposed to function in follicular maturation in mouse models. We have examined their expression in human mural (mGCs) and cumulus granulosa cells (CCs). Expression of PVR and RGS11 in mGCs decreased in medium-sized follicles compared to small follicles of IVM cycles and increased again in large follicles. Luteinization caused decreased expression of both PVR and RGS11. In vitro incubation of mGCs with progesterone-rich conditioned media decreased expression of RGS11 without affecting PVR levels. Inhibition of progesterone signaling enhanced expression of both RGS11 and PVR. Expression in CCs was examined by means of global transcriptome sequencing analysis RGS11 and EPB41L3 increased in CCs during follicular maturation while PVR levels did not change. In conclusion, during human follicular maturation there are significant changes in expression of PVR, RGS11 and EPB41L3, possibly regulated by progesterone.

Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mammalian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Introduction: There are two most popular protocols for Frozen Embryo Transfer: the natural and the E2&P4 replacement cycles. There is still a controversy whether one is superior over the other., Purpose: To compare the outcome in patient groups undergoing FET following these protocols., Methods: About 1235 FET cycles were retrospectively analyzed during a period of 12 years. In 798 cycles (group A), the natural cycle protocol was used, and in 437 cycles (group B), the exogenous E2&P4 administration protocol was used., Results: The average patient age was 32.11 ± 0.27 years in group A and 32.94 ± 0.19 years in group B (p<0.05). The endometrial thickness was 9.54 ± 0.11 mm and 8.95 ± 0.13 mm in groups A and B, respectively (p<0.001). The peak serum E2 level was 162.51 ± 8.97 pg/mL and 250.78 ± 33.67 pg/mL in groups A and B, respectively (p<0.001). The implantation, clinical pregnancy, and ongoing pregnancy rates in groups A and B were 6.47%, 12.91%, and 10.4% versus 4.26%, 8.47%, and 5.95%, respectively (p<0.05)., Conclusions: Natural endometrial preparation yields better outcome in compare with exogenous E2&P4 in FET cycles with higher endometrial thickness, implantation, and clinical pregnancy rates.

Objective: To examine whether patients with poor ovarian response (POR) during conventional IVF/intracytoplasmic sperm injection (ICSI) treatment cycle may benefit from a modified natural cycle (MNC)-IVF., Design: Cohort historic study., Setting: Tertiary, university-affiliated medical center., Patient(s): One hundred eleven patients with POR, defined according to the Bologna criteria, who underwent a subsequent MNC-IVF within 3 months of the previous failed conventional IVF/ICSI cycle. The elimination of bias in this selection, for the purposes of this study, was achieved by including only a subgroup of "genuine" poor responder patients, those who yielded up to three oocytes after controlled ovarian hyperstimulation (COH) with a minimal gonadotropin daily dose of 300 IU., Intervention(s): Modified natural cycle IVF protocol with GnRH antagonist (GnRH-a) supplementation. Gonadotropin-releasing hormone antagonist treatment was started when a follicle of 13 mm was present. Two to three ampules of hMG were coadministered daily during the GnRH-a treatment., Main Outcome Measure(s): Live birth rate, pregnancy rate (PR), number of oocytes retrieved, and number of embryos transferred., Result(s): Live birth rate in "genuine" poor ovarian responders was <1%. Furthermore, in the subgroup of patients with POR who underwent a previous conventional IVF/ICSI cycle with a yield of only one oocyte, no pregnancies were achieved during the MNC-IVF cycle., Conclusion(s): Modified natural cycle-IVF is of no benefit for genuine poor ovarian responders and the option of egg donation should be seriously considered for this population., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Cell culture techniques of human mural granulosa cells (MGCs) serve as a major in vitro tool. However, the use of luteinized MGCs has major limitations due to their luteinized state. Our aim was to establish a standardized protocol for the culture of MGCs as a model for different stages of folliculogenesis. We showed that early-non-luteinized, preovulatory-non-luteinized and luteal-MGCs have distinct gene expression pattern. After 4 days of incubation of luteinized-MGCs, ovulatory genes mRNA's achieve expression levels similar to the early non-luteinized follicles. FSH stimulation for 48 h of these 4 days cultured MGCs showed ovulatory genes mRNA's expression similar to the pre-ovulatory non-luteinized follicles. These FSH-stimulated cells responded to hCG stimulation in a pattern similar to the response of pre-ovulatory follicles. This novel model may provide a standardized research tool for delineation of the molecular processes occurring during the latter stages of follicular development in the human ovary., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Objective: To investigate the mRNA expression of genes related to steroidogenesis and OHSS in granulosa cells (GCs) of patients triggered with GnRH agonist compared to patients triggered with hCG., Design: Mural GCs were obtained at the time of oocyte retrieval and gene expression was analyzed using quantitative real time RT-PCR., Settings: Single center, case control study., Patient(s): 24 women who were treated with GnRH agonist or hCG for triggering of ovulation., Interventions: GC collection., Main Outcome Measure(s): The expression of genes related to steroidogenesis and OHSS in mural GCs., Results: The fertilization rate was similar in the two groups. The mRNA expression of CYP19A1 (0.50 vs 1, arbitrary unit), CYP11A1 (0.6 vs. 1) and 3 beta hydroxysteroid-dehydrogenase (0.39 vs 1) was significantly lower in the GnRH group. The expression of VEGF (0.74 vs. 1) and inhibin β B (0.38 vs 1) was lower in the GnRH analog triggered group., Conclusion: Expression of genes related to steroidogenesis is lower at the time of oocyte retrieval in patients triggered with GnRH agonist. The decreased expression of VEGF and inhibin β B in the GnRH agonist group can explain the mechanism of early OHSS prevention.

