scholarly journals Melatonin as an endogenous hormone to slow down the exhaustion of ovarian follicle reserve in mice–a novel insight into its roles in early folliculogenesis and ovarian aging

2020 ◽  
Author(s):  
Chan Yang ◽  
Qinghua Liu ◽  
Yingjun Chen ◽  
Xiaodong Wang ◽  
Zaohong Ran ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Primordial Follicle ◽  
High Dose ◽  
Follicle Growth ◽  
Ovarian Aging ◽  
Neonatal Mice ◽  
Primordial Follicle Activation ◽  
Follicle Activation

Abstract Previous studies have shown that long-term intake of exogenous melatonin can effectively delay ovarian aging, but the mechanism has not been fully elucidated. We observed that SNAT, the rate-limiting enzyme in the melatonin synthetic pathway, is localized in primordial and early follicle, and that granulosa cells isolated from follicle can synthesize melatonin. In vitro cultured neonatal mice ovaries with melatonin inhibited primordial follicle activation and early follicle growth. In vivo experiments further indicated that daily injections of melatonin to neonatal mice during the primordial follicle activation phase can reduce the number of activated follicles by inhibiting the PI3K-AKT-FOXO3 pathway; during the early follicle growth phase, injections of melatonin significantly suppressed early follicle growth and atresia, and transcriptome data showed that multiple pathways involved in folliculogenesis, including PI3K-AKT, were suppressed. Further, SNAT knockout in mice resulted in a significant increase in follicle activation and atresia, and eventually accelerated ovarian aging. We also demonstrated that prolonged high-dose melatonin intake had no obvious adverse effect on the health condition of mice. This study confirms that endogenous melatonin is involved in the regulation of ovarian aging, and reveals that melatonin delays ovarian aging by inhibiting primordial follicle activation, early follicle growth and atresia.

P-444 Presence of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and organotypic cultures of murine ovaries limits early primordial follicle depletion

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Terren ◽  
M Nisolle ◽  
C Munaut
Keyword(s):  
In Vitro Culture ◽  
Organotypic Culture ◽  
Primordial Follicle ◽  
Slow Freezing ◽  
Signalling Pathways ◽  
Primordial Follicle Activation ◽  
Mtor Signalling ◽  
Follicle Activation

Abstract Study question Which signalling pathways are implicated in primordial follicle activation induced by cryopreservation and/or organotypic culture? Is it possible to limit this activation through pharmacological inhibitors? Summary answer Our findings provide support for the hypothesis that mTOR and PI3K inhibitors might represent an attractive tool to delay cryopreservation- and culture-induced primordial follicle activation. What is known already Cryopreservation of ovarian tissue containing immature primordial follicles followed by auto-transplantation (OTCTP) is the only option available to preserve the fertility of prepubertal patients or patients requiring urgent therapy for aggressive malignancies. However, a major obstacle in this process is follicular loss immediately after grafting, possibly due to slow neovascularization, apoptosis and/or massive follicular recruitment. In vitro and in vivo studies indicate that the PI3K/PTEN/Akt and mTOR signalling pathways are involved in follicle activation. The transplantation process seems to be the major cause of primordial follicle activation after OTCTP but information about how cryopreservation itself impacts follicle activation is sparse. Study design, size, duration Whole murine ovaries (4-8-weeks old) were cryopreserved by slow freezing and exposed to LY294002 (a powerful PI3K inhibitor) or rapamycin (a specific mTOR inhibitor) during cryopreservation and/or organotypic in vitro culture for a 24 h or 2 days. Participants/materials, setting, methods Western Blot and immunofluorescence analyses were used to determine the activation of PI3K/PTEN/Akt and mTOR signalling pathways in murine ovaries cryopreserved and/or organotypically cultured with/without inhibitors.Follicles were quantified according to their maturation degree on H&E stained histological sections.  Main results and the role of chance Ratio of phosphorylated Akt or rps6 to total proteins (p-Akt/Akt and p-rps6/rps6) was increased in slow-frozen murine ovaries compared to control fresh ovaries, indicating an activation of the PI3K/PTEN/Akt and mTOR signalling pathways. The use of pharmacological inhibitors of follicle signalling pathways (LY294002 (25µM) and rapamycin (1µM)) during the cryopreservation process decreased p-Akt/Akt and p-rps6/rps6 ratios. In vitro organotypic culture for 24 h increased only the activation of the PI3K/PTEN/Akt pathway, as shown by increased p-Akt/Akt ratio in fresh ovaries cultured for 24 h compared to fresh non-cultured ovaries. This activation can be counteracted by cryopreservation of murine ovaries with rapamycin followed by in vitro culture for 24 h in the presence of LY294002. Follicle density quantifications indicated that when cryopreserved ovaries were maintained in culture for 2 days, a decrease of primordial follicle density concomitant with an increase of secondary and more mature follicles were found in comparison to slow-frozen/thawed ovaries without culture. Supplementation of the culture medium with LY294002 and rapamycin for 24 h or 2 days preserved primordial follicle densities compared to ovaries cultured without inhibitors. Limitations, reasons for caution This study is an in vitro study using murine ovaries. To analyze the efficiency of LY294002 and rapamycin to limit cryopreservation and transplantation induced follicle recruitment, these inhibitors should be tested in an in vivo model. Furthermore, these findings will need to be confirmed with human samples. Wider implications of the findings We showed for the first-time that the sequential use of pharmacological inhibitors, rapamycin during the slow freezing process followed by organotypic culture supplemented with LY294002, is effective to limit early primordial follicle depletion. Trial registration number /

