Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 µM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Making a good egg: human oocyte health, aging, and in vitro development.

Mammalian eggs (oocytes) are formed during fetal life and establish associations with somatic cells to form primordial follicles that create a store of germ cells (the primordial pool). The size of this pool is influenced by key events during the formation of germ cells and by factors that influence the subsequent activation of follicle growth. These regulatory pathways must ensure that the reserve of oocytes within primordial follicles in humans lasts for up to 50 years, yet only approximately 0.1% will ever be ovulated with the rest undergoing degeneration. This review outlines the mechanisms and regulatory pathways that govern the processes of oocyte and follicle formation and later growth, within the ovarian stroma, through to ovulation with particular reference to human oocytes/follicles. In addition, the effects of aging on female reproductive capacity through changes in oocyte number and quality are emphasized, with both the cellular mechanisms and clinical implications discussed. Finally, the details of current developments in culture systems that support all stages of follicle growth to generate mature oocytes in vitro and emerging prospects for making new oocytes from stem cells are outlined.

Evidence for a fragile X messenger ribonucleoprotein 1 (FMR1) mRNA gain-of-function toxicity mechanism contributing to the pathogenesis of fragile X-associated premature ovarian insufficiency.

Fragile X-associated premature ovarian insufficiency (FXPOI) is among a family of disorders caused by expansion of a CGG trinucleotide repeat sequence located in the 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene on the X chromosome. Women with FXPOI have a depleted ovarian follicle population, resulting in amenorrhea, hypoestrogenism, and loss of fertility before the age of 40. FXPOI is caused by expansions of the CGG sequence to lengths between 55 and 200 repeats, known as a FMRI premutation, however the mechanism by which the premutation drives disease pathogenesis remains unclear. Two main hypotheses exist, which describe an mRNA toxic gain-of-function mechanism or a protein-based mechanism, where repeat-associated non-AUG (RAN) translation results in the production of an abnormal protein, called FMRpolyG. Here, we have developed an in vitro granulosa cell model of the FMR1 premutation by ectopically expressing CGG-repeat RNA and FMRpolyG protein. We show that expanded CGG-repeat RNA accumulated in intranuclear RNA structures, and these aggregates were able to cause significant granulosa cell death independent of FMRpolyG expression. Using an innovative RNA pulldown, mass spectrometry-based approach we have identified proteins that are specifically sequestered by CGG RNA aggregates in granulosa cells in vitro, and thus may be deregulated as consequence of this interaction. Furthermore, we have demonstrated reduced expression of three proteins identified via our RNA pulldown (FUS, PA2G4 and TRA2ß) in ovarian follicles in a FMR1 premutation mouse model. Collectively, these data provide evidence for the contribution of an mRNA gain-of-function mechanism to FXPOI disease biology.

Potential ovarian toxicity and infertility risk following targeted anti-cancer therapies.

Unlike traditional chemotherapy agents which are generally cytotoxic to all cells, targeted anti-cancer therapies are designed to specifically target proliferation mechanisms in cancer cells but spare normal cells, resulting in high potency and reduced toxicity. There has therefore been a rapid increase in their development and use in clinical settings, including in curative-intent treatment regimens. However, the targets of some of these drugs including kinases, epigenetic regulatory proteins, DNA damage repair enzymes and proteasomes, have fundamental roles in governing normal ovarian physiology. Inhibiting their action could have significant consequences for ovarian function, with potentially long-lasting adverse effects which persist after cessation of treatment, but there is limited evidence of their effects on reproductive function. In this review, we will use literature that examines these pathways to infer the potential toxicity of targeted anti-cancer drugs on the ovary. Lay summary: Compared to traditional chemotherapy agents, anti-cancer therapies are thought to be highly effective at targeting cancer cells but sparing normal cells, resulting in reduced drug side effects. However, many of processes within the cells that these drugs affect are also important for the ovary to work normally, so suppressing them in this way could have long-lasting implications for female fertility. This review examines the potential toxicity of anti-cancer therapies on the ovary.

