Hormonal Regulation of Reproduction

Puberty is the event that switches the reproductive system on and is restrained by higher levels of central nervous system control. The earlier endocrine event of puberty is an increase in the sensitization in kisspeptin pulses at night and an increase in the pulsatile release of the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) in an episodic pattern of pulses into the hypothalamic–pituitary portal system [1]. GnRH travels through the hypophysial portal blood system to the anterior pituitary, where it binds to the GnRH receptors on the cell surface of the gonadotropes. This is reflected by an increase in FSH and LH secretion. The secretion of FSH and LH at the appropriate frequency and amplitude is critical for normal fertility in the female. Gonadal steroids are produced due to positive feedback of gonadotropin stimulation. The pubertal growth start results from an increase in growth hormone production induced by sex steroids, as well as by local production of growth factors. Positive feedback leads to the onset of ovulation and menses in girls by mid-puberty or later.

Low levels of estradiol (E₂) have little effect on LH secretion and inhibit FSH secretion. High levels of E₂ induce the LH surge at midcycle, and high steady levels of E₂ lead to sustained elevated LH secretion. Low levels of progesterone (P₄), acting at the level of the pituitary gland, enhance the LH response to GnRH and are responsible for the FSH surge at midcycle. High levels of P₄ inhibit the pituitary secretion of gonadotropins by inhibiting GnRH pulses at the level of the hypothalamus. The hypothalamic–pituitary axis is described in detail in Chapter 2.

The Vagina

The vagina is a tubular muscular organ, about 7–8 cm long, and is capable of great distention (Figure 4.2). The organ extends from its external orifice in the vestibule between the labia minora of the vulva to the cervix. It is composed mainly of smooth muscle and is lined with a mucous membrane. The vaginal mucosa contains tiny exocrine glands (Bartholin’s glands) that secrete lubricating fluid during sexual response. During sexual intercourse, the vagina triggers the ejaculation of semen and serves as a receptacle for semen. The vagina also serves as the lower portion of the birth canal.

Figure 4.2 Female reproductive anatomy.

The “normal” microbiome of the vagina in nonpregnant, healthy women predominantly includes a variety of Lactobacillus species, which provide a healthy, supportive environment [2]. In healthy individuals, Lactobacillus species dominate this ecosystem at a concentration of 10⁷–10⁸ colony forming units per gram of vaginal fluid. Broadly, the vaginal microbiota (VMB) can be classified into different community groups or grades. In an analysis of VMB samples collected from healthy, nonpregnant women, Grade I, characterized by a dominance of Lactobacillus crispatus, was found in 26.2% of the sampled population, while Grades II (6.3%), III (34.1%), and V (5.3%) were characterized by a dominance of Lactobacillus gasseri, Lactobacillus iners, and Lactobacillus jensenii, respectively. These four main groups were isolated primarily from White and Asian women. Grade IV, found mainly in Black and Hispanic women, was classified by the dominance of non-Lactobacillus species, and included Gardnerella, Prevotella, Corynebacterium, Atopobium, Megasphaera, and Sneathia [3]. Lactobacilli and other bacteria metabolize glycogen to glucose and maltose and further to lactic acid, resulting in a vaginal pH of 3.8–4.4, which is defined as normal.

The Uterus

The uterus serves as the site of embryo implantation and development. In a woman who has never been pregnant, the uterus is pear-shaped and measures approximately 7.5 cm in length, 5 cm in width at its widest part, and 3 cm in thickness. The uterus has two main parts: the body at the top, and the cervix, a lower, narrow “neck” (Figure 4.2).

The uterine wall comprises three layers: an inner endometrium, middle myometrium, and an incomplete outer layer of the parietal peritoneum. The endometrium lines the mucous membrane, and is composed of three layers: a compact surface layer of partially ciliated, simple columnar epithelial cells called the stratum compactum, a spongy middle layer of loose connective tissue called the stratum spongiosum and a dense inner layer termed the stratum basale that attaches the endometrium to the underlying myometrium. Throughout menstruation and after delivery of a baby, the compact and spongy layers slough off. The endometrium varies in thickness from 0.5 mm just after menstruation, to about 5 mm near the end of the endometrial cycle (before menstrual flow). The endometrium has a rich supply of capillaries, as well as numerous exocrine glands that produce mucus and other substances onto the endometrial surface.

The myometrium consists of three layers of smooth muscle fibers that extend in all directions, and give the uterus great strength.

The peritoneum is the serous membrane covering the abdominal cavity. This peritoneal lining of the cavity supports many of the abdominal organs and serves as a conduit for their blood vessels, lymphatic vessels, and nerves.

The cavity of the uterine body is directed downward and constitutes the internal os, which opens into the cervical canal. The cervical canal forms the external os, which opens into the vagina. The mucous glands in the lining of the cervix produce mucus that changes in consistency during the female reproductive cycle. Most of the time, cervical mucus acts as a barrier to sperm. Cervical mucus becomes more slippery and facilitates the movement of sperm through the cervix around the time of ovulation.

The uterus obtains a generous supply of blood from uterine arteries, which branch off from the internal iliac arteries. Also, the ovarian and vaginal arteries anastomose with the uterine vessels, thereby serving as an additional blood source. Arterial vessels penetrate the layers of the uterine wall as arterioles and then break up into capillaries between the endometrial glands. Uterine, ovarian, and vaginal veins return venous blood from the uterus to the internal iliac veins.

As in the vagina, the “normal” microbiome of the uterus in nonpregnant, healthy women predominantly includes a variety of Lactobacillus species. The low abundance of Lactobacillus in the endometrium is associated with poor reproductive and IVF outcomes [2].

Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome, a well-established uterine anomaly, involves an uncommon variation in the prenatal development of the female genital tract. Its incidence is approximately 1 in 4000–5000 female births, and its underlying cause is unknown. The syndrome features include an absent or very short vagina and a uterus that can be absent or immaturely formed. Females with MRKH syndrome have functioning ovaries, normal external genitalia, and the genetic appearance of 46,XX female chromosome pattern. Breast development and growth of pubic hair are also typical. Associated renal and/or skeletal abnormalities are shared. MRKH syndrome is also known as Müllerian (female internal sex organs) agenesis (no growth) syndrome. The usual external appearance of MRKH females makes it difficult to diagnose until puberty – often when a girl visits a physician because she has not started to menstruate. In some cases, a young woman may have attempted unsuccessfully to have intercourse. The average age of diagnosis usually is between 15 and 18 years, although occasionally, a girl may be diagnosed at birth or during childhood because of other health problems. A pelvic ultrasound may be used to determine the presence or absence of the uterus and its condition.

The Uterine Tubes

The uterine tubes are also called Fallopian tubes or oviducts. The first correct anatomical description of uterine tubes was in 1561 by Gabrielis Fallopius. In 1566, Fallopius described the extension of these tubes from the uterus to the ovaries. Reinier de Graaf defined the function of the Fallopian tubes in 1672. The Fallopian tubes are approximately 10 cm long and are attached to the uterus at its upper outer angles. The same three layers (mucous, smooth muscle, and serous) of the uterus compose the tubes. Inflammation of the uterine tubes may lead to scarring and partial or complete closure. The pH in the female reproductive tract is graduated, with the lowest pH in the vagina (~pH 4.4) increasing toward the Fallopian tubes (~pH 7.9), reflecting variation in the site-specific microbiome and acid–base buffering at the tissue/cellular level.

The Ovary

The female gonads, or ovaries, are homologous (in origin) to the testis in the male. The adult ovaries weigh about 4–8 g each. They are paired and located on each side of the uterus (Figure 4.2). The ovary consists of three main parts: the outer cortex (the major bulk of follicles), the inner medulla, and the hilum. The hilum is the part of the ovary which attaches along its anterior margin by a double fold of peritoneum, the mesovarian ligament (mesovarium), and broad ligament. The ovarian ligament anchors it to the uterus. It includes blood support and nerves. The rete ovarii, the ovarian analog of the rete testis, is present in the hilus of all ovaries. After menopause, the ovaries typically shrink to a size approximately one-half that has been seen in the reproductive period.

The ovary is not quiescent during childhood. Follicles begin to grow at all times and frequently reach the antral stage. The lack of gonadotropin support prevents full follicular development and function. There is no evidence that ovarian function is necessary until puberty. However, before puberty, the oocytes are active and synthesize mRNAs and protein. Ovariectomy in prepubertal monkeys has indicated that the prepubertal suppression of GnRH and gonadotropins is partially dependent on the presence of ovaries, suggesting some functional activity of the ovary in childhood.

Female fertility depends on the supply and maturation of the ovarian germ cells, i.e., the oocytes, and the differentiation and proliferation of the ovarian somatic cells, i.e., granulosa cells (GCs) and theca cells (TCs). Assembly of oocytes and somatic cells into follicular structures, a process also called initial folliculogenesis, marks the last step of ovarian differentiation. During folliculogenesis, oocytes grow in size, and are surrounded by an increasing number of GC layers; from the preantral stage onward, TCs differentiate outside the follicle. GCs are delimited by a thin basement membrane and bordered outside by mesenchymal cells.

When a primordial follicle enters the growth phase, its surrounding flat GCs become cuboidal and proliferative. Follicles reach the primary (one layer of flattened GCs), preantral (number of GCs), and antral (antral cavity) stages [4]. As maturation advances, the number of granulosa layers increases, and the cells begin secreting increasing amounts of an estrogen-rich fluid that pools around the oocyte in a space called an antrum. From puberty onward, the follicle can pass into the preovulatory follicle stage in which the oocyte resumes meiosis and becomes arrested again in metaphase II. The oocyte is then ovulated in the oviduct, rendering it ready for fertilization. Subsequently, TCs and GCs differentiate into luteal cells and form the corpus luteum (CL), which produces P₄ (Figure 4.3).

Figure 4.3 Menstrual cycle.

Follicular growth and atresia are not interrupted by pregnancy, ovulation, or periods of anovulation. This dynamic process continues at all ages, including infancy, and around menopause. At puberty, ovaries contain approximately 300 000–400 000 follicles; however, from this large reservoir, about 400–500 follicles will ovulate during a woman’s reproductive years. The antral follicle count (AFC) positively correlates with age and declines at a rate of 3.8% per year [5]. Follicular decline (atresia/apoptosis) causes the elimination of more than 99% of germ cells from a cohort of the ovary.

Folliculogenesis

During the reproductive era, primordial follicles are found in clusters in the superficial cortex. They contain a primary oocyte, measuring 40–70 µm in diameter, surrounded by a single layer of flattened, mitotically inactive GCs, resting on a thin basal lamina. When follicles enter the growth phase, they increase, both by a proliferation of GCs and by an increase in the size of the oocyte. The first morphological evidence of follicular growth is the assumption of a cuboidal to columnar shape among GCs. Although primary and preantral follicles can grow in the absence of gonadotropins, optimal development may require these hormones. In contrast, beyond the antral follicle stage, follicular growth, maturation, and survival are dependent on gonadotropins [4]. When early-growing follicles become vascularized, they are directly exposed to factors circulating in the blood. Circulating FSH is the earlier regulator of ovarian folliculogenesis. However, despite the presence of FSH receptors on GCs [6], the role of FSH in sustaining early follicular growth remains unclear, as follicles at this stage seem to be unresponsive to gonadotropins [7–9]. Indeed, follicles smaller than 2 mm do not display any change in GC proliferation upon cyclic changes in FSH levels [4]. Under conditions of FSH deprivation, the development of early-growing follicles can be continued by local factors. However, it appears possible that these factors are less efficient at sustaining growth than they are when they act in synergy with FSH.

