FEMALE REPRODUCTIVE ENDOCRINOLOGY A' THE OVARY AND THE HYPOTHALAMUSPITUITARYOVARY AXIS

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FEMALE REPRODUCTIVE ENDOCRINOLOGYA. THE
OVARYAND THE HYPOTHALAMUS-PITUITARY-OVARY AXIS

• Dr. Michal Lahav

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INTRODUCTION COMPARISON BETWEEN THE MALE AND THE
FEMALE GONAD

• Like the testis, the ovary is an endocrine gland
  which produces gametes ready for fertilization as
  well as hormones, the most important of which are
  steroids.
• In both glands, the gametes (spermatozoa and
  oocytes, respectively) originate in diploid
  proliferating cells (spermatogonia and oogonia,
  respectively), some of which are diverted to
  meiosis. However, while in males this process
  starts at the time of sexual maturation and
  continues often into old age, in the female
  oogonia appear at fetal life, give rise to cells
  committed to meiosis, and disappear before birth.

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• In the testis the gametes and the steroid hormone
  (testosterone) are produced in separate
  compartments (the seminiferous tubules and the
  interstitial Leydig cells). In contrast, in the
  ovary every gamete is enveloped by its own set of
  endocrine cells.
• An oocyte wrapped in at least one layer of
  endocrine cells is named ovarian follicle.
  Follicles are the structural and functional units
  of the ovary.
• This difference in the histological organization
  of the testis and ovary is related to the
  essential difference in the function of the two
  types of gonads while the testis produces
  hormones and gametes continuously, the ovary
  produces both of them in a cyclic manner.

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• As a rule, the regulation of cyclic processes is
  more complex than that of processes which proceed
  at a constant rate. A cycle which is as long as
  28 days (on average) is particularly hard to
  create.
• The cyclic production of gametes and hormones is
  associated with the need to support pregnancy
  (both its establishment and its continuous
  existence).
• Follicles begin to be formed before birth, and
  some of them go through the early stages of
  development before as well as after birth.
  However, it is only at puberty that some
  follicles reach the final stage of development,
  at which they are capable of undergoing ovulation.

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• Mitosis of oogonia ends on 7th month (fetal
  life)
• Atresia of oogonia from 8th week to 8th-9th
  month (fetal life)
• Meiosis (up to prophase I) from 8th week to 7th
  month (fetal life)
• Primordial follicle formation from 16th week
  (fetal life) to the end of 6th month post partum.

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Modified from Williams, 1998
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FOLLICULAR DEVELOPMENT

• Most of the follicles which reside in the ovaries
  at birth (1-2 millions) never reach maturity
  they undergo degeneration in a process named
  atresia.
• It is believed that follicles at their earliest
  stage, primordial follicles, do not undergo
  atresia, but follicles that have begun their
  development are prone to this process at any
  stage.
• Follicular development up to maturity takes about
  a year, with the earlier stages being slower.
  Once a follicle began to develop, it does not
  stop it proceeds to maturity or becomes atretic
  at some earlier stage.

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• Between birth and puberty the ovaries lose 80 of
  their follicles. The maximal number of
  ovulations in a womans lifetime is 400-500. The
  continuing atresia results in ovaries depleted of
  follicles at an average age of a little over 50,
  and this marks the end of the fertile period.

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COMMENTS TO THE SCHEMA OF FOLLICULAR DEVELOPMENT
(see below)

• Follicles are embedded in the dense connective
  tissue of the ovarian cortex.
• In primordial follicles the oocyte is surrounded
  by one layer of flat epithelial cells, the
  granulosa.
• Between the oocyte and the granulosa, a layer of
  extracellular proteins, zona pellucida, is
  observed. It is composed of 3 oocyte-produced
  proteins (ZP1, ZP2, ZP3), two of which serve as
  receptors to the spermatozoon in the process of
  fertilization.
• The granulosa layer is wrapped by basement
  membrane (basal lamina). It is composed of
  extracellular matrix proteins typifying the
  contact area between epithelial and fibroblast
  layers, with protein contributions from both cell
  types.

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• Throughout follicular development, until
  ovulation, blood vessels do not cross the basal
  lamina.
• At the onset of follicular development the
  granulosa cells become cuboidal and begin to
  proliferate. Primary follicles have one layer of
  cuboidal granulosa cells.
• Secondary follicles have several layers of
  granulosa cells. In addition, the fibroblasts
  adjacent to the granulosa begin to differentiate
  into endocrine cells (theca cells).
• All these stages of development are not affected
  by the gonadotropins (LH and FSH). Gonadotropin
  receptors begin to be expressed only in secondary
  follicles (LH-R in the theca and FSH-R in the
  granulosa).