Objective: To compare the outcome of vitrification versus slow freezing cryopreservation for cleavage stage day 2-3 embryos., Design: A retrospective observational study., Setting: All thawed embryos assisted reproduction cycles between January 2010 and December 2012 at a single IVF laboratory of a Tertiary Medical Center., Patients: Five hundred and thirty-nine cycles of day 2-3 thawed embryos., Interventions: In 327 of the thawed cycles, the embryos were vitrified and in 212 of the cycles the embryos were derived from slow freezing embryos., Main Outcomes Measure: Embryo survival rate, blastomere surviving rate and pregnancy rate., Results: Embryo survival rate was significantly higher after vitrification compared with slow freezing (81.6%, 647/793 versus 70.0%, 393/562 embryos, p < 0.0001). The clinical pregnancy rate per ET was significantly higher following vitrification compared to slow freezing, 20.0%, 63/314 versus 11.9%, 23/193, respectively (p = 0.02)., Conclusions: Vitrification of day 2-3 cleavage stage embryos yields better cycle outcome in all the parameters compared to slow freezing.

Objective: To evaluate the efficacy and safety of 3 months of vaginal mifepristone treatment on leiomyoma volume and related symptoms., Design: Prospective, open-label, two tertiary centers, phase II clinical trial., Setting: Two tertiary medical centers in Israel., Patient(s): Thirty-three enrolled women, ages 30-53 years, diagnosed with symptomatic uterine fibroids., Intervention(s): Patients received 10 mg mifepristone vaginally daily for 3 months., Main Outcome Measure(s): Reduction in uterine leiomyoma volume. Improvement in symptoms related to uterine fibroids was assessed with the use of the "Uterine Fibroid Symptoms Quality of Life Questionnaire" (UFS-QoL). The number of bleeding days, safety, and tolerability were secondary measures., Result(s): Mifepristone treatment significantly reduced leiomyoma volume from 135.3 ± 22.9 cc at enrollment to 101.2 ± 22.4 cc after 3 months of treatment. The UFS-QoL Score significantly decreased from 20.7 ± 0.7 at enrollment to 14.0 ± 0.8 after 3 months of treatment. The number of bleeding days significantly decreased by 3.5 days. Endometrial biopsies showed no evidence of endometrial hyperplasia or cellular atypia. There were no major side effects during the course of the study, and treatment was well tolerated., Conclusion(s): Vaginal mifepristone may offer an effective treatment option for women with symptomatic uterine leiomyoma and can improve the patients' quality of life., Clinical Trial Registration Number: NCT00881140., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Progesterone, the main steroid synthesized by the corpus luteum (CL), prepares the uterus for implantation, maintains the CL survival, and induces progesterone auto-secretion. However, the molecular mechanisms involving the progesterone auto-secretion pathways at the luteal phase are not fully understood, especially in humans. We aim to study the molecular mechanism of the progesterone pathway in human granulosa cells. Our model system consists of luteinized human-mural-granulosa-cells (hmGCs) obtained from follicles aspirated during in vitro fertilization (IVF) procedures. hmGCs were seeded in culture and were subjected to different hormonal treatments. mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR). Progesterone levels were measured by enzyme immunoassay (EIA). We show that exposure of luteinized hmGCs to the progesterone receptor antagonist, RU486 (mifepristone), resulted in inhibition of LHCGR, LH/hCG target genes and progesterone secretion. Exposure of hmGCs to medium that was incubated with hmGCs for 4 d - conditioned medium (CM), which contain 150 ± 7.5 nM progesterone, resulted in induction of LHCGR and LH/hCG target genes, which was blocked by RU486. In addition, RU486 inhibited some of the progesterone biosynthesis pathway genes. Our results revealed a novel mechanism of the progesterone antagonist pathway in the luteal granulosa cells and emphasis the fundamental role of progesterone in the early luteal phase.

