Female Reproduction 1 and 2: The Menstrual Cycle R.J. Witorsch, Ph.D. OBJECTIVES:

At the end of this block of lectures, the student should be able to:

1. Describe the functional anatomy of ovary with particular reference to events prior to, during and after ovulation.

2. Describe the functions of female sex accessory organs. 3. Describe and explain the events and regulatory mechanisms involved in the

human menstrual cycle. 4. Describe the events involved in the secretion, circulation, and biological half-life

of estrogen and progestin in the adult cycling female. 5. Describe the physiological actions and mechanisms of action of estrogen and

progestin. 6. Describe and explain the various srategies involved in female contraception. 7. Explain the etiologies, symptomologies, and diagnostic strategies of the following

gonadal disorders in the female:

a. hypogonadism b. precocious puberty c. menstrual disorders.

Suggested Reading: Costanzo 2nd Edition, pp. 417-424

I. OVARY

A. Female reproduction is characterized by monthly cycles, culminating in the extrusion of a single egg from the ovary by the process known as ovulation.

B. The major structures of the ovary and their functions are (Fig. 1):

Figure 1. Mammalian Ovary

1. Hilus, the area where lymphatics, nerves and blood vessels enter and exit the ovary.

2. Germinal epithelium: a misnomer, in reality a specialization of peritoneal mesothelium.

3. Primordial follicle: a single egg cell (oocyte) surrounded by a single layer of cells (granulosa cells). This is the basic reproductive unit of ovary. The primordial follicle is separated from adjacent stroma by a basement membrane surrounding the granulosa cells. Primordial follicles are found primarily in outer cortex just beneath fibrous capsule of ovary. The following are noteworthy features of the primordial follicles:

a. The oocyte is small (< 25μm) and in prophase of meiosis I. b. Primordial follicles mature and subsequently release a single

oocyte from the ovary at ovulation. Primordial follicles provide a resting pool from which follicles develop.

c. About 1-2 x 106 primordial follicles are present in each ovary at birth.

i. Groups of follicles undergo partial development and atresia (degeneration) during late fetal life and through prepubertal years.

ii. At puberty there are approximately 4 x 105 primordial follicles in each ovary. The rest have undergone development and atresia in earlier years.

d. From the beginning of female fertility (menarche) to the end of female fertility (menopause) there are approximately 400 ovulatory cycles. About 1,000 primordial follicles/ovary are mobilized (i.e., undergo development) during each cycle.

i. Only 1 follicle fully matures while the remainder undergo atresia.

ii. Recent observations (2004) suggest that mammalian ovaries contain stem cells that can generate new eggs through adulthood. This challenges the assumption that the full complement of eggs are present at birth.

4. Follicles in various stages of development

a. The primary follicle is an early stage of development beyond the primordial stage. It contains an enlarged oocyte (80-100μm) which is surrounded by a zona pellucida. In the primary follicle the granulosa cells become cuboidal (having been previously flat).

b. With further development granulosa cells proliferate and a vesicle (antrum) forms within granulosa cell region from a plasma exudate. Connective tissue layers formed around the follicle are called the theca interna and theca externa.

5. The mature (Graafian) follicle is the structure of the follicle just prior to ovulation. Fluid infiltration and antrum formation are maximal and the follicle has a diameter of 5 mm.

6. At ovulation the follicle ruptures. The oocyte (plus some granulosa cells) is extruded from the ovary, leaving behind most of the follicle.

a. Just prior to or at ovulation, meiosis I is completed and the primary oocyte becomes the secondary oocyte + first polar body.

7. The corpus hemorrhagicum is a blood-filled remnant of the ruptured follicle retained by the ovary immediately after ovulation.

8. The corpus luteum is the structure transformed from the corpus hemorrhagicum. Granulosa cells are increased in size and lipid-laden (luteinized or yellow in color). Granulosa cells are the major source of estradiol and progesterone after ovulation.

9. Regressing corpora lutea: Regression of the corpus luteum begins toward the end of an ovarian cycle and continues into the subsequent cycle.

10. Corpus albicans is scar tissue resulting from corpus luteum regression. 11. Atretic (or degenerating) follicles result from developing follicles that

never fully mature and undergo regression prior to reaching the Graafian stage.

12. Ovarian stroma is fibroelastic connective tissue and interstitial cells. Stromal cells contain lipid derived from atretic follicles. They secrete sex steroids in response to LH and human chorionic gonadotrophin (hCG).