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• The earlier development just described is
  dependent on local regulators, growth factors.
• Growth factors are small proteins that act via
  membranal receptors. In almost all cases they
  act locally (within diffusion range), namely,
  they have autocrine or paracrine effects.
• In the follicle, growth factors are secreted by
  the oocyte, the granulosa and the theca
  throughout follicular development.
• Follicles of the next stage, tertiary follicles,
  begin to develop an antrum. FSH action is
  essential for the formation of antral follicles.
• The follicular fluid contains components which
  come from the blood, as well as molecules locally
  produced by the follicular cells.

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• The volume of the follicular fluid as well as the
  number of the endocrine cells, primarily the
  granulosa, continue to grow.
• A follicle at an advanced stage of development,
  Graafian follicle, is also shown in the next
  figure.
• At this point the oocyte is held off center by a
  bridge and a wrapping of granulosa cells. These
  cell layer in named the cumulus oophorus.
• In this figure the various stages of follicular
  development are not drawn in the correct relative
  proportions. The oocyte in primordial follicles
  (40 micrometer diameter) reaches a diameter of 80
  micrometer in secondary follicles, and remains at
  this size in the following stages.

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Williams, 1998
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TIME COURSE OF THE LATER STAGES OF FOLLICULAR
GROWTH

• The next figure shows the time course of
  follicular development, beginning with the
  secondary follicle.
• These stages take 85 days the time scale is
  presented in the X axis (abscissa).
• There are two Y axes (ordinates) The right Y
  axis shows the diameter of the follicle (in mm)
  at each stage, whereas the left Y axis shows the
  corresponding number of granulosa cells. The
  theca layer is not shown for the sake of
  simplicity.
• The last 14 days shows the changes during the
  first half of the human ovulatory cycle, namely,
  the follicular phase, which is followed by
  ovulation.

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From Yen et al 1999, Fig. 6-16
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STEROIDOGENESIS IN THE OVARIAN FOLLICLE

• The main hormonal product of the ovarian follicle
  is estradiol, the most active natural estrogen.
  Like all steroid hormones, estradiol is
  synthesized from cholesterol.
• Both theca and granulosa participate in estradiol
  synthesis. The theca converts cholesterol to
  androgens, which are obligatory intermediates in
  estrogen production, and the granulosa converts
  androgens to estrogens.
• After ovulation, the endocrine follicular cells
  undergo transformation to a new structure - the
  corpus luteum (CL). In mammals, the main luteal
  steroid product is progesterone. The human CL
  produces also estradiol (via androgens).

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GENERAL COMMENTS ON STEROIDOGENESIS

• Most steroidogenic enzymes are expressed in
  several, or even all, steroidogenic cells, but
  some are unique to one cell type.
• Most steroidogenic enzymes are hydroxylases, and
  use a typical heme as a prosthetic group. Thus
  they are classified as cytochrome P450 enzymes
  (shortened to P450 or CYP).
• Hydroxylases require NADPH and molecular oxygen
  (O2).
• Some of the steroidogenic enzymes reside in the
  mitochondrial matrix, and others are embedded in
  the ER membrane, facing the cytosol. Few of the
  enzymes are soluble (see enzyme list below).

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• Most steroidogenic enzymes catalyze more than one
  biochemical reaction. In some of them the
  hydroxylated products are short-lived
  intermediates (see figure below).
• The rate-limiting step in steroidogenesis is the
  first one the conversion of cholesterol to
  pregnenolone. The enzyme (P450scc) is localized
  in the mitochondrial matrix.
• However, it is the transport of cholesterol into
  the matrix, rather than the intrinsic activity of
  the enzyme itself, which is rate limiting.
• Cholesterol transport into the mitochondrial
  matrix is facilitated by a protein named StAR
  (steroidogenic acute regulatory protein).

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STEROIDOGENESIS IN THE FOLLICLE
GRANULOSA
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EFFECTS OF THE GONADOTROPINS ON FOLLICULAR
STEROIDOGENESIS

• Theca cells express the LH receptor (LH-R). LH
  acts via adenylate cyclase (AC) activation and
  thus cyclic AMP (cAMP) accumulation.
• Cyclic AMP activates protein kinase A (PKA),
  which phosphorylates and activates StAR, thus
  rapidly stimulating the formation of
  pregnenolone, and consequently of androgens.
• Cyclic AMP also increases the transcription of
  the genes coding StAR and the enzymes P450scc,
  3ß-HSD, and P450c17 in theca cells.
• Such effects on transcription take hours rather
  than minutes.

