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 woman's 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.

Key Words::

• Anti-Müllerian hormone
• folliculogenesis
• non-growing follicular pool
• ovarian reserve

Introduction

The follicle, consisting of an oocyte surrounded by supporting somatic cells, is fundamental to the ovary's role as a reproductive organ. Oocytes are formed in the developing fetus when proliferating primordial germ cells enter meiosis from the 11th week of gestation. The earliest follicular structures, the primordial follicles, consist of an oocyte halted in prophase I of meiosis surrounded by one layer of squamous granulosa cells. They arise once oocytes recruit pre-granulosa cells after around 18 weeks of gestation. Despite there being approximately 7 × 10⁶ oogonia in fetal life, newborn girls have only 1 million primordial follicles; a figure that continues to fall during early life such that females enter their reproductive years with a pool of around 300,000 primordial follicles. The vast majority of these follicles are destined to become atretic. At the start of every menstrual cycle, a cohort, or ‘wave’, of primordial follicles is recruited to grow, with only one (in monovular species, such as humans and ruminants) or several (in polyovular species, such as mice) follicles achieving dominance by mid-cycle with subsequent release of their now mature and fertilizable, oocyte during ovulation. A myriad of factors, acting at the autocrine, paracrine and endocrine level, are involved in the complex process of folliculogenesis, which comprises follicle recruitment from the primordial pool, dominant follicle selection and, finally, ovulation. This review will examine these three stages, outlining the important biochemical players involved. The methods of assessing follicle growth will also be described.

Initiation of follicular growth

The activation of primordial follicle growth is regulated by a fine balance between stimulatory and inhibitory factors. It is this balance that helps determine the length of a woman's reproductive lifespan and understanding of its regulation is thus a key goal in reproductive biology. Once activated, primordial follicles become primary follicles (an oocyte surrounded by one layer of cuboidal granulosa cells) and then secondary follicles (with multiple layers of granulosa cells and an outermost theca cell layer) which subsequently develop a fluid filled antral cavity. Selection for ovulation occurs following further growth and with increasing gonadotropin dependence, rather than the gonadotropin sensitivity of earlier stages (Figure 1).

Figure 1. A summary of folliculogenesis. The primordial follicle pool develops into primary then secondary follicles independently of gonadotrophins, but the process is closely regulated by activating (green box) and inhibitory (red box) factors. Growth from a secondary follicle to an antral follicle is increasingly gonadotrophin-dependent, with ovulation being entirely gonadotrophin-dependent.

A critical pathway identified to be involved in the regulation of the initiation of primordial follicle growth is the phosphatidylinositide 3-kinase (PI3K) pathway, located within the oocyte. When this intracellular signalling pathway is activated by growth factors such as stem cell factor (SCF; also known as Kit ligand), a cytokine produced by the granulosa cells of the primordial follicle, an increase in PI3K results in increased phosphorylation of Akt, a serine/threonine-specific protein kinase, with the end result being increased cell survival and proliferation and inhibition of apoptosis. Follicular activation can also be stimulated via this pathway by the mammalian target of rapamycin complex (mTORC). The role of mTORC in human follicular activation has been shown by demonstrating that treatment of immature follicles with rapamycin, an inhibitor of mTORC, can lead to oocyte death [1].

The PI3K pathway is kept in control by inhibitory factors, including phosphatase and tensin homolog (PTEN) and the transcription factor forkhead box O3a (FOXO3a). PTEN inhibits the stimulation of Akt and therefore suppresses cell proliferation. Consequently, PTEN null mice demonstrate premature activation of the primordial follicle pool, thus shortening the reproductive lifespan considerably [2]. This is oocyte specific, as PTEN deficiency in granulosa cells does not induce this phenotype. FOXO3a, a member of the forkhead family of transcription factors, is another important downstream component to the PI3K pathway. Like PTEN, FOXO3a inhibits follicular activation and thus knock-out mice also demonstrate premature ovarian insufficiency [3].

