Anatomy & Physiology for Midwives 3: Third Edition

Chapter 4. Reproductive cycles

Learning objectives

• To describe follicular development, ovulation and subsequent events in the ovary.

• To describe the hormonal changes in the non-fertilized menstrual cycle.

• To outline the principles of hormonal regulation of reproduction and to identify factors that affect this regulation.

• To describe the effects of the hormonal changes on the female reproductive system.

• To relate the cyclical fluctuation in hormone levels to other changes in female physiology.

• To describe how hormonally induced contraception works.

• To describe the hormonal changes of menopause and how they affect fertility and well-being.

Introduction

The function of the ovaries is to release female gametes or ova (singular: ovum or ‘egg’; see Box 4.1) and to produce the steroid hormones oestrogen and progesterone. Relatively few oocytes (immature ova) are produced during a woman's reproductive life compared with the number of male gametes (spermatozoa). The ovarian follicles produce all the oocytes, steroid hormones and inhibin (a cytokine which inhibits synthesis and secretion of FSH). The cyclical pattern of hormone release has cyclical effects on the whole body, and behaviour, of the woman. The effects are particularly pronounced on the genital tract, facilitating its functions in gamete transport and the implantation and development of the conceptus. The first part of the cycle, the follicular phase, is dominated by the release of oestrogen produced by the developing follicles (Fig. 4.1). This oestrogen-dominant phase prepares the woman for ovulation, receipt of the sperm and fertilization of the oocyte. In the second half of the cycle, the luteal phase, the effects of progesterone are dominant. The physiological changes in this phase of the cycle prepare the woman's body for pregnancy and promote implantation and nurture of the conceptus should fertilization be successful. Progesterone is secreted from the corpus luteum; thus, this phase of the cycle is known as the luteal phase.

Box 4.1

Note about terminology

Although the terms are often used interchangeably, the term ‘oocyte’ refers to the developing gamete within the ovary, whereas the term ‘ovum’ (plural ova) refers to the gamete after ovulation had occurred. Oogenesis refers to the process of formation (mitotic division) of oocytes from oogonia (formed from the primordial germ cells). The primary oocyte is an oocyte which has undergone mitosis and entered meiosis (which is arrested in the 7th month of gestation). By the time the female infant is born, all of her oogonia are primary oocytes in meiotic arrest. The secondary oocyte is formed (together with the first polar body) when the first meiotic division is completed just before ovulation. So the ovum is the egg after the second meiotic division, when the second polar body is extruded. As fertilization triggers the completion of the second meiotic division, the newly-ovulated female gamete does not stay an ‘ovum’ very long as the sperm has already penetrated the ovum; once the chromosomes from the egg and sperm are united, the fertilized ovum is correctly known as an embryo. The one-celled embryo is also referred to as a zygote. At the 16-cell stage, the embryo is called a morula (Latin for mulberry). When the fluid-filled cavity (blastocoele) forms in the embryo separating the trophoblast cells from the inner cell mass, the embryo is called a blastocyst.

Chapter case study

Zara is now 12 weeks pregnant and has just attended the midwives' clinic to arrange her 12-week nuchal fold scan at her local maternity unit. Zara is quite excited because not only is her twin sister pregnant but her husband's sister and two of her best friends as well are pregnant and all the babies are due within the same 2 weeks. They, and their partners, had all been working abroad together doing voluntary work over a period of 1 year and had lived together in shared accommodation.

They had helped build and set up an orphanage for children whose parents had died from AIDS-related illnesses in an African country, and had only returned home a week before Zara's pregnancy was confirmed.

• What possible explanation could account for all five of the women conceiving together?

• What environmental factors could have influenced the menstrual cycles of the women and what possible physiological mechanisms may have resulted in the group apparently ovulating simultaneously?

Ovulatory cycles usually have a duration of 24–32 days; the follicular phase is 10–14 days and the luteal phase between 12 and 15 days. Longer cycles usually have a prolonged follicular phase and delayed ovulation.

The follicular phase

The stages of cell division leading to the production of the female gamete begin early in fetal life. The process is halted until puberty when ovulation begins, and then halted again until fertilization. The developing egg is called an oocyte; it differentiates into a mature haploid ovum (or egg). Although dramatic progress in the development of an oocyte takes place during the follicular phase, the development of the follicle begins about 3 months prior to the menstrual cycle in which it is released at ovulation (Fig. 4.2). Folliculogenesis begins with the recruitment of primordial follicles from the ovarian pool; the signals which initiate recruitment are unknown. At the beginning of the cycle, a number of primary follicles are recruited to undergo initial development, stimulated by luteinizing hormone (LH). These secondary follicles express follicle-stimulating hormone (FSH) receptors. One follicle is selected to continue development as the dominant follicle. It is this follicle that releases the ovum at ovulation. The remnants of the follicle then become the corpus luteum.

In the female fetus, the primordial germ cells migrate to the ovary at 21 days postfertilization and become oogonia. They proliferate by mitotic division and then differentiate into primary oocytes; by 20 weeks of gestation, in each ovary, there are about 10 million primary oocytes ready to enter meiotic division (see Chapter 5). The oocytes degenerate throughout fetal life so the female neonate is born with a finite number of 250000–500000 oocytes; this is all the oocytes she will ever have. Degeneration of the oocytes continues throughout postnatal life; there are fewer than 200000 oocytes left at puberty and numbers continue to fall. However, if one assumes a reproductive life of about 35–40 years, the maximum number of ova released from the ovaries will be no more than a few hundred. Although it was thought that the oocytes number was established during fetal development, it is now suggested that some ovarian oogonia still unassociated with follicular cells persist from the fetal period and can undergo mitosis thus acting as a stem cell population of oocytes in postnatal life (Johnson et al., 2004).

Meiosis is a specialized cell division involved in the production of gametes (see Chapter 7, p. 149, for a detailed description). In it, the number of chromosomes is reduced from 46 (known as the diploid number) to 23 (the haploid number). In the first part of meiosis, the DNA replicates, so each chromosome is composed of two chromatids. The homologous chromosomes pair up along their long axes and crossing over occurs so the chromosomes exchange genetic material. In humans, two or three exchanges of DNA occur per chromosome pair ensuring that the combination of genes will be unique (Box 4.2). This is achieved by areas of fusion (called chiasmata) forming between adjacent chromosomes (see Chapter 7). The oocyte remains arrested in the prophase stage of division I of meiosis for a number of years until sexual maturity (Fig. 4.3). During this time, the primary oocytes synthesize the extracellular matrix and the secretory vesicles. Arrested meiosis provides a specialized mechanism to allow the oocyte to grow while it contains duplicate copies of chromosomes and, therefore, twice as much DNA is available to direct synthesis of RNA as in a somatic cell. The hormones of puberty release the oocyte from the first meiotic block; this process is called oocyte maturation. The oocyte resumes division I of meiosis. The chromosomes re-condense, the nuclear envelope breaks down and the meiotic spindle forms. The chromosomes then segregate into the two daughter nuclei. The cytoplasm divides asymmetrically to form a large secondary oocyte and a small polar body, each of which contains 23 chromosomes as pairs of chromatids. Meiotic division then arrests for a second time, so the oocyte released at ovulation has still not completed meiosis. Activation of the oocyte and release from the second meiotic block occurs at fertilization. In the second meiotic division, the sister chromatids finally segregate into two nuclei and the cytoplasm again divides asymmetrically, this forming the mature ovum and a second polar body, each with 23 chromosomes. Because both divisions of the cytoplasm are asymmetrical, the ovum is large. Effectively, the polar bodies are small packages of cytoplasm which contain discarded chromosomes. During in vitrofertilization (IVF), observing the presence of the second polar body indicates that fertilization has occurred (see Chapter 6).

Box 4.2

Gamete formation

• Cell proliferation (mitosis)

• Genetic reshuffling and reduction (meiosis)

• Packaging of chromosomes

• Gamete maturation

In the female, mitosis of the gametes stops in the fetal period, whereas in the male, mitotic division of gametogenesis begins at puberty and continues until senescence (see Box 4.3). The termination of mitosis in the female, and entry into meiosis, is under the control of protein complexes, maturation-promoting factor (MPF) and cytostatic factor (CSF), which regulate the oocyte's progression through meiosis (Dale et al., 1999). MPF phosphorylates multiple proteins involved in chromosome condensation and the breakdown of the nuclear envelope. The hormones of puberty increase MPF activity at the exit of the first meiotic block. It is hypothesized that the second meiotic block occurs because the maturing oocyte accumulates CSF which inhibits cell division. Oocyte activation and the release from the second meiotic block are thought to be triggered by a component of the sperm that enters the oocyte at fertilization and causes CSF degradation.

Box 4.3

Oogenesis compared with spermatogenesis

• Mitosis: fetal in woman; after puberty in man

• Meiosis: halted in woman; can last many years

• Relatively few oocytes released

• Release is episodic at ovulation; not a continuous stream

• Organization is comparable to testis (stromal tissue containing primordial follicles, tubules) and glandular tissue (interstitial glands, Leydig cells)

• Mitotic proliferation is less

• Time-course of gamete production is much longer

Both premature arrest at the end of oocyte maturation and parthenogenic release from meiotic arrest are thought to cause infertility. As women age, the extended duration of arrested meiosis, which may last for over 50 years, probably results in the meiotic spindles (see Fig. 7.11, p. 153) becoming increasingly fragile; this leads to an increased rate of abnormalities such as Down's syndrome and failed implantation.

Development of a mature female gamete depends on complex interactions between the developing gamete and the surrounding cells forming the outer layers of the follicle. Mitosis is completed during fetal development. During the first meiotic prophase, the primordial germ cells stimulate organization of the surrounding cells to form the granulosa cells (flattened cuboidal epithelial cells) which condense and encircle them forming the primordial follicles. The follicular cells secrete a basement membrane around the outside forming a cellular unit (Fig. 4.4). These granulosa cells are connected to the oocyte by gap junctions through which nutrients can be transported. The primitive oocyte therefore has two layers and is about 18 μm in diameter. A few follicles may resume development spontaneously and incompletely throughout fetal and neonatal life. However, regular recruitment of the primordial follicles into the pool of growing follicles begins at puberty when levels of FSH increase, so the primordial follicles containing oocytes may stay arrested in meiosis for decades.

From puberty, a few primordial follicles spontaneously restart their development each day, forming a continuous stream of growing preantral or primary follicles. Most of these early follicles fail to develop fully and undergo atresia (fail to develop any further); less than 0.1% of follicles will ovulate and develop into a corpus luteum. The granulosa cells of follicles undergoing atresia accumulate lipid droplets and reduce protein synthesis. Both the granulosa cells and the oocyte become apoptotic (undergo ‘programmed cell death’). White blood cells invade the dying follicular cell mass and scar tissue forms.

As the majority of the follicles regress rather than progress through development, the ovary has a dense population of atretic follicles resulting in an irregular corrugated outer surface of the ovary. The development of primordial follicles into primary or preantral follicles takes about 85 days; the preantral phase is the longest phase of development of the oocyte. Initiation and progress through this early follicular development is independent of pituitary hormones but there may be paracrine regulation by cytokines (see Chapter 3), such as epidermal growth factor (EGF; Box 4.4). These developing follicles do not secrete significant amounts of steroid hormones. Further follicular development requires pituitary support (secretion of FSH and LH).

Box 4.4

Cytokines and growth factors

Inhibin and activin

The gonadotrophins (FSH and LH) stimulate the production of the cytokines, activin and inhibin. The cytokines modulate actions of steroid hormones and gonadotrophins. Inhibin appears to affect only reproduction (see Fig. 4.7), whereas the closely related activin affects cell growth and differentiation in other tissues. Inhibin is produced by the granulosa cells of small antral follicles in response to FSH (Roberts et al., 1993) and suppresses FSH secretion. It is also produced by the Sertoli cells of the testis. Levels peak mid-cycle but remain high in the luteal phase, because the corpus luteum produces inhibin in response to LH. Inhibin production in the pituitary has a local inhibitory effect on FSH release (Hillier, 1991). Inhibin stimulates androgen output by the thecal cells and moderates aromatizing activity of the granulosa cells (Hillier, 1991). Activin is produced by granulosa cells, which also secrete follistatin, which may modulate the effects of activin. The thecal cells of the dominant follicle also produce activin (Roberts et al., 1993). The anterior pituitary gland also produces activin, which is co-secreted with the gonadotrophins and enhances FSH production. It inhibits pituitary production of growth hormone GH, ACTH and PRL. It may also have a role in embryogenesis (Hamilton-Fairley and Johnson, 1998). Activin is present in follicular fluid but is inhibited by follistatin. Activin suppresses the androgen output by thecal cells but stimulates aromatizing capacity of granulosa cells. It therefore inhibits progesterone production. Activin is present early in the cycle, and inhibin later in the cycle, thereby producing a balance between androgen output and conversion.

