Follicle Recruitment Research Articles

Antioxidant supplementation partially rescues accelerated ovarian follicle loss, but not oocyte quality, of glutathione-deficient mice†.

The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80mM NAC in drinking water from postnatal day (PND) 21-30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γH2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.

Crosstalk between PTEN/PI3K/Akt Signalling and DNA Damage in the Oocyte: Implications for Primordial Follicle Activation, Oocyte Quality and Ageing.

The preservation of genome integrity in the mammalian female germline from primordial follicle arrest to activation of growth to oocyte maturation is fundamental to ensure reproductive success. As oocytes are formed before birth and may remain dormant for many years, it is essential that defence mechanisms are monitored and well maintained. The phosphatase and tensin homolog of chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt) is a major signalling pathway governing primordial follicle recruitment and growth. This pathway also contributes to cell growth, survival and metabolism, and to the maintenance of genomic integrity. Accelerated primordial follicle activation through this pathway may result in a compromised DNA damage response (DDR). Additionally, the distinct DDR mechanisms in oocytes may become less efficient with ageing. This review considers DNA damage surveillance mechanisms and their links to the PTEN/PI3K/Akt signalling pathway, impacting on the DDR during growth activation of primordial follicles, and in ovarian ageing. Targeting DDR mechanisms within oocytes may be of value in developing techniques to protect ovaries against chemotherapy and in advancing clinical approaches to regulate primordial follicle activation.

Serum anti-Müllerian hormone levels during estrus and diestrus in mares

Anti-Müllerian hormone (AMH) is a homodimeric glycoprotin secreted by granulosa cells in postnatal females. AMH plays an important role in follicle recruitment. The aim of the study was to analyse AMH concentrations under the guidance of circulating estradiol and progesterone levels in mares. The study was conducted on 25 non-lactating mares with regular estrous cycles. Blood samples were collected during estrus and diestrus to analyse estradiol, progesterone and AMH concentrations. Estradiol and progesterone concentrations were significantly different (P < 0.001) between estrus and diestrus. AMH concentrations in estrus were significantly higher than those in diestrus (P < 0.05). According to the authors’ knowledge, this is the first report to suggest that the sexual cycle stage affects AMH concentrations in mares. Further investigations are needed to reveal factors affecting the AMH production and secretion pattern throughout the sexual cycle.

Vitamin D3 supplementation influences ovarian histomorphometry and follicular development in prepubertal albino rats

Objective: To evaluate the development of ovarian follicles in female albino rats following vitamin D3 supplementation. Methods: Eighteen prepubertal female albino rats, aged 3-4 weeks, weighing (70.25±9.16) g, were assigned to three groups (n=6 in each group). Group A was treated with 5.00 mL/kg of distilled water and served as the control group, group B was treated with 0.025 mg/kg of vitamin D3 dissolved in distilled water, and group C was treated with 0.125 mg/kg of vitamin D3 dissolved in distilled water. All treatments were administered orally, twice weekly for 28 days. Blood and ovaries were harvested under anaesthesia. Serum vitamin D3 levels were determined by using spectrophotometric method. Ovaries were processed for histology and every10th hematoxylin and eosin stained-section was selected for histomorphometry. The number of follicles at each developmental stage was estimated.Results: Both 0.025 mg/kg and 0.125 mg/kg of vitamin D3 significantly increased serum concentrations of vitamin D3 and calcium (P<0.05), but did not alter inorganic phosphorus concentration (P>0.05). The control group had fewer growing follicles (primary, secondary and antral follicles) and more non-growing follicles (primordial and atretic follicles) when compared with the vitamin D3-supplemented groups (P<0.05). Vitamin D3 at 0.025 mg/kg significantly increased antral follicles and corpora lutea counts (P<0.05). Vitamin D3 at 0.125 mg/kg significantly increased total, primordial and atretic follicles counts (P<0.05), but significantly decreased primary, secondary, antral follicles count, ovarian weight, relative ovarian weight, and ovarian surface area when compared with the control group and rats treated with 0.025 mg/kg of vitamin D3 (P<0.05).Conclusions: Vitamin D3 supplementation at 0.025 mg/kg can enhance optimal ovarian follicle recruitment and development in female rats.

