Hormonal regulation of folliculogenesis

Gonadotropins are the main regulators of follicular development and steroid hormone production in the ovary. Follicle-stimulating hormone (FSH) and LH are gonadotropic hormones secreted by the pituitary gland that coordinate antral folliculogenesis and ovulation. They are heterodimeric glycoproteins that consist of an α-subunit and a unique β-subunit. The α-subunit is a component of FSH, LH, thyroid stimulating hormone (TSH) and human chorionic gonadotropin (hCG) (Ruman et al., Reference Ruman, Pollak, Trousdale, Klein and Lustbader2005). Positive and negative feedback loops in the hypothalamic–pituitary–gonadal axis coordinate follicle maturation with sexual behaviour and preparation for pregnancy. The ovary produces growth factors such as activin, inhibin and follistatin for modulation of FSH secretion and local regulation of follicle development (Edson et al., Reference Edson, Nagaraja and Matzuk2009).

The main signalling pathway is activated by FSH binding to specific membrane receptors located on granulosa cells. This interaction alters G-protein complex conformation, which is followed by activation of adenylate cyclase (the enzyme that catalyzes cAMP synthesis from ATP). In the presence of cAMP, protein kinase A (PKA) phosphorylates and activates transcription factors that regulate gene activity, including aromatase, α- and β-subunits of inhibin, the receptor for luteinizing hormone and human chorionic gonadotropic (LHCGR), and many other molecules (Richards et al., Reference Richards, Russell, Ochsner, Hsieh, Doyle, Falender, Lo and Sharma2002).

LH stimulates androgen synthesis by theca cells (Liu et al., Reference Liu, Qiao, Jiang, Jiang, Han, Wang and Ren2015). LHCGR is expressed at high levels in the granulosa cells of preovulatory follicles, this expression allows cells to respond to a surge of LH, which initiates a cascade of events leading to the resumption of meiosis in oocytes, an increase in the number of cumulus cells, rupture of the follicle and the formation of the corpus luteum (luteinization) (Edson et al., Reference Edson, Nagaraja and Matzuk2009).

The LH surge activates genes that are responsible for the formation and stabilization of the extracellular matrix of the cumulus oophorus, i.e. the region of the antral follicle wall that carries the oocyte. The LH surge causes a rapid increase in the expression of epidermal growth factor (EGF) family genes (Areg, Ereg and Btc) in cumulus cells. These ligands are integral membrane proteins and are released from the cell surface by ectodomain cleavage. These ligands then bind to EGF receptors (EGFRs) and activate them. In addition, this binding leads to the activation of mitogen-activated protein kinase (MAPK)3 and MAPK1, also known as extracellular signal-regulated kinases 1 and 2 (ERK1/2), respectively. ERK1/2 can stimulate oocyte maturation. However, the specific roles of the EGF network and ERK1/2 in ovulation regulation, oocytes maturation and further follicular luteinization are not yet sufficiently clear (Fan et al., Reference Fan, Liu, Shimada, Sterneck, Johnson, Hedrick and Richards2009; Zhang et al., Reference Zhang, Liu, Ji, Sha, Zhang and Fan2015).

As with many other protein hormones, FSH consists of a family of isoforms that differs in size, charge and elimination rate (Roche, Reference Roche1996). FSH is encoded by a single gene, but the pituitary gland is able to produce more than 17 different FSH isoforms due to post-translational modifications. These isoforms differ in their oligosaccharide structure and in the degree of terminal sialylation and sulfonation. A change in the carbohydrate patterns due to sulfonation (by addition of sulfonic acid groups – SO₃H) and sialylation (by attachment of sialic acid residues) can reduce the pH of the protein and increase the half-life of the isoforms. Remodelling of carbohydrate complexity can significantly affect the biological activity of hormones (Trousdale et al., Reference Trousdale, Yu, Pollak, Husami, Vidali and Lustbader2009). A change in FSH isoform expression occurs during the ovarian menstrual cycle. The least acidic FSH isoforms were observed during the late follicular phase, and in the mid-cycle. These isoforms have a smaller half-life period, indicating the existence of regulatory mechanisms that control the intensity and duration of FSH signal exposure (Macklon et al., Reference Macklon, Stouffer, Giudice and Fauser2006). Less sialylated forms of FSH more actively stimulate estradiol and progesterone production by granulosa cells than the acidic isoforms (Loreti et al., Reference Loreti, Ambao, Andreone, Trigo, Bussmann and Campo2013b).