The molecular mechanisms that underlie fragile X-associated premature ovarian insufficiency: is it RNA or protein based?

The FMR1 gene contains a polymorphic CGG trinucleotide sequence within its 5' untranslated region. More than 200 CGG repeats (termed a full mutation) underlie the severe neurodevelopmental condition fragile X syndrome, while repeat lengths that range between 55 and 200 (termed a premutation) result in the conditions fragile X-associated tremor/ataxia syndrome and fragile X-associated premature ovarian insufficiency (FXPOI). Premutations in FMR1 are the most common monogenic cause of premature ovarian insufficiency and are routinely tested for clinically; however, the mechanisms that contribute to the pathology are still largely unclear. As studies in this field move towards unravelling the molecular mechanisms involved in FXPOI aetiology, we review the evidence surrounding the two main theories which describe an RNA toxic gain-of-function mechanism, resulting in the loss of function of RNA-binding proteins, or a protein-based mechanism, where repeat-associated non-AUG translation leads to the formation of an abnormal polyglycine containing protein, called FMRpolyG.

Analysis of expression of candidate genes for polycystic ovary syndrome in adult and fetal human and fetal bovine ovaries†.

Polycystic ovary syndrome (PCOS) appears to have a genetic predisposition and a fetal origin. We compared the expression levels of 25 PCOS candidate genes from adult control and PCOS human ovaries (n = 16) using microarrays. Only one gene was potentially statistically different. Using qRT-PCR, expression of PCOS candidate genes was examined in bovine fetal ovaries from early stages when they first developed stroma through to completion of development (n = 27; 60-270 days of gestation). The levels of ERBB3 mRNA negatively correlated with gestational age but positively with HMGA2, FBN3, TOX3, GATA4, and DENND1A.X1,2,3,4, previously identified as correlated with each other and expressed early. PLGRKT and ZBTB16, and less so IRF1, were also correlated with AMH, FSHR, AR, INSR, and TGFB1I1, previously identified as correlated with each other and expressed late. ARL14EP, FDFT1, NEIL2, and MAPRE1 were expressed across gestation and not correlated with gestational age as shown previously for THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX, and KRR1. LHCGR, because of its unusual bimodal expression pattern, had some unusual correlations with other genes. In human ovaries (n = 15; <150 days of gestation), ERBB3.V1 and ERBB3.VS were expressed and correlated negatively with gestational age and positively with FBN3, HMGA2, DENND1A.V1,3,4, DENND1A.V1-7, GATA4, and FSHR, previously identified as correlated with each other and expressed early. Thus, the general lack of differential expression of candidate genes in adult ovaries contrasting with dynamic patterns of gene expression in fetal ovaries is consistent with a vulnerability to disturbance in the fetal ovary that may underpin development of PCOS.

Reduced retinoic acid synthesis accelerates prophase I and follicle activation.

In female mammals, reproductive potential is determined during fetal life by the formation of a non-renewable pool of primordial follicles. Initiation of meiosis is one of the defining features of germ cell differentiation and is well established to commence in response to retinoic acid. WIN 18,446 inhibits the conversion of retinol to retinoic acid, and therefore it was used to explore the impact of reduced retinoic acid synthesis on meiotic progression and thus germ cell development and subsequent primordial follicle formation. e13.5 mouse fetal ovaries were cultured in vitro and treated with WIN 18,446 for the first 3 days of a total of up to 12 days. Doses as low as 0.01 µM reduced transcript levels of the retinoic acid response genes Stra8 and Rarß without affecting germ cell number. Higher doses resulted in germ cell loss, rescued with the addition of retinoic acid. WIN 18,446 significantly accelerated the progression of prophase I; this was seen as early as 48 h post treatment using meiotic chromosome spreads and was still evident after 12 days of culture using Tra98/Msy2 immunostaining. Furthermore, ovaries treated with WIN 18,446 at e13.5 but not at P0 had a higher proportion of growing follicles compared to vehicle controls, thus showing evidence of increased follicle activation. These data therefore indicate that retinoic acid is not necessary for meiotic progression but may have a role in the regulation of its progression and germ cell survival at that time and provide evidence for a link between meiotic arrest and follicle growth initiation.

