Association between the length of in vitro embryo culture, mode of ART, and the initial endogenous hCG rise in ongoing singleton pregnancies.

STUDY QUESTION: Is there an association between the length of in vitro culture, mode of ART and the initial endogenous hCG rise, in cycles with a foetal heartbeat after single embryo transfer (ET) and implantation? SUMMARY ANSWER: Both the length of in vitro culture and the mode of ART have an impact on the initial endogenous rise in hCG in singleton pregnancies. WHAT IS KNOWN ALREADY: Different factors have been identified to alter the kinetics of hCG in pregnancies. Current studies show conflicting results regarding the kinetics of hCG after different types of ART (fresh vs frozen ET (FET)), the inclusion or not of preimplantation genetic testing (PGT), and the length of time in in vitro culture. STUDY DESIGN, SIZE, DURATION: This was a multicentre cohort study, using prospectively collected data derived from 4938 women (5524 treatment cycles) undergoing IUI (cycles, n = 608) or ART (cycles, n = 4916) treatments, resulting a in singleton ongoing pregnancy verified by first-trimester ultrasound scan. Data were collected from the Danish Medical Data Centre, used by the three participating Danish public fertility clinics at Copenhagen University hospitals: Herlev Hospital, Hvidovre Hospital, and Rigshospitalet, from January 2014 to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The fresh ET cycles included cleavage-stage (2 or 3 days in vitro) and blastocyst (5 days in vitro) transfers. FET cycles included cleavage-stage (3 days in vitro before cryopreservation) or blastocyst (5 or 6 days in vitro before cryopreservation) transfers. The IUI cycles represented no time in vitro. To attain a comparable interval for serum-hCG (s-hCG), the ovulation induction time was identical: 35-37 h before oocyte retrieval or IUI. The conception day was considered as: the insemination day for pregnancies conceived after IUI, the oocyte retrieval day for fresh ET, or the transfer day minus 3 or 5 as appropriate for FET of Day 3 or 5 embryos. Multiple linear regression analysis was used, including days post-conception for the hCG measurement as a covariate, and was adjusted for the women's age, the cause of infertility, and the centre. For FET, a sensitivity analysis was used to adjust for endometrial preparation. MAIN RESULTS AND THE ROLE OF CHANCE: The study totally includes 5524 cycles: 2395 FET cycles, 2521 fresh ET cycles, and 608 IUI cycles. Regarding the length of in vitro culture, with IUI as reference (for no time in in vitro culture), we found a significantly lower s-hCG in pregnancies achieved after fresh ET (cleavage-stage ET or blastocyst transfer). S-hCG was 18% (95% CI: 13-23%, P < 0.001) lower after fresh cleavage-stage ET, and 23% (95% CI: 18-28%, P < 0.001) lower after fresh blastocyst transfer compared to IUI. In FET cycles, s-hCG was significantly higher after blastocyst transfers compared to cleavage-stage FET, respectively, 26% (95% CI: 13-40%, P < 0.001) higher when cryopreserved on in vitro Day 5, and 14% (95% CI: 2-26%, P = 0.02) higher when cryopreserved on in vitro Day 6 as compared to Day 3. Regarding the ART treatment type, s-hCG after FET blastocyst transfer (Day 5 blastocysts) cycles was significantly higher, 33% (95% CI: 27-45%, P < 0.001), compared to fresh ET (Day 5 blastocyst), while there was no difference between cleavage-stage FET (Days 2 + 3) and fresh ET (Days 2 + 3). S-hCG was 12% (95% CI: 4-19%, 0.005) lower in PGT FET (Day 5 blastocysts) cycles as compared to FET cycles without PGT (Day 5 blastocysts). LIMITATIONS, REASONS FOR CAUTION: The retrospective design is a limitation which introduces the risk of possible bias and confounders such as embryo score, parity, and ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS: This study elucidates how practices in medically assisted reproduction treatment are associated with the hCG kinetics, underlining a potential impact of in vitro culture length and mode of ART on the very early embryo development and implantation. The study provides clinicians knowledge that the type of ART used may be relevant to take into account when evaluating s-hCG for the prognosis of the pregnancy. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. AP has received consulting fees, research grants, or honoraria from the following companies: Preglem, Novo Nordisk, Ferring Pharmaceuticals, Gedeon Richter, Cryos, Merck A/S, and Organon. AZ has received grants and honoraria from Gedeon Richter. NLF has received grants from Gedeon Richter, Merck A/S, and Cryos. MLG has received honoraria fees or research grants from Gedeon Richter, Merck A/S, and Cooper Surgical. CB has received honoraria from Merck A/S. MB has received research grants and honoraria from IBSA. MPR, KM, and PVS all report no conflicts of interest. TRIAL REGISTRATION NUMBER: The study was registered and approved by the Danish Protection Agency, Capital Region, Denmark (Journal-nr.: 21019857). No approval was required from the regional ethics committee according to Danish law.

