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 µm in diameter expand to more than 120 µm (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 (~

25µm 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). Müllerian 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 ~ 25µm to ~ 120µm. 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 paracrine-

autocrine regulators of follicular growth and atresia are identified (4).

FSH enhances the secretion of most of them by granulose cells. Insulin-

like 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 FSH-

receptor 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 high-

molecular-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 ; E2 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 E2 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 up-

regulation). 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, two-

gonadotropin concept for adequate E2 synthesis but also demonstrate

convincingly that increased E2 production is not mandatory for normal

follicle growth up to the preovulatory stage (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 C17,20-

lyase activity) converts both progestins (pregnenolone and

progesterone) to androgens [dihydroepiandrosterone and

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 8–10 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

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 <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 LH-

stimulated 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 3rd 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. The

ultrashort GnRH agonist stimulation protocol yields results inferior to those

obtained with the short and long protocols (Ron et al., 2005).

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 18-

20mm (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 evidence-

based 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 today’s 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 ~2-

fold (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 8–10 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- Mu¨llerian

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 PI=

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

woman’s 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 so-

called 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 hypothalamo-

pituitary 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 luteal–follicular 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 female’s 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 woman’s 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-Müllerian 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 luteal–follicular 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 1·1 years to 7·3 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 0·1 ng/ml, AMH had a

sensitivity of 87·5% and a specificity of 72·2% 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 1·13 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

Expected response to injectable ovarian stimulating drug

(FSH product) and chances for success

Less than

4

Extremely 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.

4-7 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

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.

8-10

Somewhat reduced count.

Higher than average rate of IVF cycle cancellation.

Slightly reduced chances for pregnancy as a group.

11-14

Normal (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.

15-26

Normal (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 26

High 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 Expected fertility potential for women under 37

of antral

follicles

For 37 and older we need to be more cautious -

low antrals and late 30's or early 40's is

significantly worse

Less than 4 Extremely lowcount.

I think that there is a high risk of poor fertility

potential.

5-7 Very low count.

I think fertility issues are possible - either soon, or

within several years (very hard to predict).

8-11 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

we'd like...

12-14 Low end of the average range.

I am not worried at all yet.

However, as antral counts drop over time, fertility

issues might develop.

Over 14 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 37˚C in 5% CO2, then washed and placed into organ culture

dishes containing the same medium and incubated at 37˚C in 5% CO2 for

approximately 1 hour. Then placed in a 100 µl drop of buffered HTF

(Human tubal factor) containing 80 IU hyaluronidase/ml for 30-45 seconds,

then the oocyte was removed and placed in 100µl 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 100µl

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,

prophase 1; Fig. 21a)

Dense and compact appearing cumulus cells,

tightly packed all around the oocyte

Shows a centrally located germinal vesicle

on light microscopy.

No polar body present.

Grade 2 (nearly mature,

metahase 1: Fig 21b)

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)

Grade 3 (mature/

preovulatory, metphase

11: Fig 21c)

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

Grade 4 (postmature; Fig.

21e)

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.

Grade 5 (atretic nonviable; Atresia oocurs in all oocytes from early

Fig. 21f) immature to postmature stages

Cumulus cell mass is missing

Corona radiata is present, clumped and

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 CaCl2-2H2O 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 µl of HTF supplement with

0.5%HSA. A small amount of 10% Polyvinyl Pyrrolidone (PVP) wormed at

37˚C 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 double-

depression glass slide using holding pipettes and injection needle. The

mature oocyte was contained in a 10 µl 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-µl drop of the 105 PVP solution with a

1-µl 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 37˚C 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 Characteristics

Grad A (high-

quality embryo;

Fig. 23a)

Blastomeres evenly sized, nearly pherical

Cytoplasm uniform, slightly granulated

four blastomeres (48 h after

insemination), eight blastomeres (72 h

after insemination) blastomeres

symmetrically located within the zona

pellucida.

No fragmentation

Zona pellucida looks pale; some corona

cells may remain; some spermatozoa are

visible within the zona.

Grade B (good

embryo; Fig. 23b)

Blastomeres slightly uneven or irregularly

shaped

Gytoplasm with granules reduced

blastomere adherence up to 10%

fragmented blastomeres

Grade C (sufficient

embryo; Fig. 23c)

Blastomeres of uneven size and

appearance reduced blastomer adherence.

