Our concept of human ovarian reserve presumes that the ovary develops several million nongrowing follicles (NGFs) at around 5 months of gestational age. Over the life span of a female, it undergoes a monthly cycle of oocyte maturation as well as integrated endocrine function, which results in a gradual decline of these NGFs. This process continues up to the age of menopause, around 50–51 years, when approximately 1000 NGFs remain ( Fig. 4.1 ). In light of this fact, several biomarkers have evolved to predict as well as evaluate the existing ovarian oocyte pool and foresee the procreative capacity of a human female. More importantly, advanced and assisted reproductive techniques (ARTs) depend heavily upon this background information. Hence, it is a matter of concern to understand the current scientific concepts and available evaluation parameters for ovarian reserve. To make matters even more complex, several hereditary diseases and systemic conditions are associated with decreased ovarian reserve (DOR), which needs to be thoroughly entailed in the clinical situation as well.
Overview of ovarian tissue
Nature has designed ovaries to provide a composite endocrine and reproductive function in a human female. The cascade of events happening during a menstrual cycle is the interplay of various hormones, which help in the fulfillment of these functions. These hormones originate not only from the ovaries but involve the hypothalamus and pituitary gland. However, there are certain autocrine and paracrine factors working alongside the pituitary ovarian axis that have critical roles in reproductive function. These include transforming growth factor-β (TGF-β) family peptides: inhibin A, inhibin B, activin, and the anti-Mullerian hormones.
A clear description of cortex and medulla in the ovaries is not universally defined, but for functional understanding, cortex includes an area of developing and maturing germ cells, while the medulla is a composition of loose connective tissue. Further details of cortex include surface germinal epithelium, followed by loose mesenchymal cells interspersed between cortical sex cords. This close placement of germ cells and mesangium is meant to provide a well-nurtured and interactive environment for the timely maturation of ovarian follicles. One end of each ovary is designated as the hilum, which aligns the blood vessels and lymphatics to and from the ovaries and provides a surface attachment in the form of mesovarium to the broad ligament.
Ovaries are surrounded by a cuboidal germ cell epithelium. This is formed during embryogenesis by the differentiation of somatic cell lineage into primordial germ cells. Several growth factors and ligand interaction lead to the final settlement of these primordial cells on the genital ridge, after which they are referred to as oogonia . From there starts the development of primordial follicles with oogonia transforming into mitotic stage oocytes . These primordial oocytes then differentiate further into primary oocytes within the primary follicles ( Fig. 4.2 ). Finally, any progenitor cell’s capability to differentiate into primordial cells is stopped at the stage when oocytes enter into meiosis, and subsequently, further maturation is arrested. This landmark developmental step usually happens around the 8th week of gestation. In reproductive biology, this checkpoint has been conceptualized to mark the final oocyte pool in a human female for her lifetime. However, several experiments over the last decade have negated this concept. On the contrary, clear confirmation about ovarian stem cell reserve and its role in forming an essential component of ovarian reserve is still under development.
After having laid down the essential cellular pillar for human development, it is important to understand its functional chemistry. Several intraovarian paracrine factors and genes take part in the recruitment of early primordial follicles for the developing cohort as detailed in Table 4.1 .
