Human Ovarian Follicles Research Articles

Proteome of fluid from human ovarian small antral follicles reveals insights in folliculogenesis and oocyte maturation.

STUDY QUESTIONIs it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)?SUMMARY ANSWERThe largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes.WHAT IS KNOWN ALREADYProtein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis.STUDY DESIGN, SIZE, DURATIONProteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed.PARTICIPANTS/MATERIALS, SETTING, METHODSWe collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation.LIMITATIONS, REASONS FOR CAUTIONA possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons.WIDER IMPLICATIONS OF THE FINDINGSThis study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation.STUDY FUNDING/COMPETING INTEREST(S)The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors.TRIAL REGISTRATION NUMBERN/A

Identification of a unique epigenetic profile in women with diminished ovarian reserve

To investigate whether epigenetic profiles of mural granulosa cells (MGC) and leukocytes from women with diminished ovarian reserve (DOR) differ from those of women with normal or high ovarian reserve. Prospectively collected material from a multicenter cohort of women undergoing fertility treatment. Private and university-based facilities for clinical services and research. One hundred and nineteen women of various ages and ovarian reserve status (antimüllerian hormone level) who provided blood samples and MGC. None. Measures of epigenetic aging rates from whole-genome methylation array data: DNA methylation variability, age acceleration, DNA methylation telomere length estimator (DNAmTL), and accumulation of epimutations. Comparison of DOR or high ovarian reserve samples to controls (normal ovarian reserve) showed differential methylation variability between DOR and normal samples at 4,199 CpGs in MGC, and 447 between high and normal (false-discovery rate < 0.05). Variable sites in MGC from DOR were enriched in regions marked with the repressive histone modification H3K27me3, and also included genes involved in folliculogenesis, such as insulin growth factor 2 (IGF2) and antimüllerian hormone (AMH). Regardless of ovarian reserve, very few signals were detected in leukocytes, and no overlaps with those in MGC were found. Furthermore, we found a higher number of epimutations in MGC from women with DOR (Kruskal-Wallis test, difference in mean = 3,485). The somatic cells of human ovarian follicles have a distinctive epigenetic profile in women with DOR. A high frequency of epimutations suggests premature aging. Ovarian reserve status was not reflected in the leukocyte epigenetic profile.

Impairment of Pol β-related DNA base-excision repair leads to ovarian aging in mice

The mechanism underlying the association between age and depletion of the human ovarian follicle reserves remains uncertain. Many identified that impaired DNA polymerase β (Pol β)-mediated DNA base-excision repair (BER) drives to mouse oocyte aging. With aging, DNA lesions accumulate in primordial follicles. However, the expression of most DNA BER genes, including APE1, OGG1, XRCC1, Ligase I, Ligase α, PCNA and FEN1, remains unchanged during aging in mouse oocytes. Also, the reproductive capacity of Pol β+/- heterozygote mice was impaired, and the primordial follicle counts were lower than that of wild type (wt) mice. The DNA lesions of heterozygous mice increased. Moreover, the Pol β knockdown leads to increased DNA damage in oocytes and decreased survival rate of oocytes. Oocytes over-expressing Pol β showed that the vitality of senescent cells enhances significantly. Furthermore, serum concentrations of anti-Müllerian hormone (AMH) indicated that the ovarian reserves of young mice with Pol β germline mutations were lower than those in wt. These data show that Pol β-related DNA BER efficiency is a major factor governing oocyte aging in mice.

Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival.

SummaryClassically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Ovarian tissue cryopreservation and novel bioengineering approaches for fertility preservation.

