Ovulation Stimulation with Gonadotropins, 1st ed. 2015

12. Multifollicular Stimulation

Jean-Claude Emperaire1

(1)

Bordeaux, France

For in vitro fertilization (IVF), the process of ovarian stimulation includes some unique yet essential contrasts to the methods for classic mono- or pauci-follicular stimulations.

First and foremost, this process is a multifollicular stimulation. Even if only a single embryo is to be transferred, IVF typically seeks to collect several oocytes in order to provide an optimal choice of embryo for transfer, as well as to enable frozen preservation of supernumerary high quality embryos for possible transfer at a later time.

Therefore, this procedure should be considered as a deliberate ovarian hyperstimulation. It should be fully expected, if hCG is administered, that the excessive quantity of follicles of all sizes, developing in both ovaries, together with the resulting estrogen secretion, will expose the patient to well-identified risks for an ovarian hyperstimulation syndrome. A safe process is made possible solely because oocyte harvesting evacuates the follicular contents and diminishes the chain of events leading to an OHSS. This procedure is in effect a controlled expansion of the safety limits.

Ovarian stimulation is only one of several aspects necessary for a successful outcome from IVF. Chances for pregnancy are very dependent on high standards and quality in the embryology laboratory. While a high-grade laboratory can compensate to an extent for suboptimal oocyte quality, the reverse is not true. The best-run stimulation protocol will be of little value in the face of inadequate embyrology procedures. For this reason, stimulation quality in IVF correlates less well with successful pregnancy rates than do most classic stimulations.

This multifollicular stimulation, or rather, this controlled ovarian hyperstimulation (COH) is realized by manipulating two of the usual rules that control follicular recruitment and development (Fig. 12.1): (1) Administration of supra-physiologic gonadotropin doses as soon as the first days of the cycle, which quickly exceeds the FSH threshold and recruits more elements of the follicular pool. (2) Continuation of these high doses throughout the stimulation period to prevent closure of the FSH window, thereby sustaining a complete development and maturation of most of the recruited follicles.

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Fig. 12.1

Comparison of the classical and multifollicular ovarian stimulation

12.1 Evolution of Ovarian Stimulation Protocols for IVF

Having abandoned the use of clomiphene for ovarian stimulation, Edwards and Steptoe turned next to a low dose gonadotropin stimulation, but left this approach as well because they felt that it led to implantation failures. Their first successful IVF pregnancy in 1978 was actually the result of a spontaneous ovulation that simply involved monitoring the urinary peak of LH to identify the proper moment for oocyte harvest [1]. Once the feasibility of IVF was demonstrated, additional teams decided to stimulate ovulation with the goal of producing more oocytes and more suitable embryos for transfer.

12.1.1 Stimulation Protocols

Initially, two distinct stimulation protocols came into use [2]:

·               The Clomiphene-HMG protocol, more commonly used in Europe: Oral clomiphene citrate, 100 mg daily, was administered for 5 days, starting on CD2, followed by injections of 225 IU HMG on CD 3, 5, and 7, and with the initial first monitoring assessment conducted on CD 9.

·               The HMG-Only protocol, more commonly adopted in the USA: This model was notably developed and used by Drs. Georgiana and Howard Jones, and consisted simply of injections with 150–225 IU HMG daily from CD 2 or 3, and initial monitoring controls made on CD 7–8.

In fact, both protocols had a pair of blatant drawbacks: one was that it resulted in the production of asynchronous follicles of many sizes. In addition, untimely LH surges promoted by supra-physiologic estrogen secretion from the numerous follicles led to premature luteinization and/or to an ovulatory process fraught with difficulties in choosing the precise timing for oocyte harvest. As a result many cycles had to be cancelled.

Furthermore, management of these protocols was somewhat challenging. hCG administration should ideally be done at the precise moment when sufficient numbers of follicles are mature, but before a spontaneous gonadotropin surge provoked by the most advanced follicle begins to halt the development of intermediary follicles. This difficult task often led to a disappointing oocyte yield or to an overly high rate, some 15 %, of cancelled cycles. Successful pregnancy rates, taking into account all degrees of hCG elevation, were only 15–25 % per transfer, although poor performance of embryology laboratories during this era undoubtedly played a role in overall results. At the present time one should expect rates at least twice as high, now that these protocols are enhanced by GnRH antagonists.

12.1.2 Use of GnRH Agonists

The appearance of GnRH agonists in 1986 revolutionized protocols of ovarian stimulation for IVF purposes because the agonists suppress the potential for LH surges and thereby improve management of the stimulation process. Out of this principle, two new protocol designs appeared at virtually the same moment:

·               The Short Agonist Protocol, that exploited the initial “flare-up” effect of a short-lived agonist. It quickly fell from favor because its pregnancy rates were only marginally better than those of previous protocols not using GnRH analogs. While the long agonist protocol rates are now clearly superior, the short agonist approach remains as a possible last resort for certain poor responders.

·               The Long Agonist Protocol, that can be considered as an ovarian stimulation after a preliminary desensitization. This approach has resulted in a clear augmentation of successful pregnancy rates, and has now risen to become the gold standard of ovarian stimulation for IVF purposes.

In contrast to earlier protocols, a more homogeneous follicular cohort results from this long agonist protocol. To be sure, a distinctly greater total gonadotropin dose is necessary to stimulate development and maturation of follicles in a previously desensitized ovary. However, the absence of premature LH surges together with the homogeneous follicle cohort allows selection of more oocytes and leads to higher pregnancy rates. The period for follicle recruitment is also extended. It must also be acknowledged that follicular cohort sizes and plasma estradiol levels are much higher than previously known during stimulation protocols, and this is accompanied by a higher risk for ovarian hyperstimulation.

Because of these enhanced risks for hyperstimulation and also multiple pregnancies following the transfer of several embryos, and also because of modern success with embryo freezing, clinicians are now being urged to consider a single embryo transfer (SET) and to freeze the supernumerary good quality embryos. This reduces the need for multifollicular stimulation cohorts and results in milder (“soft,” “friendly”) stimulation protocols.