Purpose: To prospectively study the AMH expression and secretion pattern in mural granulosa cells (GCs) and follicular fluid (FF) from small follicles and medium follicles that were collected from normo-ovulatory (NO) and polycystic ovary syndrome (PCOS) patients undergoing in vitro maturation (IVM) treatments., Methods: FF AMH levels and mRNA expression of mural GCs were measured in small (≤ 10 mm) and medium size follicles (11-15 mm) obtained from IVM treatments and large size follicles (≥ 16 mm) obtained from in vitro fertilization treatments., Results: First, we show that AMH expression and protein level in the FF of NO patients were significantly higher in the small size follicles than in the medium and large size follicles (p < 0.003). We could not demonstrate these differences in PCOS patients. Second, we found significantly higher levels of AMH protein and mRNA in the large and medium (but not small) size follicles of PCOS patients compared to follicles from NO patients (p < 0.02). Finally, we observed a positive correlation between FF AMH of small and medium size follicles from NO patients and serum AMH (p < 0.03 and p < 0.0002, respectively)., Conclusions: Our data demonstrate a pathological dysregulation of AMH expression and secretion in follicles from PCOS patients and emphasize the association between the physiological downregulation of AMH and follicular antral health.

Purpose: To assesse circulating levels of Anti-Müllerian hormone (AMH) as a predictor of oocyte number and their potential to mature in vitro in both normo-ovulatory (NO) women and in women with Polycystic Ovary Syndrome (PCOS) undergoing in vitro maturation (IVM) treatments., Methods: We prospectively studied NO women and women diagnosed with PCOS, (age range 21-39 years) underwent IVM treatments at our center. Serum AMH levels were quantified before each cycle and correlated to oocytes number, maturation and fertilization during in vitro maturation., Results: 104 NO and 30 PCOS IVM cycles were followed with retrieval of a total of 672 and 491 oocytes, respectively. In NO women, the serum AMH level positively correlated with the number of oocytes retrieved, (R = 0.6; P <0.0001) the number of M2 oocytes at 24 and 48 h (R = 0.4; P <0.01; R = 0.26 p < 0.007, respectively) and with the total number of M2 oocytes (R = 0.47; P < 0.0001). In the PCOS group, the serum AMH level positively correlated only with the number of oocytes retrieved (R = 0.43; P <0.03). Receiver operating characteristic (ROC) analyses showed that a cutoff AMH level of 1.56 (ng/ml) could identify patients with 5 or more oocytes at OPU with a sensitivity of 83 % and a specificity of 75 %. An AMH level of 1.63 (ng/ml) was the threshold for 5 or more matured oocytes (sensitivity = 81 %, specificity = 53 %)., Conclusions: Serum AMH may be used as a marker to identify candidates for IVM treatment in both NO and PCOS women.