Could perturbed fetal development of the ovary contribute to the development of polycystic ovary syndrome in later life?

Polycystic ovary syndrome (PCOS) affects around 10% of young women, with adverse consequences on fertility and cardiometabolic outcomes. PCOS appears to result from a genetic predisposition interacting with developmental events during fetal or perinatal life. We hypothesised that PCOS candidate genes might be expressed in the fetal ovary when the stroma develops; mechanistically linking the genetics, fetal origins and adult ovarian phenotype of PCOS. In bovine fetal ovaries (n = 37) of 18 PCOS candidate genes only SUMO1P1 was not expressed. Three patterns of expression were observed: early gestation (FBN3, GATA4, HMGA2, TOX3, DENND1A, LHCGR and FSHB), late gestation (INSR, FSHR, and LHCGR) and throughout gestation (THADA, ERBB4, RAD50, C8H9orf3, YAP1, RAB5B, SUOX and KRR1). A splice variant of FSHB exon 3 was also detected early in the bovine ovaries, but exon 2 was not detected. Three other genes, likely to be related to the PCOS aetiology (AMH, AR and TGFB1I1), were also expressed late in gestation. Significantly within each of the three gene groups, the mRNA levels of many genes were highly correlated with each other, despite, in some instances, being expressed in different cell types. TGFß is a well-known stimulator of stromal cell replication and collagen synthesis and TGFß treatment of cultured fetal ovarian stromal cells inhibited the expression of INSR, AR, C8H9orf3 and RAD50 and stimulated the expression of TGFB1I1. In human ovaries (n = 15, < 150 days gestation) many of the same genes as in bovine (FBN3, GATA4, HMGA2, FSHR, DENND1A and LHCGR but not TOX3 or FSHB) were expressed and correlated with each other. With so many relationships between PCOS candidate genes during development of the fetal ovary, including TGFß and androgen signalling, we suggest that future studies should determine if perturbations of these genes in the fetal ovary can lead to PCOS in later life.

Dazl determines primordial follicle formation through the translational regulation of Tex14.

Deleted in azoospermia-like (DAZL) is a germ cell RNA-binding protein that is essential for entry and progression through meiosis. The phenotype of the Dazl knockout mouse has extensive germ cell loss because of incomplete meiosis. We have created a Dazl hypomorph model using short interfering RNA knockdown in mouse fetal ovary cultures, allowing investigation of Dazl function in germ cell maturation. Dazl hypomorph ovaries had a phenotype of impaired germ cell nest breakdown with a 66% reduction in total follicle number and an increase in the proportion of primordial follicles (PMFs), with smaller oocytes within these follicles. There was no significant early germ cell loss or meiotic delay. Immunostaining of intercellular bridge component testis-expressed protein (Tex)14 showed ∼59% reduction in foci number and size, without any change in Tex14 mRNA levels. TEX14 expression was also confirmed in the human fetal ovary across gestation. Using 3'UTR-luciferase reporter assays, translational regulation of TEX14 was demonstrated to be DAZL-dependant. Dazl is therefore essential for normal intercellular bridges within germ cell nests and their timely breakdown, with a major impact on subsequent assembly of PMFs.-Rosario, R., Crichton, J. H., Stewart, H. L., Childs, A. J., Adams, I. R., Anderson, R. A. Dazl determines primordial follicle formation through the translational regulation of Tex14.

MicroRNA profiling of ovarian granulosa cell tumours reveals novel diagnostic and prognostic markers.

