Menstrual physiology

Menstrual cycle physiology may be considered at different levels: hypothalamic, pituitary, ovarian, and endometrial (Figure 40.1). Peptide and steroid hormones provide cross-talk and feed­back between these levels to regulate menstruation.

Hypothalamic-pituitary regulation of ovarian function

The classical model of the menstrual cycle starts with the hypothal­amus and GnRH (Figure 40.2). GnRH is produced and released from neurons in the arcuate nucleus of the medial-basal hypothal­amus and the preoptic area of the ventral hypothalamus. It is re­leased by axonal transport into the capillaries of the hypophyseal portal system for delivery to the anterior pituitary gland, where GnRH stimulates the synthesis and secretion of LH and FSH from the gonadotrophs.

GnRH is secreted in a pulsatile manner, which is an absolute prerequisite for normal adult reproductive function, including pu­bertal maturation and fertility (9, 10). The frequency and amplitude of GnRH secretion vary depending on the stage of reproductive life and across the menstrual cycle. In the normal cycling woman,

Figure 40.1 Schematic representation of pituitary and ovarian hormones across the menstrual cycle and the response of the ovary and endometrium.

GnRH pulses are of low amplitude, but increase in their frequency during the follicular phase, to a frequency of every 60 minutes during late follicular phase. High GnRH pulse frequency favours LH release, which is also secreted in pulses reflecting those of GnRH and is predominant over FSH in the late follicular phase necessary for ovulation.

During the menstrual cycle GnRH and gonadotropin activity is highly regulated by ovarian feedback loops. In the follicular phase, oestrogen exerts negative feedback at the level of hypothalamus to suppress LH and FSH secretion. Inhibin B more selectively sup­presses FSH secretion. However, in the late follicular phase, by yet unclear mechanisms, negative oestrogen feedback switches to positive oestrogen feedback, culminating in the preovulatory LH surge (and to a lesser extent a rise in FSH) necessary for ovulation. Following ovulation, progesterone from the corpus luteum mediates negative feedback to slow down GnRH pulse frequency and subse­quently LH secretion. During the luteal phase, inhibin A also sup­presses FSH levels. With demise of the corpus luteum in the absence

Table 40.1 Principal hormones of the menstrual cycle

Figure 40.2 The hypothalamic-pituitary-ovarian axis. Hypothalamic gonadotrophin-releasing hormone (GnRH) signals to the anterior pituitary which in turn results in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion.

of pregnancy, gonadal steroid secretion declines, resulting in men­struation and stimulation of FSH secretion in response to a loss of negative sex-steroid feedback.

Prepubertal development of the HPO axis and menarche

The ovarian germ cells (oogonia) appear at around day 25, and then migrate to the developing gonad at around day 30 of gestation. They undergo meiosis and differentiate into primary oocytes, having ar­rested during the first meiotic division before entering prophase I.

Pulsatile GnRH secretion is observed during fetal life from around the 16th week of gestation with sustained gonadotropin and sex-steroid secretion. With advancing gestation, LH and FSH secre­tion is suppressed due to the negative sex-steroid feedback from the developing ovary. Withdrawal of placental steroids following de­livery results in rebound GnRH and gonadotropin activity and per­sists into the early neonatal period. To distinguish this phenomenon from the true puberty, this phase is referred to as ‘mini-puberty’, where despite the secretion of gonadotropins and sex steroids, ovu­lation is not present. After the postnatal rise, pulsatile GnRH and go­nadotropin secretion become quiescent throughout childhood, with some irregular and low-amplitude pulses, suppressing any activity of the now developed and responsive gonads.

With the onset of puberty there is a nocturnal increase in GnRH secretion and enhanced responsiveness of the pituitary. Slow GnRH pulsatility initially stimulates FSH secretion, resulting in ovarian fol­licular development and a multicystic appearance of the ovaries, and with the acceleration of GnRH pulsatility LH is secreted.