Rapamycin treatment maintains developmental potential of oocytes in mice and follicle reserve in human cortical fragments grafted into immune-deficient mice

The ovarian follicle pool size is limited; it decreases with age and following germ cell-damaging chemo- or radiation therapies. Due to a trend of delaying child-bearing age in the modern society, it is important to investigate the possibility to maintain the follicle reserve for middle-aged women and cancer-bearing patients subject to therapies. Earlier studies demonstrated the important role of the mammalian targets of the rapamycin (MTOR) signaling pathway in the activation of primordial follicles and suggested that treatment with the MTOR inhibitor rapamycin could maintain the follicle pool in rodents. Here, we confirmed the ability of rapamycin treatment for 3 weeks to suppress primordial follicle development and to maintain follicle pool size in mice. We further demonstrated that the developmental potential of oocytes was not affected by rapamycin treatment and the effect of rapamycin to decrease initial follicle recruitment is reversible. Using human ovarian cortical fragments grafted into immune-deficient mice, we demonstrated the ability of rapamycin to suppress follicle growth from the primordial stage. Our studies provide the basis for further studies on the possibility of using MTOR inhibitors to maintain follicle reserve in middle-aged women and cancer patients before/during germ cell-damaging therapies.

Neonatal Exposure to Agonists and Antagonists of Sex Steroid Receptors Affects AMH and FSH Plasma Level and Their Receptors Expression in the Adult Pig Ovary.

Simple SummaryThe ovarian development and the establishment of ovarian reserve during fetal and/or neonatal life is critical for future reproductive success. Many environmental chemicals are known to negatively affect development and physiology of human and animal ovaries by interfering with endocrine systems, resulting in aberrant reproductive functions. The present study shows the long-term impact of neonatal exposure to agonists and antagonists of sex steroid receptors on AMH and FSH signalling in the ovary of adult pigs. Our findings suggest alteration in ovarian follicle recruitment from ovarian reserve arising from neonatal disruption of androgen/estrogen signalling induced by environmental endocrine active compounds. Everyday use of many endocrine disruptors is already prohibited after their harmful impacts on normal physiology have become known. Nevertheless, market introduction of new chemicals with potential deleterious influence on reproductive physiology has continued. Our outcomes confirm that a neonatal window plays an essential role in the physiological programming of ovarian function in adult pigs. The influence of environmental chemicals on this critical neonatal window needs to be investigated in order to gain a comprehensive view of deleterious interactions between endocrine disrupting chemicals and ovarian function.In this study piglets were injected with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen) or corn oil (controls) between postnatal days 1 and 10 (N = 5/group). Then plasma anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) concentration and the expression of their receptors were examined in the adult pig ovary. TP and FLU decreased plasma AMH and FSH concentration. In preantral follicles, TP resulted in upregulation of AMHR2 and FSHR expression, but decreased AMH protein abundance. FLU upregulated AMHR2 expression, while OP increased FSHR mRNA. In small antral follicles, OP upregulated ACVR1 and BMPR1A expression, while FLU increased BMPR1A mRNA. FLU and ICI resulted in upregulation of AMHR2 expression. TP and FLU upregulated AMH expression, while it was downregulated in response to OP or ICI. Moreover, OP and ICI resulted in downregulation of FSHR expression, while FLU decreased FSHR protein abundance. In conclusion, neonatal exposure to either agonist or antagonist of androgen receptor affected AMH and FSH signalling systems in preantral follicles. In small antral follicles these systems were influenced by compounds with estrogenic, antiestrogenic, and antiandrogenic activity. Consequently, these hormonal agents may cause an accelerated recruitment of primordial follicles and affect the cycling recruitment of small antral follicles in pigs.

Regulation of AMH, AMHR-II, and BMPs (2,6) Genes of Bovine Granulosa Cells Treated with Exogenous FSH and Their Association with Protein Hormones.

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3–8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3–8 mm), medium (9–12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.

WOMEN IN REPRODUCTIVE SCIENCE: Anti-Müllerian hormone: a look back and ahead.

Anti-Müllerian hormone (AMH) is a member of the TGF-β family secreted by immature Sertoli cells and by granulosa cells of growing ovarian follicles. In males, it induces the regression of fetal Müllerian ducts and represses androgen synthesis through receptors located on the Leydig cell membrane. In female mice, AMH inhibits primary follicle recruitment and sensitivity to FSH. Measurement of circulating AMH is of value to pediatric endocrinologists allowing them to detect the presence and functional activity of testicular tissue without resorting to stimulation by human chorionic gonadotropin. In women, AMH levels are correlated with the size of the ovarian follicle pool and provide information on the likelihood of spontaneous or induced pregnancy.