BACKGROUND: The aim of this study was to explore the clinical utility of microRNAs (miRNAs) as improved markers of ovarian granulosa cell tumours (GCTs) for cancer diagnosis and prognosis prediction. Current histopathological and genetic markers, such as the presence of a FOXL2 gene mutation to distinguish between the two major subtypes are not wholly accurate and as such novel biomarkers are warranted. METHODS: The miRNA expression profiles of five formalin-fixed, paraffin-embedded (FFPE) adult-GCTs and five juvenile-GCTs were assessed using Affymetrix miRNA 3.0 Arrays and compared for differential expression. Ten miRNAs were assessed in an additional 33 FFPE tumours and four normal granulosa cell samples by quantitative RT-PCR, and their expression correlated to clinical information. RESULTS: MicroRNA array found 37 miRNAs as differentially expressed between the two GCT subtypes (p < 0.05, fold change ≥2 and among these, miRs -138-5p, -184, -204-5p, -29c-3p, -328-3p and -501-3p were validated by RT-qPCR as differentially expressed between the two GCT subtypes (p < 0.05). In addition, the expression of miR-184 was predictive of tumour recurrence in adult-GCTs, specifically for patients diagnosed with stage I and II and stage I only disease (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: This study is the first to report on global miRNA expression profiles of human ovarian GCTs using FFPE tumour samples. We have validated six miRNAs as novel markers for subtype classification in GCTs with low levels of miR-138-5p correlating with early tumour stage. Low miR-184 abundance was correlated with tumour recurrence in early stage adult-GCT patients as a candidate predictive biomarker. Further studies are now needed to confirm the clinical utility of these miRNAs as diagnostic and recurrence markers, and understand their possible roles in the pathogenesis of GCTs.

RNA-binding proteins in human oogenesis: Balancing differentiation and self-renewal in the female fetal germline.

Primordial germ cells undergo three significant processes on their path to becoming primary oocytes: the initiation of meiosis, the formation and breakdown of germ cell nests, and the assembly of single oocytes into primordial follicles. However at the onset of meiosis, the germ cell becomes transcriptionally silenced. Consequently translational control of pre-stored mRNAs plays a central role in coordinating gene expression throughout the remainder of oogenesis; RNA binding proteins are key to this regulation. In this review we examine the role of exemplars of such proteins, namely LIN28, DAZL, BOLL and FMRP, and highlight how their roles during germ cell development are critical to oogenesis and the establishment of the primordial follicle pool.

RNA immunoprecipitation identifies novel targets of DAZL in human foetal ovary.

