Ovarian Cycle Review

Modest suppression of pituitary gonadotropin

secretion during pill intake and recovery of FSH release

during the pill-free week creates a situation resembling the

early follicular phase of the normal menstrual cycle and

allows for substantial residual ovarian activity.

Resting primordial follicles continuously enter the growing

pool throughout life (for review see Refs. 1–3). The magnitude

of depletion of the primordial follicle pool is dependent

on age and is most pronounced during fetal

development. Oocytes are detectable in fetal ovaries after 16

weeks of gestational age. The great majority of oocytes are

lost after the fifth month of intrauterine life, when a maximum

of approximately 7 million germ cells have been reported

(3). The presence of growing follicles in fetal ovaries

has been substantiated extensively (4). At birth, both ovaries

contain approximately 1 million primordial follicles. Reproductive

life starts with approximately 0.5 million primordial

follicles at menarche. Thereafter, loss of follicles takes place

at a fixed rate of around 1000 per month, accelerating beyond

the age of 35. Studies in the rat model suggest indeed

that follicle loss is inversely related to the number of primordial

follicles present in the ovaries (9). Once follicles are

stimulated to grow, they can either reach full maturation and

ovulate or become atretic. Follicles are present in the ovary

at different stages of development, and large numbers of

follicles of different sizes can be observed at any given point

of the menstrual cycle (10). The distribution of developmental

stages of follicles entering atresia may vary with age (11).

It is generally believed that, especially at an early age, loss of

follicles is largely due to atresia of primordial follicles (12).

It is unknown as yet which factors regulate initiation of

growth of primordial follicle (12, 13) and whether maturing

follicles may enter atresia at all developmental stages.

When primordial follicles enter the growth phase they

enlarge by an increase in size of the oocyte together with

granulosa cell proliferation (primary follicle). Transition into

the secondary follicle stage involves alignment of stroma

around the basal lamina and the development of an independent

blood supply. The stroma subsequently differentiates

into a theca externa (similar to surrounding stroma cells)

and a theca interna layer. Theca interna cells express LH

receptors early on (15). Development of an antral cavity (at

a follicle size ;100 to 200 mm) divides granulosa cells in cells

surrounding the oocyte (cumulus) and cells that border the

basement membrane. During early preantral follicle development,

FSH receptors also become detectable on granulosa

cells. The time span between a primary and an early

antral follicle in the human is unknown but is proposed to

be several months. Subsequent stages from early antral to

preovulatory follicles exhibit clear morphological characteristics,

and the time interval is assessed to be approximately 3 months.

An increase in the

number of granulosa cells is critically important for the advancement

in developmental stages of the follicle.

Under normal conditions, only about 400 follicles reach the

mature preovulatory stage and ovulate in a lifetime. Hence,

loss of follicles due to atresia — with apoptosis [i.e. programmed

cell death (18)] as the underlying cellular mechanism—

rather than growth and subsequent ovulation should

be considered the normal fate of follicles. The importance of

oxidative stress in inducing atresia (19) and gonadotropins

and various growth factors (‘survival factors’) to suppress

apoptosis (20, 21) has been emphasized recently. FSH decreases

apoptosis in granulosa cells obtained from hypophysectomized

rats (22) and prevents apoptotic changes of cultured

preovulatory follicles.

In the human the process of initiation of follicle growth

and subsequent exhaustion of the resting pool of primordial

follicles appears to be regulated independently of stimulation

by gonadotropins (24). Follicles become dependent on

stimulation by FSH only at an advanced developmental

stage, as will be discussed later.

For instance,

follicles grow up to the early antral stage in long-term hypophysectomized

animals

It appears in the human that follicle development

up to the antral stage continues throughout life until

depletion of follicles around menopause, even under conditions

in which endogenous gonadotropin release is diminished

substantially (5, 29). Such conditions include prepubertal

childhood (30 –33), pregnancy (34 –37), and the use of

steroid contraceptives (see Section IV). In addition, follicle

growth up to the early antral stage has been described in

women with absent gonadotropin secretion, either due to

hypophysectomy, as discussed by Block (1), or to hypothalamic/

pituitary failure.

