Manual of Ovulation Induction & Ovarian Stimulation Protocols Gautam N Allahbadia, Rubina Merchant
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1Fundamentals of Stimulation
  1. Regulation of the Menstrual Cycle and the Effect of Controlled Ovarian Hyperstimulation on Cycle Characteristics
  2. Regulation of Gonadotropin Secretion
  3. The Role of Gonadotropins in Follicular Development and their Use in Ovulation Induction Protocols for Assisted Reproduction
  4. Predictors of Ovarian Response to Controlled Ovarian Stimulation: Are they Useful?
  5. Clinical Significance of Antral Follicle Count and Anti-Müllerian Hormone in Predicting the Stimulation Outcome
  6. Human Ovulation and Transvaginal Sonography
  7. The Significance of Monitoring Folliculogenesis
  8. Identification of Patients at High Risk for Excessive Response to Ovarian Stimulation
  9. The Impact of Repeated Ovarian Stimulation on the Ovarian Reserve
  10. Luteinizing Hormone Activity in Ovarian Stimulation for IVF: Is it Indispensible?
  11. Luteal Phase Support in Controlled Ovarian Hyperstimulation Protocols: Why and How?

Regulation of the Menstrual Cycle and the Effect of Controlled Ovarian Hyperstimulation on Cycle CharacteristicsCHAPTER 1

Peter Kovacsara
 
INTRODUCTION
The ovaries are paired pelvic organs with dual functions. They act as endocrine glands and secrete hormones in a cyclic fashion and also serve as the source of oocytes for reproduction. Its functional unit is the follicle that is made up of the oocyte, granulosa and theca cells. The ovary contains a finite number of follicles 4and when their number is reduced to a few thousand, only the ovary ceases its cyclic activity. Successful reproduction, using own eggs, requires regular ovarian activity but pregnancy rates decline with age and reach very low levels over 40 despite the monthly cycles. The treatment of the infertile patient during ART is determined by the presenting clinical scenario and the results of the infertility evaluation; sometimes, it is the irregular ovarian activity that needs to be regulated or more commonly, COH is applied to recruit several follicles simultaneously in otherwise regularly cycling women. In order to provide proper treatment, one has to understand the normal physiologic process first. New data about the control of spontaneous follicle development allows us to modify, individualize stimulation protocols and therefore, to improve the treatment outcome.
This chapter will review the normal process of folliculogenesis and the accompanying endometrial changes and will also discuss the reproductive impact of malfunctions at various levels of the hypothalamic-pituitary-ovarian axis. Finally, the significance of the changes induced during COH will be reviewed.
 
CLINICAL DISCUSSION
 
Ovarian Organogenesis and the Initial Stages of Folliculogenesis
The primitive gonad develops as a thickening of the coelomic epithelium covering the mesonephros and forms the gonadal ridge at 5 weeks gestation. Primordial germ cells migrate from the yolk sac to here under the influence of growth factors such as bone morphogenic proteins (BMP).1 Upon arrival, they increase their number by mitosis and by the 7th week, they colonize the primitive gonad. Further mitotic divisions rapidly increase the number of oogonia to a peak number of 6 to 7 million by mid-gestation.1,2
As DNA synthesis, leading to up to meiosis is initiated, the oogonia turn into primary oocytes. This process starts around week 8.1 The precursors of granulosa cells surround the oocytes from week 15 of embryonic life. The primary oocyte, surrounded by one layer of flat granulosa cells, is called the primordial follicle. There are numerous locally produced growth factors that guide this transition. The number of primordial follicles and the rate by which they enter the growth phase determine the duration of a woman’s reproductive period.
The recruitment of these primordial follicles into the growth phase is regulated by the complex interaction of the autocrine and paracrine effects of growth factors secreted by the oocyte itself, the granulosa and theca cells. Some of these growth factors inhibit the follicles from entering the growth phase (e.g. tumor suppressor tuberous sclerosis complex I, PTEN, p27, FOXL2, AMH) and therefore, their effect is the maintenance of a large follicle pool.1,3,4 The initially flat granulosa cells turn into cuboid shaped cells as an early sign of follicle activation and later, they form multiple layers. The oocyte, surrounded by layers of granulosa cells, is called the secondary follicle. At the same time, the oocyte undergoes changes as well and increases its size and the zona pellucida is formed around it. As the follicle continues to grow fluid filled spaces (antrum) appear and coalesce in it (preantral, antral stage). It is the preantral follicle that first expresses follicle-stimulating hormone (FSH) receptors. 5These early developments are gonadotropin-independent but local growth factors (BMP, GDF-9, activin, etc.) influence it.1,3,4 Parallel with the follicular growth, the surrounding stroma differentiates as well and theca cells form. Some of the growth factors that regulate follicle growth are secreted by these theca cells. Theca cells also act as the source of androgen for estradiol synthesis so they are an equally important component of the whole complex. The theca layer brings blood supply to the follicle as the granulosa cells and the oocyte have no direct blood supply.
Pituitary gonadotropins, FSH and luteinizing hormone (LH) play an increasing role from the preantral and antral stages of development and promote the final stages of follicle maturation, the emergence of the dominant follicle and the control ovulation.
As some of the primordial follicles enter the growth phase, others undergo atresia. The two processes together will determine the number of follicles in the ovary. By birth, the number of follicles is reduced to 1 to 2 million and by puberty, to 400,000 to 500,000, primarily as a result of atresia. This process continues throughout life until menopause when the number of follicles is down to 1000 to 2000 and ovarian follicular activity ceases.2
 