The appearance of GnRH antagonists in 1999 could not have come at a better time to enhance this strategy because daily antagonist administration only prevents premature LH surges. Without the preliminary desensitization, the follicular cohort becomes more heterogeneous and smaller in number than with GnRH agonists. Despite a smaller number of oocytes, pregnancy rates remain comparable to those from agonist use but are made possible within a more moderate stimulation protocol. Moreover, risks for ovarian hyperstimulation are dramatically reduced because ovulation may be triggered when necessary using a short lived GnRH agonist.

Use of GnRH antagonists finally brings in the advantages of earlier multifollicular stimulation protocols that were in use before the appearance of GnRH agonists. Thanks to adequate control of the LH levels, it is once again possible to develop a sufficient number of mature oocytes and realize customary pregnancy success rates, but without the drawbacks of the long agonist protocol. On the other hand, management of antagonist stimulation is somewhat more challenging than with agonist protocols. Deciding on the precise moment to introduce the antagonist, at least in the flexible protocol, becomes crucial.

12.2 The Multifollicular Stimulation Protocol for IVF

At the present time, follicular stimulation for IVF purposes (controlled ovarian hyperstimulation, COH) should use both gonadotropins and a GnRH analogue in tandem to prevent a misplaced ovulation or a premature luteinization.

12.2.1 Preliminary Steps

Evaluations and preparations for stimulation are not different from those for a classic stimulation; numerous factors such as tobacco use and body weight should be taken into consideration and corrected whenever possible. Similarly, the clinician must be aware of any chronic medical conditions that could be affected by this type of procedure or by pregnancy itself. One should also investigate for a patient or family history of clotting disorders.

Evaluation of the patient’s ovarian reserve is mandatory for COH, because this assessment helps to anticipate excessive and/or untoward ovarian responses, and it provides guidance for selection of the starting gonadotropin dose. The two cornerstone indices of ovarian reserve are the AFC and plasma AMH. These measures usually vary in the same direction, but there is not yet a true consensus of normal ranges for these parameters. That said, an excessive response to stimulation should be anticipated when the total AFC is more than 24 follicles and/or the AMH level is above 6 ng/ml. Conversely, an insufficient response is likely when the total AFC is less than six follicles and/or the AMH level is lower than 1.0 ng/ml. It should be noted that while the AMH level correlates to some extent with the size of the recruited follicular cohort, it cannot predict oocyte quality or the chances for a successful pregnancy [3].

Several algorithms with multifactorial calculations have been proposed to establish the proper starting dose for each patient, but these typically approach only a probabilistic level [4]. Nevertheless the initial dose, which will be maintained for the first 5–6 stimulation days, is highly important for a harvest of 6–15 mature oocytes, the optimal number for achieving a live birth [5]. An insufficient starting dose will recruit a low follicle number that will not be corrected by further increases of posology; an excessive dose will provoke growth of too many follicles and a decrease of subsequent dosing may become very difficult to manage.

12.2.2 GnRH Analogues

Gonadotropin releasing hormone (GnRH, gonadorelin) is a decapeptide secreted in a pulsatile manner by hypothalamic neurons located primarily in the preoptic nucleus that project to the median eminence for secretion into portal vessels. Arcuate neurons project dopaminergic fibers that modulate GnRH release and suppress prolactin release. Upon transport via hypothalamic-pituitary portal vessels, GnRH binds to specific receptors on pituitary gonadotrophs. A pulsatile neuropeptide secretion signals a similar pattern of FSH and LH release that controls plasma gonadotropin levels, their circulating ratio, and also their relative bioactivity according to variations of their isoform composition.

The pituitary gonadotroph GnRH receptor is a G-coupled protein whose function follows the basic principle of desensitization. In order to exert a stimulating effect upon the gonadotropin secretion, the occupation of these receptors must be episodic, as is precisely the case with endogenous pulsatile GnRH secretion. When receptor occupation is maintained by continuous administration of GnRH or a high-affinity analogue, a tachyphylaxis occurs that terminates additional gonadotropin release. The intracellular subunits of the G-coupled GnRH receptor must reassemble in order to respond to another stimulus, but cannot do so while the peptide ligand remains bound to the outside binding domain. In order to be effective, GnRH administration must be pulsatile and this is accomplished through the use of a programmed pump. Continuous administration of GnRH is also possible if one wishes to terminate or down-regulate further gonadotropin release in order to reduce gonadal activity (Fig. 12.2).

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Fig. 12.2

The opposite effects of gonadorelin in accordance with its mode of administration

Substitution of certain amino-acids of the GnRH decapeptide sequence creates analogues having different biological properties than the native hormone. However, the three first amino acids of the sequence are necessary for biological activity and the eight residue is essential for high affinity receptor binding (Fig. 12.3).

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Fig. 12.3

Gonadorelin structure

12.2.3 GnRH Agonists

A simple substitution of the l-alanine residue at position 6 or the l-glycine at position 10 with the respective dextrorotary enantiomer creates a potent agonist molecule because it gains an enhanced affinity for the GnRH receptor and the structure resists degradation by plasma peptidases. These modified peptides retain a longer bioactive half-life and are effective at lower doses, which explains the contradictory effects of long acting agonists on gonadotropin secretion. An initial potent stimulation of gonadotropin release (the “flare up” effect) is quickly followed by a secondary inhibition due to residual occupation of receptors by the agonist. Continuous administration of native GnRH also produces a refractory desensitization of gonadotrophs. Similarly, prolonged administration of these modified agonists, either daily (in nasal sprays or by subcutaneous administration) or as injected depot preparations, results in FSH-LH suppression and a concomitant anovulation and reduction of ovarian steroids. This property is being used to advantage in various estrogen-dependent clinical conditions, from precocious puberty to endometriosis to hormone-dependent malignancy (Table 12.1).

Table 12.1

Structures of gonadotropin releasing hormone (GnRH, gonadorelin) and several analogs

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12.2.4 GnRH Antagonists

Substitution of the amino acids at positions 1, 2 and 3 of the decapeptide sequence results in loss of most of the biological activity, but the property of high-affinity receptor binding is preserved. This type of analogue acts as a GnRH antagonist: it binds to the receptor without an initial flare up effect while it prevents native GnRH from gaining receptor access (Fig. 12.4). A single administration has only a brief duration of action (about 24 h) and is reversed by use of GnRH itself or with one of the synthetic short lived agonists.