In the rat

model it has been suggested that theca cell differentiation

and early preantral follicle growth is dependent on subtle

stimulation byLH.

In contrast to early follicle development, stimulation by

FSH is an absolute requirement for development of large

antral preovulatory follicles. Duration and magnitude of FSH.

stimulation will determine the number of follicles with augmented

aromatase enzyme activity and subsequent E2 biosynthesis.

High FSH levels usually occurring during the luteo-

follicular transition give rise to continued growth of a

limited number (cohort) of follicles. Subsequent development

of this cohort during the follicular phase becomes dependent

on continued stimulation by gonadotropins.

in the human only a single follicle from the cohort is

selected to gain dominance and ovulate every cycle. Remaining

cohort follicles enter atresia due to insufficient support

by reduced FSH levels. The only exception to this rule is

familial dizygotic twins in which ongoing growth and ovulation

of multiple follicles occur (46, 47). A reduced rate of

follicle atresia due to altered intrafollicular steroidogenesis

independent from gonadotropins has recently been proposed

as the underlying cause

Intrafollicular endocrine changes: The majority of enzymes involved in the biosynthesis of

ovarian steroids belong to the cytochrome P-450 gene family

(for review see Refs. 49 and 50). This group of enzymes

includes: 1) Cholesterol side-chain cleavage enzymes (P-

450SCC), which convert cholesterol to pregnenolone. 2) The

P-450C17 enzyme (involving both 17a-hydroxylase and

C17,20-lyase activity) converts both progestins (pregnenolone

and progesterone) to androgens [dihydroepiandrosterone

and androstenedione (AD), respectively]. 3) The aromatase

enzyme complex (P-450A ROM), converts androgens [AD

and testosterone (T)] to estrogens (estrone and E2, respectively)

Two enzymes that are not members of the P-450 gene

family are also important for gonadal steroid synthesis: 3bhydroxysteroid

dehydrogenase, converting D5-steroids

(such as pregnenolone) to D4-steroids (such as progesterone),

and 17 ketosteroid reductase converting AD to T and estrone

to E2.

The cholesterol side-chain cleavage enzyme represents the

major rate-limiting step in steroid hormone synthesis. Moreover,

proteins involved in the acquisition of cholesterol (including

lipoprotein receptors and enzymes involved in de

novo cholesterol synthesis) have also been shown to be im-

In vitro studies using cells isolated from human ovarian

follicles have demonstrated convincingly that theca cells are

the source of follicular androgens (54, 55) — predominantly

AD(56, 57)—whereas granulosa cells only produce E2 when

androgens are added to the culture medium (58–60). In the

human ovarian follicle, immunocytochemistry (with the use

of antibodies against specific enzymes, allowing direct visualization

of the distribution of the enzyme in tissue) as well

as Northern blot analysis of RNA has shown the P-450C17

enzyme to be restricted to the theca cell layer (61, 62), consistent

with the notion that these cells are the major site of

intrafollicular androgen production. mRNA levels for

P-450C17 are increased dramatically in preovulatory follicles

(63), which correlate well with augmented 17a-hydroxylase

activity of human theca cells in culture

. However,

appreciable quantities of mRNA (63, 65, 66) and the aromatase

enzyme (62, 67) were observed in dominant follicles

in the late follicular phase. These observations are in keeping

with the high level of aromatase enzyme activity expressed

in vitro by granulosa cells obtained from preovulatory follicles

(59, 68). In addition, mRNA expression is in good agreement

with immunolocalization of the aromatase enzyme

(66). Synthesis of the P-450AROM enzyme could also be

induced by FSH administration to human granulosa cells in

culture (69). When follicles mature, granulosa cells also exhibit

elevated mRNA levels for P-450SCC, LH receptor, activin,

and inhibin (70).

The theca interna layer of developing follicles responds to

LH and synthesizes androgens (71, 72). AD and its immediate

metabolite T are transferred from the theca layer to the

intrafollicular compartment. For this reason these steroids

are present in large quantities in ovarian follicles of all sizes

and represent the main steroid produced by early antral

follicles (73–75). Atretic follicles of all sizes (between 2 and

13 mm diameter) also contain high androgen levels (57, 76)

and low E2 concentrations (77). Granulosa cells become responsive

to FSH only at more advanced stages of development

and are capable of converting the theca cell-derived

substrate AD to E2 by induction of the aromatase enzyme.