Abnormal Ovarian Development, Damage to the Follicle Pool and its Reproductive Consequences
The migration of the germ cells into the primitive gonad is required for normal gonadal development. If the germ cells fail to reach the gonad or upon their arrival, other mechanisms disrupt gonadal development, the primitive gonad will regress leading to gonadal dysgenesis. The most common etiology of gonadal dysgenesis is the absence of one of the X chromosomes (Turner syndrome). Gonadal dysgenesis affects the reproductive potential as well as the normal development of the primary and secondary sexual characteristics.5
It is now understood that there are several inhibitory factors that prevent follicles from entering the growth phase. If they malfunction, the follicle pool is reduced rapidly and the ovary fails prematurely. Certain genetic mutations (e.g. FOXL2) have been linked to premature ovarian failure (POF).6
In the postnatal life, the ovary may be exposed to gonadotoxic effects (chemotherapy, radiation therapy) that destroy follicles and therefore, shorten the reproductive life span of the ovaries.7 Once the follicles are lost, they can no longer be replaced and reproduction becomes possible only through the involvement of donor oocytes.
 
Folliculogenesis
Recruitment of the follicles into the growth phase occurs in waves from puberty until menopause. It takes about 85 days from follicle activation until ovulation. It is always a group of follicles that enter this phase and through various regulatory mechanisms, typically only one will get to ovulation. The majority of this 85-day maturation is gonadotropin-independent and is controlled by the autocrine and paracrine interaction of growth factors [epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), insulin-like growth 6factor (IGF), fibroblast growth factor (FGF), growth differentiation factor-9 (GDF-9), platelet derived growth factor (PDGF), BMPs, transforming growth factor beta (TGF-β), etc.].1-4
 
Neuroendocrine Control of Folliculogenesis
The primary regulators of follicle development (last 2–3 weeks) are FSH and LH from the antral stage. These peptide hormones are released from the anterior pituitary in response to hypothalamic GnRH pulses. The rate of synthesis and release are affected by the ovarian steroid and peptide hormone feedback. GnRH is a decapeptide that is produced and released by the hypothalamus. Its synthesis is under to control of stimulatory and inhibitory signals (steroid hormones, opioids, neuropeptide Y, GABA, serotonin, prolactin, noradrenalin, etc.). Release into the portal vessels is pulsatile (every 60–90 minutes in the follicular phase and 120–240 minutes in the luteal phase; besides the frequency, the amplitude changes as well).8,9 Upon reaching the anterior lobe of the pituitary it binds to its surface receptors and initiates the release of stored FSH and LH from there. The receptor-ligand complex is then internalized and upon dissociation the GnRH receptors can be recycled to the cell surface. FSH and LH will reach the ovary through the systemic circulation.
The primary target of FSH is the granulosa cell where it stimulates granulosa cell proliferation, increases aromatase activity, increases granulosa cell FSH receptor expression and theca cell LH receptor expression. The primary target for LH is the theca cell where it augments androgen production. Androgens diffuse over to the granulosa cells and are converted to estradiol by aromatase. Estradiol is released into the blood stream and upon reaching the hypothalamus and pituitary it exerts its negative feedback and will lower FSH release.10 This negative feedback will decrease the FSH and LH output from the mid-follicular phase as the dominant follicle increases its estradiol release. As the estradiol synthesis reaches a sufficiently high level and if its production is maintained, it will induce the positive feedback that ultimately leads to ovulation and the release of the oocyte at midcycle (positive feedback).
 