Follistatin

Follistatin was identified in 1987 as an inhibitor of FSH secretion (Roberts et al., 1993). It is synthesized in the ovary and inhibits activin activity by acting as an activin-binding protein.

Interleukins

Interleukins are a group of cytokines, small protein molecules, that communicate between the immune system cells and between immune and other cells. They are usually secreted by immune cells and usually have a local action. Interleukins include histamine, prostaglandin, tumour necrosis factor (TNF), IL-1 and IL-6. IL-1 is a polypeptide cytokine, usually produced by activated macrophages, which induces an acute phase reaction in the liver in response to inflammation. However, it is also produced by granulosa cells in a hormone-dependent manner with a peak production mid-cycle. IL-1 affects follicular maturation and a number of aspects of ovulation, including increasing production of prostaglandins, collagenase, nitric acid and hyaluronic acid (Hurwitz et al., 1992) and steroidogenesis. It is not known whether other members of the interleukin family are involved in follicular development.

Epidermal growth factor (EGF)

EGF is produced by many tissues including granulosa cells. It appears to inhibit FSH-stimulated oestrogen and inhibin production and proliferation and differentiation of granulosa cells. EGF may be involved in the selection of the dominant follicle (Hamilton-Fairley and Johnson, 1998).

Transforming growth factors (TGF)

Transforming growth factors, TGF-α and TGF-β, have been identified in thecal cells (Adashi et al., 1989). TGF-α has similar properties to EGF and suppresses granulosa cell differentiation. It also regulates differentiation of other cell types including fetal ovaries and ovarian carcinoma cells. Members of the TGF-β family are structurally similar to inhibin and increase FSH receptor expression, positively modulating granulosa cell proliferation and differentiation.

Insulin-like growth factors (IGFs)

Insulin-like growth factors stimulate mitotic division and cell differentiation. Their effects are mediated by insulin-like growth factor binding proteins (IGF-BP). In follicular development, they appear to coordinate the production of steroid hormones from the granulosa and thecal layers of the follicle (Giudice, 1994). IGF-I enhances follicular development and hormone production. IGF-II enhances the response to insulin. IGF-BP bind to the IGFs decreasing the concentration of free growth factor. Decreased levels of binding proteins are associated with follicular growth and increased concentrations of binding proteins are found in atretic follicles (Mason et al., 1993). The large follicles from women with polycystic ovaries have lower concentrations of growth factors and higher concentrations of binding proteins (Mason et al., 1994). IGFs seem therefore to have an important role in follicular growth, maturation and ovulation. In pregnancy, IGFs and their binding proteins play essential roles in modulating fetal growth and development (see Chapter 9).

Tumour necrosis factor (TNF)

TNF was initially identified as having a role in inflammation and in inhibiting tumour growth. It is produced by follicular cells and stimulates steroidogenesis, and may have a role in ovulation.

Early in the cycle, the concentration of FSH is sufficient to support the further development of some preantral follicles. The preantral follicles that are optimal for further development are of the appropriate size and maturity to respond and have adequate FSH receptors. Recruitment of the follicles is related to the interaction between the FSH concentration and the number of FSH receptors on the developing follicles. Therefore, the number of follicles surviving is related to the amount of FSH present. The antral development phase, ovulation and luteal phase comprise one cycle in the humans. In other mammalian species, antral expansion occurs in the luteal phase of the previous cycle, thus shortening non-fertile cycles.

Usually about 15–20 preantral follicles are rescued from atresia each month and undergo initial stages of development and marked enlargement in response to the increasing FSH concentration at the beginning of each cycle. Several components contribute to the growth of the follicle: the oocyte enlarges, the follicular cells divide and further stromal cells are recruited to form the expanded outer layers of the follicle. The oocyte itself increases in diameter to 60–120 μm. It synthesizes large amounts of ribosomal RNA (rRNA) and messenger RNA (mRNA) to increase its protein stores ready for the maturation of the oocyte and fertilization, but does not resume meiosis. The follicular cells divide into several layers of granulosa cells, which secrete an amorphous and acellular translucent jelly, the zona pellucida. The zona pellucida is formed from condensation of glycoprotein and accumulates between the granulosa cells and the oocyte, acting as an extracellular coat of the oocyte. It has an important role in sperm binding and penetration during fertilization (see Chapter 6). Although the zona pellucida acts to separate the oocyte from the avascular granulosa cells, cytoplasmic processes penetrate the zona pellucida forming gap junctions at the oocyte surface. These allow delivery of low molecular weight substrates, such as nucleotides and amino acids, and cellular signalling molecules into the oocyte. Gap junctions also exist between granulosa cells.

The third component of follicular growth is condensation of ovarian stromal cells on the basement membrane (membrana propria) of the follicle. These recruited cells form a loose matrix of spindle-shaped cells around the follicle, known as the thecal layer. The cells differentiate into two layers: the theca interna, an inner layer of highly vascular glandular cells, and the theca externa, a poorly vascularized fibrous capsule.

There is critical bidirectional paracrine and juxtacrine communication between the oocyte and the surrounding somatic or follicular cells of the ovary, the granulosa and thecal cells. FSH is required both for granulosa cell differentiation and division (Richards and Pangas, 2010). The granulosa cells acquire receptors for FSH and oestrogen and the theca interna cells acquire receptors for LH. Synthesis of steroid hormones by the follicle requires cell cooperation (Fig. 4.5; Hillier et al., 1994). Interstitial glands lie within the stroma and between the developing follicles. They are formed of steroidogenic cells and produce androgens for secretion and aromatization to oestrogen in follicles. LH stimulates the theca interna cells to synthesize androgens (testosterone and androstenedione) from acetate and cholesterol but these cells initially have limited capacity to synthesize oestrogens. Androgens from the theca interna cells diffuse to the avascular granulosa cells. The granulosa cells are unable to synthesize androgens but can aromatize androgens to oestrogens (oestradiol-17β and oestrone). The enzyme aromatase (CYP19) is involved in the steroid biosynthesis pathway leading to increased oestrogen production. FSH stimulates production of insulin-like growth factor I (IGF-I), which stimulates aromatase activity and hence oestrogen production. Activins and oestradiol enhance the actions of FSH (Richards and Pangas, 2010). Small amounts of LH are required to amplify follicular oestrogen production. The steroids are secreted into the bloodstream, where they have a systemic effect, and into the follicular fluid, where they may have a paracrine role. Androgens also stimulate aromatase. Oestrogens stimulate granulosa cells to proliferate and express further oestrogen receptors. Therefore, oestrogen further stimulates oestrogen output, an example of positive feedback. This increases the amount of circulating oestrogen from the most advanced or ‘dominant’ follicle (therefore, monitoring oestrogen output is a guide to the maturity of the most mature follicles). The dominant follicle, which undergoes the most growth, will enlarge from 20 to 200–400 μm diameter. The dominant follicle produces oestradiol, which inhibits FSH secretion. However, the dominant follicle develops exquisite sensitivity to FSH and can continue to respond to the decreasing concentration of FSH. The smaller follicles, destined to become atretic, lose their responsiveness to FSH and do not develop LH receptors (Scheele and Schoemaker, 1996).

Early in the antral phase under the influence of FSH, the granulosa cells produce inhibin B and activin which suppresses androgen output and increases the aromatizing capability of the granulosa cells, thus promoting oestrogen synthesis. Later in the antral phase under the influence of both FSH and LH, the granulosa cells switch to producing inhibin A which stimulates androgen output and attenuates the aromatizing activity. Thus, androgen output is regulated by activin and inhibins and the ratio of inhibin A:inhibin B acts as a marker of follicular growth

The dominant antral follicle

The single follicle emerging as dominant undergoes preovulatory growth. This dominant follicle produces more oestrogen, which inhibits production of FSH from the pituitary gland. This is an example of negative feedback where a product limits its own production. The effect of oestrogen inhibiting production of FSH is that further development of the other follicles is limited. These follicles with fewer FSH receptors exposed to a diminishing supply of FSH are least able to respond to FSH and therefore undergo a downward spiral and become atretic. The dominant follicle continues to produce inhibin A which stimulates androgen production by the thecal cells and aromatization by the granulosa cells, thus leading to the surge of oestrogen. The dominant follicle also has a high ratio of insulin-like growth factor 2 (IGF-2) to IGF-BP which mediates LH-stimulated androgen output and FSH-dependent aromatization.

Angiotensin II (A-II), the product of the renin–angiotensin system (RAS, see Chapter 2), may be involved in oocyte maturation and ovulation. There is a RAS that is specific to the ovary and A-II occurs in high concentrations in preovulatory follicles. This ovarian RAS is implicated in the pathogenesis of ovarian tumours, ovarian hyperstimulation syndrome, ectopic pregnancy and hypertension (Hassan et al., 2000). Symptoms of ovarian hyperstimulation syndrome include serious metabolic and fluid disturbances which are associated with abnormal control of the RAS (note that ovarian hyperstimulation syndrome is a recognized complication of IVF treatment – see Chapter 6). It has also been suggested that A-II may have a role in the formation and maintenance of the corpus luteum, regulation of progesterone production and angiogenesis (Hamilton-Fairley and Johnson, 1998). The biggest or dominant follicle, which is best able to respond to FSH, further develops on the pathway to expansion and ovulation. Oestrogen and FSH stimulate the mid-cycle expression of LH receptors on the outer layers of granulosa cells of the dominant follicle, which means that it will be able to respond to the mid-cycle surge of LH secretion. Entry into the preovulatory phase depends on both the expression of these receptors and a surge of LH from the anterior pituitary gland.

The granulosa cells continue to divide and increase in size. However, most of the increase in follicular size is due to accumulation of follicular fluid formed from mucopolysaccharides, secreted from granulosa cells, and serum transudate. The fluid coalesces forming an antrum (or cleft) filled with follicular fluid. The antrum separates the granulosa cells into two regions: the corona radiata (a rim of granulosa cells) around the oocyte and the outer membrana granulosa. The oocyte becomes isolated and suspended in the fluid connected to the rest of the granulosa cells by a thin strand of cells, the cumulus oophorus (egg stalk). The oocyte does not increase in size but continues to synthesize RNA and protein.

Follicular development is dependent on pituitary support. Removal of the pituitary gland (hypophysectomy) results in the cessation of follicular growth and the death of the oocyte. This can be halted by adding back LH and FSH, which stimulate further growth. It takes 8–12 days for the primary follicle to grow into the antral follicle. Failure of follicular growth for any reason results in a restarting of the cycle of follicular development, and hence both a longer first phase of the cycle and a longer cycle. It seems likely that women who regularly have a longer than normal menstrual cycle have either a slow rate of follicular development and increasing oestrogen secretion or the dominant follicle starts developing but fails, so the next most appropriate follicle takes over the role as the dominant follicle.

Although the increasing concentration of oestrogen (predominantly oestradiol, E₂) initially has a negative feedback on the hypothalamus and pituitary, there is a critical concentration of oestrogen that is stimulatory provided it lasts for a critical duration. When the diameter of the follicle is 18–22 μm and the oestradiol concentration reaches 600–1200 pmol/L, there is positive feedback on the anterior pituitary gland leading to a sudden increase or ‘surge’ of LH release (Hamilton-Fairley and Johnson, 1998; Fig. 4.6).

The effect of the LH surge is twofold. First, it stimulates the terminal growth phase of the preovulatory follicle and the meiotic and cytoplasmic maturation of the oocyte, culminating in expulsion of the oocyte from the ovary. These effects include re-initiation of oocyte meiosis and expansion of the cumulus cell–oocyte complex (Richards and Pangas, 2010). Second, it causes luteinization, a series of endocrine changes within the follicular cells that result in a different hormone secretory profile in the second half of the cycle.

Within a few hours of the LH surge, there are dramatic changes in the oocyte, which resumes meiotic division. There may also be a positive signal from the granulosa cells or a reduction of gap junction communication, which decreases the flow of meiosis-arresting substances to the oocyte. Progression through the remainder of the first meiotic division results in half the chromosomes (as paired chromatids) and almost all the cytoplasm being enclosed in the secondary oocyte, which is destined to become the ovum. The remaining chromosomes and very little cytoplasm are enclosed in a membrane forming a very small cell, known as the first polar body (see Fig. 4.3). Thus, the secondary oocyte keeps the bulk of the materials that were synthesized earlier in follicular development; these are conserved for the zygote. The chromosomes of the secondary oocyte enter the second meiotic division and go on to the next stage of division, called metaphase (see Fig. 7.11), where they align on the spindle. However, meiosis is then immediately arrested for a second time; this is regulated by CSFs. Meiosis resulting in the production of a mature female pronucleus will not resume until successful fertilization following ovulation. By this time, the oocyte will already contain the sperm nucleus. Thus, there is actually no time when the oocyte is a true gamete in the sense of being a cell with only 23 chromosomes, as is the case of a spermatozoon.