The intrafollicular concentrations of biologically active cortisol in women rise abruptly shortly before ovulation and follicular rupture.

STUDY QUESTION: What is the temporal activity and the concentration in follicular fluid (FF) of the anti-inflammatory steroid cortisol during the ovulatory process in humans? SUMMARY ANSWER: Intrafollicular concentrations of cortisol become massively upregulated close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. WHAT IS KNOWN ALREADY: Ovulation has been described as a local, controlled inflammatory process resulting in the degeneration of the follicle wall which facilitate oocyte extrusion. Ovulation also affects the glucocorticoid metabolism of granulosa cells (GCs) and although de novo synthesis of cortisol only occurs in the adrenal cortex, the mid-cycle surge has been shown to induce a change from high expression of HSD11B2, inactivating cortisol to cortisone, to high expression of HSD11B1 which reversibly catalyses cortisol production from cortisone. Furthermore, high concentrations of progesterone and 17OH-progesterone within follicles may cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women donated FF and GCs from one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles: T = 0 h, T = 12 h, T = 17 h, T = 32 h. A second sample was collected at oocyte pick up at T = 36 h. The concentration of cortisol and cortisone together with a range of sex steroids was measured by LC-MS/MS in FF collected at the five time points mentioned above. Whole genome microarray data, validated by q-PCR analysis, was used to evaluate gene expression of CYP11B1, CYP21A2, HSD11B1, HSD11B2, and NR3C1 in GCs at the same time points. MAIN RESULTS AND THE ROLE OF CHANCE: The concentration of cortisol was significantly increased from a few nM at 0 h to around 100-140 nM (P ≤ 0.0001) at 32-36 h, whilst cortisone was almost constant from 0 to 17 h at a concentration of between 90 and 100 nM being significantly reduced to 25-40 nM (P ≤ 0.0001) at 32-36 h. This was paralleled by a 690-fold upregulation of HSD11B1 from 0 to 12 h increasing to a more than 20.000-fold change at 36 h. HSD11B2 was quickly downregulated 15- to 20-fold after ovulation induction. Concentrations of progesterone and 17OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CBP due to similar binding affinities. Furthermore, a significant decrease in 11-deoxycortisol expression was seen, but CYP11B1 expression was below detection limit in GCs. LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and results may differ from the natural cycle. More observations at each specific time point may have strengthened the conclusions. Furthermore, we have not been able to measure the actual active biological concentration of cortisol. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study collectively evaluated the temporal pattern of cortisol and cortisone concentrations during human ovulation, rendering a physiological framework for understanding potential dysregulations in the inflammatory reaction of ovulation. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, and Novo Nordisk Foundation grant number NNF21OC00700556. Interreg V ÔKS through ReproUnion (www.reprounion.eu); Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-ß family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.

Rare genetic variants suggest dysregulation of signaling pathways in low- and high-risk patients developing severe ovarian hyperstimulation syndrome.

PURPOSE: To investigate if rare gene variants in women with severe ovarian hyperstimulation syndrome (OHSS) provide clues to the mechanisms involved in the syndrome. METHODS: Among participants in a prospective randomized study (Toftager et al. 2016), six women with predicted low and six women with predicted high risk of OHSS developing severe OHSS (grades 4 and 5, Golan classification) were selected. In the same cohort, six plus six matched controls developing no signs of OHSS (Golan grade 0) were selected. Whole-exome sequencing was performed. Analysis using a predefined in silico OHSS gene panel, variant filtering, and pathway analyses was done. RESULTS: We found no convincing monogenetic association with the development of OHSS using the in silico gene panel. Pathway analysis of OHSS variant lists showed substantial overlap in highly enriched top pathways (p value range p < 0.0001 and p > 9.8E-17) between the low- and high-risk group developing severe OHSS, i.e., "the integrin-linked kinase (ILK) signaling pathway" and the "axonal guidance signaling pathway," both being connected to vasoactive endothelial growth factor (VEGF) and endothelial function. CONCLUSION: Rare variants in OHSS cases with two distinct risk profiles enrich the same signaling pathways linked to VEGF and endothelial function. Clarification of the mechanism as well as potentially defining genetic predisposition of the high vascular permeability is important for future targeted treatment and prevention of OHSS; the potential roles of ILK signaling and the axonal guidance signaling need to be validated by functional studies.

