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2 The objectives of this chapter are: To provide an understanding of the various stimulation protocols used in IVF To enable the student to understand the factors affecting the choice of protocol based on the patient characteristics and To help the student understand the various factors affecting response to stimulation and help choose the best dose for that individual patient. 2

3 The first IVF baby was born during a natural (unstimulated) IVF cycle. However, it was soon recognized that the success rate of IVF in natural cycles was low, primarily due to the low number of oocytes retrieved. Ovarian stimulation using urinary gonadotropins was adopted to deal with this problem, resulting in a significant increase in both the number of eggs retrieved, as well as the success rate of IVF. 3

4 With the increasing use of stimulation in IVF cycles, various problems were recognized. Premature luteinization and failure of synchronous follicular recruitment due to early dominant follicle selection were the two main problems resulting in reduced success rates. Also, ovulation could occur at any time of the day necessitating intensive monitoring and oocyte retrieval at inconvenient times of the day. Gonadotropin-releasing hormone agonists (GnRHa) were demonstrated to result in pituitary desensitization and successfully dealt with these problems, becoming the next major breakthrough in IVF treatment. More recently gonadotropin releasing hormone antagonists (GnRHant), which have a similar function but take a shorter period of time to achieve pituitary suppression (and prevent the premature LH surge) are increasing being used for the same purpose. 4

5 In the natural cycle, follicular dominance is achieved by the estradiol induced negative feedback on the pituitary gland, causing a decline in FSH below threshold levels. In IVF cycles, exogenous gonadotropins are used to achieve supra-threshold levels of gonadotropins during the phase of follicular recruitment to interfere with this process of dominant follicle selection and enable multiple follicular recruitment. Pituitary desensitization by using either GnRH agonists or GnRH antagonists, is an important part of IVF stimulation as it eliminates the possible interference by endogenous hormones, enabling synchronous follicular development. This prevents premature luteinization, and helps to reduce intensive and frequent monitoring allowing control over the timing of oocyte retrieval. The LH surge is substituted by exogenous hcg, enabling clinics to time oocyte retrieval. 5

6 Various protocols have been used to achieve the aforementioned aims of IVF stimulation. Some protocols use the GnRH agonists while the others use antagonists to achieve pituitary desensitization. The protocols also vary in the gonadotropins used for ovarian stimulation. Either recombinant or highly purified follicular stimulating hormone (rfsh or HP-FSH) are used. Administration of GnRH agonists result in an initial flare-up effect and hence needs to be started much ahead, either in the mid-luteal phase of the previous cycle (long protocol) or in the early follicular phase (short protocol). In contrast, the GnRH antagonists result in rapid pituitary desensitization and hence need to be given in the mid-follicular phase, just prior to the rise in the LH levels. They can be used as a single dose or as multiple doses given daily. 6

7 The long protocol is the oldest and still the most commonly used regimen for ovarian stimulation. It starts in the mid-luteal phase (day 21) of the previous cycle with GnRH agonist being administered daily for about two weeks or until down-regulation is complete. Suggested criteria for this are estradiol (E2) levels below 180 pmol/l, luteinizing hormone (LH) below 2 IU/L and P4 below 2 nmol/l. Once down-regulation is achieved, gonadotropins are administered subcutaneously to stimulate follicular growth with the GnRH agonist being continued at a lower dose. The hmg/fsh dose is subsequently adjusted according to follicular growth, as monitored by serum E2 levels and/or transvaginal ultrasonography. Human chorionic gonadotropin (hcg), either urinary or recombinant, is given once the follicular cohort consists of at least two follicles more than 18mm in diameter. Oocyte retrieval follows hours later. This protocol provides excellent cycle control making it the protocol of choice for first time patients as well as for those with previous normal response. However, longer duration of treatment, greater consumption of gonadotropins, higher cost and the uncertainty of pregnancy at the start of GnRH agonist treatment are its main disadvantages. 7