Concurrently, the LH surge promotes maturation of the cytoplasmic compartment of the oocyte. The cytoplasmic processes between the oocyte and the granulosa cells withdraw and contact is lost. The Golgi apparatus (see Table 1.1) synthesizes lysosome-like cortical granules, which align under the surface of the oocyte. Protein synthesis continues but the profile of the proteins synthesized changes as the oocyte prepares for fertilization. The gonadotrophin surge stimulates the cumulus cells surrounding the oocyte (see Fig. 4.4) to secrete hyaluronic acid, which disperses the cumulus cells embedding them in a mucus-like matrix.

Ovulation is triggered by the mid-cycle surge of LH, which occurs in response to sustained high levels of oestrogen released from the developing dominant follicle. The single mature preovulatory follicle has a diameter of 2–2.5 cm in an ovary that is approximately 3 cm long. It was this structure that de Graaf identified and named in 1672. The increased size and changed position of the follicle mean that it protrudes from the surface of the ovary (see Fig. 4.2). This results in the thinning of the layer of epithelial cells between the wall of the follicle and the peritoneal cavity. As expansion continues, the wall becomes thinner and avascular, and the cells appear to dissociate.

About 36 h after the LH surge, ovulation occurs and the oocyte is expelled from the ovary (see Fig. 4.2). The LH surge stimulates the production of a cascade of proteolytic enzymes, including renin and other trypsin-like enzymes from thecal cells, which digest the follicle wall. The biochemical changes, including generation of oxygen free radicals, that precede ovulation are similar to those seen in inflammation. Plasminogen activator, which converts procollagenase to collagenase, is produced by granulosa cells resulting in the breakdown of the connective tissue. Progesterone production rises immediately after the LH surge and the preovulatory increase in progesterone may be important in follicular rupture as it decreases formation of collagen. Prostaglandins increase vascular permeability, which maintains the intrafollicular pressure as fluid begins to leak through the eroded follicular wall. Small contractile waves also ripple across the ovary increasing the intrafollicular pressure. The force is cushioned by the follicular fluid, so the pressure generated is targeted at the weakened ovarian surface, causing it to rupture. Some women experience abdominal pain around the time of ovulation on the same side of the ovary producing the ovum. This is referred to as Mittelschmerz pain (derived from German ‘middle pain’). As the follicle ruptures and the ovarian surface is breached, the fluid washes out the oocyte, which is surrounded by the granulosa (cumulus) cells, from the ovary to the exterior. The oocyte is swept into the uterine tube by the fimbria. It is then propelled towards the uterus by peristaltic muscular activity and cilia movements of the epithelial cells lining the tube. After taking years (12–50 years) to complete maturation, the oocyte is then viable and fertilizable for only about a day.

The luteal phase

Within 2 h of the LH surge, there is a transient rise in the oestrogen and androgens secreted by the follicle as the thecal layers become stimulated and hyperaemic. The outer granulosa cells with their newly expressed receptors for LH no longer convert androgens to oestrogen but synthesize progesterone instead. The cells no longer bind oestrogen or FSH. The result is a marked increase in progesterone secretion, which begins several hours before ovulation.

After ovulation, the residual parts of the follicle remaining in the ovary collapse into the space and form the corpus luteum (‘yellow body’; see Fig. 4.4). There is some bleeding and fibrotic activity in the cavity, which allows formation of a fibrin core around which the remaining granulosa cells congregate. The structure is enclosed by a capsule of fibrous thecal cells. The basement membrane between the granulosa cells and thecal cells breaks down allowing vascularization of the interior. This allows increased transport of cholesterol precursor to the luteinizing granulosa cells to maintain a high rate of progesterone secretion. A few of the thecal cells disperse to the stroma tissue. The granulosa cells first luteinize, then stop dividing and hypertrophy into large luteal cells. The luteal cells are rich in mitochondria, endoplasmic reticulum and Golgi bodies and have numerous lipid droplets and lutein, a yellow carotenoid pigment.

Luteinization is associated with a progressive increment in progesterone secretion from the corpus luteum. The outer thecal cells form a stem cell population of smaller luteal cells which have numerous LH receptors and produce progesterone and androgens. Levels of progesterone rise until the middle of the luteal phase (see Fig. 4.6). The corpus luteum produces oestrogen and inhibin as well as progesterone. All three hormones inhibit secretion of FSH from the anterior pituitary gland and therefore prevent further development of follicles.

It has been suggested that a cause of fertility problems may be inadequate production of progesterone at the time of ovulation and during the subsequent luteal phase. However, exogenous administration of progesterone or human gonadotrophin (hCG) has had limited success in clinical practice. It appears that most women have a proportion of their cycles with a low progesterone output without their overall fertility being affected (Hamilton-Fairley and Johnson, 1998). A shortened luteal phase can lead to intermenstrual bleeding, premenstrual ‘spotting’ and short cycles.

The corpus luteum also synthesizes relaxin, secretion of which peaks in the middle of the luteal cycle (Johnson et al., 1993), probably regulated by LH. Relaxin may be involved in promoting the growth of the myometrium and cervix and growth and secretory activity of the endometrium (Huang et al., 1991).

Effectively, the corpus luteum is an endocrine gland producing oestrogen and progesterone. The LH surge stimulates its growth and activity. Unless fertilization occurs, the life of the corpus luteum is very short, and it undergoes spontaneous luteolysis (degeneration and regression) after about 6 days. The corpus luteum appears to have an age-related decrease in responsiveness to LH (Zeleznik and Hillier, 1996) and so requires progressively more LH for survival. Following the LH surge, LH concentration in the luteal phase is low, so luteolysis will occur. Blood flow to the corpus luteum falls and the follicular tissue becomes ischaemic. The concentrations of oestrogen and progesterone begin to fall as the degenerating corpus luteum stops hormone production. Thus, luteolysis terminates a non-fertile cycle. As the level of oestrogen falls, the inhibition on the hypothalamus will be abrogated and FSH secretion will resume, ready for the next cycle. The atrophying corpus luteum loses its yellow pigment, so it becomes known as a corpus albicans (‘white body’). It gradually contracts over a period of months, leaving a white scar tissue which is absorbed into the stromal tissue of the ovary.

If fertilization occurs, then hCG, which has structural similarities to LH, rescues the corpus luteum from luteolysis, stimulating its further growth and production of steroid hormone up to the 10th week when placental endocrine function becomes established. Human chorionic gonadotrophin has a longer half-life than LH, so it provides a sustained and more intense stimulus. If fertilization occurs, then the concentration of relaxin also continues to rise until the end of the first trimester.

Regulation of gonadotrophin secretion

The brain controls and regulates the ovarian cycle. The gonadotrophs in the anterior pituitary secrete the glycoprotein hormones LH and FSH (which together are known as gonadotrophins). There appear to be two distinct populations of cells in the anterior pituitary, each producing one particular type of hormone; however, fluorescent labelling shows that some cells contain both hormones. Synthesis and secretion of LH and FSH are dependent on gonadotrophin-releasing hormone (GnRH) from the hypothalamus, which acts as the common mediator of influences via the CNS. (GnRH is also known as luteinizing hormone releasing hormone, or LHRH.) The hypothalamus releases GnRH into the hypophysial portal circulation that runs to the pituitary gland.

This pathway means that control of the reproduction can be modulated and affected by other inputs from the higher brain centres. The GnRH neurons convert neural signals into endocrine signals. Stress, nutritional status and environmental influences, for instance, affect the timing and success of the reproduction. Ovulation appears to be seasonally regulated in populations experiencing seasonal variation in food availability; suspending reproductive function when nutrition is poor favours maternal health and outcome of pregnancy.

There are two levels of regulation. First, the GnRH neurons of the hypothalamus have an inherent pulsatile activity. The steroid hormones, the gonadotrophins (LH and FSH) and GnRH feedback on the hypothalamic–pituitary axis exert a second level of endocrine control. Prolactin (PRL) also has an effect on the control of reproduction.

GnRH and gonadotrophins are released in a pulsatile manner (see Box 4.5). The cells releasing GnRH appear to be widely and diffusely distributed (Rance et al., 1991) but are remarkably synchronized to produce pulses of GnRH. During the follicular phase, the pulses are of low amplitude and high frequency, occurring every 60 min (Clarke, 1996). In the luteal phase, they are more irregular and have high amplitude and occur with a low frequency of about every 2 h. The output of the gonadotrophins, LH and FSH, is changed by increasing or decreasing the amplitude or frequency of the pulses or by modulating the response of the gonadotrophs to the pulses. Prior to the LH surge, gonadotroph GnRH receptor density increases and the cells become more sensitive to GnRH. Inhibin and activin affect secretion of FSH without affecting secretion of GnRH. Two phenomena are observed: first, a depressant effect on output of gonadotrophins by increased oestrogen, progesterone and inhibin; and second, an increased surge of LH and FSH secretion induced primarily by oestradiol (Fig. 4.7). The pattern of pulsatile secretion of GnRH is regulated by a complex mechanism that allows multiple signals, such as neurotransmitters and sex steroids, to determine ovulation.

Box 4.5

Biological rhythms

The study of biological rhythms is termed ‘chronobiology’. All living cells, organs, organisms and groups of individuals demonstrate rhythmical changes within their internal (endogenous) physiology that can also result in external changes in behaviour. The rhythms can be categorized according to the length of the cycle or period of oscillation.

• Ultradian: the rhythm is less than 1 day, for example, rapid eye movement in sleep.

• Circahordal: the period of oscillation is around 1 h.

• Circadian: the period of oscillation is about 1 day; levels of many hormones such as cortisol fluctuate on a daily basis.

• Infradian: the rhythm is repeated in a cycle greater than 1 day, for example, menstrual and oestrus cycles.

• Circaseptram: the period of oscillation is about 1 week.

• Circatidal: the rhythm relates to tidal movement of water.

• Circalunar (synodic): the rhythm relates to the cycle of the moon.

• Circannual: the rhythm has a cycle of about 1 year.

These fluctuations are often affected by the external environment and appear to enable the individual to respond to forthcoming changes within the environment. Factors that influence or reset the cycle are described as entraining the cycle. In the human brain, the suprachiasmatic nuclei of the hypothalamus may influence daily fluctuations. The pineal gland, which produces the hormone melatonin at night, appears to affect the suprachiasmatic nuclei, acting as an entrainer. The circadian pattern of melatonin secretion is achieved through inhibition via a neural pathway (outside the optic nerve) that is activated by light stimulation upon the retina. Therefore, within temperate zones, light acts as an entrainer on a daily basis and, because of the fluctuation of the photoperiod (length of daylight exposure), entrainment of annual cycles can also be achieved.

In the early part of the follicular phase, rising levels of FSH stimulate oestrogen production from the developing follicles. Rising concentrations of oestradiol have a negative feedback effect on gonadotrophin production from the anterior pituitary, so FSH secretion falls. Activin and inhibin also affect FSH secretion. Inhibin is involved in the negative feedback of FSH secretion. However, the dominant follicle is exquisitely sensitive to even a diminishing concentration of FSH and continues to produce oestrogen, which markedly increases by two- to fourfold. These concentrations are maintained for 48 h, which produces a positive feedback effect resulting in the dramatic surge of LH and FSH release seen in mid-cycle prior to ovulation. The effect of oestradiol is very sensitive: a low concentration has a marked and rapid effect that is evident within 1 h and maximal within 4–6 h. During the luteal phase, increased progesterone concentrations reinforce the negative feedback effects of oestradiol. The production of both LH and FSH secretion is very low; therefore, the positive effect of oestradiol is blocked.

Cyclical effects of oestrogens and progesterone

Organs that respond to hormonal changes have receptors for the hormones. Responses can change because hormone levels fluctuate or because receptor density on the target organs alters. The principal actions of oestrogen and progesterone during the monthly cycle are on the endometrium which is one of the tissues most sensitive to ovarian steroid hormones. The endometrium undergoes cyclical changes: the growth of the uterine wall in expectation of an embryo, and its degeneration if fertilization does not take place. In the first half of the cycle, the uterus goes through a proliferative phase. Oestrogen stimulates the epithelial cells of the basal layer of the endometrium to divide and proliferate, forming a thick mucosal wall with numerous endometrial glands (Fig. 4.8). Oestrogen also stimulates proliferation of the stoma and glands and angiogenesis (growth of new blood vessels): extensive vascular tissue, spiral arteries and veins develop within the endometrium. Within the space of a few days, the effect of oestrogen is to increase the height of the wall from 0.5 to 5 mm, a remarkable 10-fold increase. The thickness of the endometrium is monitored by ultrasound in assisted conception (see Chapter 6) to assess whether it is optimal for implantation; the insertion of embryos into a uterine cavity with an endometrium less than 5 mm thick is unlikely to be successful. The myometrium does not grow extensively during the menstrual cycle. During the proliferative phase, oestrogen primes the endometrial cells by inducing the synthesis of progesterone receptors.

