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Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Dijkzigt Academic Hospital and Erasmus University Medical School, Rotterdam, The Netherlands
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Abstract |
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 Top Abstract I. IntroductionII. Dynamics of Normal...III. Gonadotropin Induction of...IV. Steroid Contraception and...V. Conclusions and Future...References |
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I. Introduction |
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TopAbstractI. IntroductionII. Dynamics of Normal...III. Gonadotropin Induction of...IV. Steroid Contraception and...V. Conclusions and Future...References |
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New insight in the interplay between systemic and intraovarian factors regulating development and atresia of follicles may have significant implications. Relevant clinical conditions include ovarian ageing as well as chronic anovulation in patients presenting with serum FSH and estradiol (E2) hormone levels within the normal range, frequently diagnosed as polycystic ovary syndrome (PCOS). More effective and safe protocols for stimulation of ovarian function for infertility therapy may be developed. This involves both gonadotropin induction of ovulation (aiming at single dominant follicle growth in anovulatory patients) and so-called ‘controlled’ ovarian hyperstimulation for in vitro fertilization (IVF) (aiming at interfering with single dominant follicle selection to induce ongoing multiple follicle development in ovulatory women).
Due to ongoing concern regarding the potential for side effects and long-term health hazards, doses of combined estrogen/progestin steroid contraceptive pills have been decreased continuously since their introduction in the 1960s. It has been noticed subsequently that tolerance for omission of pill intake, especially around the pill-free interval, has diminished substantially in women using regimens presently on the market. 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.
Concepts involved in regulation of follicle growth during gonadotropin induction of ovulation (attempting to enhance fertility) as well as during steroid contraception (aiming at inhibiting fertility) are derived from recent findings regarding regulation of ovarian function under physiological circumstances. Therefore, these three conditions have been selected as the major focus of the present review.
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II. Dynamics of Normal Human Follicle Growth and Selection |
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TopAbstractI. IntroductionII. Dynamics of Normal...III. Gonadotropin Induction of...IV. Steroid Contraception and...V. Conclusions and Future...References |
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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 µm) 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 (7, 15, 16). 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 (for review
see Ref.12) (Fig. 1
). An increase in the number of
granulosa cells is critically important for the advancement in
developmental stages of the follicle. The time interval required for a
given follicle to pass these different developmental stages can
therefore also be assessed by calculating the granulosa cell-doubling
time (duration of mitotic activity in vitro) (17).
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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 (Section II.E). For instance, follicles grow up to the early antral stage in long-term hypophysectomized animals (25, 26). Similar numbers of maturing follicles, as compared with controls, have been found in anencephalic fetuses (27, 28), and exposure of ovaries to high gonadotropin levels has failed to result in accelerated follicle loss (12). 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, 31, 32, 33), pregnancy (34, 35, 36, 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 (38). However, observations in hypogonadal mice suggest that gonadotropins do play a role in initiation and continuation of follicle growth (39). In the rat model it has been suggested that theca cell differentiation and early preantral follicle growth is dependent on subtle stimulation by LH (40, 41). In addition, assessment of ovarian morphology of term infant monkeys showed a reduced number of primordial and primary follicles and increased follicle atresia after hypophysectomy (42). In conclusion, the question of whether the extent and rate of early follicle growth is dependent on exposure to minute amounts of gonadotropins remains unsolved (43, 44). Improved knowledge regarding mechanisms regulating initiation of primordial follicle growth as well as atresia of early stages of follicle development may shed more light on clinical conditions such as ovarian ageing and premature ovarian failure, as well as the great individual variability in menopausal age.
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 contrast to other primate species such as the Booroola sheep (14, 45), 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 (48).
B. 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 17
-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). Moreover, a specific DNA sequence,
termed Ad4, has recently been identified as a transcription factor
regulating the expression of steroidogenic P450 genes. The expression
of Ad4-binding protein (a zinc finger DNA-binding protein also known as
steroidogenic factor-1) has been shown to correlate with the
immunolocalization of steroidogenic enzymes in the human ovary (51).