scholarly journals Peroxisome Proliferator-Activated Receptor Gamma Modulator Promotes Neonatal Mouse Primordial Follicle Activation In Vitro

2020 ◽  
Vol 21 (9) ◽  
pp. 3120
Author(s):  
Sook Young Yoon ◽  
Ran Kim ◽  
Hyunmee Jang ◽  
Dong Hyuk Shin ◽  
Jin Il Lee ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Primordial Follicle ◽  
Neonatal Mouse ◽  
Peroxisome Proliferator ◽  
Follicle Growth ◽  
Peroxisome Proliferator Activated Receptor ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation

Peroxisome proliferator-activated receptor gamma (PPARγ) is known as a regulator of cellular functions, including adipogenesis and immune cell activation. The objectives of this study were to investigate the expression of PPARγ and identify the mechanism of primordial follicle activation via PPARγ modulators in mouse ovaries. We first measured the gene expression of PPARγ and determined its relationship with phosphatase and tensin homolog (PTEN), protein kinase B (AKT1), and forkhead box O3a (FOXO3a) expression in neonatal mouse ovaries. We then incubated neonatal mouse ovaries with PPARγ modulators, including rosiglitazone (a synthetic agonist of PPARγ), GW9662 (a synthetic antagonist of PPARγ), and cyclic phosphatidic acid (cPA, a physiological inhibitor of PPARγ), followed by transplantation into adult ovariectomized mice. After the maturation of the transplanted ovaries, primordial follicle growth activation, follicle growth, and embryonic development were evaluated. Finally, the delivery of live pups after embryo transfer into recipient mice was assessed. While PPARγ was expressed in ovaries from mice of all ages, its levels were significantly increased in ovaries from 20-day-old mice. In GW9662-treated ovaries in vitro, PTEN levels were decreased, AKT was activated, and FOXO3a was excluded from the nuclei of primordial follicles. After 1 month, cPA-pretreated, transplanted ovaries produced the highest numbers of oocytes and polar bodies, exhibited the most advanced embryonic development, and had the greatest blastocyst formation rate compared to the rosiglitazone- and GW9662-pretreated groups. Additionally, the successful delivery of live pups after embryo transfer into the recipient mice transplanted with cPA-pretreated ovaries was confirmed. Our study demonstrates that PPARγ participates in primordial follicle activation and development, possibly mediated in part by the PI3K/AKT signaling pathway. Although more studies are required, adapting these findings for the activation of human primordial follicles may lead to treatments for infertility that originates from poor ovarian reserves.

P–444 Presence of pharmacological inhibitors of the PI3K/PTEN/Akt and mTOR signalling pathways during cryopreservation and organotypic cultures of murine ovaries limits early primordial follicle depletion

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C Terren ◽  
M Nisolle ◽  
C Munaut
Keyword(s):  
In Vitro Culture ◽  
Organotypic Culture ◽  
Primordial Follicle ◽  
Slow Freezing ◽  
Signalling Pathways ◽  
Primordial Follicle Activation ◽  
Mtor Signalling ◽  
Follicle Activation