The normal response of the follicles to gonadotropins requires the presence of both these hormones and their receptors. It has been shown that mice that lack follicle-stimulating hormone receptor (FSHR) are infertile (Dierich et al., Reference Dierich, Sairam, Monaco, Fimia, Gansmuller, LeMeur and Sassone-Corsi1998). Moreover, many isoforms of human FSHR are known. About 2000 single nucleotide polymorphism (SNPs) in the FSHR have been reported, however only few of these affect fertility (Casarini et al., Reference Casarini, Santi and Marino2015) because of impaired binding with FSH and FSHR or a decrease in FSHR production. Genetic analysis performed to detect FSHR isoforms may be used as a predictive marker of diminished ovarian reserve and infertility (Ilgaz et al., Reference Ilgaz, Aydos, Karadag, Taspinar, Eryilmaz and Sunguroglu2015).

Gonadotropins are the key regulators of follicular development and steroid hormone production in the ovary. Large numbers of internal ovarian regulators, however, especially insulin-like growth factor (IGF), EGF, transforming growth factor (TGF)-α and TGF-β, can modulate the cellular response to gonadotropic stimulation. Inhibins and activins also have a local effect in the ovaries, in addition to their endocrine and paracrine functions. They suppress FSH secretion by the pituitary gland. IGF-I stimulates the proliferation of granulosa cells and increases their aromatase activity. In theca cells, IGF-I has a positive influence on 17α-hydroxyprogesterone production; IGF-I and IGF-II together with LH stimulate androgen synthesis. EGF and TGF-α are structurally similar and can bind to one receptor. These factors promote granulosa cell proliferation, but inhibit FSH-induced expression of aromatase and estradiol synthesis. TGF-β also stimulates the proliferation of granulosa cells. In pre-antral follicles, activin increases the number of granulosa cells and elevates FSH receptor and aromatase expression levels that in turn lead to increased estradiol production (Macklon et al., Reference Macklon, Stouffer, Giudice and Fauser2006). Inhibins stimulate LH-induced androgen synthesis by theca cells (Roche, Reference Roche1996).