Malfunction of the Central Nervous System Regulatory Axis and its Reproductive Significance
If GnRH neurons do not reach the hypothalamus (Kallmann’s syndrome) during organogenesis or if they are destroyed (tumor, surgery, hemorrhage), the pituitary FSH and LH release will be minimal and insufficient to initiate and maintain follicle growth.11 Clinically, a hypogonadal state is seen during which, steroid hormone production is minimal and follicles do not enter the final stages of folliculogenesis. A similar picture can be seen if the hypothalamus is intact but the stalk is destroyed and the portal circulation carrying GnRH to the anterior lobe of the pituitary is disrupted. One has two options to restore regular ovulatory cycles in these cases. A GnRH pump can be used (when the pituitary circulation is intact) to stimulate the pituitary to release FSH and LH. The pump is set to release adequate boluses of GnRH at certain frequency (75 ng/kg every 60 minutes) that mimic the physiologic follicular and luteal phase.12 This approach typically results in the growth of a single follicle and mono-ovulation. The alternative method is to use gonadotropins FSH and LH to induce folliculogenesis. FSH alone is sufficient to promote follicle growth 7but in the absence of LH, estradiol synthesis will be minimal lacking the androgen precursors for it.13 Therefore, in the case of hypogonadotropic hypogonadism both FSH and LH are required for successful stimulation.14
During COH, a hypogonadotropic state is induced on purpose with the aim to prevent premature luteinization, ovulation. Two methods are used to prevent the spontaneous LH release from the pituitary. Synthetic analogs of the GnRH are chemically modified variants of the naturally occurring hormone by substituting the amino acids at position 6 and 10. These modifications extend the molecules half-life and when bound to the receptor, it results in its desensitization and down-regulation after an initial flare effect.15 GnRH antagonists are also obtained following modifications of the GnRH molecule. When bound to the receptor, it does not lead to receptor down-regulation but its effect is competitive inhibition of the natural GnRH. This inhibition can be overcome with a bolus of GnRH. GnRH antagonist use is not associated with a flare phenomenon; its effect is profound and immediate.15
 
