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FOLLICULOGENSIS

1. ANATOMY
Ovary
The ovaries are a pair of pale white glands with an irregular scarred surface during sexual life due to the presence of follicles, corpora lutea and the shrinkage of the corpora albicantes. The size of each ovary varies in different individuals but is about 4x3x2cm. It weights 6-8 g before the menopause, much less after that due to atrophy. The long axis is vertical so that there is an upper pole, to which is attached the infundibulopelvic fold of the peritoneum or suspensory ligament while the lower pole, to which is attached the ovarian ligament connecting the ovary to the uterine cornu; an anterior border, to which is attached the mesovarium, a double layer of peritoneum from the posterior aspect of the broad ligament; a free posterior border; a lateral surface in contact withy the ovarian fossa lined by peritoneum on the lateral pelvic wall; and a medial surface facing inward towards the rectovaginal pouch of the peritoneum (Basic Science in Obstetrics and Gynecology, 2005).

Relations:
In the nulliparous woman each ovary lies in its fossa just below the bifurcation of the common iliac artery a short distance in front of the ureter as it enters the pelvis (Basic Science in Obstetrics and Gynecology, 2005). Anteriorly is the broad ligament. Posteriorly lie the ureter and the

internal iliac artery and vein. Superiorly the upper pole is in relation to the ampulla of the uterine tube, which curls round the top of the ovary so that the abdominal ostium and fimbriae come to lie on its medial surface. Medially is the uterovaginal pouch containing coils of the ileum; on the right side, sometimes, is the appendix. Laterally is the peritoneum of the ovarian fossa, separating the ovary from the external iliac vein above, the superior vesical, obliterated umbilical and obturator vessels and obturator nerve running forwards on the obturator internus muscle laterally, and the ureter and internal iliac artery behind. The ovary is pulled upwards by the enlarging uterus in pregnancy and may not quite regain its normal position afterwards (Basic Science in Obstetrics and Gynecology, 2005).

Uterus:
The uterus is a pear-shaped hollow organ 8 cm long, 5 cm wide at the fundus and 3cm from front to back. Its walls are 1-2cm thick. It lies between the bladder in front and the recto-uterine pouch (of Douglas) and the rectum behind. The lumen is connected to the peritoneal cavity by the uterine tubes above and to the exterior by the cervical canal and vaginal below. It is divided into a triangular body (or corpus) above and a fusiform cervix below, joining at the isthmus. The part of the uterine body between the uterine tubes is known as the fundus (Basic Science in Obstetrics and Gynecology, 2005). The cavity of the body has a smooth lining and is triangular in shape, but because the anterior and posterior walls are in apooistion the cavity on sagittal section is seen only as a cleft. The cavity of the cervix is fusiform in shape. It joins the cavity of the body at the internal os and the vagina at the

external os (Basic Science in Obstetrics and Gynecology, 2005).

Blood supply of the ovaries and uterus


Ovarian arteries: the ovarian arteries arise anterolaterally just below the renal, running retroperitoneally to leave the abdomen by crossing the common or external iliac artery in the infundibulopelvic fold. They cross the corresponding ureter and may supply twigs to it but have no other abdominal branches. The right artery crosses the inferior vena cava and is crossed by the middle colic vessels, the caecal, terminal ileal and ileocolic veins. The left is crossed by the left colic and sigmoid branches of the inferior mesenteric vessels and the descending colon. Lymphatics and veins accompany the arteries, the left vein ending in the left renal vein and the right in the inferior vena cava (Basic Science in Obstetrics and Gynecology, 2005). The uterine artery runs medially on the levator ani and above the transverse cervical condensation above and in front of the ureter and above the lateral vaginal fornix. Having supplied the ureteric and vaginal branches it runs up the side of the uterus in the broad ligament supplying the uterus and anastomoses with the ovarian artery (Basic Science in Obstetrics and Gynecology, 2005).

Figure (xx): The divisions of the anterior branch of the internal iliac artery and the ovarian artery in the pelvis

2. THE FOLLICULOGENSIS
A. Morphology and Physiology of folliculogensis The follicle is an essential functional unit of the ovary. Folliculogenesis is the development of the follicle from the primordial stage through a series of morphologically defined stages: primary, preantral, antral and Graafian or preovulatory follicle stage culminating in the release of the egg during ovulation, and the remaining cells of the follicle transform into a transient endocrine organ, the corpus luteum, that produces progesterone necessary to support early pregnancy. Follicle growth from the primordial follicle stage to the preovulatory stage in humans is a lengthy process and is estimated to take almost 1 year. During this time, oocytes that begin at a size

<20 m in diameter expand to more than 120 m (Kumar and Matzuk, 2000). The process of follicular development and survival depends on autocrine and paracrine signaling involving growth factors from granulosa cells, theca cells, stromal- interstitial cells, and the oocytes. These factors include several molecules such as bone morphogenetic protein (BMP)-4, survivin, growth determinant factor-9 (GDF-9), integrin and gonadotrophins (Nilsson and Skinner, 2003). Folliculogenesis begins with the recruitment of a primordial follicle into the pool of growing follicles and ends with either ovulation or death by atresia. Follicles are present in the ovary at different stages of development, and large numbers of follicles of different sizes can be observed at any given point of the menstrual cycle (Gougeon., 2004).

Folliculogenesis can be divided into two phases (Fig.5, 6): Gonadotropin-independent and -dependent follicle growth: The first phase, termed the preantral or gonadotropin-independent phase, which is controlled by locally produced growth factors through autocrine and paracrine mechanisms, is characterized by the growth and differentiation of the oocyte (Zeleznik., 2004). 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. Such conditions include prepubertal childhood, pregnancy, and the use of steroid contraceptives. Follicle growth up to the early antral stage has been described in women with absent gonadotropin secretion, either due to hypophysectomy, or to hypothalamic/pituitary failure (Fauser and van Heusden, 1999). The preantral or (Class 1) phase is divided into three major stages: the primordial, primary, and secondary follicle stages. Altogether, the development of a primordial to a full-grown secondary follicle requires 290 days or about 10 regular menstrual cycles (Erickson, 2003). Locally produced growth factors (GFs) are critically involved in controlling preantral follicle development during the gonadotropin-independent period through autocrine and paracrine mechanisms (Erickson and Shimasaki, 2001). The primordial follicle Histologically, a primordial follicle contains a small primary oocyte (~ 25m in diameter) arrested in the dictate stage of meiosis, a single layer of flattened or squamous granulosa cells and a basal lamina. The basal lamina, the granulosa and oocyte exist within a microenvironment in which direct contact with other cells does not occur. Primordial follicles do not have an independent blood supply and thus have limited access to the endocrine system (Fortune et al., 2000). The initiation of follicle growth is defined as the transition of primordial follicles from the quiescent to the growth phase. It has been shown that follicle growth initiation consists of two distinct, consecutive

phases. The first phase characterized by the transformation of granulose cells from flattened to cuboidal in shape and by their proliferation. During the second phase, an increase in the number of granulose cells is accompanied by an increase in the size of the oocyte (Jamnongjit and Hammes, 2005). All primordial follicles (oocytes) are formed in the human fetus between the sixth and the ninth month of gestation. The number of eggs or primordial follicles in a woman's ovaries constitutes her ovary reserve (OR) (Zeleznik, 2004). Recruitment The entry of an arrested primordial follicle into the pool of growing follicles is termed recruitment or the primordial-to-primary follicle transition. Start soon after their formation in the fetus and continue until the pool of primordial follicles is exhausted after menopause. Recruitment occurs at a relatively constant rate during the first three decades of a woman's life; however, when it reaches a critical number of ~25,000 at 37.5 1.2 years of age, the rate of loss of primordial follicles accelerates ~2-fold (Gougeon, 2004). 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 (Fox et al., 2001).

A change in shape from squamous to cuboidal, and the acquisition of mitotic potential in the granulosa cells are histological hallmarks of

recruitment. This is followed by gene activation and subsequent growth of the oocyte. The primary mechanisms that control recruitment involve the granulosa cells and the oocyte is a responding tissue to the primary activation event (Fortune et al., 2000). Three activators of recruitment are known, namely, granulosa-derived kit ligand, theca-derived Bone Morphogenetic Protein-7 (Lee et al., 2001), and high plasma levels of pituitary FSH (Fortune et al., 2000). Mllerian Inhibiting Substance (MIS) has been found to inhibit recruitment (Durlinger et al., 2002; Erikson 2003; Gruijters et al., 2003). The primary follicle A primary follicle is defined by the presence of one or more cuboidal granulosa cells that are arranged in a single layer surrounding the oocyte. The major developmental events that occur in the primary follicle include FSH receptor expression by granulosa cell and oocyte growth and differentiation. Primary follicle development is also accompanied by striking changes in the oocyte. During the preantral period, the oocyte increases in diameter from ~ 25m to ~ 120m. This enormous growth occurs as a consequence of the reactivation of the oocyte genome (Bachvarova, 1985). Some of the oocyte mRNAs are translated and the resulting proteins contribute to oocyte growth and differentiation (Teixeira et al., 2002, Hreinsson et al., 2002; Gougeon, 2004). The secondary follicle The major changes occur during secondary follicle development include: The primary-to-secondary transition Secondary follicle development begins with the acquisition of a second layer of granulosa cells. This step is termed the primary-to-secondary follicle

transition. It involves a change in the arrangement of the granulosa cells from a simple cuboidal epithelium to a stratified or pseudostratified columnar epithelium. Cx43 like GDF-9 and BMP-15, coupling plays an indispensable role in the mechanisms controlling the formation of a secondary follicle (Erikson, 2003). The antral (Graafian) or gonadotropin-dependent phase: It is regulated by FSH and Luteinizing hormone (LH) as well as by growth factors, and characterized by the tremendous increase of the size of the follicle itself (up to approximately 25mm). Stimulation by FSH is an absolute requirement for development of large antral preovulatory follicles (Erickson, 2000). Duration and magnitude of FSH stimulation will determine the number of follicles with augmented aromatase enzyme activity and subsequent estradiol (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 (Gougeon, 2004). After antrum formation, the follicle becomes dependent on FSH stimulation for continued growth and development; however, it is becoming increasingly clear that long-term homeostasis of developing Graafian follicles also depends on positive influences evoked by GF-dependent signaling (Erickson and Shimasaki, 2001). The Graafian follicle: A Graafian follicle is characterized by a fluid filled cavity at one pole

of the oocyte. This process is termed cavitation or beginning antrum formation, a cavity or antrum containing a fluid termed follicular fluid or liquor folliculi. Follicular fluid is an exudate of plasma and is conditioned by secretory products from the oocyte and granulosa cells. It is the medium in which the granulosa cells and oocyte reside and through which regulatory molecules must pass on their way to and from this microenvironment (Erickson, 2000; Gougeon, 2004). Graafian follicle growth and development are divided into four stages based on size. Each dominant follicle has a destiny to complete the transition from the small (1-6 mm), medium (7-11 mm), large (12-17 mm), to the preovulatory state (18-23 mm) in women. An atretic follicle usually fails to develop beyond the small to the medium stage (1-10 mm). The relative abundance of Graafian follicles and their sizes vary as a function of age and the menstrual cycle (Erickson, 2003). After cavitations, the basic plan of the Graafian follicle is established, and all the various cell types are present in their proper position awaiting the stimuli that lead to gradual growth and development. A Graafian follicle is a member of the heterogeneous family of relatively large follicles measures 0.4 to ~23 mm in diameter (Erickson, 2000). The size of a Graafian follicle is determined largely by the size of the antrum, which in turn is determined by the volume of follicular fluid, which varies between 0.02 to 7 ml., and the proliferation of the granulosa and theca cells which proliferate extensively (as much as 100-fold). Cessation of follicular fluid formation and mitosis that limits the size of the atretic follicle (Gougeon, 2004).

The theca interstitial cells possess cell receptors for LH and insulin. In response to LH and insulin stimulation, they produce high levels of androgens, most notably androstenedione. The theca interna is richly vascularized by a loose capillary network that surrounds the Graafian follicle during its growth (Zeleznik 2004).

The way in which the granulosa cells differentiate in the Graafian follicles appears to be controlled by a morphogen gradient emanating from the oocyte. Two known oocyte morphogens are Growth differentiation Factor 9 (GDF-9) and Bone Morphogenetic protein 15 (BMP-15). Consequently as a Graafian follicle develops, the morphogens, GDF-9 and BMP-15, function as gradient signals for the generation of distinct classes of functionally different granulosa cells (Frindlay et al., 2002; Yamamoto et al., 2002; Hreinsson et al., 2002). Selection In normal cycling women, the dominant follicle is selected from a cohort of class 5 follicles at the end of the luteal phase of the menstrual cycle (Gougeon, 1996). The rate of granulosa mitosis appears to increase sharply (~2 fold) in all cohort follicles after the mid-luteal phase, suggesting that luteolysis contributes somehow to an increase in mitosis in the granulosa cells in the pool of small Graafian follicles. The first indication that the selection has occurred is that the granulosa cells continue dividing at a relatively fast rate in one cohort follicle while proliferation slows in the granulosa of the other cohort follicles. This effect is observed about the time

of menses. Thereafter, the mitotic rate of the granulosa and theca cells remains high through the rest of Graafian follicle development. As the follicular phase proceeds, the dominant follicle grows rapidly, reaching 6.9 0.5 mm at days 1 to 5, 13.7 1.2 mm at days 6 to 10, and 18.8 0.5 mm at days 11 to 14. Conversely, growth proceeds more slowly in the other Graafian follicles of the cohort (Zeleznik, 2004; Hreinsson et al., 2002). The underlying mechanism of selection involves the secondary rise in plasma FSH. During the menstrual cycle, the secondary FSH rise in women begins a few days before plasma progesterone falls to basal levels at the end of luteal phase. FSH levels remain elevated through the first week of the follicular phase of the cycle. Increased and sustained levels of circulating FSH are obligatory for selection and female fertility. It is believed that decreased estradiol and inhibin A production by the corpus luteum (CL) are the major causes for the secondary rise in FSH and dominant follicle selection (Gougeon, 2004).

Fig. 5:

Diagram illustrating the different stages of folliculogenesis (Rabe et al., 2002).

Fig. 6: The timetable of normal folliculogenesis in women (Gougeon, 2004) In each menstrual cycle, the dominant follicle that ovulates originates from a primordial follicle that was recruited almost one year earlier. The preantral or Class 1 phase is divided into three major stages: the primordial, primary, and secondary follicle stages. Altogether, the development of a primordial to a full-grown secondary follicle requires = 290 days or about 10 regular menstrual cycles. The antral phase is typically divided into four stages: the small (Class 2, 3, 4, 5), medium (Class 6), large (Class 7), and preovulatory (Class 8) Graafian follicle stages. After antrum formation occurs at the Class 3 stage (~0.4mm in diameter), the rate of follicular growth accelerates. The time interval between antrum formation and the development of a 20 mm preovulatory follicle is about 60 days or about 2 menstrual cycles. A dominant follicle is selected from a cohort of class 5

follicles at the end of the luteal phase of the cycle. About 15 to 20 days are therefore required for a dominant follicle to grow to the preovulatory stage. Atresia can occur after the Class 1 or secondary follicle stage, with the highest incidence occurring in the pool of small and medium (Class 5, 6, and 7) Graafian follicles (Erickson, 2003; Gougeon, 2004). 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) (Fauser and van Heusden, 1999). Atresia Atresia can occur after the Class 1 or secondary follicle stage, with the highest incidence occurring in the pool of small and medium (Class 5, 6, and 7) Graafian follicles. Under normal conditions, only about 400 follicles reach the mature preovulatory stage and ovulate in a lifetime. Hence, loss of follicles due to atresia-with apoptosis i.e. programmed cell death, as the underlying cellular mechanism -rather than growth and subsequent ovulation should be considered the normal fate of follicles. The importance of oxidative stress in inducing atresia and gonadotropins and various growth factors (survival factors) to suppress apoptosis, has been emphasized recently (Tilly and Tilly 1995; Hsueh et al., 1994; Erikson, 2003) . Apoptosis is an essential component of ovarian function and development. Indeed, it is the mechanism that makes the female biological clock tick. During fetal life, apoptosis mainly involves the oocyte. Alternatively, it involves the granulosa cells of the growing follicle during the adult life. Hypothetically, mechanisms underlying the exhaustion of the ovarian reserve of follicles

include: (i) quality control leading to the elimination meiotic anomalies; (ii) a deficit in survival factors produced by somatic neighboring cells; (iii) a self-sacrifice or altruistic death (Monniaux, 2002). This classical view of a finite primordial follicle pool has been challenged recently by Johnson et al., who showed that germline stem cells can repopulate a germ cell-depleted postnatal ovary and renew the primordial follicle pool. However, it remains unknown to what extent this process delays the onset of menopause (Johnson et al., 2004; Johnson et al., 2005).

B. Endocrinology of folliculogenesis

Intrafollicular homeostasis:
Intra-ovarian peptides play important roles in modulating gonadotropin effects on ovarian function. 33 putative paracrineautocrine regulators of follicular growth and atresia are identified (4). FSH enhances the secretion of most of them by granulose cells. Insulinlike growth factor I (IGF-I) augments FSH-mediated aromatization, granulose cell mitogenesis, and the induction of LH receptors. Inhibin, in addition to its endocrine negative effect on FSH secretion, inhibits aromatization and stimulates LH-induced androgen production by theca cells (5). Activin has a positive effect on aromatization (6,7), granulose cell mitogenesis (8,9), and a negative paracrine action on LH-induced androgen production by theca cells (5). Activin is also involved in the regulation of apoptosis in the ovary (10). Follistatin, the third member in the inhibin/ activin family, is antagonistic to activin (11). Vascular endothelial growth factor (VEGF) and growth factors such as epidermal growth factor (EGF) and transforming growth factors (TGF), also play important roles in modulating gonadotropin effects on ovarian function (12, 13). The LH surge initiates luteinization and the beginning of progesterone production by the granulose cells of the dominant follicle. It is also responsible for the resumption of meiosis in the oocyte (14). Activin promotes and inhibin inhibits the LH surge and superovolution

in a rat model (15). LH stimulates the synthesis of cytokines, the best known of which is interleukin-1 (IL-1) which modulates activation of prostaglandins (16) and the proteolytic cascade that are essential for follicular rupture (17). Ovulation occurs 24-36 hr after the onset of the LH surge, when the follicle, which is about 20mm, ruptures and the oocyte is released from the ovary. After ovulation, the dominant follicle becomes the corpus luteum. Producing progesterone, E2, and inhibin, which suppress the growth of new follicles in the ovary. At the end of the cycle, luteolysis causes decline in both steroids and inhibin.

Intra-Ovarian Growth Factors:


Ovarian follicles produce a number of TGF-related proteins. Anti-mullerian hormone, TGFs, activins, and inhibins are produced by granulose cells. Both bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are expressed exclusively by the oocyte of several species (18-20) BMP15 and GDF9 stimulate granulose cell mitogenesis (21). BMP15 is a potent inhibitor of FSHreceptor expression and participates in negative feedback influencing granulose cell mitosis (22). BMP6 is also expressed in the oocyte and inhibits FSH action, probably by downregulation of adenylate cyclase (233). There is a rapid decrease in BMP6 concentration in granulose cells around the time of dominant follicle selection. Members of the TGF superfamily signal through the activin/

TGF and/ or BMP pathways (24). The BMP receptors (BMPRIA/ ALK3, BMPRIB/ ALK6, and BMPR2) are transmembrane serine/ threonine kinases closely related to the transforming growth factor beta receptors (TGFBRI/ ALK5, TGFBR2) and activin receptors (ACVR1, ACVR1B, ACVR2, and ACVR2B). BMP receptors are expressed in granulose cells and oocytes (25) and the BMPs exert their biological actions by forming heteromeric complexes with type I and II receptors (26). Ligands bind to the type II receptors leading to transphosphorylation of the type I receptor. The type I kinase activates proteins which migrate to the nucleus and together with other proteins regulate expression of target genes. GDF9, BMP15, BMP4, and BMp7 all use BMP2 as a binding receptor (27). BMP15 signals through interaction of BMPR1B and BMPR2 activating the SMAD1/5/8 pathway (28). Consequently, BMP proteins appear to interact with a limited number of receptors to activate two downstream Smad pathways. Moreover, several high-affinity binding proteins including follistatin, noggin, and gremlin antagonize BMP signaling (29). How granulose cells and other cell types in the ovary differentiate between signals from multiple ligands in this pathway remains unclear. OHSS is the major serious and potentially life-threatening complication of ovulation induction in IVF-ET treatment. It is characterized by transudation of protein-rich fluid from the vascular space into the peritoneal cavity and to a less extent, pleural and pericardial cavities. The basic pathophysiologic event in OHSS is an

acute increase in capillary permeability; however, the exact factors responsible for this phenomenon have, until recently, not been clear. Because intensity of the OHSS is related to the degree of ovarian response to ovulation induction therapy, OHSS is probably an exaggeration of normal ovarian physiology. Part of the angiogenic response, which occurs in the follicle at the time of ovulation, is increased vascular permeability VEGF. VEGF stimulates endothelial cell mitogenesis and renders capillaries highly permeable to highmolecular-weight protein (59). VEGF has been identified in rat (60) and primate ovaries predominantly after the LH surge. Luteal-phase treatment with GnRH agonist, to suppress LH secretion, decreased VEGF messenger-RNA expression, implying such expression is dependent on LH. We first reported the role of VEGF in OHSS (61). We have demonstrated that VEGF is the major capillary permeability factor in OHSS ascites. Although other capillary permeability factors may not have been detected. 70% of the capillary permeability activity in OHSS ascites was neutralized by recombinant human VEGF antiserum. The incidence of OHSS after induction of ovulation varies between 1% and 30%, as reported in various publications. This variation is probably due to the difference in the definition of OHSS. OHSS is classically divided into three categories. The treatment of OHSS is conservative. Bed rest and symptomatic relief are usually sufficient for mild and moderate OHSS.

In mild cases, symptoms subside usually within a few days, whereas in moderate cases, symptoms can require up to 3 wk to subside. When pregnancy occurs, OHSS will last longer.
The estrogens need for follicle development: In Vitro studies have shown for the rat model that E 2 plays important autocrine roles in stimulating FSH-induced granulosa cell proliferation, aromatase enzyme induction, production of inhibin, increase in E2 and FSH receptors, and formation of LH receptors on granulosa cells ; E 2 exhibits a paracrine action on adjacent theca cells by inhibiting androgen production. Estrogens have also been shown to inhibit apoptotic changes of ovarian follicles (Billig et al., 1993). This may not be the case for higher species, including the human. Under normal conditions, augmented E 2 levels may merely be associated with normal follicle development. Follicles can mature fully without a concomitant rise in E2 (which was believed to be responsible for the decreased need for stimulation by FSH through autocrine short loop upregulation). This 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( Simoni et al., 2002). A 2.5-fold difference in maximum early follicular phase FSH serum concentrations 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 (Simoni etal., 1997).

The majority of growth factors, such as insulin-like growth factors (IGF), transforming growth factor, fibroblast growth factor, and activin, enhance FSH action in vitro. Other growth factors inhibit FSH-stimulated E2 biosynthesis including inhibin, epidermal growth factor, and IGF binding protein (IGFBPs) (Mason et al., 1992). A deficiency of the 17_PRIVATE "TYPE=PICT;ALT={alpha}"_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. 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 (Fauser et al., 1999; Gougeon 2004). 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. Subsequent IVF and cleavage rates were not different from normal (Pellicer et al., 1991). Two unrelated females have been described 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. Large ovarian cysts have been described in both patients, suggesting that growth of antral follicles can occur in the absence of intraovarian estrogen biosynthesis (Morishima et al., 1995).

A study on safety and pharmacokinetic properties of human recombinant FSH, 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 (Fauser 1997). 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. A normal rise in immunoreactive serum inhibin levels in the majority of these women excluded the possibility of granulosa cell abnormalities per se (Schoot et al., 1994). 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). When urinary FSH was combined with long-term GnRH agonist comedication suppressing the endogenous release of LH and FSH, similar observations were reported. 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) (Latronico et al.,1996; Toledo et al., 1996). These observations in the human confirm the two-cell, twogonadotropin concept for adequate E2 synthesis but also demonstrate convincingly that increased E2 production is not mandatory for normal follicle growth up to the preovulatory stage (Zeleznik 2004).