Two enzymes that are not members of the P-450 gene family are also
important for gonadal steroid synthesis: 3ß-hydroxysteroid
dehydrogenase, converting 
5-steroids (such as pregnenolone) to
4-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 important for sufficient steroid biosynthesis (50). Patients have been described with mutations in DNA encoding for a protein involved in cholesterol transport within the cell (so-called steroid acute regulatory protein) (52) or encoding for specific enzymes involved in the steroid synthesis pathway (for review see Ref.53). The significance of each step for normal steroid biosynthesis and subsequent ovarian function has been clarified by the careful description of underlying gene abnormalities and the phenotype expression in the event that certain steroids are lacking.
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, 59, 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 17-hydroxylase activity of human theca cells in
culture (64). Small antral follicles were shown to lack P-450AROM mRNA.
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, 74, 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 (54, 78, 79, 80, 81, 82).
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, 84, 85, 86, 87). Intrafollicular
E2 concentrations were up to 40,000-fold higher than those
in peripheral plasma, and 20-fold higher concentrations of
E2 have been observed in venous blood draining the ovary
containing the dominant follicle as compared with the contralateral
side (88, 89). 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
(90). After enucleation of the largest follicle no further differences
were found in steroid levels in blood draining both ovaries (91). A
correlation between intrafollicular E2 concentrations and
follicle diameter has been substantiated in large dominant follicles
(75, 77, 83). All studies show low E2 levels in relatively
small (<10 mm diameter) nondominant follicles (57, 68, 76, 77, 83),
and the absence of a correlation between follicle size and
E2 levels in this size range (Fig. 2) was
emphasized recently (75). 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. Moreover, lower doses of FSH
induce similar E2 production by cultured rat granulosa
cells obtained from larger follicles, again indicating that cells
obtained from more mature follicles exhibit augmented sensitivity for
stimulation by FSH (93). Finally, a distinct relationship was observed
between follicle diameter and the number of granulosa cells that was
recovered at each size (94).
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C. Are estrogens needed for follicle development?
As discussed above, dominant follicle development in the human is
closely associated with increased follicular estrogen biosynthesis.
E2 receptors have been shown to be present in rat granulosa
cells, as studied by ligand-binding assays (95). Numerous in
vitro studies have shown for the rat model that E2
plays important autocrine roles in stimulating FSH-induced granulosa
cell proliferation (76, 96), aromatase enzyme induction (97, 98, 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 (72). Estrogen
(diethylstilbestrol) treatment of immature hypophysectomized rats
stimulates growth of large numbers of follicles. Human chorionic
gonadotropin (hCG) and FSH-induced follicle development could be
inhibited by the administration of estradiol antiserum (104),
suggesting again autocrine stimulatory roles for endogenous estrogens.
Estrogens have also been shown to inhibit apoptotic changes of ovarian
follicles (20). 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 (105).
Several lines of evidence, however, gave strong support to the notion
that this may not be the case for higher species, including the human.
Under normal conditions, augmented E2 levels may merely be
associated with normal follicle development. A deficiency of the
17-hydroxylase enzyme due to a specific gene defect affects both
adrenal steroidogenesis and androgen and estrogen production by the
ovary. This condition is characterized by hypergonadotropic
hypoestrogenic primary amenorrhea, with arrest of follicle development
at the early antral stage (106). However, normal follicle development
could be induced in these patients by FSH treatment for IVF (after GnRH
agonist suppression of endogenous gonadotropin release) despite
extremely low intrafollicular levels of AD, T, and E2.
Oocytes could be obtained and fertilized in vitro resulting
in normal early embryo development (107, 108). 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 (109).
Subsequent IVF and cleavage rates were not different from normal.
Moreover, two unrelated females have been described recently with
mutations in the CYP19 gene (consisting of 10 exons, and localized on
chromosome 15, q21.1 region), resulting in the total absence of
aromatase enzyme activity (110, 111). Large ovarian cysts have been
described in both patients, suggesting that growth of antral follicles
can occur in the absence of intraovarian estrogen biosynthesis. Recent
experiments in monkeys treated with an aromatase inhibitor between day
8 and 10 of the follicular phase have also excluded the possibility
that increased levels of circulating E2 in the late
follicular phase is required to sustain follicle maturation (112).