|FIGLA; factor in the germline-α||NOBOX; newborn oogenesis homeobox gene|
|Foxl2; forkhead box L2||BMP15; bone morphogenetic protein 15 gene|
|KIT; kit receptor||BRCA1; breast cancer type 1 susceptibility protein gene|
|KITL; kit ligand||LHCGR; luteinizing hormone/choriogonadotropin receptor gene|
|IGF; insulin-like growth factor||STAR; steroidogenic acute regulatory protein gene|
|GDF9; growth differentiation factor 9||CYP11A1; side-chain cleavage enzyme gene|
|AMH; anti-Mullerian hormone||HSD3B2; 3β-hydroxysteroid dehydrogenase isomerase type 2 gene|
|NGF; nerve growth factor||CYP17A1; 17-hydroxylase/17,20-lyase gene|
|BDNF; brain-derived neurotrophic factor|
|NT-3 and NT-4; neurotrophin-3 and neurotrophin-4|
|GDNF; glial cell line-derived neurotrophic factor|
|Inhibin A and inhibin B|
Stages from primary, secondary, and tertiary follicular growths are therefore follicular-stimulating hormone (FSH)-independent and occur over several menstrual cycles after puberty. Maturation of oocytes beyond the meiotic stage comes under the prepubertal FSH effect when each oocyte becomes surrounded by granulosa cell layer. This layer eventually converts into a selected Graafian follicle at the time of puberty when FSH levels are raised in a critical time frame. In each menstrual cycle, the estradiol levels reach their peak near mid-cycle resulting in the complete maturation of a dominant ovarian follicle to mark the process of ovulation . But it is only after the luteinizing hormone (LH) surge that ovulation occurs, releasing a mature ovum for fertilization.
The transition of the Graafian follicle to corpus luteum after ovulation, a process called luteinization , and its sustainability are dependent on the continuous supply of LH or its surrogate human chorionic gonadotropin. The key function of constant large production of progesterone from corpus luteum depends on low density lipoprotein (LDL)-cholesterol and functional mitochondrial steroidogenic acute regulatory (STAR) protein. If pregnancy does not take place, then the life of corpus luteum is around 14 days; subsequently, it converts into corpus albicans .
Indicators of ovarian reserve
Follicle-stimulating hormone and follicle-stimulating hormone receptors
Gonadotropins belong to the family of peptide hormones with differences in β polypeptide chains. The FSH is different from other peptide hormones, not only because it is a heterodimer with a different β chain, but it also has different isoforms, which work differently in the normal menstrual cycle. These isoforms are regulated by inhibin B as well as estradiol, which is secreted from granulosa cells inside the growing antral follicle, and are formed in early and late/preovulatory stages of the follicular phase, respectively. The postulation behind this differential activity is to allow maximum follicular growth in the early part of the follicular phase and to enhance more estradiol secretion and therefore support later ovulation. In the context of this hypothesis and its role in follicular growth, there is no doubt that FSH has served as a menopausal as well as ovarian reserve marker for more than a decade. However, recent developments in the field of infertility have revealed its clinical utility limitations at least in assessing ovarian oocyte pool and predicting future ART options. More recently, the polymorphisms of genes encoding FSH and FSH receptors have been investigated to show some role in the outcomes of ovarian stimulation therapies and to serve as ovarian reserve markers. Although no universal marker is yet completely determined to be sensitive or specific, elevated FSH levels continue to be a valuable tool at least in certain clinical settings.
Anti-Mullerian hormone and AMH receptor
The anti-Mullerian hormone (AMH), being a glycoprotein, belongs to the TGF-β superfamily. It induces the Mullerian ducts to regress during the male sex differentiation. In cryptorchidic males, it serves as a biochemical marker for testicular tissue. The hormone acts through serine/threonine kinase receptors (AMHRI and AMHRII). The AMH and AMHRII gene defects in men cause one of the conditions of differences of sex differentiation called “persistent Mullerian duct.” Recent developments in reproductive medicine have disclosed its role not only on the fetal external genital differentiation but also on the postnatal reproductive life of women. Its levels in women after puberty are similar to those in males and it can serve as an ovarian reserve marker. The granulosa cells of the preantral and small growing follicles are the ovarian sources of AMH. It is responsible for the gonadotropin-independent growth of primary follicles and the selection of ovulating follicles from the oocyte pool throughout the lifetime of a human ovary. Particularly, it appears to have a suppressive effect on the collection of primordial follicles and simultaneously stimulates the growing cohort of follicles by increasing their responsiveness to FSH.