At this stage of growth, a system of gap junctions, i.e., intercellular channels that allow nutrients, inorganic ions, second messengers, and small metabolites to pass from cell to cell, appear in the GC layer. These channels are composed of connexins (Cx), with connexin 43 (Cx43) being the most abundant Cx in the ovary and expressed in the GCs from the start of folliculogenesis.

Paracrine factors and intercellular gap junctions ensure complex bidirectional communication between the oocyte and its surrounding somatic cells during folliculogenesis, for the coordinated development of both somatic cell and germ cell compartments [10–11]. In this dialog, the oocyte plays a vital role in the early stages of follicular growth on somatic cells [12]. Among the oocyte-specific factors potentially involved in GC proliferation are three members of the transforming growth factor-beta (TGF-β) superfamily, i.e., growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15) and BMP-6 [13]. Oocyte factors potentially involved in the initiation of follicular growth include KIT, a receptor activated by GC-derived KIT ligand (KL), as well as fibroblast growth factor 2 (FGF2) [14–15]. Other molecules have been reported capable of stimulating the initiation of follicular growth, with a body of evidence demonstrating a role for neurotrophic factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neutrophins [16–18]. Oocytectomy of rabbit preovulatory follicles led to the early luteinization of follicles and increased P₄ production [19]. Similarly, GCs from rat antral follicles transform into luteal cells after oocytectomy [20]. Gradually, follicles become secondary follicles, i.e., follicles with three or more complete layers of GCs surrounding the oocyte that are served by one or two arterioles, terminating in an anastomotic network just outside the basal lamina. The importance of this event is highlighted by the fact that the follicle becomes directly exposed to factors circulating in the blood. At this stage, an acellular layer, the zona pellucida (ZP), arises and encases the oocyte. Its formation is usually attributed to the GC, but the oocyte may also play a role.

Simultaneously, the surrounding ovarian stroma cells near the basal lamina become aligned parallel to each other and constitute the theca layer. As the follicle enlarges, the TCs differentiate into two parts. The outer part, named theca externa, is composed of cells that do not differ from undifferentiated TCs. In the inner part, named theca interna, some fibroblast-like precursor cells assume the appearance of typical steroid-secreting cells, also referred to as epithelioid cells. From the time of appearance of epithelioid cells, the secondary follicle is defined as a preantral follicle and constitutes the first class of growing follicles, with classification based on morphological aspects and the total number of GCs in each follicle [4]. GCs of preantral, small antral, and mid-antral follicles (5–8 mm) exclusively produce anti-Müllerian hormone (AMH) [21] and make the greatest contribution (~60%) to serum AMH. In more developed follicles, follicles >10 mm in diameter, the GCs fail to produce AMH. AMH was first discovered in follicular fluid (FF) in 1993. AMH inhibits follicular development and recruitment and suppresses primordial follicle activation. Both AMH gene expression and concentrations in FF negatively correlate with the E₂ concentration [22]. Absolute serum levels of AMH do not necessarily reflect the number of ovarian follicles; although detection of serum AMH indicates the presence of secondary or early antral follicles, one cannot rule out the presence of non-AMH-secreting primordial follicles. Patients with undetectable AMH levels have responded to hormonal treatment due to the presence of residual primordial follicles. Thus, the detection of serum AMH ensures the presence of secondary follicles but does not accurately reflect ovarian reserve.

The first evidence of antrum formation occurs in follicles measuring 200–400 µm in diameter. When small fluid-filled cavities merge to form the antrum, the follicle enters the early antral phase and possesses FF with a composition similar to that of the blood serum. From this time, the GCs surrounding the oocyte proliferate and differentiate to form the “cumulus oophorus,” which contains the oocyte in its center and protrudes into the antrum. Since there is great importance of wide interactions between the oocyte and follicular tissues, the GC–oocyte gap junctions play a critical role during folliculogenesis. Also, within the large antral follicle, the oocyte plays a role in the establishment of the various functions of the two GC subpopulations, i.e., mural GC and granulosa-derived cumulus cells (CCs). After gonadotropin stimulation, mural GCs express LH receptors [23–24], while CCs do not express LH receptors, P450, or P₄ receptors, but do synthesize hyaluronic acid, an extracellular matrix component enabling the expansion of the cumulus within the follicular cavity [25]. LH receptor concentrations are highest in the cells closest to the basement membrane and are lowest in those that surround the oocyte [26]. Growing oocytes accelerate follicular development, suggesting that the oocyte may act as a “folliculogenesis clock.”

With the accumulation of fluid in the antral cavity and proliferation of GCs and TCs, the follicle progresses at an increasing rate through following stages of development, until it reaches a size of 2–3 mm, and becomes a selectable follicle. The early-growing follicle reaches the preantral stage within 2–3 months, and 70 additional days are necessary for the preantral follicle to become early antral reaching a size of approximately 2 mm [27].

Healthy selectable follicles are detected at all stages of the menstrual cycle. During the late luteal phase, their number rises in response to elevating peripheral FSH levels [28]. At this point, there are between 3 and 11 selectable follicles per ovary in women between the ages of 24 and 33 years [29], but counts significantly decline with age. The follicle intended to ovulate during the subsequent cycle will be selected from this bank of follicles [28; 30]. Whereas early-growing follicles are unresponsive to cyclic hormonal changes, selectable follicles are more receptive to these alterations, owing to the increased expression of LH receptors [31], which heightens sensitivity to an elevating LH pulse frequency (every 90 minutes) during the early follicular phase [32], and later stages of the follicular phase (every 60 minutes) [33], as well as to the positive effect of GDF-9 on androgen production (via activity of enzymes involved in androgen production) by TCs [34]. When follicles reach the selectable phase, their GCs are characterized by increasing concentrations of FSH receptors, which are responsive to the proliferation-related FSH effects, but not to its impact on estrogen production.

