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THE NORMAL MENSTRUAL CYCLE AND THE CONTROL OF OVULATION

Menstruation is the cyclic, orderly sloughing of the uterine lining, in response to the interactions of hormones produced by the hypothalamus, pituitary, and ovaries. The menstrual cycle may be divided into two phases: (1) follicular or proliferative phase, and (2) the luteal or secretory phase (Fig. 1).

The length of a menstrual cycle is the number of days between the first day of menstrual bleeding of one cycle to the onset of menses of the next cycle. The median duration of a menstrual cycle is 28 days with most cycle lengths between 25 to 30 days.1-3 Menstrual cycles that occur at intervals less than 21 days are called polymenorrheic, and menstrual cycles, which are prolonged more than 35 days, are called oligomenorrheic. The menstrual cycle is typically most irregular around the extremes of reproductive life, menarche and menopause, due to anovulation and inadequate follicular development.4-6 The luteal phase is relatively constant with a duration of 14 days. The variability of cycle length is usually derived from varying lengths of the follicular phase of the cycle, ranging from 10 to 16 days.

Figure 1. Hormonal, Ovarian, endometrial, and basal body temperature changes and relations throughout the normal menstrual cycle. (From Carr BR, Wilson JD. Disorders of the ovary and female reproductive tract. In: Braunwald E, Isselbacher KJ, Petersdorf RG, et al, eds. Harrison's Principles of Internal Medicine. 11th ed. New York: McGraw-Hill, 1987: 1818-1837.

THE FOLLICULAR PHASE

The follicular phase begins from the first day of menses until ovulation. Lower temperatures on a basal body temperature chart and more importantly, the development of ovarian follicles characterize this phase. Folliculogenesis begins during the last few days of the preceding menstrual cycle until the release of the mature follicle at ovulation. Declining steroid production by the corpus luteum and the dramatic fall of Inhibin A and B levels allows for FSH to rise during the last few days of the menstrual cycle.7 This event allows for the recruitment of a cohort of ovarian follicles in each ovary, one of which is destined to ovulate during the next menstrual cycle. Once menses ensues, FSH levels decline due to the negative feedback of estrogen and the negative effects of inhibin produced by the developing follicle.8,9 FSH activates the aromatase enzyme in granulosa cells, which converts androgens to estrogen. A decline in FSH levels leads to the production of a more androgenic microenvironment within adjacent follicles to the growing dominant follicle. Also, the granulosa cells of the growing follicle secrete a variety of peptides that may play an autocrine/paracrine role in the inhibition of development of the adjacent follicles.

Development of the dominant follicle has been described in three stages: (1) Recruitment, (2) Selection, and (3) Dominance (Fig.2). During days 1 through 4 of the menstrual cycle, a cohort of follicles is recruited from a pool of nonproliferating follicles in response to FSH. Between cycle days 5 and 7 and only one follicle is selected from the cohort of recruited follicles to ovulate and the remaining follicles will undergo atresia. By cycle day 8, one follicle exerts its dominance by promoting its own growth and suppressing the maturation of the other ovarian follicles.

Figure 2. Time course for recruitment, selection, and ovulation of the dominant ovarian follicle (DF) with onset of atresia among other follicles of the cohort (N-1). (From Hodgen GD. The dominant ovarian follicle. Fertil Steril 1982; 38:281-300).

During the follicular phase, serum estrogen levels rise in parallel to the growth of follicle size as well as to the increasing number of granulosa cells. FSH receptors exist exclusively on the granulosa cell membranes. Increasing FSH levels during the late luteal phase leads to an increase in the number of FSH receptors and ultimately to an increase in estradiol secretion by granulosa cells. The increase in FSH receptor numbers is due to an increase in the population of granulosa cells and not due to an increase in the concentration of FSH receptors per granulosa cell. Each granulosa cell has approximately 1500 FSH receptors by the secondary stage of follicular development and FSH receptor numbers remains relatively constant for the remainder of development.10 The rise in estradiol secretion appears to increase the number of estradiol receptors.11 In the presence of estradiol, FSH stimulates the formation of LH receptors on granulosa cells allowing for the secretion of small quantities of progesterone and 17-hydroxyprogesterone (17-OHP) which may exert a positive feedback on the estrogen-primed pituitary to augment LH release .12 FSH also stimulates several steroidogenic enzymes including CYP19, aromatase, and 3-B-hydroxysteroid dehydrogenase.13,14 In Table 1, the production rates of sex steroids during the follicular phase, luteal phase, and at the time of ovulation are presented.