Recently, another intracellular signalling pathway, the Hippo signalling pathway, has been suggested as potentially having a role in follicle activation [4]. The Hippo pathway is a highly conserved kinase cascade, activated by cell membrane-bound regulators in areas of high cell density. Activation leads to phosphorylation and inactivation of a cell proliferation transcription factor called yes-associated protein (YAP). Therefore, in cell dense tissue, cell growth and proliferation is impeded, whilst in areas of low cell density, the pathway remains inactivated and YAP promotes cell growth: this mechanism has been implicated in determining organ size, and may explain why fragmentation of ovarian cortex, and hence disruption of the Hippo pathway, leads to increased initiation of follicle growth [4].

Several other factors have been shown to activate primordial follicles to form primary follicles [5]. Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are members of the transforming growth factor β (TGFβ) family, with proven roles in early follicular development. Furthermore, BMP4 is necessary for the survival of oocytes. Leukaemia inhibitory factor (LIF), a granulosa cell-secreted cytokine, and fibroblastic growth factor (bFGF), produced by the oocyte, also appear to stimulate the transition of primordial follicles to primary follicles in various species [5].

Conversely there are important inhibitory factors present in the ovary to control the rate of primordial follicle activation, one of which is anti-Müllerian hormone (AMH). Like the BMPs, AMH is a member of the TGFβ superfamily; however, a key role appears to be suppression of primordial follicle recruitment [6]. AMH is produced by the granulosa cells of pre-antral and small antral follicles, with a sharp decline in larger antral follicles [7]. It is possible that AMH produced by the cohort of growing preantral and early antral follicles inhibit the growth of their neighbouring primordial follicles, thereby restraining the number of follicles activated at any one time. In addition to its direct inhibitory role in early follicle development, AMH can also suppress activation indirectly, by reducing the expression of SCF and bFGF [5].

After activation from primordial follicles to primary follicles, the next step is development into secondary, or pre-antral, follicle stages. This process requires further oocyte growth and proliferation of the granulosa cells. Once again, members of the TGFβ superfamily play a role, with the dimeric protein activin enhancing granulosa cell proliferation [8]. At this point, follicular growth is gonadotrophin-sensitive rather than dependent; activin also promotes the responsiveness of granulosa cells to follicle-stimulating hormone (FSH) [8], and, consequently, further follicular growth relies on gonadotrophins. Growth differentiation factor 9 (GDF9) and BMP15 are both expressed by the oocyte and are essential in the development of follicles past the primary stage [9]. They work synergistically to regulate cell proliferation within the follicle and regulate SCF expression. AMH has a further inhibitory role at this stage of folliculogenesis, by reducing FSH-sensitivity. The downregulation of AMH expression as follicles grow coincides with an equally sharp upregulation of aromatase expression, and thus the capacity of the follicle to make estradiol [7]. AMH is thus not only involved in controlling the rate at which follicles leave the primordial pool, but it may play a role in the selection of the dominant follicle. While these growth factors have increasingly defined roles, there are likely to be other, as yet, unrecognized pathways of key importance.

A crucial aspect to the whole process is the ability for the oocyte and its surrounding somatic cells to communicate with each other. It is now clear that this communication is bi-directional, via gap junctions and transzonal projections, which allow essential molecules to pass between the oocyte and granulosa cells (reviewed in [10]).

Follicle selection and ovulation

As follicles grow, they become progressively more dependent on gonadotrophins for continued development and survival. FSH promotes granulosa cell proliferation and differentiation, allowing the follicle to increase in size. By the time the follicle forms an antrum, from follicular fluid produced by the granulosa cells, it is entirely FSH-dependent for further development. Both the granulosa cells and theca cells express gonadotrophin receptors and become responsible for sex steroidogenesis, with theca cells becoming LH responsive (thus producing androgen) and granulosa cells responding to FSH (and converting theca cell-derived androgen to oestradiol by aromatization: the two-cell, two-gonadotrophin hypothesis). By now, granulosa cells can be compartmentalized into two groups: cumulus cells surrounding the oocyte and promoting its maturation, and mural granulosa cells around the inner aspect of the follicle, producing sex steroids. The follicle destined for when ovulation occurs is now ∼10–12 mm in diameter and can be accurately monitored by ultrasound.

