THE MENSTRUAL CYCLE & OVULATION

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 pelvis2. 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 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 (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 9th 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 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 (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 (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 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 countExpected response to injectable ovarian stimulating drug (FSH product) and chances for success

Less than 4Extremely low count, very poor (or no) response to stimulation and a cancelled cycle expected.Should seriously consider not attempting IVF at all.Rare pregnancies if IVF attempted.

4-7Low 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 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.

8-10Somewhat reduced count.Higher than average rate of IVF cycle cancellation.Slightly reduced chances for pregnancy as a group.

11-14Normal (but intermediate) count, the response to drug stimulation is sometimes low, but usually good.Slight increased risk for IVF cycle cancellation.Pregnancy rates as a group only slightly reduced compared to the "best" group.

15-26Normal (good) antral count, should have an excellent response to ovarian stimulation.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.

Over 26High count, watch for polycystic ovary type of ovarian response.Likely to have a high response to low doses of FSH product.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 folliclesExpected fertility potential for women under 37For 37 and older we need to be more cautious - low antrals and late 30's or early 40's is significantly worse

Less than 4Extremely lowcount.I think that there is a high risk of poor fertility potential.

5-7Very low count.I think fertility issues are possible - either soon, or within several years (very hard to predict).

8-11Intermediate 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 we'd like...

12-14Low end of the average range.I am not worried at all yet.However, as antral counts drop over time, fertility issues might develop.

Over 14Normal 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 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.

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