We have recently participated in a study on safety and pharmacokinetic
properties of human recombinant FSH (113, 114) in hypogonadotropic
female volunteers. The complete absence of endogenous as well as
exogenous LH in these subjects did provide the unique opportunity to
study effects of FSH alone on ovarian steroid production and follicle
growth (115). 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 (Fig. 3) (87). A normal
rise in immunoreactive serum inhibin levels in the majority of these
women excluded the possibility of granulosa cell abnormalities
per se (38). A discrepancy between serum E2
levels and follicle development has also been observed in
hypogonadotropic women comparing purified FSH of urinary origin and
human menopausal gonadotropin (HMG; 1:1 ratio of LH to FSH activity)
(116). When urinary FSH was combined with long-term GnRH agonist
comedication suppressing the endogenous release of LH and FSH, similar
observations were reported (117). It is of special interest to note
that large antral follicles were also observed in the ovaries of two
amenorrheic patients described with inactivating mutations of the LH
receptor (and consequently low E2 production) (118, 119).
These observations in the human confirm the two-cell, two-gonadotropin
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. These
observations are fully supported by more recent similar studies in the
monkey. Follicular growth and oocyte maturation in LH-deficient
macaques did occur with FSH alone (120), and fertilization rates of
oocyte obtained from recFSH/GnRH antagonist-treated monkeys were not
compromised (121).
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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 (see also Section II.F). This concept may also bear significance for our thinking regarding underlying causes of anovulation, in particular in polycystic ovaries. Follicles may cease to mature due to defective intraovarian regulatory mechanisms rather than the absence of aromatase enzyme induction per se (129).
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 (130, 131, 132).
D. In vivo regulation of follicle maturation in the monkey
A series of in vivo studies in the monkey has
systematically addressed endocrine factors regulating follicle growth
(for comprehensive reviews see Refs. 133–137). A significant
proportion of these experiments have subsequently been confirmed in the
human (see Section II.E). Surgical ablation of the dominant
follicle in the late follicular phase of the cycle blocked the midcycle
gonadotropin surge and ovulation. These observations indicate that no
other follicles from the recruited cohort were capable of replacing the
dominant follicle, presumably due to atretic changes. New follicle
recruitment occurred in response to a rise in endogenous FSH levels,
similar to ovarian response after removal of the corpus luteum. The
duration until the next ovulation was 12 days, which equals the normal
follicular phase of the cycle. Therefore ovulation was delayed after
follicle cautery and advanced after luteectomy (138). 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 (139).
By repeated cautery of the dominant follicle, it was also shown that
the midcycle gonadotropin surge of the preceding cycle plays no role in
follicle recruitment for the subsequent cycle (140).
Dominant follicle selection, and subsequent asymmetrical ovarian
estrogen output, occurs around the midfollicular phase (141, 142). The
dominant follicle requires continued though reduced support by FSH. In
fact, growth of a single dominant follicle could be sustained in GnRH
antagonist-treated monkeys by the administration of exogenous FSH in
decremental doses (Fig. 4) (143), suggesting enhanced
sensitivity for FSH when the dominant follicle matures (see also
Sections II.B and II.E2). The dominant follicle
continued its development despite relatively low late follicular phase
FSH concentrations, incapable of stimulating growth of less mature
follicles. Subsequent experiments in the monkey model have addressed
the significance of FSH for single dominant follicle selection. 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 (145, 146).
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E. The FSH threshold and window concept for in vivo follicle growth
1. 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.
Follicle growth will eventually cease and follicles will enter atresia
if the appropriate endocrine signal is lacking. Although each follicle
may have an equal potential to reach full maturation, 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 (152, 153). Moreover, multifollicular
growth was shown to be associated with higher FSH concentrations above
the threshold (153) (Fig. 5), using a low-dose incremental
protocol for FSH induction of ovulation. Each growing follicle has a
threshold requirement for stimulation by circulating FSH. 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 (12) (see also Section II.A).
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Elegant experiments in the human have further substantiated the FSH threshold concept and have generated additional support for the notion that follicles ready to be recruited are present throughout the menstrual cycle. Removal of the dominant follicle in the late follicular phase, or luteectomy in the luteal phase during gynecological surgery, results in new follicle recruitment and subsequent ovulation (157, 158, 159). 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 agreement with observations in the monkey model after similar intervention and indicate indeed that suppressed gonadotropin secretion (due to corpus luteum or dominant follicle steroid production) is responsible for inhibition of more advanced follicle maturation. Moreover, these observations are in keeping with the notion that final and gonadotropin-dependent follicle growth preceding ovulation takes approximately 14 days, coinciding with the follicular phase length of the menstrual cycle. 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 (160, 161).