8 In the short protocol, the administration of GnRH agonist is started in the early follicular phase (day 2 of menses) and gonadotropins are started on the following day (day 3). The monitoring, timing of hcg injection and oocyte retrieval is as for the long protocol. This protocol tries to derive benefit from an initial "flare-up" response due to endogenous FSH release from the pituitary gland that usually occurs in the first few days of GnRH agonist administration. As there is no preceding pituitary suppression, this protocol results in a better response than the long protocol. However, the most important disadvantage is the high progesterone level during the early follicular phase, likely caused by the rescue of the preceding corpus luteum. Studies have confirmed a lower pregnancy rate using this protocol compared to the long protocol (Daya, 2009). Hence, in practice it is used only in patients with poor ovarian reserve or those with a previous poor response in the long protocol cycle. 8

9 Several different GnRH agonists, buserelin, leuprorelin, nafarelin, and triptorelin are routinely used in ART. The preparations differ in their potency and route of administration. Nafarelin and buserelin are available as a nasal spray, which needs to be given two to six times a day while buserelin, leuprorelin and triptorelin are given as subcutaneous injections once a day. With the intranasal route the absorption of the GnRH agonist fluctuates resulting in an unpredictable response. Nevertheless, in most patients it is sufficient to prevent the spontaneous LH surge. More recently single injections of depot preparations are being tested. Meta-analyses of trials comparing the different preparations showed no difference in either the efficacy of pituitary suppression or IVF outcomes (number of oocytes collected and pregnancy rate). However, women treated with nafarelin required fewer ampoules of gonadotropins for ovarian stimulation and fewer days of stimulation, compared to leuprorelin, triptorelin and buserelin. On the other hand, the use of depot preparations of GnRH agonists was associated with increased requirements for gonadotropins and a longer time required for ovarian stimulation even though the IVF outcomes were not significantly different. Wong JM, Forrest KA, Snabes SZ et al Efficacy of nafarelin in assisted reproductive technology: a meta-analysis. Hum Reprod Update Jan- Feb;7(1):

10 Although GnRH agonist treatment is very effective, it has several pitfalls. There is an initial stimulation of GnRH receptors before pituitary desensitization is achieved. Hence, 7-14 days are required for adequate downregulation, menopausal symptoms are not unusual, and unless a depot preparation is used, daily injections or multiple daily intranasal administrations are required for 2 to 4 weeks. In contrast, GnRH antagonists, being competitive inhibitors of endogenous GnRH due to their receptor binding property, rapidly inhibit secretion of gonadotropin and steroid hormones with a reduction of FSH and LH secretion within 8 hours after administration a potential advantage over GnRH agonists. In the GnRH-ant protocol, the gonadotropins are started on day 2 of the cycle and GnRH-ant are added in the mid-follicular phase to prevent the premature LH surge. Two different compounds, cetrorelix and ganirelix are available and are equally efficacious. They can be used in two different protocols, the single and multiple dose protocol. The day of start of the GnRH-ant may be fixed or flexible, as explained in the next slides. 10

11 The multiple-dose GnRH-ant protocol involves the daily subcutaneous injections of 0.25 mg of either cetrorelix or ganirelix from day 5 or 6 of stimulation (the fixed start) until administration of human chorionic gonadotropin (hcg). The single-dose protocol involves a single subcutaneous injection of 3mg GnRH-ant on day 7 or 8 of stimulation. This single dose provides 4 days of pituitary suppression. If the patient needs more days of stimulation, the daily 0.25mg of GnRH-ant injections are required until the hcg trigger. The monitoring, criteria for hcg administration and oocyte retrieval are similar to the agonist protocols. 11

12 In the flexible start protocol, the day of start of GnRH-ant is not fixed. Instead, it is started when the diameter of the leading follicle is 14mm or more. In the multiple-dose GnRH-ant protocol daily subcutaneous injections of 0.25 mg of either cetrorelix or ganirelix are given until the administration of hcg. In the single-dose protocol a single subcutaneous injection of 3 mg GnRH-ant is injected when the diameter of the leading follicle is 14mm or more. This single dose provides 4 days of pituitary suppression. If the patient needs more days of stimulation, the daily 0.25 mg of GnRH-ant injections are required until the hcg trigger. The monitoring criteria for hcg administration and oocyte retrieval are similar to the agonist protocols. 12