From the above discussion, it is clear that AMH is also a reliable prognosticator of time to menopause and is a useful adjunct in the evaluation of a woman’s “reproductive age.” Besides, higher levels of AMH are associated with a higher count of oocytes or antral follicles retrieved during controlled ovarian stimulation in the in vitro fertilization (IVF) cycles. Hence, AMH is also believed as a surrogate marker of the ovarian reserve and is used in pretreatment evaluation of infertile patients for guiding hormonal stimulation. Although there is only a slight variation in the AMH level throughout the natural menstrual cycle, optimal timing of measurement during the cycle is still not determined. In comparison with other markers described later, AMH was shown to have 100% sensitivity in one of the IVF trials, which assessed markers for poor responders in IVF treatment. Recent reports have also explored the use of AMH in the estimate of ovarian aging in women before or after chemotherapy, predicting the risk of cardiovascular disease near menopause, estimation of long-term reproductive potential following ovarian surgery, in patients with various medical illnesses, and also screening for polycystic ovaries. Furthermore, AMH can be used as a biochemical parameter for the diagnosis as well as a tumor marker in therapeutic monitoring for granulosa cell tumors.
Inhibin A and inhibin B
Inhibins belong to the members of the broader TGF-β family of autocrine, paracrine, and endocrine factors. Along with other peptide hormones, they are produced by the ovarian granulosa cells under the influence of FSH. While the production of inhibin may occur in many other body tissues including adrenal, pituitary, and placenta, most of it is derived from the gonads. Inhibins have been classified into two heterodimeric isoforms: inhibin A and inhibin B. Their α-subunit is identical, but the β-subunits (βA and βB) are distinct, as they are encoded by separate genes. The main role of inhibins remains to suppress FSH production in the pituitary gland.
Over the past two decades, several useful avenues regarding the inhibin physiology and their clinical significance in reproductive medicine have been discovered. It is now well recognized that the two inhibin isoforms have different secreting patterns during the menstrual cycle. Moreover, inhibins may be involved in the physiological adaptation of pregnancy. Clinically, inhibins may serve as sensitive tumor markers in postmenopausal women, as a useful tool for evaluating ovarian reserve in conditions associated with premature ovarian failure and infertility as well as predicting response to ART. Furthermore, inhibins act as a biochemical marker for prenatal screening of Down syndrome as it is a component of the quadruple test, and it also serves in the diagnosis of other feto-maternal disorders.
Activin and follistatin
Activin is structurally homologous to inhibin but has functionally reverse actions. Activin is a heterodimer of two subunits that are similar to the β-subunits of inhibins A and B. The three activin isoforms have therefore various combinations of these β-subunits resulting in activin A (β A β A ), activin B (β B β B ), and activin AB (β A β B ). At the pituitary level, activin stimulates the release of FSH, whereas in the ovaries, it augments FSH action. Interestingly, activin is produced in both granulosa cells and pituitary gonadotrophs; however, it is the local activin in the pituitary that regulates the FSH.
Follistatin is a monomeric protein produced in the pituitary gland as well as ovaries. It appears to neutralize the biological functions of activin. Therefore, the local follistatin levels in tissues modify the effects of activin. This explains the ultimate suppressive effects that follistatin exerts on pituitary FSH secretion.
The biological role of these TGF-β proteins in determining ovarian reserve as well as other gonadal disorders has been studied for decades but has little development to become clinically applicable.
Antral follicle count (AFC)
The assessment of the exact number of antral follicles by vaginal ultrasound has long been considered the best test to assess the ovarian reserve and was reviewed in a recent publication. Decreased AFC is one of the major causes of unexplained infertility in a prospective cohort study. In usual practice, on day 3 of the menstrual cycle, a vaginal ultrasound is performed to determine the number of antral follicles. There is no standard definition of an antral follicle. Some investigators considered an antral follicle size between 2 and 5 mm, while others have used the criteria of 10 mm. A review of the literature showed that AFC had a significant positive correlation with levels of serum AMH. An AFC of less than 3–7 indicates a decrease in the ovarian reserve, subsequently reflecting a poor ovarian response to IVF cycles. Recent research has shown a stronger correlation between AFC and oocytes retrieved, as compared to age and FSH levels.