Direct effects have been described of the antiestrogen clomiphene citrate on E2 synthesis by cultured human granulosa cells (Olsson and Granberg 1990). 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. 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 (Fauser 1994; Gougeon 2004). 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 (Danforth 1995; Fauser et al., 1999; Zeleznik 2004). The Role of FSH: The concept that FSH is obligatory for dominant follicle (DF) selection and development was arrived at almost 60 years ago. In a very real sense, it represents the cornerstone of our understanding of ovary physiology. The increase in plasma FSH that occurs during the late luteal and early follicular phases of the menstrual cycle is the basis for DF selection in women. The stringent requirement for this FSH rise in the selection process is

demonstrated by the fact that in its absence, there is no DF and no ovulation (Zeleznic, 1993). The physiological consequence of this FSH rise is that a critical threshold concentration of FSH is achieved within the microenvironment of the chosen follicle. There is a consensus that the threshold level of FSH results in the expression of E2, which in turn suppresses plasma FSH levels; this in turn causes the concentration of FSH in developing cohort follicles to fall below threshold levels. It is widely accepted that this FSH withdrawal phenomenon in cohort follicles is involved in the massive apoptosis of the granulosa cells that occurs during atresia. There is evidence that mitosis in cohort follicles can be markedly stimulated by treatment with human menopausal gonadotropin (hMG) during the early follicular phase (Gougon, 1990). One implication of this observation is that hMG-treatment might increase the number of presumptive DFs in women by rescuing cohort follicles from atresia. Chronology of the process of folliculogenesis in human ovaries. Evidence from histomorphometric studies suggests that changes in granulosa mitosis might constitute one mechanism by which selection occurs (Erickson, 2000). Shortly after the midluteal phase, the granulosa cells in all cohort follicles appear to show an increase (approximately two fold) in the rate of mitosis. One of the first indications that a DF has been selected is that the granulosa cells in the chosen follicle continue proliferating at a fast rate while the rate of proliferation slows in the non-DFs. Because this distinguishing event appears in the late luteal phase, it is likely that the DF is

selected at this time of the cycle. Given the importance of FSH in the selection process, it is not unreasonable to assume that the basic mechanism underlying these changes in granulosa proliferation are functionally related to the relatively high threshold level of FSH in the microenvironment of the chosen follicle (Richard et al., 1998). A fundamental question concerns the number of potential selectable follicles in any given cohort. The simple truth is that we do not know the answer to this basic question. There is evidence in women that death of the DF or corpus luteum (CL) leads to the immediate selection of a new DF. This observation supports the conclusion that human ovaries always contain a pool of small Graafian (class 4 and 5) follicles (see Fig. 1) from which another DF can be selected. Although the precise number is unknown (Erickson and Shimasaki, 2001). Gougeon, 2004 suggests that the ovaries of normal young women may contain a cohort of approximately four to six healthy class 4 to 5 follicles. In this regard, it is likely that the size of the pool is variable, being correlated with age and ovary reserve. Considerable effort has been devoted to understanding the mechanism of FSH action in the DF. The fact that the granulosa cells are the only cell types known to express FSH receptors targets them as physiologically important in mediating FSH action in the ovary. The accumulated data from a large number of studies demonstrate that FSH receptor signaling plays a fundamental role in the growth and differentiation of the DF through its ability to promote follicular fluid formation, cell proliferation, E2 production, and LH receptor expression (Richard, 1994).

The temporal pattern and level of expression of these FSH-dependent genes are crucial for the expression of the normal physiological functions ascribed to the DF. It should be mentioned that the FSH stimulation of LH receptors in the granulosa cells is required for LH/ hCG to induce ovulation and luteinization (Richard et al., 1998). A key feature of the temporal pattern of LH receptor expression is that it is suppressed throughout most of folliculogenesis. A high level of LH receptor expression is not induced the granulosa cells until the DF reaches the preovulatory stage (Minegishi et al., 1997). This fact supports the possibility that when LH enters the follicular fluid during the late follicular phase it may be an important effector of granulosa function, perhaps even replacing FSH as the principle regulator of cyto-differentiation (Erickson and Shimasaki, 2001). The Role of LH: Although FSH is the central regulator of DF survival and development, LH/ hCG signaling pathways play fundamental physiological roles. Physiologically, LH-dependent signaling pathways in the theca interstitial cells elicit changes in gene expression that are critical for E2 production (Erickson, 1985). Specifically, activation of the LH receptors in theca cells leads directly to the stimulation of high levels of androstenedione production. The major physiological significance of this LH response is to provide aromatase substrate to the granulosa cells where it is metabolized by P450 aromatase to E2; this is the two gonadotropin-two cell concept of DF estrogen biosynthesis (Fig. 7). Because E2 production is unique to DFs, the level of

plasma E2 is a useful marker for monitoring the physiological responses of endogenous or exogenous gonadotropins in women (Erickson and Shimasaki, 2001).

Fig. 7: Diagram illustrating the two gonadotrophin-two cell concept of follicular


estradiol production. (Erickson, 2001)

There are three additional physiologically important func- tions of LH/hCG in the DF and CL. First, the ovulatory dose of LH/hCG is responsible for ovulation and CL formation. Second, LH is essential for P4 and E2 production by the CL during the early and midluteal phases of the menstrual cycle. And third, hCG is obligatory for transforming the CL of the

cycle into the CL of pregnancy (Erickson and Shimasaki, 2001).

Intrafollicular endocrine changes:


The majority of enzymes involved in the biosynthesis of ovarian steroids belong to the cytochrome P-450 gene family (Strauss and Miller, 1991; Fauser et al., 1999; Zeleznik, 2004). This group of enzymes includes: 1- Cholesterol side-chain cleavage enzymes (P-450SCC), which convert cholesterol to pregnenolone. The cholesterol side-chain cleavage enzyme represents the major rate-limiting step in steroid hormone synthesis. Proteins involved in the acquisition of cholesterol have also been shown to be important for sufficient steroid biosynthesis (Fauser, 1999; Erikson, 2003). 2- The P-450C17 enzyme (involving both 17-hydroxylase and C17,20lyase activity) converts to both progestins (pregnenolone and and progesterone) androgens [dihydroepiandrosterone

androstenedione (AD), respectively]. In vitro studies using cells isolated from human ovarian follicles have demonstrated that theca cells are the source of follicular androgens. -Predominantly AD-whereas granulosa cells only produce E2 when androgens are added to the culture medium. 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, 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 , which correlate well

with augmented 17-hydroxylase activity of human theca cells in culture (Fauser et al.,1999; Gougeon, 2004). 3- The aromatase enzyme complex (P-450A ROM), converts androgens [AD and testosterone (T)] to estrogens (estrone and E2, respectively). Small antral follicles were shown to lack P-450AROM mRNA. However, appreciable quantities of mRNA, and the aromatase enzyme 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 (Simpson et al., 1992; Zeleznik, 2004). The mRNA expression is in good agreement with immunolocalization of the aromatase enzyme. Synthesis of the P-450AROM enzyme could also be induced by FSH administration to human granulosa cells in culture .When follicles mature, granulosa cells also exhibit elevated mRNA levels for P-450SCC, LH receptor, activin, and inhibin (Fauser et al., 1999; Gougeon, 2004).

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 (Takayama et al., 1995; Erikson, 2003).

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

important

for

gonadal

steroid

synthesis:

3bata-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 (Fauser et al., 1999; Zeleznik, 2004).

The theca interna layer of developing follicles responds to LH and synthesizes androgens. 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. Atretic follicles of all sizes (between 2 and 13 mm diameter) also contain high androgen levels and low E2 concentrations. 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 (Van Dessel et al., 1996; Gougeon, 2004). Large (>8 mm diameter) follicles in the mid- and late follicular phase of the menstrual cycle contain (up to 10,000-fold) higher quantities of E2 compared with small follicles. 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. In IVF patient a correlation exists between the E2/androgen ratio in follicle

fluid and follicular health and fertility potential of oocytes (Van Dessel et al., 1996; Gougeon, 2004). After enucleation of the largest follicle no further differences were found in steroid levels in blood draining both ovaries. A correlation between intrafollicular E2 concentrations and follicle diameter has been substantiated in large dominant follicles. All studies show low E2 levels in relatively small (<10 mm diameter) nondominant follicles, and the absence of a correlation between follicle size and E2 levels in this size range. 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 810 mm in diameter. 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, suggesting increased sensitivity. A distinct relationship was observed between follicle diameter and the number of granulosa cells that was recovered at each size (Fauser et al., 1999; Zeleznik, 2004). Enhanced E2 biosynthesis is closely linked to preovulatory follicle development and that high estrogen output of the dominant follicle is regulated by FSH-stimulated granulosa cell function. Development of smaller follicles in the early follicular phase, although dependent on FSH, is not associated with increased E2 production (Zeleznik, 2004).

OVULATION INDUCTION (ovarian stimulation)


Ovulation induction is a process of promotion of follicular growth and development culminating in ovulation. It is a frequently utilized therapeutic procedure for the management of infertility (Guttam et al., 2004). A. Indication of ovulation induction Ovarian stimulation with fertility drugs is used for treatment of: 1- Various types of ovulation dysfunction: Approximately 40% of all female infertility problems are results of ovulatory dysfunction (Baired, 2003). According to the world Health Organization ovulatory dysfunctions are classified into, three groups; Group I hypothalamic pituitary failure with lack of endogenous estrogen activity and fail to experience progestin withdrawal bleeding, Group II Hypothalamic pituitary dysfunction with oligomenorrhea, amenorrhea, hyperandrogenism and luteal phase disorders, Group III Ovarian failure with various degree of hypergonadonadotropic hypogonadal dysfunction (Barid, 2002). 2-To improve ovulation in sub fertile women: Women with apparently normal cycles have subtle cycle abnormalities such as luteal phase abnormalities, hyper-prolactinaemia and abnormal FSH and LH patterns and luteinized unruptured follicle syndrome. So induction of ovulation can improve such abnormalities (Rodin et al., 1994).

3-Imperical treatment to maximize chances of conception: with or without IUI in male infertility, endometriosis and unexplained infertility (Takeuch et al., 2000). 4- As a fundamental adjunct to increase the success of treatment with the assisted reproductive technology (ART) (Ng et al., 2001). The detailed description of ART is beyond the scope of this thesis. However, the following is a brief appraisal of these techniques. Intrauterine Insemination (IUI): Where processed semen placed into uterine cavity via catheterization at the time of spontaneous or induced ovulation. In Vitro Fertilization (IVF) and Embryo Transfer (ET): Where Meta phase two (MII) retrieved oocytes are incubated in-vitro with selected sperms waiting for spontaneous fertilization and at early stages of embryonic division, selected embryos will be transferred via special catheter (ET catheter) into the uterine cavity. Zygote Intrafallopian Transfer (ZIFT): After IVF the selected embryos at zygote stage of development is transferred to the fallopian tube through a laparoscopic approach. Gamete Intrafallopian Transfer (GIFT): Sperm and oocyte are introduced into the ampullary part of the fallopian tubes under direct laparoscopic visualization. Intracytoplasmic Sperm Injection (ICSI): where selected spermatozoon is in-

vitro placed in the MII oocyte cytoplasm. (David, 2007). B. The Mechanism of Ovarian Stimulation According to Baird's theory, several antral follicles begin to grow simultaneously; Only one follicle can achieve dominance, provided it developed to certain size and maturation level before the FSH gate (rise of serum FSH levels in the early follicular phase) and develop further as the single dominant follicle (Fig. 8 a) (Baird, 1987) Alternatively, this period can be extended (the FSH gate can be widened), this will enable several antral follicles to grow simultaneously to a size and develop to a level required for entrance through the widened FSH gate. There are two options for circumventing this process of follicular selection and development of several follicles (Fig. 8 b, c). Prolonged elevation of FSH can be achieved by direct administration of exogenous FSH. Alternately, administration of the anti-estrogens clomiphene and tamoxifen as well administration of an aromatase inhibitor, in the presence or absence of exogenous FSH, also can result in ovarian stimulation presumably by diminishing the negative feedback effects of estrogen on FSH secretion. (Rabe et al., 2002).

Fig.8:

Selection of the dominant follicle in (A) spontenous cycle when only one follicle can enter the FSH gate. (B) to increase the number of dominant follicles one can increase the number of follicles entering the FSH gate or ; (C) widen the FSH gate (Rabe et al., 2002).

Physiological basis of controlled ovarian stimulation: One of the inherent difficulties in this approach to ovarian stimulation is that follicular maturation is likely to be asynchronous due to the asynchronous nature of the development of preantral follicles; oocytes collected from these follicles could differ in their maturational states as well. One possible way of reducing the variability of differing maturational states of follicles could be by providing a sequential FSH and LH treatment regimen to limit follicular recruitment to a group of follicles. Switching from FSH to LH would maintain the growth of follicles with LH receptors on granulosa cells but would prevent the additional maturation of less mature follicles. In addition, administration of LH in the absence of FSH may actually reduce the number of smaller follicles, possibly by elevating intrafollicular androgen levels (Filicori 2002; Zeleznik 2004). Fig.(9) Summarizes the therapeutic options for increasing serum FSH levels by influencing the hypothalamo-pitutary-ovarian axis at different levels to induce multiple follicular development (Rabe et al., 2002).

Fig.9:

Summary of different possibilities for ovarian stimulation for IVF (Rabe et al., 2002).

The antiestrogenic effect of Clomiphene Citrate on the central nervous system increases FSH and LH pulse frequency, giving a moderate gonadotrphin stimulus to the ovary and thus increasing the cohort of follicles reaching ovulation. On the other hand, gonadotrophins induce multifollicular development by directly increasing FSH levels above threshold values and consequent stimulation of follicular growth. However, in about 15% of cycles stimulated with gonadotrphins and /or CC, the exaggerated estradiol levels due to the multifollicular response provoke high LH concentrations during the follicular phase or an untimely spontaneous LH surge (Fig.10). This may lead to impaired oocyte quality or, more often, to cycle cancellation. For this reason, to avoid interference from endogenous gonadotrphin secretion; a combined therapy of gonadotrophins and GnRH agonists has been gradually introduced (Tarlatzis and Grimbizis, 2002).

Fig. 10:

Occurrence of premature LH surge and premature lutenization in a value critical for induction of LH surge in an earlier phase of the follicular phase than stimulated cycle. (B) Rapidly increasing serum E2 reaches the during (A) spontaneous cycle (Rabe et al., 2002).

C. Ovarian stimulation regimen The philosophy of stimulation is dependent on the goals of ovulation induction depending on the medical condition of each couple, and can be grouped in two major categories; Firstly, procedures conducted to restore ovulation in patients with menstrual and ovulatory disorders. Secondly, stimulation of multiple folliculogenesis in normal women undergoing assisted reproductive procedures ART (Paulson, 2005). D. The ovarian stimulation regimen for IVF The ideal ovarian stimulation regimen for IVF should have a lower cancellation rate, minimize drug costs, risks and side effects, required limited monitoring, and maximize singleton pregnancy rates (Leon and Marc, 2005). Numerous regimens for ovarian stimulation have been described ranging from no stimulation (Natural cycle), to minimal stimulation (clomiphene citrate), or mild stimulation (sequential stimulation with clomiphene citrate and low dose exogenous gonadotropins) [Frindlly IVF], to aggressive stimulation (high dose exogenous gonadotropins, alone or in combination with GnRH agonist or antagonist) Because the egg yield is greater, large number of embryos, and probability of having an optimal number of embryos for transfer and cryopreservatin (Leroy et al., 2005). Natural cycle: The first birth resulting from IVF derived from an oocyte collected in a natural unstimulated cycle. Cancellation rate are high (25-75%), success rate per cycle start are very low, and there is no opportunity to select or cryopreserved embryo. It remains an option for women who respond poorly to ovarian stimulation, and those with medical conditions in whom the risks

of ovarian stimulation are best avoided (Fahy et al., 1995). Exogenous hCG is administrated when the leading follicle reaches a size of maturity, frequent monitoring of endogenous serum LH level (to detect the LH surge) is better defining the time of oocyte retrieval (Rongieres et al., 1999). Clomiphene citrate: Clomiphene is a nonsteroidal triphenylethylene derivative with both estrogen agonist and antagonist properties. However, in almost all circumstances, clomiphene acts purely as an antagonist; its weak estrogenic action is clinically apparent only when endogenous estrogen levels are very low (Clark et al., 2005). Clomiphene competes for and binds to estrogen receptors throughout the reproductive system and remains bound for an extended interval of time and ultimately depletes receptor concentrations by interfering with receptor recycling. At the hypothalamic level, estrogen receptor depletion prevents accurate interpretation of circulating estrogen levels, which are lower than they truly are. Reduced estrogen negative feedback triggers normal compensatory mechanisms that alter the pattern of GnRH secretion and stimulate increased pituitary gonadotropins release, which in turn drives ovarian follicular development (Mikelson et al., 2005). Gonadotrophins: Exogenous gonadotropins have been used to induce ovulation in gonadotropin deficient women and those with clomiphene resistance. These

potent medications are very effective, but also costly and associated with risks including multiple pregnancy and ovarian hyperstimulation syndrome (Van de Weijer et al., 2003). Preparations of gonadotropins: The following is the most commonly used preparation. Human menopausal Gonadotrophin (HMG) is extracted from the urine of postmenopausal women. Residual urinary proteins create the need for administration by intramuscular injection. Each ampoule consists of equal amount of FSH and LH eg. 75 IU FSH and 75 IU LH (Dor et al., 2002). Subsequently Urofolletropin (uFSH), a preparation of 75 IU FSH and < 0.7 IU LH per ampoule, was developed by removing most of the LH using an immunoaffinity column of antibodies against (hCG). The presences of significant amounts of urinary protein in the preparation require intramuscular injection (Felberbaum et al., 2000). Highly purified FSH, developed with an immunoaffinity column of antihuman FSH, has < 0.001 IU LH in each ampoule and much lower levels of contaminating urinary proteins, enabling subcutaneous injection (Daya, 2001). The in vitro production of recombinant human FSH (rFSH) was achieved through genetic engineering. Which contains less acidic isoform that have a shorter half-life than urinary FSH but stimulate estrogen secretion as or even more efficiently. Its advantages include the absence of urinary proteins, more consistent supply and less patch to patch variation in biologic activity (Fleberbaum et al., 2000; Filicori et al., 2003).

A recombinant from of human LH having physicochemical, immunologic, and biologic activities comparable to those of human pituitary LH has been developed and was approved for use in Europe in 2000 (Iecotomec et al., 2003). Modalities of ovulation induction with Gonado-trophins: The three most common modalities of stimulation protocols: the fixed, the step-down, and the low-dose step up regimens. The fixed dose regimen: using a fixed dose of gonadotrophins according to the requirement of the patient to reach a successful ovulation (usually 150 IU/day) for 2 weeks (Andoh et al., 1998). The step down regimen: is designed to more closely approximate the pattern of serum FSH concentrations observed in spontaneous cycles, development of only the more sensitive dominant follicle while withdrawing support from the less sensitive smaller follicles in the cohort (Homburg et al., 1999). It consisted of 225 IU/d of hMG for the first 2 days followed by 150 IU/d until the follicular diameter reached 9mm, after which the dose was decreased to 75 IU/d for the next 7 days. When follicular development was not observed by U/S, the dose of hMG was increased to 150 IU/d after the 9 th day (Andoh et al., 1998). The low dose step up regimen: In both women with hypogonadotropic hypogonadism (WHO group I) and those with clomiphene-resistant anovulation (WHO group II) initial attempts to induce ovulation should begin with a low daily dose (75 IU daily). It consisted of 75 IU/d of HMG for the first 7 days, and if the follicular diameter did not exceed 9mm, the

dose increased by 37.5 IU every 7 days. Dosages should be adjusted according to the frequently monitored ovarian response (Chong et al., 2005). Because women with polycystic ovary syndrome (PCO) syndrome often are sensitive to low doses of gonadotropin stimulation, early and frequent monitoring is generally wise. Ovarian hyperstimulation syndrome (OHSS), multiple pregnancy, and canceled cycles usually can be avoided by using a "low-slow" treatment regimen involving low doses (37.5-75 IU daily), and a longer duration of time (Calaf et al., 2003). Insulin-resistant women may be less sensitive to gonadotropin. Metrformin treatment before and during gonadotropin stimulation can help to improve response, limit the number of smaller developing ovarian follicles (De Leo et al., 2005). Gonadotrophin releasing hormone agonists / antagonist: In about 15% of cycles stimulated with gonadotrphins and/or CC, the exaggerated estradiol levels due to the multifollicular response provoke high LH concentrations during the follicular phase or an untimely spontaneous LH surge. This may leads to impaired oocyte quality or, more often, to cycle cancellation. For this reason, to avoid interference from endogenous gonadotrphin secretion; a combined therapy of gonadotrophins and GnRH agonists and antagonists has been gradually introduced (Tartralatzis and Grimbizis, 2002). Gonadotrophin releasing hormone agonists

GnRH agonist administration leads to prolonged agonistic action on the GnRH receptors due to their higher affinity to the receptors and their higher biological stability. The initial increase gonadotrophin secretion from pituitary cells, a phenomenon known as the flare-up effect, which results from activation of mechanisms that are identical to those observed after natural GnRH agonist administration. However, the prolonged administration of agonists with there chronic action on pitutary gonadotrophs suppresses pituitary function. This is due to down-regulation of the GnRH receptors and the inhibition of post-receptor mechanisms (pitutary desensitization) that are responsible for the synthesis and release of gonadotrophins which block the positive oestradiol (E2) feedback to the pituitary and the resulting untimely LH surges (Borm and Mannaerts, 2002). The GnRH agonist treatment may suppress endogenous LH levels below those necessary for normal follicular development in some women. Because only about 1% of LH receptors need to be occupied to support normal follicular steroidogenesis, these low levels of LH are sufficient to meet the need in most women stimulated with uFSH or rFSH alone (Balasch J et al., 2001). The only disadvantage is the GnRH agonist treatment sometimes blunts the response to subsequent gonadotropin stimulation and increases the dose and duration of gonadotropin therapy required to stimulated follicular development, which increase the total cost of treatment (Meldrum DR et al., 2005). It is known that in a suppressed pituitary gland the dose of GnRH agonist needed to maintain suppression gradually decreases with the length

of treatment. On the other hand, as ovarian stimulation with gonadotrophins progresses, the suppression of pituitary gonadotrophin secretion becomes more effective and the concentrations of endogenous LH decrease (Fabregues et al., 2005). Modification of GnRH decapeptyl enables the development of GnRH antagonists, which competitively inhibit the natural gonadotrophin secretion (Paul and Caroline, 2004). GnRH antagonists offer several potential advantages over agonists; Duration and dose of treatment is shorter, as antagonist treatment can be postponed until after estradiol levels are already elevated, thereby eliminating the estrogen deficiency symptoms that can emerge in women treated with an agonist (Olivennesf et al., 2000), for the same reasons this stimulation protocols may benefit poor responder women (Albano et al., 2000). By eliminating the flare effect of agonists, GnRH antagonists avoid the risk of stimulating development of a follicular cyst and decrease the risk of OHSS (Fleberbaum et al., 2000). The two GnRH antagonists available for clinical use are Ganirelix and Citrorelix, they are equally potent and effective. For both the minimal effective dose to prevent premature LH surge is 0.25mg/day (Akman et al., 2001). Four major protocols that combine exogenous gonadotrophins and GnRH agonists are currently employed:

Fig 11:

Combination of GnRH agonist and gonadotropins in stimulation protocols for ART: ultrashort, short, long follicular, long luteal and fast desensitization protocols (Rabe et al., 2002).

Long protocol: The "long protocol" is the preferred ovarian stimulation regimen for ART Because GnRH agonists has more advantages than disadvantages. This is the most traditional and widely employed protocol (reports for the year 2000 that more than 80% of stimulated cycles were performed according to long protocol) (Wang et al., 2002). Because the egg yield is greater, the large number of embryos the probability of having an optimal number of embryos for transfer and excess embryos for cryopreservatin is greater (Meldrum et al., 2005). Modalities of long protocol: Long luteal phase protocol: These regimens provide improved clinical results (greater number of preovulatory follicles and embryos, increased pregnancy rate) (Surry et al.,

2004). It consists of GnRH agonists administration started in the mid-luteal phase of the cycle preceding gonadotrophin ovulation induction and continued until hCG administration (Peter R. 2006). In the typical cycle, GnRH agonist treatment begins during the midluteal phase, approximately 1 week after ovulation, at a time when endogenous gonadotropin levels are at or near their nadir. The acute release of stored pituitary gonadotropins in response to the agonist, known as the "flare" effect, is least likely to stimulate a new wave of follicular development (Urbancsek et al., 2005). GnRH agonist treatment may be scheduled to begin on cycle day 21 (assuming a normal cycle of approximately 28 days duration), but monitoring basal body temperature (BBT) or urinary LH excretion to more precisely determine when ovulation occurs helps to ensure that treatment begins during the midluteal phase (approximately 8 days after the LH surge or rise in BBT), as intended (Pellicer et al., 2005).

Fig. 12:

Diagram illustrating long luteal phase protocol (Rabe. et al., 2002).

The fast desensitization protocol involves GnRH agonist administration from the mid luteal phase of the cycle then stimulation with gonadotrophins from the thered day of the nexist cycle. This regimen combineds the advantages of long and short desensitization protocols. In particular, the GnRH agonist started in the mid luteal phase prontlly inhibit the pituitary gonadotrophins secretion. Moreover, although the GnRh agonist is administered over a relatively breef period only, this method also precludes the initial increase of gonadotrophin secretion of the beginning of the follicular phase (Lounaye et al., 2004). Treatment may also begin in the early follicular phase 'Long follicular protocol' (first day of the cycle), but the time required to achieve pituitary down-regulation is longer (as indicated by low FSH and LH levels and or E2 <50pg/ml and or lack of presence of antral follicles (with diameter exceeding 4mm).), and the prevalence of cystic follicles is higher. Gonadotropin stimulation also yields more follicles and oocytes when agonist treatment begins during the luteal phase, possibly because LHstimulated androgen production and circulating androgen levels are more effectively suppressed throughout folliculogenesis Gonadotropin administration in conjunction with agonist is continued until hCG administration (Cedars, 2005). The dose and duration of gonadotropin treatment required to induce successful ovulation vary among women, even among cycles within a woman. Whereas many women are extremely sensitive to relatively low

doses of gonadotropins (75-225 IU daily), others require substantially greater stimulation (300-450 IU daily) (Olive, 2005). Typical starting dose of gonadotropins range between 225 and 300 IU of uFSH, uHMG or rFSH daily, depending on age, weight, results of ovarian reserve testing and the response observed in any previous trial. Either a step up or step down may be used, but the latter approach is generally preferred. This dose is adjusted according to the patient's response to stimulation from cycle day8 (5 days from stimulation) as assessed by TVus and or E2 level (Stelling et al., 2003). In women who respond poorly to stimulation using the standard daily GnRH agonist treatment regimens, decreasing the doses of agonist by half or more (Kawalik et al., 2005) or discontinuing agonist treatment early (after5 days of gonadotropin stimulation) or completely (when stimulation begins) helps to improve response and overall results (Schachter et al., 2001). Oocyte retrieval using TVUS under sedation is generally performed approximately 36 hours after hCG administration. Mostly longer intervals do not substantially increase the risk of ovulation or adversely affect oocyte quality fertilization rates or overall results in GnRH agonist down-regulated cycles, but earlier retrieval may yield fewer mature oocytes (Tureck et al., 2005).