Pituitary and Ovarian Hormonal Control of Folliculogenesis
The final stages of follicle development are initiated as the activity of the corpus luteum diminishes (reduction in estradiol, progesterone and inhibin A levels) and the hypothalamus and pituitary are released from their state of suppression. As the negative feedback diminishes the pituitary starts to release increasing amounts of gonadotropins and it is this inter-cycle rise in FSH that is responsible for follicles entering the final stages of folliculogenesis. Besides the rise and fall of steroid hormone levels, numerous other growth factors are actively involved in the regulation of these steps.
Activins and inhibins are secretory products of growing follicles. Activin and inhibin are made up of two chains (alpha and beta chains). There are two types of inhibin. Inhibin A is made up of an alpha and a beta A chain, while inhibin B is made up of an alpha and beta B chain. Inhibin A is the main product of the corpus luteum and inhibin B is the main product of smaller follicles. Activins are dimers of the beta chains (activin A: beta A-beta A; activin B: beta B-beta B; activin AB: beta A-beta B) and stimulate FSH production, enhance granulosa cell proliferation, increase granulosa cell FSH receptor expression, improve responsiveness to FSH and increase aromatase enzyme activity.1,3,4 Follistatin binds activin and therefore, antagonizes its effects. As the follicles mature, they produce increasing amount of inhibin B. Inhibin exerts a negative effect on pituitary FSH release. Inhibin however, also has important paracrine effects within the ovary as it augments theca cell androgen synthesis that is primarily under the control of LH.1,3,4
Follicle-stimulating hormone also stimulates IGF production and IGF is another positive regulator of theca cell androgen synthesis.
There are however, numerous other growth factors (BMPs, GDF-9) that affect cell proliferation, enzyme activity and therefore, steroid synthesis, cell sensitivity to FSH and LH and through these effects, fine tune the growth of the follicle.
In response to the rising estradiol and inhibin levels, the pituitary decreases its FSH output and therefore, only those follicles survive that have the highest FSH receptor expression. These follicles also express LH receptors on their theca cells and from the mid-follicular phase, on the granulosa cells as well.
8
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Fig. 1.1: The figure displays the complex interactions of the various endocrine, paracrine and autocrine effects regulating the hypothalamic-pituitary-ovarian axis, symbol +: stimulatory effect, symbol –: inhibitory effect
In addition to maintaining their ability to respond to the falling FSH levels, these follicles are now able to grow, and synthesize steroid hormones in response to LH as well.4 As the follicle grows, its secretory capacity changes, favoring inhibin production instead of activin. Inhibin is a known negative regulator of FSH release. Follistatin is also produced by the larger follicles and by counteracting activin’s effect, it also reduces FSH release. In addition to this peptide control of FSH release, estradiol that is produced in increasing amounts by the growing follicles has a strong negative feed-back effect at the level of the hypothalamus and pituitary as well. The net effect of these endocrine changes is a gradual reduction in FSH levels during the follicular phase. Those follicles that have not expressed enough FSH receptors cannot survive this shortage of FSH and will undergo atresia. The follicle that has the most FSH and later, LH receptors will maintain its growth and activity despite the reduction in gonadotropin stimulus and will emerge as the dominant follicle.1,2,4,16,17 The complex interaction of peptide and steroid hormones and growth factors is shown in Figure 1.1. The two cells—two gonadotropins theory behind the function of the follicular unit and its connection to the hypothalamus, pituitary are also evident based on Figure 1.1.18,19
 
Clinical Application of the Regulators of Folliculogenesis and Potential Alternative Management Options during COH
 
Ovarian Function Markers
Some of the hormones and growth factors that participate in the control of follicle development are also used to assess the functional capacity of the ovaries and 9based on them, stimulation protocols can be individualized. The assessment of ovarian function and the ability to predict response to stimulation is important to avoid both under- and over-response to gonadotropins. Hormonal and ultrasound parameters, as well as results of dynamic studies, are used for this purpose. Most commonly, early follicular phase FSH, estradiol levels, inhibin B, anti-Müllerian hormone (AMH), ovarian volume and antral follicle counts (AFC) are measured in everyday clinical practice. Elevated levels of FSH (> 10–12 IU/L) or estradiol (> 75 pg/mL) at the beginning of the cycle indicate diminished ovarian reserve. Inhibin B is produced by small follicles and when its value is under 45 pg/mL it indicates a reduced, small follicle pool. Low AMH levels correlate with low response to stimulation while elevated levels predict hyper-response.20,21 As the number of follicles decline, the ovarian volume will be reduced as well and fewer small (2–10 mm) antral follicles can be seen at the onset a cycle. These parameters are useful when the patient’s stimulation protocol is decided upon.22
 
Potential Management Options of Poor Responders
The treatment of women who are expected to have low response to stimulation, either based on age, on any of the above markers or on previous poor response to stimulation is challenging. In the majority of the cases, the follicle pool is reduced and therefore, the number of follicles that enter the final stages of folliculogenesis is low. Several groups evaluated adjuvant therapies with the aim of increasing this initial follicle pool. Observations of the normal physiologic process and experimental data based on animal models have suggested a positive effect of androgens on follicle growth. Androgen treatment in animals was shown to increase follicle count, theca layer thickness and FSH responsiveness.23,24 There are some promising reports with androgen pretreatment prior to COH with dehydroepiandrosterone sulfate (DHEAS) or testosterone though further randomized studies should confirm the existing results.4,25 Aromatase inhibitors that prevent androgen to estrogen conversion (and therefore, increase local, intraovarian androgen levels) have also been successfully used to improve the stimulation outcome.26 A similar effect was expected from the prestimulation administration of LH. Luteinizing hormone was supposed to increase theca cell androgen synthesis and the androgen in return, was expected to improve response to stimulation with FSH. Currently available data however, do not show improved clinical outcome.27,28
 
