Cheryl E. Dunlop

Ovarian stem cells—Potential roles in infertility treatment and fertility preservation

One of the principal beliefs in reproductive biology is that women have a finite ovarian reserve, which is fixed from the time they are born. This theory has been questioned recently by the discovery of ovarian stem cells which are purported to have the ability to form new oocytes under specific conditions post-natally. Almost a decade after their discovery, ovarian, or oogonial, stem cells (OSCs) have been isolated in mice and humans but remain the subject of much debate. Studies in mice have shown that these cells can be cultured to a mature oocyte stage in vitro, and when injected into germ-cell depleted ovary they can form follicles and have resulted in the birth of healthy offspring. There are few data from human OSCs but this finding would open the door to novel fertility preservation strategies for women with both age-related and premature ovarian insufficiency (POI). As the number of girls and young women surviving cancer increases worldwide, POI secondary to gonadotoxic treatments, such as chemotherapy, is becoming more common. The ideal fertility preservation approach would prevent delays in commencing life-saving treatment and avoid transplanting malignant cells back into a woman after treatment: OSCs may offer one route to achieving this. This review summarises our current understanding of OSCs and discusses their potential clinical application in infertility treatment and fertility preservation.

Prospective study into the value of the automated Elecsys antimüllerian hormone assay for the assessment of the ovarian growing follicle pool

OBJECTIVE To evaluate a new fully automated assay measuring antimüllerian hormone (AMH; Roche Elecsys) against antral follicle count in women of reproductive age. DESIGN Prospective cohort study. SETTING Hospital infertility clinics and academic centers. PATIENT(S) Four hundred fifty-one women aged 18 to 44 years, with regular menstrual cycles. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) AMH and antral follicle count (AFC) determined at a single visit on day 2-4 of the menstrual cycle. RESULT(S) There was a statistically significant variance in AFC but not in AMH between centers. Both AFC and AMH varied by age (overall Spearman rho -0.50 for AFC and -0.47 for AMH), but there was also significant between-center variation in the relationship between AFC and age but not for AMH. There was a strong positive correlation between AMH and AFC (overall spearman rho 0.68), which varied from 0.49 to 0.87 between centers. An agreement table using AFC cutoffs of 7 and 15 showed classification agreement in 63.2%, 56.9% and 74.5% of women for low, medium, and high groups, respectively. CONCLUSION(S) The novel fully automated Elecsys AMH assay shows good correlations with age and AFC in women of reproductive age, providing a reproducible measure of the growing follicle pool.

Uses of anti-Müllerian hormone (AMH) measurement before and after cancer treatment in women

There is a growing body of evidence supporting the use of anti-Müllerian hormone (AMH) serum levels as a biomarker of the growing follicle pool, in turn taken to reflect the ovarian reserve, in patients being treated for cancer. Many cancer therapies are gonadotoxic, often inducing premature ovarian insufficiency (POI) and thus rendering such patients infertile. The degree of ovariotoxicity is related to the type of treatment, dosage and patients age. As survival rates from cancer improve, post-treatment reproductive health is becoming increasingly important to affected girls and women of reproductive age to allow them to have biologically-related children. AMH levels taken post-treatment may be able to guide advice regarding remaining reproductive lifespan and aid decision-making on suitable adjuvant hormonal treatments such as in hormone receptor-positive breast cancer. Furthermore, pre-treatment AMH levels are now shown to be predictive of long-term ovarian function. The development of prognostic scoring and classification methods including the use of pre-treatment AMH, as well as other patient factors including age, to determine the likelihood of return of menses may allow better individualisation of advice regarding the use of fertility preservation strategies prior to commencing cancer treatment. AMH may also be a useful marker of cancer therapy-related ovarian damage in pre-pubertal children, although there are very limited data on the relationship between AMH and the ovarian reserve in children and adolescents. AMH is proving to be of increasing value in assessing ovarian function and advising patients before and after cancer treatment.

Ovarian germline stem cells.

It has long been established that germline stem cells (GSCs) are responsible for lifelong gametogenesis in males, and some female invertebrates (for example, Drosophila) and lower vertebrates (for example, teleost fish and some prosimians) also appear to rely on GSCs to replenish their oocyte reserve in adulthood. However, the presence of such cells in the majority of female mammals is controversial, and the idea of a fixed ovarian reserve determined at birth is the prevailing belief among reproductive biologists. However, accumulating evidence demonstrates the isolation and culture of putative GSCs from the ovaries of adult mice and humans. Live offspring have been reportedly produced from the culture of adult mouse GSCs, and human GSCs formed primordial follicles using a mouse xenograft model. If GSCs were present in adult female ovaries, it could be postulated that the occurrence of menopause is not due to the exhaustion of a fixed supply of oocytes but instead is a result of GSC and somatic cell aging. Alternatively, they may be benign under normal physiological conditions. If their existence were confirmed, female GSCs could have many potential applications in both basic science and clinical therapies. GSCs not only may provide a valuable model for germ cell development and maturation but may have a role in the field of fertility preservation, with women potentially being able to store GSCs or GSC-derived oocytes from their own ovaries prior to infertility-inducing treatments. Essential future work in this field will include further independent corroboration of the existence of GSCs in female mammals and the demonstration of the production of mature competent oocytes from GSCs cultured entirely in vitro.