This so-called ‘two-gonadotropin, two-cell’ concept emphasizes

that adequate stimulation of both theca cells by LH and

granulosa cells by FSH is required for adequate E2 biosynthesis,

as has been recognized since the 1940s

Large (.8 mm diameter) follicles in the mid- and late

follicular phase of the menstrual cycle contain appreciable

(up to 10,000-fold) higher quantities of E2 compared with

small follicles, as has been shown by numerous authors (60,

75, 76, 83–87). Intrafollicular E2 concentrations were up to

40,000-fold higher than those in peripheral plasma, and 20

It has

been demonstrated in IVF patients that a correlation exists

between the E2/androgen ratio in follicle fluid and follicular

health and fertility potential of oocytes

The magnitude of E2 synthesized by granulosa cells in vitro

is dependent on the size of the follicle from which cells were

obtained, with AD metabolized to E2 only by granulosa cells

from follicles beyond 8–10 mm in diameter (59, 68, 92). Follicle

fluid E2 concentrations are also correlated with the

amount of aromatase activity expressed in vitro (60). In addition,

granulosa cells in culture produce larger quantities of

E2 in response to similar doses of FSH if cells were obtained

from larger (.8 mm) follicles (59, 68, 92), suggesting increased

sensitivity

Numerous in vitro studies have shown for the rat model

that E2 plays important autocrine roles in stimulating FSHinduced

granulosa cell proliferation (76, 96), aromatase enzyme

induction (97–99), production of inhibin (100), increase

in E2 and FSH receptors (101), and formation of LH receptors

on granulosa cells (102, 103). In addition, E2 exhibits a paracrine

action on adjacent theca cells by inhibiting androgen

production.

Based on these observations,

the concept has arisen that augmented intrafollicular

E2 production is a conditio sine qua non for ongoing

follicle maturation. In fact, absent induction of aromatase

enzyme activity has been widely accepted as the underlying

cause of follicle maturation arrest and subsequent anovulation

in PCOS.

In another patient suffering from a

partial P-450C17 (17, 20-lyase step) deficiency, follicle growth

could also be achieved after the administration of exogenous

FSH despite low intrafollicular E2 levels.

Despite a significant increase in serum

FSH levels, in the same order of magnitude as the intercycle

rise in FSH during the normal menstrual cycle, serum E2

levels remained low. However, development of multiple preovulatory

follicles emerged within 14 days. In a single subject,

three large follicles between 13 and 18 mm in diameter

were aspirated, and extremely low intrafollicular levels of

AD and E2 were found

These observations in the human confirm the two-cell, twogonadotropin

concept for adequate E2 synthesis but also

demonstrate convincingly that increased E2 production is not

mandatory for normal follicle growth up to the preovulatory

stage. It is still uncertain whether estrogen receptors are present

on granulosa cells from higher primates, including the human.

Collectively, these data suggest that in the human, E2 is not

required for follicle development. It appears that, under normal

conditions, augmented E2 synthesis is merely associated

with dominant follicle development, where growth of the

follicle is, in fact, driven by other nonsteroidal (growth) factors.

During the follicular phase of the normal menstrual cycle

E2 is clearly important for other crucial physiological processes

such as stimulation of endometrial proliferation, cervical

mucus production, and induction of the midcycle LH

surge and subsequent ovulation. Whether oocyte maturation

in the human requires exposure to estrogens remains unclear

at this stage

Ovarian response to exogenous

gonadotropins (as estimated by rising serum E2 levels)

was equal, regardless of whether gonadotropins were administered

in the follicular or midluteal phase of the cycle

The dominant follicle requires continued

though reduced support by FSH. In fact, growth of a single

dominant follicle could be sustained in GnRH antagonisttreated

monkeys by the administration of exogenous FSH in

decremental doses (Fig. 4) (143), suggesting enhanced sensitivity

for FSH when the dominant follicle matures.