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Fig. 12.4

Modes of action of GnRH agonists and antagonists

12.2.5 COH with GnRH Agonists

Use of GnRH agonists was introduced into multifollicular stimulation protocols for IVF purposes with the aim of preventing inappropriate LH surges. This controls the number of follicles reaching full maturity without risk that the more advanced ones would trigger a spontaneous ovulation. At present there are two protocols that differ in the timing of GnRH use:

The Short Protocol (Fig. 12.5). This utilizes only short-lived agonists, administered daily. Treatment starts on CD2 with the agonist alone for two days, then gonadotropin injections are added from CD3 until ovulation. The two important effects of the agonist are at work:

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Fig. 12.5

Design of the short agonist protocol

·               The initial stimulatory action provokes an FSH – LH surge that initiates follicular recruitment, roughly equivalent to a daily administration of 150 IU FSH.

·               The subsequent pituitary tachyphylaxis permits exogenous ovarian stimulation through a full maturity of the follicular cohort but removes risk for a premature endogenous LH surge.

The first monitoring control should be done on CD6, the 5th stimulation day of four days GnRH agonist with two days gonadotropin administration added. Thereafter, assessments should be conducted at 2–3 day intervals according to the ovarian response (Table 12.2).

Table 12.2

Management of short agonist multifollicular protocol with a stimulation target of 6–14 follicles after 2 days of FSH (see StimXpert)

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This table illustrates the types of decisions that should be made with various combinations of follicular response (FD mm, top row) and plasma estradiol (E2 pg/ml, left side column)

Within each box, The Circle Number indicates the optimal number of days to continue stimulation, if triggering criteria have not yet been met after 2 days of stimulation. The Circle Color indicates the suggested percentage change for FSH dosing in relation to the previous (starting) dose. A White Circle with S indicates the cycle should be stopped or abandoned with no further stimulation. Boxes with HCG identify the follicle + estrogen criteria that permit ovulation triggering. Boxes with a number in parentheses refer to 1 of 3 footnotes listed below

When a patient is unable to come in for assessment after the indicated number of days, it remains possible to conduct the monitor one day earlier or later, possibly including a change of the FSH dose

The number of estradiol ranges has been purposely limited in order to simplify the table’s construction, and should be considered as approximate when interpretations are made. In particular, when values are close to the indicated upper or lower value of E2 within a range, the clinical decision should consider options indicated for the adjacent hormone level

Footnotes

1. Follicular growth that progresses faster than the rise of plasma estradiol levels may be related to an insufficient LH effect; in this case it is better to repeat the stimulation with an FSH preparation that includes LH

2. Caution: Risks for OHSS increase to the degree that plasma estradiol surpasses 2,500 pg/ml

3. A stimulation that progresses too rapidly (<7 days) may harm oocyte quality

The Long Protocol (Fig. 12.6). This design introduces a new concept in ovarian stimulation, by exploiting even more the principle of pituitary desensitization. Stimulation itself is begun only after the ovaries have been put to rest by the prolonged withdrawal of endogenous gonadotropins following the agonist-induced shutdown of pituitary FSH and LH. The initial GnRH-induced flare up effect is not exploited in this protocol (Table 12.3). Ovarian activity ceases following daily administration of a short-lived GnRH agonist, or after a single injection of a long-acting GnRH agonist that lasts upwards of 1 month. In the latter case, gonadotropin administration should begin during the second half of the agonist effect, so that a possible embryo is not exposed to the GnRH analogue. Accordingly, gonadotropin administration begins 15–20 days after the agonist is administered, when ovarian shutdown has been established, and the injections continue under agonist control until the ovarian triggering.

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Fig. 12.6

Design of the long agonist protocol

Table 12.3

Management of long agonist multifollicular protocol with a stimulation target of 6–14 follicles after 5 days of FSH (see StimXpert)

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This table illustrates the types of decisions that should be made with various combinations of follicular response (FD mm, top row) and plasma estradiol (E2 pg/ml, left side column)

Within each box, The Circle Number indicates the optimal number of days to continue stimulation, if triggering criteria have not yet been met after 5 days of stimulation. The Circle Color indicates the suggested percentage change for FSH dosing in relation to the previous (starting) dose. A White Circle with S indicates the cycle should be stopped or abandoned with no further stimulation. Boxes with HCG identify the follicle + estrogen criteria that permit ovulation triggering. Boxes with a number in parentheses refer to 1 of 3 footnotes listed below

When a patient is unable to come in for assessment after the indicated number of days, it remains possible to conduct the monitor one day earlier or later, possibly including a change of the FSH dose

The number of estradiol ranges has been purposely limited in order to simplify the table’s construction, and should be considered as approximate when interpretations are made. In particular, when values are close to the indicated upper or lower value of E2 within a range, the clinical decision should consider options indicated for the adjacent hormone level

Footnotes

1. Follicular growth that progresses faster than the rise of plasma estradiol levels may be related to an insufficient LH effect; in this case it is better to repeat the stimulation with an FSH preparation that includes LH

2. Caution: Risks for OHSS increase to the degree that plasma estradiol surpasses 2,500 pg/ml

3. A stimulation that progresses too rapidly (<7 days) may harm oocyte quality

Long-acting agonists may be administered on the first day of the cycle (a “follicular long-agonist” protocol), with the desensitization control and gonadotropin administration beginning on CD15. Alternatively, it may be started following ovulation (CD20) in the previous cycle (a “luteal long-agonist” protocol); in that case, desensitization control and gonadotropin administration should begin on CD 10 of the following cycle.

Both protocols produce comparable results. Observations of functional ovarian cysts on the day of desensitization control are undoubtedly less frequent after the luteal administration protocol. On the other hand, the follicular administration protocol is the approach of choice in cases of irregular cycles, because administration on CD 20 of the previous cycle might intervene just before a late spontaneous ovulation and thus disorganize the following menses.