The irregular nocturnal pattern of LH secretion gradually in­creases until regular LH pulses are established, occurring every 90 minutes irrespective of time of day. This results in the production of gonadal steroids and contribution to the development of external sexual characteristics (thelarche—onset of breast development). As circulating concentrations of oestrogens rise, positive feedback develops to induce a preovulatory LH surge with resultant ovula­tion and the first experience of menstruation (menarche). Of note, adrenarche (axillary and pubic hair growth) reflects the maturation of the hypothalamic-pituitary-adrenal axis. Many of the initial cycles may be anovulatory, which can contribute to the heavy and ir­regular menstrual bleeding that can affect some girls in adolescence. After 4-5 years the number of anovulatory cycles reduces from 90% to 20% with the consequent experience of more regular menstrual bleeds.

Menopause

Menopause marks the end of the female natural reproductive life and is a retrospective diagnosis based on the absence of periods for 1 year. It occurs when the supply of primordial follicles available for recruitment is exhausted. This process of follicle loss commences while the fetus and her developing gonads are still in utero (16-18). At the time of menopause there is a corresponding decline in circu­lating oestrogen levels and gonadotropin levels (FSH and LH) are elevated.

Ovarian function

Ovarian function within the menstrual cycle can be broadly div­ided into the follicular phase, ovulation, luteal phase, and finally the luteal-follicular transition.

Follicular phase

Throughout ovarian life the oocytes reside within follicles. At any time point in the reproductive years there are follicles at different developmental stages within the ovary.

Initially, they are found in primordial follicles, consisting of a primary oocyte surrounded by a single flattened layer of granulosa cells. At the beginning of each menstrual cycle, a cohort of prim­ordial follicles is activated to initiate growth, regulated by both stimulatory and inhibitory molecular signals. The follicles transition into primary follicles, with the granulosa cells becoming cuboidal in shape, before developing into larger preantral follicles. At this stage, the zona pellucida develops between the oocyte and granulosa cells and surrounding stromal cells differentiate to form the theca. Subsequently, the follicles enlarge, developing a fluid-filled cavity termed the antrum. Preantral and early antral follicles produce anti-Mullerian hormone, which appears to have an inhibitory ef­fect on the growth of nearby primordial follicles, thus preventing their activation. This prevents the ovarian reserve being exhausted too quickly. Anti-Mullerian hormone is also therefore an indirect measure of ovarian reserve as it reflects how many growing follicles there are within the ovary—t he higher the serum concentration of anti-Mullerian hormone, the greater the remaining number of follicles (19).

As the follicles grow they acquire FSH and LH receptors and be­come increasingly gonadotropin dependent, with antral follicles completely dependent on FSH for granulosa cell proliferation and LH for theca cell sex steroidogenesis.

Ovulation

Following the LH surge and just prior to ovulation, the oocyte re­sponds to its hormonal environment and re-enters meiosis, ar­resting at metaphase II resulting in a secondary oocyte and a polar body. The granulosa cells decrease oestradiol secretion as a result of decreased sensitivity to FSH. Progesterone production is initiated as a result of LH-driven increased cholesterol side-chain cleavage (P450scc) levels. This enzyme is critical to steroidogenesis, particu­larly the conversion of cholesterol to pregnenolone. The rise in LH and FSH causes an increase in antral blood flow. This increased vas­cularity and local secretion of prostaglandins causes an increase in size of the follicle and it distends the surface of the ovary. Proteolytic enzymes are synthesized in the theca and activated by prostaglan­dins, causing degradation of the distended follicular wall followed by rupture of the follicle capsule. The oocyte, surrounded by the zona pellucida and attached granulosa cells, is ejected from the ruptured follicle. This occurs on approximately day 14 of the men­strual cycle. Under the influence of oestrogens prior to ovulation, increased tubal motility and elevated activity of the densely ciliated fimbriae allows approximation of the aperture of the distal fallopian tube to the ovary. This promotes passage of the expelled oocyte into the tube to be met by the ascending spermatozoa. If fertilization oc­curs, meiosis is completed.

Luteal phase

Following ovulation, the walls of the ovarian follicle collapse. Under the influence of LH both the theca and the granulosa cells prolif­erate and the latter develop into luteal cells. There is an influx of lipid droplets and lutein, which gives the corpus luteum its characteristic yellow appearance. Overall, the corpus luteum enlarges to around 15 mm. The oestrogen and progesterone secreted by the luteal cells negatively feed back to the anterior pituitary and levels of FSH and LH decrease. The corpus luteum is reliant on LH for progesterone production and luteolysis occurs in the absence of pregnancy as the mature corpus luteum becomes less sensitive to the remaining LH. This process remains incompletely understood, particularly with re­gard to the initiating trigger. In addition to reduced gonadotropins, luteolysis is likely a product of decreased LH sensitivity, altered pro­gesterone receptor isoform ratios, and oestrogen receptor (ERα) levels (20, 21). Demise of the corpus luteum begins at around day 24 and the corpus luteum is replaced by whitish scar tissue, the corpus albicans. Over subsequent cycles it is replaced by connective tissue, then absorbed.