After ovulation, the cells of the enlarging corpus luteum begin to secrete progesterone, which has a dramatic effect on the secretory activity of the endometrial glands. In this secretory phase, the effects of progesterone are dominant, although oestrogen is still secreted from the corpus luteum. The spiral arteries continue growing and thus become more prominent and coiled as the height of the endometrium remains unchanged. The endometrial glands become dilated and convoluted with secretions rich in glycogen, mucus, proteins, sugars, amino acids and enzymes. The secretory products are important for the survival and nutrition of the zygote and blastocyst prior to implantation. Failure of conception results in diminishment of the corpus luteum and decreased steroid hormone production. By the 7th postovulatory day, the secretory process ceases and the glands become exhausted and regress.

Cyclical effects are particularly evident within the female reproductive tract. Activity of the myometrium is inversely related to progesterone secretion. During menstruation, when progesterone levels are low, the uterine contractions, mediated by prostaglandins, have a higher frequency and strength than in labour (Lyons et al., 1991). These uterine contractions are responsible for dysmenorrhoea (period pains). Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin inhibit prostaglandin synthesis, and are therefore effective in reducing pain. After menstruation, there is a slow decline in myometrial activity during the follicular phase; it reaches negligible activity mid-cycle. Thus, the uterus is at its most quiescent (still) at the time of implantation. Uterine quiescence is maintained until the late luteal phase when levels of progesterone fall. If pregnancy intervenes, levels of progesterone remain high and the myometrium remains inactive. Uterine blood flow, on the other hand, correlates positively with the pattern of oestrogen secretion. Because of low levels of oestrogen, blood supply to the endometrium during menses and early follicular phase is reduced. A marked increase occurs just prior to ovulation followed by a slight nadir. A secondary peak occurs in the luteal phase which mirrors the rise in oestrogen production. This means that endometrial blood flow is relatively high at the time of implantation but lower at menstruation, which helps to limit the blood loss at the latter time.

Uterine contractile activity varies with the stage of the menstrual cycle (Kunz and Leyendecker, 2001). These periovulatory waves of muscle contraction are directional, moving inwards from the cervix to the fundus, encouraging the semen to travel towards the egg (Bulletti and de Ziegler, 2005). In the preovulatory period, uterine peristaltic activity within the uterine tube also directs sperm transport preferentially to the uterine tube lateral to the ovary containing the dominant follicle. The uterine activity is relatively quiescent in the late luteal phase which favours implantation. However, there are still some gentle peristaltic waves which may promote high fundal implantation. Uterine peristalsis causes some retrograde menstruation. It is thought that this may be of evolutionary benefit and help preserve body iron (Kunz and Leyendecker, 2001). Dysfunction of uterine activity may be involved in the development of endometriosis, uterine adenomyosis and infertility.

Uterine tubes

Oestrogen stimulates epithelial cell activity, increasing cilia movement and secretion. This facilitates the movement of the ovum along the uterine tubes following ovulation. These effects are reversed by progesterone, which inhibits the peristaltic activity of the uterine tube smooth muscle.

Cervix

Oestrogen relaxes the myometrial fibres supplying the cervix and increases stromal vascularization and oedema. Collagenase is activated, which causes some dispersal of the tightly bound collagen bundles into a looser matrix. The result is that the cervix becomes softer to touch. The external os everts prior to ovulation. Progesterone causes the cervical muscle to retract and the stroma to become more compact as the collagen matrix reforms. The external os becomes tighter. The change in texture of the cervix is used as part of natural family planning (see Box 4.6). The cervix is softer at ovulation and a few days before, coinciding with the fertile period. At this stage, it has the consistency of lips compared with the harder ‘nose-like’ cartilaginous consistency of the cervix later in the cycle when the effects of progesterone are dominant.

Box 4.6

Concepts of natural family planning

• On the basis of periodic abstinence

• Also known as ‘rhythm method’ or ‘safe period’

• Assumes that the interval between ovulation and menstruation is constant

• On the basis of recognition of signs of ovulation and fertile phases of menstrual cycle

– temperature rise after ovulation

– increased cervical mucus and watery vaginal secretions

– softer consistency of cervix

• Probably most effective for birth spacing

The cyclical changes in blood flow are reflected by the composition of cervical mucus, which is copious and receptive to sperm penetration in mid-cycle (Table 4.1). When progesterone levels are high, small volumes of thick cervical mucus are secreted that are hostile and impenetrable to sperm. The increased viscosity of the mucus in the latter half of the menstrual cycle reduces the risk of ascending infection at the time of implantation.

Both oestrogen and progesterone are secreted from the corpus luteum in the second half of the cycle. The concentrations of oestrogen and progesterone will continue to rise if successful fertilization results in secretion of hCG and consequent survival of the corpus luteum. Therefore, the effects of the hormones in the second half of the cycle on the female body portend the changes that would take place in pregnancy.

Vagina

Oestrogen increases mitotic activity and secretion in the vaginal epithelial cells. Stimulation by progesterone results in an increased size of the nucleus of vaginal epithelial cells. It is important when examining cervical cells, obtained from a smear, to relate the morphological differences to the stage of a woman's menstrual cycle. Earlier in the cycle, cells appear flatter, whereas under the influence of progesterone they tend to become clumped and folded. There are also cyclical changes in the pH of the vagina as oestrogen stimulates the growth of commensal lactobacilli (Döderlein's bacilli). These lactobacilli metabolize glycogen from the cervical secretions producing lactic acid as a metabolic by-product, which decreases pH to a level that protects the reproductive tract from opportunistic pathogenic microorganisms.

The resident flora of the vagina also produces volatile aliphatic acids, which have distinctive odours. The profile of acids changes throughout the cycle under the influence of the changing hormones, and may result in changed sexual behaviour. It is suggested that male responses to their partners are affected by the cyclical fluctuations in olfactory stimuli stimulating sexual responsiveness and interaction. These olfactory signals do not seem to be consciously perceived in humans. Another example is women who live together are often observed to demonstrate menstrual synchrony: they ovulate and menstruate at the same time. Recently, it has been demonstrated that humans have the potential to communicate by pheromones. Odourless body secretions from women in different phases of the menstrual cycle can advance or delay the phases of other women (Stem and McClintock, 1998). Sexual desire, sexual activities and sexual satisfaction are all reported to increase around ovulation (see Chapter 5).

There are additional effects and benefits of oestrogen on women's health. Oestrogens appear to protect the cardiovascular system; thus, women of reproductive age and normal endocrine function have a lower incidence of hypertension and a reduced risk of cardiovascular disease owing to higher levels of high-density lipoproteins (HDL), which lower circulating levels of cholesterol. Oestrogens stimulate osteoblasts, the cells involved in bone formation, thereby maintaining bone mass. Oestrogens may depress appetite and are mildly anabolic. There is a preovulatory drop in food intake; this decrease in female ‘foraging’ behaviour is hypothesized to allow the woman more time for activity, including ‘shopping’ for alternative mates (Fessler, 2003). This hypothesis is also supported by the suggestion that because ovulation is concealed in humans, the woman is able to select her mate at ovulation as opposed to other female mammals that become sexually attractive to males around ovulation (Ridley, 1994). Increased consumption of food is observed in the late luteal phase.

Postovulatory levels of progesterone are high, causing a slight increase in the basal metabolic rate. The basal body temperature rises owing to the influence of progesterone on the thermoregulatory centre of the hypothalamus. A temperature rise of 0.2–0.6°C confirms ovulation has taken place but does not predict it. In the second half of the cycle, the skin may appear more pigmented and acne may worsen as progesterone increases constriction of sebaceous glands. Progesterone also increases appetite during the luteal phase (Buffenstein et al., 1995). Women with premenstrual syndrome (PMS) may report cravings for carbohydrate, which are often associated with feelings of depression (Dye and Blundell, 1997; see Box 4.7). It is frequently reported that common medical and mental health disorders are exacerbated at specific phases of the menstrual cycle (Pinkerton et al., 2010). Patterns of fluctuations in energy intake, appetite and depression may be associated with low serotonin or dopamine activity or other hormonally induced alterations in the brain which influence responses to pleasure and desire and thus affect food ingestion (Van Vugt, 2010). Changes in appetite and cravings can influence energy intake and expenditure (Davidsen et al., 2007). Changes in metabolic responses due to ovarian hormones can influence exercise performance (Oosthuyse and Bosch, 2010). Metabolism of drugs and alcohol may also cyclically alter during the menstrual cycle (Terner and de Wit, 2006).

Box 4.7

Premenstrual syndrome (PMS)

PMS is very common, with physical and psychological symptoms occurring in about 50% of women of reproductive age during their lifetime. Premenstrual dysphoric disorder (PMDD) is a more severe form of PMS that is associated with the luteal phase of the menstrual cycle. PMDD is classified as a depressive disorder by the American Psychiatric Association and affects about 3–5% of women. It is characterized by anxiety, anger and severe irritability. It is more severe than PMS and usually requires treatment to allow an affected woman to function in her environment. Both PMS and PMDD are characterized by mood swings including depression, but clinical depression occurs in PMDD. Some women have been acquitted of crimes conducted when they were affected by PMS and PMDD.

The cause of PMS is not clear. It may be due to a hormonal imbalance and low progesterone secretion in the luteal phase, abnormal neurotransmitters response, disorganized aldosterone function leading to water retention, deficient adrenal hormone secretion due to abnormal hypothalamic–pituitary–adrenal function, carbohydrate intolerance, a nutrient deficiency, stress or a combination of these factors (Girman et al., 2003). Women may be prescribed antidepressants such as serotonin reuptake inhibitors. Many women select lifestyle modification and complementary or alternative medicine approaches rather than conventional medicine. Women with PMS tend to consume more dairy products, refined sugar and high-sodium foods, so many clinicians recommend reducing intake of these. As fat contributes to oestrogen levels and fibre helps to reduce the effects of oestrogen on gut flora, high-fibre low-fat diets may be recommended. Reducing caffeine intake, from coffee, tea and caffeinated soft-drinks, can also be helpful. Vitamin B₆ can affect neurotransmitter release but the evidence that it improves PMS symptoms is inconclusive. As excess vitamin B₆ can cause nerve damage before symptoms of toxicity are evidenced, the daily dose should be limited. There is some evidence that calcium supplements can be beneficial. Of the herbal preparations more commonly used to relieve symptoms, the evidence is stronger for chasteberry (vitex) and ginkgo than for black cohosh, kava and St John's wort; the effects of evening primrose oil are thought to be a placebo effect. Exercise, such as yoga, helps reduce depression and anxiety symptoms. Many women find that keeping a symptom diary helps to identify exacerbating and relieving strategies.

Oestrogen and progesterone affect connective tissue oedema and hyperaemia and can cause increased breast size and tenderness. Progesterone binds to renal aldosterone receptors, causing natriuresis (sodium excretion) and blocking aldosterone occupation. Aldosterone increases to restore sodium retention, so there is a net effect of sodium retention. Oestrogen stimulates angiotensinogen production, which also tends to enhance sodium retention. Thus, in the luteal phase of the cycle, salt and water retention may be increased causing generalized weight gain and premenstrual feelings of bloatedness. The fluctuations in oestrogen and progesterone throughout the menstrual cycle affect the skin including skin structure (thickness, collagen production and breakdown and fluid retention), hydration, pigmentation, elasticity, wound healing and vasodilation (Farage et al., 2009). Thermoregulation, immune function and sleep patterns also exhibit a cyclical pattern in parallel with hormonal changes. In addition, reproductive hormones impact on psychoneurological processes affecting cognitive, emotional and sensory functions even at the level of hormone fluctuations that occur during the menstrual cycle (Farage et al., 2008).

Menstruation

Menstruation is the loss of most of the decidual (superficial) layers of the endometrium accompanied by some blood loss that occurs after withdrawal of steroid hormones at the end of each menstrual cycle. During the menstrual cycle, the spiral arteries supply the endometrial stroma in preparation for implantation of the blastocyst; they have a remarkable ability to vasoconstrict during menstruation in order to limit blood loss. In humans, menstrual loss usually lasts about 5–7 days. Humans and other primates, together with elephant-shrews and some types of bats, are the only animals that menstruate. In these species there is marked progesterone-related proliferation of the endometrium and implantation is invasive. It was suggested that menstruation evolved as a protective mechanism (cleansing process) against sperm-borne pathogens by shedding any infected endometrial tissue and delivering immune cells to the uterine cavity. However, an alternative hypothesis is that cyclical regression and proliferation of the endometrium is energetically more economical in term, of reproductive costs than constantly maintaining a receptive endometrium (Strassmann, 1996). In many species, regression of the endometrium is accompanied by reabsorption of tissue debris. It is suggested that the copious menstrual bleeding in humans relates to the relatively large size of the uterus and the organization of the microvasculature (Strassmann, 1996). It has also been suggested that cyclical menstruation protects the uterine tissue from hyperinflammation and oxidative stress associated with deep placentation (Brosens et al., 2009).