Study question: Can novel meiotic RNA targets of DAZL (deleted in azoospermia-like) be identified in the human foetal ovary? Summary answer: SYCP1 (synaptonemal complex protein-1), TEX11 (testis expressed 11) and SMC1B (structural maintenance of chromosomes 1B) are novel DAZL targets in the human foetal ovary, thus DAZL may have previously unrecognised roles in the translational regulation of RNAs involved in chromosome cohesion and DNA recombination in the oocyte from the time of initiation of meiosis. What is known already: The phenotype of Dazl deficiency in mouse is infertility in both sexes and DAZL has also been linked to infertility in humans. Few studies have explored targets of this RNA-binding protein. The majority of these investigations have been carried out in mouse, and have focussed on the male thus the basis for its central function in regulating female fertility is largely unknown. Study design size, duration: We carried out RNA sequencing after immunoprecipitation of endogenous DAZL from human foetal ovarian tissue (17 weeks of gestation, obtained after elective termination of pregnancy) to identify novel DAZL targets involved in meiosis (n = 3 biological replicates). Participants/materials, setting, methods: Using quantitative RT-PCR, we examined the expression of selected RNAs identified by our immunoprecipitation across gestation, and visualised the expression of potential target SMC1B in relation to DAZL, with a combination of in situ hybridisation and immunohistochemistry. 3' untranslated region (3'UTR)-luciferase reporter assays and polysome profile analysis were used to investigate the regulation of three RNA targets by DAZL, representing key functionalities: SYCP1, TEX11 and SMC1B. Main results and the role of chance: We identified 764 potential RNA targets of DAZL in the human foetal ovary (false discovery rate 0.05 and log-fold change ≥ 2), with functions in synaptonemal complex formation (SYCP1, SYCP3), cohesin formation (SMC1B, RAD21), spindle assembly checkpoint (MAD2L1, TRIP13) and recombination and DNA repair (HORMAD1, TRIP13, TEX11, RAD18, RAD51). We demonstrated that the translation of novel targets SYCP1 (P = 0.004), TEX11 (P = 0.004) and SMC1B (P = 0.002) is stimulated by the presence of DAZL but not by a mutant DAZL with impaired RNA-binding activity. Large scale data: The raw data are available at GEO using the study ID: GSE81524. Limitations, reasons for caution: This analysis is based on identification of DAZL targets at the time when meiosis starts in the ovary: it may have other targets at other stages of oocyte development, and in the testis. Representative targets were validated, but detailed analysis was not performed on the majority of putative targets. Wider implications of the findings: These data indicate roles for DAZL in the regulation of several key functions in human oocytes. Through the translational regulation of novel RNA targets SMC1B and TEX11, DAZL may have a key role in regulating chromosome cohesion and DNA recombination; two processes fundamental in determining oocyte quality and whose establishment in foetal life may support lifelong fertility. Study funding and competing interest(s): This study was supported by the UK Medical Research Council (grant no G1100357 to R.A.A. and an intramural MRC programme grant to I.R.A.). The authors declare no competing interests.

The transcriptional responses of cultured wound cells to the excretions and secretions of medicinal Lucilia sericata larvae.

Maggots, through their excretions and secretions (ES), promote wound healing by removing necrotic tissue, counter bacterial infection, and activate wound associated cells. We investigated the effects of a physiological dose of maggot ES on four wound-associated cell types in vitro with Affymetrix gene expression arrays; keratinocytes, endothelial cells, fibroblasts, and monocytes. Keratinocytes showed the fewest (n = 5; p < 0.05, fold-change ±2) and smallest fold-changes (up to 2.32×) in gene expression and conversely THP1 monocytes had the most (n = 233) and greatest magnitude (up to 44.3×). There were no genes that were altered in all four cell-lines. Gene pathway analysis identified an enrichment of immune response pathways in three of the treated cell-lines. Analyses by quantitative RT-PCR found many genes dynamically expressed in ES dose dependent manner during the three day treatments. Phenotype analyses, however, found no effects of ES on cell viability, proliferation, migration and angiogenesis. ES was 100× less potent at triggering IL-8 secretion than fibroblasts treated with purified bacterial lipopolysaccharide (LPS; in equivalent amounts to that found in ES; ∼40 EU/ml). Furthermore, co-treatment with LPS and ES decreased the LPS-alone triggered IL-8 secretion by 13%. Although ES had no direct effect on wound cell phenotypes it did partially reduce the immune response to bacterial LPS exposure. These observations were consistent with the profile of transcriptional responses that were dominated by modulation of immune response genes. Maggot therapy may therefore improve wound healing through the secondary effects of these gene changes in the wound cells.

FMRP Associates with Cytoplasmic Granules at the Onset of Meiosis in the Human Oocyte.