The ovarian cycle can be readily assessed via serial ovarian ultrasound scans and serum hormone measurements throughout the menstrual cycle (Figure 4.4). During the menstruation, there are numerous antral follicles in the ovaries measuring 4–8 mm in diameter. These follicles develop from primordial follicles through both gonadotropin-independent and gonadotropin-sensitive phases of growth [35]. It is not very easy to determine how long this process takes, but evidence from tissue transplantation studies proposes that it is longer than 3 months [36]. These antral follicles are gonadotropin dependent and will not continue to grow without gonadotropin stimulation.

Figure 4.4 Folliculogenesis.

The rate of follicle growth can vary from cycle to cycle and from woman to woman (Figure 4.5). Differences in the length of a menstrual cycle depend on the variations in the length of early- to mid-follicular phases. However, when the leading follicle reaches 12 mm in diameter, generally on day 9 or 10 of the menstrual cycle, it continues to grow at an average of 2 mm in diameter each day. This growth is associated with rapidly increasing E₂ concentrations that exert negative feedback on the pituitary to reduce FSH secretion. Follicles larger than 12 mm in diameter will already be expressing LH receptors on their GCs, and LH will maintain follicle growth and function in the presence of declining FSH concentrations. This machinery is responsible for the follicular selection and uni-follicular ovulation; however, it means that follicles reaching 12 mm or more may also ovulate in response to an LH surge. Increasing E₂ secretion from the dominant follicle promotes a switch to positive feedback at the hypothalamus and pituitary that results in a gonadotropin surge. The dominant follicle will generally measure between 17 and 23 mm in diameter at the time of the LH surge, which usually occurs on days 14–15 of a regular menstrual cycle. Generally, the length of the menstrual cycle is 28 days (22–44 days), time to ovulation is close to 14.8 days (9–33 days), and the postovulatory phase is close to 13.2 days (7–17 days).

Figure 4.5 Folliculogenesis.

At the late follicular phase, LH secretion frequency accelerates to occur every 65 minutes. The major effect of LH binding is the activation of G protein, which, in turn, activates adenylate cyclase and increases the production of cAMP [37]. The cAMP-dependent protein kinase A (PKA) pathway is significant; however, LH receptor binding also activates Phospholiase C/inositol phosphate signaling independent of the cAMP/PKA pathway. Other cellular pathways involving ERK and AKT are associated with LH receptor signaling and may have a part in non-steroidogenic processes, such as cell proliferation, differentiation, and cell survival in the follicle [38]. At least 20–30 isoforms of both FSH and LH circulate in the blood during the menstrual cycle [39], each of which may bear different bioactivity.

The dominant healthy follicle appears to be the selected follicle. It grows at a quicker rate than do other subordinate follicles [27], contains a detectable level of FSH [40], and differs substantially from selectable follicles by a concomitant increase in E₂ receptors within the GC and the E₂ levels within the FF.

In addition to E₂, FF contains a broad profile of active compounds such as classical hormones (FSH, LH, growth hormone, prolactin, P₄, prostaglandins [PGs], corticoids), TGF-β superfamily members (inhibin, activin, AMH, BMP-15), growth factors (insulin-like growth factor-I [IGF-I], IGF-II, tumor necrosis factor alpha, FGF2), interleukins (IL-1, IL-2, IL-10, IL-12), proteins and peptides (α-fetoprotein, leptin, endothelin, oxytocin, vasopressin, oocyte maturation inhibitor, homocysteine, β-endorphin, lactoferrin, angiotensin II, prorenin), and others.

At ovulation, the cumulus–oocyte complex is released from the ruptured follicle. As the oocyte does not express LH receptors, its maturation is associated with ovulation and mediated by the neighboring GCs and CCs. The LH surge causes a breakdown of cellular communications between the oocyte, CCs and GCs, and because GCs are involved in the maintenance of oocyte arrest, maturation is thereby stimulated [41].

Oocyte Growth

The oocyte, which is present in the ovary of the mammalian female from birth, is arrested at prophase (diplotene stage with four full sets of chromosomes) of the first meiosis. During early follicle development the oocyte grows at the quickest rate, increasing its diameter from approximately 40–70 μm in large primary follicles to around 100 μm in early antral follicles. Next to this stage of development, the oocyte diameter increases at a prolonged rate, reaching approximately 140 μm (120–150 μm) in the preovulatory follicle.

The oocyte can already control follicular growth during early follicle development. Deletion of Pten in the oocyte leads to enhanced activation of the primordial follicle, while the deletion of Pdk1 reduces primordial follicle survival and accelerates ovarian aging [42]. The oocyte, which might possess FSH receptors [43–44], begins to produce factors such as GDF-9 and BMP-15 that are specific regulators of follicle development. Both GDF-9 and BMP-15 bind to specific TGF-β superfamily receptors located on the membrane of GCs or CCs. The oocyte can control its growth through GDF-9, which downregulates KL expression in GCs, which, in turn, regulates BMP-15 expression in the oocyte [45]. Just as the oocyte controls follicular development, follicular cells can also control oocyte growth. Activin secreted from GCs can negatively control oocyte development.

The oocyte is encased by the ZP. The human ZP is a matrix composed of four glycoproteins (ZP1, ZP2, ZP3, and ZPB) [46], which surrounds all mammalian oocytes and mediates several essential roles such as binding sperm in a species-specific manner, inducing the acrosome reaction, preventing polyspermia, and protecting the embryo before implantation. The space formed between the oocyte membrane (oolemma) and the ZP of the oocyte is called the perivitelline space. This space does not develop in the oocyte at the germinal vesicle (GV) stage and could be seen after the oocyte completes its maturation. CCs communicate with the oocyte via transzonal projections (microvilli and cilia), which penetrate the ZP to form oocyte–CC junctions [47].