When complete ovarian rest is assured and gonadotropin administration is started, it is possible to reduce the daily dose of a short-lived agonist by half without risking a pituitary “escape” before triggering ovulation. Although this method was initially proposed for cases of poor ovarian response, it may be extended to all the patients.

12.2.6 Choosing a GnRH Agonist

Long-acting depot agonists produce a more “depressed” pituitary gland than the ovarian rest obtained with daily injections of short-acting agonists. The latter usually leaves a basal level of gonadotropin secretion that daily agonist injections maintain at a minimal but not absent level. For this reason a greater total quantity of gonadotropins will be necessary to complete a stimulation cycle when long-acting formulations are used, because there is not even a low-level endogenous pituitary contribution.

None of the available GnRH agonists has been established as superior to any other insofar as pregnancy rates are concerned, and there are no reports of systematic prospective studies comparing one to another. On the other hand, patient responses may vary from one agonist to another, and a particular agonist may produce different results in different patients. This difference can be appreciated by examining the gonadotropin surge profile of the flare up effect, or by the comparative ability of various agonists to suppress FSH and LH in different patients. Thus one may consider changing to a different agonist in the same patient, from one COH to another.

Regardless of the ovarian appearance at ultrasound, pituitary suppression may be considered complete when plasma estradiol falls below 30 pg/ml. Ovarian ultrasound should reveal a number of follicles <6–8 mm accompanied by a thin linear uterine endometrium. Complete pituitary shutdown may not be reached by the time of the first examination. In these cases, continuation of the short agonist, or waiting several days longer after a depot long-acting agonist injection, may bring a more satisfactory ovarian rest.

It remains however possible that estradiol values will level off, or even increase while awaiting the moment to commence gonadotropin injections. In these cases, one may consider prolonging the cycle by immediate administration of another agonist and then repeating the assessment 15 days later. On rare occasions a paradoxical stimulatory effect of a depot agonist may occur, and a true ovarian hyperstimulation still remains possible. If ultrasound reveals numerous mature or pre-mature follicles in the presence of elevated serum estradiol levels, moving ahead with triggering the ovulation and retrieving oocytes seldom results in a successful pregnancy, and usually leads to a diminished chance for pregnancy in the patient [6]. In this situation it is preferable to cancel the cycle and prescribe 20 days of a potent oral progestin, whose anti-gonadotropic effect will return the ovaries to a resting state.

Once the desensitization is accomplished, ovarian stimulation may start, with an initial monitoring control made after 5–6 days of gonadotropin administration. Additional controls, basically intended to decide possible modifications of posology, are scheduled every two to three subsequent days. Two or three evaluations following the desensitization control will usually be necessary and sufficient.

12.2.7 Choosing a Gonadotropin Preparation

All of the available commercial preparations with an FSH effect produce essentially comparable pregnancy rates. The only question for clinicians is whether to add LH during the protocols, because use of GnRH agonists will suppress pituitary release of both gonadotropins. In fact, a large majority of patients maintain a residual, sufficient LH secretion, so administration of FSH alone should provide adequate ovarian stimulation. An essential minimal level for LH is estimated to be 1.2 IU/l. In theory, this identifies a need for serum LH monitoring, if a borderline endogenous LH secretion becomes insufficient for optimal follicular maturity. However, most LH assay kits are inadequately sensitive for accurate measures below 2 IU/l. Insufficient LH secretion may also be suspected when the follicular growth rate observed at ultrasound is rising faster than estradiol levels, but this kind of discrepancy may be very difficult to identify with a multifollicular cohort. The problem is manageable by changing the gonadotropin preparation and turning to an FSH + LH effect for successive cycles.

In summary, while the systematic supplementation of FSH with LH in agonist stimulation protocols does not yield better conception results overall, save for some older patients, the addition also has no recognized detrimental effect. There seems to be no overall difference in results obtained using pure FSH preparations versus those combining FSH and LH.

12.2.8 Choosing a Starting Dose

A starting dose of 150 IU seems best for the younger, normally-responsive patient. Higher doses are recommended for patients over 38 years of age, in those who are substantially overweight, or in those with a low ovarian reserve established by measures of AFC and/or AMH. Dosing level should also be diminished in the opposite situations.

Several algorithms have been proposed to calculate a priori the starting dose by means of several measured parameters, but it appears they only offer statistical data that may actually lead to erroneous assumptions for some patients [45]. The number of possible starting doses being rather low, this dose may be calculated rather easily for most cases by listing the patient’s age, her AFC or AMH level, and her BMI in a tabular fashion that increases or decreases the dose (Table 12.4).

Table 12.4

A simplified calculation schematic for the starting gonadotropin dose based on patient age, ovarian reserve (AFC or AMH level) and BMI

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Each of the three parameters can induce a 50 % increase or a 33 % reduction of the average starting dose (150 IU). The maximum range of changes remains between 75 IU (50 % reduction) and 300 IU (100 % increase)

12.2.9 Ovarian Responses to Monitor

Ovarian reactions to the gonadotropin administration may be evaluated according to two different approaches:

1.

2.

12.2.10 Criteria for Triggering

Among all the designed COH protocols, the long agonist protocol entails the greatest risk for ovarian hyperstimulation, if for no other reason that the process may be pursued without theoretical limits. There are likewise no generally acknowledged safety limits for the targeted number of follicles or the level of serum estradiol. Fertility teams tend to define their own limits in accordance with their own experiences and perceptions of various hazards. This question is further complicated in cases of COH because plasma estradiol levels, typically viewed as the best marker for OHSS risk, are poorly correlated with the follicular number due to the numerous (and safer) small follicles that still contribute to the estrogen pool. Estradiol level per follicle >14 mm diameter has been estimated to vary between 70 and 140 pg/ml [9], thus a maximum of 15 mature follicles together with an estradiol level ≤2,500 pg/ml would appear to be a reasonable upper limit. A higher estradiol level in the presence of numerous mature follicles would appear to be less hazardous than that same level in the presence of only a few mature follicles accompanied by a greater number of small and intermediate follicles.