Germ cell development and primordial follicle formation during fetal life is critical in establishing the pool of oocytes that subsequently determines the reproductive lifespan of women. Fragile X-associated primary ovarian insufficiency (FXPOI) is caused by inheritance of the FMR1 premutation allele and approximately 20% of women with the premutation allele develop ovarian dysfunction and premature ovarian insufficiency. However, the underlying disease mechanism remains obscure, and a potential role of FMRP in human ovarian development has not been explored. We have characterised the expression of FMR1 and FMRP in the human fetal ovary at the time of germ cell entry into meiosis through to primordial follicle formation. FMRP expression is exclusively in germ cells in the human fetal ovary. Increased FMRP expression in germ cells coincides with the loss of pluripotency-associated protein expression, and entry into meiosis is associated with FMRP granulation. In addition, we have uncovered FMRP association with components of P-bodies and stress granules, suggesting it may have a role in mRNA metabolism at the time of onset of meiosis. Therefore, this data support the hypothesis that FMRP plays a role regulating mRNAs during pivotal maturational processes in fetal germ cells, and ovarian dysfunction resulting from FMR1 premutation may have its origins during these stages of oocyte development.

Is there a role for DAZL in human female fertility?

The RNA binding protein deleted in azoospermia-like (Dazl) is a key determinant of germ cell maturation and entry into meiosis in rodents and other animal species. Although the complex phenotype of Dazl deficiency in both sexes, with defects at multiple stages of germ cell development and during meiosis, demonstrates its obligate significance in fertility in animal models, its involvement in human fertility is less clear. As an RNA binding protein, identification of the in vivo mRNA targets of DAZL is necessary to understand its influence. Thus far, only a small number of Dazl targets have been identified, which typically have pivotal roles in germ cell development and meiotic progression. However, it is likely that there are a number of additional germ cell and meiosis-relevant transcripts whose translation is affected in the absence of Dazl. Efforts to identify these RNA targets have mainly been focused on spermatogenesis, and restricted to mouse. In women, prophase I occurs in fetal life and it is during this period that the ovarian follicle pool is established, thus factors that have a role in determining the quality and quantity of the ovarian reserve may have significant impact on reproductive outcomes later in adult life. Here, we suggest that DAZL may be one such factor, and there is a need for greater understanding of the role of DAZL in human oogenesis and its contribution to lifelong female fertility.

New Zealand University students' knowledge of fertility decline in women via natural pregnancy and assisted reproductive technologies.

Female fertility declines with age. University students are the group of people most likely to postpone parenthood, yet several international studies have shown that they overestimate their fertility. We designed a questionnaire based on a previous study in Israel, where university students were asked to answer questions related to their awareness of fertility decline in spontaneous and in vitro fertilisation (IVF) pregnancies, and methods they considered would prolong their reproductive lifespan. Our study has shown that New Zealand University students overestimated the rates of pregnancy for both spontaneous natural and IVF pregnancies. Students are mainly aware of the availability of assisted reproductive technologies (ARTs), but overestimate their effectiveness. Few students mentioned non-medical or well-being initiatives as measures to prolong parenthood. It is important that university students are aware of the rate of fertility decline in women, as although ARTs can be effective at times, they are not a guaranteed solution to an ageing woman's fertility. New Zealand University students, like other cohorts, overestimated the chances of a woman getting pregnant and predicted the fertility decline to occur much later than it does in reality.

The role of FOXL2 in the pathogenesis of adult ovarian granulosa cell tumours.

OBJECTIVE: It has been four years since the discovery of the FOXL2 402C>G mutation in adult ovarian granulosa cell tumours. Yet to date, there have been few studies which have investigated the precise role of the mutation in tumour pathogenesis. This review aims to summarise the research in this area, proposes a mechanism of action for the mutation, and explores the implications for clinical practice and future therapeutics. METHODS: A literature search was performed with the keywords 'granulosa cell tumour' and 'FOXL2' on PubMed. RESULTS: Although the search returned 52 articles, of these only nine publications investigate the pathogenic effect of the mutant FOXL2 allele. Mutant FOXL2 maintains some of the transcriptional activity of the wildtype allele, but there is a subtle alteration of the expression in a unique suite of cancer-related genes. The mutation appears to deregulate the anti-proliferative transforming growth factor beta (TGF-ß) pathway and this may contribute to the pathogenesis of adult GCTs. The inability of mutant FOXL2 to elicit an effective apoptotic signalling cascade may also be important in GCT pathogenesis. CONCLUSION: The 402C>G mutation in FOXL2 is central to the development of adult granulosa cell tumours. Based on the evidence, we suggest that FOXL2 is an oncogene or tumour suppressor depending on the genetic context that is the GCT subtype.