Small molecules are transferred in both directions and impact both the oocyte and the follicle. To maintain meiotic arrest, GCs produce natriuretic peptide precursor type C (NPPC) that binds NPPC receptors (NPR2) on CCs, resulting in the production of cGMP, which is then transferred to the oocyte via gap junctions to inhibit phosphodiesterase 3 A activity, thereby preventing hydrolysis of cAMP, ensuring meiotic arrest. CCs also produce cAMP in response to FSH and LH stimulation. In turn, cAMP activates PKA, which inhibits transition of the oocyte from the GV to prophase of the first meiosis (M I) stage, ensuring the meiotic arrest.

Follicular Steroidogenesis

Steroidogenesis, beginning with the uptake of cholesterol from circulating lipoproteins and storage of cholesterol as esters in cellular lipid droplets, is crucial for the synchronization of follicle growth and oocyte development. The theca interna utilizes cholesterol for de novo synthesis of pregnenolone, which is then enzymatically converted into P₄, and then into androstenedione [48]. Androgen secretion by TCs under LH stimulation appears to result from the activity of mitochondrial enzymes, such as cholesterol side-chain cleavage (P450scc), which converts cholesterol to pregnenolone. Then, 3β-hydroxysteroid dehydrogenase (3β-HSD) converts pregnenolone to P₄, which is then converted to androstenedione and testosterone by 17ɑ-hydroxylase/lyase (P450c17ɑ/lyase), respectively. Androgen receptor-mediated events (increased expression of FSH receptor, IGF-I receptor, and IGF-I in GCs) allow the growing follicle to interact with FSH, growth factors, and oocyte-derived factors to promote GC differentiation into mural and cumulus cell layers. The androgens cross the basement membrane into the GC, where they are converted to estrogens by the actions of aromatase (CYP19A1) [49], which is absent in TCs. These changes lead to maximal intrafollicular E₂ concentrations that coincide with the plasma E₂ peak. From the early to late follicular phase, E₂ levels in human FF increase from 658 ng/ml to 2583 ng/ml. At the same time, intrafollicular concentrations of 17ɑ-OH progesterone and P₄ increase; these progestins are mainly produced by TCs, since 3β-HSD is only slightly expressed in GCs [50] and P450c17ɑ/lyase is absent. This two-cell (TC–GC) steroidogenic process, facilitated by GC-derived paracrine factors that promote TC P450c17 activity, ensures adequate aromatizable androgen production for continuous E₂ synthesis, despite declining serum FSH levels with follicle growth (Figure 4.6). E₂ is crucial for proper oocyte development because immature human oocytes have E₂-dependent, calcium-mediated mechanisms of cytoplasmic maturation that are susceptible to inhibition of androgen levels. Immature human oocytes from small, hyperandrogenic polycystic ovary syndrome follicles exhibit lower rates of in vitro maturation, fertilization, and embryo development.

Figure 4.6 Steroidogenesis of the estrous cycle.

Atretic Follicles

Of the original primordial follicles present at birth, approximately 0.1% mature to the point of ovulation. The remaining 99.9% undergo atresia, a process that begins before birth and continues throughout adolescent and reproductive life, but which is most intense immediately after birth and during puberty and pregnancy [51–52]. Factors that initiate atresia and determine which follicles will ultimately undergo atresia are unknown. Atresia of early follicles (primordial and preantral) begins with degeneration of the oocyte, manifested by nuclear changes. Degeneration of the GCs soon follows and the follicle disappears without a trace. In contrast, atresia of follicles that have reached the antral stage of development is more complex and variable but ultimately leads to obliterative atresia and the formation of scar, corpus fibrosum [53]. The earliest evidence of this process is the mitotic inactivity of the GC. Some follicles may persist as atretic cystic follicles for an indefinite time interval.

Maturation of the Preovulatory Follicle

From the time it is selected, the follicle destined to ovulate enlarges significantly, from 6.9 ± 0.5 mm (diameter, counting from 2 to 5 × 10⁶ GCs) during the early follicular phase, to 18.8 ± 0.5 mm (50 to 100 × 10⁶ GCs) during the late follicular phase, which demands a high growth rate during the early follicular phase. This growth is accompanied by a massive generation of radicals that require protection against free radicals. During this final stage of growth, the GCs are subjected to marked morphological alterations, resulting from modulation of the cytoskeleton organization [54].

From the time it is selected, the follicle destined to ovulate shows marked changes in its steroidogenic activity. On the one hand, the production of androgens by the TCs is enhanced in response to increasing production of inhibin A [55–56], which strongly stimulates P450c17ɑ/lyase. On the other hand, aromatase expression, which is detected only in GCs of follicles ≥10 mm [57], is stimulated by increased production of IGF-II [58], while NGF stimulates both FSH receptor synthesis and E₂ production [59]. As the follicle matures, the circulating levels of FSH decline in response to E₂ and inhibin produced by the follicle itself. With the rise in plasma LH levels, the concentration of LH receptors within the GCs, and the binding ability of the preovulatory follicle is elevated [31; 60]. The highest concentrations of LH receptors were found in mural GCs close to the basement membrane, while lower concentrations were measured in GCs located closer to the antrum; CCs and the oocyte lack LH receptors. At this stage, the preovulatory follicle continues to mature under the influence of intrafollicular FSH and E₂ [61–62], thereby maintaining a high E₂:androstenedione ratio [63].

During maturation, the preovulatory follicle becomes a highly vascularized structure [64]. The considerable increase in thecal vascularization occurs as a result of active endothelial cell proliferation in thecal layer blood capillaries, induced by angiogenic factors, such as vascular endothelial growth factor (VEGF) [65].

Shortly before ovulation, the preovulatory follicle reaches a diameter of 15–25 mm [62] and partially protrudes from the ovarian surface at a point that represents the eventual rupture point, or stigma.