In addition, the decision to trigger ovulation or to abandon the cycle is challenged by the unpredictable character of OHSS. On the one hand complications may not occur outside the safety limits, and on the other they may well happen within the safety range. The usual reason for pushing toward an OHSS is that clinicians are reluctant to cancel a cycle in uncertain circumstances when it can lead to further disillusionment for couples who have already invested so much.

Serum progesterone in excess of 1.5 ng/ml, on the morning of the scheduled triggering, may decrease the chances for pregnancy, undoubtedly because it disturbs the implantation window, even as fertilization rates and embryonic development would appear unchanged. An elevated progesterone result should cause the cycle to be stopped, or alternatively the embryos should be frozen for implantation at a better moment, because the implantation ability of such embryos appears unaffected in another cycle [10]. Also, a rapid and short stimulation (less than 7 days) may result in suboptimal oocyte quality, and reduce the chances for success.

12.2.11 Comparing Short Versus Long Protocols

Both protocols were commenced as soon as the GnRH agonists appeared on the market, and their respective advantages and disadvantages became quickly recognized.

The short protocol, no matter the chosen strategy of use, has suffered from comparatively inferior rates of pregnancy. This deceptive advantage is explained in part by the protocol’s tendency to develop smaller and less homogeneous follicular cohorts. Indeed, results are little improved over those observed with earlier protocols that did not include co-treatment with GnRH analogues.

The long protocol, however, has also been beset with some drawbacks, balanced by the critical benefit of pregnancy success to rates that were previously unknown. Among its major drawbacks is the need for a significantly larger quantity of administered gonadotropin per cycle. Higher FSH doses are necessary in order to reactivate the resting ovaries and also to drive the stimulation in the total absence of endogenous FSH. In addition, the prolonged pituitary-ovarian shutdown induces a form of menopausal syndrome of estrogen deprivation. Some patients may suffer hot flashes with sweats, headaches, and numerous other symptoms before the slowly developing stimulated follicles begin to replenish her estrogen. Many patients will recall this as the most difficult part of the entire IVF experience.

Nonetheless the advantages to the long protocol are also significant: the ovarian desensitization step of putting the ovaries to rest prior to beginning the stimulation induces a better, more homogeneous follicular cohort than with any of the other COH protocols in use. This poorly understood phenomenon results in greater numbers of high quality oocytes and embryos to select for fresh transfer or for freezing. Secondly, the monitoring process is made simpler because the ovaries have been basically disconnected from the pituitary and are responding only to the administered gonadotropins. A truly linear, almost monotonous stimulation is certainly the easiest to manage, save for the omnipresent risk for an OHSS. Thus, and perhaps most importantly, the rate of successful pregnancy was significantly higher than with any other COH protocol in use at that time, even as possible side effects, particularly on the endometrium, could not be ruled out. The long agonist protocol quickly became the gold standard for COH, and remains so for numerous IVF teams despite the evolution of other approaches following the appearance of GnRH antagonists.

12.2.12 COH with GnRH Antagonists

The availability of GnRH antagonists has permitted another approach for prevention of premature LH surges during the multifollicular simulation. This has also led to a more flexible protocol management because of the crucial differences in agonist-versus-antagonist effects on the pituitary gland.

First of all, an antagonist produces no initial stimulation of FSH-LH, as gonadotropin secretion ceases immediately. The effect can be reversed by an agonist within hours, whereas even a single administration of a short agonist is followed by a pituitary tachyphylaxis that persists for several days. Antagonists do not result in pituitary desensitization, even following a prolonged administration, and a normal pituitary response can be restored quickly by administration of gonadorelin or a synthetic congener. Antagonist protocols are of the short type, with gonadotropin administration starting as soon as the first days of the menstrual cycle.

12.2.13 Pre-treatment

The stimulation cycle may be prepared with a combination oral contraceptive during the cycle prior to stimulation, in order to prevent an inter-cycle rise of endogenous FSH. An alternate strategy is for the patient to take 4 mg oral micronized 17-β estradiol beginning on day 20 of the cycle prior to the planned stimulation. This has no recognized effect on the pregnancy rates, but it helps for the programming of oocyte retrieval. For example, one can begin gonadotropin administration on Wednesday through Friday of a week and thereby avoid ovarian punctures and laboratory work over the weekends [11].

12.2.14 Starting the Antagonist Administration

Gonadotropin stimulation can begin as soon as CD 2–3, or one to two days after discontinuation of the oral hormone pre-treatment, using a fixed dose for the first five stimulation days, until the first programmed monitoring control. At this point, two different antagonist protocols are possible depending upon the chosen moment of antagonist introduction.

1.

2.

Both protocols yield comparable results in terms of pregnancy rates: the fixed protocol usually results in more antagonist injections, but still remains as easy to manage as a short agonist stimulation. On the other hand, the flexible antagonist protocol usually requires fewer injections but needs more experience to conduct successfully, particularly to avoid starting the antagonist too late [13].

At present one can choose from two short lived antagonists for daily administration of 0.25 mg. A third preparation (cetrorelix 3 mg) designed for depot use and a 3-day duration effect is no longer available.

12.2.15 Revisiting Other Protocols with Antagonists

Suppression of LH surges without pituitary desensitization by using GnRH antagonists has led to improved management of some earlier developed protocols:

·               The clomiphene-HMG/FSH protocol retains a main advantage of simplicity and a reduction of gonadotropin consumption. Criteria for introducing the GnRH antagonist remain the same, e.g., at the time of the initial monitoring control on CD9 [14].

·               The natural cycle protocol tampers with the principal follicle only after it has been spontaneously recruited and selected. Concomitant daily administration of FSH and LH, usually as 150 IU HMG, plus a GnRH antagonist, can begin as soon as the follicular diameter reaches 14 mm. This helps sustain the selected follicle growth during the few days necessary to achieve optimal maturity, but without running the risk of a premature gonadotropin surge [15].

12.2.16 Recommendations for Management of the Stimulation (Table 12.5)

12.2.16.1 Selecting the Gonadotropin: Antagonist Combination

No particular combination of the various gonadotropin preparations and the two available antagonists has ever been established as superior. On the other hand, one combination may work best in certain patients, and for this reason it is best to consider changing the combination from one stimulation cycle to the next in the same patient.

