Introduction
Reproductive endocrinology, a relatively new subspecialty of obstetrics and Gynecology, came of age during the 1980s. The discipline has benefited
greatly from substantial recent advances in reproductive biology and allied fields and technologic improvements in computers, ultrasonography, and surgical instrumentation. All of these developments have been applied to clinical practice at an unprecedented
rate.(1)
The work of Steptoe and Edwards resulted in the birth of the first in-vitro fertilization (IVF.) infant; the conception was the product of a spontaneous
cycle.(2)
During their initial effort human menopausal gonadotropins (hMG) were utilized for ovarian stimulation to produce multiple follicles for ovum
retrieval.(3)
Their technique using the spontaneous cycle was based on endocrine abnormalities and luteal phase
defects (LPD) associated with stimulation.(4)
However, in view of the relatively low pregnancy rate, due not only to the presence of a single preovulatory follicle in a spontaneous cycle but also to the difficulty of monitoring a spontaneous cycle and performing the oocyte retrieval 24 hours a
day.(5)
Several groups of investigators again adopted the use of ovulation inducing agents.
(6) (7) Indeed, these workers soon demonstrated that not only was it possible to produce pregnancies in cycles in which the patient received ovulation - inducing agents, but actually the percentage of patients successfully undergoing oocyte recovery and ultimately embryo replacement and pregnancy was substantially
higher.(7)
Accordingly, all established groups today rely on the use of ovulation-inducing agents to increase the number of preovulatory follicles, and thus, ultimately the number of embryos available for replacement.
(5) (8) (9) (10) (11)
Ovarian simulation
The first pregnancy obtained by IVF and embryo transfer was obtained using ovarian stimulation but it proved to be an ectopic
pregnancy(3). The first full-term pregnancies were achieved with oocytes from unstimulated
cycles (2). Subsequent studies, however, have shown that ovarian stimulation is associated with better
results(6). Hence, most centres are now performing IVF and other assisted reproduction techniques in stimulated cycles
(12). The following agents are used to stimulate the ovaries:
(i) clomiphene citrate, alone or in combination with hMG (concomitantly or sequentially),
(ii) hMG,
(iii) purified FSH, alone or in combination with hMG,
(iv) highly purified urinary FSH (13) (14) and
(v) recombinant FSH (15).
GnRH agonists are administered intra-nasally, s.c. or i.m., in the long, short or
ultra short protocol and in combination with hMG and/or purified FSH (16)
(17).
GnRH antagonists are involved in the final steps of oocyte maturation, which is achieved by the administration of HCG or by the endogenous luteinizing hormone (LH) surge
(12). Oocyte aspiration is performed 34-38 h after HCG injection or 26-28 h after the detection of an endogenous LH surge
(18).
Follicular development can be monitored by serial hormonal measurements (oestradiol, LH, progesterone) and by ultrasonography
(12) (18). The use of these indices may optimize ovarian stimulation and lower the incidence of OHSS. On the other hand, the application of GnRH agonists requires less
monitoring.(12) (18)
Physiology
Spontaneous cycle
A cohort of primordial follicles are continuously initiating follicular growth independent of gonadotropin stimulation. Once the growing follicle reaches the preantral stage, however, appropriate levels of gonadotropins, particularly follicle stimulating hormone (FSH) are required for development to the preovulatory stage. The presence of FSH induces an increase in estrogen production from the follicle and synergistically, the estrogen and FSH increase the FSH receptor content of the growing follicle
(10) (19) (20).
In a spontaneous cycle the levels of FSH are rising immediately prior to and during menses
(10) (21).
The follicle that is at the appropriate preantral stage of development when the FSH begins increasing is selected to become the sole surviving or dominant follicle. As this soon-to-be dominant follicle begins growing and producing increasing amounts of estrogen, FSH production decreases through negative feedback, thus heralding the death (or atresia) of the less developed follicles.
The role of ovulation-inducing agents for in vitro fertilization is to disturb this normal relationship by increasing the amounts of FSH available to follicles other than the dominant follicles and thus to increase the total number of follicles that reach the preovulatory stage.
(5) (22).
Clomiphene Citrate (CC)
The single agent most commonly used for enhanced follicular recruitment for in-vitro fertilization has been
clomiphene citrate (5).