Menstruation is an inflammatory process which results in tissue remodelling. The endometrial wall is described as being in a state of ‘secretory exhaustion’ (Clancy, 2009) and begins to breakdown because there is no embryonic signal. The mechanism of menstruation is thought to be either tissue destruction following necrosis and/or an inflammatory response (Salamonsen, 2003). In the destructive model, anoxia causes necrosis of the endometrium. The degeneration of the corpus luteum results in a fall in oestrogen and progesterone levels, which causes a modest but significant decrease in endometrial tissue height so the spiral arteries are coiled tighter and compressed. This results in a reduced blood flow, ischaemia and denudement of the endometrial tissue and interstitial haemorrhage. The withdrawal of progesterone stimulates the production of prostaglandins which are released by the spiral arteries stimulating vasoconstriction and vasodilatation resulting in rhythmic waves of contraction and relaxation in the latter. (The effect is like breaking a wire by rhythmically bending it backwards and forwards.) The waves become longer and more profound causing the decidual endometrium to break away along the natural plane of cleavage. The straight arteries in the basal layer maintain the blood supply. It is from these that new spiral arteries will regenerate. Alternatively, menstruation can be regarded as an inflammatory response. There is a marked increase in the number of leukocytes, particularly mast cells, immediately before menstruation as progesterone levels fall. These are probably attracted by chemokines produced by the endometrial cells. Mast cell degranulation may trigger the extracellular activation of matrix metalloproteinases, proteases that have the capacity to degrade components of the tissue. Prostaglandins are also involved in stimulating uterine contractions, which aid the removal of endometrial debris and blood.

Within 12 h, the height of the endometrium falls from 4 to 1 mm. At the end of the secretory phase, there is an ischaemic phase followed by the menstrual phase leading to the next proliferative phase. Endometrial repair is very rapid and occurs without scarring.

Menstrual flow is usually between 35 and 95 mL and consists of endometrial debris and blood. Blood loss is limited by vasoconstriction of the spiral arteries and formation of thrombin–platelet plugs in the terminal portions of the straight arteries. When oestrogen secretion resumes at the beginning of the next cycle, it stimulates healing and new tissue growth. Menstrual blood does not coagulate in the pattern seen normally. The damaged endometrial cells secrete proteolytic and fibrinolytic enzymes, which inhibit the formation of fibrin and therefore clot formation. The average volume of blood lost is 50 mL, which accounts for 0.7 mg of iron, a loss that is just matched by dietary iron absorption.

Case study 4.1 looks at the problem of calculating the length of gestation from the date of the last menstrual period.

Case study 4.1

Njuka is in England with her husband who is a diplomatic representative of a central African country. When they first meet, the midwife asks Njuka when her baby is due and is informed that four full moons are left to pass before the baby will come. The midwife, intrigued by this answer, asks Njuka how she knows this. Njuka explains that six full moons have passed since her last period and that is how she knows.

• How accurate is Njuka's calculation of her gestation?

• Why is it important for a midwife to be able to estimate the length of gestation?

• What other information can help in this estimation?

Hormonal causes of infertility

Hormonal causes of infertility account for about a third of the known causes (Box 4.8).

Box 4.8

Hormonal causes of infertility

• Hypogonadotrophic hypogonadism

• Anorexic states

• Weight fluctuations

• Obesity

• Hyperprolactinaemia

• Polycystic ovary syndrome (PCOS)

Hypogonadotrophic hypogonadism is due to malfunction of the hypothalamic–pituitary axis and is characterized by low levels of oestrogen. Women with normal pituitary functions can be successfully treated with pulsatile exogenous GnRH from a small infusion pump. The hypothalamus is frequently entrained by the pump so normal rhythms of pulsatile secretion continue after the pump is removed. Alternatively, women can be treated with exogenous gonadotrophins. Human menopausal gonadotrophin (hMG) extracted from the urine of postmenopausal women (the effects of ovariectomy or the menopause are to decrease oestradiol concentration, which results in raised circulating levels of FSH and LH) is used because it contains both FSH and enough LH to stimulate synthesis of androgenic precursors for oestrogen production. Ultrasound monitoring of follicular development is important to assess the development of excess follicles and the risk of multiple pregnancy. Ovarian stimulation can cause ovarian hyperstimulation syndrome which has serious implications because vascular permeability can suddenly increase, resulting in a movement of fluid out of the vascular system (McClure et al., 1994). In many respects, hypogonadotrophic hypogonadism resembles menopause (see below).

Weight loss can also disrupt the hypothalamic–pituitary axis. Anorexic patients often have disrupted menstrual cycles, but acute weight loss or disruptions in energy intake (such as those associated with ‘crash’ dieting) even within a normal body weight range may disrupt hormone secretion. A body mass index (BMI) greater than 19 kg/m² and at least 22% fat as a proportion of body weight seem to be necessary for the maintenance of normal ovulatory cycles. It has been suggested that the critical fat mass for fertility is equivalent to the energy requirements of pregnancy (Frisch, 1990). Low body fat delays puberty and the menarche. Weight loss particularly affects LH secretion and can result in an abbreviated luteal phase.

Appetite is stimulated by the orexigenic neuropeptide Y (NPY) from the hypothalamus. NPY is involved in the regulation of food intake and energy balance. It has both stimulatory and inhibitory effects at the pituitary gland. In the well-nourished state, NPY release is acute and intermittent, a mode of secretion that potentiates GnRH-induced LH release. However, fasting decreases plasma glucose concentrations and extremes of exercise result in chronic secretion of NPY and continuous NPY receptor activation, which is inhibitory to LH release and thus fertility.

Eating causes storage of triacylglycerides in adipose cells, which stimulates the cells to release leptin. Leptin seems to be the satiety signal, which modulates the release of NPY. In starvation, leptin levels are low and NPY levels are high, which inhibits GnRH. The nutritional control of reproduction probably had an important evolutionary role in suppressing fertility at times of poor food supply. Suspending reproductive function at times of food shortage is protective.

Case study 4.2 looks at the problem of underweight in the calculation of gestation.

Case study 4.2

Lisa, a 17-year-old primipara, presents herself at the midwives' clinic, giving a vague history and saying that she thinks she might be pregnant. On palpation and abdominal examination, Lisa seems to be 26 weeks' pregnant and this is supported by a fundal height of 26 cm. The presence of fetal heart sounds confirms that Lisa is indeed pregnant. On questioning Lisa, the midwife discovers that Lisa has had only two scanty periods in the last 2 years and does not know the date of her last menstrual period. Lisa smokes 60 cigarettes a day, and has the appearance of being very underweight.

• Can you identify any possible reasons why Lisa might have irregular periods?

• Why must the midwife not assume that Lisa is 26 weeks' pregnant?

• Are there any clues that the midwife may investigate in order to estimate more precisely the actual gestation of Lisa's pregnancy?

Paradoxically, obesity also affects fertility; obese women are over-represented in fertility clinics and have increased incidence of menstrual abnormality and a higher risk of miscarriage. The effects of obesity may persist even after weight loss has occurred. One of the reasons is that the adipose tissue is metabolically active, producing altered ratios of oestrogens and androgens. Obesity also affects insulin secretion (obese people are more likely to demonstrate insulin resistance) and affects production of leptin.

Hyperprolactinaemia can result from PRL-secreting tumours, which are usually benign. However, other factors including stress, breast stimulation or examination, hypothyroidism, polycystic ovary syndrome (PCOS) and dopaminergic antagonists can also raise circulating PRL levels. Hyperprolactinaemia can cause oestrogen deficiency, amenorrhoea and galactorrhoea (milk production). The management of hyperprolactinaemia is usually by administration of bromocriptine, a dopamine agonist, although tumours may be surgically removed.

Polycystic ovary syndrome (PCOS) affects 5–10% of women; it is the most common endocrine disorder in women of reproductive age and is one of the leading causes of infertility. It is usually suspected from clinical signs and symptoms, and confirmed by ultrasound examination that shows enlarged ovaries containing more than 10 large cysts. Some women exhibit symptoms of disrupted cycles, central obesity and hyperandrogenism, which can cause acne, alopecia and hirsutism. The endocrine causes are hypersecretion of LH, glucose intolerance and increased levels of testosterone, insulin and PRL. Oestrogen levels are high but not cyclical and ovulation frequently does not occur. The follicles retain the oocyte, forming ovarian cysts which may take on a string-of-pearls appearance. Weight loss often improves the hormonal profile and alleviates the symptoms. Women with PCOS are at greater risk of developing impaired glucose tolerance and type II diabetes (Legro et al., 1999). Pregnant women with PCOS are at greater risk of developing gestational diabetes and therefore should be screened (Radon et al., 1999 and Vollenhoven et al., 2000).

Clomifene citrate (Clomid) is an anti-oestrogenic drug that can be used to re-establish a normal pattern of ovulation in women with PCOS; however, its use is associated with an increased risk of multiple pregnancies. In cases where women fail to respond to clomifene citrate, especially if the BMI is above 25 kg/m², combined treatment with metformin and clomifene citrate improves ovulation and pregnancy rates (National Collaborating Centre for Women's and Children's Health, 2004).

Artificial control of fertility

The first oral contraceptives were extracts from yam. Although yams are rich in progesterone-like compounds, the active ingredient was actually mestranol, an oestrogenic agent. The combination of progestogen and mestranol was essential for good cycle control. Natural progesterone and most other steroid hormones are digested in the gastrointestinal tract and are usually effective only if injected. Chemically modified hormones are resistant to proteolytic digestion in the gut but retain their biological activity. Many synthetic steroid hormones have been developed that have similar biological activity as the naturally occurring hormones and are metabolized very slowly by the liver, increasing their half-life. The term progestogens is used to describe the family of natural and synthetic progesterone-like compounds.

The first contraceptive pills, used in Britain since 1961, were combined oral contraceptive pills (COC), combinations of an oestrogen and a progestogen. Currently, the most common progestogen and oestrogen combinations used are norethisterone and ethinyloestradiol, respectively (Fig. 4.9). A course of COC pills is taken for 21 days followed by 7 pill-free (or placebo) days when hormone levels fall, mimicking natural hormonal cycles and allowing a withdrawal bleed. Monophasic pills have a constant concentration of the active agents whereas biphasic and triphasic preparations attempt to mimic the characteristic fluctuations in oestrogen and progesterone throughout the cycle. Alternatively, progesterone-only preparations, known as ‘minipills’, containing small doses of only progesterone are taken on a continuous basis. Progesterone can also be administered as a depot injection or as a slow-releasing preparation from a subcutaneous or uterine source.

It takes at least one complete menstrual cycle for the pill to become effective. Some drugs reduce the effect of the pill and can cause breakthrough bleeding, or even permit pregnancy. These include barbiturates, antibiotics and some anti-epileptic drugs.

Emergency contraception (‘the morning-after pill’) usually consists of two or four high-dose pills which can be combinations of oestrogen and progesterone or only progesterone. The morning-after pill has strong side-effects and relatively low reliability so it is not recommended as the main method of birth control (Halpern et al., 2010); it also does not protect against sexually transmitted diseases (STDs). Use of the morning-after pill is controversial. For those who believe that pregnancy begins at implantation, the morning-after pill prevents pregnancy, but for those who believe that pregnancy begins at conception, prevention of implantation is classified as abortion because fertilization has already occurred. The morning-after pill should not be confused with mifepristone, which is a synthetic steroid used as an abortifacient for the chemical termination of early pregnancy.

Synthetic oestrogens feedback on the hypothalamus during the antral phase of the menstrual cycle, reducing the levels and the rate of pulsatile secretion of GnRH. Therefore, the release of FSH is inhibited and follicular maturation and expression of LH receptors do not occur. The oestrogens also prevent the LH surge and subsequent ovulation. Production of endogenous oestrogen is reduced.