A distinctive epigenetic ageing profile in human granulosa cells.

STUDY QUESTION: Does women's age affect the DNA methylation (DNAm) profile differently in mural granulosa cells (MGCs) from other somatic cells? SUMMARY ANSWER: Accumulation of epimutations by age and a higher number of age-related differentially methylated regions (DMR) in MGCs were found compared to leukocytes from the same woman, suggesting that the MGCs have a distinctive epigenetic profile. WHAT IS KNOWN ALREADY: The mechanisms underlying the decline in women's fertility from the mid-30s remain to be fully elucidated. The DNAm age of many healthy tissues changes predictably with and follows chronological age, but DNAm age in some reproductive tissues has been shown to depart from chronological age (older: endometrium; younger: cumulus cells, spermatozoa). STUDY DESIGN, SIZE, DURATION: This study is a multicenter cohort study based on retrospective analysis of prospectively collected data and material derived from healthy women undergoing IVF or ICSI treatment following ovarian stimulation with antagonist protocol. One hundred and nineteen women were included from September 2016 to June 2018 from four clinics in Denmark and Sweden. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were obtained from 118 healthy women with varying ovarian reserve status. MGCs were collected from 63 of the 119 women by isolation from pooled follicles immediately after oocyte retrieval. DNA from leukocytes and MGCs was extracted and analysed with a genome-wide methylation array. Data from the methylation array were processed using the ENmix package. Subsequently, DNAm age was calculated using established and tailored age predictors and DMRs were analysed with the DMRcate package. MAIN RESULTS AND ROLE OF CHANCE: Using established age predictors, DNAm age in MGCs was found to be considerable younger and constant (average: 2.7 years) compared to chronological age (average: 33.9 years). A Granulosa Cell clock able to predict the age of both MGCs (average: 32.4 years) and leukocytes (average: 38.8 years) was successfully developed. MGCs differed from leukocytes in having a higher number of epimutations (P = 0.003) but predicted telomere lengths unaffected by age (Pearson's correlation coefficient = -0.1, P = 0.47). DMRs associated with age (age-DMRs) were identified in MGCs (n = 335) and in leukocytes (n = 1) with a significant enrichment in MGCs for genes involved in RNA processing (45 genes, P = 3.96 × 10-08) and gene expression (152 genes, P = 2.3 × 10-06). The top age-DMRs included the metastable epiallele VTRNA2-1, the DNAm regulator ZFP57 and the anti-Müllerian hormone (AMH) gene. The apparent discordance between different epigenetic measures of age in MGCs suggests that they reflect difference stages in the MGC life cycle. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: No gene expression data were available to associate with the epigenetic findings. The MGCs are collected during ovarian stimulation, which may influence DNAm; however, no correlation between FSH dose and number of epimutations was found. WIDER IMPLICATIONS OF THE FINDINGS: Our findings underline that the somatic compartment of the follicle follows a different methylation trajectory with age than other somatic cells. The higher number of epimutations and age-DMRs in MGCs suggest that their function is affected by age. STUDY FUNDING/COMPETING INTEREST(S): This project is part of ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS, the Danish National Research Foundation and the European Research Council. The authors declare no conflict of interest.

Two waves of transcriptomic changes in periovulatory human granulosa cells.

STUDY QUESTION: How does the human granulosa cell (GC) transcriptome change during ovulation? SUMMARY ANSWER: Two transcriptional peaks were observed at 12 h and at 36 h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. WHAT IS KNOWN ALREADY: The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. STUDY DESIGN, SIZE, DURATION: This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12 h, 17 h or 32 h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36 h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12 h that resembled the initiation of an inflammatory response, and one peak at 36 h that resembled the effector functions of inflammation such as vasodilation, angiogenesis, coagulation, chemotaxis and tissue remodelling. Genes involved in cell-matrix interactions as a part of cytoskeletal rearrangement and cell motility were also upregulated at 36 h. Predicted activated upstream regulators of ovulation included FSH, LH, transforming growth factor B1, tumour necrosis factor, nuclear factor kappa-light-chain-enhancer of activated B cells, coagulation factor 2, fibroblast growth factor 2, interleukin 1 and cortisol, among others. The results confirmed early regulation of several previously described factors in a cascade inducing meiotic resumption and suggested new factors involved in cumulus expansion and follicle rupture through co-regulation with previously described factors. LARGE SCALE DATA: The microarray data were deposited to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/gds/, accession number: GSE133868). LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and the findings may therefore differ from a natural cycle. However, the results confirm significant regulation of many well-established ovulatory genes from a series of previous studies such as amphiregulin, epiregulin, tumour necrosis factor alfa induced protein 6, tissue inhibitor of metallopeptidases 1 and plasminogen activator inhibitor 1, which support the relevance of the results. WIDER IMPLICATIONS OF THE FINDINGS: The study increases our understanding of human ovarian function during ovulation, and the publicly available dataset is a valuable resource for future investigations. Suggested upstream regulators and highly differentially expressed genes may be potential pharmaceutical targets in fertility treatment and gynaecology. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by EU Interreg ÔKS V through ReproUnion (www.reprounion.eu) and by a grant from the Region Zealand Research Foundation. None of the authors have any conflicts of interest to declare.