Early follicular phase administration of E2

caused a significant reduction in serum FSH and a lengthening

of the follicular phase (144). Moreover, administration

of antiestrogen antibodies in the early to midfollicular phase

gives rise to elevated serum FSH levels, which interferes with

single dominant follicle selection resulting in ongoing maturation

of additional cohort follicles

provide in vivo evidence for

the concept that gonadotropin-responsive follicles are maintained

throughout the entire cycle. Follicles can be stimulated

to ongoing and gonadotropin-dependent development

when the appropriate endocrine signal (i.e. elevated serum

FSH levels) is operative. Under normal conditions, elevated

FSH concentrations are present during the luteo-follicular

transition only. Augmented E2 production by the most mature

(dominant) follicle starting around the midfollicular

phase causes a subsequent decrease in FSH levels due to

negative feedback effects of E2 on the hypothalamic-pituitary

axis. The dominant follicle restricts ongoing maturation of

other, less mature follicles from the cohort since FSH levels

drop below their threshold for stimulation of gonadotropindependent

growth. The dominant follicle is spared from the

inhibitory influence of reduced FSH stimulation because of

increased sensitivity to FSH

FSH threshold and follicle recruitment. Due to the demise of

the corpus luteum and the subsequent decrease in estrogen

production (148), FSH levels rise at the end of the luteal phase

of the human menstrual cycle (149). This intercycle rise is

closely synchronized with ovulation, and FSH levels start to

increase 12 days after the preceding LH surge (150). As

mentioned previously, initiation of growth of primordial

follicles occurs continuously and in a random fashion

only follicles that happen to be at a more advanced

stage of development during the intercycle rise in

FSH will gain gonadotropin dependence. The concept that

FSH concentrations above a certain level, referred to as the

‘FSH threshold,’ are needed for ovarian stimulation was first

introduced by Brown in 1978 (151) and substantiated more

recently by Schoemaker and colleagues. The individual variation

in FSH serum levels at which follicle growth was initiated

could be assessed to be between 5.7 and 12.0 IU/liter

with the use of intravenous administration of gonadotropins

in PCOS patients.

The threshold level should be

surpassed to ensure ongoing preovulatory follicle development.

This process of rescue of a cohort of follicles from

atresia by FSH stimulation is referred to by most authors as

‘recruitment.’ The recruited cohort represents a group of

follicles at a comparable (but not identical) developmental

stage. This group of follicles, by chance, happened to leave

the pool of resting follicles around the same period of time

several months before. In contrast, other investigators reserve

this term for the initiation of growth of primordial

follicles. Morphological and endocrine studies suggest that healthy

early antral follicles less than 4 mm in diameter are present

throughout the cycle (89), in keeping with the concept that follicles are continuously available for stimulation by FSH

At the end of the luteal phase, the largest healthy follicles

observed by morphological criteria have been described to be

between 2 to 5 mm in diameter (10, 89, 154), and the number

of recruitable follicles present is believed to be between 10

and 20 for both ovaries.

The largest healthy

follicles at the start of the follicular phase of the cycle have

been reported to exhibit a diameter between 4 and 8 mm (94,

155), and no morphological differences exist between these

follicles. These observations strongly suggest that the dominant

follicle is selected at a later stage of the follicular phase

of the cycle. Indeed, exogenous HMG administered during

different phases of the menstrual cycle is most effective in

stimulating follicle recruitment if administered during the

late luteal or early follicular phase.

Enucleation of the corpus luteum in 10 women

was followed by an immediate and rapid decline of E2 and

progesterone levels. This was followed by rising FSH levels,

renewed follicle growth, and ovulation within 16–19 days

after enucleation (159). These experimental results are in full

If the intercycle rise in

serum FSH is shortened by the early to midfollicular phase

administration of GnRH antagonist, follicle growth is arrested

and new follicle recruitment will follow once medication

is withdrawn

It may

be proposed that the follicle selected to gain dominance is the

one that has most rapidly acquired the highest sensitivity for

FSH. This may be the follicle that was at the most advanced

developmental stage when recruited.

with more pronounced E2 production

by cells obtained from larger follicles (59, 68, 92, 162).