Synthetic progestogens interfere with the pulsatile secretion of GnRH and decrease the production of LH. Small doses of progesterone may not suppress ovulation but large doses do inhibit maturation of follicles and ovulation. Norethisterone also slows down the breakdown of natural progesterone by the liver. It can be used in low doses because it specifically binds to the progesterone receptors, rather than to androgen receptors as well. Progestogens also reduce secretory activity of the endometrium, so it is not favourable to implantation. Under the influence of progestogens, the cervical mucus is thick and tenacious and so is unreceptive to sperm. The peristaltic muscle activity and cilia movement of the uterine tube become uncoordinated, so transport of the ovum and sperm are affected; this may directly affect successful fertilization. This effect on tubal motility is the reason why there is a slight increase in risk of ectopic pregnancy (implantation in the uterine tube) associated with progesterone preparations.

COCs have their effect by inhibiting ovulation (interrupting feedback on the hypothalamic–pituitary–ovarian axis and reducing FSH and LH), preventing follicular maturation, reducing sperm penetrability of the mucous and affecting endometrial growth and receptivity (Biswas et al., 2008). The progestogen-only pill works by reducing sperm penetrability of the mucous, reducing endometrial receptivity and reducing ovulation. It is well-tolerated but has a higher pregnancy rate as timing of pill taking is more important.

Reported side-effects of oral contraceptives include weight gain, headaches and nausea, depression, vaginal infection or discharge, urinary tract infection, breast changes, skin problems and gum inflammation. Oestrogens affect coagulation factors and promote intravascular coagulation. They also tend to increase plasma lipid levels. Therefore, they can be used safely in young, healthy, motivated women who have no history of circulatory disease. However, smoking and obesity significantly increase the risk of side-effects, particularly thromboembolic complications. Although chemical contraceptive agents have been linked to an increased risk of breast cancer, the doses of synthetic hormone used in current contraceptive preparations are now extremely low, so it is difficult to assess their risk. Contraindications to COC use include arterial disease, smoking, hypertension, migraine, stroke, venous thromboembolism and some cancers (Biswas et al., 2008). Although hormonal treatment has known thromboembolic health concerns, the morbidity complications from pregnancy and labour far outweigh the risks of using oral contraceptives.

The popularity of non-oral hormonal contraception is increasing because it is convenient and efficient and safer for women at risk. Methods include the vaginal ring and contraceptive patches which contain sustained release oestrogen preparations (Black and Kubba, 2008). Progestogen-only methods include contraceptive implants and injectable depo-preparations. Intrauterine methods such as the copper intrauterine device and long-lasting plastic devices such as the T-shaped levopnorgestrel intrauterine system are also effective. Barrier methods of contraception include male and female condoms, diaphragms and caps and spermacides. Sterilization for both men and women is used in many countries as a permanent method of contraception (Melville and Bigrigg, 2008) and worldwide is the most common method of contraception. Female sterilization is more common, although male sterilization is safer, more effective and cheaper. Regret following sterilization is reported to be rare, particularly when individuals are at an older age at sterilization, but requests for reversal following vasectomy are fairly commonplace and usually related to new partnerships or death of children.

Puberty describes the morphological, physiological and behavioural changes that occur as the gonads change from infantile to adult condition. The most obvious sign of sexual maturation in women is menarche (the first menstrual cycle), which indicates that the levels of oestrogen and progesterone are adequate to induce development of the uterus. The equivalent step in men is the first ejaculation, which is often nocturnal. However, menarche or the first ejaculation does not necessarily mean that the adolescent body is able to reproduce; early menstrual cycles are frequently anovulatory and the ejaculate of pubertal boys may be mostly seminal plasma, lacking sperm. The underlying hormonal changes are initiated 2–4 years before menarche and the first ejaculation. The sequence of pubertal changes is constant in that the changes occur in the same order (described as the harmony or ‘consonance’ of puberty) but the starting age and the time for the changes to take place vary. The hormonal changes at puberty lead to a growth spurt and attainment of adult height. In girls, the pubertal growth spurt occurs early in puberty, whereas in boys the growth spurt occurs late in puberty (Fig. 4.10). There is thought to be an evolutionary advantage to the differential timing of adult appearance (attainment of adult height) and sexual development (fertility). Bigger girls may be treated as equals by other females in society and taught ‘female life-skills’, whereas smaller males will not be construed as competitive by other males in society while they undergo development; indeed, becoming fertile while not appearing to be an adult may allow an adolescent male to sneakily mate (Bogin, 1999a).

Physical changes include the development of secondary sex characteristics, the adolescent growth spurt and marked changes in height, psychological states and fertility. All muscle and skeletal dimensions change and the body composition also alters. The earliest changes are measurable in young girls from the age of 6 years. Secondary sex characteristics become evident as secretion of oestrogen (from ovaries) and androgens (from ovaries and adrenal glands) increases. Changes are seen in the breasts, genitalia, pubic hair and voice.

The hypothalamic–pituitary–gonadal axis, which has been developing since fetal life, is activated in early infancy but inhibited in childhood before being reactivated (‘re-awoken’) at puberty. Immediately after birth, levels of hCG and placental steroid hormones fall. LH and FSH levels increase; they are released in a pulsatile pattern, with nocturnal dominance, throughout infancy and childhood. FSH levels are higher in females and LH levels are higher in males. In the prepubertal period, FSH levels are relatively high compared with LH levels.

At puberty, the pulse amplitude of GnRH increases, resulting in dramatically increased magnitude and frequency of LH secretion, particularly during sleep. In late puberty, day-time secretion of LH also increases until the adult pattern of higher basal LH levels is reached. The ‘on’ switch for puberty is unknown but may be related to body mass, energy metabolism, leptin secretion or other nutritional factors. There is a progressive change in pituitary responsiveness to GnRH, and a lifting of the restraint on the hypothalamus, which may be related to maturation of the central nervous system and hypothalamus. One of the important signals appears to be activation of the kisspeptin system (Tena-Sempere, 2010). Kisspeptins are a family of peptides which bind to the kisspeptin receptors in the hypothalamus which are closely associated with the GnRH neurons. They have recently been identified as essential neuropeptide regulators of reproductive maturation which influence the onset of puberty, ovulation and the metabolic regulation of fertility. Adrenal function matures independently before gonadal function. Adrenal secretion of sex steroids increases. Adrenal androgens stimulate pubic and axillary hair growth and have a small effect on growth and bone development. The timing of gonadal maturity and the onset of puberty correlate more closely with bone development than with chronological age. The central nervous system can restrain the onset by affecting the hypothalamic GnRH pattern.

Delayed puberty may result from Kallman's syndrome, hypogonadotropic hypogonadism, which is due to a genetic abnormality that results in a deficiency of GnRH from the hypothalamus. Precocious puberty, when puberty is initiated at a very early age, is usually unexplained and probably due to natural variation but it can also be a result of brain injury or a tumour such as a hypothalamic or pituitary tumour and unusually high production of GnRH or gonadotrophins. Whatever the cause, precocious puberty has serious consequences for social and psychological development and can influence adult height (as the pubertal hormone surges cause the early closure of the bone epiphyses and the cessation of subsequent growth). The increasing incidence of childhood obesity has tended to result in girls entering puberty earlier and boys entering puberty later (Ahmed et al., 2009). It has been suggested that the earlier timing of puberty over the last few decades has resulted in a dissociation between biological maturity which occurs at an earlier age and psychosocial maturity which lags behind and that this mismatch of modern puberty is instrumental in some of the social issues faced by adolescents today (Gluckman and Hanson, 2006).

Over half of early menstrual cycles are anovulatory and do not result in the release of an ovum. Ovulation usually occurs about 10 months after menarche. After a period of 5 years, the incidence of anovulatory cycles has decreased to about 20%. There are secular trends in the age of menarche which got progressively earlier, particularly in the early to mid 20th century. The average age of menarche in Europe is currently 12–13 years compared with 14–15 years a century ago. However, with the marked increase in childhood obesity over the last 20 years, one might expect the age of menarche to have fallen in parallel with increased body size; this has not happened.

Various influences on the age of menarche have been investigated, such as photoperiod and body mass. One suggestion was that the earlier age of menarche has coincided with the introduction of electricity, increasing the photoperiod and the individual's exposure to light (Bullough, 1981). However, a more plausible theory is that it is related to better nutrition. Women seem to have a critical body mass for successful reproduction; if their body mass falls much below, the menstrual cycle becomes erratic and stops. Body fat levels seem to play a role. Anorexic women have lower levels of FSH and LH (see p. 86). Moderate obesity is associated with earlier menarche but severe obesity delays it. The combination of heavy exercise and undernutrition is synergistic, as can be observed in ballet dancers and athletes (see Chapter 12). Chronic illness can also delay menarche; the exact mechanisms are unknown. An alternative view is that there are genetic factors influencing the rate of development and that body fatness and age at menarche are both driven by the ‘blue-print’ or genetic plan rather than age at menarche being a consequence of fat deposition. Children who are taller tend to have earlier pubertal development and the age at menarche correlates better with height than with weight (Cole, 2003). The age at menarche is clearly affected by body mass, exercise, stress, nutrition and altitude (Frisch, 1990).

The normal sequence in females is breast budding (at 8–13 years), growth of pubic hair, peak growth velocity (9.5–14.5 years) and then menarche (10–16.5 years). The pattern in boys is testicular growth, pubic hair growth, penile growth and growth spurt. These characteristic changes in pubic hair distribution and breast development in girls and external genitalia in boys have been classified as Tanner stages and are used to assess pubertal development (Table 4.2).

From about 6 months of age, childhood growth depends on adequate growth hormone (GH) secretion. Growth declines progressively and reaches its slowest velocity just before the onset of the pubertal growth spurt. In boys, the growth spurt starts slightly later, and is faster (9.4 cm/year compared with 8.3 cm/year in girls) with delayed fusion of the epiphyseal plates, so men attain a higher adult height. The pubertal growth spurt depends on sex hormones secreted from the gonads. Optimal growth depends on both sex steroids and GH; GH levels are highest in the pubertal period.

Menopause

Women continue to have menstrual cycles until the finite population of oocytes in the ovary is exhausted. The term ‘menopause’ is literally the cessation of menstrual cycles. It is defined as permanent cessation of menstruation because of the loss of ovarian follicular activity after 12 consecutive months of amenorrhoea without another cause and, hence, can only be determined retrospectively 12 months after the final menstrual period. However, the term ‘menopause’ is frequently applied to the climacteric (perimenopausal phase), which is the transitional decline of reproductive activity over a period of 2–3 years leading up to the final menstrual period, usually occurring between the ages of 45 and 55 years (median 51 years). The climacteric begins when fertility is already rapidly declining and continues until the ovaries cease secreting oestrogen. Ovarian senescence is actually a gradual process beginning from around 35 years of age; it is marked by a progressive decline in fertility and increased rate of menstrual irregularity and miscarriage. The hormonal changes affect particularly the tissues which have a high density of oestrogen receptors such as skin, epithelium of the vagina and bladder, neuronal tissue (changes in neurotransmitter release affect libido, irritability, mood, sleep, concentration and memory) and factors which influence cardiovascular and bone health. Thermoregulation is also affected.

The decline in oestrogen production is related to the number of remaining primordial follicles, the number of recruitable follicles in each cycle and the proportion of follicles that reach maturity before ovulation (Al Azzawi and Palacios, 2009). As the pool of oocytes gets smaller, hormonal changes occur; these precede the final depletion of follicles. Defective follicular phases result in fewer granulose cells in the follicle and therefore reduced oestrogen production. As oestrogen exerts a negative feedback effect at the hypothalamic–pituitary axis, the drop in oestrogen level causes FSH level to rise from about 35 years onwards. The first notable hormonal change preceding the climacteric is a drop in inhibin secretion, which results in a lowering of the negative feedback on the hypothalamic–pituitary gonadal axis. Therefore FSH secretion increases, which means more follicles are recruited at this early stage of the climacteric. The increased level of follicular development results in enhanced oestrogen production from the greater number of follicles; twin ovulations (and pregnancies) are more common. This, paradoxically, means that fertility towards the end of reproductive life is increased (reflected in an increased rate in twinning in older women) but it also increases the rate at which the dwindling pool of oocytes are recruited. So the hormonal changes move from being compensated to decompensated as there follicles are rapidly depleted below a critical number. The compromised hormonal status then results in a decreased follicular phase of the cycle and a shorter cycle length. Functioning of the ovary becomes more erratic with a variable cycle length and an increased number of anovulatory cycles. Luteinization does not occur; there is no increase in progesterone secretion but oestrogen drives endometrial proliferation which can be excessive, as can be menstrual loss when there is a cycle with successful luteinization. Eventually, as the follicles are depleted, oestrogen and progesterone levels fall and menstrual cycles cease. The loss of steroid hormone negative feedback results in a gradual rise in FSH secretion which tends to fluctuate from cycle to cycle.