Follicular hormone dynamics during the midcycle surge of gonadotropins in women undergoing fertility treatment.

Changes in concentrations of intra-follicular hormones during ovulation are important for final oocyte maturation and endometrial priming to ensure reproductive success. As no human studies have investigated these changes in detail, our objective was to describe the dynamics of major follicular fluid (FF) hormones and transcription of steroidogenic enzymes and steroid receptors in human granulosa cells (GCs) during ovulation. We conducted a prospective cohort study at a public fertility clinic in 2016-2018. Fifty women undergoing ovarian stimulation for fertility treatment were included. From each woman, FF and GCs were collected by transvaginal ultrasound-guided follicle puncture of one follicle at two specific time points during ovulation, and the study covered a total of five time points: before ovulation induction (OI), 12, 17, 32 and 36 h after OI. Follicular fluid concentrations of oestradiol, progesterone, androstenedione, testosterone, 17-hydroxyprogesterone, anti-Mullerian hormone, inhibin A and inhibin B were measured using ELISA assays, and a statistical mixed model was used to analyse differences in hormone levels between time points. Gene expression of 33 steroidogenic enzymes and six hormone receptors in GCs across ovulation were assessed by microarray analysis, and selected genes were validated by quantitative reverse transcription PCR. We found that concentrations of oestradiol, testosterone, progesterone, AMH, inhibin A and inhibin B (P < 0.001) and gene expression of 12 steroidogenic enzymes and five receptors (false discovery rate < 0.0001) changed significantly during ovulation. Furthermore, we found parallel changes in plasma hormones. The substantial changes in follicular hormone production during ovulation highlight their importance for reproductive success.

Concentration of soluble urokinase plasminogen activator receptor (suPAR) in the pre-ovulatory follicular fluid is associated with development of ovarian hyperstimulation syndrome during ovarian stimulation.

PURPOSE: Investigating whether pre-ovulatory follicular fluid (FF) levels of selected proteins differ between women who do or do not develop severe ovarian hyperstimulation syndrome (OHSS) and evaluate whether they potentially could guide a "freeze-all" strategy. METHODS: FF was collected during a randomized controlled trial comparing OHSS in antagonist versus agonist protocol including 1050 women in their first assisted reproductive technology (ART) cycle during year 2009-2013. The present sub-study is a matched case-control study comparing FF levels of soluble urokinase plasminogen activator receptor (suPAR), C-reactive protein, placental growth factor, vascular endothelial growth factor, and angiopoietins 1 and 2 in OHSS cases (n = 25, severe OHSS, and ≥ 15 oocytes), high-risk controls (n = 25, no OHSS, and ≥ 15 oocytes), and low-risk controls (n = 25, no OHSS, and 5-8 oocytes). RESULTS: FF level of suPAR differed significantly between the three groups (p = 0.018) with mean (SD) levels of 2.3 (0.4) µg/L, 2.6 (0.8) µg/L, and 2.8 (0.6) µg/L in OHSS cases, high-risk controls, and low-risk controls, respectively. Receiver operating characteristic curve analysis demonstrated that suPAR levels could predict severe OHSS (AUC 0.678; 95% CI 0.553-0.803) with a sensitivity of 64% and a specificity of 66%. None of the other investigated proteins differed between the three groups or between OHSS cases and combined controls. CONCLUSION: The pre-ovulatory FF level of suPAR was significantly lower in women developing severe OHSS, indicating that the plasminogen activator system could be involved in the pathophysiology of OHSS. However, suPAR did not provide a satisfying predictive value for the prediction of OHSS.

Dormancy and activation of human oocytes from primordial and primary follicles: molecular clues to oocyte regulation.