12.2.16.2 Starting Doses?

In theory, initial FSH dosing should be set lower than those used in the long agonist protocols designed to “awaken” a resting ovary. In current practice, however, the same 150 IU starting dose seems best for all young normal responders. Dosing may need to be adjusted with respect to the patient’s age, ovarian reserve, and BMI.

12.2.16.3 Schedule for Monitoring

An initial assessment should be done on the day of the first gonadotropin administration, to examine for a functional cyst, or an abnormally high progesterone level. A second ultrasound and hormonal sampling should be done on the sixth stimulation day, followed by additional assessments at two-to-three day intervals. Three or four evaluations should suffice. Although LH measures are not useful in the long agonist protocol, they become mandatory in this situation because of the risk for a pituitary “escape.” While a single, isolated rise of LH (remaining under 10 IU/l) may still be effectively managed by administration of a GnRH antagonist, and not impact continuation of the cycle, a higher LH increment followed by a progesterone rise, no matter the reason, should cause the cycle to be stopped.

In addition, a rising progesterone level (>1.5 ng/ml) on the morning of the planned triggering, even in the absence of an LH surge, signals a possible ovulation escape, and could result in failure to harvest an oocyte during follicular puncture. In this situation one may choose to cancel the cycle or to continue with ovum retrieval and, if oocytes are harvested, to freeze all embryos for a later transfer. This avoids fresh transfers with an endometrium that is too mature.

12.2.16.4 Criteria for Triggering Ovulation

Criteria for triggering are the same as with the long agonist protocol, and are designed to yield a good follicular cohort (8–15 mature follicles) without risking an OHSS. Using a GnRH antagonist is very advantageous in this instance because the antagonism can be quickly reversed by administration of a short-acting agonist. This action results in an adequate final maturation of the follicles with resumption of meiosis, the only step of the ovulatory process that is still required when conducting IVF: follicular puncture replaces follicular rupture and luteal gland secretion is systematically supported by administration of progesterone. Subcutaneous injection of 0.2 mg triptorelin in place of hCG brings a substantial reduction of risk for early hyperstimulation, and does not appear to hamper oocyte harvest and quality. For these reasons, oocyte donor protocols consistently favor this agonist/antagonist approach.

However, triggering ovulation with an agonist in an antagonist protocol does have several drawbacks:

·               Follicular puncture may fail to retrieve an oocyte despite careful follicular rinsing, if serum progesterone is < 3.5 mg/ml on the following morning [16].

·               It may not prevent a late ovarian hyperstimulation, in the presence of an implanted embryo.

·               It can be followed by a defective luteal phase and a dramatic decrease of implantation rate [17]. Progesterone administration alone or in association with micronized estradiol may not be sufficient, and co-administration of a small dose of hCG (750–1,500 IU) on the evening following follicular puncture will be necessary to rescue chances for pregnancy. Of course, the administration of hCG also restores a certain risk for early OHSS [18].

On the other hand, triggering ovulation with a GnRH agonist in an antagonist protocol provides better management of some hyperstimulation risks:

·               If “reasonable” risk of hyperstimulation: trigger ovulation with hCG and complete the transfer.

·               If “high” risk: trigger with a short-acting GnRH agonist, administer 750–1,500 IU hCG on the day of oocyte retrieval, and transfer. In questionable cases, hCG administration may be delayed as late as the 5th–6th day after ovum retrieval; this permits an assessment for early hyperstimulation before deciding on going ahead with the transfer.

·               If “highly critical” risk: trigger ovulation with a short-acting agonist, do not support the luteal phase, refrain from completing the embryo transfer in the present cycle and freeze them for a later transfer. This policy does presume that the fertility center or clinic has satisfactory pregnancy rates with frozen embryos. Otherwise this situation could end with diminished chances of success for the patient.

Effective execution of these different strategies, which are becoming widely accepted among the IVF community, should result in a significant reduction, and possibly a total elimination, of OHSS secondary to COH, and to create at last a “hyperstimulation-free clinic.”

12.2.17 The Unique Situation Regarding Corifollitropin Alpha

Corifollitropin is a “long acting” FSH preparation with effects lasting up to seven days following a single injection. Thus it is possible to start a stimulation protocol with a single FSH administration, conduct an initial monitoring assessment on Day 8, and continue the protocol with another gonadotropin preparation if triggering criteria have not been met at the first assessment. Use of this product contrasts with other FSH preparations in several ways:

·               At present, corifollitropin is indicated only for patients with a normal ovarian reserve as determined by the usual criteria. There are not enough data yet on the use of this preparation in either hypersensitive or poor responders.

·               The initial dose is determined by the patient’s weight and age rather than her BMI, and only two dose sizes are available: 100 IU for patients <60 kg and up to 36 years of age; and 150 IU for patients ≥60 kg whatever their age, or >50 kg when they are >36 years of age.

·               At the present time, only the fixed-antagonist protocol can use corifollitropin, because the initial monitoring control does not occur until the eight day following stimulation; at this moment, a gonadotropin surge may have already begun in some patients, so the GnRH antagonist needs to be introduced as soon as the sixth day following beginning of stimulation.

Depending upon the ultrasound and hormonal parameters observed on the eight day, the protocol proceeds from there in the same manner as would a fixed antagonist protocol conducted with any other FSH preparation.

12.2.18 Long Agonist and Antagonist Protocols

After an initial tumultuous period that included many enthusiastic reports following the introduction of this new stimulation tool, GnRH antagonist protocols began to disappoint many clinicians who were conducting controlled hyperstimulation procedures. Pregnancy rates calculated by oocyte retrieval or embryo transfer seemed significantly inferior to those of the long agonist protocol. Furthermore, antagonist management strategies seemed to bring more new questions (e.g., indications, product choice, preferred FSH preparation, and triggering criteria) to clinicians who were typically accustomed to the linear, almost monotonous process of long agonist protocols that usually produced more satisfaction than concerns [19]. It took the perseverance of a few groups dedicated to sustaining the antagonist protocols to begin convincing clinicians who were understandably reluctant to adopt changes that could threaten their pregnancy rates. Indeed, as with any new methodology, a certain learning curve is necessary to begin recognizing that an antagonist protocol can be managed with success in the same way as other multifollicular stimulation treatments [20]. At present, the two types of COH with GnRH analogues are recognized as offering rather the same chance for pregnancy, and it is now easy enough to evaluate their respective advantages and drawbacks [21].