Cytoplasm shows large dark granules and

vacuoles.

Blastomere membrane appears ‘patchy’

blastomeres located nonsymmetrically

within the zona, enlarged perivitelline

space up to 50% fragmented blastomeres

Grade D (bad

embryo; Fig. 23d)

At least on blastomere shows be visible

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 µl

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. Basal (day 3) E2.

1.5. 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. Number of mature follicles ≥ 20 mm.

2.2. Endometrial thickness.

2.3. E2 level at day of hCG administration (E2 hCG).

2.4. Number of Meta phase (MII) oocytes.

2.5. Total number of embryos (fertilization rate).

2.6. Number of embryos transferred.

2.7. Number of patient to embryo transferred.

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

(pregnancy rate).

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

Short

protoco

l

n=30

Microdo

se

protocol

n=30

Antago

nist

protoco

l

n=30

P

Value

Age

(years)

26.7±

3.2

29.3± .8 29.2 ±

2.9

27.2 ±

3.7

0.005*

BMI(kg/

m2)

25 ±

2.9

24.5±2.

6

25 ± 3.2 25 ± 2.5 0.8

FSH

(m IU/ml)

6.3

±0.9

7.4 ± 1 7.2 ± 0.8 6.4 ±0.8 <0.0001

**

E2 (pg/ml) 43.5±1

3.5

42.6±1.

5

49.5±11.

4

46.6±

10.2

0.1

Cause of infertility

Tubal 12 (40) 11 11(36.7 12(40)

(36.7) )

Unexplaine

d

9 (30) 9 (30) 8(26.7) 8 (26.7)

Male 6(20) 7(23.3) 6(20) 6(20)

Male&

Female

3(10) 3(10) 5(16.7) 4(13.3)

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.

Variables#

Long

protoco

l

n=30

Short

protoc

ol

n=30

Microd

ose

protoco

l

n=30

Antago

nist

protoco

l

n=30

P

Value

Days of

HMG

13.4 ±

1.7

9.5 ±

1.8

10.2

±1.9

7.5 ±1.1 <0.0001*

Dose of

HMG

2555±9

30

1678±2

61

1944±4

83

1796±1

569

0.003**

Endometrial

Thickness

13±2.4 11± 2.7 11 ± 2.8 11 ± 2 0.004***

E2 at hCG 2823±5

85

2997±4

58

2675±5

58

1524±3

72

0.001***

*

n.of oocyte

retrieved

13±2.4 11± 2.7 11 ± 2.8 11 ± 2 0.004***

**

n.of MII

oocyte

8.7 ± 2 7.2 ±

1.7

6.4 ±2.2 6.1 ±

1.3

<0.0001*

*****

n. of MI

oocyte

2.8 ±

1.4

2.1 ±

0.8

2.4 ±

0.9

2.2±

0.96

0.07

n.

degenerated

oocyte

2±0.8 2.4±

1.1

2.3± 0.7 2.4± 0.7 0.34

n. of 7.3 ± 5.7 ± 5 ± 1.7 4.5 ± <0.0001*

embryos 1.8 1.6 1.5 ******

Number of

ET

2.6 ±

0.7

3.2

±0.6

3.4 ±

0.5

2.6± 0.6 <0.0001*

*******

Pregnant

(/ET)

13

(46.4)

13

(48.2)

11

(42.3)

11

(37.9)

Not pregnant 15

(53.6)

14

(51.8)

15

(57.7)

18

(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

protocol

n=30

Short

protoc

ol

n=30

Microd

ose

protocol

n=30

Antagon

ist

protocol

n=30

P

value

Not cancelled 28

(93.3)

27

(90)

26

(86.7)

29 (96.7)

Cancelled 2 (6.7) 3 (10) 4 (13.3) 1 (3.3)

Cause of cancellation

Poor

responder

1 (50) 2

(66.7)

3 (75) 1 (100)

Negative

fertilization

1 (50) 1

(33.3)

1 (25) 0

#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

Short protocol

n=30

P

Value

Age (years) 26.73 ± 3.3 29.3 ± 3.8 0.008*

BMI (kg/m2) 25 ± 3 24.5 ± 2.6 0.41

FSH(mIU/ml) 6.3 ± 0.9 7.4 ± 1 <0.001*

E2(pg/ml) 43.5 ± 13.5 42.6 ± 11.6 0.79

Cause of infertility

Tubal 12 (40) 11 (36.7)