The effect of intra-ovarian androgen priming on ovarian reserve parameters in Bologna poor responders

What are the effects of long-term androgen priming in Bologna criteria poor ovarian reserve (POR) patients undergoing IVF? This open-label pilot study was conducted at IVFMD, My Duc Hospital, Ho Chi Minh City, Vietnam. It included consecutive patients aged 18-41 years who fulfilled Bologna criteria for POR undergoing intra-ovarian androgen priming and ultra-long down-regulation with a gonadotrophin-releasing hormone agonist (GnRHa), followed by stimulation with gonadotrophins and GnRH antagonist co-treatment for IVF (n = 30). Priming consisted of low-dose recombinant human chorionic gonadotrophin (rHCG) 260 IU every second day plus letrozole 2.5 mg/day, both for 8 weeks; priming stopped on the first day of ovarian stimulation. The primary endpoint was serum anti-Müllerian hormone (AMH) concentration 8 weeks after priming. Secondary endpoints included antral follicle count (AFC) (2-10 mm), serum human chorionic gonadotrophin (HCG), testosterone and progesterone levels. Circulating testosterone, progesterone, oestradiol and HCG levels remained unchanged during androgen priming; the mean AMH level decreased steadily from 0.49 ng/ml (baseline) to 0.33 ng/ml (8 weeks). AFC was 4-5 throughout the study. A mean of 1.1 ± 0.9 good transferable embryos were obtained; embryo transfer was performed in 15 patients; no ongoing pregnancies were obtained. Long-term intra-ovarian androgen priming in the current set-up had no significant effect on hormone levels, AFC and recruitable follicles after ovarian stimulation in Bologna POR patients undergoing IVF. Further studies are needed to explore other androgen priming protocols and the clinical value of priming regimens in IVF.

SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and mTOR transcription.

In female mammals, the majority of primordial follicles (PFs) are physiologically quiescent, and only a few of them are activated and enter the growing follicle pool. Specific molecules, such as mammalian target of rapamycin (mTOR) and the serine/threonine kinase Akt (AKT), have been proven to be important for PF activation. However, how the transcription of these genes is regulated is not clear. Although activators of mTOR or AKT have been successfully used to rescue the fertility of patients with premature ovarian insufficiency, the low efficacy and unclear safety profile of these drugs hinder their clinical use in the in vitro activation (IVA) of PFs. Here, sirtuin 1 (SIRT1), an NAD-dependent deacetylase, was demonstrated to activate mouse PFs independent of its deacetylase activity. SIRT1 was prominently expressed in pregranulosa cells (pGCs) and oocytes, and its expression was increased during PF activation. PF activation was achieved by either up-regulating SIRT1 with a specific activator or overexpressing SIRT1. Moreover, SIRT1 knockdown in oocytes or pGCs could significantly suppress PF activation. Further studies demonstrated that SIRT1 enhanced both Akt1 and mTOR expression by acting more as a transcription cofactor, directly binding to the respective gene promoters, than as a deacetylase. Importantly, we explored the potential clinical applications of targeting SIRT1 in IVA via short-term treatment of cultured ovaries from mice and human ovarian tissues to activate PFs by applying the SIRT1 activator resveratrol. RSV-induced IVA could be a candidate strategy to develop more efficient procedures for future clinical treatment of infertility.-Zhang, T., Du, X., Zhao, L., He, M., Lin, L., Guo, C., Zhang, X., Han, J., Yan, H., Huang, K., Sun, G., Yan, L., Zhou, B., Xia, G., Qin, Y., Wang, C. SIRT1 facilitates primordial follicle recruitment independent of deacetylase activity through directly modulating Akt1 and mTOR transcription.

CFP1-dependent histone H3K4 trimethylation in murine oocytes facilitates ovarian follicle recruitment and ovulation in a cell-nonautonomous manner.

CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.

MRNA and miRNA Transcriptome Profiling of Granulosa and Theca Layers From Geese Ovarian Follicles Reveals the Crucial Pathways and Interaction Networks for Regulation of Follicle Selection.