STUDY QUESTION: Do specific transcriptome dynamics in human oocytes from primordial and primary follicles identify novel pathways in oocyte activation? SUMMARY ANSWER: The transcriptomic profiles in oocytes from primordial and primary follicles, respectively, revealed several new canonical pathways as putative mediators of oocyte dormancy and activation. WHAT IS KNOWN ALREADY: Cellular signaling pathways including PI3K/AKT and AKT/mTOR as well as TGF-ß and IGF signaling are known to regulate the primordial-to-primary transition in mammalian follicle development. STUDY DESIGN, SIZE, DURATION: We performed a class comparison study on human oocytes from primordial (n = 436) and primary (n = 182) follicles donated by three women having ovarian tissue cryopreserved before chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA was extracted from oocytes from primordial and primary follicles isolated by Laser Capture Microdissection, and submitted to the HiSeq Illumina platform. Data mapping, quality control, filtering and expression analysis were performed using Tophat (2.0.4), Cufflinks (2.0.2), BWA (0.6.2) and software R. Modeling of complex biological systems was performed using the IPA® software. Finally, qPCR and immunohistochemistry were employed to explore expression and localization of selected genes and products in human ovarian tissue. MAIN RESULTS AND THE ROLE OF CHANCE: We found 223 and 268 genes down-regulated and up-regulated, respectively, in the oocytes during the human primordial-to-primary follicle transition (P < 0.05 and/or FPKM fold-change >2). IPA® enrichment analysis revealed known pathways ('mTOR Signaling', 'PI3K/AKT Signaling' and 'PTEN Signaling') as well as enriched canonical pathways not previously associated with human ovarian follicle development such as 'ErB Signaling' and 'NGF Signaling' in the down-regulated category and 'Regulation of eIF4 and P70S6K Signaling' and 'HER-2 Signaling in Breast Cancer' in the up-regulated group. Additionally, immunohistochemistry on human ovarian tissue explored the intraovarian localization of VASA, FOXO1 and eIF4E. LARGE SCALE DATA: http://users-birc.au.dk/biopv/published_data/ernst_2017/. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis and no functional studies were performed. The study was based on a limited number of patients and the experimental design could not take into account the natural biological variance in human samples. Therefore, qPCR was used to confirm selected genes alongside immunohistochemical stainings. WIDER IMPLICATIONS OF THE FINDINGS: This study shows, for the first time, a detailed molecular description of global gene transcription activities in oocytes from primordial and primary follicles, respectively. Knowing the global transcription profiles of human oocyte dormancy and activation are important in developing new clinical applications. STUDY FUNDING/COMPETING INTEREST(S): E.H.E. was supported by Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.H. and S.F. were supported by an MRC (UK) project grant MR/M012638/1. K.L.H. was supported by grants from Fonden til Lægevidenskabens Fremme, Kong Christian Den Tiendes Fond. K.L.H. and L.S. were supported by the IDEAS grant from Aarhus University Research Foundation (AUFF). There are no conflicts of interest.

Proteolytic processing of anti-Müllerian hormone differs between human fetal testes and adult ovaries.

From early embryonic life, anti-Müllerian hormone (AMH) is produced by Sertoli cells and is essential for male sex differentiation. In females, AMH is produced by immature granulosa cells (GCs) but a definitive function in females is uncertain. We have assessed the cellular localization and specificity of a panel of five novel high-affinity AMH monoclonal antibodies. Two recognize the mature C-terminal form of AMH, whereas three recognize the active pro-mature form of AMH in human tissue. The antibodies were tested on fetal male testicular and mesonephric tissue aged 8-19 weeks post conception (pc), fetal male serum aged 16-26 weeks pc and human immature GCs by immunofluorescence, immunohistochemistry, ELISA and western blotting. The active pro-mature forms of AMH were expressed in both Sertoli cells from human fetal testis and human immature GCs. In contrast, the mature C-terminal form of AMH was hardly detected in Sertoli cells, but was readily detected in GCs. This particular form was also located to the nucleus in GCs, whereas the other investigated AMH forms remained in the cytoplasm. Interestingly, the distribution of the AMH forms in the fetal serum of boys showed that the fraction of inactive precursor AMH only accounted for 4.5% ± 0.6 (mean ± SD) of the total AMH measured, and the remaining AMH was the active pro-mature form. Furthermore, western blot analysis demonstrated a number of previously unrecognized molecular forms of AMH. The present findings suggest that processing of AMH is a tightly regulated process, which is likely to be important for the function of AMH and which differs between the two sexes.