The controversial existence and functional potential of oogonial stem cells

The regenerative potential of the mammalian ovary has been a controversial area over the last decade. Isolation of cells, termed oogonial stem cells (OSCs), from adult rodent and human ovaries has been reported, with these cells exhibiting both germ and stem cell markers in culture. When re-introduced into an ovarian somatic environment these cells have generated follicles capable of producing healthy offspring in rodents, and there is some evidence of human OSCs being able to form oocyte-like structures in a xenotransplant model. Importantly, there are no data on their potential physiological role within the ovary, and specifically no evidence that they contribute to the primordial follicle pool and thus to later stages of follicle development. The cues required for oocyte differentiation from these cells are not well understood either in vivo or in vitro, and these will need to be further elucidated to maximise their potential for therapeutic intervention. OSCs may also be of value as a model to investigate normal human germ cell differentiation. It is likely that their interactions with ovarian somatic cells and/or extracellular signals will be important in these processes. This review summarises our current knowledge on the isolation and characterisation of mammalian oogonial stem cells.

Isolation, purification, and culture of oogonial stem cells from adult human and bovine ovarian cortex

Abstract Background The longstanding belief that women are born with a finite ovarian reserve has been debated for almost a decade, ever since the discovery, and subsequent isolation, of purported oogonial stem cells (OSCs). These cells, described in both mice and man, seemingly have the ability to generate oocytes when cultured in vitro under specific conditions, resulting in live offspring in the case of mice. The aims of this study were to verify independently the existence of OSCs in human ovarian tissue and to determine whether they could be isolated from bovine tissue. Methods We isolated OSCs from disaggregated bovine and human ovarian cortex using a fluorescence-activated cell sorting (FACS)-based technique, with cells being sorted for the presence of VASA, a germ-cell-specific protein. The cells were subsequently cultured in vitro without the need for a feeder layer, and mRNA expression of germline-specific markers was analysed with RT-PCR. The cells were transduced to express green fluorescent protein (GFP) by use of a lentivirus for the purposes of tracking. Findings A rare population of mitotically active VASA-positive cells was isolated from both bovine and human tissue and they have been grown in vitro for several months. Up to November, 2013, cells were retrieved from three human samples, but isolation has been replicated on five separate occasions in bovine ovary samples. RT-PCR demonstrated consistent expression of several germline markers, including IFITM3, DPPA3, PRDM1, POU5F1 , and KIT . It has been possible to cryopreserve the cells with successful re-initiation of growth on thawing, and stable expression of GFP was achieved after lentivirus transduction. Interpretation The expression of germline markers indicates that these cells have characteristics of germline (oogonial) stem cells. To our knowledge, this is the first report of such cells being isolated from bovine ovarian cortex and corroborates a previous report showing the isolation of OSCs from human ovary. These cells provide a novel model for investigating germ-cell development; future experiments will involve injection of OSCs into ovarian cortex to assess whether they have the capability to undergo follicle formation and oocyte development in an in-vitro environment. If their potential can be harnessed, OSCs might have a future role in clinical applications—eg, in fertility preservation. Funding UK Medical Research Council.

The regulation and assessment of follicular growth.

Abstract Folliculogenesis is the process by which waves of small primordial follicles possessing immature oocytes are recruited to undergo development into large antral follicles, with one then being selected for ovulation of a fully competent oocyte. Folliculogenesis can be divided into three stages: follicle recruitment, selection and ovulation, and has two phases: the initial gonadotrophin-independent phase and the later gonadotrophin-dependent phase. It involves an elaborate array of biochemical signalling factors, both stimulatory and inhibitory, and the regulation of follicle growth relies on these being tightly controlled. Their increasing understanding allows reproductive biologists to attempt manipulation of folliculogenesis, which can be useful in clinical areas such as assisted reproduction and contraception. The rising average age of childbearing in many developed countries is bringing an additional focus on the importance of assessing a womans non-growing follicular pool; i.e. her ovarian reserve. This review examines the important regulatory players in the different stages of folliculogenesis and describes some of the currently available measures of ovarian reserve.

Initial characterisation of adult human ovarian cell populations isolated by DDX4 expression and aldehyde dehydrogenase activity

The existence of a population of putative stem cells with germline developmental potential (oogonial stem cells: OSCs) in the adult mammalian ovary has been marked by controversy over isolation methodology and potential for in-vitro transformation, particularly where cell sorting has been based on expression of DEAD box polypeptide 4 (DDX4). This study describes a refined tissue dissociation/fluorescence-activated cell sorting (FACS) protocol for the ovaries of adult women which results in increased cell viability and yield of putative OSCs. A FACS technique incorporating dual-detection of DDX4 with aldehyde dehydrogenase 1 (ALDH1) demonstrates the existence of two sub-populations of small DDX4-positive cells (approx. 7 µm diameter) with ALDH1 activity, distinguished by expression of differentially spliced DDX4 transcripts and of DAZL, a major regulator of germ cell differentiation. These may indicate stages of differentiation from a progenitor population and provide a likely explanation for the expression disparities reported previously. These findings provide a robust basis for the further characterisation of these cells, and exploration of their potential physiological roles and therapeutic application.