Another important molecule involved in hormonal regulation of folliculogenesis is anti-Müllerian hormone (AMH). AMH is a dimeric glycoprotein and a member of the TGF-β gene family (Knight & Glister, Reference Knight and Glister2006). AMH is commonly used as a diagnostic and prognostic clinical marker of fertility that can indicate the size of the ovarian reserve (Bhide & Homburg, Reference Bhide and Homburg2016; Łebkowska & Kowalska, Reference Łebkowska and Kowalska2017). It has been shown that AMH is expressed in the granulosa cells of primary, secondary, pre-antral and early antral follicles but not in primordial follicles, late antral follicles and oocytes (Lee et al., Reference Lee, Donahoe, Hasegawa, Silverman, Crist, Best, Hasegawa, Noto, Schoenfeld and MacLaughlin1996; Rajpert-De Meyts et al., Reference Rajpert-De Meyts, Jorgensen, Graem, Muller, Cate and Skakkebaek1999; Weenen et al., Reference Weenen, Laven, Von Bergh, Cranfield, Groome, Visser, Kramer, Fauser and Themmen2004). AMH inhibits the initiation of primordial follicle growth (Durlinger et al., Reference Durlinger, Kramer, Karels, de Jong, Uilenbroek, Grootegoed and Themmen1999), and premature ovarian failure (POF) was found in AMH-null mice (Visser & Themmen, Reference Visser and Themmen2005). Moreover, AMH suppresses FSH-induced follicle growth. This effect is probably mediated through the induction of miR-181b miRNA that, in turn, blocks adenylate cyclase 9, thus decreasing FSH-induced cAMP levels and leading to suppression of FSH signalling. AMH, through the induction of another miRNA (miR-181a), targets activin receptor 2A, thus leading to impairment of the effects of activin on folliculogenesis, and preventing follicular growth and proliferation of granulosa cells (Hayes et al., Reference Hayes, Kushnir, Ma, Biswas, Prizant, Gleicher and Sen2016). Interestingly, AMH receptors are expressed not only in the ovary but also in the pituitary gland (Bedecarrats et al., Reference Bedecarrats, O'Neill, Norwitz, Kaiser and Teixeira2003). It has been shown that LH secretion is induced by AMH through stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) neurons, which express AMH receptors (Cimino et al., Reference Cimino, Casoni, Liu, Messina, Parkash, Jamin, Catteau-Jonard, Collier, Baroncini, Dewailly, Pigny, Prescott, Campbell, Herbison, Prevot and Giacobini2016). Thus, mutations in the gene coding of AMH receptors can lead to infertility (Li et al., Reference Li, Zhou, Wang, Wang, Zhang, Wang, Cao and Kee2016). Additionally, it has been shown that AMH receptor II is overexpressed in granulosa cells of oligo/anovulatory, but not in normo-ovulatory, women with polycystic ovary syndrome (PCOS; Pierre et al., Reference Pierre, Peigne, Grynberg, Arouche, Taieb, Hesters, Gonzales, Picard, Dewailly, Fanchin, Catteau-Jonard and di Clemente2013). Therefore, AMH/AMHR signalling is crucial for regulation of folliculogenesis at both the pituitary and ovarian levels.

After implantation, the embryo produces hCG that influences the maternal organism, for example it regulates progesterone production by the corpus luteum. When the placenta becomes capable of progesterone production, hCG starts to control and maintain the fetal blood supply. Human chorionic gonadotropin stimulates angiogenesis by binding to LHCGRs located on uterus spiral arteries. In addition, hCG is involved in placental cell differentiation and stimulation of uterine growth together with growth of the fetus (Ezcurra & Humaidan, Reference Ezcurra and Humaidan2014). When LHCGR binds to its ligand its conformation changes and activates G-protein, which in turn activates adenylate cyclase (AC). Adenylate cyclase increases the intracellular cAMP pool. Induction of the cyclic AMP/protein kinase A (PKA) pathway eventually results in ovulation and steroid biosynthesis. LHCGR stimulation also activates the phospholipase C/inositol phosphate pathway. This signalling pathway is activated only during the preovulatory LH surge and during pregnancy. Phospholipase C acts as a mediator of terminal granulosa cell differentiation (Choi & Smitz, Reference Choi and Smitz2014).

Human chorionic gonadotropin has a greater affinity for this receptor and has a greater effect on follicular development than does LH (Ezcurra & Humaidan, Reference Ezcurra and Humaidan2014). LH and hCG bind to the same receptor but activate different signalling pathways (Fig. 1). LH has a greater influence on the phospholipase C/inositol 1,4,5-trisphosphate (PLC/IP3) pathway (Choi & Smitz, Reference Choi and Smitz2014), leading to the phosphorylation and inactivation of forkhead box O3 (FOXO3), which is an inhibitor of primordial follicle activation. Phosphoinositide-3 kinase (PI3K) catalyzes the conversion of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) to a second messenger – phosphatidylinositol 3,4,5-trisphosphate (PIP3). IP3 activates protein kinase B (PKB) that phosphorylates FOXO3 (Edson et al., Reference Edson, Nagaraja and Matzuk2009). hCG promotes the accumulation of cAMP, which activates the PKA signalling pathway (Choi & Smitz, Reference Choi and Smitz2014). After the LH peak, the level of cAMP reaches a plateau after 10 min, by comparison in response to hCG the plateau is reached after 1 h (Ezcurra & Humaidan, Reference Ezcurra and Humaidan2014).