Follicle development is characterized by the recruitment, growth, selection, and dominance of follicles, and follicle selection determines the lifetime reproductive performance. However, in birds, the molecular mechanisms underlying follicle selection still remain elusive. This study analyzed genome-wide changes in the mRNA and miRNA expression profiles in both the granulosa and theca layers of geese ovarian follicles before selection (4–6- and 8–10-mm follicles) and after selection (F5). The sequencing results showed that a higher number of both differentially expressed (DE) mRNAs and DE miRNAs were identified between 8–10-mm and F5 follicles compared with those between the 4–6- and 8–10-mm follicles, especially in the granulosa layer. Moreover, a Short Time-series Expression Miner analysis identified a large number of DE mRNAs and DE miRNAs that are associated with follicle selection. The functional enrichment analysis showed that DE genes in the granulosa layer during follicle selection were mainly enriched in five pathways related to junctional adhesion and two pathways associated with lipid metabolism. Additionally, an interaction network was constructed to visualize interactions among protein-coding genes, which identified 53 junctional adhesion- and 15 lipid regulation-related protein-coding genes. Then, a co-expression network between mRNAs and miRNAs in relation to junctional adhesion was also visualized and mainly included acy-miR-2954, acy-miR-218, acy-miR-2970, acy-miR-100, acy-miR-1329, acy-miR-199, acy-miR-425, acy-miR-181, and acy-miR-147. Furthermore, miRNA–mRNA interaction pairs related to lipid regulation were constructed including acy-miR-107, acy-miR-138, acy-miR-130, acy-miR-128, and acy-miR-101 during follicular selection. In summary, these data highlight the key roles of junctional adhesion and lipid metabolism during follicular selection and contribute to a better understanding of the mechanisms underlying follicle selection in birds.

Pentachloronitrobenzene alters progesterone production and primordial follicle recruitment in cultured granulosa cells and rat ovary†.

Pentachloronitrobenzene (PCNB) is an organochlorine fungicide widely used for crop production and has become an environmental concern. Little is known about the effect of PCNB on ovarian steroidogenesis and follicular development. We found that PCNB stimulated Star expression and progesterone production in cultured rat granulosa cells in a dose-dependent manner. PCNB activated mitogen-activated protein kinase (MAPK3/1) extracellulat regulated kinase (ERK1/2), thus inhibition of either protein kinase A (PKA) or MAPK3/1 signaling pathway significantly attenuated progesterone biosynthesis caused by PCNB, suggesting that PCNB induced progesterone production by activating the cyclic adenosine monophosphate (cAMP/PKA) and MAPK3/1 signaling pathways. Further investigation demonstrated that PCNB induced Star expression and altered MAPK3/1 signaling in ovary tissues of immature SD rats treated with PCNB at the dose of 100, 200, or 300mg/kg by daily gavage for 7days, while serum progesterone level was dose-dependently decreased. We demonstrated that PCNB exposure accelerated the recruitment of primordial follicles into the growing follicle pool in ovary tissues, accompanied by increased levels of anti-Mullerian hormone (AMH) in both ovary tissues and serum. Taken together, our data demonstrate for the first time that PCNB stimulated Star expression, altered MAPK3/1 signaling and progesterone production in vivo and in vitro, and accelerated follicular development with a concomitant increase in AMH in ovary tissues and serum. Our findings provide novel insight into the toxicity of PCNB to animal ovary function.

Ultrastructure and intercellular contact-mediated communication in cultured human early stage follicles exposed to mTORC1 inhibitor.

The reproductive lifespan of a woman is determined by the gradual recruitment of quiescent follicles into the growing pool. In humans, ovarian tissue removal from its in vivo environment induces spontaneous activation of resting follicles. Similarly, pharmacological activation of the PI3K/Akt pathway leads to accelerated follicle recruitment, but has been associated with follicular damage. Recent findings demonstrate that everolimus (EVE), an mTORC1 inhibitor, limits primordial follicle activation. However, its potential benefit regarding growing follicle integrity remains unexplored. Ovarian cortical fragments were exposed to ± EVE for 24h and cultured for an additional 5days. After 0, 1 and 6days of culture, fragments were either processed for ultrastructural analysis or subjected to follicular isolation for gene expression and immunofluorescence assessments. Data from transmission electron microscopy showed that growing follicles displayed similar ultrastructural features irrespective of the conditions and maintained close contacts between germinal and stromal compartments. Establishment of intra-follicular communication was confirmed by detection of a gap junction component, Cx43, in both groups throughout culture, whereas transzonal projections, which physically link granulosa cells to oocyte, formed later in EVE-treated follicles. Importantly, levels of GJA1 mRNA, encoding for the Cx43 protein, significantly increased from Day 0 to Day 1 in the EVE group, but not in the control group. Given that EVE-treated follicles were smaller than controls, these findings suggest that EVE might facilitate the establishment of appropriate intercellular communications without impairing follicle ultrastructure. Therefore, mTORC1 inhibitors might represent an attractive tool to delay the culture-induced primordial follicle activation while maintaining follicles in a functionally integrated state.