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• Granulosa cells converts androgens
  (androstenedione and testosterone) to estrogens
  (estrone and estradiol, respectively).
• Granulosa cells express FSH receptors (FSH-R) in
  secondary and antral follicles.
• FSH activates AC, and cAMP stimulates the
  expression of the gene coding for P450 aromatase.
• FSH stimulates also the expression of several
  protein regulators, like inhibin and insulin-like
  growth factor 2 (IGF-2). (Comment in many other
  mammalian species granulosa cells produce IGF-1,
  which acts via the same receptor as IGF-2).
• IGF-2 acts synergistically with cAMP in
  increasing the expression of aromatase (an
  autocrine effect).

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• IGF-2 also diffuses to the theca layer and
  synergizes with LH (namely, with cAMP) in the
  expression of StAR and P450scc mRNAs (paracrine
  effects).
• At an advanced stage of follicular growth, FSH
  plus the high concentrations of estradiol
  produced at that time in the follicle induce the
  expression of LH receptors in granulosa cells.
• This is very important, since ovulation is
  triggered by an LH surge (LH peak), namely, a
  large, transient increase in LH in the blood. In
  the process of ovulation, both theca and
  granulosa cells are greatly affected by this LH
  surge.

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• As shown above, granulosa cells proliferate
  rapidly during follicular development. This is
  brought about by several growth factors, produced
  by the theca and the granulosa cells. IGF-2 is
  one of these growth factors.
• The next figure (lowest graph on the left) shows
  the gradual increase in plasma estradiol during
  the follicular phase.
• Progesterone concentration remains low, since in
  the follicle (theca) progesterone is an
  intermediate, and is rapidly converted to other
  steroid metabolites.
• The next figure (top graph on the left)
  demonstrates the LH surge at midcycle.

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From Yen el al, 1999, Fig. 7-5
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OVULATION

• The process of ovulation includes several
  components
• The stepwise release of the oocyte from the
  follicle.
• The advancement of meiosis in the oocyte before
  it leaves the follicle.
• The luteinization of the endocrine cells (theca
  and granulosa), which includes both structural
  and functional changes.
• The very large increase in cyclic AMP, resulting
  from the LH peak, underlies these changes.

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OOCYTE RELEASE FROM THE FOLLICLE

• The LH surge induces the formation of hyaluronic
  acid by the cumulus cells, and this polymer,
  which expands extensively by absorbing water (see
  next figure), breaks this cell layer apart.
• In the developing follicle, the granulosa cells,
  cumulus cells, and the oocyte are interconnected
  by gap junctions, so that they form a network in
  which small molecules can travel.
• It was found that oocytes of large follicles are
  retained at the stage of prophase I due to
  inhibition exerted by the surrounding cells.
• Ample evidence supports the hypothesis that the
  inhibitory molecule is cAMP, which reaches the
  oocyte via gap junctions (oocytes lack AC).

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HYALURONIC ACID (HYALURONAN) IS A
SIMPLE-STRUCTURED, VERY LONG GLUCOSAMINOGLYCAN.
SINCE IT EXPANDS EXTENSIVELY IN WATER, IT IS USED
IN THE BODY IN VARIOUS CONTEXTS (EMBRYONIC
DEVELOPMENT, WOUND HEALING) AS A SPACE FILLER.
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• This can explain the finding that soon after
  cumulus expansion, when the gap junctions within
  the oocyte-cumulus complex are disrupted, meiosis
  is resumed. The oocyte (still within the
  follicle) completes the first meiotic division
  and reaches metaphase II. The resulting polar
  body dies soon.
• Also resulting from the LH surge is a cascade of
  events leading to increased activity of various
  proteolytic enzymes. The hydrolysis of
  extracellular matrix proteins in the follicular
  wall is essential in the formation of a hole
  through which the oocyte leaves the follicle.
• The oocyte enters the oviduct on its route to the
  uterus, but, if not fertilized, it dies within
  less than 24 hours.