Aim: To evaluate the effect of local injury to the endometrium during spontaneous menstrual cycles before in vitro fertilization (IVF) treatment on implantation and pregnancy rates in women with recurrent implantation failure (RIF)., Methods: In a prospective randomized controlled trial (RCT), a total of 36 patients, with RIF undergoing IVF, were randomized to two groups. In 18 patients, endometrial biopsies were performed using a pipelle curette on days 9-12 and 21-24 of the menstrual cycle preceding IVF treatment. In 18 control patients, a cervical pipelle was performed., Results: The implantation rate (2.08% versus 11.11%; p = 0.1), clinical (0% versus 31.25%; p < 0.05) and live births rates (0% versus 25%; p = 0.1) were lower in the experimental group compared with controls., Conclusion: Our RCT did not find any benefit from local injury to the endometrium in women with a high number of RIFs. Further studies are warranted to better define the target population of patients who may benefit from this procedure.

Early pregnancy loss is common among women treated with assisted reproduction treatment, but whether it is a prognostic factor for success in subsequent IVF cycles is not well established. The aim of this study was to determine whether a biochemical pregnancy (BP) or spontaneous abortion (SA) affects the pregnancy rates in the following cycle. A retrospective study of 2687 women undergoing 6678 cycles between January 1998 and March 2010 was performed. Ongoing pregnancy rate (PR) per cycle was compared between patients with a pregnancy loss versus a negative β-HCG in their previous cycles. Multivariate analysis of factors affecting ongoing pregnancy rate was performed. BP and/or SA in the first three cycles did not significantly alter the chances to conceive (16.9% patients with BP and/or SA in the previous cycle versus 16.5% patients with no previous pregnancy). From cycle 4 onwards, the presence of a previous abortion (either BP or SA) was associated with better ongoing PR (23.0% versus 11.2%, P<0.001). In conclusion, BP and/or SA in a previous cycle appears to be a positive marker for success in subsequent cycles in patients with repeated IVF failures. These results should be further investigated in this challenging group of patients., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

This study investigated anti-Müllerian hormone (AMH) expression and secretion from cumulus granulosa cells (GC) and steroidogenesis in follicular fluids (FF) with relation to oocyte maturational stages and fertilization capacity in large preovulatory follicles. This prospective study included 53 ovulatory women undergoing intracytoplasmic sperm injection. FF and cumulus GC from 140 large preovulatory follicles were individually obtained during oocyte retrieval. Main outcome measures were oocyte maturation, fertilization and embryo quality. FF were assayed for AMH, progesterone, 17β-oestradiol and testosterone. Cumulus GC were assayed for AMH mRNA expression. AMH mRNA expression and secretion in cumulus GC in preovulatory follicles containing germinal-vesicle (GV) and metaphase-I (MI) oocytes were significantly higher than follicles containing MII oocytes (P<0.01 and P<0.0001, respectively). In addition, FF AMH concentrations from atretic oocytes were significantly higher than from MII oocytes. No correlation was found between AMH expression and secretion to fertilization or embryo quality. FF of MI and GV oocytes had higher concentrations of testosterone and lower progesterone/oestradiol ratios than MII oocytes, and FF of atretic oocytes contained higher testosterone concentrations than FF of MII oocytes. AMH is highly expressed in and secreted from cumulus GC of preovulatory follicles containing premature and atretic oocytes. Anti-Müllerian hormone (AMH) is produced in the female exclusively by granulosa cells. AMH has recently been shown to be one of the most important markers of ovarian reserve and it is highly associated with ovarian follicular development. This study investigates AMH expression and secretion from cumulus granulosa cells (GC) and steroidogenesis in the follicular fluids (FF) with relation to oocyte maturational stages, and fertilization capacity in large preovulatory follicles. We conducted a prospective study with 53 ovulatory women undergoing intracytoplasmic sperm injection. FF and cumulus GC from 140 large preovulatory follicles were individually obtained during oocyte retrieval. The main outcome measures were oocyte maturation, fertilization and embryo quality. FF were assayed for AMH, progesterone, 17β-oestradiol and testosterone. Cumulus GC were assayed for AMH mRNA expression. AMH mRNA expression in cumulus GC and AMH concentrations in FF of preovulatory follicles containing premature oocytes (germinal vesicle (GV) and metaphase I (MI)) were significantly higher than preovulatory follicles containing mature oocytes (MII oocytes). In addition, FF AMH concentrations of atretic oocytes were significantly higher than FF AMH of MII oocytes. No correlation was found between AMH expression and secretion for fertilization or embryo quality. FF of preovulatory MI and GV oocytes had higher levels of testosterone and lower progesterone/oestradiol ratios than MII oocytes, and FF of atretic oocytes contained higher testosterone levels than FF of MII oocytes. This study shows that AMH is highly expressed in and secreted from cumulus GC of preovulatory follicles containing premature and atretic oocytes., (Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Aim: To determine the incidence of recurrent empty follicle syndrome (EFS) and to analyse the factors associated with this phenomenon., Methods: Retrospective analysis comparing all EFS cycles with cycles in which oocytes were retrieved in our in vitro fertilization (IVF) unit between 1998 and 2006., Results: Of 8292 IVF cycles, 163 (2.0%) resulted in empty follicles. Risk factors for EFS included advanced age (37.7 ± 6.0 years vs. 34.2 ± 6.0 years, p < 0.001), longer infertility (8.8 ± 10.6 years vs. 6.3 ± 8.4 years, p < 0.05), higher baseline follicle-stimulating hormone levels (8.7 ± 4.7 IU/L vs. 6.7 ± 2.9 IU/L, p < 0.001) and lower E2 levels before the human chorionic gonadotropin injection (499.9 ± 480.9 pg/mL vs. 1516.3 ± 887.5 pg/mL, p < 0.001) compared with cases in which ova were retrieved. Among patients with EFS, recurrent EFSs occurred in 15.8% of subsequent cycles., Conclusion: The EFS is a sporadic event in the majority of patients. However, in about 16% of the patients, EFS may recur. These cases may be a variant form of poor response and patients with repetitive EFS syndrome should be counseled concerning their chances to conceive.