Alternative Management Options of the Follicular Phase
It was discussed above that larger follicles express LH (as well as FSH) receptors on the granulosa cells from the mid-follicular phase and therefore, at this stage, LH is able to maintain the growth of these follicles too. The use of LH or human chorionic gonadotropin (hCG) (that also binds to the LH-receptor) from the mid-follicular phase onwards was tested in several clinical trials. A positive effect on larger follicles and a negative impact on smaller follicles was seen. This approach could be applied during the stimulation of hyper-responder patients when the regression of smaller follicles is desired at the same time when the larger follicles maintain their growth towards ovulation.29
 
The Role of Luteinizing Hormone during Controlled Ovarian Hyperstimulation
10There is an ongoing debate about the benefits of LH during COH for otherwise normogonadotropic patients. A negative impact of deeply suppressed as well as elevated LH levels has been described (threshold effect, ceiling effect).30 Currently, there is no consensus on which patient, if any, undergoing COH, would benefit from LH supplementation.31 There are a lot of confounding factors in the studies exploring this problem and they make it difficult to compare them for firm conclusions (different GnRH agonist or antagonist use, r-FSH vs HP-hMG vs hMG use, which lower LH cut-off is used, age dependent differences, etc.).
The addition of GnRH agonist or antagonist to the COH regimen alters the endogenous hormone profile and typically leads to suppressed LH levels, sometimes to very low levels. It is sufficiently proven that LH is needed for stimulation, since women with hypogonadotropic hypogonadism only achieve successful stimulation when LH is added to FSH.13,14 FSH alone is capable of inducing follicle growth but estradiol production will remain minimal. Estradiol synthesis can, however, be corrected by the daily administration of 75 IU LH during stimulation.32,33 Those women, however, who have intact hypothalamic-pituitary function, do produce some LH even when GnRH analogs are used during COH and in the majority of the cases, these low LH levels are sufficient to maintain proper multifollicular development. Several groups reported fewer follicles and oocytes when LH levels were very low (<0.5–1.2 IU/L) during the follicular phase but pregnancy rates did not seem to be affected.34-37 It appears though, that older women (> 35 years), poor responders, those with a slow initial response to stimulation, and those with deeply suppressed LH levels (< 0.5 IU/L) may benefit from additional LH.38-41
 
Ovulation
Throughout the process of folliculogenesis, the follicle increases its size, which is mainly due to fluid accumulation. The growing follicle increases its synthesizing capacity and releases increased amount of estradiol. The dominant follicle typically reaches a size of 22 to 24 mm prior to ovulation. A mature follicle is able to maintain high steroid output (estradiol level has to exceed 150 pg/mL over 48–50 hours), which is needed for ovulation. Premature luteinization is also controlled by secretory products of the oocytes (GDF-9, BMPs) that prevent early progesterone synthesis by the follicle.1 While in the early follicular phase, estradiol exerts a negative feedback and lowers FSH and LH release, the continuous elevation of estradiol will elicit a positive feedback and will induce the midcycle surge of FSH and LH. The follicle ruptures and releases the oocyte 24-36 hours after the onset of the surge and 12-24 hours after the LH peak. It is at the time of ovulation that the oocyte completes meiosis I and enters the prophase of meiosis II. The egg released at the time of ovulation is picked up by the Fallopian tube and is transported towards the uterus.
Prior to the actual ovulation numerous biochemical changes can be observed in the follicle. The levels of vasoactive substances [histamine, angiotensin, platelet activating factor (PAF), vascular endothelial growth factor (VEGF) are increased. They are responsible for the formation of the capillary network surrounding the 11follicle and for its increased permeability. At the same time, the level of cytokines (prostaglandins, leukotrienes, plasminogen activator) and certain enzymes (e.g. collagenase) are increased within the follicle as well. Collagenase breaks down the follicle wall and extracellular matrix. These vascular changes and the accumulation of the follicular fluid by this stage lead to significant follicle expansion prior to ovulation (often noted as midcycle pain). These biochemical changes will ultimately result in the rupture of the dominant follicle.2,42
The enzymatic activity of the follicle changes as well and it shifts is synthesizing capacity towards progesterone synthesis. The main product of the corpus luteum is progesterone, which is responsible for the induction of endometrial changes that allow implantation and normal progress of pregnancy. Progesterone is the product of the luteinized granulosa and theca cells. It is the midcycle LH surge that induces the luteinization of granulosa and theca cells. As part of luteinization, these cells increase their LH receptor expression, increase the production of activating factors required for progesterone synthesis and the enzymes needed for normal luteal function are induced. The main product of the corpus luteum, progesterone, is synthesized from cholesterol (mainly by increasing the uptake of LDL-cholesterol by endocytosis). Proteins that are responsible for the uptake and transfer of cholesterol to the inner membrane of mitochondria are upregulated (e.g. StAR).43,44 Cholesterol is then converted into progesterone by multiple enzymatic steps. The activity of enzymes required for this (3β-hydroxysteroid dehydrogenase) is enhanced while the activity of enzymes (17–20 lyase) that would take the cholesterol on a different path is blocked. In addition to progesterone, the luteinized granulosa cells produce estradiol and inhibin too.44,45 In case of successful implantation, the syncytiotrophoblast will secrete hCG and it will maintain the hormonal support of the enzyme pathway required for extended luteal function in early gestation. Without hCG, the hormone synthesizing capacity of the corpus luteum regresses. This, in turn, increases the GnRH pulsatility, leading to an increase in pituitary FSH release and the recruitment of a new group of follicles into the final stages of their development. As steroid hormone production decreases, the synthesis of prostaglandin F2α, tumor necrosis factor α (TNF-α), reactive oxygen species, matrix metalloproteinases (MMPs) increases and luteolysis is initiated.46 This leads to the breakdown of the endometrium and ultimately to menstruation, which signals the end of the cycle.
 