Luteinizing hormone receptors are located on theca cells during all stages of the menstrual cycle in contrast to granulosa cells. LH principally stimulates androstenedione production and to a lesser degree testosterone production in the theca cells. In the human, androstenedione is then transported to the granulosa cells where it is aromatized to estrone and finally converted to estradiol by 17-b-hydroxysteroid dehydrogenase type I. This is known as the two-cell, two-gonadotropin hypothesis of regulation of estrogen synthesis in the human ovary (Fig 3).

Figure 3. Two-cell, two-gonadotropin hypothesis of regulation of estrogen synthesis in the human ovary. Adapted by Carr, BR. Diseases of the ovary and Reproductive Tract. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR, eds. Williams Textbook of Endocrinology 9th edition. WB Saunders, Philadelphia, p.751-817.

The primordial follicle is surrounded by a single layer of granulosa cells and is arrested in the diplotene stage of the first meiotic division. After puberty, the primordial follicle enlarges and develops into a preantral follicle. It then develops a cavity and is known as an antral follicle. Finally, it becomes a preovulatory follicle on its way towards ovulation. Due to the presence of 5-a-reductase, preantral and early antral follicles produce more androstenedione and testosterone in relation to estrogens.15 5-a-reductase is the enzyme responsible for converting testosterone to dihydrotestosterone (DHT). Once testosterone has been 5- -reduced, DHT can not be aromatized. However, the dominant follicle is able to secrete large quantities of estrogen, primarily estradiol, due to high levels of CYP19. This shift form an androgenic to an estrogenic follicular microenvironment may play an important role in selection of the dominant follicle from those follicles that will become atretic.
Development of the follicle to the preantral stage is gonadotropin independent and any follicular growth beyond this point will require gonadotropin interaction. Gonadotropin secretion is regulated by gonadotropin releasing hormone (GnRH), steroid hormones, and various peptides released by the dominant follicle. FSH is elevated during the early follicular phase and then begins to decline until ovulation. In contrast, LH is low during the early follicular phase and begins to rise by the midfollicular phase due to the positive feedback from rising estrogen levels. For the positive feedback effect of LH release to occur, estradiol levels must be greater than 200 pg/ml for approximately 50 hours in duration.16 The gonadotropins are secreted in a pulsatile fashion and the frequency and amplitude of the pulses vary according to the phase of the menstrual cycle (Table2). During the early follicular phase, LH secretion occurs at a pulse frequency of 60 to 90 minutes with relatively constant pulse amplitude. During the late follicular phase prior to ovulation, the pulse frequency increases and the amplitude may begin to increase. In most women, the LH pulse amplitude begins to increase after ovulation.17

There are numerous substances found in follicular fluid that regulate the microenvironment of the ovary and that regulate steroidogenesis in granulosa cells, such as steroids, pituitary hormones, plasma proteins, proteoglycans and non-steroidal ovarian factors. Growth factors such as insulin-like growth factor 1 and 2 (IGF1, IGF2) and epidermal growth factor (EGF) are recognized as playing important roles in oocyte development and maturation.18,19 The concentration of ovarian steroids is much higher in follicular fluid in comparison to plasma concentrations. There are 2 populations of antral follicles: (1) large follicles, which are greater than 8mm in diameter, and (2) small follicles, which are less than 8mm. In the large follicles, the concentrations of FSH, estrogen, and progesterone are high while prolactin concentration is low. In the small follicles, prolactin and androgen levels are higher than in large antral follicles.20

OVULATION

Ovulation occurs approximately 10-12 hours after the LH peak.21 The LH surge is initiated by a dramatic rise of estradiol produced by the preovulatory follicle (Figure 4). To produce the critical concentration of estradiol needed to initiate the positive feedback, the dominant follicle is almost always >15mm in diameter on ultrasound.22 The LH surge occurs 34 to 36 hours prior to ovulation and is a relatively precise predictor for timing ovulation. The LH surge stimulates luteinization of the granulosa cells and stimulates the synthesis of progesterone responsible for the midcycle FSH surge. Also, the LH surge stimulates resumption of meiosis and the completion of reduction division in the oocyte with the release of the first polar body. It has been demonstrated in cultured granulosa cells that spontaneous luteinization can occur in the absence of LH. It is hypothesized that the inhibitory effects of factors such as oocyte maturation inhibitor or luteinization inhibitor are overcome at ovulation.23

Figure 4. Changes in gonadotropins and ovarian steroids at midcycle, just prior to ovulation. The initiation of LH surge is at time 0. Abbreviations: E2, estrogen; P, progesterone (From Hoff JD, Quigley ME, Yen SCC. Hormonal dynamics at midcycle: A re-evaluation. J Clin Endocrinol Metab. 57:792, 1983.