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FORMATION OF THE CORPUS LUTEUM

• In the human corpus luteum the two layers of
  endocrine cells can still be observed. However,
  extensive growth of capillaries into the
  granulosa layer occurs, mainly due to an
  increase in VEGF (produced primarily in the
  granulosa).
• Theca cell luteinization, i.e., their
  differentiation into theca-lutein cells, includes
  acquisition of epithelial morphology, but no
  change in steroidogenesis.
• In contrast, granulosa cell luteinization
  includes massive increase in cell size, and a
  sharp increase in the expression of genes
  required for progesterone synthesis (StAR,
  P450scc, 3ß-HSD).
• On the other hand, the granulosa loses the FSH-R,
  so that LH is the regulator of both luteal cell
  types.

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STEROIDOGENESIS IN THE VARIOUS OVARIAN CELL
TYPES THECA, GRANULOSA, THECA-LUTEIN AND
GRANULOSA-LUTEIN CELLS
GRANULOSA LUTEIN CELLS
THECA LUTEIN CELLS
RANGES IN BLACK FOLLICLE. RANGES IN
VIOLET CORPUS LUTEUM
GRANULOSA LUTEIN CELLS
GRANULOSA
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LUTEAL REGRESSION (LUTEOLYSIS)

• In the absence of fertilization, the corpus
  luteum is functional for about two weeks (the
  luteal phase).
• It is stimulated by LH, which, via cAMP, supports
  steroidogenesis both by activating StAR and by
  increasing the expression of the relevant genes).
• However, during the luteal phase the LH support
  weakens (see below). Moreover, in the absence of
  pregnancy, factors that act to suppress luteal
  function are synthesized. One of the most
  important luteolytic factors is prostaglandin F2a
  (PGF). Luteolysis is a multistep, prolonged
  process, starting with a fall in steroid hormone
  production and ending with apoptosis of luteal
  cells.

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HOW DOES PREGNANCY SAVE THE CORPUS LUTEUM?

• The embryo reaches the uterus 4-5 days after
  ovulation, and implantation begins about 2 days
  later. On the following day, a hormone of
  placental origin, human chorionic gonadotropin
  (hCG), becomes detectable in plasma. hCG is
  structurally homologous to LH and activates the
  LH receptor.
• Thus, the stimulatory influence of hCG on the CL
  overcomes the inhibitory effects.
• How can the tiny amount of placental tissue
  produce such an effective amount of hCG? hCG is
  much richer with sugar chains than the pituitary
  glycoprotein hormones. This extends hCG
  half-life to 36 h, compared to 30 min for LH.

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From Yen el al, 1999, Fig. 7-5
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THE HYPOTHALAMUS-PITUITARY-OVARY AXIS

• The above figure, top left, shows the
  concentrations of LH and FSH during the ovulatory
  (menstrual) cycle in women.
• The general pattern is relatively low
  concentrations throughout most of the cycle, with
  sharp peaks in the middle. The LH peak is very
  large, whereas the FSH peak is small. There are
  also modest changes outside the peaks, which are
  physiologically important all the same, as
  explained below.
• Regulation of such a cyclic pattern is complex,
  and a component of positive feedback is essential
  for creating such pattern.

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Emotional Stress
Higher Neural Centers
Physical stress
Monoamines (NE,E,DA)
Via opioids
-
-
,-
-
Inadequate food intake
Opioids
Hypothalamus
-
-
Via opioids (weak)
-
Pulsatile GnRH
-
more on LH

Pituitary
only on FSH
-

(weak)
LH
FSH
Progesterone
Inhibin
Ovary
Estradiol
Corpus Luteum
Stroma
Theca
Granulosa
Testosterone
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Modified from Greenspan and Gardner,2001.
Fig.13-10
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HYPOTHALAMUS-PITUITARY-OVARY AXIS

• HYPOTHALAMUS
• Stimulatory hormone Gonadotropin-releasing
  hormone (GnRH). Another name Luteinizing
  hormone (LH)-releasing hormone.
• Structure A peptide of 10 amino acids.
• Signaling PI-PLC? activation.
• ANTERIOR PITUITARY (ADENOHYPOPHYSIS)
• Hormones Follicle-stimulating hormone (FSH) and
  luteinizing hormone (LH) group name
  gonadotropins (gonadotrophins).
• Structure Glycoproteins (heterodimers ? and ?
  chains ? is hormone-specific, and ? is identical
  in TSH, FSH and LH).
• Signaling AC activation for both.
• Cell type producing both FSH and LH gonadotroph
  (or gonadotrope).