Clomiphene citrate was first synthesized in 1956, introduced for clinical trails in 1960, and approved for clinical use in the United States in 1967. Clomiphene is available in 50 mg. tablets, under the trade names of Clomid, and Serophene
(10).
It is a mixture of two stereo-chemical isomers which have anti- and weak oestrogenic properties (the En and Zu isomers, respectively). The anti-oestrogenic properties effect ovarian activity via an increase in endogenous
gonadotropin secretion from the pituitary. Current clinical preparations contain about 40% Zu and 60% En isomer
(23).
There are problems associated with CC use:
- Its effects are long-lasting (24). After a standard five-day course of treatment (100 mg daily, starting between the third and the fifty day of spontaneous or induced bleeding), binding activity was detected on day 14 and, in some patient, on day 22 of the cycle.
- The Zu (but not En) isomer is long acting. Significant plasma concentrations of the Zu isomer were detected up to one month after treatment
(25) (26).
- The En isomer is the active component in initiating follicular development
(27). The Zu isomer does not significantly affect the number of follicles present, or oestradiol or luteal phase
progesterone.
- There is a reported higher incidence of subclinical loss in clomiphene citrate-induced pregnancies compared to the normal population
(28).
- The induced increase in luteinsing hormone (LH) secretion can far exceed that for follicle stimulating hormone (FSH)
(29), which is further exaggerated in a polycystic ovaries (PCO) patient. High LH has been associated with
miscarriage(30), which in a PCO patient can be best corrected by the use of a
gonadotropin-releasing hormone (GnRH) agonist (31) (32).
- There is a high reported incidence of luteinised unruptured follicle (LUF) syndrome in patients with unexplained infertility
(33).
The anti-oestrogenic effects are at the level of the cervix and endometrium
(34).
- There is an increased incidence of ectopic pregnancies in in vitro fertilisation (IVF)
(35) (36).
- There is substantial literature support for a possible direct adverse effect at the level of the rat, rabbit and human
oocyte (37).
Clomiphene citrate in IVF
Saunders et al (1992) (38) have associated the use of clomiphene citrate in superovulation cycles with a higher miscarriage rate than GnRH
agonist(38).
Corson and Batzer (1986) (39) and Cohen et al (1986) (35) have suggested a relationship between clomiphene citrate use and ectopic pregnancy. However, Grab et al (1992)
(40) believe that the higher rate of ectopic pregnancies in patients treated with clomiphene citrate is more likely to be associated with the diagnosis of infertility
(40). Harrison et al (1993) (36) have reported a trend towards an increasing ectopic pregnancy rate with increasing daily doses of clomiphene citrate
(36). (This could be related to clomiphene citrate effects on tubal transport
(42). They state that, although clomiphene citrate “remains a valuable tool in the treatment of infertility ... until its does-effect relationship with pregnancy loss is clarified, it would seem prudent to use the minimum possible
dose”.(42)
Gonen and Casper (1990) (41) found that the endometrium was thinner following the use of clomiphene citrate with
hMG compared to hMG alone in IVF patients who had a thin endometrium in a previous clomiphene
citrate/hMG cycle (41). This may be due to the anti-oestrogenic effect of clomiphene citrate on the endometrium.
Most published series on the use of clomiphene citrate alone for enhanced follicular recruitment report a mean of less than 2 oocytes recovered per patient undergoing follicular aspiration
(43) (44).
A report in 1983 demonstrated that 50 mg. per day of CC, when given on cycle days 5 - 9 produced statistically identical degrees of enhanced follicular recruitment (size and number) compared with higher dosage
(43).
In 1995 Benadiva et al (1995) (45) reported that: selected patients who failed previous IVF attempts with gonadotropins with or without GnRH analogues may benefit from the addition of CC to their ovarian stimulation protocol
(45).
In 1998 an open randomized study of IVF in natural cycles or with clomiphene citrate (CC) in the more fertile younger patients and those with normal ovulatory function was done, and the authors were concluded that CC was an acceptable alternative to GnRH-a and FSH yielding a comparable success rate per embryo transfer, but with a low twin rate and if patients accept the increased cycle cancellation rates (40% in natural and 20% in CC cycles), CC may replace GnRH a in selected patient groups in clinics with otherwise high implantation rates, whereas natural cycles IVF seems to be too inefficient for routine use. A negative anti-oestrogenic effect of CC on Oocyte fertilization, embryo development or implantation rates was not detected
(46).