Luteal-follicular transition

As the corpus luteum degenerates, circulating concentrations of oestradiol and progesterone rapidly decrease. As a result, FSH and LH plasma concentrations rise and a fresh group of follicles are re­cruited. The withdrawal of progesterone initiates menses.

Maintenance of the corpus luteum in pregnancy

In the event of conception, the developing embryo secretes human chorionic gonadotropin (hCG) from the syncytial trophoblast. This is detectable in maternal serum from around 8 days following con­ception. hCG rescues the corpus luteum, preventing menstruation and supporting early embryonic growth. Between 7 and 12 weeks' gestation, oestrogen and progesterone production shifts to the pla­centa and once the second trimester is entered, the corpus luteum slowly involutes and hCG levels decline.

If the corpus luteum is removed prior to 7 weeks' gestation then miscarriage almost inevitably occurs as a result of progesterone and oestrogen withdrawal. A similar effect can be induced by the use of progesterone antagonists such as mifepristone which underpins its use in the medical management of unwanted pregnancy.

Endometrial response to ovarian function

The endometrium consists of two functionally separate layers overlying the myometrium: functional and basal layers. The func­tional layer is shed during menses and the basal layer is preserved throughout. Both layers comprise glandular epithelial and stromal cells and the functional layer is covered by a surface epithelial layer. The constituent cells may express receptors for progesterone, oes­trogen, and androgens as well as receptors for the glucocorticoid hormone, cortisol (22, 23). The expression of the receptors varies depending on the stage of cycle. Occasional lymphoid follicles may be observed as well as a variety of leucocytes. The endometrium has a complex blo o d supply arising from the radial branches of the uterine artery and the ovarian vessels. There are both short, straight arteries which supply the basal layer and longer, spiral vessels supplying the whole of the endometrium. The spiral vessels connect with the venous system though capillary networks and direct arteriovenous communications.

The endometrial aspect of the menstrual cycle is divided into proliferative, secretory, and menstrual phases, broadly corres­ponding to the follicular, luteal, and luteal-follicular transition phases of the ovarian cycle. The classical histological descrip­tion of the cycling endometrium dates from the 1950s (24) but for research purposes dating may be more robustly determined utilizing histological features combined with the date of the re­ported last menstrual period and measurement of serum oes­tradiol and progesterone. More recently, advances in molecular phenotyping have added a greater depth of understanding to the molecular and cellular events that underpin menstrual cycle stage and function (25).

Proliferative phase

Following menstruation, the exposed basal layer of the endomet­rium proliferates rapidly under the influence of rising oestradiol levels. Concurrently, the glandular cells within the endometrium expand: in the early proliferative phase the glandular cells are ini­tially cuboidal and the glands themselves are small but by the late proliferative phase the glands are tortuous and the individual epithe­lial cells appear columnar. Proliferation is brisk and mitotic figures are observed (Figure 40.3). The stroma is compact throughout the proliferative phase and angiogenesis commences with elongation of the spiral arteries.

Secretory phase

Following ovulation, secretion of progesterone from the corpus luteum inhibits proliferation. The morphological changes associ­ated with progesterone exposure develop between ovulation and approximately 48 hours after progesterone withdrawal (due to demise of the corpus luteum). The endometrial glands become more tortuous and acquire increased secretion of glycoproteins, evident as subnuclear vacuolation (a feature of the early secretory phase). Glandular nuclei move to the centre of cells and mitosis is supressed. The endometrial spiral arterioles undergo remodelling to become increasingly coiled.

The spiral arterioles have a pivotal role at this time because if im­plantation is successful, the interaction with invading trophoblasts will play a crucial role in the onward pregnancy outcome.