Preeclampsia, a severe unpredictable complication of pregnancy, occurs in 6% of pregnancies, usually in the second or third trimester. The specific etiology of preeclampsia remains unclear, although the pathophysiological hallmark of this condition appears to be an inadequate blood supply to the placenta. As a result of the impaired placental blood flow, intrauterine growth restriction (IUGR) and consequential fetal oxidative stress may occur. Consistent with this view, pregnancies complicated by preeclampsia and IUGR are characterized by up-regulation of key transcriptional regulators of the hypoxic response including, hif1α and as well as p53 and its target genes. Recently, the presence of circulating cell-free fetal RNA has been documented in maternal plasma. We speculated that pregnancies complicated by preeclampsia and IUGR, will be associated with an abnormal expression of p53 and/or hif1α related genes in the maternal plasma. Maternal plasma from 113 singleton pregnancies (72 normal and 41 complicated pregnancies) and 19 twins (9 normal and 10 complicated pregnancies) were collected and cell free RNA was extracted. The expression of 18 genes was measured by one step real-time RT-PCR and was analyzed for prevalence of positive/negative expression levels. Results indicate that, among the genes examined, cell free plasma expressions of p21 and hif1α were more prevalent in pregnancies complicated by hypoxia and/or IUGR (p<0.001). To conclude, we present in this manuscript data to support the association between two possible surrogate markers of hypoxia and common complications of pregnancy. More work is needed in order to implement these findings in clinical practice.

Aims: Ovarian follicular development and ovulation in mammals is a complex and highly regulated process. Most advances in the understanding of the ovulatory process have come from animal models. However, translational research in humans is of crucial importance for improving fertility treatment and control., Methods: IVM/IVF procedures allow us to obtain follicular fluid and granulosa cells (GC) from follicles in different developmental stages with and without hCG priming., Results: Using the cells and fluids obtained in IVM/IVF procedures allowed us to characterize human ovulatory gene expression during antral folliculogenesis and ovulation, examine gene expression in luteinized and non-luteinized GC in vivo and in vitro and to use cumulus GC genes as biomarkers for oocyte and embryo maturity and competence., Conclusion: Biological material obtained during IVM/IVF procedures is an important tool to study the human ovulatory cascade and can serve to improve IVM techniques and fertility treatment and control.