II. FEMALE SEX ACCESSORY ORGANS (Figure 2)

Figure 2. Female Reproductive Organs

A. The female sex accessory organs provide a proper environment for the post-ovulatory egg, its fertilization and the development of the fetus. The major female sex accessory organs and their roles are:

1. The fallopian tube (or oviduct) which receives the egg after being extruded from the ovary at ovulation and is the organ where fertilization occurs.

2. The uterus is composed mucosa (endometrium) and muscle wall (myometrium). The uterus is the site where the fertilized egg

(blastocyst) implants, the placenta forms and embryo develops and is the organ involved in parturition.

3. The cervix is the constricted structure of the lower uterus and serves as the boundary between uterus and vagina. The cervix plays a passive role in conception and is the major site of spermatozoan storage, post-coitus.

4. The vagina is the organ of sexual receptivity. The "sweating" response of the vagina is the earliest response to sexual stimulation.

III. HUMAN MENSTRUAL CYCLE (Figure 3)

A. The human menstrual cycle is a recurring pattern of physiological changes in the female reproductive system. The primary goal of the cycle is to prepare the uterus for implantation of the fertilized ovum. On average the menstrual cycle is of 28 days duration, although on an individual basis cycles usually range from 25-35 days.

B. The menstrual cycle exhibits the following phases: menses (menstrual flow) which occurs from day 1 to 4 or 5 of the cycle; the proliferative phase (referring to uterine events) or follicular phase (referring to ovarian events) which occurs from days 5 to 14 of the cycle; ovulation or extrusion of the egg from the ovary which occurs on day 14 of the cycle; and the secretory phase (referring to uterine event) or luteal phase (referring to an ovarian event which occurs during the last half (days 15 to 28) of the cycle.

C. Cyclic changes are observed in pituitary hormone levels.

1. FSH exhibits a slow decline in days 1-14. At midcycle (day 14) a small surge in serum FSH occurs. From days 15-28, FSH is maintained at levels lower than at days 1-14. An abrupt small increase in FSH occurs on days 28-1 (at the end of the old cycle and the start of the new cycle.

2. LH levels are stable but low during the first half of the cycle (days 1-14). On day 14, a large ovulatory surge in LH occurs which lasts about 24 h. A slow decline in LH levels occurs from days 15 to 28 followed by an abrupt small increase between days 28 and 1.

3. PRL (prolactin) levels are higher during days 15-28 than days 1-14 (not shown), a pattern which is of questionable consistency.

Figure 3. Human Menstrual Cycle

D. Cyclic changes in ovarian histology

1. Days 1-14 (follicular phase) is characterized by development of primordial follicles . About 1,000 primordial follicles are mobilized however usually only one is destined to become the Graafian follicle.

2. Days 14-15 is the time when ovulation occurs. This is 36-38 hr after the start of the LH surge.

3. Days 15-21 is the time when a portion of the follicle retained in the ovary becomes the corpus hemorrhagicum for a 1-2 day period. This structure then becomes the corpus luteum for the duration of the period.

4. Days 21-25 is the time when peak corpus luteum development and function occurs.

5. Days 26-28 is the time when regression of corpus luteum initiated and the cycle ends.

6. After 28 days (subsequent cycles) luteal regression continues and the corpus luteum ultimately becomes a corpus albicans.

E. Cyclic changes in plasma steroid levels

1. Estradiol

a. From days 1-14 of the cycle a gradual increase in plasma estradiol level occurs. The estradiol is produced by the developing follicle. The theca interna makes testosterone which is then aromatized to estradiol by the granulosa cells.

i. Thirty to sixty hrs prior to the LH surge (during the second week of cycle) plasma estradiol rises at a more rapid rate.

b. Days 14-15 exhibits an elevation in estradiol corresponding to the LH surge and post-ovulation, estradiol levels drop.

c. Days 15-25 exhibits a second rise in estradiol. This estradiol is now being produced by the corpus luteum.

d. Days 26-28 is characterized by a decline in estradiol levels which is concomitant with the regression of the corpus luteum.

2. Progesterone

a. On a molar basis, the output of progesterone exceeds that of estradiol. However, there are no dramatic changes observed during the follicular phase of the menstrual cycle. From Days 1-14 serum progesterone levels are low.

b. A periovulatory increase is seen in plasma 17α-hydroxyprogesterone (not shown in Fig. 3).

c. On days 15-25 (post-ovulation) gradual increase in progesterone production from corpus luteum occurs.

i. Peak levels of progesterone occur between days 21-25 concomitant with maximal corpus luteum activity.

d. On days 26-28 progesterone levels decline concomitant with corpus luteum regression. The post-ovulatory pattern of 17α-hdroxyprogesterone is similar to that of progesterone.