The loss of follicles means that there is no oestrogen production from granulose cells and no recruitment of thecal cells which produced androgens. Although the postmenopausal ovary no longer synthesizes oestradiol, there is some peripheral conversion of androstenedione by adipose tissue providing a source of oestrone. So body fat essentially acts like inbuilt hormone replacement therapy (HRT). The adrenal gland produces a small amount of progesterone and some testosterone. DHEA production by the ovaries falls which means ovarian androstenedione production falls (although some amount is still produced by the adrenal gland), so testosterone levels fall. The secretion of sex hormone binding protein (SHBP) from the liver diminishes because its production is stimulated by oestrogen and inhibited by androgens (and obesity). So postmenopausally levels of SHBP fall and bioavailability of free testosterone is enhanced. However, overall the net effect is one of androgen deficiency, which can affect sense of well-being, muscle mass and strength, sexual desire and sexual receptivity, sexual arousal and orgasm, memory and cognition, and cause depression, adding to the effect of oestrogen deficiency.

Premature menopause is defined as menopause before 40 years of age; it is usually a result of autoimmune disorders, genetics, or problems with ovarian development or chromosomal abnormalities. Menopause can also be induced, for instance by surgery (removal of ovaries usually with a hysterectomy) or by treatment for cancer. With recent increases in longevity, an average woman may spend about 35–40% of her lifespan in the postmenopausal period. Menopause is a complex and natural process of ageing. It is unique to humans (and a few other species such as the short-finned pilot whale and the Asian elephant) and is thought to have an evolutionary advantage. There are advantages to the species in preventing late childbearing and ensuring that the dependent human offspring are more likely to have the care and protection of their mother (and she to have the support of her mother; Shanley and Kirkwood, 2001). The Grandmother Hypothesis (Kuhle, 2007) suggests that the presence of postmenopausal women is beneficial for the species; compared to other species, human infants are altricial (very helpless and dependent), maturation is delayed (because childhood is an important learning time for a species with a big brain), infant mortality rates are high and inter-birth intervals are short. Grandmothers are socially established, reliable and skilled, possess specialized knowledge and have a vested interest in the survival of their grandchildren. Indeed, there are examples of grandmothers (who have their own infants), able to take over breastfeeding of their grandchildren (Scelza, 2009).

A number of factors affect the age of menopause. These include leanness and nutritional status, ethnicity and genetics. Smokers, women who have not had children and women of lower socio-economic status tend to have an earlier menopause as do women who have shorter menstrual cycle length. There is no relationship between age at menarche and age at menopause.

Morphologically, the ovaries at menopause appear smaller and relatively devoid of follicles (Santoro and Chervenak, 2004). There is a finite number of ova; however, hormonal changes precede the depletion of follicles. There are about 25000 follicles remaining at the time of menopause. One of the earliest changes is a decrease in inhibin production by the granulosa cells. This results in decreased negative feedback at the hypothalamic–pituitary axis and an increase in GnRH level, which promotes secretion of FSH and follicular development. This is the reason for the paradoxical increase in twinning rate that is observed in women conceiving late in reproductive life. After this brief increase in follicular development, the menstrual cycles tend to become shorter, particularly in the follicular phase. This results in oestrogen secretion diminishing, so the production of androgens increases. Menstrual cycles become increasingly erratic with variable cycle lengths and an increase in anovulatory cycles. Anovulation is associated with progesterone deficiency, which is associated with prolonged or irregular vaginal bleeding. As the cycles become less frequent, there is increased time for endometrial proliferation, which can lead to excessive menstrual blood loss. Ultimately, oestrogen and progesterone levels decrease and cycling ceases.

From menopause onwards, levels of FSH and LH are high and levels of oestrogen and inhibin are decreased. FSH increases because of the lack of a negative feedback from oestrogen influencing the anterior pituitary gland. The postmenopausal ovary continues to produce considerable amounts of androgens and some progesterone; thus, natural menopause is not equivalent to the effects of a surgically induced menopause following oophorectomy (removal of ovaries). There is some oestrogen production by the adipose tissue; this (and the protective cushioning provided by fat) is the reason why fatter women are protected, at least partially, from osteoporosis.

In addition to the reproduction tract itself, there are many other target organs bearing oestrogen receptors, which respond to the fall in circulating oestrogen levels. The resulting vasomotor instability produces symptoms of hot flushes (or ‘hot flashes’), sweats and palpitations. The thermoregulatory centre in the hypothalamus falsely signals that body temperature is too high. It is thought that the decreased oestrogen level abrogates the catechol–oestrogen inhibition of tyrosine hydroxylase so noradrenaline levels are increased. This results in physiological processes such as increased peripheral vasodilation that attempt to reduce core body temperature. Hot flushes are not always visible but may be extreme; skin temperature may increase by as much as 7–8 °C for a few minutes accompanied by a rapid increase in heart rate. Emotional and psychological problems such as anxiety, depression, loss of libido and mood swings may occur. Insomnia is also a frequently cited problem.

All the tissues of the female reproductive tract have a high density of oestrogen receptors and are profoundly affected by oestrogen withdrawal. The uterus shrinks. The vaginal epithelium diminishes and becomes less elastic. The vaginal cells decrease production of glycogen, affecting lactobacillus colonization, so the pH increases, resulting in increased susceptibility to vaginal infections. Vaginal atrophy causes vaginal secretions to diminish, which may result in painful intercourse. Menopausal women have an increased frequency of urinary problems, which is probably related to oestrogen withdrawal as there are many oestrogen receptors in the urinary tract (which shares the same embryonic origin as the lower reproductive tract). The walls of the lower bladder and urethra become thinner and the urethral muscles weaken, which increases the risk of stress incontinence.

Oestrogen protects the cardiovascular system; the risk of cardiovascular disease doubles in postmenopausal women. The incidence of coronary heart disease in premenopausal women and postmenopausal women treated with HRT is much lower than in men. Oestrogen inhibits the uptake and degradation of low-density lipoprotein (LDL) by the coronary blood vessel endothelium. It may also inhibit coronary vasospasm. Oestrogen has been shown to decrease vascular resistance (and therefore blood pressure), increase cardiac output and increase synthesis of nitric oxide (NO, a potent locally acting vasodilator). Postmenopausal women have significantly higher levels of serum cholesterol and triacylglycerides. Oestrogen inhibits endothelial hyperplasia, smooth muscle cells growth and platelet activation. Oestrogen withdrawal is associated with raised levels of certain blood-clotting factors and an increased tendency for thrombosis, and thus with increased risk of myocardial infarction and cerebrovascular accident (stroke). Insulin resistance is more common in postmenopausal women.

Skeletal changes occur as the decrease in oestrogen results in increased bone resorption, increasing the tendency to stoop and the likelihood of fractures. Osteoblasts (bone-producing cells) have oestrogen receptors. Osteoclast activity increases postmenopause and osteoblast activity decreases. Oestrogen deficiency uncouples bone formation and bone resorption. This effect is increased by changes in the hormones controlling calcium balance. Levels of calcitonin fall in parallel with oestrogen levels. Calcitonin inhibits the activity of osteoclasts (bone-absorbing cells). The progressive loss of calcium from the bones and the long postmenopausal lifetime mean that a woman can lose about half of her trabecular bone density and about a third of her cortical bone and is therefore predisposed to osteoporosis. Collagen is lost from the skin, tendons and bones.

There are also changes in metabolism and body composition postmenopause. Women tend to gain fat, especially visceral fat, and to lose muscle mass.

Hormone replacement therapy (HRT) aims to reduce the diverse symptoms and adverse effects of menopause (Box 4.9). HRT provides low doses of various combinations of hormones. It is effective during its use but not in the long-term. Oestrogen on its own is mitogenic and promotes endometrial hyperplasia (which is associated with increased risk of cancer). Oestrogen with progesterone is safer as progesterone abrogates cell division and increases endometrial secretory activity. The Women's Health Initiative (WHI) studies in the United States looked at long-term health outcomes of women taking HRT using randomized controlled primary-prevention trials. The WHI studies found that HRT increased the risk of heart disease, stroke, deep vein thrombosis, pulmonary embolism and some types of cancer. This was at odds with previous studies which tended to select healthy women as subjects and omitted women who smoked or might be at increased risk of vascular disease. However, the figures need to be considered in context; although the risk of disease was increased significantly, the actual numbers of women affected were very low. Rather than being a panacea for all menopausal problems, there are benefits and risks to HRT which need to be assessed, as for all medications. Some women have extreme symptoms of menopause which have a very negative influence on the quality of life.

Key points

• The ovary produces the female gametes (ova) and steroid hormones, oestrogen and progesterone.

• Relatively few female gametes are produced during a woman's reproductive life, between puberty and the menopause.

• The meiotic division of the ovum begins in the female fetus and is suspended until ovulation, halts again, and is completed at fertilization.

• Follicular development begins about 3 months prior to ovulation but key stages in development of the follicles are stimulated by FSH in the first half of the menstrual cycle in which the ovum is released. The developing follicles produce oestrogen; usually a single dominant follicle matures and ovulates.

• The first half of the menstrual cycle (follicular phase) is dominated by oestrogen and prepares the reproductive system for ovulation, for instance by stimulating growth of the endometrial lining.

• Ovulation is triggered by the surge of LH. The ovum surrounded by a rim of cumulus cells is released and swept into the uterine tube.

• Follicular cells remaining in the ovary become the corpus luteum, which produces progesterone and oestrogen.

• The second half of the cycle (luteal phase) is dominated by the effects of progesterone, which prepare the body for pregnancy.

• LH promotes secretion from the corpus luteum. However, the effect is short-lived, so the corpus luteum regresses, unless rescued by hCG from the dividing cells of the embryo, and menstruation ensues.

• Pituitary secretion of FSH and LH is under the control of pulsatile GnRH release from the hypothalamus. Oestrogen and progesterone exert negative feedback effects on the hypothalamic–pituitary axis except at mid-cycle, when oestrogen exerts positive feedback leading to the LH surge and ovulation.

• The hypothalamus integrates other signals regulating reproductive function. Fertility can be disrupted by abnormal endocrine activity such as abnormal production of GnRH and hyperprolactinaemia, abnormal follicular development and extremes of weight loss or gain.

• Understanding the hormonal regulation of reproduction has allowed manipulation of fertility using chemical analogues of the steroid hormones in contraceptives.

Application to practice

There are many environmental influences, both internal and external, that may affect the regulation of reproductive cycles. There is increasing evidence that many pollutants and chemicals in the environment can have negative effects upon human reproduction; knowledge of this is important in understanding some possible causes of subfertility and congenital abnormalities of the genitalia.

It is important to realize that the menstrual cycle prepares women for pregnancy. The physiological changes, in preparation for and support of pregnancy, are initiated prior to ovulation and conception.

An understanding of the variance in the menstrual cycle is important when considering the estimated due date. Knowledge of the reproductive cycles is essential in understanding the various methods of birth control.

Box 4.9

Hormone replacement therapy (HRT)

• Exogenous oestrogen replaces ovarian oestrogen

• Prevents long-term consequences

• Oral route or directly to genital tract or systemically (transdermal patch or subcutaneous implant)

• Abrogates flushing and sweating

• Stimulates replication of, and secretion from, vaginal epithelial cells

• Progesterone given to induce menstruation (and prevent endometrial hyperplasia)

• Progesterone-withdrawal bleeding is major reason for non-compliance

• Protects against coronary heart disease and osteoporosis

Annotated further reading

Balen In: (Editor: Balen, A.H.) Infertility in practice ed 3 (2008) Informa, UK.

A practical guide, based on the author's clinical practice, which provides an overview of human infertility problems, aetiology and evidence-based possible interventions.

Guillebaud Guillebaud, J., In: Contraception today ed 6 (2007) Informa, UK.

This book provides an evidenced based guide to all forms of contraception available. This latest edition includes information for contraceptive use in the older women.

Gougeon Gougeon, A., Human ovarian follicular development: from activation of resting follicles to preovulatory maturation, Ann Endocrinol (Paris) 71 (2010) 132–143.

An in-depth but clearly explained review of follicular growth in the human ovary which describes the interactions of hormones and local growth factors in controlling oocyte maturation and follicular growth.

Hirschberg Hirschberg, A.L., Polycystic ovary syndrome, obesity and reproductive implications, Womens Health (Lond Engl) 5 (2009) 529–540.

A recent review about polycystic ovary syndrome (PCOS) and its biological basis which covers effects on fertility, pregnancy and metabolic syndrome and discusses effective lifestyle programmes.

Glasier Glasier, A., In: Handbook of family planning and reproductive healthcare ed 5 (2007) Churchill Livingstone, New York.

A practical handbook covering all forms of contraception, pill prescribing, possible complications, advantages and disadvantages of each method and the clinical management of women with psychosexual disorders, sexually transmitted infections, menopausal symptoms and gynaecological problems.

Johnson Johnson, M.H., In: Essential reproduction ed 6 (2007) Blackwell Science, Oxford.

