Brief schematic representation of folliculogenesis. Ovarian follicles undergo atresia at every stage of development and differentiation throughout the reproductive life span. They become sensitive to gonadotropins at recruitable stage (class 5) and remain that way afterward. RFP: resting follicle pool, also known as primordial follicles
There are two stages in a reproductive-aged woman’s natural menstrual cycle when serum FSH is in abundance: early follicular phase and mid-cycle. Serum FSH level starts to rise by late luteal phase as a result of disinhibition from declining progesterone and inhibin A production by failing corpus luteum, which leads to a high-normal serum FSH level in the early follicular phase. The ovarian follicles that reach to the recruitable stage by completing their 85-day growth at this time of the cycle will therefore be successfully recruited for further growth. The endocrine environment, however, may not be favorable at mid-cycle for such recruitment despite the presence of FSH. During the first 5–6 days of this FSH abundance at early follicular phase, recruited follicles will simultaneously grow and compete for dominant follicle selection. Multiple factors may influence the number of dominant, preovulatory follicles, although it is typically one during a natural cycle. These factors include but may not be limited to the age of the woman, ovarian reserve, extent and duration of FSH abundance, existence of underlying ovulation dysfunction, and type of it. As the woman’s ovarian reserve diminishes, the number of follicles in the recruitable pool decreases, and recruitment process typically occurs sooner in the cycle [5]. Controlled ovarian stimulation allows for follicular recruitment in women with ovulation dysfunction, who are unable to produce or maintain an adequate serum FSH level to regularly and consistently initiate this process on their own. This can be achieved by either inducing intrinsic FSH secretion or using extrinsic FSH. In the next several sections, we will review the history of ovarian stimulation in a more or less chronological fashion, but let us first define ovulatory dysfunction, which constitutes the main indication for this intervention.
7.1.1 Ovulatory Dysfunction and Use of Clomiphene Citrate for Ovulation Induction
- I.
Hypogonadotropic anovulation
- II.
Eugonadotropic anovulation
- III.
Hypergonadotropic anovulation
Hypogonadotropic anovulation typically refers to a central problem, either hypothalamic or pituitary in origin, causing decreased secretion of gonadotropins into circulation. Suppression of the pacemaker cells of the reproductive clock, also known as gonadotropin-releasing hormone (GnRH) neurons in the arcuate nucleus of the hypothalamus, by neighboring autonomic centers, physical destruction of hypothalamus, or congenital defects that interfere with the migration of GnRH neurons to the arcuate nucleus, may lead to this type of ovulatory dysfunction. The secretion of both FSH and luteinizing hormone (LH) is affected, and their serum levels are typically low (<3 IU/L). The GnRH neurons gain sensitivity to serum estradiol levels with puberty, and their pulse frequency and amplitude decrease with estrogen negative feedback, which may be elucidated by either as a direct action on GnRH neurons or indirectly through other neurons or nuclei [8, 9]. Activin and inhibin, both products of the granulosa cells of the ovarian follicle, contribute to this feedback mechanism, which is typically interrupted with this type of anovulation, either by functional suppression of the GnRH neurons, their absence, or physical destruction. Therefore, hypoestrogenic state does not increase GnRH pulse frequency and amplitude in women with hypogonadotropic anovulation, like it normally would do. With this lack of change in the pacemaker activity, downstream FSH and LH secretion by the pituitary gland remains unchanged and low despite the hypoestrogenic state.
Eugonadotropic anovulation typically refers to an ovulatory dysfunction in the setting of normal serum gonadotropin levels. There are various pathophysiologic mechanisms that may lead to this type of anovulation; polycystic ovarian syndrome (PCOS) and obesity are the most common examples. These women typically require high-normal FSH levels in serum to “jump-start” folliculogenesis by recruiting available follicles as indicated above.
Hypergonadotropic anovulation refers to either severely diminished or depleted ovarian reserve, since this would typically lead to high serum gonadotropin levels. Ovarian stimulation in women with this type of ovulatory dysfunction is usually disappointing and associated with a high risk of cancellation due to the lack of ovarian response. Strategies to optimize outcome in this type of ovulatory dysfunction will be reviewed in detail in Part I of this book.
7.1.2 Clomiphene Citrate
Clomiphene citrate was first synthesized in 1956, used in clinical trials in 1960, and approved for clinical use in 1967 [10, 11]. It is a nonsteroidal triphenylethylene derivative that acts as a selective estrogen receptor modulator (SERM) with both estrogen agonist and mostly antagonist properties. It is a racemic mixture of two stereoisomers, enclomiphene and zuclomiphene [12, 13]. The former is the more potent isomer with a shorter half-life, whereas the latter stays in circulation for several weeks after a single dose [14]. Clomiphene competitively binds to nuclear estrogen receptors for an extended duration of time, interferes with their recycling, and depletes them causing perceived hypoestrogenemia at the GnRH neuron level [12]. This would effectively increase the frequency (in ovulatory women) [15], or amplitude (in women with PCOS) [16] of GnRH pulses, leading to increased FSH and LH release by pituitary gland downstream, assisting or inducing follicle recruitment as reviewed above. The exact nature of the mechanism of action of clomiphene is still uncertain [17], which may involve changes in the insulin-like growth factor (IGF) system [18].