Abstract Study question Which signalling pathways are implicated in primordial follicle activation induced by cryopreservation and/or organotypic culture? Is it possible to limit this activation through pharmacological inhibitors? Summary answer Our findings provide support for the hypothesis that mTOR and PI3K inhibitors might represent an attractive tool to delay cryopreservation- and culture-induced primordial follicle activation. What is known already Cryopreservation of ovarian tissue containing immature primordial follicles followed by auto-transplantation (OTCTP) is the only option available to preserve the fertility of prepubertal patients or patients requiring urgent therapy for aggressive malignancies. However, a major obstacle in this process is follicular loss immediately after grafting, possibly due to slow neovascularization, apoptosis and/or massive follicular recruitment. In vitro and in vivo studies indicate that the PI3K/PTEN/Akt and mTOR signalling pathways are involved in follicle activation. The transplantation process seems to be the major cause of primordial follicle activation after OTCTP but information about how cryopreservation itself impacts follicle activation is sparse. Study design, size, duration Whole murine ovaries (4–8-weeks old) were cryopreserved by slow freezing and exposed to LY294002 (a powerful PI3K inhibitor) or rapamycin (a specific mTOR inhibitor) during cryopreservation and/or organotypic in vitro culture for a 24 h or 2 days. Participants/materials, setting, methods Western Blot and immunofluorescence analyses were used to determine the activation of PI3K/PTEN/Akt and mTOR signalling pathways in murine ovaries cryopreserved and/or organotypically cultured with/without inhibitors.Follicles were quantified according to their maturation degree on H&E stained histological sections. Main results and the role of chance Ratio of phosphorylated Akt or rps6 to total proteins (p-Akt/Akt and p-rps6/rps6) was increased in slow-frozen murine ovaries compared to control fresh ovaries, indicating an activation of the PI3K/PTEN/Akt and mTOR signalling pathways. The use of pharmacological inhibitors of follicle signalling pathways (LY294002 (25µM) and rapamycin (1µM)) during the cryopreservation process decreased p-Akt/Akt and p-rps6/rps6 ratios. In vitro organotypic culture for 24 h increased only the activation of the PI3K/PTEN/Akt pathway, as shown by increased p-Akt/Akt ratio in fresh ovaries cultured for 24 h compared to fresh non-cultured ovaries. This activation can be counteracted by cryopreservation of murine ovaries with rapamycin followed by in vitro culture for 24 h in the presence of LY294002. Follicle density quantifications indicated that when cryopreserved ovaries were maintained in culture for 2 days, a decrease of primordial follicle density concomitant with an increase of secondary and more mature follicles were found in comparison to slow-frozen/thawed ovaries without culture. Supplementation of the culture medium with LY294002 and rapamycin for 24 h or 2 days preserved primordial follicle densities compared to ovaries cultured without inhibitors. Limitations, reasons for caution This study is an in vitro study using murine ovaries. To analyze the efficiency of LY294002 and rapamycin to limit cryopreservation and transplantation induced follicle recruitment, these inhibitors should be tested in an in vivo model. Furthermore, these findings will need to be confirmed with human samples. Wider implications of the findings: We showed for the first-time that the sequential use of pharmacological inhibitors, rapamycin during the slow freezing process followed by organotypic culture supplemented with LY294002, is effective to limit early primordial follicle depletion. Trial registration number /

Differentially Expressed lncRNAs After the Activation of Primordial Follicles in Mouse

2018 ◽  
Vol 26 (8) ◽  
pp. 1094-1104
Author(s):  
Liping Zheng ◽  
Ruichen Luo ◽  
Tie Su ◽  
Liaoliao Hu ◽  
Fengxin Gao ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Expression Profiles ◽  
Ovarian Follicle ◽  
Primordial Follicle ◽  
Differentially Expressed ◽  
Ovary Development ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation

The activation of primordial follicles is critical to ovarian follicle development, which directly influences female fertility and reproductive life span. Several studies have suggested a role for long noncoding RNAs (lncRNAs) in ovarian function. However, the precise involvement of lncRNAs in the initiation of primordial follicles is still unknown. Here, an in vitro culture model was used to investigate the roles of lncRNAs in primordial follicle activation. We found that primordial follicles in day 3 mouse ovaries were activated after culturing for 8 days in vitro, as indicated by ovarian morphology changes, increases in primary follicle number, and downregulation of mammalian Sterile 20-like kinase messenger RNA (mRNA) and upregulation of growth differentiation factor 9 mRNA. We next examined lncRNA expression profiles by RNA sequencing at the transcriptome level and found that among 60 078 lncRNAs, 6541 lncRNA were upregulated and 2135 lncRNA were downregulated in 3-day ovaries cultured for 8 days in vitro compared with ovaries from day 3 mice. We also found that 4171 mRNAs were upregulated and 1795 were downregulated in the cultured ovaries. Gene ontology and pathway analyses showed that the functions of differentially expressed lncRNA targets and mRNAs were closely linked with many processes and pathways related to ovary development, including cell proliferation and differentiation, developmental processes, and other signaling transduction pathways. Additionally, many novel identified lncRNAs showed inducible expression, suggesting that these lncRNAs may be good candidates for investigating mouse primordial follicle activation. This study provides a foundation for further exploring lncRNA-related mechanisms in the initiation of mouse primordial follicles.