Unexplained 9 (30) 9 (30)

Male 6 (20) 7 (23.3)

Male& Female 3 (10) 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

n=30

Short protocol

n=30

P

Value

Days of HMG 13.4 ± 1.7 9.5 ± 1.8 <0.001*

Dose of HMG 2555 ± 930 1678 ± 261 <0.001*

Endometrial

Thickness

13±2.4 11± 2.7 0.002*

E2 at hCG 2823±585 2997±458 0.21

N. of oocyte

retrieved

13 ± 2.4 11 ± 2.7 0.002*

Number of MII

oocyte

8.7 ± 2 7.2 ± 1.7 0.004*

Number of MI

oocyte

2.8 ± 1.4 2.1 ± 0.8 0.04*

n. of degenerated

oocyte

2 ± 0.8 2.4 ± 1.1 0.2

n. of embryos 7.3 ± 1.8 5.7 ± 1.6 0.0008*

Number of ET 2.6 ± 0.7 3.2 ± 0.6 0.003*

Pregnant(/ET) 13 (46.4) 13 (48.2)

Not pregnant 15 (53.6) 14 (51.8)

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.

variabels#

Long

protocol

N=30

Short

protocol

N=30

P value

Not cancelled 28 (93.3) 27 (90)

Cancelled 2 (6.7) 3 (10)

Cause of cancellation

Poor responder 1 (50) 2 (66.7)

Negative

fertilization

1 (50) 1 (33.3)

# 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.

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

Microdose

protocol

N=30

P

Value

Age (years) 26.37 ± 3.3 29.2 ± 2.9 0.002*

BMI (kg/m2) 25 ± 3 25 ± 3.2 0.99

FSH (mIU/ml) 6.3 ± 0.9 7.2 ± 0.8 <0.0001*

E2 (pg/ml) 43.5 ± 13.5 49.5 ± 11.4 0.07

Cause of infertility

Tubal 12 (40) 11(36.7)

Unexplained 9 (30) 8(26.7)

Male 6 (20) 6(20)

Male& Female 3 (10) 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

N=30

Microdose

protocol

N=30

P

Value

Days of HMG 13.4 ± 1.7 10.2 ±1.9 <0.0001*

Dose of HMG 2555±930 1944±483 0.002*

Endometrial

Thickness

13±2.4 11 ± 2.8 0.01*

E2 at hCG 2823±585 2675±558 0.33

n. of oocyte

retrieved

13 ± 2.4 11 ± 2.8 0.01*

Number of MII

oocyte

8.7 ± 2 6.4 ±2.2 0.0002*

Number of MI

oocyte

2.8 ± 1.4 2.4 ± 0.9 0.27

n.of degenerated

oocyte

2 ± 0.8 2.3 ± 0.7 0.15

Total number of

embryos

7.3 ± 1.8 5 ± 1.7 <0.0001*

Number of ET 2.6 ± 0.7 3.4 ± 0.5 0.76

Pregnant(/ET) 13 (46.4) 11 (42.3)

Not pregnant 15 (53.6) 15 (57.7)

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.

variables

Long

protocol

N=30

Microdose

protocol

N=30

P

value

Not cancelled 28 (93.3) 26 (86.7)

Cancelled 2 (6.7) 4 (13.3)

Cause of cancellation

Poor responder 1 (50) 3 (75)

Negative

fertilization

1 (50) 1 (25)

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

Antagonist

protocol

n=30

P

Value

Age (years) 26.7 ± 3.2 27.2 ± 3.7 0.6

BMI (kg/m2) 25 ± 2.9 25 ± 2.5 0.9

FSH (mIU/ml) 6.3 ± 0.9 6.4 ±0.8 0.55

E2 (pg/ml) 43.5±13.5 46.6 ± 10.2 0.3

Cause of infertility

Tubal 12(40) 12(40)

Unexplained 9(30) 8(26.7)

Male 6(20) 6(20)

Male& Female 3(10) 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.