Short protocol: The ''short or flare'' protocol is an alternative stimulation regimen that exploits both the initial brief agonistic phase of the response to a log- acting GnRH agonist and the subsequent suppression of agonistic phase of

endogenous gonadotropin secretion induced by longer-term treatment (Padilla et al., 2005). This protocol consists of the administration of GnRH agonist starting early in the follicular phase of the ovulation induction cycle (cycle day 1) then exogenous gonadotrophins starting on cycle day 3). The doses of gonadotropin stimulation are based on response and indications for hCG administration are the same as in the long protocol (Karancle et al., 2005). Ultra-short Protocol: The ultra-short protocol is a variation of short-protocol and has been designed for poor responders, regimen this scheme is bassed on the assumption that suppression of the mid-cycle LH surge can be obtained through a very short course of GnRH agonist. The GnRH agonist is used only during the first 3 days of the cyle. Gonadotropin administration is started on the 3 rd day of the cycle until HCG injection as in previous

protocols (Tan et al., 2005).

Fig. 14:

Diagrame illustrating ultra short protocol (Rabe et al., 2002).

Premature LH surge are more prevalent than in cycles stimulated with the standard short or long protocols because down-regulation of endogenous gonadotropin secretion requires longer term agonist treatment. obtained with the short and long protocols (Ron et al., 2005). The ultrashort GnRH agonist stimulation protocol yields results inferior to those

Fig 18: Diagram illustrating GnRH (Single dose protocol) (David K 2007).

The mulitiple dose protocol: From cycle day two start stimulation with gonadotrophin 150 IU HMG/day, from cycle day 7 the GnRH antagonist was administered of (0.25mg.) subcutaneously daily. On day 5, the dose of human menopausal gonadotrophins (HMG) was adjusted to the individual ovarian response of each patient as assessed by estradiol values and follicle measurement. This treatment was continued until triggering of ovulation, with 10,000 IU of HCG, when the leading follicle reached a diameter of 1820mm (measured by transvaginal ultrasound) and oestradiol values indicated a satisfactory follicular response (Fauser et al., 2002s).

Fig.19:

Diagram illustrating GnRH antagonist multiple dose protocol (David, 2007).

Higher doses of gonadotrophin stimulation may help to increase the number of follicles and oocytes (Fluker et al., 2001, Escuderoet al., 2004). Women with PCO exhibit high tonic LH secretion and are predisposed to premature LH Surge when treated with slandered ovulation

induction regimen, also they are at risk for developing OHSS when aggressively stimulated with gonadotrophin but the smaller follicular cohort observed in antagonist cycles may help to reduce these risks when tend to be high responders (Kolibianakis et al., 2003). Monitoring of ovulation induction: Monitoring of ovulation induction aims to: Evaluate the ovarian response during the stimulation period so adjustments can take place if the response is insufficient or too strong. The monitoring will identify those who have not responded adequately or poor responders (Ludwig et al., 2006), and to detect women at risk of OHSS (Ng et al., 2000), to evaluate follicular and endometrial maturation, aiming to find the optimal time for triggering ovulation with (HCG) (Wikland, 2002). Ovulation induction was first monitored by serum E2 level (Mature follicle give 150-200 pg/ml E2 serum level). However, it was not possible to draw conclusions from such measurements on how many mature follicles would ovulate (Banicsi et al., 2002). Thus, Since the follicle is a fluid filled structure, it can be easily visualized by ultrasound techniques which developed a dominant role in the area of monitoring ovulation induction, helps to acquire more knowledge about both follicular and endometrial development regarding the total number of follicles and follicular maturation (by measuring the mean diameter of the follicle) (Wikland, 2003). These first ultrasound measurements should be performed in a stimulated cycle between days 5-7, but this may vary depending on the protocol used and if the patient is at risk for OHSS (Aboulghar et al., 2003).

The additional number of measurements is also dependent on the stimulation protocol and the reason for ovulation induction. More frequent measurements may be required according to high dose protocols and the degree of uncertainly as to how the woman will respond (Wikland, 2002). US is used to determine the maximum diameter of the ovaries and the mean diameter of the dominant follicle. It has not been possible to identify a definitive size of follicle, which confirms its maturity. In fact, there seems to be a relative wide range of follicular size that can contain a mature oocyte rather than smaller or very large follicles. For this reason, a mean diameter of 17-19 mm has been arbitrarily set as the size at which ovulation should be induced (Grunfield et al., 2006). Furthermore, it has been found that if the leading follicle has reached a diameter of 15-16mm, the growth rate is approximately 2mm/24hours (Leerenttveld and Waldimiroff, 2006). This figure can then be used to predict when the largest follicle will reach the optimal day (Follicular size 17-19mm) for hCG administration. Ultrasonographic change in endometrial thickness and echogenic pattern has been described during the normal menstrual cycle. High correlation between endometral thickness and increased steroid levels in blood, as well as oestrogen and progesterone receptors (Ludwig et al., 2006). No real consensus has been reached with regard to the ideal endometrial thickness for an optimal chance of implantation in stimulated

cycles (Friedler et al., 1996). However, in ovulation induction cycles, were able to show a correlation between endometrial thickness as measured by ultrasound on the day of hCG and the pregnancy rate, no pregnancy was found if the endometrium was <7-8mm, measured by ultrasound, determines a mature endometrium which is suitable for induction of ovulation, provided that the follicles are of sufficient size. If the follicles are large enough for inducing ovulation, but the endometrium is < 7mm, it is probably better to continue stimulation for 1-2 days more or check the oestrogen production. The endometrial thickness can thus be used as an assay for oestrogen production (Narayan et al., 2004). Also three- dimensional ultrasound and colour doppler identify and quantify blood flow in small vessels of the follicular wall to study ovulation as well as the uterine artery for predication of endometrial receptivity (Steer et al., 2003).

Prediction and detection of ovarian response A. Ovarian response In assisted reproduction programs, the response of ovulating women to exogenous gonadotrophin therapy is quite variable and difficult to predict. Patient characteristics, rather than the stimulation protocol, seem to determine the individual response (Maritza et al 2000); Althrough the dose and duration of gonadotropin treatment required to induce successful ovulation vary among women, even among cycles within a woman. Whereas many women are extremely sensitive to relatively low doses of gonadotropins (75-225 IU daily), others require substantially greater stimulation (300-450 IU daily) (Olive, 2005). The optimal starting dose of Gonadotrophins during the first treatment cycle in IVF and ICSI remains controversial. The majority of fertility clinics have chosen a standard dose for a standard patient (A standard patient is <40 years of age, with two ovaries, a normal serum basal FSH and a regular menstrual cycle.). A number of studies have attempted to define an optimal standard dose (Out et al., 2001). The doses vary between 100 and 250 IU/day, reflecting the range of policies from friendly IVF with a minimal dose, to an approach where a large number of oocytes is considered a criterion of success. Irrespective of the dose used there seems to be a wide range of responses ranging from one oocyte at retrieval to more than 30 oocyte (Neuspiller et al., 2003). In young ovulating women undergoing in vitro fertilization (IVF) treatment, the standard stimulation protocol can result in either poor response or in ovarian hyperstimulation syndrome (Balasch et al 2006).

The high ovarian responder patients Ovarian hyper stimulation syndrome (OHSS) is a dangerous complication of controlled ovarian hyperstimulation (COH) for IVF, its frequency is 0.5-5 percent in the general IVF population, rather than spontenous OHSS (Edelstien et al., 2007). The clinical entity of OHSS has been described in mild, moderate and severe categories depending on the extent of clinical symptoms and signs. Although mild OHSS is relatively common, it is of low clinical relevance. In contrast, severe OHSS is infrequent but is a more serious condition characterized by significant ovarian enlargement and increased capillary permeability leading to, ascites, pleural effusion, pericardial effusion, haemoconcentration, thromboembolic phenomena, respiratory distress oliguria and renal failure. It is a potentially fatal condition requiring prompt hospitalization for therapy aimed at symptom relief, fluid management to restore plasma volume and renal perfusion (correct fluid imbalance), prevention of thrombosis and support the patient until the condition resolves. Ultrasound examination and serum oestradiol values are currently used to predict patients at risk. The ideal treatment is prevention, but there has been only limited success (Aboulghar and Mansour 2003; Paul and Caroline, 2004). The available evidence about pathophysiology would support a central role of inflammatory cytokines and angiogenic growth factors (Huger et al., 2006). Human chorionic gonadotropin (hCG) is through to play a crucial role in the development of the syndrome, because sever form are indeed restricted to cycles with exogenous hCG (to induce ovulation or as luteal phase support) or with endogenous pregnancy derived hCG (Sebaldo et al., 2007). Spontaneous forms of OHSS are very rare and are always reported

during pregnancy (hCG usually peaks between 8 and 10 weeks gestational age). Spontaneous and iatrogenic OHSS share similar pathophysiological sequences: massive recruitment and growth of the ovarian follicles, extensive lutinization, and over secretion of vasogenic molecules (e.g. vascular endothelial growth factor and angiotensin) by lutinized corpora lutea, provoking a third space fluid shift (Paul and Caroline, 2004; Leon and Marc, 2005). Identification of the at-risk patient: Women with polycystic ovarian syndrome (PCOS) or PCOS-like patients are very vulnerable to developing OHSS because they appear to have a greater sensitivity to gonadotropins resulting in the recruitment of large numbers of follicles at varying stages of maturity. The presence of follicles of intermediate maturity and those that are immature is associated with an increased risk of OHSS. Hence, ultrasonography is important for monitoring the response to treatment during ovarian stimulation. It is also important to identify women with polycystic ovaries because they tend to have a brisk response to ovarian stimulation (Deckey et al., 2007). Increased ovarian volume, and increased number of antral follicles and the necklace or string of black pearls appearance of the ovaries is a negative prognostic sign indicating an increased sensitivity to gonadotropins (Lass, 2002; Chan et al., 2005). Young women and those with lean body mass are also more vulnerable to OHSS. Women with a previous history of OHSS are also at higher risk of developing OHSS in a subsequent treatment cycle. Despite taking great care to carefully monitor patients with risk factors, it is well

recognized that a good proportion of women who develop OHSS cannot be identified as being at risk before ovarian stimulation is commenced, the condition only becoming apparent once treatment has begun (Aboulghar and Mansour 2003). Patients at high risk for OHSS undergoing COH for IVF with classic ovulation induction protocols for IVF may show a decrease in oocyte and embryo quality in spite of a high number of oocytes collected. The introduction of regimes in 'hyper responding' patients should be evidencebased using a carefully planned and controlled strategy (Paul and Caroline, 2004). The Poor Ovarian Responder Patients The management of the poor ovarian responder in controlled ovarian hyper stimulation (COH) around the world has been a long-standing challenge. Although there is no clear, universal definition of the poor responder patient, they tend to represent about 10 % of patients undergoing COH treatment for of ART., despite advances in ovarian stimulation protocols and IVF laboratory techniques (Gautam. et al., 2004; Leon and Mark, 2005). Definition of Poor Responders: The original definition of poor response to COH was based only on low oestradiol concentrations, those patients who stimulated with 150 IU of human menopausal gonadotrophin (HMG) IM, and had a peak oestradiol concentration of <300 pg/ml. But most authors define the poor ovarian response in patients that develop less than four mature oocytes by the time

of human chorionic gonadotropin (hCG) administration, or a peak estradiol (E2) of less than 500 pg/ml during IVF or the patient having undergone a previous IVF cycle with a poor stimulation outcome (Gautam et al., 2004; Roest et al., 2006). Definitions of poor response should include the degree of ovarian stimulation used. A low oocyte number is only detrimental if the cumulative dose is >3000 IU FSH. Cancellation at 300 IU FSH/day is associated with a significantly worse prognosis and could define poor response (Kailasamet et al., 2004). This definition generally implies failure to achieve a certain number of mature follicles or a certain estrogen level in relation to the amount of ovarian stimulation that has been given. It is possible that women who do not respond well to a relatively low dose of gonadotrophin will response better to a higher dose, but it has been shown that increasing the dose beyond a certain level rarely improve the outcome (Leon and Mark, 2005; Perez et al., 2007). Despite these differences in definition 'poor responders' represent a heterogeneous group of patients who can be divided clinically into; Patients with low ovarian reserve and patients with normal ovarian reserve who are inherently low responders to gonadotrophin stimulation. Advanced age, previous ovarian surgery , pelvic adhesions and high body mass index may be associated with poor ovarian response (Keay et al., 2002; Akande et al., 2002).

Proper classification of the poor ovarian responder before treatment begins allows the clinician to appropriately counsel the patient on accurate prognosis and realistic chances of pregnancy; and in determining proper treatment protocols for the poor ovarian responder (Kupker et al., 2006). B. Ovarian reserve More than 100 years ago, population studies clearly documented a decrease in fertility with increasing age. In todays culture of widely available birth control and workforce equality, women often delay childbearing to pursue a career. As a result, the childbearing age for women has been delayed from the 20s to the 30s and even into the early 40s (Diczfalusy, 2002). This societal shift has resulted in an increase in the number of women who are interested in fertility and have regular cycles, but who are subfertile due to a reduction in their oocyte (egg) supply. Recognition of the profound adverse effect of a reduction in oocyte supply on fertility led to the concept of ovarian reserve and the moniker of diminished ovarian reserve ( Sun et al., 2008). The term was coined by Navot et al. in 1987 for women having an exaggerated FSH level of 26 IU/L or more (>2 SD above control value) during a clomiphene citrate (CC) challenge test. Women with diminished ovarian reserve have no overt clinical symptoms other than subfertility but do demonstrate subtle changes in baseline hormone levels (Sun et al., 2008). Ovarian reserve is a term used to describe the functional potential of the ovary and reflects the number and quality of oocytes within it. The accurate determination of ovarian reserve contributes to be a great challenge for

reproductive physicians (Macklon and Fauser, 2005).

The concept of

ovarian reserve, defined as the size and quality of the remaining ovarian follicular pool. All primordial follicles (oocytes) are formed in the human foetus between the sixth and the ninth month of gestation. The number of eggs or primordial follicles in a woman's ovaries constitutes her ovary reserve (OR) (Zeleznik, 2004). Recruitment occurs at a relatively constant rate during the first three decades of a woman's life; however, when it reaches a critical number of ~25,000 at 37.5 1.2 years of age, the rate of loss of primordial follicles accelerates ~2fold (Gougeon, 2004). Resting primordial follicles continuously enter the growing pool throughout life. The magnitude of depletion of the primordial follicle pool is dependent on age and is most pronounced during fetal development. Oocytes are detectable in fetal ovaries after 16 weeks of gestational age. The great majority of oocytes are lost after the fifth month of intrauterine life, when a maximum of approximately 7 million germ cells have been reported. At birth, both ovaries contain approximately 1 million primordial follicles. Reproductive life starts with approximately 0.5 million primordial follicles at menarche. Thereafter, loss of follicles takes place at a fixed rate of around 1000 per month, accelerating beyond the age of 35 (Erickson, 2003). Competent follicles produce inhibin-B which exerts negative feedback effects on pituitary FSH secretion. As age increases, the shrinking follicular pool secretes progressively less inhibin-b and FSH levels rise progressively, most notably in the early follicular phase. Increasing intercycle FSH concentration stimulate earlier follicular recruitment, resulting in advanced

follicular development early in the cycle and an earlier acute rise in serum estradiol levels, a shorter follicular phase, and decreasing over all cycle length . This age related physiologic mechanisms form the basis for all contemporary tests of ovarian reserve (Seifer et al., 2005). Ovarian reserve can be considered normal in conditions where stimulation with the use of exogenous gonadotrophins will result in the development of at least 810 follicles and the retrieval of a corresponding number of healthy oocytes at follicle puncture (Fasouliotis et al., 2000). With such a yield, the chances of producing a live birth through IVF are considered optimal (Broekmans et al., 2006). There is a need to identify women of relatively young age with clearly diminished reserve, as well as women around the mean age at which natural fertility on average is lost (41 years) but still with adequate OR. In clinical terms, we aim to identify women with a high risk of producing a poor response to ovarian stimulation and/or a very low probability of becoming pregnant through IVF, as well as those who still produce enough oocytes to have a good chance of becoming pregnant even if female age is advanced (Broekmans et al., 2006). If it appears possible to identify such categories of women, then management could be individualized, for instance by stimulation dose or treatment scheme adjustments (Tarlatzis et al., 2003), by counseling against initiation of IVF treatment or pertinent refusal to accept initiation, or by indicating the necessity of early initiation of treatment before reserve has diminished too far (Aboulghar and Mansour, 2003). Ovarian reserve tests help to predict the response to exogenous gonadotropin stimulation and the likelihood of success of IVF and are

widely accepted as an essential element of the evaluation of IVF candidates. Concedring the associated coasts, logistics, and risks, accurate prognostic information is very helpful to couples how may considering IVF (Leon and Marc, 2005). To date, no clear-cut predictors of ovarian responsiveness to gonadotropins have been identified. Several parameters have been postulated as predictors of the ovarian response, Screening tests studied include: age, biochemical markers (FSH, estradiol-E2, inhibin B, anti- Mullerian hormone, FSH-LH ratio) (Tremellen et al., 2005) but serum FSH remains the most widely used (Akande et al., 2003). However, intercycle variation limits both sensitivity and specificity of a single serum FSH level (Scott et al., 1996), growth hormone, insulin-like growth factor-I (Keay et al., 2003), ovarian morphometric markers (ovarian volume, antral follicle count, and mean ovarian diameter) (Bancsi et al., 2002) that are assessed in the early follicular phase (basal) of the menstrual cycle (Kupesic et al., 2002), evaluation of ovarian stromal blood flow (Kupesic et al., 2002), cigarette smoking (Kailasamet et al.,2004). Dynamic assessment of OR by methods such as the clomiphene citrate challenge test, exogenous FSH ovarian response test (Kwee, 2004) , and the GnRH-analogue stimulation test (Frattarelli et al.,2000) improves sensitivity of OR assessment, albeit at the expense of inconvenience and increasing cost (Bowen et al., 2007).

IDENTIFICATION OF DOPPLER ULTRASOUND


Doppler is a form of ultrasound, which measure the speed of the red blood cells moving alone blood vessels. It takes two principle forms, one where a color map of the blood vessels is shown on the conventional ultrasound image (Color Doppler); and another where tracing of the flow is shown on a graph so, that the speed of the flow can be measured (Spectral Doppler) (Ziadi et al., 1996). Blood flow is important because it is the method by which oxygen is transported to body organs. During a women fertility years there is a fluctuation of the blood flow during menstrual cycle. With a more blood flow to the uterus in the second half of the cycle to aid implantation of the embryo. An increase in the blood flow is also found before ovulation around healthy follicles which give an indication of the health of the oocyte (eggs) i.e. there are a dramatic changes in the ovarian volume associated with follicular growth and atresia, as well as development and regression of corpus luteum (Ziadi et al., 1996). Follicular growth are followed by an increase in the per follicular capillary net work volume. Finding strongly suggests that the vascular supply plays a critical role in the selection of the dominant follicle that is destined to mature and ovulate (Dickey et al., 1997). After the menopause blood flow to the uterus decrease due to fall in the estrogen and when this occurs the effectiveness of hormone

replacement therapy can be initiated by measurement the increase in the blood flow (Zaidi et al., 1998).

Diagnostic power of Doppler ultrasound examination


Doppler ultrasound assessment of endometrium predicts successful embryo implantation in IVF cycles as successful implantation depends on multiple factors including embryo quality and endometrial receptivity, although the contribution of embryo quality to implantation has been studied extensively, the non invasive assessment of the endometrial receptivity is much more difficult, there is no consensus in the literature as on the predictive value of endometrial thickness or morphology on implantation rates. Transvaginal pulsed and color Doppler emerged as a useful tool in the non invasive evaluation of the endometrial receptivity. Various workers have confirmed the predictive value of uterine artery impedance indices on implantation rates, measured after pituitary suppression, on the day of hCG and on the day of embryo transfer. Street and Co-workers (1992) were the first to show that an increase in the uterus artery impedance, as measured by transvaginal color flow imaging is associated with poor implantation and when uterine artery PI is greater than 3.0 there is an absent sub endometrial blood flow (Riccabona et al., 1996; Chein et al., 2004). Recent advances in ultrasound technology have made accurate non invasive assessment of the pelvic organ feasible. Transvaginal

color and pulsated Doppler ultrasonography has become an important non invasive tool in the evaluation of utero-ovarian perfusion during menstrual cycle and in vitro fertilization treatment (IVF) (Ng et al., 2006). Adequate ovarian blood flow is an important precondition for normal physiological ovarian function. The use of transvaginal color Doppler and pulsed Doppler ultrasound now permit a non invasive assessment and prediction of ovarian response. Women with polycystic ovaries syndrome have an increased ovarian stromal blood flow velocity in the early follicular phase of the normal menstrual cycle. This increase in the ovarian stromal blood flow velocity had also been observed after pituitary suppression and after controlled superovulation in women undergoing IVF treatment. It has also been shown that women with polycystic ovarian syndrome have a higher serum concentration of vascular endothelial growth factor (VEGF) which may account for the increase ovarian vascularity seen in these patients. The increased ovarian vascularity may, in turn, partly explain the increase sensitivity to gonadotropin stimulation and the increased rate of OHSS observed in these women. Furthermore significant rise in the serum VEGF concentration after human chronic gonadotropin (hCG) administration appears to be the single most important predictor of OHSS (Engmann et al., 1999).

Three- dimensional ultrasound

Three- dimensional ultrasound technology has the ability to visualize planes orthogonal to the transducer face, which had not been possible with conventional two- dimensional ultrasound. The development of three dimension equipment allows the acquisition of volume data, reconstruction of the volume image and simultaneous viewing of the three orthogonal planes. These developments are associated with important advantages over two dimensional ultrasound. The ability to visualize the oblique or coronal plane allows accurate volume measurements, especially or irregularly shape objects, because individual variations in structure can be accurately broken during the measuring process. These measurements are therefore reliable and highly reproducible. Storage and subsequent detailed evaluation of acquired volume data and image projection in any orientation may help to resolve diagnostic uncertainties, for example, for the diagnosis of congenital uterine anomalies (Kupesic et al., 2002; Jurkovic et al., 1995). Assessment of uterine morphology and exclusion of endometrial pathology are essential before commencement of treatment during assisted reproduction treatment (Kupesic et al., 2002; Kyei et al., 1995). Three- dimensional- ultrasound allows a non- invasive and accurate assessment of congenital anomalies because it provides a more accurate spatial visualization and quantitative information on endometrial cavity and quantitative information on endometrial cavity

and myometrium than two- dimensional ultrasound. In a study by Jurkovic and colleagues suing three-dimensional ultrasound, they were able to diagnose all major uterine anomalies and to distinguish between sub-septate and bicornuate uteri. One major pitfall, however, is that the presence of large uterine fibroids may prevent adequate assessment of uterine morphology (Al-Took et al., 1999; Jurkovic et al., 1995). Three- dimensional ultrasound may improve diagnostic accuracy of polycystic ovaries before commencement of assisted conception treatment in order to determine the appropriate starting dose of gonadotropins. Increased ovarian stroma is an essential criterion for the morphological diagnosis of polycystic ovaries. Accurate objective assessment of ovarian stromal volume can be made using three dimensional ultrasound by stracting the volume of the follicles from the total ovarian volume, which was not previously possible with conventional two dimensional ultrasound. Using this technique, women with polycystic ovaries have an increased ovarian stromal volume, the total follicular volume is not significantly different from that of women with normal ovarian morphology and the increased, ovarian stromal volume is associated with increased production of the retrieved steroids. The increase mean ovarian volume found in women with polycystic ovaries is, therefore, a reflection of increased ovarian stromal volume rather than increased cyst volume. Other groups have also shown a higher degree of accuracy for three dimensional assessment of ovarian stromal and total volumes when compared with

two dimensional ultrasound. Increased stromal echogenicity observed in women with polycystic ovaries compared with normal ovaries reflects the appearance cause by an increased total stromal volume and lower mean echogenicity of the entire ovary rather than any actual increase in mean stromal echogenicity. Ultrasound monitoring of follicular response during ovarian stimulation is an integral part of assisted reproduction technologies. It is well recognized that the follicular size and follicular fluid volume are related to oocyte maturity, oocyte retrieved rate. It is imperative, therefore, that actual follicular measurements are contained in order to increase the likelihood of obtained mature oocyte and estimation of follicular volume is more accurate using three dimensional ultrasound measurements which is not influenced by the shape or the size of the follicles (Kupesic et al., 2002; Merce et al., 2006; Feichtinger et al., 1998) The measurements of follicular volume obtained by three dimensional technique were all within 1 mL of the true follicular volume, as determined by the volume of aspirates, while the limits of agreements using two dimensional ultrasound were 2.5 mL below or 3.5 mL above the true volume. Follicular aspiration using three dimensional ultrasounds has been reported, but at the moment is unlikely to become routine. The value of measuring the endometrial thickness and assessing its morphological appearance to predict the likelihood of implantation is somewhat controversial. One of the reasons may be the subjective

natures of assessing the thickness and the appearance of the endometrium. Endometrial volume measurement by three dimensional ultrasound is highly reproducible, but it remains to be seen whether objective assessment of endometrial reflectively and volume by three dimensional ultrasound is useful in predicting the chances of implantation (Wu et al., 1998).