Breast cancer patients who cannot delay treatment or for whom hormone stimulation and egg retrieval are contraindicated require alternative methods of fertility preservation prior to gonadotoxic treatment. Ovarian tissue cryopreservation is an alternative approach that may offer patients the opportunity to preserve fertility and carry biologically-related children later in life. Various experimental approaches are being explored to obtain mature gametes from cryopreserved and thawed ovarian tissue for fertilization and implantation using biomimetic tissue culture in vitro. Here we review the most recent developments in ovarian tissue cryopreservation and exciting advances in bioengineering approaches to in vitro tissue and ovarian follicle culture. Slow freezing is the most widely accepted method for ovarian tissue cryopreservation, but efforts have been made to modify vitrification for this application as well. Numerous approaches to in vitro tissue and follicle culture are in development, most prominently two-step culture systems for ovarian cortical tissue and encapsulation of ovarian follicles in biomimetic matrices for in vitro culture. Refinements to slow freeze and vitrification protocols continue to address challenges associated with cryopreservation, such as ice crystal formation and damage to the stroma. Similarly, improvements to in vitro tissue and follicle culture show promise for utilizing patients' cryopreserved tissues to obtain mature gametes after disease treatment and remission. Development of an effective and reproducible culture system for human ovarian follicles will serve as a broad assisted reproductive technology for cancer survivors who cryopreserved tissue prior to treatment.

In vitro Culture of Early Secondary Preantral Follicles to Obtain MII Oocyte Developmental Competence from CD-1 Outbred Mice

Background: The ovarian follicle is the fundamental functional tissue unit that consists of mammalian ovary. In humans, it has been known that females are born with a maximum number of follicles or oocytes that are not only non-renewable, but also undergoing degeneration with time with a sharply decreased oocyte quality after the age of 35. Methods: Here, we demonstrate that successful isolation of primary, early secondary and late secondary follicles from the ovaries of CD-1 outbred female mice and in vitro culture system to successfully induce the development of MII oocytes. Result: The 9 days of in vitro culture of early secondary follicles showed significant higher rates in growth and maturation displaying higher numbers of antral follicles and MII oocytes developed from early secondary follicles compared to those cultured for 11 days. However, there was no visible difference induced by the size of initial follicles in the rates of growth and maturation. MII oocytes derived from in vitro culture of early secondary follicles following in vitro fertilization developed into two-cell embryos. These observations demonstrate that developmentally competent MII oocytes can be obtained by in vitro culture of preantral follicles derived from the ovaries of CD-1 mice and reveal a crucial role for CD-1 mice as a novel model for research on human ovarian follicles. Furthermore, this study proposes an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation of humans for assisted reproductive medicine.

Maternally contributed Nlrp9b expressed in human and mouse ovarian follicles contributes to early murine preimplantation development.

The aim of the study is to investigate presence and role of the gene encoding the maternally contributed nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD)-containing protein 9 (NLRP9) in human and mouse ovaries, respectively, and in preimplantation mouse embryo development by knocking down Nlrp9b. Expression levels of NLRP9 mRNA in human follicles were extracted from RNA sequencing data from previous studies. In this study, we performed a qPCR analysis of Nlpr9b mRNA in mouse oocytes and found it present. Intracellular ovarian distribution of NLRP9B protein was accomplished using immunohistochemistry. The distribution of NLRP9B was explored using a reporter gene approach, fusing NLRP9B to green fluorescent protein and microinjection of in vitro-generated mRNA. Nlrp9b mRNA function was knocked down by microinjection of short interference (si) RNA targeting Nlrp9b, into mouse pronuclear zygotes. Knockdown of the Nlrp9b mRNA transcript was confirmed by qPCR. We found that the human NLRP9 gene and its corresponding protein are highly expressed in human primordial and primary follicles. The NLRP9B protein is localized to the cytoplasm in the blastomeres of a 2-cell embryo in mice. SiRNA-mediated knockdown of Nlrp9b caused rapid elimination of endogenous Nlrp9b mRNA and premature embryo arrest at the 2- to 4-cell stages compared with that of the siRNA-scrambled control group. These results suggest that mouse Nlrp9b, as a maternal effect gene, could contribute to mouse preimplantation embryo development. It remains to investigate whether NLRP9 have a crucial role in human preimplantation embryo and infertility.

Roles of insulin-like growth factor II in regulating female reproductive physiology.