Figure 1

Theoretical pathway of divergence in receptor-mediated signal transduction of LH and hCG (according to Choi & Smitz, Reference Choi and Smitz2014). Abbreviations: AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; hCG, human chorionic gonadotropin; IP3, inositol 1,4,5-trisphosphate; LH, luteinizing hormone; LHCGR, receptor for luteinizing hormone and chorionic gonadotropin hormone; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PLC, phospholipase C.

The application of gonadotrophic hormones in assisted reproductive technology

Hormonal stimulation is an essential part of modern assisted reproductive technology (ART). Gonadotropins are applied to stimulate folliculogenesis in humans and animals. This kind of treatment leads to an increase in the number of oocytes or embryos that can be used for ART (Edwards et al., Reference Edwards, Kind, Armstrong and Thompson2004).

Human chorionic gonadotropin (hCG) was first isolated from the blood and urine of pregnant women in 1927 and is one of the most commonly used hormones in clinical practice. The action of hCG is very similar to that of Prolan B (LH) and, in 1932, it was released under the commercial name Pregnil (Lunenfeld, Reference Lunenfeld2004).

At the start of the introduction of ART, PMSG was used for ovarian follicle growth stimulation. PMSG is secreted by the endometrial cups (ring-like structures on the surface of the endometrium around the fetus's attachment point) of pregnant mares. PMSG can be detected initially at 37–42 days of pregnancy; it reaches its highest concentration between 50 and 70 days, and almost completely disappears after the fourth month of pregnancy. At present, PMSG is not administered to humans, as this drug can cause an immune response.

In 1941, a ‘two-step protocol’ was proposed. The main idea of the new protocol was to stimulate follicle growth and ovulation sequentially. In the first step, follicles are stimulated by gonadotropins (PMSG or pituitary gonadotropins from pigs or sheep) and, in the second step, ovulation is induced by human chorionic gonadotrophin (HCG). At this time, a hyperstimulation syndrome was described in which ovaries were dotted with numerous haemorrhagic follicles that lacerated and bled at the slightest touch (Lunenfeld, Reference Lunenfeld2004).

Gonadotropin, isolated from the human pituitary gland in 1958, has been the most suitably used hormone for oogenesis stimulation over time. From 1958 to 1988, it has been used successfully in several medical centres around the world to induce ovulation in the treatment of ovulation disorders. The source of this drug, however, has been too limited. In addition, some patients have shown symptoms of Creutzfeldt–Jakob disease (the primary manifestation of spongiform encephalopathy, or prion disease) more than 20 years after the initiation of pituitary gonadotropin treatment (Lunenfeld, Reference Lunenfeld2004).

In the early 1970s, the idea of individual treatment protocols for FSH and LH administration for each patient became paramount. Attempts to separate these two hormones were not successful as the proposed methods were too cumbersome, complicated or expensive, or were not sufficiently precise or effective. With the advent of genetic engineering, these problems have been overcome. In 1995, the commercial drug Gonal-F was licensed for use (Lunenfeld, Reference Lunenfeld2004).

FSH in its recombinant and purified form is currently used to support ovarian follicle growth in ART. Recombinant human FSH has a short half-life (approximately 24 h after injection), therefore it needs to be injected daily (subcutaneously or intramuscularly) over an average period of 8–12 days. The manufacture of rhFSH-carboxy-terminal peptide (CTP), which has a two to three times greater half-life than rhFSH, made it possible to reduce the number of injections needed, compared with the standard rhFSH protocol (Ruman et al., Reference Ruman, Pollak, Trousdale, Klein and Lustbader2005). Moreover, in the mouse model, it has been shown that the efficiency (the quantity of oocytes after stimulation) of rhFSH-CTP treatment was significantly higher than that of rhFSH application (Trousdale et al., Reference Trousdale, Yu, Pollak, Husami, Vidali and Lustbader2009).