Effect on human oocytes and embryo development
More information is now available on the effects of clomiphene citrate on gametes. Yoshimura et al (1988)
(47) demonstrated no effect of clomiphene citrate administrated to perfused rabbit ovaries on either ovulation or fertilisation rates, but a significant reduction in the number
offspring resulting from embryo transfer (47). Administration of oestrogen to the perfusate reversed this effect, suggesting that the anti-oestrogenic effects of clomiphene citrate may affect post-fertilisation development. Clomiphene citrate also decreases the fertilisation rate in mouse oocytes
(48). In the human, Oelsner et al (1987) (49) have measured high concentrations of clomiphene citrate isomers, particularly the Zu isomer, in follicular fluid obtained at the time of oocyte recovery in women undergoing IVF using clomiphene citrate for superovulation
(49). They reported a direct relationship between the rate of degeneration of blastocysts and the concentration of clomiphene citrate. Wramsby et al (1987)
(50) reported a 50% incidence of abnormal chromosome karyotype in 23 human oocytes obtained at laparoscopy from women treated with clomiphene citrate
(50). While reports to date are largely preliminary, they suggest that clomiphene citrate has a widespread effect which may help to explain the low pregnancy
rate.(49)
Safety
Side-effects
Minor side-effects do occur, but they rarely interfere with treatment. About 10% of women complain of hot flushes during administration; the concomitant administration of oestrogen does not alleviate these
(51). Among almost 4,000 women reviewed by Kistner (1968) (52) less than 2% complained of other minor side-effects such as nausea, vomiting, breast tenderness, dizziness, mild skin reaction and reversible hair loss
(52). Some women (1.6%) noted mild visual disturbances which resolved once the drug was withdrawn.
Two other major side-effects of clomiphene citrate administration are those associated with ovarian stimulation and ovulation induction. Clomiphene citrate induces multiple follicular development, and ovarian hyperstimulation can occur. It occurs less often, however, than following ovulation induction with conventional
gonadotropin therapy, although chronic, low-dose, gonadotropin protocols reportedly cause significantly less ovarian hyperstimulation syndrome and multiple births
(53). Rust et al (1974) (54) reported ovarian cysts in 6.7% of women studied
(54). The duration of therapy is probably more important than the dose of clomiphene citrate used
(55) cysts usually resolve spontaneously in a few weeks, and cases of full-blown hyperstimulation with nausea, vomiting, ascites and hydrothorax are rare
(57) (58). Additionally, bilateral adnexal torsion has been reported after CC therapy
(85). As a consequence of multiple follicular development, multiple pregnancy does occur after ovulation induction with CC (6-7%
(59), 17.8% (60)). While the majority are twin pregnancies, triplets and higher multiples have be reported.
A recent study, which considered 3837 women treated for infertility, between 1974 and 1985, has highlighted that long-term CC use may increase the risk of ovarian cancer
(61), however as only eight women with cancer were identified in this study, more powerful studies are needed to confirm or
refutes these results. The pregnancy rate in both long term (>12 cycles) and short-term CC users was similar. Thus it is recommended that a patient’s cause of infertility should be reassessed if she has not conceived after a maximum of six treated cycles
(61).
Human menopausal gonadotropin (hMG)
hMG Alone
Low Dose
The principal experience with the “physiologic” use of hMG for follicular recruitment comes from the Eastern Virginia Medical School.
(62). Those investigators adapted their extensive experience with
hMG for ovulation induction in anovulatory women to its use for enhanced follicular recruitment. Typically, two ampules of
hMG were administered daily beginning on the third or fifth cycle day, depending upon the length of the preceding cycle. Based on the clinical response of the patients (cervical mucus changes and vaginal cytology) as well as the measured levels of serum estradiol,
hMG administration was continued until the appropriate degree of follicular development was achieved, at which time the preovulatory dose of hCG was given. In their initial experience with this regimen, 2.0
(63) and 2.4 (64) oocytes were recovered per patient
undergoing laparoscopy.