Adult granulosa cell tumours (GCT): clinicopathological outcomes including FOXL2 mutational status and expression.

OBJECTIVES: The aim of this research was to use nucleic acids isolated from formalin-fixed paraffin-embedded (FFPE) tissue to investigate the diagnostic potential and prognostic significance of FOXL2 in adult-type GCTs, particularly as a marker of identifying early stage patients that are likely to relapse. METHODS: We performed a retrospective review of GCT patients referred to the Auckland Gynae-Oncology Multidisciplinary Team from 1955 to 2012. Baseline characteristics, clinical course, histopathology and survival data was recorded. Using nucleic acids extracted from FFPE tumour blocks, FOXL2 mutation status and expression was determined by DNA sequencing and RT-qPCR, respectively, and correlated with clinical data. RESULTS: 57 adult GCT patients were identified, however FFPE tumour blocks were available for only 37 of these patients. Sequencing results confirmed the presence of the FOXL2 mutation in 70% of patients. FOXL2 mutation positive adult tumours showed a trend towards higher FOXL2 expression than wildtype adult tumours, particularly in stage I patients (p=0.051). In addition, patients with homozygous FOXL2 mutations had a significantly higher relapse rate (p=0.04). There was no significant correlation between FOXL2 mutation status or FOXL2 expression and any other clinical variables. CONCLUSIONS: FFPE tumour blocks are a valuable resource of molecular information, especially when studying rare tumours such as GCTs. The FOXL2 mutation appears to have some diagnostic potential, however additional work in a larger cohort needs to be completed to confirm the prognostic significance of this gene mutation, and its expression.

Comparative study of microRNA regulation on FOXL2 between adult-type and juvenile-type granulosa cell tumours in vitro.

OBJECTIVES: Despite their distinct biology, granulosa cell tumours (GCTs) are treated similarly to other ovarian tumours. Predominantly expressed in granulosa cells, the transcription factor Forkhead Box L2 (FOXL2) is near absent in juvenile-type GCTs. This research aimed to investigate miRNAs as a mechanism of suppression of FOXL2 expression in juvenile-type GCTs. METHODS: The miRNA abundance of two GCT cell lines COV434 and KGN was profiled using Affymetrix miRNA GeneChip arrays. Luciferase assays were used to confirm miRNA binding to the 3'UTR of FOXL2. Identified as promising candidates, the miR-17 miRNA family was targeted for knockdown with a miRNA sponge. Additionally, individual family members miR-17, miR-20b and miR-106a were knocked down using Anti-miR™ inhibitors. Subsequently, FOXL2 expression was analysed using RT-qPCR and Western blotting. RESULTS: The profiling of COV434 and KGN cells revealed unique miRNA signatures, with COV434 expressing miR-17 family miRNAs whilst KGN expressed members of the let-7 miRNA gene family. Luciferase assays confirmed miRNA binding to FOXL2's 3'UTR. Reduction of miR-17 family miRNAs increased FOXL2 mRNA expression, however luciferase assays performed in combination with the sponge suggested this is an indirect effect. As no changes in protein were observed, we propose another miRNA is repressing the translation of FOXL2 mRNA. CONCLUSION: Through miRNA profiling we have begun to unravel the profiles of GCTs, showing that juvenile and adult derived-cell lines are biologically distinct. By expanding on this discovery we may further elucidate the miRNA-mRNA pathways involved in GCT initiation and progression with potential for novel therapeutics for these cancers.