The long agonist protocol has two main advantages: (1) a better, more homogeneous follicular cohort that results in a higher oocyte yield, (2) a simple management design that works well even with some approximations. The protocol also has significant drawbacks: (1) a longer duration of treatment that includes 10–15 days of agonist preparation prior to stimulation, (2) the agonist may induce an unpleasant period of estrogen withdrawal, (3) there is always a risk for OHSS that can prove difficult to manage, because an agonist triggering is not possible with this protocol.

The antagonist protocol includes many of the reverse characteristics. The main disadvantages are: (1) a smaller, less homogeneous follicular cohort that should still be sufficient to provide cumulative pregnancy rates comparable to those of the long agonist protocol, perhaps due to better oocyte quality, (2) the monitoring is less forgiving of management inaccuracies, insofar as the flexible protocol is concerned. Among the advantages are: (1) a shorter treatment duration with lower total doses of gonadotropins, (2) a better patient tolerance for the stimulation, due to the shorter treatment period and the absence of estrogen deprivation symptoms, and also with fewer injections when corifollitropin alpha is used, and (3) an easier management of risks for early ovarian hyperstimulation, principally because of the possibility of turning to a GnRH agonist in order to trigger ovulation. Increased use of the antagonist protocol has resulted in a better definition and refinement of the concept of the mild stimulation [2223].

12.2.19 Which COH Protocol Is Best?

This question remains difficult to answer directly, because it is generally acknowledged that all types of agonist and antagonist protocols yield comparable pregnancy rates when correctly managed. Some patients will become pregnant with almost any stimulation protocol while others will need a more customized approach. It is important to keep in mind that the patient should not be subject to the specifics of a protocol; rather, the protocol must submit to the specific aspects of each patient. Treatments should be adjusted according to the specific context, and other considerations:

·               Each clinician should draw on his/her own experiences and greatest familiarity with the various protocols.

·               For each patient, a particular FSH preparation should be chosen to match precisely a given GnRH analogue. This critical match may be difficult to foresee.

·               For each IVF cycle to remain true to its objectives. Continuing progress in embryo technology permits the number of transferred embryos to be reduced, which of course lowers the risk of multiple pregnancies. The increasing use of the single embryo transfer strategy, with the same rate of successful pregnancy, makes it pointless, not to mention hazardous, to harvest a large number of oocytes, save for special situations like oocyte donation or fertility preservation.

For all these reasons, it seems less useful to search for the most ideal protocol in general, and more important to choose the best infertility work-up. Rather than repeatedly using the same FSH preparations with the same GnRH analogue, and with the same protocol for several IVF attempts, it seems preferable to follow a diversified strategy that encompasses the best chance to match a protocol correctly with each patient’s problems and characteristics.

The Antagonist Protocol. Except for certain situations, co-treatment with a GnRH antagonist is perhaps the best stimulation approach for the first one or two attempts leading to in vitro fertilization:

·               Most all patients who “implant well” will become pregnant no matter the stimulation protocol, and large follicular cohorts are not even needed.

·               The antagonist protocol is a “kinder, gentler” stimulation that leaves fewer bad memories than does the long agonist treatment. This point is far from insignificant because too many patients with a good prognosis abandon their IVF program along with their hopes for pregnancy because of fear or the reality of uncomfortable stimulation procedures.

·               The antagonist protocol is more applicable to all patient ages and levels of ovarian reserve. In patients with a lower follicular count, the long agonist treatment results in a difficult and less productive ovarian awakening in spite of higher FSH doses.

·               The sequence of an antagonist protocol plus an agonist for triggering is becoming widely used when a large oocyte harvest is sought without a fresh transfer, such as for oocyte donation or fertility preservation programs.

The Long Agonist Protocol may become a better choice after one or two unsuccessful antagonist attempts, as the potential beneficial and harmful effects of the different GnRH analogues on the endometrium and implantation remain largely unknown. This approach may also be chosen as a first line stimulation in two different situations: (1) In case of endometriosis: the preliminary phase of estrogen deprivation, which may be prolonged for several weeks or even months, can have a positive effect on the symptoms of endometriosis as well as on the protocol and the chance for pregnancy. (2) In case a larger follicular cohort is desired for a fresh transfer: this could be useful for prolonged in vitro culture for blastocysts, or when a limited quantity of spermatozoa has been obtained by testicular epididymal sperm extraction (TESE).

Regardless of the protocol used, if the first IVF attempt fails, it is quite reasonable to consider changing it for the next COH, even if it is acknowledged that the chances for success do not rest on the sole ovarian stimulation. One can change FSH preparation and/or GnRH analogue with respect to starting doses and treatment management of the previous cycle. On the other hand, one may choose to repeat the same protocol when a clinical pregnancy of any duration was achieved previously.

These sorts of strategies undoubtedly have an empirical nature, but would any other strategy be totally devoid of this? If all attempts at IVF fail at last, the changes of protocol have the advantage of presenting the entire spectrum of available treatment options to the patient. In addition, those patients who explore the internet will be able to verify on their own the variety of possibilities that were undertaken.

Finally, clinicians should always be sensitive to the patient’s comfort, and of ways to diminish untoward experiences by, for example, encouraging self-administered subcutaneous injections. Along the same line, there is little reason to impose a long agonist protocol of daily injections of triprorelin or buserelin, followed by two more daily injections (FSH and a GnRH agonist) when a single depot administration, or even nasal sprays of nafarelin or buserelin should yield the same results. Even when an IVF attempt fails, a COH achieves a certain bit of success when it does not leave the patient with unpleasant memories of this most difficult period of the IVF process, nor with anxiety about the next attempt. Keep in mind that giving up remains the primary cause of failure for patients undertaking IVF procedures.