Variables#

Long

protocol

N=30

Antagonist

protocol

n=30

P

Value

Days of HMG 13.4± 1.7 7.5 ±1.1 <0.0001*

Dose of HMG 2555±930 1796±1569 <0.026*

Endometrial

Thickness

13±2.4 11 ± 2 0.0006*

E2 at hCG 2823±585 1524±372 <0.0001*

n. of oocyte

retrieved

13±2.4 11 ± 2 0.0006*

n. of MII oocyte 8.7 ± 2 6.1 ± 1.3 <0.0001*

n. of MI oocyte 2.8 ± 1.4 2.2 ± 0.96 0.05

n. of degenerated

oocyte

2 ± 0.8 2.4 ± 0.7 0.05

n. of embryos 7.3 ± 1.8 4.5 ± 1.5 <0.0001*

Number of ET 2.6 ± 0.7 2.6 ± 0.6 0.6

Pregnant (/ET) 13 (46.4) 11 (37.9)

Not pregnant 15(53.6) 18 (62.1)

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.

Variables

Long

protocol

n=30

Antagonist

protocol

n=30

P

Value

Not cancelled 28 (93.3) 29 (96.7)

0.38

Cancelled 2 (6.7) 1 (3.3)

Cause of cancellation

Poor responder 1 (50) 1 (100)

1.0

Negative

fertilization

1 (50) 0

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

Microdose

protocol

n=30

P value

Age (years) 29.3 ± 3.8 29.2 ± 2.9 1.0

BMI (kg/m2) 24.5 ±2.6 25 ± 3.2 0.42

FSH(m IU/ml) 7.4 ± 1 7.2 ± 0.8 0.6

E2 (pg/ml) 42.6 ± 11.5 49.5± 11.4 0.02*

Cause of infertility

Tubal 11(36.7) 11(36.7)

Unexplained 9(30) 8(26.7)

Male 7(23.3) 6(20)

Male& Female 3(10) 5(16.7)

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.

Variables

Short

protocol

n=30

Microdose

protocol

n=30

P

Value

Days of HMG 9.5 ± 1.8 10.2 ±1.9 0.14

Dose of HMG 1678±261 1944±483 0.01*

Endometrial

Thickness

11± 2.7 11 ± 2.8 0.77

E2 at hCG 2997±458 2675±558 0.022*

n. of oocyte

retrieved

11± 2.7 11 ± 2.8 0.77

n. of MII oocyte 7.2 ± 1.7 6.4 ±2.2 0.17

n. of MI oocyte 2.1 ± 0.8 2.4 ± 0.9 0.17

n.of degenerated

oocyte

2.4 ± 1.1 2.3 ± 0.7 0.9

n. of embryos 5.7 ± 1.6 5 ± 1.7 0.1

Number of ET 3.2 ± 0.6 3.4 ± 0.5 0.12

Pregnant(/ET) 13 (48.2) 11 (42.3)

Not pregnant 14 (51.8) 15(57.7)

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.

Variables

Short

protocol

n=30

Microdose

protocol

n=30

P

Value

Not cancelled 27 (90) 26 (86.7)

Cancelled 3 (10) 4 (13.3)

Cause of cancellation

Poor responder 2 (66.7) 3 (75)

Negative

fertilization

1 (33.3) 1 (25)

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

Antagonist

protocol

n=30

P

Value

Age (years) 29.3 ± 3.8 27.2 ± 3.7 0.38

BMI (kg/m2) 24.5 ± 2.6 25 ± 2.5 0.4

FSH(m IU/ml) 7.4 ± 1 6.4 ±0.8 <0.0001*

E2(pg/ml) 42.6±11.5 46.6± 10.2 0.2

Cause of infertility

Tubal 11(36.7) 12(40)

Unexplained 9(30) 8(26.7)

Male 7(23.3) 6(20)

Male& Female 3(10) 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

Antagonist

protocol

n=30

P

Value

Days of HMG 9.5 ± 1.8 7.5 ±1.1 <0.0001*

Dose of HMG 1678±261 1796±1569 0.68

Endometrial

Thickness

11± 2.7 11 ± 2 0.95

E2 at hCG 2997±458 1524±372 <0.0001*

n. of oocyte

retrieved

11 ± 2.7 11 ± 2 0.95

n. of MII oocyte 7.2 ± 1.7 6.1 ± 1.3 0.01*

n.of MI oocyte 2.1 ± 0.8 2.2± 0.96 0.83

n. of degenerated

oocyte

2.4± 1.1 2.4± 0.7 0.9

n. of embryos 5.7 ± 1.6 4.5 ± 1.5 0.003*

n. of ET 3.2 ±0.6 2.6± 0.6 0.0001*

Pregnant (/ET) 13 (48.2) 11 (37.9)

Not pregnant 14 (51.8) 18 (62.1)

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.