More sialylated rhFSH stimulates inhibin B synthesis; less sialylated analogues are not capable of stimulating inhibin B synthesis, but have a significant stimulatory effect on the inhibin A synthesis. An increase in inhibin serum concentration was observed in the early follicular phase of the menstrual cycle that coincided with the presence of more acidic forms of FSH. In contrast, the less acidic forms detected in the late follicular phase were associated with the appearance of inhibin A (Loreti et al., Reference Loreti, Fresno, Barrera, Andreone, Albarran, Fernandez, Larrea and Campo2013a).

The improved forms of recombinant FSH (with a long half-life), however, were still less effective than PMSG. PMSG is the most glycosylated pituitary glycoprotein of placental mammals: both subunits of this hormone contain N-linked and O-linked glycosylation sites. The high sialic acid content greatly increases its half-life compared with other glycoprotein hormones. In mice, only multiple doses of purified or recombinant FSH are equal to a single dose of PMSG for inducing ovulation. PMSG also has luteinizing activity. It is important for the optimal development of follicles in the preovulatory phase (Ruman et al., Reference Ruman, Pollak, Trousdale, Klein and Lustbader2005).

A significant increase in progesterone blood concentration was observed after stimulation with PMSG (5 IU) in comparison with rhFSH application (2.5 IU), but the estradiol concentration did not change. In mammals, the progesterone/estradiol ratio is more important for implantation than the concentration of each hormone. Estrogen and progesterone have an antagonistic effect on the endometrium. Progesterone is important during preparation of the endometrium for implantation. Gonadotropic stimulation causes changes in the spatial distribution of the progesterone receptor that accelerate the maturation of the endometrium (Kelley et al., Reference Kelley, Kind, Lane, Robker, Thompson and Edwards2006); a high level of FSH or estrogen may increase the susceptibility of granulosa cells to LH. This change allows luteinization to be induced even at low LH levels. Luteinization leads to an early increase in progesterone levels, and causes early maturation of the endometrium (Kelley et al., Reference Kelley, Kind, Lane, Robker, Thompson and Edwards2006).

Estradiol and progesterone regulate the expression of five genes that have a function during implantation: Lif, Vegf, Hoxa10, Esr1 and Pgr. In mice, oogenesis stimulation by gonadotropin reduces Lif expression levels in the uterus on the fourth day post coitum (dpc). Leukemia-inhibiting factor (LIF) is essential for implantation. In the absence of Lif expression, the mouse endometrium does not respond to blastocyst attachment. LIF is an interleukin-6 family cytokine, and its expression in the mouse endometrium is induced by estrogen. Typically, expression of this gene peaks on the fourth day of pregnancy, and decreases on the fifth day of gestation. Thus, reduced Lif expression indicates that exogenous gonadotropins accelerate endometrial maturation. Expression of other key implantation genes in stimulated mice did not differ from that of control mice because expression of these genes does not normally differ at days 4 or 5 of gestation (Kelley et al., Reference Kelley, Kind, Lane, Robker, Thompson and Edwards2006).

At present, it is commonly assumed that the use of recombinant human gonadotropins is the most effective method of ART. However, current methods of purification have allow high quality fractions of gonadotropins from human menopausal gonadotropin (HMG) to be obtained. HMG is derived from the urine of postmenopausal women (Wolfenson et al., Reference Wolfenson, Groisman, Couto, Hedenfalk, Cortvrindt, Smitz and Jespersen2005). The drugs Bravelle and Menopur are the two most commonly used examples of this class. The urine fraction that contains FSH activity is used for Bravelle preparation, whereas the fractions that have FSH and LH activity are used for Menopur. The ratio of FSH and LH activity in Menopur is 1:1. The LH activity of Menopur is mainly due to hCG because most LH molecules are lost during purification (Wolfenson et al., Reference Wolfenson, Groisman, Couto, Hedenfalk, Cortvrindt, Smitz and Jespersen2005).