Doppler studies:
Doppler studies were performed concomitantly with ultrasonography, using the same ultrasound machine. Ovarian and uterine vascularity were studied. The following indices were measured: resistance index (RI), Pulsatility index (PI), and (PSV) peak systolic velocity.

Doppler indices
Because of inherent difficulties in quantitatively evaluating blood flow the blood flow velocity waveform has commonly been interpreted to distinguish patterns associated with high and low resistance in the distal vascular tree (Fig. 16). Three indices are in common use, the systolic/ diastolic ratio (S/D ratio), the pukatility index (PI, also called the impedance index), and the resistance index (RI, also called the pourcelot ratio). (Zalud et al., 1994) The S/D ratio is the simplest but it is irrelevant when diastolic velocities are absent, and the ratio become infinite.

Definitions of RI and PI are as follows: Resistance index RI= Pulsatility index


S D S

PI=

S D (Mean)

The RI is moderately complicated but the appeal of approaching 1.00 when diastolic velocities are abnormally low and does, therefore, reflect the relative impairment of flow by high resistance. These indices are ratios, independent of the angle between the ultrasound beam and the insonated blood vessel, and therefore not dependent on absolute measurement of true velocity (Zalud et al., 1994). The PI requires computer assisted calculation of mean velocity. The three indices are highly correlated (3, 4). There are intrinsic error in all that have been quantifies and lie between.10 and 20%. There may be advantages to the RI or PI where flow is markedly abnormally or in early pregnancy, when a very low end diastolic velocity can be a normal finding (Zalud et al., 1994).

Figure (16): Spectrum parts used in the calculation of RI and PI

Instrumentation for Doppler measurements There are two basic technological methods of reapplication of the Doppler effect in medicine (Fig. 17). It is possible to transmit and receive ultrasound waves continuously with a probe that contains a transmission transducer and a reception transducer (continuous wave in Fig. 17). Another possibility is to transmit in the form of pulses whose Doppler shift is measured after the time necessary for ultrasound to reach a defined depth in the body (pulse wave in Fig. 17).

Figure (17): Continuous wave (CW) and pulse (PW) Doppler

If, however, one must measure the flow in a single blood vessel, the PW system used (Derchi et al., 1992).

Methods of Assessing Ovarian Reserve: and its impact on IVF results 1-Chronological Age (Maternal age) Natural fertility rates, decline as maternal age increases (Fertility in women peaks between the ages of 20 and 24 and then steadily decreases, by 4-8% for ages 25-29, 15-19% for ages 30-34, 26-46% for ages 35-39, and by as much as 95% after the age of 40 (Maroulis et al., 2005). In an IVF program, ovarian aging is characterized by decreased ovarian responsiveness to gonadotropin administration and lowered pregnancy rates (Hendriks et al., 2005). It is well established that a womans advancing age is directly correlated with lower ovarian response to ovarian stimulation and to declining pregnancy prognosis, a 94% pregnancy rate in patients less than 25 years old. This declined to 57% in women between the ages of 36 and 40y (Hull et al., 2005). The introduction in the 1960s of reliable methods of contraception has led to the birth of fewer children per family. Driven by increasing levels of female education, a growing participation in labor force and career demands, postponement of childbearing has been a secondary consequence of the socalled sexual revolution (Leridon, 1998). These societal changes in family planning have caused a significant increase in the incidence of unwanted infertility due to female reproductive ageing (Ventura et al., 2001). The precise reason for this loss of fertility is not understood. There are thought to be a number of factors, including a decline in the frequency of intercourse, decreasing numbers of primordial follicles, poor oocyte quality, and problems in the uterus and embryo loss sometimes due to chromosomal abnormalities (Ventura et al., 2001).

Advancing maternal age can adversely affect implantation rates, and increase the risk of miscarriage (Spandorfer et al., 2000; Leon and Marc, 2005). The studies looked at IVF outcomes prospectively found a stronger impact of diminished ovarian reserve in patients compared to the effects of their chronological age in terms of implantation, clinical pregnancy and live birth rates (Eltoukhy et al., 2002). Specifically, patients with diminished ovarian reserve were recruited to show that this has a more significant impact on IVF outcomes than age alone (Gautam et al., 2004). Age and regularity of menses alone are unreliable ways of predicting ovarian reserve. Biological age is more reliable than chronological age. In the aging process, the ovaries become progressively less responsive to exogenous gonadotropins, until they are totally refractory at the time of menopause. Oddly the ovaries cease to respond to stimulation even though some follicles still remain in the stroma (McVeigh and Lass, 2004).Age alone is a fairly reasonable predictor of fecundity in patients with normal ovarian reserve, but that it is a poor prognostic indicator in patients with any degree of diminished ovarian reserve (Scott et al., 1991). Many studies point to 40 years of age as a significant cut-off for effectiveness of IVF (Lergo et al., 1997). The concept of poor response as a feature of chronological and ovarian aging has been supported by many studies linking poor response to ovarian hyperstimulation to subsequent early menopause (Lawson et al., 2003).

A major individual variability exists in follicle pool depletion within the normal range of menopausal age and complete follicle pool exhaustion may occur between 40 and 60 years (teVelde and Pearson, 2002). Evidence from many lines of investigation strongly suggests that the primary cause of these age-dependant changes in reproductive performance is an increasing prevalence of aneuploidy in aging oocytes resulting from disordered regulatory mechanisms governing meitotic spindle formation and function (Pellestor et al., 2003; Leon and Marc, 2005). In addition to the decline in number of oogonia with age, there is evidence to show that is also a decline in oocyte quality with increasing maternal age. For women less than 34y the rate of genetic aberrations was 24%. Between the ages of 35y and 39y, the rate was 52% and in women 40 years and older the rate was 95.8% (Hull et al., 2005). Patients presenting for IVF treatment cannot be counseled on the basis of age alone. A large number of these patients may have some degree of diminish ovarian reserve regardless of age and require more accurate prognostic tests before treatment is initiated (Van Zonneveld et al.,2003).

2-Laboratory tests a. Cycle Day 3 FSH Levels As women ages, FSH becomes elevated in an attempt to force the aging ovary to respond. However, the exact mechanism responsible for this adaptive response remains unknown (Mukherjee et al., 1996). Once the ovary is more or less exhausted, increased pituitary production of FSH follows. These events take place a few years before the actual menopause (Toner et al., 1991). Basal FSH has been reported to be a better predictor than age of ovarian response in IVF cycles stimulated with gonadotropins (Akira 2005). The monotropic rise of FSH in association with ageing is the result of a decline in ovarian hormonal feedback, in particular that of inhibin B (Welt et al., 1999; Klein et al., 2004). Also in younger subfertility patients with elevated FSH, lower inhibin levels are found, indicating limited ovarian function. This limitation is the result of a quantitative and qualitative demise of available follicles. In subfertility patients with elevated FSH it has been shown that the threshold for FSH of the follicle is slightly increased (Pal et al., 2004) which suggests that the ovary is less sensitive to FSH. Theoretically such patients may have FSH receptors which are less sensitive to FSH (Van Montfrans et al., 2004; van Rooij et al., 2004). Early follicular phase fluctuations in FSH are a reflection of the balance between ovarian steroid and peptide inhibition and the hypothalamopituitary drive during the period just before the selection of the dominant follicle. Day 3 FSH is an indirect measure of the size of the follicle cohort (from which early antral follicles can be recruited to ovulate) and is

regulated by various factors, including inhibins, activins, estradiol and follistatins (teVelde and Pearson, 2002). The basal FSH level can show marked intercycle fluctuation and that patients with baseline values in the normal range may have a diminished ovarian reserve (Akira, 2005). More than 50 percent of patients with an initial basal FSH value > 12 mlU/ ml remaining elevated in a subsequent cycle. Some reports suggest that a distinction should be made between younger and older patients with elevated FSH in the early follicular phase. In younger subfertility patients with elevated FSH, lower inhibin levels are found; indicating limited ovarian function (Klein, 2004). The current opinion is that the decline in the ovarian follicle pool is reflected by a drop in granulosa cell inhibin production, which leads to a loss of restraint of FSH. FSH levels rise and accelerate follicle growth in the diminished but still responsive follicles, causing an increase in E2 secretion as well. Thus, high basal FSH and E2 levels in the early follicular phase negatively correlate with the number of recruited follicles and the number of oocytes retrieved (Dumesic et al., 2001). From a pathophysiological point of view, large inter-cycle variations in basal FSH remain a frequent problem. Appropriate timing of FSH measurement is difficult for women with irregular periods, such as those with polycystic ovary syndrome (PCOS). Despite appropriately timed methods of sample collection, inter-cycle variations and inter-sample variations (within assay and between assays) may result in disparate FSH measurements (Lambalk and de Koning, 1998). The ovarian response to COH may be strongly dependent on the FSH receptor genotype (Lambalk and de Koning, 1998). The different variants of

receptor genotype have been related to different basal FSH levels and the different numbers of FSH ampouls needed to achieve ovarian response. In a variant of the FSH receptor protein, the amino acid asparagine is reolaced by serine at position 680 (Sudo et al., 2002). This change leads to a slightly less active FSH receptors that requires higher FSH levels for function and is probably not related to a decreased ovarian reserve ( Lambalk and de Koning, 1998). Although basal FSH concentration measured prior to the treatment cycle is widely used in many IVF programms, The limitation of FSH in estimating ovarian reserve and counseling patients has been recognized (Sharara et al., 1998), and the usefulness of FSH as a routine test in the prediction of IVF outcome has been questioned before (Bancsi et al., 2003). There is some evidence to support the predictive value of FSH in a population of women at high risk (women >40 years of age, women with poor response to ovarian stimulation and women who have failed to conceive in previous cycles) in terms of the likelihood of achieving pregnancy through assisted reproduction (Barnhart and Osheroff, 1999). In contrast, the role of day 3 FSH in the evaluation of young healthy women is extremely limited (Wolff and Taylor, 2004). A meta-analysis by (Bancsi et al., 2003) showed that the performance of basal FSH concentration for predicting poor response was moderate and the performance for predicting no pregnancy was poor. A normal basal FSH level (<10miu/ml) and young chronological age (<35 years) are generally acknowledged as the two most promising prognostic factors, reflecting ovarian function in women initiating fertility treatment (Van Rooij et al., 2004).

The Day 3 FSH levels above 15mIU/ml showed a significant declined in pregnancy rate and very few pregnancies were seen with level 25 mIU/ml. Patient with a low basal FSH level concentration <15mIU/ml, had an ongoing pregnancy rate of 9.3 %. Ongoing pregnancy rates of only 3.6 % were seen in patients with basal levels 25mIU/ml (Hansen et al., 1996). It seems, however, that the predictive value of basal FSH in the general subfertility patient is of much less value and is unable, even with high threshold values, to distinguish clearly between those patients who will have a baby and those who will not (van Rooij et al., 2004). A comparison of FSH levels in patients with one ovary to those with two ovaries, showed statistically similar ovarian responses to gonadotropins, pregnancy rates and delivery rates after controlling for the higher basal FSH levels initially found in the patients with one ovary (Lass et al., 2000). Serum markers such as basal FSH: LH ratios have not been shown to be of an added benefit over other serum markers in predicting pregnancy outcomes in IVF (Barroso et al., 2003). Weghofer et al., 2005 postulated that, as long as patients are still capable of producing a minimal number of oocytes of acceptable quality, they will also produce adequate numbers of good quality embryos for a single embryo transfer consequently high basal FSH levels especially in young patients, should not serve as exclusion criteria from fertility treatment, but as a guidance to individual patient counseling and should be interpreted according to the patient age and not in absolute terms , even within the generally considered normal range of < 10miu/ml. Basal FSH is simple to perform but does not diagnose poor ovarian reserve until high thresholds are used. Combined with other markers, such as age and antral follicle count (AFC), FSH can be useful for counseling

regarding poor ovarian response. As a test, it does not predict pregnancy and should not be used to exclude people from assisted reproduction technology (ART), especially regularly cycling young women (Maheshwari et al., 2006). Several studies have attempted to correlate the frequency distribution of FSH receptor polymorphisms and ovarian function. In most studies no association between FSH receptor variant and pathological ovarian function was shown in women with PCOS compared with control subjects (Tong et al., 2001). However, recent studies based on larger numbers of subjects identified a significant correlation (Sudo et al., 2002), and between the homozygous Ser at position 680 type II amenorrhoea. Therefore, it is still unclear whether the polymorphisms in exon 10 play a pathogenic or even only a permissive role in chronic anovulation. Significantly higher serum FSH levels in women with homozygous Ser at position 680 have been reported both in normal ovulatory subjects and in anovulatory patients (Sudo et al., 2002), suggesting that this receptor genotype might result in a mild `resistance' to the gonadotrophin. In any case, since FSH receptor variants appear to respond differently to FSH stimulation in vivo, they might play some role in determining ovarian response to pharmacological stimulation with FSH. (Sudo., 2002). Ovarian response to FSH stimulation in different allele carriers: Gromoll and Simoni in 2001 reported that 2 allelic variants in the FSHR gene display either an alanine or threonine at position 307 and an asparagine or serine at position 680. The allelic variants are equally present and distributed according to mendelian laws in Caucasians. The authors further reported that functional studies in vitro of the 2 receptor variants

thr307/asn680 and ala307/ser680 have shown no significant differences for hormone binding and cAMP production ( Simoni et al., 1999). The type of the FSHR variant does, however, determine the ovarian response to FSH stimulation in women undergoing in vitro fertilization, with the ser680 variant displaying the lowest sensitivity to FSH (Gromoll and Simoni., 2001). Recently a polymorphic variant of the FSH receptor was found in which the amino acid asparagine (Asn) at position 680 is replaced by serine (Ser) (N680S). The N680S variant was associated with higher FSH levels in the follicular phase starting from lutealfollicular transition and more FSH was needed to obtain normal follicular response in IVF patients (Mayorga et al., 2000; Sudo et al., 2002). The latter findings suggest that this receptor variant is less sensitive to FSH and that higher endogenous FSH levels may represent a natural compensation, which is needed to enable normal follicle growth. In a group of normogonadotropic anovulatory women, the homozygous N680S variant was found to be more prevalent with higher basal FSH levels (Banicsi et al., 2002). Greb and colleagues investigated the influence of FSHR genotype on menstrual cycle dynamics in 12 women homozygous for asn680 and 9 for ser 680, all with normal menstrual cycles. The study showed that the FSH receptor ser680/ser680 genotype was associated with higher ovarian threshold to FSH, decreased negative feedback of luteal secretion to the pituitary during the intercycle transition, and longer menstrual cycles (Greb et al., 2005). Basal serum LH and FSH/LH ratios: Typically, patients with normal LH and FSH levels and those with a

high LH: FSH ratio respond as normal and high responders respectively, often yielding an adequate number of mature oocytes available for fertilization. On the other hand, patients with high FSH (or elevated E2 levels) respond poorly both in terms of oocyte numbers and quality (Muasher, 1988). There is a clear relationship between females chronological age and ovarian reserve, and both indices are used to counsel patients at the time of IVF. However, a group of young patients with normal FSH levels sometimes respond poorly to standard ovarian stimulation protocols. In this group of patients, several hypotheses have been proposed to explain the low ovarian response, but none has been proved (Pellicer et al., 1998). The identification of such patients to perform ovarian stimulation regimens using more adequate, tailored protocols represents a constant effort for physicians in order to avoid frustrating and disappointing outcome of infertility treatments. The two-cell theory suggests that both FSH and LH are needed for normal follicular growth and maturation, but until now the main role had been attributed to FSH (Taymor et al., 1996). Mukherjee et al., 1996 suggested that an elevated day 3 FSH: LH ratio >3.6, in the presence of a normal day 3 FSH is predictive of a poor response to ovarian stimulation. Similarly Noci et al., 1998 stated that low basal serum LH values < 3 IU/L, predict reduced response to ovarian stimulation as judged by decrease peak E2 and a lower number of preovulatory follicles in ovulation induction cycles. It was speculated that when early follicular LH levels are low there may be reduced activity of one or more of the known ovarian regulators (i.e., steroids or proteins such as inhbin, activin, follistin or insulin-like growth factors), which can influence follicular growth through actions by autocrine or paracrine routes.

Barroso et al., 2001 reported that IVF patients previously identified as normal responders but with a high FSH: LH ratio and low basal LH levels (and in the presence of a normal basal FSH) had a significantly lower ovarian response in terms of follicular development and a trend toward poorer implantation and pregnancy rates (suggestive of a compromised oocyte quality) when stimulated with a combination of GnRHa and pure FSH. In this group of patients a high FSH: LH ratio >3 may be used as an early biomarker of poor response to controlled ovarian hyperstimulation. A recent meta-analysis has confirmed that measuring serum LH during ovarian stimulation in ART cycles is at present of no value Kolibianakis et al., 2006. Also Kassab et al., 2007 show that the basal serum LH has no useful predictive value for IVF/ICSI clinical pregnancy and live birth outcome. Further data will be needed to determine whether evaluation of this relationship will provide clinically meaningful information. b. Follicular Phase Inhibin Levels As direct products of the granulosa cells. Inhibins are dimeric glycoproteins that is made by the ovary and named for its role in inhibiting follicle stimulating hormone (FSH), the hormone responsible for the development of ovarian follicles, theoretically might better reflect ovarian reserve as a marker of secretory capacity and follicle number ( Yong et al., 2003). Follicular granulosa cells secrete both dimmers of Inhibin hormone; inhibin A secreted in the luteal phase and inhibin B in the follicular phase (Groome et al., 1996). Inhibin A increases in the late follicular phase after the rise in serum E2 and is secreted by the dominant follicle (Hall et al., 2005). Hence, inhibin A is thought to be a marker of follicular maturity and

decreases with increasing age, which may be reflective of the fewer granulose in older women (Seifer et al., 2002). Inhibin B is a direct product of small, developing follicles in the ovary and, as such, indicates a womans ovarian reserve. The amount of inhibin B measured in serum during the early follicular phase of the menstrual cycle (days 2-6) directly reflects the number of follicles in the ovary; in other words, the higher the inhibin B, the more ovarian follicles are present (Penarrubia et al., 2000). There is a significant decline of inhibin B levels in the early follicular phase with increasing serum FSH levels and decrease further with increasing FSH concentrations and increasing age (Klein et al., 2002). Inhibin B concentrations increase during the late luteal phase and early follicular phase. Inhibin B has been postulated to represent the quantity or quality of the developing follicles in that cycle (Hall et al., 2005). Research studies have shown that the amount of inhibin B in the follicular phase of the menstrual cycle indicates the number of oocytes that will be retrieved after hormonal stimulation treatments. A higher follicular phase inhibin B level is associated with a better ovarian reserve and a higher number of follicles (oocytes) that develop in response to hormone stimulation. Moreover, it has been reported that women with very low inhibin B levels (<20 pg/ml) often have such a poor ovarian response that the IVF cycle must be cancelled ( Hazout et al., 2002). A Day 3 inhibin B level is a better predictor of IVF cancellation than age alone (Balasch et al., 1996), and it is useful in detecting women with diminished ovarian reserve who have normal day 3 FSH values. Decreases in inhibin B often precede serum FSH changes as ovarian reserve declines (Seifer et al., 2002; Walt et al., 1999). Inhibin B levels on cycle day 3 can be used as a direct measure of

ovarian reserve, with concentrations < 45 pg/ml correlating with lower E2 responses and fewer oocyte retrieved. Higher IVF cancellation rates and lower clinical pregnancy rates were also seen in women with day 3 inhibin B levels < 45 pg/ml. (Akira, 2005). Day 5 inhibin B levels were measured after 4 days of stimulation and were found to correlate with the number of mature follicles >14 mm, number of oocytes retrieved, and number fertilized oocytes. Women with levels < 400 pg/ml had poorer outcomes in all of the IVF outcome parameters, compared to those with levels > 400 pg/ml. beneficial role in early detection of either the poor responder for cancellation, or the hyperresponder for reduction of mdication dose. day 5 inhibin B levels < 100 pg/ml may be an indication for cancellation of that cycle, and that levels > 1000 pg/ml may warrant reduction in the gonadotrophin dose and close monitoring for ovarian hyperstimulation syndrome (OH). Day 5 inhibin B levels measured during treatment cycles correlated well with lack of ovarian response, but not with pregnancy outcome (Broekmans et al., 2003; Lambalk et al., 2006). b. Serum Estradiol Levels This test is indirect estimate of ovarian reserve. Basal E2 values are

beneficial in screening for the potential poor ovarian responder in the context of a "normal" FSH value, It has been shown that a day 3 E2 level can vary as much as 40% compared to day 2 or 4 values, while the FSH value only shows an 18% variance between these days. Thus, while the FSH value alone is a more accurate predictor of ovarian reserve, the E2 level has value in interpreting the FSH results. Because of the negative feedback

of elevated E2 levels on FSH secretion, a "normal" value of FSH on day 3 of a cycle may be falsely low in the face of elevated E2 levels. E2 determination with day 3 FSH assessment was superior to either test alone. (Brown et al., 1995). This particular hormone is most attractive because it is a direct product of the ovary. The theoretical principle that supports basal E2 screening is the ability to detect patients with shortened follicular phases who may have progressed far enough into their follicular phases to invalidate the evaluation of their basal FSH levels (Frattarelli et al., 2000). The early follicular phase E2 level can vary widely between days 2 to 4 and elevated levels may be present due to an early recruitment or development of a dominant follicle. This early luteal recruitment may occur when a diminished cohort of follicles produces less inhibin (Kligman et al., 2001). It is possible that the higher E2 level might suppress FSH levels into the "normal" range even when a patient has diminished ovarian reserve. Elevated follicular phase E2 levels may also be seen in the perimenopause. Regardless of age, elevated day 3 E2 levels and FSH levels have also been associated with an increased risk of recurrent pregnancy loss (Kligman et al., 2001;Trout and Seifer, 2000). It was found that low cycle day 3 E2 level combined with normal cycle day 3 FSH level have been associated with improved stimulation response, higher pregnancy rates and lower cycle cancellation rates (Evers et al., 1998). It is suggested that elevated early follicular phase estradiol levels may indicate an inappropriately advanced stage of follicular development,

consistent with ovarian aging. However, it may simply reflect the presence of functional ovarian cysts (Lockwood et al., 2004). The addition of E2 may allow clinician to identify patients who are at increased risk for cycle cancellation. Patients with basal E2 levels that are undetectable or above the normal early follicular range should not be counseled that they have diminished ovarian reserve. Although cycle outcome was poorer, elevated basal E2 levels would not seem to be a reason to cancel or postpone a patient stimulation cycle ( Frattarelli et al., 2000). Patient with basal E2 levels > 30 pg/ml had a poor ovarian response to stimulation and had a low pregnancy rate, while those with basal E2 levels> 75pg/ml had no pregnancy (Kligman et al., 2001). d. Anti-Mllerian hormone level In the adult ovary, AMH is likely to have an inhibitory effect on primordial follicle recruitment, as well as on the responsiveness of growing follicles to FSH. In contrast to most hormonal markers of the follicular status, AMH is exclusively produced by the granulosa cells of a wide range of follicles (primary to early antral stages), presumably independently of FSH and with little susceptibility to disorders of antral follicle growth during the lutealfollicular transition. This characteristic makes it a promising parameter in the evaluation of ovarian follicular reserve (Feyerisen et al., 2006). Serum AMH levels have been measured at different times during the menstrual cycle, suggesting extremely subtle or nonexistent fluctuation (Cook et al., 2000). One single hormone measurement for AMH seems sufficient and remains relatively constant during the follicular phase

and entire menstrual cycle (David, 2007). Minimal fluctuations in serum AMH levels may be consistent with continuous noncyclic growth of small follicles (La Marca et al., 2004). Serum levels on day 3 of the menstrual cycle show a progressive decrease over time in young normoovulatory women and to correlate with age, FSH and the number of antral follicles. In a study in 2005, a group of women was studied twice and the interval between the two visits ranged from 11 years to 73 years. A reduction in mean AMH levels of about 38% was observed, whereas the number of antral follicles and the levels of FSH and inhibin B did not change (van Rooij et al., 2005). With respect to other known markers, AMH seems to better reflect the continuous decline of the oocyte/follicle pool with age. The decrease in AMH with advancing age may be present before changes in currently known ageing-related variables, indicating that serum AMH levels may be the best marker for ovarian ageing and menopausal transition (van Rooij et al., 2004).