Subsequent ovulation is triggered by a surge of LH and an antral follicle will only become preovulatory if both its granulosa cells and theca cells express LH receptors at the time of this surge. It has become clear that the effect of the LH surge is mediated through EGF family members including epiregulin and amphiregulin produced by mural granulosa cells and acting on the cumulus [11]. Most mammals control their ovulation rate in order to regulate the number of offspring they can conceive at one time. In humans, this requires that only one growing follicle attains ‘dominance’, whilst the rest become ‘subordinate’ and, eventually, atretric. The importance of the oocyte in regulating this process is demonstrated by the effect of heterozygosity for mutations in BMP15 and GDF9 in sheep which can result in higher ovulation rates, whereas homozygosity leads to sterility [12].

The basis for the model for the regulation of ovulation rate was first proposed almost 30 years ago [13]. It is thought that, during a follicular wave, there is a figurative ‘gate’ or ‘window’ of time during which FSH concentrations are above the threshold necessary for large, gonadotrophin-dependent follicles to escape atresia. The length of the FSH ‘window’ thereby dictates how many follicles become ovulatory: one would hypothesize that monovular species have a short window, thus allowing only one follicle to undergo ovulation following the necessary LH surge.

Clearly, folliculogenesis is complicated and not yet fully understood. However, could some of the major players involved be useful clinically in assessing the follicular pool?

Assessment of the follicular pool

It is widely believed that women are born with a finite supply of oocytes (although this has been challenged in the last decade (reviewed in [14]). As such, the size of the non-growing follicular (NGF) pool determines the length of a woman's reproductive lifespan. For clinical purposes, it is useful to know the size of a woman's available follicular pool to aid assessment and management of infertility. Furthermore, the increasing trend of delaying having children in women in western societies indicates that accurate prediction of ovarian reserve is important, not only medically, but sociologically, as women seek to find out how long they have left on their ‘biological clock’. These two pools should be distinguished: the term ‘ovarian reserve’ is often used in this context although more correctly should be reserved for the primordial follicle pool. What is often meant is the potential ovarian response to exogenous gonadotrophins (most commonly in superovulation for IVF), which consists of follicles already at the antral stage of development. At present there are no known biomarkers of the primordial follicle pool, and the available markers have, at best, indirect relationships with the number of primordial follicles in the ovary based on the assumption that the size of the pool of follicles that are potentially recruitable is related to the primordial follicle number, allowing prediction of reproductive lifespan. Histological analysis of primordial follicle number has been correlated with ultrasound and serum assessment of ovarian reserve markers [15] showing that there are indeed useful relationships, but the indirectness of these relationships must always be borne in mind in interpretation. This has been recently demonstrated in an analysis of AMH in children with newly diagnosed cancer, which showed that AMH was generally low compared to age-matched controls, and correlated negatively with markers of ill health such as C reactive protein [16].

One physical method of evaluating the ovarian reserve is measurement of the antral follicle count (AFC) by transvaginal ultrasonography (TVUSS). The AFC is the number of follicles less than 10 mm in diameter in the early follicular phase of the menstrual cycle and has a close inverse correlation with age. However, AFC shows some variation from one menstrual cycle to the next and there is inter-observer variability by the sonographer. Additionally improvements in ultrasound technology have had an impact on image quality and thus follicle recognition: this is of importance where the AFC is used for diagnostic purposes, e.g. in polycystic ovary syndrome [17].

Various serum biomarkers have also been proposed for the prediction of the size of the remaining follicular pool. Three hormones that have been assessed as biomarkers are FSH, inhibin B and AMH; however, the ‘perfect’ biomarker is yet to be found. Early follicular phase FSH has been the most extensively used biomarker to date as it has long been measurable and indicates ovarian feedback on the hypothalamic-pituitary axis. As women age and their ovarian reserve declines, FSH concentrations rise. The major drawback is the marked cycle-to-cycle variation in FSH, as well as the need for the sample to be taken in the early follicular phase.