scholarly journals Distinct Signaling Pathways Distinguish in vivo From in vitro Growth in Murine Ovarian Follicle Activation and Maturation

2021 ◽  
Vol 9 ◽  
Author(s):  
Mahboobeh Amoushahi ◽  
Karin Lykke-Hartmann
Keyword(s):  
Ovarian Tissue ◽  
Ovarian Follicle ◽  
Primordial Follicle ◽  
Ovarian Tissue Cryopreservation ◽  
Ros Production ◽  
Primordial Follicles ◽  
Expression Of Genes ◽  
Follicle Activation

Women with cancer and low ovarian reserves face serious challenges in infertility treatment. Ovarian tissue cryopreservation is currently used for such patients to preserve fertility. One major challenge is the activation of dormant ovarian follicles, which is hampered by our limited biological understanding of molecular determinants that activate dormant follicles and help maintain healthy follicles during growth. Here, we investigated the transcriptomes of oocytes isolated from dormant (primordial) and activated (primary) follicles under in vivo and in vitro conditions. We compared the biological relevance of the initial molecular markers of mature metaphase II (MII) oocytes developed in vivo or in vitro. The expression levels of genes involved in the cell cycle, signal transduction, and Wnt signaling were highly enriched in oocytes from primary follicles and MII oocytes. Interestingly, we detected strong downregulation of the expression of genes involved in mitochondrial and reactive oxygen species (ROS) production in oocytes from primordial follicles, in contrast to oocytes from primary follicles and MII oocytes. Our results showed a dynamic pattern in mitochondrial and ROS production-related genes, emphasizing their important role(s) in primordial follicle activation and oocyte maturation. The transcriptome of MII oocytes showed a major divergence from that of oocytes of primordial and primary follicles.

scholarly journals In-vitro fragmentation of ovarian tissue activates primordial follicles through the Hippo pathway

2020 ◽  
Vol 2020 (4) ◽  
Author(s):  
C De Roo ◽  
S Lierman ◽  
K Tilleman ◽  
P De Sutter
Keyword(s):  
Tissue Culture ◽  
Ovarian Tissue ◽  
Hippo Pathway ◽  
Primordial Follicle ◽  
Akt Pathway ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
The Hippo Pathway ◽  
Follicle Activation