Prostaglandins and proteolytic enzymes such as collagenase and plasmin, are increased in response to LH and progesterone. Although the precise mechanism is not known, proteolytic enzymes and prostaglandins are activated and digest collagen in the follicular wall, leading to an explosive release of the oocyte-cumulus complex.24. Prostaglandins may also stimulate ovum release by stimulation of smooth muscle within the ovary. The point of the dominant follicle closest to the ovarian surface where this digestion occurs is called the stigma. There is no evidence to support the theory that follicular rupture occurs as a result of increased follicular pressure, although precise measurements precisely at rupture have not been performed.25 In humans, ovulation probably occurs randomly from either ovary during any given cycle, not preferentially in the contralateral ovary during the next menstrual cycle as in the primate model.26,27
The concentrations of prostaglandins E and F series and hydroxyeicosatetraenoic acid (HETE) reach a peak level in follicular fluid just prior to ovulation.28,29 Prostaglandins may stimulate proteolytic enzymes while HETEs may stimulate angiogenesis and hyperemia.30 Patients treated with high dose prostaglandin synthetase inhibitors such as indocin, can block prostaglandin production and effectively block follicular rupture. This gives rise to what is known as the luteinized unruptured follicle syndrome and it presents in fertile and infertile patients equally.31 Therefore, infertility patients are advised to avoid taking prostaglandin synthetase inhibitors, as well as cyclo-oxygenase (COX)-2 inhibitors, especially around the time of ovulation.32 A schematic diagram illustrating the proposed mechanisms involved in follicular rupture is presented in figure 5.

Figure 5. Proposed mechanisms involved in follicular rupture. From Tsafriri A, Chun S-Y. Ovulation. In: Adashi E, Rock JA, Rosenwaks Z. Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott-Raven, 1996:236-249.

Estradiol levels fall dramatically immediately prior to the LH peak. This may be due to LH downregulation of its own receptor or because of direct inhibition of estradiol synthesis by progesterone. Progesterone is also responsible for stimulating the midcycle rise in FSH. Elevated FSH levels at this time are thought to free the oocyte from follicular attachments, stimulate plasminogen activator, and increase granulosa cell LH receptors. The mechanism causing the postovulatory fall in LH is unknown. The decline in LH may be due to the loss of the positive feedback effect of estrogen, due to the increasing inhibitory feedback effect of progesterone, or due to a depletion of LH content of the pituitary from downregulation of GnRH receptors.33

LUTEAL PHASE

After ovulation, the granulosa cells continue to enlarge, become vacuolated in appearance, and begin to accumulate a yellow pigment called lutein. The luteinized granulosa cells combine with the newly formed theca-lutein cells and surrounding stroma to become what is known as the corpus luteum. The corpus luteum is a transient endocrine organ that predominately secretes progesterone and its primary function is to prepare the estrogen primed endometrium for implantation of the fertilized ovum. The basal lamina dissolves and capillaries invade into the granulosa layer of cells in response to secretion of angiogenic factors by the granulosa and thecal cells.34 Eight or nine days after ovulation approximately around the time of expected implantation, peak vascularization is achieved. This time also corresponds to peak serum levels of progesterone and estradiol. The central cavity may accumulate with blood and become a hemorrhagic corpus luteum. The life span of the corpus luteum depends upon continued LH support. Corpus luteum function declines by the end of the luteal phase unless human chorionic gonadotropin is produced by a pregnancy. If pregnancy does not occur, the corpus luteum undergoes luteolysis under the influence of estradiol and prostaglandins, and forms a scar tissue- the corpus albicans.