Ovulation

Ovulation is the release of an oocyte–cumulus complex from the stigma of the follicle from a single ovary. After the oocyte–cumulus complex is released, it travels down the Fallopian tube, where fertilization by a sperm cell may occur. Ovulation typically occurs in the middle of a woman’s menstrual cycle, but the timing of the process varies for each woman, and it may even vary from month to month.

High circulating E₂ levels, released by the dominant follicle, initiate a preovulatory surge of plasma LH [66]. Serum E₂ peaks approximately one day before the LH surge and 37 hours before ovulation. The LH surge generally occurs on days 14–15 of a typical 28-day menstrual cycle (may occur between day 9 and 30 of a cycle). LH surges are extremely variable in configuration (one peak or few peaks), amplitude, and duration (1–10 days) in healthy, contraceptive-free, regularly cycling women with proven fertility [67–69]. The day of the LH surge is believed to be the day of maximum fertility. Ovulation has been estimated to occur 36 ± 5 hours after the onset of the LH surge, 24–36 hours after the E₂ peak, and 10–12 hours after the LH peak [66; 70]. Ovulation is believed to have occurred if the follicle reached a mean diameter of 18 mm to 26 mm and changed in shape or sonographic density [71]. The preovulatory LH rise appears to have a circadian rate, occurring in the early morning in the majority of women. Within 6 hours of the start of the LH surge, the GCs show signs of early luteinization and begin to secrete P₄, initially into the FF, but later into the ovarian vein and general circulation. Circulating P₄ impacts the temperature regulation center of the brain, resulting in a rise in body temperature. The primary reason for this phenomenon is an increase in the production and secretion of norepinephrine, which is a thermogenic neural hormone.

The LH surge induces distinct processes involved in ovulation, which can be split into three components. One component of ovulation is a reactivation of oocyte maturation wherein the oocyte, which has been maintained in the diplotene stage of prophase, progresses to metaphase of the second meiotic division. Another component is GC luteinization, whereupon the GCs develop the enzymatic machinery to synthesize P₄. The third component of the ovulatory response induced by the LH surge is follicular rupture, which is an inflammatory process involving the breakdown of the apical follicular wall (Figure 4.7) [72].

Figure 4.7 Ovulation.

The breakdown of the apical follicle wall depends on several events, one of which is constriction [73–74], which can be induced by PGs [75–76]. The LH surge upregulates PG synthesis in GCs, reaching peak concentrations just before ovulation [77]. Oxytocin is another great constrictor and its concentrations are elevated in the FF in response to luteinization [78–79]. Metalloproteinases have also been proposed to be involved in this process [80].

The LH peak induces a significant disruption and decline in gap junctions [54], leading to dissociation of mural GCs and expansion of the cumulus–oocyte complex, which constitutes a highly specialized inflammatory-related process involving activation of a variety of genes, and that is obligatory for successful ovulation [81–82]. LH-induced gap junction gating in ovarian follicles comprises two steps. First, the phosphorylation status of Cx43 protein (gap close/open) is modified immediately upon the LH surge. Later, Cx43 protein concentrations are reduced due to the attenuation of its gene expression at the transcriptional, translational, and post-translational levels [83]. Only gap junctions between GCs and CCs, which are composed of Cx43, are affected, whereas gap junctions between CCs and oocyte remain open because they are formed of Cx37. Gap junction closure between GCs–GCs and GCs–CCs reduces the supply of cGMP to the oocyte, thereby activating intra-oocyte phosphodiesterase that breaks down intra-oocyte cAMP. Disconnection of the gap junctions reduces the cAMP supply to the oocyte and, therefore, reduces the level of cAMP and switches up maturation-promoting factor, which then reinitiates meiosis and oocyte maturation. LH also inhibits adenylate cyclase and reduces the production of cAMP, which has an added effect on oocyte maturation.

Cumulus–oocyte complex expansion at ovulation requires the action of local factors produced by GCs and the oocyte in response to indirect actions of LH. These include epidermal growth factor (EGF) family members such as amphiregulin, epiregulin [84], and β-cellulin, which are produced by the mural, but not cumulus, GCs in response to LH and are mandatory for CC expansion [85]. Prostaglandin E (PGE) is also involved in this process [86]. Oocyte release of both BMP-15, whose production may be upregulated by FSH [87], and GDF-9, which stimulates, among others, expression of hyaluronan synthase and cyclo-oxygenase 2 [88], is associated with cumulus expansion.

Extragonadal influence on the ovulation process has also been reported. For example, triiodothyronine (T₃) was found to be an important agent of ovulation. It was shown that women with T₃ concentrations >80 ng/dl fail to ovulate [89].

Ruptured released oocyte–cumulus complex at ovulation is transferred to the infundibulum of the Fallopian tube (oviduct), and then moves to the ampulla region through the surface of the ciliated oviductal epithelium and awaits the sperm cells. The FF released by ovulation into the oviduct and peritoneal cavity may have an essential physiological function through its direct stimulatory effect on the oviduct (constriction and epithelial cilia motility) that assists in oocyte–cumulus complex advancement.

Maturation of the Preovulatory Follicle during the Gonadotropin Surge

After the midcycle gonadotropin surge, dramatic metabolic and morphological changes simultaneously occur within the preovulatory and ovulatory follicle that switches from an E₂-producing to a P₄-producing structure. Progesterone receptors appear in GCs, whose proliferation is arrested at this stage [90]. Also, some hours before ovulation and in response to angiogenic factors produced by GCs [65], the granulosa wall, which was avascular before the midcycle gonadotropin surge, appears to be changed after invasion of blood vessels originating from the theca layer.