AMH levels are also seen to decline gradually during FSH administration as part of controlled ovarian hyperstimulation (COH) (La Marca et al., 2004). The reduction in AMH levels observed during FSH administration may be due to a negative role of FSH on AMH secretion (Lukas-Croisier et al., 2003. Alternatively, the reduction in AMH levels could be due to the supraphysiological increase in oestradiol levels observed when exogenous FSH is administered. Indeed, oestradiol has been implicated in the down-regulation of AMH and AMHRII mRNA in the ovary. Moreover, the decrease in AMH in FSH-treated women might be the

result of a growth stimulation by FSH of the follicles that enlarge , with dramatic reduction in the number of small antral follicles, and confirming the scarce AMH expression by larger follicles. This was confirmed in a recent study in which AMH levels in follicular fluid were evaluated. Small follicles (8-12 mm in diameter) secreted AMH at levels that were approximately three times as high as those of large follicles (16-20 mm in diameter) thereby losing their AMH expression.(Fanchin et al., 2005), AMH acts as a paracrine rather than a systemic factor, and thus is not part of a negative feedback loop with involvement of gonadotropins. COH resulting in a rise of endogenous FSH and LH, does not affect AMH serum levels (van Rooij et al., 2002). Similarly, in conditions where FSH levels are suppressed, such as pregnancy, AMH levels remain constant (La Marca et al., 2005). Thus, AMH is not influenced by the gonadotropic status and reflects only the follicle population (Fanchin et al., 2003). AMH serum levels were shown to be highly correlated with the number of antral follicles before treatment and number of oocytes retrieved upon ovarian stimulation. (van Rooij et al. 2004). Furthermore, AMH may offer greater prognostic value than other currently available serum markers of ART (Hazout et al., 2004). Multiple studies carried out concerning the efficacy of AMH in prediction of ovarian reserve (DeVet et al., 2002; Seifer et al., 2002; VavRooij et al., 2002; Fanchin et al., 2003 ). High day 3 AMH concentration 1.1ng/ml is associated with a greater number of mature oocytes, a greater number of embryos, and ultimately a higher clinical pregnancy rate. Furthermore (Hazout et al., 2004). serum AMH measurements were reported to have greater prognostic value than age,

serum FSH, inhibin B or oestradiol, also it seems to be a better marker in predicting a cancelled cycle, using a cut-off of 01 ng/ml, AMH had a sensitivity of 875% and a specificity of 722% in the prediction of cancellation (Tremellen et al., 2005). However; the application of AMH to predict ongoing pregnancy seems limited, although day 3 serum AMH levels are higher in patients that become pregnant after IVF treatment than in those who do not (Hazout et al 2004). It appears that there is a strong association between early follicular AMH and number of oocytes retrieved. Midluteal and early follicular AMH may offer a better prognostic value for clinical pregnancy than other currently available markers of ART outcome ( Elgindy et al., 2008).

AMH levels have also been shown to be 10-fold lower in the cancelled cycles compared with patients who had a completed IVF cycle. In about 75% of cancelled cycles, AMH levels were below the detection limit (Muttukrishna et al., 2004)This finding has been confirmed in a large prospective study conducted on 238 women undergoing IVF. Using a cut-off value of 113 ng/ml, AMH assessment was shown to predict ovarian reserve with a sensitivity of 80% and a specificity of 85% (Tremellen et al., 2005). Other study done by La Marca et al. (2007) included 48 women attending the IVF/ICSI programme. Blood withdrawal for AMH measurement was performed in all the patients independently of the day of the menstrual cycle. They found that women in the lowest AMH quartile (<0.4 ng/ml) were older and required a higher dose of recombinant FSH than women in the highest quartile (>7 ng/ml). All the cancelled cycles due to absent response were in the group of the lowest AMH quartile, whereas the

cancelled cycles due to risk of ovarian hyperstimulation syndrome (OHSS) were in the group of the highest AMH quartile. This study demonstrated a strong correlation between serum AMH levels and ovarian response to gonadotrophin stimulation. So, clinicians may have a reliable serum marker of ovarian response that can be measured independently of the day of the menstrual cycle.

7. Ovarian biopsy Ovarian biopsy has not been found to be a useful routine test of ovarian reserve. Apart from being invasive and posing unknown future adverse effects, ovarian biopsy is not a reliable test to assess reproductive ageing on fertility, as there is a highly varied distribution of the follicles throughout the ovary. The use of ovarian biopsy in predicting pregnancy has not been tested (Lambalk et al., 2004). 3- Clinical tests for ovarian response (Dynamic Ovarian Reserve Tests): Another approach towards identifying ovarian reserve involves dynamic testing. This involves taking a baseline serum sample, stimulating the ovaries (FSH/ Clomiphene/ GnRH agonist) and then retesting the serum level again for the same marker. All the dynamic tests are more expensive, invasive and associated with the side effects of administered stimulation regimens (Maheshwari et al., 2006) a. Clomiphene Citrate Challenge Test: The clomiphene challenge test is a good predictive value for poor response in IVF/ICSI. The use of the clomiphene challenge test may improve the predictive value of basal FSH alone (Jain et al., 2004).

The clomiphene citrate challenge test (CCCT) was originally described by Navot et al in 1987 as a means of assessing ovarian reserve in women 35 years of age or older (Navot et al., 1987). It is a more reliable predictor of diminished ovarian reserve than FSH values alone when predicting response to COH (Tanbo et al., 1992). The test checks hormone levels on the 3rd (basal) and 10th day of a patient's cycle in which 100 mg of clomiphene citrate has been taken orally from days 5 through 9. An abnormal test is defined as an abnormally high FSH on day 10 (Bukman and Heineman, 2001). This test is a dynamic assessment of the ovarian reserve indirectly. The premise of the test is that in women with normal ovarian reserve, will have enough metabolic activity from a cohort of developing follicles and the overall increase in estradiol and inhibin production by the developing follicles should be able to overcome the impact of the clomiphene citrate on the hypothalamic-pituitary axis and suppress FSH levels back into the normal range by cycle day 10 ( Sharara et al., 1998). In contrast, if FSH levels remain elevated, this is considered as an indirect sign of diminished ovarian reserve due to insufficient feedback from the ovary (Scott and Hofmann, 1995). It is considered normal when it is 9.6 mlU/ml. Values between 10 and 15mlU/ml are considered indeterminate and pregnancy is possible, but lower pregnancy rates are seen and more aggressive stimulation protocols may be required. Patients with day 3 or day 10 FSH values >or = 17 mlU/ml with a CCCT rarely become pregnant and exhibit higher miscarriage rate (Hofman et al., 2000).

The test has been shown to be of value in unmasking poor responders to controlled ovarian hyperstimulation (COH) who would not have been detected by basal screening alone. Moreover, an abnormal test is associated with a reduced chance of pregnancy (Hendriks et al., 2005). It has been suggested that the CCCT may be better than basal FSH for predicting infertility treatment outcome because two levels of FSH are obtained, and the addition of clomiphene citrate may serve to reveal women who might not be detected by basal FSH screening alone (Sharara et al.,1998). Jain et al., 2004 found that basal FSH and the CCCT were found to be of similar value in predicting a clinical pregnancy in women undergoing infertility treatment. With either test a normal result was of little predictive value, but an abnormal result predicted poor outcome from infertility treatment. Given that the CCCT offers no clear advantage compared with a single basal FSH measurement, and is it associated with potential adverse effects, basal FSH is preferred. It is important to understand that the CCCT lacks positive predictive value, it is up to 94 percent accurate in detecting patients with diminished ovarian reserve; however, it does not provide direct information concerning the ovarian response using exogenous FSH/gonadotropin in IVF (Hofmann et al., 2002).

In CCCT, stimulated day `10 FSH levels are strongly predictive of decreased IVF success even when day 3 FSH levels are normal. Results of CCCT are useful for patient counseling before the IVF cycle and for choosing the optimal gonadotropin regimen (Yanushpolsky et al., 2003). Recently, a study stated that performing CCCT (single or repeated) has a rather good ability to predict poor response in IVF. However, it appears that the predictive accuracy and clinical value of the CCCT is not clearly

better than that of basal FSH in combination with an antral follicle count (Hendriks et al., 2005). Decreased inhibin B of women with an abnormal CCCT leads to the elevated FSH value seen on cycle day 10. The CCCT detects as many as 2-3 times more women with diminished ovarian reserve than the day 3 FSH value alone (Yanushpolsky et al., 2004). The CCCT combines the day 3 FSH and E2 prognostic values with the dynamic ovarian response seen by day 10. It is important to obtain E2 values on both days to place the FSH values in context on day 3. The E2 levels drawn on day 10 help to identify patients that are unresponsive to clomiphene citrate, such as those with hypothalarnic amenorrhea. Cycle day 10 progestrone levels 1.1 ng/ml with the CCCT might be indicative of a short follicular phase and poor reproductive performance (Gutam et al., 2004). b. The exogenous FSH ovarian reserve test (EFORT): The FSH test is an effective method not only for predicting poor responders to stimulation using gonadotropin but also for estimating the necessary doses of gonadotropin (Gautam et al., 2004). A good correlation between this test and the subsequent quality of the ovarian response in IVF was observed, and the predictive value of this test for good and poor responders was higher than that of basal FSH alone (David, 2007). However, the duration of administration of exogenous gonadotropin in IVF usually ranges from 7 to 10 days, and some normal responders have

slow follicular growth and E2 development. Therefore, it might be difficult to conclude that E2 response 24 hours after 1 injection of gonadotrophins reflects the ovarian response in IVF (Pull and Carollin, 2004). It is a dynamic test for assessment of ovarian reserve evaluating the estradiol serum concentration change from cycle day 2 to day 3 after the administration of a supraphysiological dose of a GnRH agonist. The latter causing a temporary increase in pituitary secretion of FSH and LH. In response the ovaries will produce E2. The test is dependent on the pituitary production of gonadotrophins and the response of the ovary to stimulation (i.e. follicle reserve) (Ranieri et al., 1998). Originally, the test was developed to improve the predictive value of day 3 FSH values in COH for IVF. Specifically, the E2 level is recorded on cycle day 3 before and 24 hours after the administration of 300 IU of purified FSH. It was postulated that the dynamic increase in E2 =30 pg/ml would be predictive of a good response in a subsequent IVF cycle (Kwee et al., 2004). The GAST test can also measure dynamic inhibin B response. Measuring the rise in inhibin B after GnRHa administration was found to be better than age and basal FSH in predicting IVF response in a group of unselected patients (Ravhon et al., 2000). Both E2 and inhibin B are produced by granulose cells. When measuring the basal concentration of these hormones in the early follicular phase different concentrations are considered as predictor of ovarian reserve. Higher inhibin B predicts better ovarian reserve; in contrast higher basal E2 concentrations predicts lower ovarian reserve (Seifer et al., 1997). The dynamic assays of E2 and inhibin B have similar predictive

properties for ovarian response to gonadotrophin stimulation (with E2 being slightly more accurate). Combining E2 and inhibin B slightly improves the power of prediction comparing with using E2 alone. However, measuring E2 is much simpler and cheaper than measuring inhibin B and it seems that for clinical practice measuring only E2 in a dynamic test is reliable enough (Ravhon et al., 2000). There is a significant prognostic value of the 24-hour change in inhibin B serum levels with the EFORT. The poor responder showed a less increase in inhibin B levels as compared with the good responders. Thus, this provocative serum marker may be useful in identifying poor ovarian responders before IVF (Dzik et al., 2000). Earlier ART studies did not show any significant benefit in the prediction of ovarian response (Padilla et al., 1990; Winslow et al., 1991); however, later studies did (Hendriks et al., 2005). Although, when compared with the predictive accuracy and clinical value of the day 3 AFC and inhibin-B measurement, GAST did not perform better. In addition, its predictive ability towards ongoing pregnancy is poor (Hendriks et al., 2005). While others confirmed the importance of GAST and stated that performing a GnRHa stimulation test allows for the accurate prediction of ovarian response to stimulation (Ravhon et al., 2000). (Scheffer et al., 2003) demonstrated that, The GAST is a superior test in the prediction of outcome in assisted reproduction treatment. It may be considered the second best single test to predict reproductive aging. Compared with the exogenous FSH ovarian reserve test, the HMG test is more practical. They assessed E2 response to the administration of 150 IU

of HMG for 5 days from the second or third day as a predictor of cycle cancellation in IVF. Their results demonstrated that the HMG test showed a better correlation with cycle cancellation than basal FSH (Kwee et al., 2004). c. GnRH- Stimulation Test (GAST): This test was introduced as a screening test for good and poor responders in IVF cycles. Day 3 FSH and E2 serum concentrations are determined, as well as the E2 response following a 300IU FSH injection on day 3. The addition of the dynamic component (E2) to the cycle day 3 FSH concentrations might be an improvement of the predictive value of good response to ovarian stimulation, not only determine poor responders, but a predictor for the cohort size as well (Fanchin et al., 1994). The test valutes the change in serum E2 levels between cycle day 2 and 3 after l mg of subcutaneous leuprolide actate is administered. Different patterns of E2 levels will be noted. Patients with E2 elevations by day 2 and declines by day 3 had better implantation and pregnancy rates than those patients with either no rise in E2, or persistently elevated E2 levels (Winslow et al., 2002; Fanchin et al., 2007). The ovarian response to this timed stimulation could help not only to predict future ovarian stimulation results, but also with adjusting the initial dose of exogenous gonadotrophin required. It was evolved due to the fact that stated FSH concentrations during the early follicular phase can show marked intercycle fluctuations. Furthermore, plasma FSH concentration on cycle day 3 does not provide direct information concerning the responsiveness of the ovaries to the exogenous gonadotrophins used in ovarian stimulation for IVF (Fanchin et al., 1994).

The EFFORT is a simple and effective method for detecting good and poor responders in IVF and provides a useful complement to the classical basal FSH measurements by improving the specificity and sensitivity of this later test (Dzik et al., 2000). Another study discusses the inhibin-B response to EFFORT in an attempt to predict ovarian response to hyperstimulation in IVF. It measured inhibin-B levels before and 24 hours after administrating a fixed dose of 300 IU FSH on cycle day 3. The results showed that the good responders had 67% increases from the baseline, while those poor responders had only 70% increase from the base line. These data indicate that women with higher baseline inhibine-B and a greater inhibine-B response to EFFORT have no diminished ovarian reserve. Conversely, women whose IVF cycles were cancelled because of failed oocyte retrieval had a low inhibin-B level, both at baseline and in response to EFFORT ( Eldar-Geva et al., 2000 and Yong et al., 2003). The intercycle variability of the inhibin-B increment and the E2 increment in the EFFORT is stable in consecutive cycles, which indicates that this reproducible test is a more reliable tool for determination of ovarian reserve than other tests. It is the endocrine test, which gives the best prediction of ovarian capacity (Kwee et al., 2003). The most recent comparison of the GAST with other tests of ovarian reserve found that the test to be the least sensitive, and less accurate than all the other tests. The GAST has not been evaluated in non-IVF populations and perhaps further studies are needed before it is accepted as a standard test of ovarian reserve (Gulekli et al., 1999). 4-Sonographic Assessment: Transvaginal ultrasonography has proved to be an easy and

noninvasive method to provide essential information on the ovarian responsiveness before the initiation of gonadotrophin stimulation (Kupesic et al., 2003). Ultrasound is essential in the modern management of couples undergoing IVF treatment because it is used to predict and monitor the ovarian response, assess endometrial receptivity, and guide the transvaginal aspiration of oocytes and subsequent transcervical transfer of embryos to the uterus. Several ultrasound parameters have been examined to predict the ovarian response to gonadotrophins, including ovarian volume ( Syrop et al., 1999), antral follicle count (Bancsi et al., 2002) and ovarian stromal blood flow (Popovic-Todorovic et al., 2003). Basal mean ovarian volume (MOV): The test is simple to perform and shows little inter-observer variation. Ovarian volume assessment is done in the luteal phase or early follicular phase (Tomas et al., 1997; Syrop et al., 2005). One caveat to the assessment of ovarian volume is that the ovaries should not contain cysts or large follicles (only follicles < 10-15 mm were allowed) (Jarvela et al., 2003). The ellipsoid formula (length x height x width), which simplifies to 0.526 x length x height x width. Probably the simplest and most accurate test of ovarian reserve is the measurement of total ovarian volume as measured by high-resolution ultrasound (Wallace and Kelsey, 2004). As the bulk of the ovary is made up of antral follicles in the absence of a corpus luteum, total volume relates closely with total antral follicles, MOV correlates with the ovarian reserve (Yong et al., 2003). The mean ovarian volume increases from 0.7 ml at 10 years to 5.8 ml at 17 years of age. It has been suggested that there are no major changes in ovarian volume during reproductive years until the premenopausal period. In women > 40 years old, there is a dramatic drop in ovarian volume, which is not related to parity. Thereafter, there is a

further sharp decline in size in postmenopausal women which seems mostly related to the time when menstruation ceases, rather than merely to age, because when oestrogen treatments were given, there appeared to be no decrease in ovarian volume with age (Scheffer et al., 2003). Mean ovarian volume was 6.6 ml in women <30 years old, 6.1 ml in women 30-39 years, 4.8 ml in women aged 40-49 years, 2.6 ml in women 50-59 years old and 2.1 ml in women aged 60-69 years old. Mean ovarian volume was 4.9 ml in premenopausal women and 2.2 ml in postmenopausal women (Pavlik et al., 2000). (Erderm et al., 2004; Ozkaya et al., 2004 ) demonstrated that mean ovarian volume estimation by transvaginal ultrasonography might be more useful than basal FSH values, CCCT, and GnRH agonist stimulation test for predicting ovarian response. With age and smoking status accounted ovarian volume is a better measure of ovarian reserve than basal FSH values (Syrop et al 2005). Although MOV correlated with IVF stimulation parameters, its use as an adjunct in counseling patients during IVF appears to be of limited value. A MOV < 2cm was associated clinically with a higher cancellation rate. Small ovaries are associated with poor response to gonadotrophins and a very high cancellation rate during IVF. Ovarian volume of < 3cm, was significantly predictive of higher IVF cancellation rates (> 50%) compared with patients who's smallest ovarian volume was > 3 cm and and a lower pregnancy rate in those cycles not cancelled regardless patients ages, and excluding polycystic ovarian syndrome patients. These patients required more ampoules of gonadotropins during stimulation, had poorer follicular development and yielded fewer oocytes. There was no absolute MOV that was predictive of pregnancy outcome or cycle cancellation ( Frattarelli et

al., 2004). (Tomas et al., 1997; Syrop et al., 2005). The mean ovarian diameter measured in the largest sagital plane is also useful. A comparison showed it to be a quick, yet reliable estimate of the measured ovarian volume. Assessment of ovarian volume sonographically can be a useful modality in identifying and counseling patients that may have a poor ovarian response before they undergo COH (Frattarelli et al., 2002). Antral follicle counts AFC, as visualized by transvaginal ultrasound scan, has attracted considerable interest as a test of ovarian reserve (van Rooij et al., 2005). It may be considered the test of first choice when estimating quantitative ovarian reserve before IVF (Hendriks et al., 2007). Tomas et al. (1997) and Chang et al. (1998) introduced the antral follicle count (AFC) as an easy-to-perform and noninvasive method to provide essential information on ovarian responsiveness before initiation of gonadotropin stimulation in IVF. It is the antral follicles that respond to stimulation and was defined as the number of follicles smaller than 10 mm (follicles 2-5 mm) in diameter detected by transvaginal ultrasound in early follicular phase (Ilkka et al., 2003). Inactive ovaries with < 5 follicles in both ovaries (Fig ), normal ovaries" with 5-10 follicles total,(Fig ). (, and "polycystic ovaries" with > 15 follicles counted (Fig ) ( Frattarelli et al., 2002). (Table 2) show the correlation between total antral follicle count and expected ovarian response (Advanced Fertility Center of Chicago, 2005 ). ( Table 3) explains the correlation between the total antral follicle count and expected fertility potential for women under 37 years ( Advanced Fertility Center of Chicago, 2005).

Table 2: Comparison between total antral follicle count and expected ovarian response (Advanced Fertility Center of Chicago, 2005). Total antral follicle count Extremely low count, very poor (or no) response to stimulation Less than 4 and a cancelled cycle expected. Should seriously consider not attempting IVF at all. Rare pregnancies if IVF attempted. Low count, we are concerned about a possible/probable poor response to the stimulation drugs. Likely to need high doses of FSH product to stimulate ovaries 4-7 adequately. Higher than average rate of IVF cycle cancellation. Lower than average pregnancy rates for those cases that make it to egg retrieval. The reduction in success rates is more pronounced beyond age 35. Somewhat reduced count. 8-10 Higher than average rate of IVF cycle cancellation. Slightly reduced chances for pregnancy as a group. Normal (but intermediate) count, the response to drug stimulation is sometimes low, but usually good. 11-14 Slight increased risk for IVF cycle cancellation. Pregnancy rates as a group only slightly reduced compared to the "best" group. Expected response to injectable ovarian stimulating drug (FSH product) and chances for success

Normal (good) antral count, should have an excellent response to ovarian stimulation. 15-26 Likely to respond well to low doses of FSH product. Very low risk for IVF cycle cancellation. Some risk for ovarian overstimulation. Best pregnancy rates overall as a group. High count, watch for polycystic ovary type of ovarian response. Likely to have a high response to low doses of FSH product. Over 26 Higher than average risk for overstimulation. Very good pregnancy rate overall as a group, but some cases in the group have egg quality issues and lower chances for pregnancy.

Table 3: Comparison between total antral follicle count and expected fertility potential for women under 37 years (Advanced Fertility Center of Chicago, 2005). Total number of antral follicles Less than 4 Expected fertility potential for women under 37 For 37 and older we need to be more cautious low antrals and late 30's or early 40's is significantly worse Extremely lowcount. I think that there is a high risk of poor fertility 5-7 potential. Very low count. I think fertility issues are possible - either soon, or 8-11 within several years (very hard to predict). Intermediate count.

It is possible that some fertility issues are already present. I am concerned about fertility issues sometime in the future. It appears that the clock is ticking faster than 12-14 we'd like... Low end of the average range. I am not worried at all yet. However, as antral counts drop over time, fertility Over 14 issues might develop. Normal count. I expect excellent fertility potential. At least for now, the clock seems to be ticking at a normal rate.

Fig. 3: Ultrasound image of an ovary at the beginning of a menstrual cycle; there are numerous antral follicles, 16 are seen in this image, This is a polycystic ovary, with a higher than average antral count and volume. This woman had very irregular periods and was a "high responder" to injectable FSH medication. (Advanced Fertility Center of Chicago, 2005).

Normal ovarian volume and "normal" antral follicle counts

Fig. 4 Ultrasound image of an ovary at the beginning of a menstrual cycle; 9 antral follicles are seen. The ovary has normal volume (cursors measuring ovary = 30 by 17.8mm). This woman had regular periods and a normal response to injectable FSH drugs. (Advanced Fertility Center of Chicago, 2005). Low ovarian volume and low antral follicle counts

Fig. 5: Both ovaries are small; the left ovary showing only one antral follicle. While the right ovary showing two antral follicles. This woman had regular periods and a normal day 3 FSH test. She only had 3 antral follicles total - from both ovaries. Attempts to stimulate her ovaries for IVF were not successful. (Advanced Fertility Center of Chicago, 2005).