Distribution and function of 3',5'-Cyclic-AMP phosphodiesterases in the human ovary.

The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary. Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found that PDE3, PDE4, PDE7 and PDE8 are the major families present while PDE11A was not detected. PDE8B was differentially expressed during folliculogenesis. In cultured GCs, inhibition of PDE7 and PDE8 increased basal progesterone secretion while PDE4 inhibition increased forskolin-stimulated progesterone secretion. In conclusion, we identified PDE3, PDE4, PDE7 and PDE8 as the major PDEs in the human ovary.

Identification of new ovulation-related genes in humans by comparing the transcriptome of granulosa cells before and after ovulation triggering in the same controlled ovarian stimulation cycle.

STUDY QUESTION: Which genes and molecular mechanisms are involved in the human ovulatory cascade and final oocyte maturation? SUMMARY ANSWER: Up-regulated genes in granulosa cells (GC) represented inflammation, angiogenesis, extracellular matrix, growth factors and genes previously associated with ovarian cancer, while down-regulated genes mainly represented cell cycle and proliferation. WHAT IS KNOWN ALREADY: Radical changes occur in the follicle during final follicle maturation after the ovulatory trigger: these range from ensuring an optimal milieu for the oocyte in meiotic arrest to the release of a mature oocyte and remodeling into a corpus luteum. A wide range of mediators of final follicle maturation has been identified in rodents, non-human primates and cows. STUDY DESIGN, SIZE, DURATION: Prospective cohort study including 24 women undergoing ovarian stimulation with the long gonadotrophin-releasing hormone agonist protocol during 2010-2012 at Holbæk Fertility Clinic. Nine paired samples of GC and 24 paired samples of follicular fluid (FF) were obtained before and after recombinant human chorionic gonadotrophin (rhCG) administration. PARTICIPANTS/MATERIALS, SETTING, METHODS: Nine paired (nine arrays before rhCG and nine arrays after rhCG) samples of GC mRNA were amplified and hybridized to Affymetrix Human Gene 1.0 ST GeneChip arrays, compared and bioinformatically analyzed. Eleven selected genes were validated by quantitative reverse transcriptase PCR. FF hormones were analyzed by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven hundred and eighty-six genes were differentially expressed (>2-fold, P<0.0001, false discovery rate <0.0012) when comparing GC isolated before and 36 h after hCG, among those were genes known to be expressed at ovulation, i.e. ADAMTS1 and HAS2. Many new ovulation-related genes were revealed, such as CD24, ANKRD22, CLDN11 and FBXO32. FF estrogen, androstenedione and anti-Müllerian hormone decreased significantly while progesterone increased, accompanied by radical changes in the expression of steroidogenic genes (CYP17A, CYP19A, HSD11B1 and HSD11B2, StAR). Genes related to inflammation, angiogenesis, extracellular matrix formation, growth factors and cancer were up-regulated while cell cycle genes were massively down-regulated. Seventy-two genes previously described in connection with ovarian cancer were among the highly regulated genes. In silico analysis for top upstream regulators of the ovulatory trigger suggested--besides LH--TNF, IGF1, PGR, AR, EGR1 (early growth response 1), ERK1/2 (extracellular signal regulated kinase 1/2) and CDKN1A (cyclin-dependent kinase inhibitor 1A) as potential mediators of the LH/hCG response. LIMITATIONS, REASONS FOR CAUTION: The present dataset was generated from women under hormonal stimulation. However, comparison with a macaque natural cycle whole follicle ovulation dataset revealed major overlap, supporting the idea that the ovulation-related genes found in this study are relevant in the human natural cycle. WIDER IMPLICATIONS OF THE FINDINGS: These data will serve as a research resource for genes involved in human ovulation and final oocyte maturation. Ovulation-related genes might be good candidate biomarkers of follicle and oocyte health. Further, some of the ovulation-related genes may serve as future ovarian cancer biomarkers. STUDY FUNDING/COMPETING INTEREST(S): Grants from the Research Fund of Region Sjælland are gratefully acknowledged. None of the authors declared any conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Specific genes are selectively expressed between cumulus and granulosa cells from individual human pre-ovulatory follicles.