Application of exogenous gonadotropins has a negative effect on early embryonic development and implantation. Ovarian stimulation leads to a decrease in the number of microvilli on the surface of the blastocyst in comparison with unstimulated control murine blastocysts and leads to delay in implantation, caused by a reduction in vascular endothelial growth factor (VEGF) expression levels. Furthermore, the duration of ovarian stimulation reduces pregnancy duration in mice (Edwards et al., Reference Edwards, Kind, Armstrong and Thompson2004). The frequency of pregnancy in stimulated mice decreased because of asynchrony between embryo development and endometrial maturation (Kelley et al., Reference Kelley, Kind, Lane, Robker, Thompson and Edwards2006). There is an evidence that the number of abnormal embryos increases with rising doses of gonadotropins. The largest percentage of defective embryos was observed after stimulation with PMSG, mainly due to its long half-life and high levels of luteinizing activity (Edwards et al., Reference Edwards, Kind, Armstrong and Thompson2004).

Hormonal stimulation of folliculogenesis in vitro

Ovarian follicles in in vitro culture require optimum conditions, particularly regarding the composition of the culture medium. The most promising results for follicles cultured in vitro were obtained by constant addition of hormones to the culture medium (Hu et al., Reference Hu, Ma, Bao, Li, Chen, Gao, Lei, Yang and Wang2011).

FSH is the main hormone that determines the survival and growth of a follicle in culture. Moreover, there is a time dependence between survival rate and hormone addition. Late addition of FSH to the culture medium leads to a decrease in the quantity of live follicles. The rate of granulosa cell proliferation depends on the presence of FSH. The antral cavity starts to form not earlier than 4 days after hormone addition to the medium (Adriaens et al., Reference Adriaens, Cortvrindt and Smitz2004). As mentioned above, FSH has several different isoforms; those that have a lower acidity are more active in vitro, but have a short half-life (Roche, Reference Roche1996).

During in vitro culture, follicular survival rate does not depend significantly on medium FSH concentration, but experimental data demonstrate that follicular growth is more intensive in medium with high hormone content. In the Kreeger experiments, FSH effects were already observed on the second day of culture. The diameter of follicles cultured in medium that contained 10 and 25 mIU/ml FSH was markedly larger than follicles in medium that contained 0 or 5 mIU/ml FSH. FSH dose escalation also increased the accumulation of lactate, which indicates activated granulosa cell metabolism. Very high doses of FSH (50 mIU/ml), however, led to a decrease in follicle survival, because products of metabolism accumulated too quickly. A positive correlation between FSH dose and estradiol level was observed in the in vitro culture system. Estradiol accumulation was observed only in follicles cultured with added FSH, but not in follicles cultured without this supplement (Kreeger et al., Reference Kreeger, Fernandes, Woodruff and Shea2005).

Investigation of the influence of LH on follicle maturation in vitro showed that follicle survival rate was significantly higher in medium supplemented with LH than without (Park et al., Reference Park, Ku, Jung, Liu, Kim, Kim, Kim, Choi, Kim and Moon2013). Moreover, LH bioactivity had an effect on antral cavity formation (Cortvrindt et al., Reference Cortvrindt, Hu, Liu and Smitz1998). Follicles cultured in medium with low doses of LH had larger diameters than those cultured in medium with high LH concentrations (Park et al., Reference Park, Ku, Jung, Liu, Kim, Kim, Kim, Choi, Kim and Moon2013). Granulosa cell proliferation is inhibited by LH concentrations in the medium of more than 5 IU/L, and the follicle undergoes atresia or luteinization. In addition, high levels of LH led to a decrease in production and sensitivity of LHCGR due to accelerated degradation of Lhcgr mRNA (Ezcurra & Humaidan, Reference Ezcurra and Humaidan2014). Researchers have used various approaches for ovarian follicle in vitro culture. A list of the main types of medium applied in in vitro culture systems is presented in Table 1.

Table 1

Types of medium for ovarian follicles in vitro culture

α-MEM, α-Minimal Essential Medium; BSA, bovine serum albumin; FCS, fetal calf serum; ITS, insulin–transferrin–selenium; IU, International Units; LH, luteinizing hormone; rhFSH, recombinant human follicle-stimulating hormone.

Medium supplemented with hCG is used to stimulate oocyte in vitro maturation, because hCG has a more significant luteinizing effect than LH (Skory et al., Reference Skory, Xu, Shea and Woodruff2015).