Variables

Short

protocol

n=30

Antagonist

protocol

n=30

P

Value

Not cancelled 27 (90) 29 (96.7)

Cancelled 3 (10) 1 (3.3)

Cause of cancellation

Poor responder 2 (66.7) 1 (100)

Negative

fertilization

1 (33.3) 0

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

Antagonist

protocol

n=30

P

Value

Age (years) 29.2 ± 2.9 27.2 ± 3.7 0.2

BMI (kg/m2) 25 ± 3.2 25 ± 2.5 0.96

FSH(m IU/ml) 7.2 ± 0.8 6.4 ±0.8 0.0001*

E2 (pg/ml) 49.5± 11.4 46.6 ± 10.2 0.2

Cause of infertility

Tubal 11(36.7) 12(40)

Unexplained 8(26.7) 8(26.7)

Male 6(20) 6(20)

Male& Female 5(16.7) 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.

Variables

Microdose

protocol

N=30

Antagonist

protocol

N=30

P

Value

Days of HMG 10.2 ±1.9 7.5 ±1.1 <0.0001*

Dose of HMG 1944±483 1796±1569 0.6

Endometrial

Thickness

11 ± 2.8 11 ± 2 0.7

E2 at hCG 2675±558 1524±372 <0.0001*

n. of oocyte

retrieved

11 ± 2.8 11 ± 2 0.7

n.of MII

oocyte

6.4 ±2.2 6.1 ± 1.3 0.53

n. of MI

oocyte

2.4 ± 0.9 2.2± 0.96 0.28

n.of

degenerated

oocyte

2.3± 0.7 2.4± 0.7 0.68

n. of embryos 5 ± 1.7 4.5 ± 1.5 0.2

Number of ET 3.4 ± 0.5 2.6± 0.6 <0.0001*

Pregnant(/ET) 11 (42.3) 11 (37.9)

Not pregnant 15 (57.7) 18 (62.1)

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.

Variables

Microdose

protocol

N=30

Antagonis

t protocol

N=30

P

Value

Not cancelled 26 (86.7) 29 (96.7)

cancelled 4 (13.3) 1 (3.3)

Cause of cancellation

Poor responder 3 (75) 1 (100)

Negative

fertilization

1 (25) 0

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* OR** 95% CI

Long protocol 4.7 1.6 - 14.1

Short protocol 1.1 0.4 – 3.2

Microdose protocol 1.2 0.4 – 3.6

Antagonist protocol 0.5 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 * OR** 95% CI

Long protocol 8.6 2.5 – 29.1

Short protocol 3.2 0.9 – 11.0

Microdose

protocol

1.7 0.5 – 6.2

Antagonist

protocol

0.24 0.09 – 0.7

* 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 * Pregnant Not

pregnant

OR** 95% CI

Long

protocol

13 15 1.4 0.5 –4.03

Short

protocol

13 14 1.6 0.48 - 4.9

Microdose

protocol

11 15 1.3 0.36 – 4.5

Antagonist

protocol

11 18 0.70 0.35 –1.8

* 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 * Cancelled Not

Cancelled

OR** 95% CI

Long

protocol

2 28 2.1 0.18 – 23.9

Short

protocol

3 27 3.3 0.3 – 35.3

Microdose

protocol

4 26 4.7 0.47 – 46.7

Antagonist

protocol

1 29 0.3 0.04 – 2.6

* 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.

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 * P.

ValueR2**

Age 0.26 0.06 0.13

BMI 0.2 0.1 0.1

Short protocol

Age 0.14 0.3 0.04

BMI 0.09 0.6 0.008

Microdose protocol

Age -0.4 0.07 0.13

BMI -0.02 0.9 0.0005

Antagonist protocol

Age -0.04 0.7 0.005

BMI -0.06 0.7 0.007

* means regression coefficient.

** R2 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. Bo-

Abbas 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 of IVF patients as it induces a mild, reversible

hypophysectomy and prevents the premature LH surge resulting in less cycle

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.

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 6th 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.

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