During folliculogenesis, the granulosa cells differentiate into two cell types: cumulus cells (CCs) and mural granulosa cells (MGCs). The objective of the study was to generate and compare the transcriptomes of MGCs and CCs from the pre-ovulatory follicle to characterize the detailed profile of the two cell populations shortly before ovulation. Twenty-one IVF/ICSI patients undergoing controlled ovarian stimulation (COS) donated CCs and MGCs from individual follicles containing metaphase II oocytes. Cells were prepared immediately after recovery and mRNA was isolated for whole-genome gene expression analysis and reverse transcriptase-polymerase chain reactions. Paired (within the individual follicle) comparisons between the CC and MGC expression profiles were performed and corrected for multiple comparisons. A total of 1562 genes were differentially expressed by >2-fold (P < 0.01) in the two cell types. Of these, 156 genes were >8-fold changed and represented specialized cellular functional categories such as inflammatory response, extracellular matrix and cell-cell communication, whereas the 1406 genes were 2-8-fold changed and represented functional categories such as proliferation and lipid metabolism. Transcripts not previously linked to the follicle were found to be differentially expressed between CCs and MGCs, suggesting specialized function in these compartments, e.g. pepsinogen A was selectively expressed in MGCs, whereas ryanodine receptor-2 (RYR2) was selectively expressed in CCs. Positive correlations were present between expression levels of RYR2 and the amphiregulin and gap-junction proteins. In conclusion, the transcriptomes of corresponding CCs and MGCs from individual pre-ovulatory follicles clearly revealed two distinct cell types. New as well as known genes representing specific cell functions close to ovulation were highlighted.

Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

Anti-Müllerian hormone remains highly expressed in human cumulus cells during the final stages of folliculogenesis.

This study evaluated whether anti-Müllerian hormone (AMH) was differentially expressed in cumulus (CC) and granulosa (GC) cells from large antral and pre-ovulatory follicles collected from individual follicles in women undergoing in-vitro maturation (IVM) or IVF treatment. Expression studies of AMH, AMH receptor 2, FSH receptor, aromatase and androgen receptor were performed in CC in IVM patients where cumulus-oocyte-complex had expanded, CC in IVM patients where cumulus-oocyte-complex remained compacted, GC from immature follicles and CC and GC from IVF patients. Microarray data on corresponding GC and CC from follicles from IVF patients was included. AMH expression was significantly higher in CC than in GC from both mature and immature follicles and in CC from immature follicles than in CC from pre-ovulatory follicles from IVF patients (P < 0.05). AMH expression was significantly higher in CC that remained compacted compared with those that had expanded (P < 0.008). AMH was correlated to the expression of FSH receptor, androgen receptor and AMH receptor 2 but not to aromatase expression. The expression pattern of AMH receptor 2 reflected that of AMH. AMH may exert intrafollicular functions even in human large antral and pre-ovulatory follicles and may be related to follicular health.

Gene expression profiles of single human mature oocytes in relation to age.

BACKGROUND: The development competence of human oocytes declines with increasing age. The objective of this study was to investigate the effect of age on gene expression profile in mature human oocytes. METHODS: mRNA was isolated for whole genome gene expression microarray analysis from metaphase II (MII) oocytes donated by IVF or ICSI patients [10 women aged <36 years (younger) and five women aged 37-39 years (both inclusive) (older)] undergoing controlled ovarian stimulation. The oocytes were donated and prepared immediately after recovery from the follicle. RT-PCR on additional four younger and two older oocytes confirmed the array analysis. RESULTS: On the basis of 15 independent replicates of single MII oocytes, 7470 genes (10 428 transcripts) were identified as present in the MII oocytes. Of these, 342 genes showed a significantly different expression level between the two age groups; notably, genes annotated to be involved in cell cycle regulation, chromosome alignment (e.g. MAD2L1 binding protein), sister chromatid separation (e.g. separase), oxidative stress and ubiquitination. The top signaling network affected by age was 'cell cycle and organism development' (e.g. SMAD2 and activin B1 receptor). CONCLUSION: There is a substantial difference between younger and older oocytes in the transcriptional level of genes involved in central biological functions of the oocytes, thus providing information on processes that may be associated with the ageing phenomenon and possibly contributing to decreased fertility.

Hormonal characteristics of follicular fluid from women receiving either GnRH agonist or hCG for ovulation induction.

