A New Understanding, Guided by Single-Cell Sequencing, of the Establishment and Maintenance of the Ovarian Reserve in Mammals.

BACKGROUND: Oocytes are a finite and non-renewable resource that are maintained in primordial follicle structures. The ovarian reserve is the totality of primordial follicles, present from birth, within the ovary and its establishment, size, and maintenance dictates the duration of the female reproductive lifespan. Understanding the cellular and molecular dynamics relevant to the establishment and maintenance of the reserve provides the first steps necessary for modulating both individual human and animal reproductive health as well as population dynamics. SUMMARY: This review details the key stages of establishment and maintenance of the ovarian reserve, encompassing germ cell nest formation, germ cell nest breakdown, and primordial follicle formation and activation. Furthermore, we spotlight several formative single-cell sequencing studies that have significantly advanced our knowledge of novel molecular regulators of the ovarian reserve, which may improve our ability to modulate female reproductive lifespans. KEY MESSAGES: The application of single-cell sequencing to studies of ovarian development in mammals, especially when leveraging genetic and environmental models, offers significant insights into fertility and its regulation. Moreover, comparative studies looking at key stages in the development of the ovarian reserve across species has the potential to impact not just human fertility, but also conservation biology, invasive species management, and agriculture.

Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation†.

The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signaling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system, which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR. Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1, and ZFX, was investigated with in-situ immunolocalization in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signaling pathway's role in primordial follicle activation.

A scoping review of the information provided by fertility smartphone applications.

The growth of smartphone application use across areas of female reproductive health has led to increased interest into their functions and benefits. This scoping review aims to determine the nature and extent of the peer-reviewed literature presented on fertility-based apps, to identify the reliability of the information within the apps, and to determine the ability of this information to educate users. A systematic search of six databases was conducted in April 2020, returning a total of 21,158 records. After duplicate removal, title and abstract screening exclusionary steps, 27 records were reviewed and charted. Records covered a variety of reproductive health themes including contraception, sexual health, and family planning, and used a range of methodologies. The accuracy of fertility information within the apps reported in these studies was variable, but overall there was a lack of depth in the coverage of content in apps. It was common for studies in this review to base fertile window algorithms on stringent cycle length and variability requirements, limiting the applicability of information delivered to users. Furthermore, studies from app affiliates often lacked collaborations with researchers, minimising the potential for fertility knowledge improvements integrated across the suite of female reproductive health apps.

The association between reproductive health smartphone applications and fertility knowledge of Australian women.

BACKGROUND: Previous studies have identified that women living in developed countries have insufficient knowledge of factors which may be contributing to the increasingly high global infertility rates such as maternal age and assisted reproductive technologies. There is a large market of reproductive health smartphone applications, yet little is known about the advantages these apps may confer to users in regards to reproductive health knowledge. METHODS: An anonymous, online survey of women living in Australia aged 18 and above was open March-June 2018, until ≥200 responses were acquired for statistical power. Respondents answered questions regarding knowledge about general fertility and related factors (age, cyclic fertility, smoking, obesity, miscarriage rate, and success of assisted reproductive technologies). Fertility knowledge was compared in respondents who did or did not use apps relating to female reproductive health. Additionally the functions preferred in reproductive health apps was described by app using respondents. Sociodemographic information was also collected, and relevant data within the dataset was subject to multivariable modelling for the outcome of the fertility knowledge questions. RESULTS: Of the 673 respondents that completed the survey, 43.09% reported using mobile phone applications relating to female reproductive health. On average, respondents answered only three of the six fertility knowledge questions correctly. App using respondents were more likely to score better on one question, related to fertility during the menstrual cycle (p < 0.001). App users most commonly reported using the menstrual tracking function in apps (82.4%), which may account for the increased knowledge of cyclic fertility. CONCLUSIONS: This data provides preliminary evidence toward the usefulness of smartphone applications as a medium for providing information about fertility to women. A limited understanding of one's own fertility was demonstrated despite being essential for the decision-making of women throughout their reproductive years.

Advances in human primordial follicle activation and premature ovarian insufficiency.

In women, the non-growing population of follicles that comprise the ovarian reserve is determined at birth and serves as the reservoir for future fertility. This reserve of dormant, primordial follicles and the mechanisms controlling their selective activation which constitute the committing step into folliculogenesis are essential for determining fertility outcomes in women. Much of the available data on the mechanisms responsible for primordial follicle activation focuses on a selection of key molecular pathways, studied primarily in animal models, with findings often not synonymous in humans. The excessive induction of primordial follicle activation may cause the development of premature ovarian insufficiency (POI), a condition characterised by menopause before age 40 years. POI affects 1-2% of all women and is accompanied by additional health risks. Therefore, it is critical to further our understanding of primordial follicle activation in order to diagnose, treat and prevent premature infertility. Research in primordial follicle activation has focused on connecting new molecules to already established key signalling pathways, such as phosphatidylinositol 3-Kinase (PI3K) and mammalian target of rapamycin (mTOR). Additionally, other aspects of the ovarian environment, such as the function of the extracellular matrix, in contributing to primordial follicle activation have gained traction. Clinical applications are examining replication of this extracellular environment through the construction of biological matrices mimicking the 3D ovary, to support follicular growth through to ovulation. This review outlines the importance of the events leading to the establishment of the ovarian reserve and highlights the fundamental factors known to influence primordial follicle activation in humans presenting new horizons for female infertility treatment.

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

STUDY QUESTION: Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? SUMMARY ANSWER: JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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). PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING AND COMPETING INTEREST(S): 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.