2. FSH window and single dominant follicle selection. In
follicles less than 10 mm, the aromatase enzyme is poorly expressed
(62) and intrafollicular E2 levels are low (Fig. 3
) (57, 75, 162). This also holds true for follicles in the early follicular
phase of the menstrual cycle. E2 production, however, can
be stimulated rapidly in vitro by adding FSH to the culture
medium (59, 68, 92). It cannot be readily explained why E2
levels remain low despite maximum FSH stimulation in the early
follicular phase (163). Intraovarian modification of FSH action may be
involved (see Section II.F.3). Under normal conditions, the
fate of developing antral follicles is closely associated with their
ability to create an estrogen-rich intrafollicular environment, as
discussed previously. 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. Indeed, FSH responsiveness
of cultured granulosa cells (obtained from follicles at various stages
of development) has been shown to be dependent on follicle size, 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 (166).
Consequently, the FSH threshold for a given follicle is not fixed but is dependent on its developmental stage and therefore changes over time. Indeed, experiments applying GnRH antagonist for 3 consecutive days in the mid- or late follicular phase of the cycle have shown convincingly that the developing follicle becomes more resistant to gonadotropin withdrawal as it becomes more mature (160). Midfollicular administration of GnRH antagonist may induce a transient follicular arrest without triggering new folliculogenesis (167) or complete follicle maturation arrest and new follicle recruitment (161), depending on the magnitude and duration of gonadotropin suppression.
FSH serum levels steadily decrease during the mid- to late follicular
phase of the menstrual cycle. The follicle that has gained dominance is
less dependent on continued support by high early follicular phase FSH
levels. However, circulating FSH levels are suppressed to a
concentration below the threshold for remaining follicles from the
recruited cohort. These follicles will therefore cease to mature and
undergo atresia. Hence, development of the most mature follicle,
closely associated with increased E2 production, secures
selection of a single dominant follicle. 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. Previous studies by our own group in 16
female volunteers have characterized follicular phase patterns of FSH
serum levels and investigated correlations between decremental FSH
levels and dominant follicle development (163) (see also Table 1 and Fig. 7, where the number of volunteers
has been extended to 42). This decrease may be due to negative estrogen
feedback on the hypothalamic-pituitary axis (168). 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 (163).
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) (171) (I.
Schipper and B. C. J. M. Fauser, unpublished observations). Moreover,
selection of a single dominant follicle is also prevented if high FSH
levels are sustained in hyperstimulation protocols for IVF. 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. We have
recognized the crucial role of decremental serum FSH levels for single
dominant follicle selection under normal conditions and have attempted
to develop a decremental (‘step-down’) dose regimen for gonadotropin
induction of ovulation for treatment of anovulatory infertility (see
Section III.D) (Fig. 6, middle panel). It could
be demonstrated, indeed, that growth of the dominant follicle is
sustained despite reduced late follicular phase stimulation by
decremental doses of exogenous FSH (173).
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-subunit combined with ßB
constitutes inhibin B) or against ßA (inhibin A). 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. Elevated early follicular phase FSH levels in elderly
ovulatory women were shown to be associated with decreased inhibin B
secretion, which may be due to a reduced number of recruitable
follicles in women of advanced reproductive age (176). 3. Dominant follicle development. The ability to monitor growth of a large antral follicle, by means of transabdominal pelvic ultrasound, was originally described in an anovulatory patient during gonadotropin induction of ovulation (177). This noninvasive technique has allowed large-scale characterization of dominant follicle growth during the normal menstrual cycle (178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200). Follicles could be visualized from 8–10 mm onward (181), and usually two to three follicles per ovary could be identified (188, 189). Follicle size assessed by ultrasound has been compared with follicle volume as determined by the amount of fluid collected after puncture (182), or with follicle size during laparoscopy (190). Interobserver variability has been shown to be limited (200). Timing of ovulation could be predicted using ultrasound (180, 186, 187, 193), and the mean size of the preovulatory follicle reported in various studies ranged between 20 and 27 mm (201). On an individual basis, a high correlation was observed between follicle size and E2 serum levels (179, 185). However, major individual variability in size of preovulatory follicles (191) results in linear regression, but low overall correlations between follicle size and serum E2 levels (75, 163, 185, 188, 195, 199). Growth of the dominant follicle is generally mentioned to be linear, with a mean daily growth rate around 2–3 mm (163, 183).