Inhibin B is produced by the granulosa cells of small antral follicles and is a key physiological inhibitor of FSH secretion, limiting the inter-cycle FSH rise. There was considerable interest in inhibin B as a predictor of ovarian response in assisted reproduction but it has now largely been replaced by measurement of AMH.

AMH has now been used in clinical practice for some years for the assessment of response to superovulation regimens used in in vitro fertilization (IVF), and its role and potential value is increasingly recognized [18]. It is useful as a predictor of those likely to respond poorly, but unlike FSH can also identify women at high risk of ovarian hyperstimulation. AMH has the advantage of low inter- and intra-cycle variability so can be measured at any time of the menstrual cycle. It is also being explored as a predictor of time to menopause, provided a woman's age is accounted for when interpreting the measurement [19]. It is, however, affected by suppressed gonadotrophin levels, with lower AMH concentrations seen during pregnancy and long-term gonadotrophin-releasing hormone (GnRH) analogue treatment. In addition, there is a pressing need for standardization of AMH quantification between the available commercial assays [18].

While AMH shows much promise as a serum biomarker, in general it is of very similar predictive value as AFC and the clinical situation will therefore at present determine which test is preferred. There remains a need for more biomarkers for the earliest stages of follicle number, and ultimately of primordial follicle number. Increasing molecular analysis of follicle populations using human samples may well provide insights of value in this quest [20].

Conclusion

It is evident that a multitude of signalling factors are required, at the right concentrations and at the right time, in order for a competent, mature oocyte to be produced on a monthly basis. Furthermore, a fine balance of activating and inhibitory factors is essential to regulate the rate at which follicles are recruited and ovulated. Clinically, knowledge of these factors is important if we wish to understand the regulation that allows follicles formed before birth to suffice for 50 years of ovarian activity. Additionally, it is necessary for manipulation of folliculogenesis for use in assisted reproductive techniques and new contraceptives. Although AMH is showing promise in the assessment of ovarian reserve, there is still much to learn about the process of folliculogenesis and further research may yet uncover a new, more accurate biomarker of ovarian reserve.

Questions and answers

Q (Villa): Does the relationship between AMH (anti-Müllerian hormone) and follicular growth vary in different pathological conditions?

A (Anderson): There is a little data on this but not much. Recently a group from Rotterdam published a paper in the journal Human Reproduction, which showed that at the point of diagnosis, AMH concentrations were lower than they ought to be in children with cancer. Also the concentrations were inversely correlated to CRP concentrations and to hemoglobin concentrations, so the more ill the child, the lower the AMH.

Comment (Carmina): There are also data in patients with Klinefelter's disease and in patients with delayed puberty. AMH is used by pediatric oncologists to differentiate patients who will go on to regular puberty from those who will not.

Comment (Anderson): Related to this is the gonadotropin dependence of AMH. Long-term gonadotropin suppression treatment will lead to decreased AMH.

Comment (Burney): There are other situations in which AMH is decreased such as endometriosis, inflammatory bowel disease and pelvic inflammatory disease.

Q (Burney): Is there any progress towards a reference standard for AMH?

A (Anderson and Beastall): There has been talk about this and moves towards it. There is growing recognition that AMH is a biomarker which is of use and for which there is a future. If it is to be used for clinical purposes, it is essential that assays are good and standardization is a prerequisite for this. There are other possible markers but the data on them is not robust and the evidence is sometimes indirect.

Q (Ballieux): My professor in Obstetrics and Gynaecology says that ultrasound is better than AMH which is thus unnecessary. Is AMH useful for patient care or for research?

A (Anderson): Ultrasound examinations are expensive and in primary care a blood test is much more useful. AMH concentration measurements are likely to become of much more clinical use in the future.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

The authors’ work in this field is supported by MRC grant G1100357.