Transvaginal ultrasound measurement of antral follicle count is quick, accurate and cost effective and permits the identification of a group of patients for which ovarian stimulation will not be effective (Scheffer et al., 1999). A single ultrasonographer assessed AFC's during the early follicular phase without any pituitary suppression, is the single best predictor for poor ovarian response in women undergoing their first IVF cycle (Banicsi et al., 2002). Measuring the number of antral follicles on ultrasound just prior to the start of the stimulation with gonadotrophins is a simple procedure, with a good intra- and inter-observer reproducibility (Scheffer et al., 2002). It provides important information on what to expect from the subsequent IVF treatment. When a low number of antral follicles are found, the patient is at high risk of developing a poor response and a high cancellation rate supporting the concept of reduced numbers of primordial follicles delivering a small antral follicle cohort, whereas a high number of antral follicles predict not only good response but also sometimes an increased risk for ovarian hyperstimulation syndrome (Chan et al., 2005). Several publications have suggested that the AFC could be used to optimize stimulation protocols in IVF (Kupesic et al., 2003). l follicles are associated with decreased ovarian response during controlled ovarian hyperstimulation for IVF,

Moreover, Chang et al. (1998) reported a trend toward lower pregnancy rates in women with few antral follicles. High ovarian volume and high antral follicle counts. The number of antral follicles decreases proportionately with age and day 3 FSH levels. Before the age of 37 years the AFC showed a mean yearly decline of 4.8 %, compared with 11.7% thereafter. Hence, the AFC in both ovaries could be related to reproductive age and could well reflect to reproductive age and could well reflect the size of the remaining primordial follicular pool (Scheffer et al., 2003). The explanation for this correlation is believed to be that antral follicles are the main origin of inhibin B secretion, which decreases the release of pituitary FSH into the blood stream. A decrease in the ovarian cohort of antral follicles increases the serum FSH level. This suggests that an early menstrual antral follicle count may be available biomarker of ovarian reserve (Chang et al., 1998). Total follicle count correlates positively with the number of oocytes retrieved and negatively with day 3 FSH and ampoules of gonadotrophins, with fewer than 10 total follicles predicting an increased chance of cancellation (Fratterlli et al., 2000). By multivariate analysis, antral follicle count was found to be the best single predictor of ovarian response and therefore prognosis, with FSH having a small additive effect ( Bancsi et al., 2002). Klinkert et al., 2005 demonstrated that AFC has been suggested to be a better marker than age and FSH for distinguishing between older patients with good and poor pregnancy prospects because it shows a better correlation with the number of oocytes at oocyte retrieval (Bancsi et al., 2002). Ovarian volume did correlate with the AFC. Three-dimensional (3D)

ultrasound, might be superior for ovarian volume measurement, and more sensitive in detecting smaller antral follicles (Orvieto, 2005). The increase in ovarian power Doppler signal during gonadotrophin stimulation is related to the antral follicle count observed after pituitary suppression. The number of small follicles present before ovarian stimulation was a better predictor of IVF outcome than ovarian volume alone (Akira et al., 2005). Also, Muttukrishna et al., 2005 demonstrated a close relation of AFC with age in various fertile and IVF-treated populations (Ng et al., 2003; Hendriks et al., 2005a). The addition of computer-aided programs that analyze the endometrial echogenicity digitally to 3D transvaginal ultrasonography, may remove any variation in human assessment, and may improve its prognostic value in IVF cycles (Fanchin et al., 2000). Application of virtual organ computer-aided analysis (VOCAL) improved accuracy of ascertained endometrial volumes (Bordes et al., 2002; Filicori et al., 2002). Basal ovarian stromal blood flow: Folliculogenesis in the human ovary is a complex process regulated by a variety of endocrine and paracrine signals (McGee and Hsueh, 2000). It has been suggested that the availability of an adequate vascular supply to provide endocrine and paracrine signals may play a key role in the regulation of follicle growth (Redmer and Reynolds, 1996). It is postulated that increased ovarian stromal blood flow may lead to a greater delivery of gonadotrophins to the granulosa cells of the developing follicles. Ovarian stromal blood flow can be assessed by colour Doppler and power Doppler ultrasound (Guerriero et al., 1999). There has been much interest regarding the potential role of trans-

vaginal Doppler ultrasound measurement of intraovarian blood flow in the early follicular phase and its relation to subsequent ovarian responsiveness in ART program. Several studies have shown that ovarian stromal blood flow at the baseline transvaginal ultrasound scan is correlated with subsequent follicular response and may be an indicator for predicting ovarian responsiveness in ART treatment (Engmann et al., 1999). Mean ovarian stromal peak systolic blood flow velocity significantly correlated with the follicular response. (Zaidi et al., 1996). Significantly lower in the poor-response group. The adjusted odds of a poor response increased significantly by an estimated 22% per cm/second decrease in velocity The follicular blood flow plays a major role during the growth and development of the follicle containing the oocyte. The follicle acquires a vascular sheet of its own at the antral stage. Recently it has found that, blood flow in the vessels that supply blood to the follicles in the ovaries in the early follicular phase correlates significantly with ovarian response (Altundag et al., 2002). Combining the color Doppler facility in ultrasonography has enabled the detection and measurement of the follicular blood flow. According to two-dimensional color Doppler studies, peak systolic velocity of individual follicles on the day of human chorionic gonadotropin (hCG) injection and egg collection correlates with oocyte recovery, development potential of the oocyte, quality of the embryo, and even with the pregnancy rate during IVF therapy. High stromal peak systolic velocity or low resistance index before the initiation of gonadotrophin stimulation seems to be associated with retrieval of a higher numbers of oocytes (Ilkkay et al., 2004). Another study showed that Doppler ultrasonographic pulsitility index (PI) of

the ovarian stromal arteries may be useful for predicting the success of IVF treatment in infertile patients (Kim et al., 2002). Color power angiography can assess follicular blood flow and predict the development of healthy oocytes. Pulsed color Doppler has shown that the intraovarian pulsatility index (PI) is significany lower in FSH-treated patients compared with spontaneous cycles, suggesting that multiple follicular development is related to a reduction in the impedance of perifollicular blood flow (Orvieto, 2005). Color power angiography can assess follicular blood flow and predict the development of healthy oocytes. Pulsed color Doppler has shown that the intraovarian pulsatility index (PI) is significany lower in FSH-treated patients compared with spontaneous cycles, suggesting that multiple follicular development is related to a reduction in the impedance of perifollicular blood flow (Orvieto, 2005). A strong correlation between follicular size in women undergoing COH and their peak perifollicular velocity and resistance index, but this did not correlate to the maturity of the oocytes, also there is a correlation in the ovarian stromal flow index and number of mature oocytes retrieved in an IVF cycle and pregnancy rates (Kupesic et al., 2003). In two-dimensional color Doppler studies, the information concerning the vascularization and blood flow in the organ is obtained from a single artery lying in a two-dimensional plane. To accurately measure the blood flow velocity, the angle of insonation to the blood vessels should be known. In the ovary the arteries are thin and tortuous, which makes the measurement difficult. A recent technical achievement, three-dimensional power Doppler ultrasonography, is less angle-dependent and enables the mapping and

quantifying of the power Doppler signal within the entire volume of interest, basically making it possible to detect the total vascularization and blood flow in the organ (Jarvela et al., 2003; Ben-Ami et al., 2007) . 5- Future identification of ovarian reserve The advances in cellular and molecular biology techniques have improved our current serological markers of ovarian reserve. They have also given future prospect for other markers being studied. The future of identifying the poor ovarian responder before COH may lie in these new molecular ovarian markers (Ying et al., 2007). Gonadotropin surge-attenuating factor (GnSAF) is an ovarian factor not yet well characterized. It is involved in the ovarian-pituitary axis, reducing responsiveness of the pituitary to GnRH without affecting LH or FSH scrtionPatients with low ovarian reserve may have less GnSAF production, and this may be involved with the premature luteinization that occurs more frequently in these patients. A preliminary study has shown that poor ovarian response patients have significantly lower circulating levels of GnSAF and a significantly blunted GnSAF rise following FSH timulation GnSAF levels are however, still investigational at this point due to th lack of an available immunoassay (Martinez et al., 2002; Shimasaki et al., 2007). Molecular advances are also being used to study other ovarian factors, such as vascular endothelial growth factor (VEGF) and their receptors. It appears that a dlicate balance between VEGF and its soluble tyrosine receptor, sVEGFR-l, is essential for an adequate ovarian response to gonadotropin stimulation. An initial study has found an excess of sVEGFR-l

in patients with poor ovarian response to COH correlating with reduced conception. Further development in this field is required before this test becomes a clinically useful marker of poor ovarian response (Ravindranth et al., 2006). It has been shown that women with PCO have a higher serum concentration of VEGF wich may account for the increase vascularity seen in these patients. The increase sensitivity to gonadotrophin stimulation and the increased rate of OHSS observed in these women. Furthermore, significant rise in the serum VEGF concentration after hCG administration appears to be single most important pridictor of OHSS (Ostuka et al., 2004)

METHODOLOGY This prospective study was designed to determine the predictive value of FSH&E2, AMH and AFC to ovarian response for controlled ovarian hyperstimulation in intracytoplasmic sperm injection cycles and to find out the best single predictor for poor ovarian response, among 250 infertile couple with tubal, male and unexplained infertility, requesting assisted fertilization attending the ART unit-International Islamic Center for Population Studies and Research, Al-Azhar University Subjects were selected from July 2007 to November 2008.. The sample size was calculated according to the last annual statistical report in this center (2007); where the pregnancy rate per retrieved cycle was 21%, with a precision was assumed to be 0.05. Diagnosis of the couples will be confirmed by basic infertility work up and investigations. The inclusion criteria were: 1- Patients had to be 35-40 years old. 2- The body mass index (BMI) 30kg/m2. 3- Ovulating women with regular menstrual cycle and having both ovaries. 4- Normal basal (day 3) FSH, LH and E2 serum levels. 5- Normal prolactin serum level. 6- First ICSI cycle. 7- Coupels with primary inferetility The exclusion criteria were: 1. Day 3 FSH >15 mIU/ml.

2. Patients diagnosed with Asospermia as a cause of male factor of infertility and causes of infertility other than tubal, male or unexplained infertility. 3. Patients having uterine anomalies such as submucous fibroid, intrauterine synechiae and endometrial polyps. 4. Basal day 2 US show ovarian cyst. 5. Patient having previous ovarian surgery.

Each patient will receive a full explanation of the purpose of the study. All data will be manipulated confidentially and anonymously. Through well-designed structured questionnaire, full data were collected from the eligible patients including detailed personal, menstrual and obstetric history. BMI was calculated by dividing body weight in Kg by the height in squared meters. Also general and local examinations as well as ultrasound (pelvic and transvaginal) on 2nd day of the cycle examination were done for each studied patient using Pie Meidica ultrasound GAIA 8500 MT 7.5 MHS

vaginal 3.5-5.5 abdominal, excluding the presence of ovarian cyst, uterine myomas or endometrial polyp and for counting the antral follicles (small follicles <10 mm) in both ovaries. On day three of the cycles preceding ovarian stimulation, blood sample (10 cc) was collected throw vein puncture at early morning, then left for about one hour for coagulation then centrifuged to obtain serum that stored at (-20) until assessment of basal hormonal profiles (FSH, LH and E2 Serum level), serum prolactine level by RIA and AMH level by ELISA. Determination of serum FSH, LH and prolctin by RIA: coat-A- counted FSH, LH and prolactine are solid phase RIA based on mono and polycolonal

antibody immobilized to the well of a polypropylene tube. Unbound I125 anti-FSH, anti-LH and anti-prolactin antibody is removed by decanting the reaction mixture and washing the tube. Their concentration is directly proportional to the radioactivity present in the tube after the wash step using gamma counter. (National committee for Clinical Laboratory Standards; 1998). Determination of serum Estradiol by RIA: E2 the coat-A- counted procedure is solid phase RIA based on E2 specific antibody immobilized to the well of a polypropylene tube. I125-labeled E2 compete for affixed time to separate bound form free and counted in gamma counter. The amount of E2 present in the patient sample is determined from a calibration curve (National committee for Clinical Laboratory Standards; 1998). Determination of serum AMH by ELISA: The active MIS/AMH ELISA is enzymatically ampliphied two-site immunoassay. In the assay, Standard, Controls, and AMH serum samples are incubated in micro titration wells witch have been coated with anti- MIS/AMH antibody. After incubation and washing, the wells well are treated with another anti- MIS/AMH detection antibody labeled with biotin. After a 2nd incubation and a washing step, the wells are incubated with streptavidine-horseradish peroxidase (HRP). After a 3rd incubation and washing step the wells are incubated with the substrate tetramethylbenzidinen (TMB). An acidic stopping solution is then added and the degree of enzymatic turnover of the substrate is determined by dual wavelength absorbance measurement at 450 and 620nm. The absorbance measured is directly proportional to the concentration of MIS/AMH standard is used to plot a standard curve of absorbance versus MIS/AMH concentration is the AMH can be calculated (Gruijter et al., 2003).

All patients were under COH with long luteal protocol in which they received daily SC triptorelin acetate (0.1 mg) on cycle day 21 of previous cycle, when the serum E2 level was < 50 pg/ml when, the stimulation with HMG (in dose of 225-300 IU), daily according to the age and BMI (women aged < 40 and those with BMI <30 started with 225 IU HMG and for women with age 40 and BMI >30 started with 300 IU HMG). All patients were monitored by serum E2 and trance-vaginal ultrasound. Starting from the 6th day of stimulation, every other day, then when the leading follicle exceeded 14 mm in diameter daily ultrasound assessment until at least two follicle reach 18 mm in diameter with serum E2 level between 150-200 pg/ml per mature follicle (and it is within acceptable range for the mature follicles present), hCG 10,000 IU was given IM for triggering of ovulation. The dose of HMG was adjusted according to the patient's response either by step up if the follicle number was less than 5 or the follicles failed to increase in size as expected by the 8 th stimulation day, or step down if the follicle number was > 20 or the follicles increased in size more than expected by the 8th stimulation day. Oocyte retrieval was performed 36 hours after the hCG by transvaginal ultrasound-guided needle aspiration under general anesthesia. Follicular fluid was aspirated into sterile tubes. The oocyte-cumulus were identified and washed in fresh HTF and equilibrated at 37C in 5% CO2, then washed and placed into organ culture dishes containing the same medium and incubated at 37C in 5% CO2 for approximately 1 hour. Then placed in a 100 l drop of buffered HTF

(Human tubal factor) containing 80 IU hyaluronidase/ml for 30-45 seconds, then the oocyte was removed and placed in 100l drop of buffered HTF with 0.5% HAS (Human serum albumin). The corona cells were removed by gentle aspiration of the oocyte in and out of a sterile drown pipette. When stripping was completed, the oocyte was washed in equilibrated BM1 (Bullentine DE control milieu) Menezo Media, and then placed in 100l microdrops of B2 medium in Petri dishes, covered with 3ml of sterile equilibrated mineral oil. The oocytes then were assessed quickly for maturity (quality) according to Hill et al., 1989 grading system using an inverted microscope equip,.ped with Hoffman optics. (Table) (Fig).

Fig 21a-f:

Oocyte grading: (A)grade1, immature, prophase. (B)grade2, metaphase, nearly mature. (C)grade 3, mature, metaphase II. (D) grade3, preovulatory, metaphase II. (E) grade 4, post mature.(F) grade5, non viable (Rabe et al., 2002)

Table 1. Oocyte grading (Hill et al. 1989) Grade Characteristics Grade 1 (immature oocyte, Dense and compact appearing cumulus cells, prophase 1; Fig. 21a) tightly packed all around the oocyte Shows a centrally located germinal vesicle on light microscopy.

Grade 2 (nearly mature, metahase 1: Fig 21b)

No polar body present. Oocyte exhibits an expanded cumulus mass, but corona radiata is closely appesed to the zona pelucida. No polar body, no germinal veside granular Diameter of extended, dissociated cumulus complex of extended, dissociated cumulus complex is 400 600 um (equivalent 3 5 oocyte diameters) Very expanded cumulus, looking fluffy in a thin web of fibrils of matrix mass. Corona radiata is still associated to the zona. Sometimes appearing loosely aggregated extruded polar body (often hardly to visualize), no nucleus. Clear ooplasm, homogeneously granulated Cumulus is clamped, sometimes absent Corona may be extremely expanded, partly missing or clumped: darkened and irregular cells. Polar body is still intact or fragmented Ooplasm may be slightly darkened, mainly granulated. Oocyte is still round and even. Atresia oocurs in all oocytes from early immature to postmature stages Cumulus cell mass is missing Corona radiata is present, clumped and

Grade 3 (mature/ preovulatory, metphase 11: Fig 21c)

Grade 4 (postmature; Fig. 21e)

Grade 5 (atretic nonviable; Fig. 21f)

irregular Polar body and nucleus are degenerated, if present. Ooplasm is dark and vacuolated. Uneven surface and very irregular shape of the oocyte; a preivitelline space is obvious Clearly visible dark (brush-like) zona pellueida.

Semen was applied to a Percoll gradient and centrifuged at 1,800Xg for 15 minutes. The separated semen fraction was removed and washed twice in HTF. Immediately before injection, 100ml of the washed semen was placed into 4ml of HTF with CaCl 2-2H2O to a final concentration of 5 mmol and centrifuged for 5 minutes 1,800Xg. The supernatant then was removed and the pellet was resuspended in 50 l of HTF supplement with 0.5%HSA. A small amount of 10% Polyvinyl Pyrrolidone (PVP) wormed at 37C and then dilutes the semen specimen. Intracytoplasmic sperm injection was performed according to the protocol of Van Steirteghem (Van Steirteghem et al., 1993). The injection procedure was carried out in a sterilized doubledepression glass slide using holding pipettes and injection needle. The mature oocyte was contained in a 10 l drop of buffered HTF with 0.5% HSA and covered by 5% CO2 equilibrated mineral oil, in one depression, the other depression contained in a 4-l drop of the 105 PVP solution with a 1-l drop of the centrifuged sperm suspension. The injection procedure was carried out on Axiovert 135, equipped with Hoffman optics, 10x, 20x and 40x objectives with 10x eye pieces and nourishing micromanipulators. The oocyte was attached to holding pipette using slight negative pressure. The injection needle containing the sperm and PVP was brought into the focal plan and a single sperm was positioned just at the tip of the microinjection needle. The next step was a slow, steady and consistent

movement into the cytoplasm of the MII oocyte. The sperm then was deposited into the cytoplasm with approximately 1 to 3 pl medium. The injected oocyte then washed twice in B2 medium covered with sterile warm equilibrated mineral oil at 37C in a 5% CO2 in a 100% humidity environment. Approximately 18 hours after injection, the oocytes were checked for signs of fertilization (two pronuclei or two distinct polar bodies).(fig)

Fig 22a-c:

Oocyte 16-18h after insemination. (a). Unfertilized oocyte, one polar body, no pronucleus; (b) Fertilized oocyte, two pronuclei, two polar bodies; (c) Polyspermy, three pronuclie Veek (1986).

After 48 hours, embryos that had cleaved to the two or three cell stage were identified and embryo grading was done according to Hill et al., 1989 garding system (Table 2), (Fig. 23). Grade A: Blastomere symmetrically arranged to zona pellucida with no fragmentation. Grade B: Blastomere slightly irregular with some fragmentation. Grade C: 50% fragmentation. Grade D: Total fragmentation.

Fig 23a-d:

Grading of embryos 48h after insemination. (a) Grade A, no fragmentation; (b) Grade B, some fragmentation; (c) Grade C,

50% fragmentation; (d) Garde D, total fragmentation (Rabe et al., 2002).

Table 2. Embryo grading (Hill et al. 1989). Grade Grad quality Fig. 23a) Characteristics (high- Blastomeres evenly sized, nearly pherical embryo; Cytoplasm uniform, slightly granulated four after pellucida. No fragmentation Zona pellucida looks pale; some corona cells may remain; some spermatozoa are Grade B visible within the zona. (good Blastomeres slightly uneven or irregularly shaped Gytoplasm blastomere with granules up reduced to size 10% and adherence blastomeres insemination) (48 h after insemination), eight blastomeres (72 h blastomeres symmetrically located within the zona

embryo; Fig. 23b)

fragmented blastomeres Grade C (sufficient Blastomeres of uneven embryo; Fig. 23c)

appearance reduced blastomer adherence. Cytoplasm shows large dark granules and vacuoles. Blastomere membrane appears patchy blastomeres located nonsymmetrically within the zona, enlarged perivitelline

Grade

space up to 50% fragmented blastomeres (bad At least on blastomere shows be visible

embryo; Fig. 23d)

blastomeres are very uneven Cytoplasm shows large dark granule and vacuoles. Blastomere membrane looks patchy, reduced blastomere adherence. Extensive fragmentation.

Then up to 3 (grade A) embryos were transferred to the uterus in 30 l of HTF containing 0.5% HSA using soft ET catheter. Luteal phase support was given to the patients for 14 days in the form of daily IM (dose) progesterone in oil, and then beta hCG titer was performed for detection of pregnancy and, then it was confirmed by ultrasound examination at 5-6 weeks gestation by visualization of gestational sac.

Study variables 1. Independent variables: 1.1. Age. 1.2. BMI. 1.3. Basal (day 3) FSH. 1.4. 1.5. Basal (day 3) E2. Day 3 AMH.

1.6. Cause of infertility. 1.7 Days of HMG. 1.8 Total dose of HMG per treated cycle. The age, BMI and basal FSH was used as confounding variables in adjusting the logistic regression models. All these variables are studied as continuous variables, except cause of infertility studied as categorical variable. 2. Outcome variables 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. Number of mature follicles 20 mm. Endometrial thickness. E2 level at day of hCG administration (E2 hCG). Number of Meta phase (MII) oocytes. Total number of embryos (fertilization rate). Number of embryos transferred. Number of patient to embryo transferred. (pregnancy rate).

2.8. Number of pregnancies to number of patient to embryo transferred

2.9.

Number of total cycle cancelled (cancellation rate).

2.10. Number of cancelled cycle due to poor responder. 2.11. Number cancelled cycle due to empty follicle. 2.12. Number cancelled cycle due to empty follicle. 2.13. 2.13. Number cancelled cycle due to degenerated PN. All these outcome variables are studied as continuous variables expect pregnancy rate (number of pregnancies/patients to ET) and cycle cancellation rates (total cancellation, cancellation due to poor responder and negative fertilization) are studied as categorical variables. In the logistic regression models the continuous outcome variables (total oocyte number, number of mature oocytes, total number of embryos) was categorized into two categories based on the median value of each variable according to their distribution in all studied patients.

Statistical Analysis Data were expressed as range and mean SD for continuous variables and number and percent distribution for categorical variables. In order to compare the studied variables in different protocols, Chi-square, Fisher exact, t tests and analysis of variance (ANOVA) were used as appropriate. P values are two-sided, and a P value < 0.05 was considered the limit of statistical significance. To estimate the probabilities of outcome variables as well as, positive pregnancy outcome and cancellation in different protocols multiple logistic regression analysis was used, where the antagonist protocol was the reference for the other protocols. To estimate these probabilities in antagonist protocol, the other protocols (long, short and microdose protocol) were used as the reference. To include the continoues outcome variables (total oocyte number, number of mature oocytes, total number of embryos) in logistic regression models, these variables were categorized into two categories based on the median value of each variable according to their distribution in all studied patients (total oocyte number <11 and >11, number of mature oocytes <7 and >7, total number of embryos <5 and >5). All models were adjusted by age, BMI for outcome variables and age, BMI, number of day 3 FSH and embryos transferred for pregnancy outcome and cancellation. In addition, the linear regression models was used to detect the association between age and BMI and the number of retrieved oocytes in different protocols to assess the predicted average number of total oocyte with respect to age and BMI and how these variables explain the variation in

total oocyte number. The collected data were analyzed by using the SAS software package (SAS). RESULTS A total of 120 ovulating woman were included in the study with 1ry infertility attributable to tubal, unexplained or male factor infertility. There were 30 patients recognized for each protocol; long, short, microdose GnRH agonist and multiple dose GnRH antagonist protocol. Table 1: Patients demographic data (age & BMI), day 3 hormonal profile (FSH & E2) and cause of infertility between GnRH agonist protocols (long, short and microdose) and GnRH antagonist multiple dose protocol. Variables # Long protoc ol n=30 Age (years) BMI(kg/m 2 ) FSH (m IU/ml) E2 (pg/ml) 26.7 3.2 25 2.9 6.3 0.9 43.51 Short protoco l n=30 29.3 .8 24.52. 6 7.4 1 42.61. 5 11 (36.7) 7.2 0.8 6.4 0.8 49.511. 4 11(36.7 ) 46.6 10.2 12(40) <0.0001 ** 0.1 Microdo Antago se n=30 29.2 2.9 25 3.2 nist l n=30 27.2 3.7 25 2.5 0.005* 0.8 protocol protoco P Value

3.5 Cause of infertility Tubal 12 (40)

Unexplaine d Male Male&

9 (30) 6(20) 3(10)

9 (30) 7(23.3) 3(10)

8(26.7) 6(20) 5(16.7)

8 (26.7) 6(20) 4(13.3)

Female Data represented by mean SD number (%). * There is statistical significant difference as regard age between long, antagonist protocol and other two protocols. ** There is statistical significant difference as regard basal FSH (day 3) between long and antagonist and other protocols. There is no statistical significant difference as regard BMI, basal e2 and cause of infertility between protocols.

Table 2:

Clinical and hormonal data and pregnancy rate of ICSI cycles between GnRH agonist protocols (long, short and microdose) and GnRH antagonist multiple dose protocol.

Long Variables# protoco l n=30 Days of HMG Dose of HMG Endometrial Thickness E2 at hCG n.of oocyte retrieved n.of MII oocyte n. of MI oocyte n. degenerated oocyte n. of embryos Number of ET Pregnant 7.3 1.8 2.6 0.7 13 13.4 1.7 25559 30 132.4 28235 85 132.4 8.7 2 2.8 1.4 20.8

Short protoc ol n=30 9.5

Microd ose l n=30 10.2

Antago nist l n=30 7.5 1.1 17961 569 11 2 15243 72 11 2 6.1 1.3 2.2

P Value

protoco protoco

<0.0001* 0.003** 0.004*** 0.001*** * 0.004*** ** <0.0001* ***** 0.07 0.34

1.8 1.9 16782 19444 61 83 11 2.7 11 2.8 29974 26755 58 58 11 2.7 11 2.8 7.2 1.7 2.1 0.8 2.4 1.1 5.7 1.6 3.2 0.6 13 5 1.7 3.4 0.5 11 6.4 2.2 2.4

0.9 0.96 2.3 0.7 2.4 0.7

4.5 1.5 2.6 0.6 11

<0.0001* ****** <0.0001* *******

(/ET) Not pregnant

(46.4) 15

(48.2) 14

(42.3) 15

(37.9) 18

(53.6) (51.8) (57.7) (62.1) Data represented by mean SD, number (%) for pregnancy rate. * ** *** There is statistical significant difference as regard days of HMG stimulation between long protocol and other protocols. There is statistical significant difference as regard dose of HMG between long protocol and other protocols. There is statistical significant difference as regard endometrial thickness at day of hCG between long and short protocols, and long and antagonist protocols. **** There is statistical significant difference as regard E2 level at day of hCG between long and short protocols, and long and antagonist protocols.

*****There is statistical significant difference as regard total number of oocytes retrieved between long protocol and other protocols. ****** There is statistical significant difference as regard number of MII oocytes between long protocol and other protocols. There is no statistical significant difference as regard number of MI (immature) oocytes, number of degenerated oocytes among protocols. ******* There is statistical significant difference as regard total number

of embryos between long and other protocols.

********There is statistical significant difference as regard number of embryo transferred between ultrashort, long and antagonist protocols and short porocol and long and antagonist protocols. There is no statistical significant difference as regard number of pregnancies to number of patients embryo transferred between protocols.

Table 3:

Cycle cancellation between GnRH agonist protocols (long, short and microdose) and GnRH antagonist multiple dose protocol.

Variable#

Long n=30

Short ol n=30 27 (90) 3 (10)

Microd ose n=30 26 (86.7) 4 (13.3)

Antagon ist n=30 29 (96.7) 1 (3.3)

P value

protocol protoc

protocol protocol

Not cancelled Cancelled

28 (93.3) 2 (6.7)

Cause of cancellation Poor responder Negative 1 (50) 1 (50) 2 (66.7) 1 3 (75) 1 (25) 1 (100) 0

fertilization (33.3) #Data represented by number (%). There is no statistical significant difference with regard to number of patient to embryo transferred (not cancelled), total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization among protocols.