Clomiphene is typically more effective in type II, eugonadotropic anovulation. In the case of an already suppressed or compromised GnRH neuron population, as may be the case for type I anovulation, clomiphene would typically not elucidate an increase in the gonadotropin secretion by the pituitary gland, and folliculogenesis would fail to resume in the ovary. However, due to its low cost and the presence of occasional patients with type I anovulation responding to clomiphene, it is relatively common to try this medication for such cases.
The use of clomiphene on patients with diminished ovarian reserve (DOR) has gained popularity in recent years, which is discussed extensively in the section of minimal stimulation for IVF in women with DOR. This is both due to its low cost and reports of IVF outcome with low-dose clomiphene containing stimulation protocols being comparable to traditional stimulation protocols. Again, these protocols and their effectiveness are reviewed in detail in Part II.
7.1.3 Use of Pituitary Gonadotropins for Ovulation Induction
Initial evidence of an endocrine pituitary-gonadal axis appeared by the observation of genital atrophy following lesions of the anterior pituitary gland [19]. Fevold et al. first confirmed the existence of two separate gonadotropins in 1931, formerly named as prolans A and B, by isolating, purifying, and renaming them as FSH and LH [20]. Even prior to this discovery, the capacity of urine from pregnant women was known to stimulate gonadal function [21], as well as the capacity of daily fresh implants of anterior pituitary gland tissue from various species of animals to stimulate precocious sexual maturity, marked enlargement of ovaries, and superovulation in immature female mice [22]. By 1930, swine pituitary extracts were started to be used clinically to treat patients. This was followed by the use of gonadotropins extracted from various species for the same purpose in the United States and Europe until the early 1960s, when a new phenomenon was discovered, the “antihormones” [23].
Several investigators reported that during chronic treatment with gonadotropins from animal origin, the ovary maintained its response only for a limited period of time; then the response became increasingly weaker and finally disappeared. Antihormones, formation of which was evoked by chronic gonadotropin treatment, were capable of inactivating gonadotropin hormone both in vivo and in vitro. This was effectively the very early description of antibody formation a few decades before the nature of immunological phenomena was fully recognized [24].
Carl Gemzell extracted gonadotropins from human pituitary gland in 1958 and reported clinical results [25]. These preparations remained in clinical use until 1988 throughout the world. However, the reservoir of human pituitaries was too small to meet the growing demand for gonadotropin preparations. Moreover, their use was linked to cases with iatrogenic Creutzfeldt-Jakob disease, which were identified in Australia, France, and the United Kingdom [26, 27]. Thus, these preparations were subsequently withdrawn from the market.
7.1.4 The Discovery of hCG
Blood and urine of pregnant women were discovered to contain a gonad-stimulating substance, as stated above; when immature female mice were injected by either of these fluids, their ovaries showed follicular maturation, luteinization, and bleeding into the ovarian stroma [21]. Initially, this gonadotropic substance was believed to be produced by the anterior pituitary gland; however, Seeger-Jones et al. showed that this gonadotropin was produced in vitro in placental tissue culture, reaching to the conclusion that the placenta was the source [28]. It was then named as human chorionic gonadotropin (hCG). Purified urinary preparations became available in 1940, from the urine obtained during the first half of pregnancy [29]. It was soon discovered that when hCG was administered during follicular phase of the cycle, no visual evidence of follicle stimulation, ovulation, or corpus luteum formation was present in the ovaries [30].
7.1.5 Use of Urinary hMG for Ovulation Induction
Gonadotropins are also readily available in the urine of postmenopausal women, which is a less expensive and more abundant resource than human pituitary glands. Methods were developed to obtain gonadotropins from this resource for clinical use in the early 1950s [31, 32]. Just like pituitary extracts, gonadotropins obtained from urinary source were a mixture of FSH and LH; the preparations were appropriately named as human menopausal gonadotropins (hMG). Despite the purity of available preparations being around 5%, hMG was in clinical use by 1960 [33], and successful ovulation was reported in type 1 (hypogonadotropic) anovulatory women with the use of hMG [34]. The Steelman and Pohley assay, a bioassay on hCG-primed immature rats, became the gold standard for FSH estimation in the hMG preparations [35]. Another problem with both hMG and human pituitary gonadotropin preparations was that the FSH/LH ratio varied from batch to batch. Variations within the range of 0.1 to 10 were deemed acceptable for clinic use in the 1970s. This opened the new challenge in the manufacturing of hMG: purification of FSH from LH and other urinary proteins. With the advancement in immunology, passive and active immunofiltration methods were developed to produce highly purified FSH preparations, which now contained <0.1 IU of LH activity and <5% of unidentified urinary proteins. The purity was also now 95%, raised from the original range of 1–2%. This enhanced purity also allowed subcutaneous administration instead of the traditional intramuscular one. The so-called highly purified FSH virtually eliminated the batch-to-batch variability and enabled the analysis of the end product by physicochemical methods in addition to the classical bioassay [23].
Cross-contamination of communicable diseases cannot be avoided
No regulatory control available
Poor quality control
Impossible-to-trace donor source
Still limited source
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