Biopsying, fragmentation and autotransplantation of fresh ovarian cortical tissue in infertile women with diminished ovarian reserve.

Can ovarian biopsying per se and/or autotransplantation of fragmented ovarian cortical tissue activate dormant follicles and increase the number of recruitable follicles for IVF/ICSI in women with diminished ovarian reserve (DOR)? Ovarian biopsying followed by immediate autotransplantation of fragmented cortical tissue failed to increase the number of recruitable follicles for IVF/ICSI 10weeks after the procedure either at the graft site or in the biopsied ovary, but 12 of the 20 women subsequently had a clinical pregnancy during the 1-year follow-up. Infertile women with DOR constitute a group of patients with poor reproductive outcome mainly due to the low number of mature oocytes available for IVF/ICSI. Recent studies have shown that in vitro activation of residual dormant follicles by both chemical treatment and tissue fragmentation has resulted in return of menstrual cycles and pregnancies in a fraction of amenorrhoeic women with premature ovarian insufficiency. This is a prospective clinical cohort study including 20 women with DOR treated at the fertility clinic, Rigshospitalet, Denmark, during April 2016-December 2017. Non-pregnant patients were on average followed for 280days (range 118-408), while women who conceived were followed until delivery. Study follow-up of non-pregnant patients ended in September 2018. The study included infertile women aged 30-39years with preserved menstrual cycles, indication for IVF/ICSI and repeated serum measurements of anti-Müllerian hormone (AMH) ≤ 5pmol/L. Patients were randomized to have four biopsies taken from either the left or the right ovary by laparoscopy followed by fragmentation of the cortical tissue to an approximate size of 1mm3 and autotransplanted to a peritoneal pocket. The other ovary served as a control. Patients were followed weekly for 10weeks with recording of hormone profile, antral follicle count (AFC), ovarian volume and assessment for ectopic follicle growth. After 10weeks, an IVF/ICSI-cycle with maximal ovarian stimulation was initiated. No difference in the number of mature follicles after ovarian stimulation 10weeks after the procedure in the biopsied versus the control ovaries was observed (1.0 vs. 0.7 follicles, P= 0.35). In only three patients, growth of four follicles was detected at the graft site 24-268days after the procedure. From one of these follicles, a metaphase II (MII) oocyte was retrieved and fertilized, but embryonic development failed. Overall AMH levels did not change significantly after the procedure (P= 0.2). The AFC increased by 0.14 (95% CI: 0.06;0.21) per week (P< 0.005), and the biopsied ovary had on average 0.6 (95% CI: 0.3;-0.88) follicles fewer than the control ovary (P= 0.01). Serum levels of androstenedione and testosterone increased significantly by 0.63nmol/L (95% CI: 0.21;1.04) and 0.11nmol/L (95% CI: 0.01;0.21) 1week after the procedure, respectively, and testosterone increased consecutively over the 10weeks by 0.0095nmol/L (95% CI: 0.0002;0.0188) per week (P= 0.045). In 7 of the 20 patients, there was a serum AMH elevation 5 to 8weeks after the procedure. In this group, mean AMH increased from 2.08pmol/L (range 1.74-2.34) to 3.94pmol/L (range 3.66-4.29) from Weeks 1-4 to Weeks 5-8. A clinical pregnancy was obtained in 12 of the 20 (60%) patients with and without medically assisted reproduction (MAR) treatments. We report a cumulated live birth rate per started IVF/ICSI cycle of 18.4%. Limitations of the study were the number of patients included and the lack of a non-operated control group. Moreover, 9 of the 20 women had no male partner at inclusion and were treated with donor sperm, but each of these women had an average of 6.8 (range 4-9) unsuccessful MAR treatments with donor sperm prior to inclusion. Although 12 out of 20 patients became pregnant during the follow-up period, the current study does not indicate that biopsying, fragmenting and autotransplanting of ovarian cortical tissue increase the number of recruitable follicles for IVF/ICSI after 10weeks. However, a proportion of the patients may have a follicular response in Weeks 5-8 after the procedure. It could therefore be relevant to perform a future study on the possible effects of biopsying per se that includes stimulation for IVF/ICSI earlier than week 10. This study is part of the ReproUnion collaborative study, co-financed by the European Union, Interreg V ÖKS. The funders had no role in the study design, data collection and interpretation, or decision to submit the work for publication. None of the authors have a conflict of interest. NCT02792569.