Endometrial Cycle
The intact endometrium is responsive to the endocrine changes that accompany folliculogenesis in an attempt to prepare itself for successful implantation of the embryo. The endometrial cycle can be divided into proliferative (Figs 1.2A and B), secretory (Figs 1.3A and B), and menstrual/regeneration phases. The endometrium is made up of a basal and a functional layer. Most changes take place in the functional layer during the menstrual cycle. It is this functional layer that is shed at the time of menstruation and is rebuilt form the basal layer.47
As the follicles increase estradiol production at the beginning of the cycle, the endometrium undergoes regeneration. The basal layer serves as the source for the cells that differentiate into the various cell types that make up the endometrium.
12
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Figs 1.2A and B: Proliferative phase endometrium. Hematoxylin–Eosin staining. (A) HE stain 10x magnification; (B) HE stain 20x magnification
13
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Figs 1.3A and B: Secretory phase endometrium. Hematoxylin-Eosin staining. (A) HE stain 40x magnification; (B) HE stain 10x magnification
14Growth is initiated in the remnants of glands with an initial aim to cover the entire cavity and then, with further proliferation to increase the thickness of the endometrium. This intensive proliferation affects the glands, the stroma and the blood vessels as well. The proliferative phase is under the control of estrogen that acts mainly via its estrogen receptor alpha (ERα) receptor. Initially, the glands grow straight but once adequate endometrial thickness is achieved they become increasingly tortuous by the end of the follicular phase. During this process, the superficial epithelial cells differentiate into ciliated cells. The role of these cilia is to spread the endometrial secretions evenly. By the late follicular phase, progesterone receptors are induced as well under the control of estradiol. Peak endometrial thickness is achieved before ovulation.47
Following ovulation, the hormonal milieu changes as the follicle turns into the corpus luteum and instead of estradiol, progesterone becomes the dominant steroid product of the ovary. Progesterone induces important secretory changes that prepare the endometrium for implantation of the blastocyst. The endometrial changes in this phase are very characteristic and by observing them, the day of the cycle can be established fairly accurately (+/–2 days).48 Soon after ovulation, glycogen storing vesicles appear beneath the nucleus of the epithelial cells and start to migrate towards the lumen over the next few days before they empty their content into the lumen (6–7 days after ovulation). At the same time, further changes take place that prepare the endometrium for implantation [pinopode formation, cell adhesion molecule (e.g. integrin) expression]. The early luteal phase is under the control of both estradiol and progesterone but as progesterone leads to a down-regulation of estradiol receptors from the mid-luteal phase, progesterone takes over as the dominant controlling force. Progesterone ultimately down-regulates its own receptors as well in the glands but progesterone receptor expression is maintained in the stroma of the functional layer.47 In addition to well-characterized changes in the epithelial layer, important stromal processes can be observed as well. The capillary network increases its branches and the larger vessels become tortuous to form the typical spiral arteries, mainly under the control of VEGF and other angiogenic factors. The increase in vascular permeability leads to stromal edema (decidualization). There are important alterations in the endometrial gene expression profile that characterize the luteal phase. Genes that participate in the process of decidualization are up-regulated and their products can be increasingly detected (e.g. IGFBP-1). In the late luteal phase, the number of different types of white blood cells (uterine natural killer cells, macrophages, neutrophils, mast cells, etc.) is increased in the stroma and by the end of the luteal phase, red blood cells are found there too.47
If there is no implantation, the corpus luteum undergoes regression and its hormone production diminishes. The reduction in the level of progesterone is accompanied by several endometrial changes. The low progesterone level mainly affects those cells that still express progesterone receptors (stromal cells). Progesterone keeps the level of enzymes (MMPs) that would break down collagen, laminin, fibronectin at low levels but as its level decreases, the synthesis of MMPs is no longer withheld. The reduction in progesterone is also a signal for increased cytokine (prostaglandins E2, F2α) and chemokine synthesis. Chemokines will attract white blood cells (mainly neutrophils) into the endometrium where they release lytic enzymes. Prostaglandins induce vasoconstriction and myometrial 15contractions. Vasoconstriction results in hypoxia and necrosis in the endometrium. Capillary vessel injuries lead to microthrombus formation that augments the effect of ischemia and results in further tissue break down. As the endometrium breaks down, the necrotic tissue (menstrual debris) is expelled by the myometrial contractions induced by prostaglandins.47 The postovulatory endometrial events are displayed in Flow chart 1.1. At the end of this phase as a new group of follicles start their development, estradiol level rises and proliferation of the endometrial islands is started as a new cycle begins (regeneration).
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Flow chart 1.1: Postovulatory hormonal events within the endometrium leading up to menstruation
 