An integrated and well-organized research-based textbook that explores comparative reproductive physiology of mammals, including anatomy, physiology, endocrinology, genetics and behavioural studies.

Kreitzman Kreitzman, L.; Russell, F.G., Rhythms of life: the biological clocks that control the daily lives of every living thing. (2004) ; Profile Books.

A very readable text covering fascinating details about the rhythms of the natural world and how time influences life.

Nappi Nappi, R.E.; Lachowsky, M., Menopause and sexuality: prevalence of symptoms and impact on quality of life, Maturitas 63 (2009) 138–141.

A review of sexual and urogenital symptoms at menopause and their impact on quality of life.

Leung Leung, P.C.K.; Adashi, E.Y., In: The ovary ed 2 (2004) Academic Press, London.

A detailed description of ovarian structure and function at the cellular and molecular level, including normal development and pathophysiology.

Mistlberger Mistlberger, R.E.; Skene, D.J., Social influences on mammalian circadian rhythms: animal and human studies, Biol Rev Camb Philos Soc 79 (3) (2004) 533–556.

A readable review, which describes how light and social stimuli (‘zeitgebers’ or time-cues) entrain mammalian circadian rhythms.

Ridley Ridley, M., The red queen: sex and the evolution of human nature. (1994) Penguin, London .

A new and exciting approach to understanding reproductive behaviour and physiology from an evolutionary perspective.

Shuttle Shuttle, P.; Redgrove, P., In: The wise wound: menstruation and every woman ed 3 (1999) Victor Gollancz, London.

This text provides an exploration of the sociological and anthropological perspectives of human reproduction including the facts, fantasies, taboos and cultural aspects surrounding menstruation.

Szarewski Szarewski, A., Choice of contraception, Curr Obstet Gyn 16 (2009) 361–365.

A short discussion of the factors affecting choice of contraception which illustrates the issues with three case studies.

References

Adashi, E.Y.; Resnick, C.E.; Hernandez, E.R.; et al., Ovarian transforming growth factor-beta (TGF beta): cellular site(s), and mechanism(s) of action, Mol Cell Endocrinol 61 (2) (1989) 247–256.

Ahmed, M.L.; Ong, K.K.; Dunger, D.B., Childhood obesity and the timing of puberty, Trends Endocrinol Metab 20 (2009) 237–242.

Al Azzawi, F.; Palacios, S., Hormonal changes during menopause, Maturitas 63 (2009) 135–137.

Biswas, J.; Mann, M.; Webberley, H., Oral contraception, Obstet Gyn Reproduct Med 18 (2008) 317–323.

Black, K.I.; Kubba, A., Non-oral contraception, Obstet Gyn Reproduct Med 18 (2008) 324–329.

Bogin, B., Evolutionary perspective on human growth, Ann Rev Anthropol 28 (1999) 109–153.

Bogin, B., Patterns of Human Growth. ed 2 (1999) Cambridge University Press, Cambridge .

Brooker, C.G., In: Human structure and function ed 2 (1998) Mosby, St Louis; 480, 488, 489.

Brosens, J.J.; Parker, M.G.; McIndoe, A.; et al., A role for menstruation in preconditioning the uterus for successful pregnancy, Am J Obstet Gynecol 200 (2009) 615–616.

Buffenstein, R.; Poppitt, S.D.; McDevitt, R.M.; et al., Food intake and the menstrual cycle: a retrospective analysis with implications for appetite research, Physiol Behav 58 (1995) 1067–1077.

Bulletti, C.; de Ziegler, D., Uterine contractility and embryo implantation, Curr Opin Obstet Gynecol 17 (2005) 265–276.

Bullough, V.L., Age at menarche: a misunderstanding, Science 213 (1981) 365–366.

Clancy, K.B., Reproductive ecology and the endometrium: physiology, variation, and new directions, Am J Phys Anthropol 140 (Suppl. 49) (2009) 137–154.

Clarke, I.J., The hypothalamic–pituitary axis, In: (Editors: Hillier, S.G.; Kitchener, H.C.; Neilson, J.P.) Scientific essentials of reproductive medicine (1996) WB Saunders, Philadelphia, pp. 120–132.

Cole, T.J., The secular trend in human physical growth: a biological view, Econ Hum Biol 1 (2) (2003) 161–168.

Dale, B.; Marino, M.; Wilding, M., The ins and outs of meiosis, J Exper Zool 285 (3) (1999) 226–236.

Davidsen, L.; Vistisen, B.; Astrup, A., Impact of the menstrual cycle on determinants of energy balance: a putative role in weight loss attempts, Int J Obes Lond 31 (2007) 1777–1785.

Dye, L.; Blundell, J.E., Menstrual cycle and appetite control: implications for weight regulation, Hum Reprod 12 (1997) 1142–1151.

Farage, M.A.; Osborn, T.W.; MacLean, A.B., Cognitive, sensory, and emotional changes associated with the menstrual cycle: a review, Arch Gynecol Obstet 278 (2008) 299–307.

Farage, M.A.; Neill, S.; MacLean, A.B., Physiological changes associated with the menstrual cycle: a review, Obstet Gynecol Surv 64 (2009) 58–72.

Fessler, D.M.T., No time to eat: an adaptionist account of periovulatory behavioural changes, Q Rev Biol 78 (2003) 3–21.

Frisch, R.E., The right weight, body fat, menarche and ovulation, Baillière's Clin Obstet Gynaecol 4 (1990) 419–439.

Girman, A.; Lee, R.; Kligler, B., An integrative medicine approach to premenstrual syndrome, Am J Obstet Gynecol 188 (2003) S56–S65.

Giudice, L.C., Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine, Fertil Steril 61 (1994) 1–17.

Gluckman, P.D.; Hanson, M.A., Evolution, development and timing of puberty, Trends Endocrinol Metab 17 (2006) 7–12.

Halpern, V.; Raymond, E.G.; Lopez, L.M., Repeated use of pre- and postcoital hormonal contraception for prevention of pregnancy, Cochrane Database Syst Rev (2010); CD007595.

Hamilton-Fairley, D.; Johnson, M.R., The ovary, In: (Editors: Chamberlain, G.; Broughton Pipkin, F.) Clinical physiology in obstetrics ed 3 (1998) Blackwell, Oxford, pp. 396–417.

Hassan, E.; Creatsas, G.; Mastorakos, G.; et al., Clinical implications of the ovarian/endometrial renin–angiotensin–aldosterone system, Ann N Y Acad Sci 900 (2000) 107–118.

Hillier, S.G., Regulatory functions for inhibin and activin in human ovaries, J Endocrinol 131 (1991) 171–175.

Hillier, S.G.; Whitelaw, P.F.; Smyth, C.D., Follicular oestrogen synthesis: the ‘two-cell, two-gonadotrophin’ model revisited, Mol Cell Endocrinol 100 (1994) 51–54.

Huang, C.J.; Stromer, M.H.; Anderson, L.L., Abrupt shifts in relaxin and progesterone secretion by aging luteal cells: luteotrophic response in hysterectomized and pregnant rats, Endocrinology 128 (1991) 165–173.

Hurwitz, A.; Loukides, J.; Ricciarelli, E.; et al., Human intraovarian interleukin-1 (IL-1) system: highly compartmentalized and hormonally dependent regulation of the genes encoding IL-1, its receptor, and its receptor antagonist, J Clin Invest 89 (6) (1992) 1746–1754.

Johnson, M.H.; Everitt, B.J., In: Essential reproduction ed 4 (1995) Blackwell Science, Oxford; 64, 65, 108, 109.

Johnson, M.R.; Carter, G.; Grint, C.; et al., Relationship between ovarian steroids, gonadotrophins and relaxin during the menstrual cycle, Acta Endocrinol (Copenh) 129 (1993) 121–125.

Johnson, J.; et al., Germline stem cells and follicular renewal in the postnatal mammalian ovary, Nature 428 (2004) 145–150.

Kuhle, B.X., An evolutionary perspective on the origin and ontogeny of menopause, Maturitas 57 (2007) 329–337.

Kunz, G.; Leyendecker, G., Uterine peristaltic activity during the menstrual cycle: characterization, regulation, function and dysfunction, Reprod Biomed Online 4 (2001) 5–9.

Legro, R.S.; Kunselman, A.R.; Dodson, W.C.; et al., Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women, J Clin Endocrinol Metabol 84 (1999) 165–169.

Lyons, E.A.; Taylor, P.J.; Zheng, X.H.; et al., Characterization of subendometrial contractions throughout the menstrual cycle in normal fertile women, Fertil Steril 55 (1991) 771–774.

Mason, H.D.; Margara, R.; Winston, R.M.L.; et al., Insulin-like growth factor-I (IGF-I) inhibits production of IGF-binding protein-I while stimulating estradiol secretion in granulosa cells from normal and polycystic human ovaries, J Clin Endocrinol Metab 76 (1993) 1275–1279.

Mason, H.D.; Willis, D.S.; Holly, J.M.P.; et al., Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells, J Clin Endocrinol Metab 78 (1994) 1265–1267.

McClure, N.; Healy, D.L.; Rogers, P.A.W.; et al., Vascular endothelial growth factor as capillary permeability agent in ovarian hyperstimulation syndrome, Lancet 34 (1994) 235–236.

Melville, C.; Bigrigg, A., Male and female sterilization, Obstet Gyn Reproduct Med 18 (2008) 330–334.

National Collaborating Centre for Women's and Children's Health, Clinical guideline 11. Fertility: assessment and treatment for people with fertility problems. (2004) National Institute for Clinical Excellence .

Oosthuyse, T.; Bosch, A.N., The effect of the menstrual cycle on exercise metabolism: implications for exercise performance in eumenorrhoeic women, Sports Med 40 (2010) 207–227.

Pinkerton, J.V.; Guico-Pabia, C.J.; Taylor, H.S., Menstrual cycle-related exacerbation of disease, Am J Obstet Gynecol 202 (2010) 221–231.

Radon, P.A.; McMahon, M.J.; Meyer, W.R., Impaired glucose tolerance in pregnant women with polycystic ovary syndrome, Obstet Gynecol 94 (1999) 194–197.

Rance, N.E.; Young, W.S.; McMullen, N.T., Topography of neurons expressing luteinizing hormone releasing hormone gene transcripts in the human hypothalamus and basal forebrain, J Comp Neurol 339(1991) 573–586.

Richards, J.S.; Pangas, S.A., The ovary: basic biology and clinical implications, J Clin Invest 120 (2010) 963–972.

Ridley, M., The red queen: sex and the evolution of human nature. (1994) Penguin Science .

Roberts, V.J.; Barth, S.; El-Roeiy, A.; et al., Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle, J Clin Endocrinol Metab 77 (5) (1993) 1402–1410.

Salamonsen, L.A., Tissue injury and repair in the female human reproductive tract, Reproduction 125 (2003) 301–311.

Santoro, N.; Chervenak, J.L., The menopause transition, Endocrinol Metab Clin North Am 33 (4) (2004) 627–636.

Scelza, B.A., The grandmaternal niche: critical caretaking among Martu Aborigines, Am J Hum Biol 21 (2009) 448–454.

Scheele, F.; Schoemaker, J., The role of follicle stimulating hormones in the selection of follicles in human ovaries: a survey of the literature and a proposed model, Gynecol Endocrinol 10 (1996) 55–66.

Shanley, D.P.; Kirkwood, T.B., Evolution of the human menopause, Bioessays 23 (3) (2001) 282–287.

Stem, K.; McClintock, M.K., Regulation of ovulation by human pheromones, Nature 392 (1998) 177–179.

Strassmann, B.I., The evolution of endometrial cycles and menstruation, Q Rev Biol 71 (1996) 181–220.

Tena-Sempere, M., Kisspeptin signaling in the brain: recent developments and future challenges, Mol Cell Endocrinol 314 (2010) 164–169.

Terner, J.M.; de Wit, H., Menstrual cycle phase and responses to drugs of abuse in humans, Drug Alcohol Depend 84 (2006) 1–13.

Van Vugt, D.A., Brain imaging studies of appetite in the context of obesity and the menstrual cycle, Hum Reprod Update 16 (2010) 276–292.

Vollenhoven, B.; Clark, S.; Kovacs, G.; et al., Prevalence of gestational diabetes mellitus in polycystic ovarian syndrome (PCOS) patients pregnant after ovulation induction with gonadotrophins, Aust N Z J Obstet Gynaecol 40(2000) 54–58.

Zeleznik, A.J.; Hillier, S.G., The ovary: endocrine function, In: (Editors: Hillier, S.G.; Kitchener, H.C.; Neilson, J.P.) Scientific essentials of reproductive medicine (1996) WB Saunders, Philadelphia, pp. 133–146.