As a result of the breakdown of the basal lamina during the LH surge, the cholesterol substrate required by GCs to generate progestin, and which is provided to cells in the form of lipoprotein-bound cholesterol, can now reach the follicle via the blood supply [54]. After the gonadotropin surge, both P450scc [57] and 3β-HSD [50; 91] appear in GCs, where the levels of adrenodoxin and steroid acute regulatory (StAR) proteins, which support the entrance of cholesterol into mitochondria, are significantly increased. The production of steroids strongly increases, rising from a mean FF concentration of 4800 ng/ml before the surge, to 11 000 ng/ml after the surge. While follicular progestin production significantly increases, that of androgens and E₂ dramatically declines. The collapsed ovulating follicle develops into a new endocrine gland, called the corpus luteum (CL).

Follicle after the Gonadotropin Surge (Corpus Luteum)

After the LH surge, the postovulatory follicle develops into the CL, or yellow body (the color originated from carotenoids). The ovarian CL was first named by Marcello Malpighi and then described by Regnier de Graaf in the 1600s. The young CL has a cystic center filled with FF and a focally hemorrhagic coagulum (Figure 4.8). The mature CL (approximately seventh day postovulation) shows increased tissue mass, a smaller cystic center, and varies in size and appearance, but usually measures between 10 mm and 30 mm in diameter [92], and is of a volume of 4.87 cm³ [93]. Capillaries from the theca interna layer penetrate the granulosa layers and reach its central cavity. Upon potent angiogenesis the theca interna becomes incorporated into the GC layers and forms a new cellular composition, remodeled and different from the layered structure of the preovulatory follicle. The CL is the most active endocrine gland in the body and is essential for the establishment and maintenance of pregnancy in most mammals, including humans. Because of its unique role, CL formation, maintenance, and regression are tightly regulated. Survival and continued function of the CL are both required throughout the first weeks of pregnancy, after which the placenta becomes responsible for the maintenance of gestation. The CL is generally considered to go through three phases during its life cycle: formation, maintenance, and regression. One additional potential phase is rescuing (sustained function) during pregnancy.

Figure 4.8 CL development after ovulation.

The development, maintenance, and steroidogenic function of the CL during the menstrual cycle are generally thought to be LH dependent [94]. During the luteal phase, there is a reduction in the frequency of LH pulses from the pituitary, from a pulse every 100 minutes in the early luteal phase to every 200 minutes in the mid-late luteal phase just before the onset of luteal regression. LH receptors are expressed in the CL [95], and LH is responsible for initiating the differentiation of the somatic cells of the ovarian follicle, TCs and GCs, into the small luteal (SLC) and large luteal (LLC) steroidogenic cells of the CL [96–97]. FSH receptors have also been identified in the early CL, although the role of FSH in luteal function is not clear. After ovulation, peripheral LH, FSH, and E₂ levels fall, but the LH concentration (with pulses approximately every 4 hours) is sufficient to maintain the CL. The newly formed CL is a highly active gland with a marked capacity for steroid synthesis. Opposite to the follicle, CL shifts from E₂ to P₄ production. Progestogenic stimulation provides an intrauterine environment that supports implantation and pregnancy, playing a vital role in the fate of the embryo. The CL of pregnancy may be indistinguishable from the regular CL but is usually larger and bright yellow in contrast to the orange-yellow of the regular late luteal CL.

P₄ secretion is supported by external LH and internal luteotropic factors (such as prostaglandins PGI₂ and PGE₂) produced by luteal cells. P₄ raises the set-point of the hypothalamic thermoregulatory center, and serum P₄ levels raise the basal body temperature. In contrast to the ovarian follicle, where TCs primarily produce P₄, in the CL both the GC-derived LLC and TC-derived SLC produce P₄. The process includes cholesterol transport into the inner mitochondria, mediated by StAR, conversion of cholesterol to pregnenolone by the P450scc complex, and conversion of pregnenolone to P₄ by 3β-HSD. In most species, aromatase expression is absent throughout the luteal life span, whereas in primates, the CL reacquires the ability to generate moderate amounts of E₂ [98]. Insufficient P₄ secretion early in the first trimester is associated with pregnancy loss and is attributed to premature loss of luteal function.

In the absence of pregnancy, the CL will regress (luteolysis), and the next female cycle will begin. Luteolysis is defined as the loss of steroidogenic function (functional luteolysis) and the subsequent involution of the CL (structural luteolysis). During luteolysis, the CL undergoes dramatic changes in its steroidogenic capacity, vascularization, immune cell activation, extracellular matrix composition, and cell viability. The process of luteolysis is associated with a marked reduction in luteal P₄ production and the loss of luteal steroidogenic cells. Vasoconstriction of small blood vessels in the CL is associated with reduction in blood supply and then with loss of luteal blood supply. The interruption of luteolysis allows the CL to support pregnancy. This CL-supporting agent is human chorionic gonadotropin (hCG) secreted from the developing embryo and which binds to the LH receptors.

The structure and function of the CL are dependent on the development of vasculature via angiogenesis. The establishment of the luteal vascular network begins in the preovulatory follicle and is stimulated by the LH surge. Following ovulation, the CL undergoes extremely rapid growth that is only matched by the fastest-growing tumors [99], alongside intense angiogenesis, higher than that seen in the most aggressive solid tumors. Not surprisingly, the majority of the proliferating cells in the early CL are of vascular origin, with proliferation rates exceeding 25% [100]. Luteal angiogenesis requires highly coordinated interplay between endothelial and steroidogenic cells, as well as fibroblasts and pericytes, to create an extensive and complex vascular network that is essential for luteal function (Figure 4.9). FGF2 and VEGF, which are potent mitogens of vascular endothelial cells (ECs) and stimulators of EC migration and survival [101–102], are the pro-angiogenic regulators of luteal angiogenesis. Hypoxia inducible factor-1 was found to be the most potent transcription factor of VEGF [103]. However, numerous other factors are expressed by several luteal cell types and also bear important modulatory functions. VEGF is secreted from luteal steroidogenic cells and acts on receptors expressed on ECs [104]. LH-dependent VEGF expression is involved in maintaining the structural and functional integrity of the CL in the normal luteal phase. In simulated early pregnancy, hCG promotes additional VEGF synthesis, and further luteal VEGF secretion, resulting in a second wave of angiogenesis [102]. Regulation of vascular permeability is a key function of the ECs and is important in the supply of nutrients/hormones to the luteal tissue.