F. Cyclic changes in the uterus

1. During menses (Days 1-4) there is a sloughing off of necrotic endometrium with blood and uterine fluid discharged as menstrual flow. This occurs because of spasmotic closure of arteries due to local release of prostaglandin-F2α. Menses is due to withdrawal of hormonal support (estradiol and progesterone) as a consequence of regression of corpus luteum on days 26-28 of the preceding cycle.

2. The proliferative phase occurs from days 5-14 of the cycle and is characterized by proliferation of uterine epithelium, glandular tissue, stroma and vascularity. Uterine thickness increases several fold. Uterine proliferation due to estradiol secreted by developing follicle.

3. The secretory phase occurs from days 15-25 of the cycle. During this period the uterus exhibits increased vascularity (coiled arteries), stromal hypertrophy, fluid accumulation (edematous tissue), further glandular development, increased glandular secretory activity (proteins, amino acids, carbohydrates), and glycogen accumulation. Secretory stage changes that occur in the uterus are a consequence of the combined effects of estradiol and progesterone after ovulation. Secretory changes in the uterus prepare the organ for implantation of a fertilized egg, if fertilization occurs. If fertilization does not occur, the corpus luteum regresses and the withdrawal of hormonal support leads to menses on day 1 of the next cycle.

G. Cyclic changes in the cervix

1. At midcycle: changes in cervical mucus occur due to the actions of estradiol. At this time, cervical mucus is highest in quantity, most penetrable to sperm (less hostile and less acidic), most transparent, and least viscous (has a high degree of elasticity), a property called "spinnbarkeit." When smeared on a slide and allowed to dry, cervical mucus exhibits a ferning pattern.

H. Cyclic changes in the vagina

1. At midcycle, the vagina exhibits cytological (exfoliative) changes due to the effects of estradiol. Among these changes are increased mitotic activity and exfoliation of squamous epithelial cells.

I. Cyclic changes in basal body temperature (BBT)

1. BBT during luteal phase (post-ovulation) is approximately 1°F greater than that during the follicular phase. The increase in BBT is attributed to an action of progesterone on the thermoregulatory center of the hypothalamus.

IV. HORMONAL CONTROL OF THE MENSTRUAL CYCLE (Figure 4.)

Figure 4. Feedback Control of Ovulation

A. Throughout most of the menstrual cycle, estrogen and progestin exert a negative feedback effect producing suppressed and/or declining levels of FSH and LH during the follicular phase and suppression of FSH and LH during the luteal phase. This negative feedback is responsible, in part, for luteal regression which occurs at the end of the cycle.

1. The small but abrupt rise in FSH and LH at the beginning of a new cycle is due to corpus luteum regression and withdrawal of estradiol and progesterone at the end of preceding cycle.

B. A rise in plasma estradiol 30-60 hours prior to the LH surge appears to stimulate LH and FSH surge by a positive feedback mechanism. The positive feedback effect of estradiol on LH and FSH release appears to occur at the pituitary level. Anterior pituitary responsiveness to GnRH increases 10-50 fold. Therefore, estradiol increases pituitary sensitivity to pulsatile GnRH (accordingly, clomiphene, a nonsteroidal estrogen, is an inducer of ovulation).

C. The ovulatory surge in LH and FSH is controlled by a single follicle which emerges, called the dominant follicle. The dominant follicle is selected from a cohort of 8-12 cells that are most sensitive to FSH.

1. The dominant follicle (from left or right ovary) achieves dominance over other follicles in both ovaries.

2. Dominance of a single follicle is evident by day 7 of the cycle. 3. The dominant follicle is the source of most of the estradiol secreted

during the follicular phase.

a. It is responsible for the pre-ovulatory rise in estradiol which then produces the positive feedback stimulus for the ovulatory surge in LH and FSH

D. Intra-ovarian mechanisms responsible for cyclic changes that occur in the ovary

1. During the follicular phase, the theca interna develops LH receptors and produces androgens from cholesterol in response to LH.