The number of growth factors involved in female fertility has been extensively studied, but reluctance to add essential growth factors in culture media has limited progress in optimizing embryonic growth and implantation outcomes, a situation that has ultimately led to reduced pregnancy outcomes. Insulin-like growth factor II (IGF-II) is the most intricately regulated of all known reproduction-related growth factors characterized to date, and is perhaps the predominant growth factor in human ovarian follicles. This review aims to concisely summarize what is known about the role of IGF-II in follicular development, oocyte maturation, embryonic development, implantation success, placentation, fetal growth, and in reducing placental cell apoptosis, as well as present strategies that use growth factors in culture systems to improve the developmental potential of oocytes and embryos in different species. Synthesizing the present knowledge about the physiological roles of IGF-II in follicular development, oocyte maturation, and early embryonic development should, on the one hand, deepen our overall understanding of the potential beneficial effects of growth factors in female reproduction and on the other hand support development (optimization) of improved outcomes for assisted reproductive technologies.

Elucidating Decorin\u2019s role in the preovulatory follicle

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

A common variant of the pregnancy-associated plasma protein-A (PAPPA) gene encodes a protein with reduced proteolytic activity towards IGF-binding proteins

Pregnancy-associated plasma protein-A (PAPP-A) is a key regulator of insulin-like growth factor (IGF) bioactivity, by releasing the IGFs from their corresponding IGF-binding proteins (IGFBPs). The minor allele of the single nucleotide polymorphism (SNP), rs7020782 (serine < tyrosine), in PAPPA has previously been associated with recurrent pregnancy loss as well as with significant reduced levels of PAPP-A protein in human ovarian follicles. The aim of the present study was to reveal a possible functional effect of the rs7020782 SNP in PAPPA by comparing recombinant PAPP-A proteins from transfected human embryonic kidney 293 T cells. The proteolytic cleavage of IGFBP-4 was shown to be affected by the rs7020782 SNP in PAPPA, showing a significantly reduced cleavage rate for the serine variant compared to the tyrosine variant (p-value < 0.001). The serine variant also showed a trend towards reduced cleavage rates, that was not significant, towards IGFBP-2 and IGFBP-5 compared to the tyrosine variant. No differences were found when analysing cell surface binding, complex formation between PAPP-A and STC2 or proMBP, nor when analysing STC1 inhibition of PAPP-A-mediated IGFBP-4 cleavage. Regulation of IGF bioactivity in reproductive tissues is important and the rs7020782 SNP in PAPPA may disturb this regulation by altering the specific activity of PAPP-A.

Effect of the neuropeptide phoenixin and its receptor GPR173 during folliculogenesis.

Folliculogenesis is a complex process, defined by the growth and development of follicles from the primordial population. Granulosa cells (GCs) play a vital role in every stage of follicular growth through proliferation, acquisition of gonadotropic responsiveness, steroidogenesis and production of autocrine/paracrine factors. A recently discovered hypothalamic neuropeptide phoenixin is involved in the regulation of the reproductive system. Phoenixin acts through its receptor, G protein-coupled receptor 173 (GPR173), to activate the cAMP/PKA pathway leading to the phosphorylation of CREB (pCREB). Here, we demonstrated the expression patterns of phoenixin and GPR173 in human ovary and explored its role in folliculogenesis. Phoenixin and GPR173 were both expressed in the human ovarian follicle, with increased expression in GCs as the follicle grows. Phoenixin treatment at 100 nM for 24 h induced the proliferation of human non-luteinized granulosa cell line, HGrC1 and significantly increased the expression levels of CYP19A1, FSHR, LHR and KITL, but decreased NPPC expression levels. These effects were suppressed by GPR173 siRNA. The expression level of CREB1, pCREB and estradiol (E2) production in the culture medium was significantly enhanced by phoenixin treatment in a concentration-dependent manner. Phoenixin also significantly increased the follicular area in a murine ovarian tissue culture model, leading to an increased number of ovulated oocytes with a higher level of maturation. Taken together, our data demonstrate that phoenixin is an intraovarian factor that promotes follicular growth through its receptor GPR173 by accelerating proliferation of GCs, inducing E2 production and increasing the expression of genes related to follicle development.