Since 1985 the transvaginal route has been introduced for pelvic
ultrasound (202, 203), allowing enhanced imaging resolution and a more
reliable assessment of changes in number and size of small follicles
(197). Growth of dominant and nondominant follicles has been studied
extensively by our group using transvaginal sonography (TVS) (for
review see Refs. 204 and 205). 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 (163, 206). The ultrasound observation
of dominant follicle selection correlates strongly with a sudden
increase in serum E2 concentrations (r =
0.84; P < 0.001), indicating that visualization of the
dominant follicle coincides with enhanced E2 synthesis
(163), as has been shown previously by augmented E2 levels
in venous blood draining the ovary bearing the dominant follicle (88).
This in vivo ultrasound observation also agrees fully with
and extends previous studies (as discussed in Section II.B)
showing that: 1) Aromatase activity in vitro is only
observed if granulosa cells were obtained from follicles beyond 8 mm in
size. 2) Augmented intrafollicular E2 concentrations (and
positive immunostaining of the P-450AROM enzyme) only in follicles
beyond 10 mm. It could also be demonstrated that early follicular phase
FSH levels decrease before the onset of a rise in serum E2
concentrations (163, 204), which supports the notion that other ovarian
factors (like for instance inhibin B) are to be held responsible for
narrowing the FSH window.
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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. FSH heterogeneity should be considered as a continuum of molecular forms, each with distinct physiochemical characteristics. Glycoprotein isohormones with different carbohydrate side chains can be separated by their differences in charge. 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 half-life is significantly reduced (for comprehensive reviews see Refs. 208 and 209).
Effects of estrogens on the in vivo isohormone profile of FSH have been repeatedly established. In fact, changes were found during the normal menstrual cycle, as well as after menopause. In a small number of women, more basic isoforms were described to be present at midcycle (210, 211, 212). Estimates of changes in FSH heterogeneity, as assessed by in vitro bioassays, during the menstrual cycle are contradictory (210, 213, 214) and appear to be dependent on the assay system used. 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. However, the net effect of a predominance of more bioactive but shorted half-life forms on the overall in vivo biopotency is unknown at this stage, and therefore the physiological significance of described changes in FSH isoforms remains open for speculation.
2. Direct interference with FSH action. It has been proposed that low molecular weight proteins specifically interfering with FSH receptor binding are present in serum (215). In addition, a high molecular weight FSH receptor binding inhibitor was partially purified from human follicle fluid by the same group of investigators (216). However, these proteins have never been fully characterized, and the physiological relevance remains uncertain (for review see Ref.207). Cell lines transfected with the human FSH receptor may prove a valuable tool with which to study further the pathophysiological relevance of inhibition of FSH receptor activation (217–219a).
3. Intraovarian interference with FSH action by growth
factors. Serum FSH concentrations are maximal in the early
follicular phase of the menstrual cycle. In contrast, circulating
E2 levels start to rise around the midfollicular phase
coinciding with the visualization of a dominant follicle by ultrasound.
E2 production, however, can be stimulated rapidly in
vitro by adding FSH to the culture medium (59, 68, 92), and it
cannot be readily explained why early follicular phase E2
levels remain low despite maximum FSH stimulation (163). The lag period
between maximum FSH stimulation and augmented ovarian E2
output may be explained by intraovarian inhibition of FSH action early
in the follicular phase or enhancement of FSH action within the
dominant follicle (Fig. 10). The dominant follicle
continues to mature despite decreased stimulation by lower late
follicular phase FSH concentrations. This observation of decreased
dependence of the dominant follicle on FSH stimulation (as discussed
extensively in Section II.E) strongly suggests that the FSH
signal is modified within the ovary, either at the level of FSH binding
to the receptor or by interference with postreceptor signal
transduction. In addition, the intrafollicular rise in E2
levels of the dominant follicle was believed to be responsible for the
decreased need for stimulation by FSH through autocrine short loop
up-regulation (220). However, it is now clear that follicles can mature
fully without a concomitant rise in E2. This observation
strongly suggests that other (intraovarian) factors in fact drive
growth of the follicle, and disturbed intraovarian regulation may prove
to be crucially important for cessation of follicle development in PCOS
patients. Moreover, a 2.5-fold difference in maximum early follicular
phase FSH serum concentrations — not correlated with any other
follicular phase parameter, such as length or follicle growth
characteristics (163) — observed in a group of young women presenting
with normal ovarian function suggest distinct differences in the
individual FSH threshold. This observation implies differences in
intraovarian regulation under normal conditions.