Estrogen levels rise and fall twice during the menstrual cycle. Estrogen levels rise during the midfollicular phase and then drop precipitously after ovulation. This is followed by a secondary rise in estrogen levels during the midluteal phase with a decrease at the end of the menstrual cycle. The secondary rise in estradiol parallels the rise of serum progesterone and 17-hydroxyprogesterone levels. Ovarian vein studies confirm that the corpus luteum is the site of steroid production during the luteal phase.35

The mechanism by which the corpus luteum regulates steroid secretion is not completely understood. Regulation may be determined in part by LH secretory pattern and LH receptors or variations in the levels of the enzymes regulating steroid hormone production, such as 3-ß-HSD, CYP17, CYP19, or side chain cleavage enzyme. The number of granulosa cells formed during the follicular phase and the amount of readily available LDL-cholesterol may also play a role in steroid regulation by the corpus luteum. The luteal cell population consists of at least two cell types, the large and small cells.36 Small cells are thought to have been derived from thecal cells while the large cells from granulosa cells. The large cells are more active in steroidogenesis and are influenced by various autocrine/paracrine factors such as inhibin, relaxin, and oxytocin.37,38

In studies looking into the mechanisms regulating the menstrual cycle, LH was established as the primary luteotropic agent in a cohort of hypophysectomized women.39 After induction of ovulation, the amount of progesterone secreted and the length of the luteal phase is dependant on repeated LH injections. Administration of LH or HCG during the luteal phase can extend corpus luteum function for an additional two weeks.40

The secretion of progesterone and estradiol during the luteal phase is episodic, and correlates closely with pulses of LH secretion (figure 6).41 The frequency and amplitude of LH secretion during the follicular phase regulates subsequent luteal phase function and is consistent with the regulatory role of LH during the luteal phase.42 Reduced levels of FSH during the follicular phase can lead to a shortened luteal phase and the development of a smaller corpus lutea.43 Also, the life span of the corpus luteum can be reduced by continuous LH administration during the follicular or luteal phase, reduced LH concentration, decreased LH pulse frequency, or decreased LH pulse amplitude.44-46 The role of other luteotropic factors such as prolactin, oxytocin, inhibin and relaxin is still unclear.47,48

Figure 6. Episodic secretion of LH (top) and progesterone (bottom) during the luteal phase of a woman. Abbreviations: LH, luteinizing hormone: P, progsterone E2, estradiol; LH + 8, LH surge plus 8 days. (From Filicori M, Butler JP, Crowley WF Jr. Neuroendocrine regulation of the corpus luteum in the human. J Clin Invest. 73:1638 1984.

The corpus luteum function begins to decline 9-11 days after ovulation. The exact mechanism of how the corpus luteum undergoes its demise is unknown. Estrogen is believed to play a role in the luteolysis of the corpus luteum.49 Estradiol injected into the ovary bearing the corpus luteum induces luteolysis while no effect is noted after estradiol injection of the contralateral ovary.47 However, the absence of estrogen receptors in human luteal cells does not support the role of endogenous estrogen in corpus luteum regression.50 Prostaglandin F2a appears to be luteolyic in nonhuman primates and in studies of women.51-53 Prostaglandin F2a exerts its effects via the synthesis of endothelin-1 which inhibits steroidogenesis and stimulates the release of a growth factor, tumor necrosis factor alpha (TNFa), which induces cell apoptosis.54 Oxytocin and vasopressin exert their luteotropic effects via an autocrine/paracrine mechanism.55 Finally, luteinizing hormone's ability to downregulate its own receptor may play a role in termination of the luteal phase.
Not all hormones undergo marked fluctuations during the normal menstrual cycle. Androgens, glucocorticoids, and pituitary hormones, excluding LH and FSH, undergo only minimal fluctuations.56-59 Due to extra-adrenal 21-hyroxylation of progesterone, plasma levels of deoxycorticosterone are increased during the luteal phase.60,61