We have recently shown that Mimp, a mitochondrial carrier protein homologue, is induced by Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling and decreases the mitochondrial membrane potential in DA3 mammary adenocarcinoma cells. We show here that induction of Mimp leads to growth arrest in response to HGF/SF by arresting cells at the S phase of the cell cycle. Induction of Mimp or its transient expression does not lead to apoptosis. Mimp also attenuates HGF/SF-induced cellular scattering in vitro and tumor growth in vivo. The exogenous induction of Mimp at levels similar to its endogenous induction by HGF/SF increases the level of the Met protein and its phosphorylation by HGF/SF but reduces the levels of Shc and prevents the HGF/SF-induced tyrosine phosphorylation of Grb2 and Shc. In contrast, the level of phosphatidylinositol 3-kinase (PI3K) increases following Mimp induction and the level of phosphorylated PI3K in response to HGF/SF is unaffected by the exogenous induction of Mimp. Moreover, exogenous Mimp prevents the HGF/SF-induced transcription of the serum response element-luciferase reporter gene. Our results show that Mimp expression reduces Met-HGF/SF-induced proliferation and scattering by attenuating and altering the downstream signaling of Met. These data show a new link between a tyrosine kinase growth factor receptor and a mitochondrial carrier homologue that regulates cellular growth, motility, and tumorigenicity.

ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.

Background: Hepatocyte growth factor/scatter factor (HGF/SF) binds to its tyrosine kinase receptor, Met, thereby stimulating diverse cellular responses. The multifunctional docking site in the C-terminal domain mediates the signal of phosphorylated Met receptors to multiple transducers. The tyrosine at position 1356 of the Met docking site is crucial for cell motility and morphogenesis., Methods: We examined the in situ distribution patterns of the Tyr1356-phosphorylated form of Met with a novel monoclonal antibody following renal injury and renal hypertrophy in rats. Sections of the kidney following either sham operation, transient ischaemia of one kidney or unilateral nephrectomy were analysed using indirect immunofluorescence staining and confocal laser scanning microscopy analysis of total Met protein levels and Tyr1356-phosphorylated Met (Met and pMet, respectively)., Results: At 6 h post-treatment, pMet increases in ischaemic kidneys compared with sham-operated kidneys, and these changes become substantial after 48 h in both medulla and cortex of ischaemic kidneys (P < 0.001). We also show significant up-regulation of Met predominantly in the medulla of ischaemic kidneys, 48 h following injury (P < 0.009). Inter-estingly, the stimulus for hypertrophy in the remnant kidney after uninephrectomy and the contra-lateral kidney during ischaemia is not accom-panied by significant up-regulation of Met or pMet staining compared with sham operation at both time points., Conclusions: We demonstrate in this work, for the first time, in situ detection of tyrosine kinase growth factor receptor docking site activation during pathological processes in the kidney. Using this methodology, we show a significant increase in Met docking site activity in both renal medulla and cortex solely following stimulation by ischaemia and repair.

Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling plays an important role in epithelial tissue morphogenesis, lumen formation, and tumorigenicity. We have recently demonstrated that HGF/SF also alters the metabolic activity of cells by enhancing both the glycolytic and oxidative phosphorylation pathways of energy production. Using differential display polymerase chain reaction, we cloned a novel gene, designated mimp (Met-Induced Mitochondrial Protein), which is upregulated in NIH-3T3 cells cotransfected with both HGF/SF and Met (HMH cells). Northern and Western blot analyses showed that mimp is induced in several Met-expressing cell lines following treatment with HGF/SF. Mimp encodes a 33-kDa protein that shows sequence homology to the family of mitochondrial carrier proteins (MCPs). Murine Mimp (mMimp) is expressed in a wide variety of tissues, exhibiting an expression pattern similar to Met. Predominant expression is seen in liver, kidney, heart, skeletal muscle, and testis. Using immunostaining for HA-tagged mMimp and a GFP-mMimp chimeric protein as well as subcellular fractionation, we determined that Mimp is primarily localized to the mitochondria. Ectopic expression of mMimp in the Met-responsive adenocarcinoma cell line, DA3, reduced the mitochondrial membrane potential (uncoupling activity). The extent of the mitochondrial depolarization positively correlated with the level of Mimp expression. Our results demonstrate that Mimp is a novel mitochondrial carrier homologue upregulated by Met-HGF/SF signal transduction, which leads to mitochondrial depolarization, and suggest novel links among tyrosine kinase signaling, mitochondrial function, and cellular bioenergetics.