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concentrations have
been described when follicles mature (233, 234, 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 (236). Certainly, overwhelming evidence is available regarding major changes in the IGF system during follicle development in the human ovary (237). 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 estrogen-dominant follicle fluid only (242), suggesting that more bioavailable IGF-II is available to synergize with gonadotropins in the dominant follicle. It should be noted, however, that growth of follicles could be induced by exogenous FSH in a patient with Laron-type dwarphism (low endogenous IGF-I secretion due to a familial GH receptor defect) (243), suggesting that IGF-I is not required for normal ovarian function. Conclusive in vivo evidence that any of the above mentioned growth factors play a distinct role in human ovarian physiology is lacking as yet.
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III. Gonadotropin Induction of Ovulation |
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TopAbstractI. IntroductionII. Dynamics of Normal...III. Gonadotropin Induction of...IV. Steroid Contraception and...V. Conclusions and Future...References |
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The great majority of anovulatory patients presently treated with gonadotropin preparations comprise normogonadotropic (i.e. normal serum FSH concentrations; World Health Organization, class II) anovulatory infertile women who failed to conceive during previous antiestrogen medication. The aim of this treatment modality is to approach normal conditions as closely as possible; i.e. maturation and ovulation of a single dominant follicle and subsequent singleton pregnancy. Characteristics of dominant follicle development during gonadotropin induction have been documented using ultrasound and serum estrogen assays (254, 255, 256, 257). 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. Therefore, the use of the term induction of ovulation for IVF is confusing and should be abandoned.
Although gonadotropin therapy has been shown to be fairly successful in terms of ovulation rates (reported in the literature between 60–100%) and cumulative pregnancy rates (reported between 20–75%), complication rates are high. Major complications include multiple pregnancies (258), ovarian hyperstimulation (259), and a high rate of early pregnancy wastage (260). The first two complications have been shown to be related to the magnitude of multiple follicle development as estimated by serum estrogen levels (261, 262, 263) and more recently by pelvic ultrasound (264). The high abortion rate has been suggested to be related to elevated LH levels (265, 266). In addition, a significant increase in the overall prevalence of multiple pregnancies over the last 10–20 yr has been established repeatedly in the literature (267, 268, 269, 270, 271), and gonadotropin induction of ovulation is certainly involved. Inherent problems include social difficulties, ethical considerations regarding fetal reduction (272, 273), perinatal morbidity, and increased health care costs (274).
A great individual variability in ovarian response to stimulation by
FSH (so-called ‘FSH threshold’) was proposed in anovulatory patients
(151). Moreover, Brown (151) stressed that only a small margin exists
between an effective dose and a dose generating excessive ovarian
response. Unfortunately, predictors for the FSH threshold of a given
patient have not been identified. The concept of the FSH threshold in
anovulatory patients was substantiated more recently (152, 153) with
the use of intravenous administration of exogenous gonadotropins by
pump. The threshold level was arbitrarily extrapolated from the first
day a follicle beyond 12 mm could be observed by transabdominal
ultrasound or TVS. No difference in the FSH threshold was observed,
comparing HMG vs. FSH. Moreover, a 2-fold variation in
individual threshold levels was observed, and higher FSH serum levels
above the assessed threshold were found to be associated with
multifollicular growth (Fig. 5). Major individual variability in
response to stimulation by exogenous FSH underscores the need for
careful and frequent monitoring of ovarian response by ultrasound
and/or rapid serum E2 assays (257) and adjustment of doses
on an individual basis. In general, the focus is to approach the
individual threshold level prudently, to prevent serum FSH
concentrations to increase far above the threshold. Differences in the
FSH threshold level result in considerable variability in the duration
of gonadotropin administration in the event that low initial doses are
administered. Unaltered late follicular phase FSH serum levels in
gonadotropin-induced cycles differ greatly from the follicular phase of
the normal menstrual cycle. This condition may elicit growth of other
cohort follicles and, as a result, induce multiple follicle
development.