Figure 4.9 Cellular composition of the CL. ET-1, endothelin 1; PGI₂, prostaglandin I₂ (prostacyclin).

Luteolysis occurs in the nongravid uterus and it is inhibited in the presence of embryonic signals [105]. Uterine-derived (or exogenous) prostaglandin F_2α (PGF_2α) initiates luteolysis [106] through a countercurrent system between the uterine vein and the ovarian artery, and rapidly reduces luteal P₄ secretion, within several hours [107]. PGF_2α acts directly on luteal steroidogenic and endothelial cells, which express PGF_2α receptors (PGFR), or indirectly on immune cells lacking PGFR, which are activated by other cells within the CL. Significant increases in the number of leukocytes [108] and inflammatory cytokines are involved in luteal regression [109]. PGF_2α stimulates locally (intra CL) to produce factors such as endothelin-1, angiopoietins, FGF2, thrombospondins, TGF-β1, and plasminogen activator inhibitor-B1, which act sequentially to inhibit P₄ production, angiogenic support, cell survival, and extracellular matrix remodeling, and induce luteal vasoconstriction to accomplish CL regression [110–114]. There is progressive fibrotic connective tissue replacement of the cellular composition of the CL, shrinkage over a period of several months, and final conversion of the CL to a corpus albicans (white body).

Although LH concentrations and pulse frequency are reduced during the luteal phase, luteolysis occurs in the presence of maintained LH concentrations. Expression of LH receptors is maintained across the luteal phase and luteolysis is initiated in the presence of LH receptors [115]. During luteolysis, there is a decline in luteal hormones, which leads to destabilization of the endometrium and induction of menstruation [116]. The fall in luteal hormones increases pituitary gonadotropin secretion, which stimulates the growth of the small antral follicles present in the ovaries at menstruation, resulting in the initiation of the follicular phase of the ovarian cycle.

Menstruation

During the proliferative phase of the cycle, the cells of the endometrium proliferate. The preovulatory LH surge induces endometrial gland and arteriole growth and development, and coil, both factors that contribute to endometrial thickening. The increasingly coiled endometrial glands begin to secrete nutrient fluid (secretory phase), a process that is obligatory for embryo receptivity.

Physiologically, menstruation is the process whereby the superficial or functional layer of the endometrium lining the uterine cavity degenerates and is removed from the uterine lumen towards the end of the luteal phase of a nonpregnant cycle. Within 5–6 days, the old lining is removed, and a new lining is generated.

Two hypotheses have been proposed for the initiation of menstruation: the vasoconstrictor hypothesis, which emphasizes the actions of PGs and endothelins, and the inflammatory hypothesis, which underscores the role of inflammatory cells and matrix metalloproteinases (MMPs). Intrauterine PGF2α causes constriction of muscle in the walls of the tightly coiled arterioles, yielding endometrial ischemia, menses, and increased uterine contractility. In parallel, lysosomal enzymes can be secreted by macrophages at menstruation. There is also evidence of a critical role of matrix MMPs in tissue breakdown associated with menstruation.

Repair of the endometrium begins as early as 36 hours after the onset of menstrual bleeding, while menstrual desquamation is still in progress, emphasizing the highly focal nature of the degradative and repair processes. During the proliferative phase, it is estrogens that regulate endometrial regeneration, which includes cessation of bleeding, re-epithelialization of the luminal lining, initiation of stromal tissue growth, and vessel repair. Regeneration is completed within 140 hours.

Between the mid-nineteenth and mid-twentieth centuries, the average age of menarche in Europe declined steadily from 17 to 13 years. The average modern woman menstruates between 350 and 400 times. The mean menstrual blood loss per cycle is 43 ml, with 10% of women losing 100 ml blood per month. Therefore, the average woman loses more than 20 liters of menstrual blood during her reproductive life.

Incomplete Meiosis

After the LH surge, the oocyte resumes meiosis and progresses from first meiotic prophase (prophase I) to the second metaphase at the time of ovulation, and regulates both cytoplasmic and nuclear maturation.

The introductory stages of meiosis are similar to those of mitosis; the cell grows during G1, duplicates all of its chromosomes during the S phase, and prepares itself for division during G2. Upon completion of G2, the oocyte enters prophase I of meiosis. Normal gene expression during meiosis (oocyte maturation) and early embryonic development (until the zygotic gene activation) requires well-timed activation of specific maternal mRNA translation. These mRNAs accumulated in the oocyte during the first meiotic arrest at prophase I.

Cytoplasmic polyadenylation element-binding group of proteins (CPEB) is an additional agent important for oocyte meiotic maturation. The CPEB induces cytoplasmic polyadenylation of RNA molecules, thereby promoting translational repression or activation. Deletion of CPEB leads to impaired synaptonemal complex formation, and subsequently to meiotic arrest at the pachytene stage of prophase I. The preovulatory follicle holds the oocyte in a state of arrest at the diplotene stage of prophase I from the moment the fetal gonads form.

Prophase I can occupy 90% or more of the duration of meiosis. The nuclear envelope of the GV oocyte remains intact and only disappears when the meiotic spindle begins to be formed, as prophase I gives way to metaphase I (MI oocyte) (Figure 4.10).

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1. Chapter 6 – Hormonal Ovarian Treatment
2. Chapter 8 – Ovum Pickup (OPU)
3. Chapter 13 – In Vitro Maturation (IVM)
4. Chapter 14 – Biopsy of Testicles

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