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• OVARY
• Hormones Estradiol (follicle and corpus luteum
  CL) progesterone (CL) testosterone (follicle
  and CL, low concentration).
• Structure Steroids.
• Signaling Intracellular (nuclear) receptors.
• Polypeptide regulators produced in the ovary
  Inhibin, which has both endocrine and local
  effects. Insulin-like growth factor (IGF-1 in
  most species, IGF-2 in the human), which acts
  locally (autocrine and paracrine effects).

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• FEEDBACK NEGATIVE AND POSITIVE
• Estrogens act on the hypothalamus, inhibiting
  GnRH production. This negative feedback
  contributes to inhibition of FSH and LH
  production.
• Estrogens act on the pituitary (gonadotroph),
  exerting a positive feedback. This feedback
  results in increased sensitivity to GnRH. In the
  human this positive feedback by estrogens is much
  stronger with regard to LH than for FSH.
• In the human the feedback effects of progesterone
  are qualitatively similar (negative in the
  hypothalamus, positive in the pituitary), but are
  much weaker than the effects of estrogens.
• Inhibin acts on the gonadotroph and inhibits
  selectively the production of FSH.
• ADDITIONAL EFFECTS
• Severe malnutrition, extensive physical exercise,
  and various stresses, inhibit the axis at the
  hypothalamic level, namely, suppress GnRH
  production.

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OBSERVATIONS AND IMPLICATIONS RELEVANT TO THE
HYPOTHALAMUS-PITUITARY-OVARY AXIS

• The negative feedback of estradiol (or other
  biologically active estrogens) on the
  hypothalamus is most obvious in extreme
  situations, in which estrogen concentration is
  very high or very low.
• Estrogen level is very low after menopause. GnRH
  production is thus very high, and so are the
  concentrations of LH and FSH in plasma.
• In advanced pregnancy, the concentration of
  estrogens (produced in the placenta) is very
  high, and thus the concentrations of pituitary
  gonadotropins are very low. The shortage of FSH
  does not allow the development of antral
  (tertiary) follicles.

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• Most contraceptive pills comprise of synthetic
  estrogen and progestin. The estrogen suppresses
  GnRH, and thus FSH. The progestin is not
  necessary for gonadotropin suppression, but is
  included for considerations pertinent to the
  peripheral target tissues of the steroid sex
  hormones (will be explained in the second
  presentation).
• It should be stressed that in the virtual absence
  of GnRH the positive feedback has no
  contribution, since it works by increasing the
  sensitivity to the GnRH present.
• Based on the mechanisms described, how are the LH
  and FSH peaks explained?

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• As the follicles (especially the largest,
  dominant follicle) grow during the follicular
  phase, plasma concentration of estradiol
  gradually increases, and the production of GnRH
  decreases.
• Thus FSH, which (in contrast to LH) is affected
  only marginally by the positive feedback of
  estradiol, decreases as well.
• In principle, GnRH stimulates both synthesis and
  secretion of the gonadotropins, and thus the
  estradiol-induced increase in the sensitivity to
  GnRH should affect both parameters.
• However, in fact, throughout most of the
  follicular phase, LH synthesis is augmented much
  more than LH secretion by the increasing
  concentrations of estradiol. Thus LH level in
  the plasma increases very slightly.

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• Therefore LH gradually accumulates within the
  pituitary (in women FSH accumulates to a lesser
  extent). In the rat pituitary contents of LH and
  FSH was measured directly, and shown to increase
  as the follicles grow. In the human this was
  tested indirectly, by showing that a constant
  dose of exogenous GnRH elicited higher and higher
  LH peaks as the follicular phase advanced. The
  experiment is shown in the next figure.
• As the dominant follicle ripens, the
  concentration of estradiol, and thus the
  sensitivity of the gonadotrophs to GnRH, increase
  to the point in which gonadotropin secretion is
  also augmented.
• Thus, the accumulated gonadotropins (in the human
  mainly LH) are finally secreted, giving rise to
  the gonadotropin peaks.

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• What is the mechanism of dominant follicle
  selection during the follicular phase, namely, to
  the fact that only one follicle ovulates in each
  cycle, whereas the rest of the cohort of
  follicles undergoes atresia?
• For antral follicles it was shown, that the
  follicle survives only if reached by a sufficient
  concentration of FSH. Since in the follicle FSH
  reaches granulosa cells only by diffusion, rather
  than by blood vessels, the physiological amount
  of plasma FSH allows (statistically) the
  formation of only one healthy follicle.
  Administration of exogenous FSH allows the
  ovulation of multiple follicles (and thus
  multi-embryo pregnancies), and suppressing FSH
  level even to a minor extent causes menstrual
  irregularity.

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From Yen el al 1999