Table 4: Comparison between long and short GnRH agonist protocols as regard patient demographic data (age & BMI), day 3 hormonal profile (FSH & E2) and cause of infertility. Variables# Long protocol n=30 Age (years) 2 BMI (kg/m ) FSH(mIU/ml) E2(pg/ml) Cause of infertility Tubal Unexplained Male 12 (40) 9 (30) 6 (20) 11 (36.7) 9 (30) 7 (23.3) 3 (10) 26.73 3.3 25 3 6.3 0.9 43.5 13.5 Short protocol n=30 29.3 3.8 24.5 2.6 7.4 1 42.6 11.6 P Value 0.008* 0.41 <0.001* 0.79

Male& Female 3 (10) # Data represented by mean SD. * Statistically significant difference.

There is no statistical significant difference as regard BMI basal E2 level and cause of infertility between long and short protocols. On the other hand, there is statistical significant difference as regard age and basal (day 3) FSH between long and short protocols.

Table 5:

Comparison between long and short GnRH agonist protocols

among regarding clinical, hormonal data and pregnancy rate of ICSI cycles. Variables# Long protocol Short protocol n=30 Days of HMG Dose of HMG Endometrial Thickness E2 at hCG N. of oocyte retrieved Number of MII oocyte Number of MI oocyte n. of degenerated oocyte n. of embryos Number of ET Pregnant(/ET) Not pregnant 13.4 1.7 2555 930 132.4 2823585 13 2.4 8.7 2 2.8 1.4 2 0.8 7.3 1.8 2.6 0.7 13 (46.4) 15 (53.6) n=30 9.5 1.8 1678 261 11 2.7 2997458 11 2.7 7.2 1.7 2.1 0.8 2.4 1.1 5.7 1.6 3.2 0.6 13 (48.2) 14 (51.8) P Value <0.001* <0.001* 0.002* 0.21 0.002* 0.004* 0.04* 0.2 0.0008* 0.003*

Data represented by mean SD, n (%) for pregnancy rate. * Statistically significant difference. There is no statistical significant difference with regard to E2 at day of hCG, number of degenerated oocyte and pregnancy rate between long and short protocol. On the other hand, there is statistical significant difference

regarding days and dose of HMG stimulation, endometrial thickness, total number of oocyte retrieved, number of MII oocyte, total number of emberyos and number of embryos transferred between long and short protocols.

Table 6: Comparison between long and short GnRH agonist protocols concidering cycle cancellation. Long variabels# Not cancelled Cancelled Cause of cancellation Poor responder Negative fertilization # Data represented by number (%). There is no statistical significant difference as regard patient to embryo transferred, total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization between long and short protocols. 1 (50) 1 (50) 2 (66.7) 1 (33.3) protocol N=30 28 (93.3) 2 (6.7) Short protocol N=30 27 (90) 3 (10) P value

Table 7:

Comparison between long and microdose GnRH agonist as regard patient demographic data (age & BMI), day 3 hormonal profile (FSH & E2) and cause of infertility.

Variables#

Long protocol N=30 26.37 3.3 25 3 6.3 0.9 43.5 13.5

Microdose protocol N=30 29.2 2.9 25 3.2 7.2 0.8 49.5 11.4

P Value 0.002* 0.99 <0.0001* 0.07

Age (years) 2 BMI (kg/m ) FSH (mIU/ml) E2 (pg/ml) Cause of infertility Tubal Unexplained Male Male& Female

12 (40) 9 (30) 6 (20) 3 (10)

11(36.7) 8(26.7) 6(20) 5(16.7)

# Data represented by mean SD, n (%). * Statistically significant difference. There is no statistical significant difference as regard BMI basal E2 as well as cause of infertility between long and microdose protocols. But, there is statistical significant difference as regard age and basal FSH between long and microdose protocols.

Table 8: Comparison between long and microdose GnRH agonist protocols as regard Clinical and hormonal data and pregnancy rate of ICSI cycles. Variables# Long protocol Days of HMG Dose of HMG Endometrial Thickness E2 at hCG n. of oocyte retrieved Number of MII oocyte Number of MI oocyte n.of degenerated oocyte Total number of embryos Number of ET Pregnant(/ET) Not pregnant N=30 13.4 1.7 2555930 132.4 2823585 13 2.4 8.7 2 2.8 1.4 2 0.8 7.3 1.8 2.6 0.7 13 (46.4) 15 (53.6) Microdose protocol N=30 10.2 1.9 1944483 11 2.8 2675558 11 2.8 6.4 2.2 2.4 0.9 2.3 0.7 5 1.7 3.4 0.5 11 (42.3) 15 (57.7) P Value <0.0001* 0.002* 0.01* 0.33 0.01* 0.0002* 0.27 0.15 <0.0001* 0.76

Data represented by mean SD, n (%) for pregnancy rate. * Statistically significant difference. There is statistical significant difference regarding days and dose of HMG, endometrial thickness total number of oocytes retrieved, number of

MII oocytes and total number of embryos between long and microdose protocols.On the other hand there is no statistical significant difference with regard to E2 at day of hCG, number of MI oocytes, number of degenerated oocytes, number of embryos transferred and pregnancy rate between long and microdose protocols.

Table 9: Comparison between long and microdose GnRH agonist protocols among cycle cancellation. Long variables Not cancelled Cancelled protocol N=30 28 (93.3) 2 (6.7) Microdose protocol N=30 26 (86.7) 4 (13.3) P value

Cause of cancellation Poor responder Negative 1 (50) 1 (50) 3 (75) 1 (25)

fertilization Data represented by number (%). There is no statistical significant difference as regard patient to embryo transferred, total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization between long and microdose protocols.

Table 10:

Comparison between long and GnRH antagonist protocols regarding patient demographic data (age & BMI), day 3 hormonal profile (FSH & E2) and cause of infertility.

Variables

Long protocol n=30 26.7 3.2 25 2.9 6.3 0.9 43.513.5

Antagonist protocol n=30 27.2 3.7 25 2.5 6.4 0.8 46.6 10.2

P Value 0.6 0.9 0.55 0.3

Age (years) 2 BMI (kg/m ) FSH (mIU/ml) E2 (pg/ml) Cause of infertility Tubal Unexplained Male Male& Female

12(40) 9(30) 6(20) 3(10)

12(40) 8(26.7) 6(20) 4(13.3)

Data represented by mean SD, n (%) There is no statistical significant difference as regard age, BMI, basal FSH and E2, and cause of infertility between long and antagonist protocols.

Table 11:

Comparison between long and antagonist protocols regarding clinical and hormonal data and pregnancy rate of ICSI cycles. Long Antagonist protocol n=30 7.5 1.1 17961569 11 2 1524372 11 2 6.1 1.3 2.2 0.96 2.4 0.7 4.5 1.5 2.6 0.6 11 (37.9) 18 (62.1) P Value <0.0001* <0.026* 0.0006* <0.0001* 0.0006* <0.0001* 0.05 0.05 <0.0001* 0.6

Variables# Days of HMG Dose of HMG Endometrial Thickness E2 at hCG n. of oocyte retrieved n. of MII oocyte n. of MI oocyte n. of degenerated oocyte n. of embryos Number of ET Pregnant (/ET) Not pregnant

protocol N=30 13.4 1.7 2555930 132.4 2823585 132.4 8.7 2 2.8 1.4 2 0.8 7.3 1.8 2.6 0.7 13 (46.4) 15(53.6)

Data represented by mean SD, no (%) for pregnancy proportion. * Statistically significant difference. There is statistical significant difference with regard to days and dose of HMG stimulation, endometrial thickness, E2 level at day of hCG, total

number of oocytes, number of MII ooctyes and the total number of embryos between long and antagonist protocols. On the other hand there is no statistical significant difference regarding number of MI oocytes, number of degenerated oocytes, number of embryos transferred and pregnancy proportion between long and antagonist protocols

Table 24:

Comparison between long GnRH agonist protocol and GnRH antagonist (multiple dose) protocol among cycle cancellation. Long Variables Not cancelled Cancelled protocol n=30 28 (93.3) 2 (6.7) Antagonist protocol n=30 29 (96.7) 0.38 1 (3.3) Cause of cancellation Poor responder Negative 1 (50) 1 (50) 1 (100) 1.0 0 P Value

fertilization Data represented by number (%). There is no statistical significant difference as regard patient to embryo transferred, total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization between long and antagonist protocols.

Table 13:

Comparison between short and microdose protocols as regard patient demographic data (age & BMI), day 3 hormonal profile (FSH& E2) and cause of infertility. Variables Short protocol n=30 29.3 3.8 24.5 2.6 7.4 1 42.6 11.5 11(36.7) 9(30) 7(23.3) 3(10) Microdose protocol n=30 29.2 2.9 25 3.2 7.2 0.8 49.5 11.4 11(36.7) 8(26.7) 6(20) 5(16.7) 1.0 0.42 0.6 0.02* P value

Age (years) 2 BMI (kg/m ) FSH(m IU/ml) E2 (pg/ml) Cause of infertility Tubal Unexplained Male Male& Female

Data represented by mean SD, number (%). * Statistically significant difference There is no statistical significant difference as regard age, BMI, basal FSH and cause of infertility between short and microdose protocols. On the other hand, there is statistical significant difference regarding basal E2 level between short and microdose protocols.

Table 14:

Comparison between short and microdose GnRH agonist protocols regarding clinical and hormonal data and pregnancy of ICSI cycles. Short Microdose protocol n=30 10.2 1.9 1944483 11 2.8 2675558 11 2.8 6.4 2.2 2.4 0.9 2.3 0.7 5 1.7 3.4 0.5 11 (42.3) 15(57.7) P Value 0.14 0.01* 0.77 0.022* 0.77 0.17 0.17 0.9 0.1 0.12

Variables Days of HMG Dose of HMG Endometrial Thickness E2 at hCG n. of oocyte retrieved n. of MII oocyte n. of MI oocyte n.of degenerated oocyte n. of embryos Number of ET Pregnant(/ET) Not pregnant

protocol n=30 9.5 1.8 1678261 11 2.7 2997458 11 2.7 7.2 1.7 2.1 0.8 2.4 1.1 5.7 1.6 3.2 0.6 13 (48.2) 14 (51.8)

Data represented by maen SD, no (%). * Statistically significant difference. There is no statistical significant difference as regard days of HMG stimulation, endometrial thickness, total number of oocytes retrieved,

number of MII, MI and degenerated oocytes, total number of embryos, number of embryo transferred and pregnancy rate between short and microdose protocols. On the other hand, there is statistical significant difference as regard total dose of HMG and E2 at the day of hCG between short and microdose protocols.

Table 15:

Comparison between short and microdose GnRH agonist protocols among cycle cancellation. Short Variables Not cancelled Cancelled protocol n=30 27 (90) 3 (10) Microdose protocol n=30 26 (86.7) 4 (13.3) P Value

Cause of cancellation Poor responder Negative 2 (66.7) 1 (33.3) 3 (75) 1 (25)

fertilization Data represented by number (%). There is no statistical significant difference as regard number of patient to embryo transferred, total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization between short and microdose protocols

Table 16:

Comparison between short and antagonist protocols regarding patient demographic data (age & BMI), day 3 hormonal profile (FSH & E2) and cause of infertility. Variables Short protocol n=30 29.3 3.8 24.5 2.6 7.4 1 42.611.5 Antagonist protocol n=30 27.2 3.7 25 2.5 6.4 0.8 46.6 10.2 P Value 0.38 0.4 <0.0001* 0.2

Age (years) 2 BMI (kg/m ) FSH(m IU/ml) E2(pg/ml) Cause of infertility Tubal Unexplained Male Male& Female

11(36.7) 9(30) 7(23.3) 3(10)

12(40) 8(26.7) 6(20) 4(13.3)

Data represented by mean SD. *Statistically significant difference. There is statistical significant difference as regard basal FSH level between short and antagonist protocols. But no statistical significant difference was found between short and antagonist protocols as regard age, BMI, basal E2 levels as wel as cause of infertility.

Table 17: Comparison between short and antagonist protocols as regard clinical and hormonal data and pregnancy rate ICSI cycles.

Variables

Short protocol n=30 9.5 1.8 1678261 11 2.7 2997458 11 2.7 7.2 1.7 2.1 0.8 2.4 1.1 5.7 1.6 3.2 0.6 13 (48.2) 14 (51.8)

Antagonist protocol n=30 7.5 1.1 17961569 11 2 1524372 11 2 6.1 1.3 2.2 0.96 2.4 0.7 4.5 1.5 2.6 0.6 11 (37.9) 18 (62.1)

P Value <0.0001* 0.68 0.95 <0.0001* 0.95 0.01* 0.83 0.9 0.003* 0.0001*

Days of HMG Dose of HMG Endometrial Thickness E2 at hCG n. of oocyte retrieved n. of MII oocyte n.of MI oocyte n. of degenerated oocyte n. of embryos n. of ET Pregnant (/ET) Not pregnant

Data represented by mean SD, n (%) *Statistically significant difference.

There has been no statistical significant difference as regard dose of HMG, endometrial thickness, of total number of oocyte retrieved, number MI and degenerated oocytes and pregnancy rate between short and antagonist protocols. On the other hand, there is statistical significant difference as regard days of HMG stimulation, E2 level at the day of hCG, number of MII oocytes, total number of embryos and number of embryos transferred between short and antagonist protocols.

Table 18:

Comparison between short and antagonist protocols as regard cycle cancellation. Short Variables Not cancelled Cancelled protocol n=30 27 (90) 3 (10) Antagonist protocol n=30 29 (96.7) 1 (3.3) 1 (100) 0 P Value

Cause of cancellation Poor responder 2 (66.7) Negative 1 (33.3) fertilization Data represented by number (%).

There is no statistical significant difference as regard number of patient to embryo transferred, total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization between short and antagonist protocols.

Table 19:

Comparison between microdose and antagonist protocols regarding patient demographic data (age & BMI), day 3 hormonal profile (FSH & E2) and cause of infertility. Variables Microdose protocol n=30 29.2 2.9 25 3.2 7.2 0.8 49.5 11.4 Antagonist protocol n=30 27.2 3.7 25 2.5 6.4 0.8 46.6 10.2 P Value 0.2 0.96 0.0001* 0.2

Age (years) 2 BMI (kg/m ) FSH(m IU/ml) E2 (pg/ml) Cause of infertility Tubal Unexplained Male Male& Female

11(36.7) 8(26.7) 6(20) 5(16.7)

12(40) 8(26.7) 6(20) 4(13.3)

Data represented by mean SD, n (%) * Statistically significant difference. There is statistical significant difference as regard basal FSH level between microdose and antagonist protocols. But no statistical significant difference was found between microdose and antagonist protocols as regard age; BMI, basal E2 level and cause of infertility.

Table 20:

Comparison between microdose and antagonist protocols as regared clinical and hormonal data and pregnancy raet of ICSI cycles. Microdose Antagonist protocol N=30 7.5 1.1 17961569 11 2 1524372 11 2 6.1 1.3 2.2 0.96 2.4 0.7 P Value <0.0001* 0.6 0.7 <0.0001* 0.7 0.53 0.28 0.68

Variables Days of HMG Dose of HMG Endometrial Thickness E2 at hCG n. of oocyte retrieved n.of MII oocyte n. of MI oocyte n.of degenerated oocyte n. of embryos Number of ET Pregnant(/ET) Not pregnant

protocol N=30 10.2 1.9 1944483 11 2.8 2675558 11 2.8 6.4 2.2 2.4 0.9 2.3 0.7

5 1.7 3.4 0.5 11 (42.3) 15 (57.7)

4.5 1.5 2.6 0.6 11 (37.9) 18 (62.1)

0.2 <0.0001*

Data represented by mean SD, n (%). * Statistically significant difference.

There is no statistical significant difference as regard dose of HMG, endometrial thickness, total number of oocytes retrieved, number of MII, MI, and degenerated oocytes, total number of emberyos and pregnancy rate between microdose and antagonist protocols. On the other hand, there is statistical significant difference regarding days of HMG stimulation, E2 at the day of hCG and number of embryos transferred between microdose and antagonist protocols.

Table 21:

Comparison between microdose and antagonist protocols regarding cycle cancellation. Microdose Antagonis Variables Not cancelled cancelled protocol N=30 26 (86.7) 4 (13.3) t protocol N=30 29 (96.7) 1 (3.3) P Value

Cause of cancellation Poor responder Negative 3 (75) 1 (25) 1 (100) 0

fertilization Data represented by number (%). There is no statistical significant difference as regard number of patient to embryo transferred, total cancellation rate, cancellation due to poor responder and cancellation due to negative fertilization between microdose and antagonist protocols.

Table 22:

Adjusted odds ratio (OR) for the association of total number of oocyte retrieved with different protocols. Variable* Long protocol Short protocol Microdose protocol Antagonist protocol OR** 4.7 1.1 1.2 0.5 95% CI 1.6 - 14.1 0.4 3.2 0.4 3.6 0.2 1.2

The antagonist protocol is the reference group for the other protocols. Long, short, microdose protocols are the reference group for antagonist protocol.

** OR adjusted by age, BMI. This table presents the association of total number of oocyte retrieved with protocols. There has been strong significant positive association of total number of oocyte retrieved with the studied long protocol where the adjusted OR was 1.1 (95% CI= 0.4 3.2); while there has been non significant positive association with the studied other agonist protocols (short, microdose) . On the other hand, of total number of oocyte retrieved is found to be reduced in patients with antagonist protocol by about 50%. The adjusted OR was 0.5 (95% CI 0.2 1.2).

Table 23:

Adjusted odds ratio (OR) for the association of number of MII oocytes with protocols among patients. Variable * Long protocol Short protocol Microdose protocol Antagonist OR** 8.6 3.2 1.7 0.24 95% CI 2.5 29.1 0.9 11.0 0.5 6.2 0.09 0.7

protocol The antagonist protocol is the reference group for the other protocols. Long, short, microdose protocols are the reference group for antagonist protocol.

**

OR adjusted by age, BM, FSH. This table presents the association of number of MII oocytes with

protocols. There has been strong significant positive association of number of MII oocytes with the studied long protocol where the adjusted OR was 8.6 (95% CI= 2.5 29.1); while there has been non significant positive association with the studied other agonist protocols (short, microdose) . On the other hand, number of MII oocytes is found to be reduced in patients with antagonist protocol by about 76%. The adjusted OR was 0.24 (95% CI 0.09 0.7).

Table 24:

Adjusted odds ratio (OR) for the association of total number of embryos with protocols among patients.

Variable* OR** 95% CI Long protocol 13.3 3.9 45.7 Short protocol 3.5 1 11.4 Microdose protocol 1.95 0.6 6.5 Antagonist protocol 0.2 0.07 0.6 The antagonist protocol is the reference group for the other protocols. Long, short, microdose protocols are the reference group for antagonist protocol.

**

OR adjusted by age, BMI, basal FSH and ET. This table presents the association of total number of embryos with

protocols. There has been strong significant positive association of total number of embryos with the studied long protocol where the adjusted OR was 13.3 (95% CI= 3.9 45.7); while there has been non significant positive association with the studied other agonist protocols (short, microdose). On the other hand, total number of embryos is found to be reduced in patients with antagonist protocol by about 80%. The adjusted OR was 0.2 (95% CI 0.07 0.6).

Table 25:

Adjusted odds ratio (OR) for the association of positive pregnancy probability with different protocols in patients underwent ET.

Variable * Long protocol Short protocol Microdose protocol Antagonist *

Pregnant 13 13 11 11

Not pregnant 15 14 15 18

OR** 1.4 1.6 1.3 0.70

95% CI 0.5 4.03 0.48 - 4.9 0.36 4.5 0.35 1.8

protocol The antagonist protocol is the reference group for the other protocols. Long, short, microdose protocols are the reference group for antagonist protocol.

**

OR adjusted by age, BMI, basal FSH and ET. This table presents the association of positive pregnancy probability

with protocols. There has been non significant positive association of positive pregnancy probability with the studied agonist protocols where the adjusted OR was 1.4 (95% CI= 0.5 4.03); 1.5 (95% CI 0.48 4.9) and 1.3 (95% CI 0.36 4.5) for Long, short, microdose protocols respectively. On the other hand, positive pregnancy probability is found to be reduced in patients with antagonist protocol by about 30%. The adjusted OR was 0.7 (95% CI 0.35 1.8).

Table 26: Adjusted odds ratio (OR) for the association of cancellation probability with different protocols. Variable * Long protocol Short protocol Microdose protocol Antagonist * Cancelled 2 3 4 1 Not Cancelled 28 27 26 29 OR** 2.1 3.3 4.7 0.3 95% CI 0.18 23.9 0.3 35.3 0.47 46.7 0.04 2.6

protocol The antagonist protocol is the reference group for the other protocols. Long, short, microdose protocols are the reference group for antagonist protocol.

**

OR adjusted by age, BMI. This table presents the association of cancellation probability with

protocols. There has been non significant positive association with the studied agonist protocols (long, short, microdose). On the other hand, total number of cancellation probability is found to be reduced in patients with antagonist protocol by about 70%. The adjusted OR was 0.3 (95% CI 0.04 2.6).

Table 27:

Association of age and BMI with number of oocyte retrieved in different protocols. Long protocol Variable Age BMI Short protocol Age BMI 0.14 0.09 0.3 0.6 0.04 0.008 * 0.26 0.2 P. Value 0.06 0.1 R 2**

0.13 0.1

Microdose protocol Age BMI Age BMI * ** -0.4 -0.02 -0.04 -0.06 0.07 0.9 0.7 0.7 0.13 0.0005 0.005 0.007

Antagonist protocol

means regression coefficient. 2 R coefficient of determination.

The age explains about 13%of variation observed in the average number of oocyte retrieved in long protocol. The average number increases by 0.26 oocyte for increasing 1 year of age (Fig.1). While the BMI explains about 10% of variation observed in the average number of oocyte retrieved in long protocol. The average number increases by 0.2 oocyte for increasing the

BMI 1kg/m2 (Fig. 2). The age explains about 4% of variation observed in the average number of oocyte retrieved in short protocol. The average number increases by 0.14 oocyte for increasing 1 year of age (Fig.3). While the BMI explains about 0.8% of variation observed in the average number of oocyte retrieved in long protocol. The number increases by 0.09 oocyte for increasing the BMI 1kg/m2 (Fig.4). The age explains about 13%of variation observed in the average number of oocyte retrieved in microdose protocol. The number decreases by 0.4 oocyte for increasing 1 year of age (Fig.5). While the BMI explains about 0.5% of variation observed in the average number of oocyte retrieved in microdose protocol. The number decreases by 0.02 oocyte for increasing the BMI 1kg/m2 (Fig.6). The age explains about 5%of variation observed in the average number of oocyte retrieved in antagonist protocol. The number decreases by 0.4 oocyte for increasing 1 year of age (Fig.7). While the BMI explains about 0.7% of variation observed in the average number of oocyte retrieved in antagonist protocol. The number decreases by 0.02 oocyte for increasing the BMI 1kg/m2 (Fig.8).

Fig 1 : Association of age with oocyte in long protocol.

Fig 2 : Association of BMI with oocyte in long protocol.

Fig 3 : Association of age with oocyte in short protocol.

Fig 4 : Association of BMI with oocyte in short protocol.

Fig 5 :

Association of age with oocyte in microdose protocol.

Fig 6:

Association of BMI with oocyte in microdose protocol.

Fig 7:

Association of age with oocyte in antagonist protocol.

Fig 8 :

Association of BMI with oocyte in antagonist protocol.

DISCUSSION

This prospective study was designed to compare the effect of 4 protocols of ovulation induction (long, short, microdose protocols as GnRH agonist protocols and multiple dose GnRH antagonist protocol) in controlled ovarian hyperstimulation for ICSI, on cycle outcomes (days and dose of HMG stimulation, fertilization rate, pregnancy rate and cancellation rate) and oocyte quality. The study included 120 ovulating women, with primary ifertility attributable to tubal, male and unexplained infertility, attending Ain Shams University Maternety Hospital, International Islamic Center for Population Studies and Research, Assisted Reproductive Unit, Al-Azhar University and Galaa Assisted Reproductive Unit during the period from July 2006 to Febrewary 2007. In this study, women excluded when aged > 36 years and those with BMI more than 30 kg/m2 (as BMI > 30 Kg/square meter were recognized obese according to WHO classification) (WHO, 2000). Such exclusion was based on the previous recent studies reported that the rate of loss of primordial follicles is accelerated by about 2-fold among patients at 37.5 1.2 years of age (Gougeon, 2004), and the poor ovarian response to be associated with high body mass index > 30 kg/ m2 (Akande et al., 2002). Also, women with basal FSH >15 IU/ml and those with elevated basal E2 levels were excluded. Hansen et al (1996) found that the Day 3 FSH level above 15 IU/ml was significantly associated with a decline in response to ovarian stimulation and in pregnancy rate. Also, basal E2 values are found to have a beneficial role in screening for the potential poor ovarian responder in the context of a "normal" FSH value (Brown et al. 1995). In addition, patients with antral follicles count < 5 were excluded, as the

antral follicle count > 5 assessed early in the follicular phase is considered to be useful predictor of ovarian response (Ilkka et al., 2003). Patients having uterine anomalies such as submucous fibroid, intrauterine synechiae and endometrial polyps, that may affect the implantation and pregnancy, were also excluded.