Abstract STUDY QUESTION What is the role of the Hippo and PI3K/Akt pathway in follicles during ovarian tissue culture in tissue derived from oncological patients and transgender men? SUMMARY ANSWER Results highlight a Hippo pathway driven primordial follicle activation in vitro, predominantly from Day 0 to Day 4. WHAT IS KNOWN ALREADY In-vitro ovarian tissue culture aims at activating and maturing primordial follicles for fertility restoration in patients with a threatened ovarian reserve. Not all patients are eligible for ovarian cortex transplantation and therefore several groups are attempting to culture ovarian tissue in-vitro. Cortex fragmentation disrupts the Hippo pathway, leading to increased expression of downstream growth factors and follicle growth. The PI3K/Akt pathway is considered the intracellular pathway to where different extracellular factors involved in primordial follicle activation in-vivo converge. In order to optimise current ovarian tissue culture models, information on progression of these pathways during tissue culture is mandatory. STUDY DESIGN, SIZE, DURATION The first step of a multistep cortex culture system was performed using 144 ovarian cortex pieces from a total of six patients. Per patient, 24 cortical strips were cultured for 6 days and six pieces per patient were collected for downstream analysis of follicle development and Hippo and PI3K/Akt pathway targets every second day. PARTICIPANTS/MATERIALS, SETTING, METHODS Ovarian tissue was obtained from oncological (N = 3; 28.67 ± 4.51 years) and transgender (N = 3; 23.33 ± 1.53 years) patients. Follicles were analysed using haematoxylin-eosin staining and pathways were studied using immunohistochemistry and precise follicle excision by laser capture micro-dissection for RT-qPCR analysis. MIQE guidelines for RT-qPCR were pursued. Reference gene selection (GAPDH, RPL3A, 18s rRNA) was performed using GeNorm Reference Gene Selection Kit. Statistical analysis was conducted with IBM SPSS Statistics 23 (Poisson regression, negative binomial regression, ANOVA and paired t-test). MAIN RESULTS AND THE ROLE OF CHANCE Immunohistochemical analysis confirmed a Hippo pathway driven primordial follicle activation due to mechanical manipulation of the cortical strips. Ovarian tissue preparation and culture induced the inhibitory phosphorylated Yes-associated protein (pYAP) to disappear in granulosa cells of primordial follicles on Day 2. The stimulatory YAP on the contrary appeared in primordial granulosa cells over increasing culture days. Looking at the YAP target connective tissue growth factor (CTGF), a significantly up-regulated CTGF was noted in primordial follicles when comparing Day 2 and Day 4 (ratio Day 2/4 = 0.082; P < 0.05), clearly showing an effect on the Hippo pathway in primordial follicles during tissue culture. Follicle classification showed a significant drop in estimated primordial follicle counts in the oncological cohort (−78%; P = 0.021) on Day 2 and in the transgender cohort on Day 4 (−634%; P = 0.008). Intermediate follicle counts showed a non-significant increasing trend to during culture and this follicle recruitment and growth resulted in a significant rise in estimated primary follicle counts on Day 6 in oncological patients (170%; P = 0.025) and, although limited in absolute numbers, a significant increase in secondary follicles on Day 4 (367%; P = 0.021) in the transgender cohort. Subsequent antral follicle development could not be observed. LIMITATIONS, REASONS FOR CAUTION A limitation is the small sample size, inherent to this study subject, especially as a large amount of tissue was needed per patient to reduce inter-patient variation in different downstream analysis techniques. A particular and specific weakness of this study is the inability to include an age-matched control group. WIDER IMPLICATIONS OF THE FINDINGS These findings support an adapted tissue preparation for Hippo pathway disruption and a shorter first phase of tissue culture. This work may also have implications for transplantation of cryopreserved tissue as larger strips (and thus slower burnout due to less Hippo pathway disruption) could be a benefit. STUDY FUNDING/COMPETING INTEREST(S) This research was financially supported by the Foundation Against Cancer (Stichting tegen Kanker, TBMT001816N), the Flemish Foundation of Scientific Research (FWO Vlaanderen, FWO G0.065.11N10) and the Gender Identity Research and Education Society (GIRES) foundation. The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 537-549 ◽  
Author(s):  
Regislane P. Ribeiro ◽  
Antonia M.L.R. Portela ◽  
Anderson W.B. Silva ◽  
José J.N. Costa ◽  
José R.S. Passos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ovarian Tissue ◽  
Follicle Stimulating Hormone ◽  
Primordial Follicle ◽  
Nuclear Antigen ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation ◽  
Stimulating Hormone

SummaryThis study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 μg/ml – Experiment 1) or in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 μg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

In-vitro regulation of primordial follicle activation: challenges for fertility preservation strategies

2018 ◽  
Vol 36 (5) ◽  
pp. 491-499 ◽  
Author(s):  
Michael J. Bertoldo ◽  
Kirsty A. Walters ◽  
William L. Ledger ◽  
Robert B. Gilchrist ◽  
Pascal Mermillod ◽  
...  
Keyword(s):  
Fertility Preservation ◽  
Primordial Follicle ◽  
Primordial Follicle Activation ◽  
Follicle Activation

scholarly journals Current Understandings of Core Pathways for the Activation of Mammalian Primordial Follicles

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1491
Author(s):  
Yu Zhao ◽  
Haiwei Feng ◽  
Yihui Zhang ◽  
Jian V. Zhang ◽  
Xiaohui Wang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Current Knowledge ◽  
Primordial Follicle ◽  
Reproductive Capacity ◽  
Molecular Networks ◽  
Female Reproduction ◽  
Primordial Follicles ◽  
Primordial Follicle Activation ◽  
Follicle Activation

The mammalian ovary has two main functions—producing mature oocytes for fertilization and secreting hormones for maintaining the ovarian endocrine functions. Both functions are vital for female reproduction. Primordial follicles are composed of flattened pre-granulosa cells and a primary oocyte, and activation of primordial follicles is the first step in follicular development and is the key factor in determining the reproductive capacity of females. The recent identification of the phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling pathway as the key controller for follicular activation has made the study of primordial follicle activation a hot research topic in the field of reproduction. This review systematically summarizes the roles of the PI3K/PTEN signaling pathway in primordial follicle activation and discusses how the pathway interacts with various other molecular networks to control follicular activation. Studies on the activation of primordial follicles have led to the development of methods for the in vitro activation of primordial follicles as a treatment for infertility in women with premature ovarian insufficiency or poor ovarian response, and these are also discussed along with some practical applications of our current knowledge of follicular activation.

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