Figure 40.3 Endometrial morphology across the menstrual cycle. Proliferative and secretory endometrium. Surface epithelium (SE), glandular epithelium (GE), and stromal compartment (St) are marked. Note the increased tortuosity of glands in secretory phase and marked thickening of the functional layer.

Adapted from FM Horne and DL Blithe. Progesterone receptor modulators and the endometrium: changes and consequences, Hum Reprod Update 2007;13:567-580.

Menstruation

In the absence of pregnancy the corpus luteum regresses, resulting in a rapid decrease in circulating progesterone and oestradiol. It is progesterone withdrawal that initiates menstruation (Figure 40.4). A local inflammatory response within the endometrium is charac­terized by cytokine release and infiltration of leucocytes with re­sultant oedema, activation of matrix metalloproteinases (MMPs), and lysis of the extracellular matrix. This culminates in the shedding of the upper two-thirds of the endometrium (the functional layer). The lower third of the endometrium (basal layer) remains in situ but has an exposed, raw mucosal surface that requires efficient repair.

Inflammatory mediators generated within the endometrium upon withdrawal of progesterone include prostaglandins, cyclooxygenase- 2, MMPs, and interleukin-8 (Figure 40.4). Leucocyte traffic is ini­tially neutrophil dominated although macrophage numbers also increase. Neutrophils contain high levels of MMPs and can activate tissue MMPs and play a critical role in the induction of endometrial shedding. Tight regulation of local endometrial events at the time and appropriate apoptosis is important to secure satisfactory onward endometrial repair. Macrophages are involved in both cytokine pro­duction and local remodelling of the endometrium and removal of debris and thus participate in both the breakdown and repair of the endometrium. Tight regulation of localized ‘physiological' inflam­mation is critical to prevent excessive bleeding at menses (4, 26).

An intact endometrial coagulation system is also necessary for the efficient cessation of menstruation. Endometrial blood vessel injury initiates immediate activation and aggregation of platelets to form a ‘plug'. Subsequently, fibrin is formed via the intrinsic and extrinsic coagulation cascade. Each pathway culminates in the conversion of factor X to Xa, which drives production of thrombin, ultimately leading to the formation of a stable fibrin clot to seal previously bleeding vessels (Figure 40.5) (4). Disorders that interfere with this systemic haemostasis have an impact on menstrual blood loss (see ‘Overview of menstrual pathology') (Figure 40.5).

Conversely, fibrinolysis is the process of conversion of plas­minogen to active plasmin, promoting the degradation of fibrin deposits. Tissue plasminogen activator (tPA) and urokinase plas­minogen activator (uPA) drive the production of plasmin. In con­trast, plasminogen activator inhibitor (PAI) inhibits fibrinolytic activity. The human endometrium contains tPA and uPA, as well as PAI and the uPA receptor (27, 28). Tight regulation to balance co­agulation and fibrinolysis is necessary for normal menstruation.

The pivotal role of the coagulation system in menstruation is evi­denced by the association between perturbation of this system and heavy menstrual bleeding (HMB). There is evidence that an over­active fibrinolytic system interferes with haemostasis and contrib­utes to HMB. Women with HMB had raised levels of tPA activity on the second day of bleeding compared to those with normal loss (27). The efficacy of tranexamic acid as a treatment for HMB provides further evidence for overactivation of the fibrinolytic system in the endometrium of these women. The antifibrinolytic, tranexamic acid, reduces tPA and PAI levels in women with HMB and results in a 58% reduction in blood loss (29). Furthermore, up to 13% of women with a complaint of HMB will have von Willebrand disease (30).

During menstruation, the shed surface of the endometrium leads to bleeding from damaged vasculature. Factors influencing blood flow include length and radius of a blood vessel and the viscosity of blood. Of these, vessel radius is the dominant contributory factor (31). Therefore, the endometrium has evolved specialized spiral ar­terioles that have the ability to undergo intense vasoconstriction during the late secretory and menstrual phases to limit menstrual blood loss. Experiments examining the endometrial vasculature in the non-human primate (rhesus macaque) suggest that this vaso­constriction is so intense that the luminal portion of the endomet­rium becomes hypoxic during menses (32). There is mounting evidence that this hypoxia may trigger a cellular protective response to increase local repair factors and drive blood vessel and tissue re­generation (33, 34).