HORMONAL EFFECTS ON THE REPRODUCTIVE TRACT

A. Endometrium

The effects of varying concentrations of estrogen and progesterone through the course of the menstrual cycle have characteristic effects on the endometrium (Fig 7).62 This allows for histologic dating of the endometrium and is most accurately accomplished by performing an endometrial biopsy 2-3 days prior to menstruation. The proliferative phase is more difficult to date accurately in comparison to the luteal phase. The glands during the proliferative phase are narrow, tubular, and some mitosis and pseudostratification is present. The endometrium thickness is usually between 0.5 and 5mm. In a classical 28 day menstrual cycle, ovulation occurs on day 14. On cycle day 16, the glands take on a more pseudostratified appearance with glycogen accumulating at the basal portion of the glandular epithelium and some nuclei are displaced to the midportion of the cells. In a formalin fixed specimen, glycogen is solubulized resulting in the characteristic basal vacuolization at the base of the endometrial cells. This finding confirms the formation of a functional progesterone producing corpus luteum. In the luteal phase, progesterone decreases the biologic activity of estradiol on the endometrium by: decreasing the concentration of estradiol receptors, increasing the enzymatic activity of 17-ß-hydroxysteroid dehydrogenase type II, the enzyme responsible for the conversion of estradiol to estrone, and by increasing the activity of estrone sulfotransferase.63,64

Figure 7. Dating of the Endometrium. From Noyes RW, Hertig AW, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1:3.

On cycle day 17, the endometrial glands become more tortuous and dilated. On cycle day 18, the vacuoles in the epithelium decrease in size and are frequently located next to the nuclei. Also, glycogen is now found at the apex of the endometrial cells. By cycle day 19, the pseudostratification and vacuolation almost completely disappear and intraluminal secretions become present. On cycle day 21 or 22, the endometrial stroma begins to become edematous. On cycle day 23, stromal cells surrounding the spiral arterioles begin of enlarge and stromal mitoses become apparent. On cycle day 24, predecidual cells appear around the spiral arterioles and stromal mitoses become more apparent. On cycle day 25, the predecidua begins to differentiate under the surface epithelium. On cycle day 27, there is a marked lymphocytic infiltration and the upper endometrial stroma appears as a solid sheet of well-developed decidua-like cells. On cycle day 28, menstruation begins.

B. Cervix

The mucous secreting glands of the endocervix are affected by the changes in steroid hormone concentration. Immediately after menstruation, the cervical mucous is scant and viscous. During the late follicular phase, under the influence of rising estradiol levels, the cervical mucous becomes clear, copious and elastic. The quantity of cervical mucous increases 30 fold compared to the early follicular phase.65 The stretchability or elasticity of the cervical mucous can be evaluated between two glass slides and recorded as the spinnbarkeit. Under the microscope, the cervical mucous displays a characteristic ferning or palm-leaf arborization appearance. After ovulation, as progesterone levels rise, the cervical mucous once again becomes thick, viscous and opaque and the quantity produced by the endocervical cells decreases.

C. Vagina

The changes in hormonal levels of estrogen and progesterone also have characteristic affects on vaginal epithelium. During the early follicular phase, exfoliated vaginal epithelial cells have vesicular nuclei and are basophilic. During the late follicular phase, the vaginal epithelial cells display pyknotic nuclei and are acidophilic due to the influence of rising estrogen levels.66 As progesterone rises during the luteal phase, the acidophilic cells decrease in number and are replaced by an increasing number of leukocytes.

MENSTRUATION

In the absence of a pregnancy, steroid hormone levels begin to fall due to declining corpus luteum function. Progesterone withdrawal results in increased coiling and constriction of the spiral arterioles. This results in tissue ischemia due to decreased blood flow to the superficial endometrial layers, the spongiosa and compacta. The endometrium releases prostaglandins that cause contractions of the uterine smooth muscle and sloughing of the degraded endometrial tissue. The release of prostaglandins may be due to decreased stability of lysosomal membranes in the endometrial cells.67 Infusions of prostaglandin F2-a in women during the luteal phase has been shown to induce endometrial necrosis and bleeding.68 The use of prostaglandin synthetase inhibitors decreases the amount of menstrual bleeding, and can be used as therapy in women with menorrhagia. Menstrual fluid is composed of desquamated endometrial tissue, red blood cells, inflammatory exudates, and proteolytic enzymes. Within two days after the start of menstruation, estrogen stimulates the regeneration of the surface endometrial epithelium, while concomitant endometrial shedding is occurring. The estrogen secreted by the growing ovarian follicles, causes prolonged vasoconstriction enabling the formation of a clot over the denuded endometrial vessels.69 Also, the regeneration and remodeling of the uterine connective tissue is regulated in part by the matrix metalloproteinase (MMP) system.70
The average duration of menstrual flow is between four to six days, but the normal range in woman can be from as little as two days up to eight days. The average amount of menstrual blood loss is 30 ml and greater than 80 ml is considered abnormal.71