High Dose
In an attempt to further increase the number of oocytes obtained per patient, a group at Yale University pioneered the use of relatively high doses of
hMG for enhanced follicular recruitment (65). These investigators administered 3 ampules per day of
hMG from cycle days 3 through 7, followed by a stepwise increase in the hMG
dosage from cycle day 8 until there were at least two follicles 16-18 mm in mean diameter, at which time hCG was administered. Using this regimen, they reported the recovery of mean of 3.2 oocytes per patient undergoing laparoscopy. In a randomized comparison of high-dose
hMG alone compared with 50 mg of clomiphene citrate per day for cycle days 5 through 9, they recovered in the
hMG group a mean of 4.6 oocytes per patient undergoing laparoscopy (Table
1) (66). The patients in the hMG group received 4 ampules per day of
hMG, beginning on cycle day 3 and continuing until the day before hCG administration. This resulted in a marked increase in the measured levels of FSH, particularly when compared with the levels seen in spontaneous cycles. In this study hCG was administered on the evening of the day that there were at least two follicles greater than or equal to 16 mm in mean diameter.
When the length of the luteal phase in the patients receiving clomiphene alone was compared with the luteal length in the
hMG patients, there was a highly significant shortening among the hMG group
(Table
2). Edwards and co-workers (1980) (2) had previously noted an inverse correlation between the peak follicular phase estrogen secretion and luteal length in a group of patients
receiving high doses of hMG (2). They postulated that the large amount of estrogen produced by the multiple follicles developing in response to the ovarian “hyperstimulation” interfered with subsequent corpus luteum function.
Clomiphene/hMG Combination
Clomiphene and hMG were used in order to maximize the recovery of fertilizable oocytes while minimizing the degree of ovarian hyperstimulation and its associated detrimental effect on the length of the luteal phase. In a prospective, randomized comparison of clomiphene alone (50 mg/day, cycle days 5-9) against the same regimen of clomiphene plus 2 ampules/day of
hMG given on cycle days 6, 8, and 10, there was a statistically significant increase in the number of follicles developing per patient and the number of oocytes recovered per patient, and an increased, but not statistically increased, number of embryos transferred to each patient
(67). In that study a mean of 2.8 oocytes were obtained per patient in the combination group
(Table
3). Interestingly, in that study there was no statistical difference in the measured levels of FSH between the two patient groups However, by the time the daily blood samples were obtained, the additional FSH from the previous day’s
hMG injection was probably cleared, in view of the approximately 3-hour half-life of
FSH.(67)
Other groups have also reported the use of differing combinations of clomiphene and
hMG for enhanced follicular recruitment. Lopata (1983) (68) reported recovering a mean of 4.6 oocytes/laparoscopy from patients who had received several different regimens of concurrent or sequential clomiphene and
hMG.
A group at the University of Southern California (1983)
(69) reported the development of 4.5 follicles per patient, the transfer of 2.4 embryos per patient, and one pregnancy in a group of 13 patients receiving a combination of 150 mg/day of clomiphene on cycle days 3-7 and 2 ampules/day of
hMG on cycle days 3, 5, and 7-11 (69). Mandelbaum et al. (1983)
(70) reported a mean of 3.5 follicles per patient and three successful pregnancies in an unspecified number of patients receiving a combination of clomiphene, 100-150 mg/day, on days 5-9 and
hMG, 2-3 ampules/day, on days 6, 8, and 10.(70)
Another trial used 50 mg of clomiphene per day on days 5-9, plus 1 ampule of
hMG per day on cycle days 5-9, and continued the hMG at a dose of 1-3 ampules/ day based on peripheral estradiol values and the follicular size, number, and growth rate. This regimen has resulted in recovery of a mean of 3.4 oocytes per patient and a 20% clinical pregnancy rate per laparoscopy
(71).
In a retrospective study of 813 oocyte retrieval-embryo transfer cycles in women with normal follicle stimulating hormone and luteinizing hormone concentrations, the relationship between the amount of human menopausal
gonadotropin (hMG) used for ovarian stimulation and treatment outcome was investigated. Patients were divided into three groups: group A patients (495 cycles) required < 40 ampoules of
hMG and had a predicted probability for pregnancy of 25% per embryo transfer; group B patients (165 cycles) required 41-77 ampoules per cycle, with a predicted probability rate for pregnancy of 5-25% per embryo transfer; and group C patients (153 cycles) required > 77 ampoules of
hMG and the predicted probability for pregnancy was < 5% per embryo transfer. Groups C and A differed significantly (P < 0.005). The mean oestradiol concentration on the day of HCG administration in group C was 6412 pmol/1, and the mean number of eggs retrieved was seven. The highest success rates were found when up to 2.5 ampoules of
hMG were required for each egg or 4.4 ampoules for each embryo. The lowest rates were obtained when > 4.8 ampoules of
hMG were necessary for each oocyte or > 9.6 ampoules for each embryo (P < 0.005)
(72).