BACKGROUND: A recent prospective randomized study from our group compared GnRH agonist (0.5 mg buserelin) and hCG (10,000 IU) for triggering of ovulation following a flexible antagonist protocol. The agonist group showed a poor reproductive outcome despite luteal phase support with progesterone and estradiol (E(2)). In the present prospective observational study, the health status of follicles from the above study was monitored by analysing the hormonal content of frozen/thawed follicular fluid samples. The aim was to test whether the poor reproductive outcome could be related to a defective pre-ovulatory follicular maturation resulting in oocytes with a compromised developmental competence. METHODS: Hormone concentrations were measured in two individual follicular fluid samples from each of 32 women receiving buserelin and 37 receiving hCG, thus representing a subset of the follicles retrieved. RESULTS: Follicular fluid levels of LH in the agonist group as compared with the hCG group was 11.1 +/- 0.5 versus 3.6 +/- 0.3 IU/l (mean +/- SEM; P < 0.001); FSH, 6.3 +/- 0.6 versus 3.3 +/- 0.2 IU/l (P < 0.001); hCG, not determined versus 139+/-8 IU/l; E(2), 1.9 +/- 0.2 versus 1.8 +/- 0.2 micromol/l (P > 0.10); progesterone, 70 +/- 4 versus 93 +/- 6 micromol/l (P < 0.001); inhibin-A, 36.9 +/- 3.1 versus 37.1 +/- 2.5 ng/ml (P > 0.10) and inhibin-B, 35.6 +/- 2.8 versus 40.1 +/- 3.1 ng/ml (P > 0.10). Thus, pronounced hormonal differences exist in follicular fluid, and the collective concentration of all three gonadotropins and the follicular fluid concentration of progesterone were much higher in the group of women receiving hCG for ovulation induction. CONCLUSION: The study suggests that GnRH agonist results in proper pre-ovulatory follicular maturation, but the ovulatory signal--probably in synergy with the resulting pituitary down-regulation--is too low to support appropriate corpus luteum (CL) function.

GnRH agonist (buserelin) or hCG for ovulation induction in GnRH antagonist IVF/ICSI cycles: a prospective randomized study.

BACKGROUND: We aimed to determine the efficacy of ovarian hyperstimulation protocols employing a GnRH antagonist to prevent a premature LH rise allowing final oocyte maturation and ovulation to be induced by a single bolus of either a GnRH agonist or hCG. METHODS: A total of 122 normogonadotrophic patients following a flexible antagonist protocol was stimulated with recombinant human FSH and prospectively randomized (sealed envelopes) to ovulation induction with a single bolus of either 0.5 mg buserelin s.c. (n = 55) or 10,000 IU of hCG (n = 67). A maximum of two embryos was transferred. Luteal support consisted of micronized progesterone vaginally, 90 mg a day, and estradiol, 4 mg a day per os. RESULTS: Ovulation was induced with GnRH agonist in 55 patients and hCG in 67 patients. Significantly more metaphase II (MII) oocytes were retrieved in the GnRH agonist group (P < 0.02). Significantly higher levels of LH and FSH (P < 0.001) and significantly lower levels of progesterone and estradiol (P < 0.001) were seen in the GnRH agonist group during the luteal phase. The implantation rate, 33/97 versus 3/89 (P < 0.001), clinical pregnancy rate, 36 versus 6% (P = 0.002), and rate of early pregnancy loss, 4% versus 79% (P = 0.005), were significantly in favour of hCG. CONCLUSIONS: Ovulation induction with a GnRH agonist resulted in significantly more MII oocytes. However, a significantly lower implantation rate and clinical pregnancy rate in addition to a significantly higher rate of early pregnancy loss was seen in the GnRH agonist group, most probably due to a luteal phase deficiency.

Neuronal involvement in the effect of an antisecretory factor-derived peptide on induced secretion in the porcine small intestine.

The antisecretory factor is a protein inhibiting enterotoxin-induced intestinal inflammation and hypersecretion. We studied the signaling pathway of three antisecretory factor-derived peptides (A1, A3 and A4) in the proximal and distal porcine small intestine. In vivo (ligated loops), only A3 significantly reduced the cholera toxin-induced fluid accumulation and only in proximal loops. A3 and A4 reduced Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the proximal segment, whereas A1 and A3 reduced the Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the distal segment. The secretory response to intraluminally added 5-hydroxytryptamine was not significantly inhibited by the peptides. The amount of intraluminal 5-hydroxytryptamine accumulated in cholera toxin-stimulated loops from the proximal segment was significantly reduced by A3. In vitro,the effect of A3 on secretagogue-induced increases in short-circuit current was recorded from proximal small intestine by the Ussing chamber technique. A3 decreased the tetrodotoxin sensitive effect of substance P. The in vivo results suggest that the antisecretory effect may involve inhibition of the local release of 5-hydroxytryptamine induced by cholera toxin, but not inhibition of secretory reflexes induced by 5-hydroxytryptamine. The in vitro results suggest that the effect of A3 lies beyond the surface epithelium, and involves mucosal neuronal structures.