C-type natriuretic peptide signaling in human follicular environment and its relation with oocyte maturation

Studies in mice have shown that C-type natriuretic peptide (CNP) is produced by granulosa cells and contributes to ovarian follicle growth and oocyte meiotic arrest until the preovulatory LH surge. In humans, the relationship between intraovarian CNP levels and oocyte meiotic resumption is unknown. The aim of this study was to investigate whether CNP and its receptor NPR2 are expressed in human ovarian follicles and if their levels change according to the meiotic phase of oocytes. We collected follicular fluid (FF) and luteinized granulosa cells (LGC) from follicle pools (n = 47), and FF, LGC and cumulus cells (CC) from individual follicles (n = 96) during oocyte pickup for in vitro fertilization. There was a positive linear correlation between CNP levels in FF pools and basal antral follicle counting (rs = 0.458; p = 0.002), number of preovulatory follicles >16 mm (rs = 0.361; p = 0.016) and number of oocytes retrieved (rs = 0,378; p = 0.011) and a negative correlation between CNP levels in FF pools and the percentage of mature (MII) oocytes retrieved (rs = −0.39; p = 0.033). FF CNP levels in follicles containing MII oocytes were significantly lower than in follicles containing immature (MI) oocytes (median = 0.44 vs. 0.57 ng/mL, p < 0.05). Accordingly, the CNP precursor gene NPPC was 50% less expressed in LGC from follicles containing MII oocytes than in follicles containing MI oocytes (p < 0.01). In addition, NPR2 mRNA was down-regulated in CC surrounding MII oocytes (60% reduction, p < 0.01). CNP signaling is downregulated in human ovarian follicles containing mature oocytes. Further studies should clarify whether CNP signaling is essential to keep oocyte meiotic arrest in humans.

Future developments: In vitro growth (IVG) of human ovarian follicles

Removal and storage of ovarian cortical tissue is currently offered to young female cancer patients undergoing potentially sterilizing chemotherapy and/or radiotherapy. For patients at high risk of reintroduction of malignancy through auto-transplantation, the ultimate aim is to achieve complete oocyte development from this tissue in vitro. The ability to develop human oocytes from the earliest follicular stages through to maturation and fertilization in vitro would revolutionize fertility preservation practice. This has been achieved in mice where in vitro grown oocytes from primordial follicles have resulted in the production of live offspring. Systems that support growth and development of oocytes from human ovarian cortex are being developed by several groups. This review focuses on the steps required to recapitulate in vitro the process of human oocyte development from the primordial stage and the systems currently available to support this.

Transcription profile of the insulin-like growth factor signaling pathway during human ovarian follicular development.

The IGF signaling cascade exerts important regulatory functions in human ovarian folliculogenesis. The scope of this study was to evaluate the transcription profile of insulin-like growth factor (IGF) genes during human ovarian follicle development and to analyze follicle fluid levels of key IGF proteins. Gene expression profiling was performed with microarray gene analysis. The analysis was assessed from ovarian follicles and granulosa cells (GCs) obtained from isolated stage-specific human ovarian follicles, including preantral follicles, small antral follicles, and preovulatory follicles. Numerous genes involved in the IGF signaling pathway was evaluated and key genes were validated by qPCR from GCs. Protein levels of various IGF components of human follicular fluid (FF) were measured by ELISA and time-resolved immunofluorometric assays (TRIFMA). The gene expression levels of PAPPA, IGF2, IGF receptors and intracellular IGF-activated genes increased with increasing follicle size. This was especially prominent in the late preovulatory stage where IGF2 expression peaked. Protein levels of intact IGF binding protein-4 decreased significantly in FF from large preovulatory follicles compared with small antral follicles concomitant with higher protein levels of PAPP-A. The IGF modulators IGF-2 receptor, IGFBPs, stanniocalcins, and IGF-2 mRNA binding proteins were all observed to be expressed in the different follicle stages. This study confirms and highlights the importance of PAPP-A regulating bioactive IGF levels throughout folliculogenesis and especially for the high rate of granulosa cell proliferation and expression of key ovarian hormones important in the last part of the follicular phase of the menstrual cycle.