FSH Requirements for Follicle Growth During Controlled Ovarian Stimulation

Introduction: Ovarian follicle growth is a key step in the success of assisted reproductive treatment, but limited data exists to directly relate follicle growth to recombinant FSH (rFSH) dose. In this study, we aim to evaluate FSH requirements for follicular growth during controlled ovarian stimulation.Method: Single center retrospective cohort study of 1,034 IVF cycles conducted between January 2012–January 2016 at Hammersmith Hospital IVF unit, London, UK. Median follicle size after 5 days of stimulation with rFSH and the proportion of antral follicles recruited were analyzed in women treated with rFSH alone to induce follicular growth during IVF treatment.Results: Starting rFSH dose adjusted for body weight (iU/kg) predicted serum FSH level after 5 days of rFSH (r2 = 0.352, p < 0.0001), median follicle size after 5 days of rFSH, and the proportion of antral follicles recruited by the end of stimulation. Day 5 median follicle size predicted median follicle size on subsequent ultrasound scans (r2 = 0.58–0.62; p < 0.0001), and hence time to oocyte maturation trigger (r2 = 0.22, P < 0.0001). Insufficient rFSH starting dose that required >5% dose-increase was associated with increased variability in follicle size on the day of oocyte maturation trigger, and negatively impacted the number of mature oocytes retrieved.Conclusion: Weight-adjusted rFSH dose correlates with follicular growth during ovarian stimulation. Early recruitment of follicles using a sufficient dose of rFSH from the start of stimulation was associated with reduced variability in follicle size at time of oocyte maturation trigger and an increased number of mature oocytes retrieved.

Dehydroepiandrosterone supplementation attenuates ovarian ageing in a galactose-induced primary ovarian insufficiency rat model.

Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI). The unique pathophysiology of classic galactosemia, with a severely reduced follicle pool at an early age, requires a new therapeutic approach. This study evaluated the effect of dehydroepiandrosterone (DHEA) on ovarian tissue in a galactose-induced POI rat model. Pregnant rats were fed with either a normal or a 35% galactose-containing diet from day 3 of conception continuing through weaning of the litters. Galactose-exposed female offspring were further divided into 5 groups on PND21. The first group received no application. Treatment groups were fed orally by gavage once daily with sesame oil (group 2), or DHEA at doses of 0.1mg/kg (group 3), 1mg/kg (group 4) or 10mg/kg (group 5) until PND70. Fertility rates of mothers with galactosemia, body weights (BWs), and ovarian weights of the litters from PND21 to PND70 were recorded. Ovarian follicle count, immunohistochemistry for proliferation and apoptosis marker expressions and TUNEL for cell death assessment were performed in offspring ovaries. Decreased fertility, ovarian/body weights were observed under galactosemic conditions, together with decreased follicle number and increased atresia. Improved postnatal development, primordial follicle recruitment and follicular growth were observed after DHEA treatment. After DHEA treatment, the expression of Ki67 protein was found to be increased; elevated expression of cleaved-caspase-3 under galactosemia was found to be reduced. Our data suggests that DHEA treatment may be a potentially useful clinical therapy to improve ovarian ageing in women with POI-induced by galactosemia.