Comparing the four protocols (long, short and microdose GnRH agonist protocols and GnRH antagonist multidose protocol) with each others, the results of this study revealed significant increase in the total number of oocytes retrieved, and this was found in long and short protocol over the other protocols. Also, regarding number of MII and total number of embryos obtained, there has been significant increase in long protocol over other protocols. This finding is explained by the more oocyte recruitment observed in long protocol with higher number of good quality oocytes which results in more good quality embryos ; also the more degree of pituitary suppression in the long protocol patients and the availability of single depot injection which is more comfortable and with less bias for patient than daily SC injection that may not be injected in the right way and with loss of part of the active material may lowers the response of the patient to the treatment protocol. On the other hand, there has been no significant difference between protocols (long, short and microdose GnRH agonist protocols and GnRH antagonist multiple dose protocol) considering pregnancy rate and cycle cancellation, this is mainly explained by tailoring of the protocols for each patient that depends mainly on basic selection criteria which is evident in the younger age and lower basal FSH levels in long and antagonist protocol

patients. Until now, to the best of our knowlges, no available studies comparing these four protocols with each others; so comparign the results data with other studies can not be done. However, individual studies comparing the most frequently used long and short protocols are at hand. When comparing the long and short protocols, the results of the current study revealed significant increase in endometrial thickness in long protocol patients than in short protocol group. That may be attributed to more pituitary suppression by long GnRH agonist protocol that results in higher number of oocytes, which in turn produce more E2, is responsible for the difference observed in endometrial thickness between the two protocols. BoAbbas et al. (2001) studied the clinical and hormonal effect of long and short protocols on 180 patients for each protocol during ICSI cycles in a retrospective study, in agreement with the current study reported significant increase endometrial thickness in long protocol patients. With regard to the total number of oocyte retrieved and number of MII oocyte this study found significant increase in long over the short protocol in the previous outcome data, but with regard to umber of MI there was significant decrease in long over short protocol. This finding is explained by the more number of oocytes recruitment in long protocol; also the younger the age and the lower basal FSH level in long protocol patients may be explained the higher number of good quality oocytes. Cramer et al (1999) in their retrospective study included 1980 patients among normal responders, found the number of mature oocytes to be relatively fewer in short protocol patients compared with long protocol with statistically significant difference in agreement with this study.

The significant increase in total number of embryos and significant decrease in number of embryos transferred observed in long protocol patients than in short protocol group might be explained by the more mature oocytes available for injection that results in more high quality embryos available for transfer, this is in agreement with Cramer et al (1999) who observed that the number of embryos was significantly higher in long protocol patients. On the other hand, there has been no statistical significant difference with regard to pregnancy rate and cycle cancellation for long and short protocols. This may be due to the impact of other factors, such as the basic criteria for patient selection in different protocols which is well demonstrated between the two protocols in older age and higher basal FSH level in short protocol group. Also, personnel practice in injection (ICSI) procedure, ET and laboratory environment could have an impact on the current results. However, Cramer et al (1999) in their study showed no significant difference between the two protocols regarding age and basal FSH in disagreement with study. Also, they reported a slightly insignificant higher clinical pregnancy rate and non significant decrease in cycle cancellation in long protocol than short protocol patients Comparing long and microdose protocols, there has been significant increase in the endometrial thickness in long protocol patient than in microdose protocol group. This difference is attributed to the higher oocyte number observed in long protocol patients which produced higher levels of E2. Leondires et al (1999) studied the clinical and hormonal effect of long and microdose protocols on 170 patients for each protocol during ICSI cycles among poor responders in a prospective study, and found that there is

no significant difference between the microdose and long protocols regarding endometrial thickness, this is in disagreement with the current study, and that can be explained by the patients recruited for their study were poor responders. Regarding total number of oocytes retrieved, number of MII oocytes, and total number of embryos, the observed significant increase in the previous outcoms in long protocol patients over the microdose protocol ones, which might be explained by more degree of pituitary suppression, allows more oocyte recruitment. Again the younger the age and the lower basal FSH level observed in long protocol patients and the more degree of pituitary suppression can explains the higher number of good quality oocytes, which in turn results in more number of good quality embryos. Also, the high oocytes number gives rise to more E2 that can explain the difference in endometrial thickness between the two protocols. This finding is not consistent with Leondires et al (1999) they reported no significant difference between long and microdose protocols as regarded number of oocyte retrieved and number of embryos transferred. Again the poor responder patients in their study can explain the disagreement of their results. On the other hand, the current study results revealed no significant difference between long and microdose protocols considering pregnancy rate as well as cancellation rate. This may be due to the impact of other factors such as the basic criteria for patient selection in different protocols which are well evident between the two protocols in older age and higher basal FSH level in microdose protocol group. Also, personnel practice in injection (ICSI) procedure, ET technique and laboratory environment for incubation

could have an impact on such results. Consistent with Leondires et al (1999) they also found slight non significant increase in pregnancy rate in long protocol compared with microdose .However, in disagreement with Leondires et al (1999), they report no significant difference with respect to age and basal FSH between both groups. Also, they found a higher significant cancellation rate in microdose group (27.5%) compared with long protocol group (8.2%). This disagreement may be explained by that the patients recruited for the Leondires et al (1999) study were poor responders. With regard to the comparison between long and antagonist protocols, the results of the current study revealed significant increase in days and dose of HMG stimulation. This can be explained by the degree of pituitary desensitization in long protocol which needs high doses oh gonadotropin stimulation for longer period, while not presenting antagonist protocol. This is in disagreement with Barmat et al (2005), they studied the clinical and hormonal effect of long and antagonist protocols on 230 patients for every protocol during ICSI cycles among poor responder in a retrospective study. They found that, there was no no statistical significant difference in duration of stimulation and the dosage of HMG in either group. This in fact because of the oral contraceptive pills usage before starting in Barmat et al (2005) study patients; and also the patient recruited for their study were poor responder. There is also significant increase in long over the antagonist protocols regarding total number of oocytes, number of MII ooctyes, endometrial thickness, E2 level at day of hCG, and total number of embryos. The increased number of retrieved oocytes may be due to higher recruitment with higher quality oocytes that produced more E2 in long protocol group that is

due to the reflection of the initial flare up effect after down regulation with GnRH agonist, and this explains the significant difference in endometrial thickness, higher E2 level at day of hCG and higher number of embryos obtained. In agreement with these findings, Barmat et al (2005) found that E2 levels were significantly higher in long protocol. But in disagreement with the current study, they did not find any significant difference between long and antagonist protocols regarding number of oocyte retrieved, number of MII oocyte, embryo transferred.. In contrast, Cheung et al (2005), studied the clinical and hormonal effect of long and antagonist protocols on 86 patients for long protocol and 62 patients for antagonist protocol during ICSI cycles among normal responders in a prospective study, they found E2 levels were significantly higher in antagonist group patients compared with long protocol. However, for pregnancy and cancellation rates, this study was consistent with Barmat et al (2005) and Cheung et al (2005) where no significant difference was observed between the two protocols. Considering the comparison short and antagonist protocols the results of the current study found significant increase regarding days of HMG stimulation and E2 level at the day of hCG in the short protocol group, these findings can be explained by the pituitary suppression in short protocol group needs higher doses of stimulation with exogenous gonadotropins for longer period. Also, with regard to number of MII oocytes, total number of embryos and number of embryos transferred, the results of this study revealed significant increase in short over the antagonist protocols in the previous outcome results. The initial flare up effect at the early follicular phase in

short protocol ends in more oocyte recruitment, more mature oocytes which increasing the fertilization rate. Mohamed et al (2005) studied the effect of short and antagonist protocols on 234 patients in ICSI cycles in a prospective study and reported that the E2 level was higher in patients used the antagonist protocol. The difference may be explained by the excess number of growing follicles which are more in antagonist protocol in their study. These follicles produce more E2. Moreover, after down-regulation with GnRH agonists, LH levels are not similar in all patients, which lead to varying degrees of E2 production with the same follicular development and under the same FSH effect. Regarding pregnancy and cancellation rates, the results of these study revaeled no significant difference between short and antagonist protocols. Mohamed et al (2005) In agreement with the current study, they also found with regard to pregnancy rate insignificant decrease and insignificant increase in cancellation rate in antagonist group. On comparing short and microdose protocols, regarding total dose of HMG and E2 at the day of hCG, the results revealed significant increase in these parameters in short protocol group; this difference most probably due to the degree of pituitary suppression is more in short protocol which needs more exogenous HMG stimulation, also, the insignificant excess in the number of mature oocytes may be responsible for the production of more E2 in short protocol patients. On the other hand, there is no significant difference with regard to fertilization rate, pregnancy rate and cancellation rate between short and microdose protocols. Regarding the comparison between microdose and antagonist protocols

the current study results revealed significant increase. In the days of HMG stimulation, E2 at the day of hCG, and number of embryos transferred in microdose protocol group. On the other hand, there has been insignificant difference between both protocols in the pregnancy rate as well as cycle cancellation. Again, there have not been available studies in the literature comparing these short and microdose protocols, and microdose and antagonist protocols. So comparing the results of this study with other studies can not be done. Using the logistic regression analysis, allowed the examination of the association of the studied protocols with the total number of oocyte, total number of MII oocyte, total number of embryos, cycle cancellation as well as positive pregnancy outcome. Compared with antagonist protocol, the positive pregnancy outcome was found to be increase by 1.4, 1.6 and 1.3 for long, short and microdose protocol respectively. On the other hand, the positive pregnancy outcome was found to be reduce by 30% for antagonist protocol group (odds ratio (OR) = 0.70; 95% confidence interval (CI) = 0.35 1.80) compared with other protocols (long, short and microdose). All these associations were not statistically significant. Compared with antagonist protocol, there have been positive association between other studied protocols and the ability to obtain total number of oocyte >11. The highest and significant positive association was observed in long protocol (OR= 4.7; 95% CI= 1.60 14.1). This finding, however, should be interpreted cautiously because of the observed wide confidence interval. On the other hand, there was a negative association for the

antagonist protocol (other protocols were the references) in the ability to obtain total number of oocyte >11 (OR= 0.50; 95% CI = 0.20 1.20). This finding indicates that the probability to obtain total number of oocyte >11 in patients used antagonist protocol was reduced by 50% compared with agonist protocols, yet this was insignificant. Compared with antagonist protocol, the probability of having a total number of MII oocyte > 7 was found to be increase by 8.6, 3.2 and 1.7 for long, short and microdose protocol respectively. Although the positive association observed in long protocol was statistically significant (OR = 8.65; 95% CI= 2.5 29.1), this finding should be interpreted cautiously because of wide confidence interval. On the other hand, significant negative association was observed between the total number of MII oocyte > 7. The probability to obtain total number of MII oocyte > 7 in patients used antagonist protocol was found to be reduced by 76% (OR = 0.24; 95% CI= 0.09 0.7)compared with agonist protocols. Compared with antagonist protocol, the probability of having a total number of embryos > 5 was found to be increased by 13.3, 3.5 and 1.95 for long, short and microdose protocol respectively. Although the positive association observed in long protocol was statistically significant (OR = 13.3; 95% CI= 3.9 45.7), this finding should be interpreted cautiously because of wide confidence interval. On the other hand, a significant negative association was observed between the total number of embryos > 5 with the antagonist protocol, and the probability of having a total number of embryos > 5 was found to be reduced by 80% (OR= 0.20; 95% CI= 0.07 0.60) compared with other studied protocols. The probability of cycle cancellation was found to be increased by 2.1, 3.3 and 4.7 for long, short and microdose protocol respectively (antagonist

protocol is the reference). On the other hand, the cycle cancellation is found to be reduced by 70% (OR= 0.3; 95% CI= 0.04 2.6) which is statistically not significant in antagonist protocol compared with other studied protocols. Again, because of the observed wide CI, a careful interpretation of these results is mandatory. Using a linear regression analysis, the results of this study found that, the age explained about 13% of variation observed in the average number of oocyte retrieved in long protocol. The average number of oocytes increases by 0.26 for each increase in age of one year. The BMI is found to explain about 10% of variation observed in the average number of oocyte retrieved in long protocol. The average number increases by 0.2 for each increase in BMI of 1kg/m2. In the short protocol, the age explains about 4% of variation observed in the average number of oocyte retrieved. The average number increases by 0.14 for each increase in age of one year. The BMI, however, showed a negligible effect on the variation observed in the average number of oocyte retrieved among these patients In the microdose protocol, the age is found to explain about 13%of variation observed in the average number of oocyte retrieved, and the average number decreases by 0.4 for each increase in age of one year. The BMI has a negligible effect. In antagonist protocol, the age explains about 5% of variation observed in the average number of oocyte retrieved in antagonist protocol. The average number decreases by 0.4 for each increase in age of one year. The BMI has a negligible effect. Consistent with Filicori et al (2002) studied the age, BMI and antral

follicle count in the prediction of cycles outcomes on 420 patients in retrospective study and Lee et al (2001) studied the factors that can predict the umber of oocytes in stimulated cycles on 352 patients in a prospective study. They found the age and BMI to predict the cycle outcome concerning the number of oocytes. In these studies, age explaines 10% Filicori et al (2002) and 8% Lee et al (2001) of the variation observed in the average number of oocytes retrieved in patients under stimulation with long protocol, the average number of oocyts were found to increase by 0.34 Filicori et al (2002) and 0.4 Lee et al (2001) for each increase in age of one year. Also, they found that the BMI explains 7% Filicori et al (2002) and 12% Lee et al (2001) of variation observed in the average number of oocytes retrieved in long protocol patients, the average number of oocytes ewre found to decreased by 0.3 Filicori et al (2002) and 0.14 Lee et al (2001) for each increase in BMI of one kg/m2. However, there have been no available studies in the literature for the other protocols so we cannot compare the results observed in the current study with other data. In the current study the results found that microdose and short GnRH agonist protocol offers significant cost saving as they shortens the treatment period and decreases the total required dose of HMG, over the long GnRH agonist protocol. However, the long protocol results in better outcomes considering the number and quality of retrieved oocytes, the fertilization rate (total number of embryos obtained) than short and microdose protocols. The GnRH antagonist protocol appears to be the least effective compared with other GnRH agonist and results in outcome less but nearly equal to those obtained by standard long GnRH agonist protocol. It also found to offers significant cost saving over long protocol as it decreases the

treatment period as well as the total gonadotropin stimulation dose, and more over, so allowing more flexibility of treatment and more comfortability for patient. So it can be considered the ideal protocol for patients not responding to a long GnRH agonist protocol. Considering the pregnancy rate and cycle cancellation, the current study did not observe any significant differences among the studied protocols. When convenience, costs, and side effects are taken into account, a single dose of long acting GnRH agonist should probably be the first choice. This study has a number of strengths that include, the power of the study was designed to be 80% irrespective to the relative small number of the patients recruited for this study; also, being prospective and multicenter study. Unlike other retrospective studies, the problem of missing data and low quality data did not found. According to the best of our knowledge, this study can be considered the first to compare these different 4 protocols at one time. No data were available in the literature concerning the comparison of short and microdose protocols, and microdose and antagonist protocols. These shortages do not allowed comparing and discussing the current results, concerning the comparison of these protocols, with other published studies. The use of logistic as well as linear regression analyses, allowing the examination of the association of these different protocols with some cycle outcome variables and to predict the number of oocyte retrieved in each protocol according to age and BMI of the studied patients. Although this study is considered to be multicentric (including patients from three ART centers in Cairo), the generalization of its results is still questionable partly due to the exclusion criteria which we used and partly due to the relatively small number of the studied patients compared with

other multicentric studies. So, if generalization is taken into consideration, this should be cautiously at least to the patients with the same characteristics as those included in this study. Also, the wide confidence intervals observed while examining the associations of agonist (long, short and microdose) and antagonist protocols with cycle outcome variables limit the benefits obtained from these results.

Summary Ovulation of normal female is a complex process involving many organs. The Three major organs that regulate human reproduction are the hypothalamus the pituitary and the ovary. The hypothalamus pulsatile generator of reproduction, produce and secrete GnRH, which by reaching pituitary, evoke the release of FSH and LH. In response to gonadotropin stimulation, the ovaries initiate a dynamic process of steroidogenesis, which results in the formation of mature ovum ready to be fertilized. Any defect in this group of complex processes results in infertility, which affects up to one in seven couples nowadays. Proportion of these couples may be able to ultimately conceive, but for the majority conception is unlikely without some form of medical intervention. IVF-ET and, more recently, ICSI are now commonly used treatment for infertility.. Currently, most ICSI cycles are carried out under an ovarian stimulation with the goal of achieving multiple folliculogenesis to increase the fertilization rate, more embryos for transfer and cryopreservation and increase the pregnancy rate.

It was not uncommon for 10-15% of IVF cycles to be cancelled due to premature LH surges. The availability of GnRH agonist changed the management cancellation. Different GnRH-agonist regimens have been used but a major distinction is based on the duration of use before the invitation of gonadotrophin therapy. The short or flare regimen is begun during the follicular phase of the treatment cycle, 1 or 2 days before gonadotrophin administration, in ultrashort protocol GnRH is administered in the first three days of the cycle only. Long down regulation regimen, which is the preferred ovarian stimulation regimen for ART, is begun either during the luteal phase of the cycle before treatment or during the follicular phase at the treatment cycle and is continued for least 10 days before gonadotrophin administration. One of the drawbacks of the GnRH agonists; is the need for high dosages of gonadotropins; another one is longer stiomulation periods which is required for obtaining an adequate ovarian response in long agonist protocol. This had let the investigator to propose the use of a lower dose of GnRH agonists, especially for patients with a previous low responder to gonadotrophin stimulation, in an attempt to maximize ovarian response without losing the benefits of GnRH agonist down regulation. The mircrodos protocol using the lowest GnRH agonist dose that can induce pituitary downregulation 20-40 ug Leuprolide acetate twice daily. of IVF patients as it induces a mild, reversible hypophysectomy and prevents the premature LH surge resulting in less cycle

Recently, GnRH antagonist made available for clinical use, which competitively blocks pituitary gland receptors, including a rapid, reversible suppression of gonadotrophin secretion and benefit from the endogenously produced gonadotrophin. GnRH antagonist have many advantages for patients and physicians with regard to convenience and flexibility of administration, duration and dose of treatment is shorter, as antagonist, eliminating the estrogen deficiency symptoms that can emerge in women treated with an agonist. By eliminating the flare effect of agonists, GnRH antagonists avoid the risk of stimulating development of a follicular cyst and decrease the risk of OHSS. So, this prospective study is designed to compare the effect of 4 protocols of ovulation induction (long, short, microdose protocols as GnRH agonist protocols and multiple dose GnRH antagonist protocol) in controlled ovarian hyperstimulation for ICSI, on cycle outcomes (days and dose of HMG stimulation, fertilization rate, pregnancy rate and cancellation rate) and oocyte quality. The study included 120 ovulating women, with primary infertility attributed to tubal, male and unexplained infertility, attending Ain Shams University Maternety Hospital, International Islamic Center for Population Studies and Research, Assisted Reproductive Unit, Al-Azhar University and Galaa Assisted Reproductive Unit, during the period from July 2006 to February 2007. Women excluded when aged more than 36 years, BMI > 30 kg/m2, High basal FSH and E2, antral follicles < 5, and those with infertility causes other than tubal, male and unexplained infertility. Also, patients with uterine abnormality were excluded. Then, patients were classified into 4 groups;

each group included 30 eligible patients for each protocol. The dose of HMG was adjusted according to the patient's response either by step up or step down. All patients were monitored by serum E2 and trance-vaginal ultrasound. Starting from the 6 th day of stimulation, hCG

10,000IU was given IM for triggering of ovulation when at least 2 follicles reaches 18-20mm. Oocyte retrieval was performed 36 hours after the hCG by transvaginal ultrasound-guided needle aspiration under general anesthesia. ICSI was performed according to the protocol of Van Steirteghem. Comparing the four protocols (long, short and microdose GnRH agonist protocols and GnRH antagonist multidose protocol) with each others, the results of this study revealed statistically significant differences between long protocol and other protocols regarding the days and dose of HMG stimulation. With regard to total number of oocytes retrieved, there is statistically significant difference between long and short protocol and other protocols. Also, regarding number of MII and total number of embryos obtained, there has been statistically significant difference between long protocol and other protocols. On the other hand, there has been no statistically significant difference between protocols (long, short and microdose GnRH agonist protocols and GnRH antagonist multiple dose protocol) considering pregnancy rate and cycle cancellation. Comparing the short and long protocols there is statistical significant differencewere found with respect to age, basal (day 3) FSH, days and dose

of HMG stimulation, endometrial thickness, total number of oocyte retrieved, number of MII oocyte, total number of emberyos and number of embryos transferred between long and short protocols. On the other hand, there has been no statistical significant difference were observed with regard to BMI and basal E2, E2 at day of hCG, number of degenerated oocyte, pregnancy rate and cycle cancellation between the two protocols. Comparing the long and microdose protocols there is statistical significant difference were found with respect to age, basal FSH, days and dose of HMG stimulation, endometrial thickness, total number of oocytes, number of MII oocytes and total number of embryos between both protocols. On the other hand, there has been no statistical significant difference were observed regarding BMI, basal E2, E2 at day of hCG, number of MI and degenerated oocytes, number of embryo transferred, pregnancy rate as well as cycle cancellation between long and microdose protocols. Comparing the long and antagonist protocols the results of our study showed statistical significant difference with respect to days and dose of HMG, endometrial thickness, E2 at day of hCG, total number of oocytes retrieved and total number of embryos between both protocols. On the other hand, there has been no statistical significant difference were observed regarding age, BMI, basal FSH and E2, number of MI and degenerated oocytes, number of embryo transferred, pregnancy rate and cycle cancellation among the two protocols. When comparing the short and microdose protocols, the results of the

current study revealed statistically significant difference between microdose and short protocols regarding basal E2, dose of HMG and E2 at day of hCG. On the other hand, there has been no statistical significant difference were observed regarding age, BMI, basal FSH, days of HMG, endometrial thikness, total number of oocytes, number of MII and MI as well as degenerated oocytes, total number of embryos, number of embryos transferred, pregnancy rat and finally cancellation rate. Comparing the short protocol to antagonist protocol, the results of the current study observed statistically significant difference between short protocol to antagonist protocol regarding basal FSH, days of HMG, E2 at day of hCG, number of MII oocytes, total embryo obtained and number of embryo transferred. On the other hand, there has been no statistical significant difference were found with respect to age, BMI, basal E2, dose of HMG, endometrial thickness, total oocytes retrieved, number of MI and degenerated oocytes, pregnancy rate and lastly cycle cancellation among two protocols. Finally, when comparing the microdose to antagonist protocol, the results of the current study observed statistically significant difference between the microdose to antagonist regarding basal FSH, days of HMG, E2 at day of hCG and number of embryo transferred. On the other hand, there has been no statistical significant difference were found with respect to age, BMI, basal E2, dose of HMG, endometrial thickness, total number of retrieved oocytes as well as number of MII, MI and degenerated oocytes, total numer of resulting embryos, pregnancy rate and cycle cancellation. Using the logistic regression analysis, allowed the examination of the

association of the studied protocols with the total number of oocyte, total number of MII oocyte, total number of embryos, cycle cancellation as well as positive pregnancy outcome. The results of this study revealed that, there has been positive association observed between GnRH agonist protocols compared to antagonist protocol with regard to the ability to obtain total oocyte >11, MII >7 and total embryos >5, the highest and significant association was observed in long protocol patient. On the other hand, there has been negative significant association in antagonist protocol compared to other GnRH agonist protocols regarding these outcome parameters. With regard to the positive pregnancy probability as well as cycle cancellation, the results revealed they were insignificantly increased with GnRH agonist protocols, on the other hand they were found to be insignificantly decreased with antagonist protocol compared with other protocols Using the linear regression analysis, the results of this study found that, the age and BMI explained the variation observed in the total number of oocyet retrived in different protocol patients, but all with no significant values.CONCLUSION RECOMMENDATIONS Although, microdose and short GnRH agonist protocol may offer significant cost saving over the long GnRH agonist protocol as they shortens the treatment period and decreases the total required dose of HMG, the long protocol results in better outcome than short and microdose protocols considering the number and quality of retrieved oocytes, the fertilization rate (total number of embryos obtained).

The GnRH antagonist protocol appear to be the least effective as a GnRH agonist and results in outcome less but nearly equal to those obtained by standard long GnRH agonist protocol; also, it found to offer significant coast saving over long protocol as it decreases the treatment period as well as the total gonadotropin stimulation dose, allows more flexibility of treatment and more comfortable for patient, decrease the incidence of ovarian hyperstimulation syndrome, avoid risk of cyst formation and avoid side effects related to prolonged estrogen depletion which can be observed with patient under stimulation with GnRH agonist protocols, So it can be considered the ideal protocol for patients not responding to a long GnRH agonist protocol. Considering the pregnancy rate and cycle cancellation, the current study did not observe any significant differences among the studied protocols. The current study suggested that for ICSI cycles, in which fertilization is precise and high proportion of mature oocytes is required, the long GnRH agonist protocol should be used. When convenience, costs, and side effects are taken into account, a single dose of long acting GnRH agonist should probably be the first choice. Finally, we recommend that the future researches to take into consideration the limitations of this study and trying to overcome it, to include large number of patients, to use regression analysis to be able to predict and examine the association between cycle outcomes and the studied protocols. Finally, the researchers should pay more attention to compare the

cycle outcomes between short and microdose protocols as well as between microdose and antagonist protocols because of the observed shortage of data concerning the comparison of these protocols. References

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