The NADPH oxidase 4 is a major source of hydrogen peroxide in human granulosa-lutein and granulosa tumor cells

H2O2 is a reactive oxygen species (ROS), which can diffuse away from its site of generation and may act as a cell-to-cell signaling factor. The mechanisms responsible for the generation of H2O2 in human ovarian follicles and possible signaling role(s) of H2O2 are not well known. We identified a source of H2O2, the enzyme NADPH oxidase (NOX) 4, in isolated differentiated, in-vitro fertilisation-derived human granulosa-lutein cells (GCs), in proliferating human granulosa tumour cells (KGN), as well as in situ in cells of growing ovarian follicles. H2O2 was readily detected in the supernatant of cultured GCs and KGN cells. H2O2 levels were significantly lowered by the NOX4 blocker GKT137831, indicating a pronounced contribution of NOX4 to overall H2O2 generation by these cells. We provide evidence that extracellular H2O2 is taken up by GCs, which is facilitated by aquaporins (peroxiporins). We thus conclude that GC-derived H2O2 might act as autocrine/paracrine factor. Addition of H2O2 increased MAPK-phosphorylation in GCs. Moreover, reducing H2O2 production with GKT137831 slowed proliferation of KGN cells. Our results pinpoint NOX4 and H2O2 as physiological players in the regulation of GC functions.

Steroids and miRNAs in assessment of ovarian tissue damage following cryopreservation.

Ovarian cortical tissue cryopreservation is a relatively novel approach to preserving fertility in women diagnosed with cancer. However, the effects of freezing-thawing are not fully understood, mainly due to the lack of suitable methods to assess tissue's survival after thawing. Disparities in steroid production have been associated with ovarian failure by disrupting folliculogenesis, ovulation and oocyte apoptosis. Moreover, specific microRNAs, identified in human ovarian follicles, are thought to play a fundamental role in folliculogenesis. In this study, we investigated the possible interplay between the ovarian steroidal production and microRNA expression patterns in spent culture media, as potential non-invasive markers for ovarian tissue damage after cryopreservation. Cryopreservation of ovarian cortical tissue decreased (P<0.05) both steroid production (oestradiol and progesterone) and expression of microRNA-193b and 320A in spent culture media over 5 days, however, expression of microRNA-24 increased (P<0.05). The number of primordial follicles were also reduced (P<0.05) in fresh-cultured and cryopreserved-cultured cortical tissues when compared with fresh tissues. Downregulation of microRNA-193b and microRNA-320A together with upregulation of microRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production. Thus, there appears to be an interplay between these microRNAs, ovarian steroid production and cell damage, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.

Physiological roles of activins in the human ovary

Abstract Initially discovered in the pituitary as stimulators of follicle stimulating hormone, activins are homo- or heterodimers of inhibin subunits, which belong to the transforming growth factor-β superfamily. Subsequent studies have demonstrated that these growth factors play multifaceted roles in regulating various functions in multiple organs, including the ovary. The spatial and temporal expression of inhibin subunits (α, βA, βB, and βC), their cognate receptors, and activin-binding proteins (inhibins and follistatins) in the principal cells of growing follicles in human ovaries indicates that these activin isoforms are involved in ovarian biology. Information collected from animal studies and clinical samples suggests that these locally produced growth factors are crucial modulators of various ovarian functions, including primordial germ cell development, follicular growth and development, ovarian steroidogenesis, extracellular matrix remodeling, oocyte maturation, ovulation, and luteal function. Along with gonadotropins, intrafollicular activins exert synergistic and complementary effects on growing follicles to help them develop a mature, competent oocyte that is prepared for fertilization. Abnormal activin expression, an imbalanced activin/follistatin ratio, and the dysregulation of the activin signaling pathway have been observed in several ovarian pathologies, such as reproductive aging, polycystic ovary syndrome, and ovarian cancers. Recent advancements in our understanding of the molecular interactions and mechanisms that underlie activins and the development of related ovarian abnormalities have provided insights into disease pathogenesis and increased opportunities to achieve efficient and safe therapies.

Concentration of stromal cell-derived factor-1 (SDF-1/CXCL12) in the follicular fluid is associated with blastocyst development.