Contribution of recruitable follicles to circulating anti-Müllerian hormone levels following maximal gonadotrophin stimulation in patients with limited ovarian reserve

In women, the majority of anti-Müllerian hormone (AMH) measured in serum originate from small antral follicles measuring 2–10 mm. In gonadotrophin-stimulated cycles prior to assisted reproductive technology (ART), most of the recruitable follicles develop beyond 10 mm in size and thus lose their AMH secretion capacity causing declining serum AMH levels. The aim of this study was to define the residual serum AMH level after elimination of the AMH producing recruitable follicles following maximal gonadotrophin stimulation. We measured serum AMH and number of follicles according to size at several time points during a cycle of maximal gonadotrophin stimulation (fixed dose of 300 IE HP-hMG) in 107 women with low AMH (median AMH 5 pmol/L, interquartile range (IQR) 3.3–8.3). We found that AMH decreased gradually and reached a minimum level of −55.4% (95% CI −59.6; −50.7) of the baseline value four days after ovulation trigger. Our findings suggest that the residual AMH production origins from pre-antral and small antral follicles not visible by sonography and that they account for up to 40% of the circulating AMH.

Anti-Mullerian Hormone (AMH) as a Novel Marker for Ovarian Function: A Review

Background: Anti-Mullerian Hormone (AMH) is a homodimeric glycoprotein linked by disulfide bonds which belongs to the Transforming Growth Factor (TGF) beta superfamily. AMH is produced by gonadal tissue namely testicular Sertoli cells and ovarian granulosa cells especially pre-antral and antral follicles. AMH involved in embryonic sexual differentiation in male, while it has an inhibitory effect on primordial follicle recruitment and responsiveness of the growing follicles to FSH, in female. Current studies show promising clinical utilities of AMH measurement in predicting ovarian function.Objective: This review aims to explore further about the physiology of AMH and its role as a novel marker for ovarian function.Method: A review of relevant literature was performed to elaborate AMH involvement in ovarian function. A total of 14 qualified published literature of all years until 2018 were collected from several electronic database and manual search and included in this review.Results: AMH level reflected ovarian follicular reserve, an important indicator in infertility treatment (assisted reproduction technique) and a sensitive marker for ovarian aging. AMH is also a valuable tool in diagnosis and recognition of recurrence granulosa cell tumors, and a marker of ovarian dysfunction particularly polycystic ovary syndrome. A low circulating AMH level is observed in obesity and male with fertility problems.Conclusion: AMH level can be used as primary or supplementary markers to aid in the diagnosis and treatment of several reproductive related conditions in males and females. A set of guidelines about sample storage and handling and age-specific standardized reference values are needed to optimize clinical application of AMH.

Relationship between cyclic follicle recruitment and ovulation in the hen ovary

Determining whether follicle recruitment in the domestic hen is functionally linked to ovulation would inform investigations on the exact time of cyclic recruitment and subsequently, the earliest cellular event mediating recruitment. The objective of the present studies was to determine if the absence of ovulation results in the failure of cyclic recruitment. 3 groups of Hy-Line W-36 hens were studied: no-ovulation, first-ovulation, or late-ovulation within the sequence. Time and occurrence of oviposition and ovulation were documented by video recording and cloacal palpation. To determine the presence of a recently recruited follicle, ovaries were collected during the last hour of photoperiod, and follicles weighing 280 to 900mg, with yellow yolk incorporated, and diameter of 9 to 12mm, were considered recently recruited. To compare the amount of yolk uptake in recruited follicles, hens were fed Sudan IV dye 6h before ovary collection. Percent dyed yolk was quantified from cross-sections of the recruited follicles. To confirm follicle viability, granulosa cell (GC) mitotic activity was assessed via flow cytometry. Initial results indicated the presence of a recently recruited follicle in 10 of 11, 7 of 10, and 9 of 10, no-ovulation, first-ovulation, and late-ovulation ovaries, respectively, with no significant differences in weight (543 ± 62mg, 456 ± 71mg, 620 ± 75mg, respectively). There were no significant differences in yolk incorporation among the most recently recruited follicles with 19.0 ± 2.0%, 18.0 ± 3.7%, and 19.8 ± 3.2% dyed yolk in no-ovulation, first-ovulation, and late-ovulation ovaries, respectively. Finally, there were no significant differences in % GC in the S/G2-M phase of mitosis among recruited follicles (19.0 ± 5.0%, 22.0 ± 4.2%, 15.67 ± 1.0%, in no-ovulation, first-ovulation, and late-ovulation ovaries, respectively), confirming viability of all recruited follicles. We conclude that cyclic recruitment occurs independently of ovulation and propose that the order and timing of cyclic recruitment is predetermined at an earlier stage of follicle development.