Dynamic ovarian reserve tests
There are several dynamic methods employed in the past to assess ovarian reserve. A quick overview of the steps to conduct these tests is summarized in Table 4.2 . Their cumbersome performing methods and technical limitations have rendered them less common in practice.
|Step 1||Step 2||Step 3|
|CCCT||Cycle day 3: FSH and estradiol levels||Cycle days 5 and 9: clomifene citrate 100 mg daily is given||Cycle day 10: FSH level|
|EFORT||Cycle day 3: FSH and estradiol levels||Single injection of FSH 300 IU given||Cycle day 4: estradiol level|
|GAST||Cycle day 3: basal FSH, inhibin B, and estradiol levels||A subcutaneous injection of 100 μg triptorelin is given||Cycle day 4: FSH, inhibin B, and estradiol levels|
Clomifene citrate challenge test (CCCT)
Kahraman et al. had reported the sensitivity of CCCT to be 43% and a specificity of 76%, whereas the positive and negative predictive values of this test are 37% and 80%, respectively. The test is performed by checking FSH and estradiol levels on day 3 of the menstrual cycle; then, 100 mg of clomifene citrate is given daily between days 5 and 9 of the cycle, FSH level is then checked again on cycle day 10. With its limitations, it has now become less common in clinical practice. However, it does become part of the assessment of poor (as in primary ovarian insufficiency) and good responders (as in polycystic ovarian syndrome) in ovarian stimulation cycles of IVF.
Exogenous FSH ovarian reserve test (EFORT)
This test comprises cycle day 3 determination of FSH and estradiol levels; thereafter, the patient is given a single injection of FSH 300 IU. On cycle day 4, an estradiol level is checked again. Normal ovarian reserve is defined when the basal FSH is < 11 mIU/mL and the increment in estradiol is > 30 pg/mL. This test first described by Fanchin et al. emphasizes that the synergistic contribution of dynamic change in estradiol and the classically low basal FSH quantities has endorsed the prognostic value of this test. This testing has not been investigated extensively and is not extensively used in the clinical setting due to diagnostic inaccuracy, high cost, and incorrect utility. These limitations revealed in systematic reviews and meta-analysis suggest to abandon them completely.
GnRH-agonist stimulation test (GAST)
On cycle day 3, blood is taken for baseline FSH, inhibin B, and estradiol levels, followed by a single subcutaneous injection of 100 μg triptorelin. After 24 h, the same tests are repeated. The analytical precision of GAST, however, could not be determined in several studies due to discrepancies in the way of their performances.
Despite their limited fame, these tests continue to be important in assessing response to gonadotropin treatment in the IVF cycles and remain an important tool in the evaluation of the reproductive potential of a couple.
Disorders affecting ovarian reserve
Genetic abnormalities leading to decreased ovarian reserve (DOR) in females range from significant chromosome abnormalities, submicroscopic chromosome deletion and duplications, and DNA sequence variations in the genes that control numerous biological processes. From the developmental point of view, these processes are implicated in several stages of oogenesis, sustenance of oocyte pool, maintaining a hormonal milieu, and anatomical and functional integrity of female reproductive organs. Moreover, several systemic disorders are associated with early ovarian function decline or defective development from the start.
Aneuploidy: Extra chromosomes on number 13 and 18 have an association with ovarian dysgenesis and failure. Postulating the existence of some ovarian genes to be located on chromosomes 13 and 18, an area requiring further research.
Autosomal genes: In otherwise healthy women balanced autosomal translocations have been found resulting in the clinical manifestation of DOR.
Mendelian disorders: Certain Mendelian disorders are also associated with DOR leading to POI/POF. The list may be evolving; however, some of the common disorders are listed in Table 4.3 .