PurposeTo study the association between stromal cell‐derived factor‐1 (SDF‐1/CXCL12) and vascular endothelial growth factor (VEGF) concentrations in individual human ovarian follicles and IVF outcomes.MethodsConcentrations of SDF‐1 and VEGF in 261 follicular fluid samples were measured with enzyme‐linked immunosorbent assay. IVF outcome parameters were included in fertilization rate, cleavage rate, embryo morphology on day 3, and blastocyst morphology on day 5.ResultsThe follicular concentration of SDF‐1 and VEGF was not significantly associated with fertilization and cleavage outcome, and embryo morphology. The rates of full blastocysts and good‐quality blastocysts were significantly higher in follicles with an SDF‐1 concentration of 275‐350 pg/mL than in the follicles with SDF‐1 concentrations of <200 and ≥350 pg/mL (P < 0.05). The follicular concentration of VEGF was not associated with the blastocyst morphology.ConclusionOur findings showed that follicular concentration of SDF‐1, and not VEGF, may be a valuable biochemical marker of blastocyst development.

In Vitro Growth of Human Ovarian Follicles for Fertility Preservation

Young female cancer survival rates significantly increased due to the great progress of cancer therapy. In fact, cryostorage and transplantation of ovarian tissue have already resulted in the birth of healthy babies. Follicle in vitro growth (IVG) has the great potential of restoring fertility by achieving functional oocytes from the most immature stages to maturation. This is suitable for a wide range of patients, from pubertal to perimenopause women. Notable achievements have been achieved in human follicle IVG in the past decade. Mature oocytes have been successfully collected from long-term sequential follicle IVG. However, it is still a major challenge to establish a stable and efficient follicle IVG system able to generate mature and competent oocytes. Hereby, we review the approaches being taken so far using ovarian tissue to support follicle growth at different stages in vitro.

Janus kinase JAK1 maintains the ovarian reserve of primordial follicles in the mouse ovary

Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. A series of integrated, intrinsic signalling pathways (including PI3K/AKT, mTOR and KITL) are responsible for regulating the ovarian reserve of non-growing primordial follicles and ultimately female fertility. The JAK-STAT signal transduction pathway is highly conserved with established roles in cell division and differentiation. Key pathway members (specifically JAK1, STAT3 and SOCS4) have been previously implicated in early follicle development. A laboratory animal study was undertaken using the C57Bl/6 inbred mouse strain as a model for human ovarian follicle development. To determine which Jak genes were most abundantly expressed during primordial follicle activation, mRNA expression was analysed across a developmental time-course, with ovaries collected from female mice at post-natal days 1 (PND1), 4 (PND4), 8 (PND8), as well as at 6 weeks (6WK) and 7 months (7MTH) (n ≥ 4). Functional analysis of JAK1 was performed on PND2 mouse ovaries subjected to in vitro explant culture treated with 12.5 μM Ruxolitinib (JAK inhibitor) or vehicle control (DMSO) for 48 h prior to histological assessment (n ≥ 4). The expression and localization of the JAK family during ovarian follicle development in the C57Bl/6 inbred mouse strain were evaluated using quantitative PCR, immunoblotting and immunolocalisation. Functional studies were undertaken using the JAK inhibitor Ruxolitinib to investigate the underpinning cellular mechanisms via biochemical in vitro inhibition and histological assessment of intact neonate ovaries. All experiments were replicated at least three times using tissue from different mice unless otherwise stated. Jak1 is the predominant Jak mRNA expressed in the C57Bl/6 mouse ovary across all developmental time-points assessed (P ≤ 0.05). Forty-eight hour inhibition of JAK1 with Ruxolitinib of PND2 ovaries in vitro demonstrated concomitant acceleration of primordial follicle activation and apoptosis (P ≤ 0.001) and upregulation of downstream JAK-STAT pathway members STAT3 and suppressors of cytokine signalling 4 (SOCS4). N/A. Results are shown in one species, the C57Bl/6 mouse strain as an established model of human ovary development. Ruxolitinib also inhibits JAK2, with decreased efficacy. However, Jak2 mRNA had limited expression in the mouse ovary, particularly at the neonatal stages of follicle development, thus any effect of Ruxolitinib on primordial follicle activation was unlikely to be mediated via this isoform. This study supports a key role for JAK1 in the maintenance and activation of primordial follicles, with potential for targeting the JAK-STAT pathway as a method of regulating the ovarian reserve and female fertility. This project has been funded by the Australian National Health and Medical Research Council (G1600095) and The Hunter Medical Research Institute Bob and Terry Kennedy Children's Research Project Grant in Pregnancy & Reproduction (G1501433). All authors declare no conflict of interests.