12.3 In Case of Poor Ovarian Responses

In the COH process, the normal or average follicular response is generally agreed to be 6–14 ova. Fewer than six follicles is a weak response, 14–18 ova is a high response, and more than 18 is excessive. For an initial IVF attempt, a poor ovarian response (POR) is usually agreed to be three follicles or less. At a consensus conference that was recently convened in Bologna, Italy, a POR was defined when two of the following occur [24]:

·               The patient is age 40 or older, or has additional risk factors for ovarian damage

·               A POR (≤3 oocytes) observed in a previous COH with a long agonist protocol

·               A poor ovarian reserve as measured by AFC (<5–6 follicles) and/or the AMH level (<0.5–1.0 ng/ml)

Together, these criteria include the circumstances by which a POR is discovered, either a priori, from the patient’s age, history of ovarian damage, or abnormal ovarian reserve parameters; or a posteriori, following a deceptive COH. Nevertheless, these criteria can harbor surprises: while an expected POR may not occur, an inadequate response may remain fortuitous or be secondary to an erroneous starting dose of FSH.

Poor responses to ovarian stimulation typically share two characteristics: the occurrence rate increases with the patient’s age and POR significantly diminishes the patient’s chances for pregnancy. When making this diagnosis, it is very important to distinguish patients who should persevere in the process from those who will not and should turn instead to oocyte donation. Both are indeed heterogeneous populations but with different prognoses, which seems better for patients that have already conceived at least once. Quality of oocytes is not always correlated with quantity. Thus patients who fail to grow more than three oocytes during a stimulation trial can be divided into two categories [25]:

·               Those with impaired oocyte quality because of age, or because of ovarian insufficiency from various causes at a younger age. These patients typically present with shorter menstrual cycles and higher FSH levels. Outlook for continued attempts at IVF remains deceptive.

·               Those with good oocyte quality who should be encouraged to continue with IVF, in order to make good use of the few remaining oocytes that are more likely to produce an implanting embryo.

A number of approaches have been proposed in an effort to increase oocyte number in patients who demonstrate a limited number of FSH-responsive follicles per cycle.

12.3.1 In Preparation for the Stimulation

·               Oral estrogen +/− progestin. The patient takes oral contraceptives or a 4 mg dose of micronized estradiol daily prior to a short agonist or an antagonist protocol, from 10 days before menses until 1–3 days before beginning gonadotropin administration. This serves to counteract an inter-cycle rise of FSH.

·               Growth hormone injections, to stimulate synthesis of hepatic IGF1, which in turn enhances the FSH effect on ovarian granulosa cells. Daily administration of 8–12 IU should begin either 10 days before menses or at the beginning of the FSH stimulation, and should be continued until the day of triggering [26].

·               Oral dehydroepiandrosterone (DHEA), 75 mg daily, beginning 6–12 weeks before the IVF attempt, has been proposed to enhance ovarian response, and with a cumulative effect over time [27]. This weak androgen, of ovarian and adrenocortical origin, is a pro-hormone for the follicular steroidogenesis, notably for testosterone. However a mechanism of action remains largely hypothetical. It may increase IGF1 synthesis and/or production of an intra-ovarian hormonal environment similar to the micropolycystic ovary situation with attendant abnormal transition between primordial and primary follicles.

·               Oral letrozole, an inhibitor of aromatase. This compound, which lacks intrinsic hormonal activity, can stimulate ovulation and has been tested for this indication [28].

12.3.2 The Stimulation Protocol

The long agonist protocol, using a GnRH agonist administered daily, poses a challenge for low-responding ovaries to gonadotropins. One might consider reducing the agonist dose by half, or even stopping it altogether as soon as the FSH stimulation begins.

The long agonist depot protocol, which induces an even more profound ovarian shutdown, is clearly not suitable for the POR patient.

The short agonist protocol may be modified by using only microdoses, e.g., 0.05–0.025 mg triptorelin, when the regular daily dose is 0.1 mg, This should prevent an LH surge while leaving a sufficient residual of pituitary gonadotropin secretion [2930].

Antagonist protocols are generally well suited for POR treatment situations because there is no preliminary ovarian desensitization. Sequential short agonist/antagonist microdoses have also been proposed [31].

The spontaneous (naturalcycle may also be considered as a last resort in order to harvest a single oocyte with hope that the quality will be optimal after minimal hormonal manipulations [32]. This approach may be enhanced by using clomiphene 100–150 mg daily for five days starting on CD2, with a goal of growing an extra follicle.

12.3.3 The FSH Preparation

Addition of LH is believed to improve the ovarian response in these patients, even as they may show no detectable LH deficit [33]. Extracted gonadotropin preparations appear more appropriate when high doses are being administered, due to the “ceiling effect” of FSH.

12.3.4 The Starting Dose

Daily gonadotropin doses in excess of 300 IU are capable of inducing a better ovarian response in overweight patients whose POR had resulted from insufficient posology. In other patients, elevating the gonadotropin dose does not usually compensate for the lack of follicles sensitive to FSH [34]. Furthermore, increasing oocyte yield in these patients does not result in a higher pregnancy rate, perhaps because of the FSH ceiling effect. As a matter of fact, excessive FSH is counterproductive at the follicular level and actually may harm the oocyte quality. However it must be acknowledged that every clinic can report a few patients who achieved a successful pregnancy following daily administration of 450 IU or more FSH.

12.4 Conclusions

There is no single perfect multifollicular stimulation protocol for cases of POR related to the patient’s age. No method appears superior to others, and furthermore none of them has been well validated [16]. Contradictory results appear in one publication or another and, inasmuch as the POR patients represent a heterogeneous group, the definition of POR itself remains a matter of debate. This deceptive situation confirms that the problem of the POR lies neither with the gonadotropin preparations nor with their administration protocols, but with conditions at the ovarian level itself. Nevertheless, it seems justified to continue with a few IVF attempts for these patients by testing some of these strategies in selected and motivated couples, after duly informing them of the empirical nature and uncertain outlook for these methods. The goal here is to pursue oocyte quality over oocyte quantity.

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