Gonadotropin releasing hormone analogue (GnRHa)
The introduction of gonadotropin releasing hormone against (GnRHa) prior to and concomitant with human menopausal gonadotropin stimulation has provided one anticipated and one unexpected advanta1ge
(73) (74).
It eliminates the possibility of premature LH surges and in addition it has provided some increase in the success rate of IVF. The GnRH agonist given either by subcutaneous injection or by nasal spray, can in prescribed doses, cause a
down regulation of the pituitary instead of the normal stimulatory effect. FSH and LH secretion are decreased and ovarian follicle activity follows suit. The waves of oocytes that begin growth are inhibited. Therefore, when stimulation with pergonal is initiated, the ovary is in a resting state. It is uncertain why this may confer an advantage during IVF. In addition to preventing premature LH surges and premature luteinization (and
progesterone production). It may decrease LH stimulation of ovarian androgen production (which can interfere with follicular development)
(10) (22).
Hypothalamic GnRH plays a critical role in the neurohormonal control of reproduction by stimulating the secretion of the pituitary
gonadotropins LH and FSH., which support the development of gonads, gametogenesis and the production and release of gonadal steroids. At the pituitary level, GnRH interacts with specific G
protein-coupled receptors located on the surface of gonadotrophs and triggers the generation of an array of second messengers and the activation of several intracellular pathways, to regulate in an integrated manner the
synthesis and release of gonadotropins. These include the activation of phosphoinositidase C with the ensuing production of diacylglycerol and inositol-trisphosphate, which are responsible for the activation of protein kinase C and the mobilization of
intracellular Ca2+ respectively. GnRH also induces the activation of phospholipases D and A2, production of cAMP and cGMP and, under certain circumstances the activation of tyrosine kinases and the MAP kinase cascade. In addition, evidence exists suggesting the presence of extrapituitrary receptors which respond to locally produced GnRH (in gonads, placenta, mammary gland etc.). Ligand analogues which interact with the GnRH receptor, and activate or inactivate the
intracellular signaling cascade and cellular functions are widely used to treat a variety of diseases, including breast and prostatic cancer, infertility, endometriosis and precocious puberty. These analogues have been designed empirically and represent the outcome of a great number of in-vitro or in-vivo structure function studies with chemically synthesized GnRH analogues or natural
GnRHs.(75)
Following their introduction to gynecological practice in the 1980s, the indications and uses have expanded enormously, none more so than in the treatment of aspects of infertility and in particular in their use in assisted reproduction
programs.(74)
The amino acid sequencing of gonadotropin releasing hormone (GnRH) was first determined in 1971
(76). Because of its short biological activity, analogues were synthesized by substituting other amino acid bases or complex molecules.
(77). These were initially used to treat sex-hormones dependant tumours, particularly cancer of the prostate. However by inducing low levels of pituitary
gonadotropins, it was realized that the role of GnRH as could be extended to include the endocrinological manipulation of infertile patients
(78)
In 1982, Meldrum et al. (79) first suggested the use of GnRH-a to create a “medical oophorectomy” in the treatment of endometriosis and in the same year Fleming et al.
(80) described the use of GnRH-a in combination with gonadotropins
for ovulation induction. Shortly after, in 1984, the first report of GnRH-a use in in vitro fertilization
(81) was published. Their use in assisted reproduction has resulted in reduced cycle cancellation, convenient timing of treatment, and higher live birth rates
(82), as a result most units now use GnRH-a routinely despite the extra costs involved. Following increased use in assisted reproductive treatments, in 1990, Abdalla et al.
(83) described the successful use of GnRH-a in women with polycystic ovary syndrome (PCOS) and recurrent miscarriage. The role of GnRH-a in ovarian hyperstimulation syndrome (OHSS) is unclear, but in 1990, Gonen et al.
(84) used GnRH-a to induce ovulation in patients at risk of
OHSS.
|
by Mohamed
Nabil El Tabbakh, MD, OBGYN.net Egypt
Representative