2. Granulosa cells develop FSH receptors. In response to FSH, androgen is converted to estradiol by the aromatase system. Estradiol stimulates the proliferation, induces FSH receptors (and increases FSH responsiveness) of granulosa cells. Toward the end of the follicular phase (late antral stage) FSH and estradiol stimulate development of LH receptors granulosa cells. Antral fluid contains androgen, estradiol and progesterone as a result of the hormonal interactions described above. Through most of the cycle, granulosa cells are not vascularized, just prior to ovulation granulosa cells become vascular, hyperemic, and edematous.

3. Ovulation involves plasmin-assisted degradation of the wall of the follicle.

4. Post-ovulation granulosa cells become luteinized, cease to proliferate, hypertrophy and secrete estradiol and progesterone in response to LH.

5. Luteal failure (luteolysis) at day 26 occurs by a mechanism that has not been fully characterized. However, LH withdrawal via negative feedback is implicated. Intraovarian estrogens and prostaglandin F2α may also participate.

6. The GnRH surge is not necessary for ovulation. The ovulatory surge in LH and FSH requires pulsatile GnRH and positive feedback effect due to the pre-ovulatory rise of estradiol. Pulsatile GnRH (every 90 min) delivered via a portable infusion pump is used in the treatment of anovulatory conditions. Nevertheless, a GnRH surge occurs at midcycle. This surge by itself appears too small to be responsible for the surge in LH and FSH. Pulsatile GnRH may facilitate the ovulatory surge by providing a self-priming effect.

7. Pulsatile GnRH rate can be modified. Estradiol increases and progesterone tends to decrease pulse frequency. As ovulation approaches, pulse frequency increases and decreases during luteal phase.

8. The periovulatory release of 17α-hydroxyprogesterone may enhance anterior pituitary sensitivity to GnRH.

V. ESTROGENS

A. The principal circulating estrogen is estradiol-17ß which is in equilibrium with its 17-ketosteroid derivative, estrone.

B. Plasma binding: Ninety-nine % of circulating estradiol-17β is bound to plasma proteins, 48% bound to albumin and 51% to sex hormone binding globulin (SHBG which same as testosterone binding globulin TeBG).

C. Metabolism, conjugation and excretion: The major metabolite of estradiol-17β is a 16α -hydroxylated derivative, estriol (Fig. 5). Hydroxylation of estradiol-17β can also occur at the C-2 position forming catechol estrogen (Fig. 5). In humans the 16α-hydroxylation and 2-hydroxylation pathways are inversely related to one another. Tobacco smoking is associated with increased 2-hydroxylation, whereas alcohol consumption associated with increased 16α-hydroxylation. Sixteen α-hydroxylated estrogens are biologically active while 2-hydroxylated estrogen are relatively inactive. In women as well as in certain strains of mice, increased hepatic 16α-hydroxylation of estradiol is linked with increased incidence of breast cancer. Estrogens (secreted and metabolites) are excreted primarily as glucuronide and sulfate conjugates.

Figure 5. Two Major Metabolites of Estradiol-17β

D. Among the physiological actions of estrogens are the following: regulation of gonadotrophin secretion (positive and negative feedback), influences sexual behavior, cyclic changes in uterus, cervix and vagina, granulosa cell proliferation and development, maturation and maintenance of sex accessory organs and secondary sex characteristics, mild anabolic effects, maintenance of pregnancy, lowered threshold of uterus to contractile stimuli during parturition, development of the tubular mammary gland, stimulation of prolactin release, decrease in serum LDL cholesterol levels, an anti-osteoporotic effect, and induction of progestin receptors.

1. Estrogen induction of progestin receptors indicates that estrogens are required for progestin action. This is the basis for estrogen priming effect for progestin action. Steroid receptor analysis of breast biopsy specimens reveals important information about the status of a breast cancer. A tumor that is positive for both estrogen receptor (ER) + and progesterone receptor (PR) + suggests that the tumor is hormone dependent. ER+, PR- suggests that tumor is hormone independent (i.e., nonfunctional estrogen receptors).

VI. PROGESTINS

A. The principal circulating progestin is progesterone. The corpus luteum also produces 17α-hydroxy progesterone.

B. Plasma binding: Ninety-eight % of circulating progesterone is bound to plasma proteins, 48% bound to albumin and 50% to corticosteroid binding globulin (CBG).

C. Metabolism and excretion (Fig 6): The principal metabolic conversions of progesterone are A ring and 20-ketone reduction in liver which forms pregnanediol from progesterone, and pregnanetriol from 17α-hydroxy progesterone. Pregnanetriol may be converted further to etiocholanolone, a 17 keto derivative. Etiocholanolone is also a product of cortisol and testosterone metabolism. Progestin derivatives are excreted either in unconjugated form or as glucuronide or sulfate conjugates.