Responsiveness to FSH stimulation is also increased in preovulatory

follicles (164). In addition, in the late follicular

phase, steroidogenic function of granulosa cells from the

dominant follicle is also stimulated by LH (165). Finally,

observations in the monkey suggest that increased vascularization

of individual follicles (resulting in the preferential

exposure to circulating factors) may also be instrumental in

the selective maturation of preovulatory follicles

The FSH ‘gate’ (168) or

‘window’ (169, 170) (Fig. 6, upper panel) concept has been

introduced to emphasize the significance of a transient elevation

of FSH above the threshold. This concept emphasizes

the importance of time (i.e. duration of elevated FSH levels)

rather than dose (magnitude of FSH elevation) for single

dominant follicle selection

However, it seems that the initiation

of declining serum FSH levels precedes augmented

ovarian estrogen output. We have observed a clear association

between the magnitude of decrease in endogenous FSH

serum levels and the E2 rise, indicating that the duration of

FSH stimulation (duration of serum FSH above the threshold)

is a major determinant for ovarian E2 production.

The magnitude of multiple

follicle growth in IVF patients has been shown to be

proportional to the late follicular phase accumulation of FSH

in serum (172). These experiments confirm that the duration

(related to the window concept) rather than the magnitude

(threshold concept) of FSH stimulation determines the number

of developing follicles.

Inhibin levels did not change during the early

follicular phase.

Follicular phase serum patterns of

inhibin A appear to be comparable to previously used less

specific assays (175). In contrast, a profound rise in inhibin

B serum levels was observed early in the follicular phase,

suggesting that it is secreted by recently recruited cohort

follicles in response to FSH. This rapid rise in inhibin B occurs

just after the intercycle rise in FSH. It may be proposed that

inhibin B limits the duration of the FSH rise (narrowing the

FSH window) through negative feedback at the pituitary

level and may therefore be crucial for mono follicle development.

Follicles could be visualized from

8–10mmonward (181), and usually two to three follicles per

ovary could be identified. Growth of the dominant follicle is generally

mentioned to be linear, with a mean daily growth rate

around 2–3 mm

TVU: Up to 11 follicles (.2 mm in diameter)

could be observed throughout the cycle in each ovary, and

a dominant follicle could be visualized from 10 mm onward

(Fig. 9) on cycle day 9 (Table 1). The size of nondominant

follicles visualized by TVS always remains below 11 mm.

The ultrasound observation of dominant follicle

selection correlates strongly with a sudden increase in serum

E2 concentrations

1. Heterogeneity of FSH. Variant forms of FSH are synthesized

and secreted by the anterior pituitary, on the basis of differences

in oligosaccharide structure of these glycoproteins

as well as the number of incorporated terminal sialic acid

residues.

Depending on the sophistication of techniques

used, up to 20 isoforms have been characterized for

human FSH. Heavily sialylated (more acidic) FSH has been

described to exhibit reduced receptor binding and in vitro.

bioactivity, whereas circulating half-life of these forms is

extended. These forms may be desialylated in the circulation.

In contrast, basic isoforms have been described to be more

biopotent in vitro (2- to 5-fold), whereas the circulating halflife

is significantly reduced.

It has been speculated that ovarian follicles are

recruited in the early follicular phase (when gonadal steroid

feedback is low) predominantly by more acidic FSH isoforms,

whereas follicle selection and rupture later during the

follicular phase is dependent chiefly on more basic FSH

isoforms

The majority of growth factors, such as

insulin-like growth factors (IGF) (226), transforming growth

factor-b, fibroblast growth factor, and activin (227), have

been shown to enhance FSH action in vitro. In contrast, other

growth factors have been shown to inhibit FSH-stimulated E2

biosynthesis by cultured human or primate granulosa cells,

including inhibin (228), epidermal growth factor (229 –231),

and IGF binding protein (IGFBPs) (232). Decreased follicle

fluid epidermal growth factor and transforming growth factor-

a concentrations have been described when follicles mature

(233–235). Moreover, white blood cell-derived cytokines,

such as like tumor necrosis factor, interferon, or

interleukins, have been proposed to be relevant for human

ovarian physiology.