Endometrial Changes during Controlled Ovarian Hyperstimulation
16During stimulation, supraphysiologic steroid hormone levels are achieved and this has a direct effect on endometrial maturation. COH was shown to advance endometrial development and the synchrony of the glandular and stromal compartment is disrupted. Estradiol, produced in the follicular phase, upregulates both the estrogen and progesterone receptors. Compared to the natural cycle, progesterone levels rise faster following stimulation and this leads to premature down-regulation of the steroid receptors (mainly estrogen receptors and less effect on progesterone receptors).49 This leads to accelerated stromal development compared to the glands and results in stromal-glandular asynchronism.49 These findings are even more pronounced if there is a premature LH and progesterone rise at the end of the stimulation. In addition to the alterations in the endometrial steroid hormone regulation, the secretion of proteins that make the endometrium receptive prior to implantation is affected by stimulation as well. Pinopode expression seems to be advanced, cell adhesion molecules are expressed prematurely (integrins, glycodelin A) and numerous genes are differently up- or down-regulated compared to the natural cycle.5053 The induced endometrial changes are not uniform but differ based on the stimulation protocol used. The GnRH antagonist-based protocol was reported to induce changes that are more comparable to those of the natural cycle when compared to the gene expression profile induced by the GnRH agonist long protocol.51 Stimulation advances the endometrial development by 2 to 3 days.54 Adequate luteal phase support may, however, decrease the negative effects of COH on endometrial advancement.5557
 
CONCLUSION
The menstrual cycle is regulated by numerous hormones. Some of them are produced locally, while others mediate their effects by endocrine mechanisms. Technology nowadays, allows us to interfere with the normal secretion of these hormones and we can replace them individually or in combination. The use of gonadotropins and GnRH analogs, however, influences the build-up of the endometrium as well. In order to avoid the negative effect of the exogenously administered hormones, we need to understand the basic physiology of the menstrual cycle. Lessons learned from spontaneous, mono-ovulatory cycles cannot necessarily be adopted during ovarian stimulation when much higher steroid and sometimes, significantly suppressed gonadotropin levels are achieved. In addition to the effect on multi-follicular maturation, we need a much better understanding of the impact of stimulation on the endometrium. The technology to identify embryos with high implantation potential is rapidly improving so now, we have to find ways to increase endometrial receptivity. The use of gentler, individually tailored stimulation protocols may be the first step in this direction.
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