During the interphase from one menstrual cycle to the other, serum FSH
concentrations surpass the threshold for stimulation of ongoing and
gonadotropin-dependent follicle development. Serum FSH levels decrease
steadily during the follicular phase, securing the formation of a
single dominant follicle. Only this follicle reaches the full mature
state despite diminished stimulation by FSH, whereas growth of the
remaining less mature follicles in the cohort ceases due to
insufficient support by FSH. The significance of this pattern of FSH
stimulation is stressed by various intervention studies, both in the
human and in the monkey model, as discussed extensively in
Sections II.D and II.E. The threshold concept for
induction of ovulation focuses only on the magnitude of ovarian
stimulation by FSH, but ignores the element of time. In contrast, the
FSH window concept emphasizes the importance of FSH concentrations
surpassing the threshold for a limited period of time only. Decremental
dose regimens for exogenous FSH may be more effective in inducing
preferential growth of the leading follicle (Fig. 6, middle
panel). This approach may have implications for gonadotropin
induction of ovulation, as discussed later in this section. In addition
to the gonadotropin dose, many other factors may influence treatment
outcome. These conditions will first be discussed.
B. Conditions affecting treatment outcome
1. Patient-related factors. Women diagnosed as
hypogonadotropic hypogonadism, by definition, suffer from inadequate
stimulation of ovarian function. FSH serum levels are below the
threshold, and growth of follicles is arrested at a stage where further
development becomes dependent on stimulation by gonadotropins. If FSH
levels rise above the threshold, due to exogenous administration of
gonadotropin preparations, ovarian response should be normal. Success
and complication rates of gonadotropin induction of ovulation in these
patients is indeed favorable (275, 276, 277, 278, 279, 280, 281). However, the great majority of
patients presently treated with gonadotropins present with
clomiphene-resistant normogonadotropic anovulation. Serum FSH and
E2 levels in these patients are within normal limits.
Obviously, normal limits for both FSH and E2 depend heavily
on the phase of the menstrual cycle. As mentioned previously, maximum
early to mid follicular phase FSH levels are twice as high as late
follicular phase concentrations (see also Table 1). Moreover, even in
young regularly cycling women the FSH threshold varies considerably (at
least 2-fold). This variability is poorly recognized in the
classification of anovulation on the basis of serum FSH assays. 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.
Presently, the wish to establish a family is expressed later in life. Therefore, the population of women seeking help for infertility is increasing in age. It has been documented that cumulative conception rates after gonadotropin induction of ovulation are distinctly different when women under the age of 35 are compared with older women (276, 280).
Obesity frequently coincides with PCOS, and differences in pharmacokinetic characteristics of gonadotropin preparations (283), as well as clinical outcome (284, 285) related to body weight, have been reported. Moreover, other concomitant endocrine disorders such as hyperprolactemia or adrenal hyperandrogenemia may also affect treatment outcome.
2. Hormone preparation-related factors. Preparations of urinary gonadotropin have been continuously improved since its commercial introduction. HMG preparations contain similar (1:1 ratio) FSH and LH activity, as required by regulatory agencies (286, 287). The most significant improvements of HMG preparations over the years involved the introduction of 1) purified urinary FSH (with only minute amounts of LH), 2) highly purified urinary FSH (obtained through an affinity extraction procedure, removing virtually all of the contaminating proteins) (288), and 3) human recombinant FSH preparations (113, 289, 290, 291). Other recombinant glycoprotein preparations — such as recombinant hCG (292), LH (293), long-acting FSH (FSH-CTP) (294), short-acting (deglycosylated) FSH (295), and single-chain gonadotropins (296) — will be available soon. This fascinating development certainly provides the clinical investigator with a whole new set of tools with which to manipulate ovarian function. Moreover, the clinician will have the unique and challenging opportunity to tailor compounds and corresponding circulating half-lives according to the treatment goal and the individual needs of the patient. Different host cells produce recombinant FSH with different isohormone profiles (297). Therefore in vivo biopotency of a given distribution of FSH isoforms may vary. However, it is uncertain at this stage whether this approach my result in improved treatment outcome.
Since elevated LH levels are believed to be involved in poor reproductive outcome, many studies have been undertaken to test whether the administration of urinary FSH, as compared with HMG, may improve treatment outcome in PCOS patients. However, all published comparative trials have failed to show such an effect (298, 299, 300, 301). Considerable batch-to-batch differences have been observe
) is associated with
higher FSH levels above the threshold and more ampoules per day as
compared with patients presenting with monofollicle development (
).
[Reproduced with permission from M. van der Meer et
al.: Hum Reprod 9:1612–1617, 1994
(153).]
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