Expression of IGF-II and their

binding protein (IGFBPs), as well as IGF receptors, has been

shown to be dependent on the developmental stage of the

follicle (238, 239). IGFBP-3 was shown to exhibit structural

similarity with the FSH-binding inhibitor (240), and the IGFBP

profile in follicle fluid has been described to vary during

follicle development, independent from changes in serum

(241). Moreover, proteases capable of specifically decreasing

the level of IGFBP-4 could be demonstrated in estrogendominant

follicle fluid only

Ovarian manipulation

HMG preparations (FSH to LH activity ratio,

1:1), obtained from urine of postmenopausal women, are

administered to stimulate follicle growth, whereas pregnant

women provide the urine source for hCG preparations (with

LH-like activity) to induce ovulation.

It should be stressed that the goal of induction of

ovulation is completely different from ‘controlled’ ovarian

hyperstimulation for IVF, where the goal is to interfere with

selection of a single dominant follicle to obtain multiple

oocytes for IVF.

The threshold level was arbitrarily

extrapolated from the first day a follicle beyond 12mmcould

be observed by transabdominal ultrasound or TVS. No difference

in the FSH threshold was observed, comparingHMG

vs. FSH.

For a given anovulatory woman, FSH levels ‘within

the normal range’ may simply mean FSH levels below the

threshold for ovarian stimulation. Hence, only the intercycle

rise in FSH above the threshold may be lacking in these

patients.

Normogonadotropic anovulatory women frequently suffer

from PCOS. This heterogeneous group of patients is characterized

by ovarian abnormalities (polycystic ovaries) combined

with distinct endocrine features (elevated serum LH

and/or androgen levels) (282). Various lines of evidence

indicate that early follicle development is normal in these

patients, whereas anovulation is caused by disturbed dominant

follicle selection (74). This abnormal condition may be

caused by disturbed intraovarian regulation of FSH action

(129), and therefore response to exogenous FSH may be different

from normal. Hence, the presence or absence of ovarian

abnormalities in patients may influence treatment outcome

after exogenously administered gonadotropins. This

may explain major differences in the FSH threshold and

duration of stimulation needed to induce preovulatory follicle

development in these patients.

Correlations between serum

E2 levels and number and size of follicles have been studied

(194, 306), and it was shown that E2 production is the net

result of all developing follicles. This is in sharp contrast to

normal follicle development where estrogens are produced

by a single dominant follicle only.

Concomitant medication, in addition to gonadotropins,

may include: 1) dexamethasone suppression of adrenal androgen

production (310); 2) GnRH agonists to suppress endogenous

release of LH (and FSH) (311, 312); 3) dopamine

agonists therapy in case of hyperprolactinemia; 4) GH in an

attempt to improve ovarian responsiveness (313); and 5)

luteal support either by hCG or progestins.

1. Conventional step-up protocol. Conventional step-up dose

regimens for gonadotropin induction of ovulation are characterized

by initial daily doses of two ampoules (5 150 IU of

bioactive FSH). Doses may be increased after 5 days in the

event that ovarian response is judged to be insufficient.

Low dose-regimen: step-up regimen for gonadotropin induction of ovulation has

been the preferred method of stimulation in Europe since

1990. This dose regimen is characterized by low initial daily

gonadotropin doses ranging between one-half and one ampoule

(38–75 IU of bioactive FSH), and doses are only increased

by one-half ampoule per day after 14 days, in cases

of insufficient ovarian response. Pharmacokinetic studies have indicated that it takes approximately

5 days before steady state FSH levels are reached

when similar gonadotropin doses are administered daily

through the intraperitoneal route

Initial dose finding studies have generated a dose regimen

that can be used in clinical practice. We have abandoned the

use of GnRH agonists since 1992 without any loss of clinical

efficacy. A similar FSH dose regimen is applied; i.e. a twoampoule/

day starting dose shortly after a spontaneous or

progestagen-induced bleeding, followed by a decrease to one

and one-half ampoules/day once a dominant follicle can be

visualized by TVS (at least one follicle $ 10 mm). The dose

is further decreased to one ampoule/day 3 days after the first

dose reduction.

Reference

- Manipulation of Human Ovarian Function: Physiological

Concepts and Clinical Consequences-1997