Figure 6. Major Products of Progestin Metabolism

D. Among the physiological actions of progestins are regulation of gonadotophin secretion, cyclic changes in uterus, and maintenance of pregnancy. During pregnancy progestins increase the threshold of the uterus to contractile stimuli and promote development of the alveolar mammary gland.

VII. FEMALE ORAL CONTRACEPTIVE A. Female oral contraception are available in several forms.

1. Synthetic progestins (derivatives of 19-nortestosterone, such as

norethindrone) + estrogens (such as ethinyl estradiol). The original rationale for this approach was based inhibition of the ovulatory surge of LH by negative feedback. However, in the presence of estrogens, lower doses of progestins are contraceptive even when they do not block ovulation. This is because, cervical mucus becomes hostile to sperm, as well as adverse effects of progestin on the endometrium and oviduct. Thus contraception may be the result of local effects on cervical mucus and other components of the female reproductive tract.

2. RU-486 is a steroid analogue (11β-(4-dimethyl-amino phenyl)-17β-hydroxy-17α)prop-1-ynyl)-estra-4,9-dien-3-one) that has a high affinity for the progesterone receptor but appears unable to initiate transcriptional events. As a result, RU 486 would prevent trophoblastic implantation.

a. RU-486 has similar antagonistic actions on the glucocorticoid receptor system and has utility in the treatment of disorders associated with glucocorticoid excess such as Cushing's syndrome.

3. Plan B, an emergency contraceptive containing the progestin, levonorgestrel given as two tablets 12 hrs apart. The procedure is about 90% effective in preventing pregnancy if taken within 72 hrs of intercourse. Among the possible contraceptive mechanisms of this method are: 1) inhibition or delay of ovulation, 2) altered tubal transport of sperm or ova, or 3) inhibition of implantation of the blastocyst by alteration of the endometrium.

VIII. FEMALE GONADAL DISORDERS

A. Hypogonadism and precocious puberty can occur in females by the same mechanisms described previously for males. Pulsatile GnRH has utility for treatment of hypothalamic (secondary) hypogonadism. Long-acting GnRH analogues have utility for treatment of complete precocious puberty of hypothalamic origin. Adrenogenital disorders produce virilizing syndromes in females by mechanisms described in adrenal cortex lectures.

B. Menstrual abnormalities, are the most common disorders in women and are due to defects in the menstrual cycle, such as failure to ovulate, shortened follicular phase, defects in timing of luteal regression, too abrupt fall in estrogen levels at time of ovulation, hypothalamic-pituitary dysfunction, and ovarian or uterine defects.

C. Several diagnostic tests are useful in evaluating menstrual irregularities, among these are: basal body temperature (should increase 1°F during luteal phase); evaluation of cervical mucus (for viscosity, ferning), evaluation of vaginal smear (for exfoliated squamous cells), serum hormone levels (e.g., LH, FSH, gonadal steroids) and provocative tests (LH, FSH; GnRH) to evaluate target organ responsiveness.

IX. STUDY QUESTIONS

1. The dominant follicle is producing most of its estradiol during which days of the menstrual cycle:

A. Days 1-4 B. Days 5-14 C. Days 15-20 D. Days 21-25 E. Days 26-28

2. The fertilized egg goes through the morula (or pre-blastocyst) stage during

which days of the menstrual cycle?

. Days 1-4 A. Days 5-14 B. Days 15-20 C. Days 21-25 D. Days 26-28

3. The corpus luteum undergoes regression during which days of the menstrual cycle?

. Days 1-4 A. Days 5-14 B. Days 15-20 C. Days 21-25 D. Days 26-28

4. The uterus exhibits maximum secretory activity during which days of the menstrual cycle?

. Days 1-4 A. Days 5-14 B. Days 15-20 C. Days 21-25 D. Days 26-28

5. The corpus hemorrhagicum is evident during which days of the menstrual

cycle?

. Days 1-4 A. Days 5-14 B. Days 15-20 C. Days 21-25 D. Days 26-28

6. Granulosa cells are proliferating and the antrum is being formed (i.e.

follicular development), during which days of the menstrual cycle?

. Days 1-4 A. Days 5-14 B. Days 15-20 C. Days 21-25 D. Days 26-28

Answer Key: 1. B; 2. C; 3. E; 4. D; 5. C; 6. B