13 Fixed versus flexible A meta-analysis (Al-Inany et al, 2005) of randomized studies comparing the fixed and flexible approaches concluded that there was no statistically significant difference in pregnancy rate, although there was a trend towards a higher pregnancy rate with the fixed protocol. However, there was a significant reduction in the amount of recombinant FSH and the antagonist used with the flexible protocol. At the moment, there is no clear answer as to which of these two approaches is better and use of either is a matter of personal preference. Single versus multiple dose GnRH antagonist protocol The single dose GnRH antagonist protocol has the advantage of fewer injections, with only 10% of cycles requiring additional daily doses of GnRH antagonist (Olivennes et al., 2000). Owing to the larger dose, there is some concern about an excessive and potentially harmful suppression of endogenous LH. This does not cause any significant difference in pregnancy rates as shown in a multicentre study comparing the multiple and single dose protocols of cetrorelix (Olivennes et al, 2003). Another prospective randomized trial did not find a significant difference in the pregnancy rates between the ganirelix multiple dose and cetrorelix single dose protocols (Wilcox et al., 2005). 13

14 There are several advantages of the antagonist protocol over the long protocol. The shorter duration of treatment and fewer menopausal symptoms render it more patient-friendly. Cyst formation, due to the initial flare-up effect of GnRH agonists is avoided and the requirement of gonadotropins is also lower than with the agonist protocol, providing a financial benefit. An important advantage, as shown in a recently published Cochrane review (Al-Inany et al, 2007) is the significant reduction in the incidence of severe OHSS in antagonist cycles compared to the agonist cycles (p = 0.01; OR = 0.60, 95% CI ). The antagonist cycle is less programmable compared to the agonist cycle and this lack of flexibility poses a problem for some patients and IVF centers. However, the main disadvantage with this protocol is the significantly lower clinical pregnancy and ongoing pregnancy/live-birth rate in the antagonist group (p = 0.009; OR = 0.83, 95% CI and p = 0.02; OR = 0.82, 95% CI respectively) as shown in the Cochrane review. This could be due to the learning curve and hopefully with accumulating knowledge this can be improved. 14

15 The response to stimulatory drugs during an IVF cycle depends on several factors: the number of recruitable follicles, their sensitivity to FSH as well as the bioavailability of FSH. While there are no strict criteria, adequate response to conventional ovarian stimulation usually means the retrieval of 8-10 oocytes. Failure to recruit an adequate number of follicles and retrieve 4-5 mature oocytes is termed 'poor response', while recruitment of 20 or more follicles in high responder increases the risk of OHSS. Both are undesirable outcomes and hence an attempt is made to optimize ovarian stimulation to result in an adequate response. The most important strategies to optimize ovarian stimulation include: Personalizing the protocol Various treatment protocols are more suited to certain groups of women. The flare-up or antagonist protocols give better success rates in women with poor ovarian reserve (e.g. patients with low antral follicle counts or high FSH) or poor response to the long protocol. The antagonist protocol has been shown to reduce the risk of OHSS and may be the better choice in women at high risk of the same. Optimal starting dose of gonadotropin In the first cycle, the important question is: What dose of gonadotropins will give an optimal response? Recent studies have demonstrated that predictive models can be useful in determining the optimal starting dose (e.g. Popovic- Todorovic et al, 2003). Manipulating the FSH dose according to the response The use of models is as yet neither widespread nor foolproof and hence there 15

16 A standard patient' is one in whom we expect a normal response to ovarian stimulation and is usually defined as a patient aged less than 40 years, with a regular menstrual cycle (21-35 days), and a normal basal FSH. While the long protocol being the oldest and the one with the most experience is still the most commonly used protocol and gives good results in most standard patients, the GnRH antagonist protocol is more patient-friendly and is now increasingly being preferred by many centers for these standard patients. 16

17 The long protocol gives poor results in patients with poor ovarian reserve, likely due to the profound pituitary suppression. While some studies have shown better results with the antagonist and microdose flare protocol in this group of patients, others have failed to demonstrate any benefit. The presented table demonstrates the results of a comparison of various treatment protocols acquired in multiple studies. The results were combined in a meta-analysis by Shanbhag S. et al in The table shows an overview of the 4 trials, comparing clinical pregnancy rates and ongoing pregnancy rate per women for different treatment protocols. The first category shows a comparison of GnRH antagonist with conventional GnRHa long protocol. It was represented by a single trial with 30 participants in each arm (Marci 2005). The second category is a comparison of a GnRHa flare-up protocol with GnRHa long protocol. This protocol was represented by a single trial with 26 women in the flare-up protocol arm and 28 in the long protocol arm (Dirnfeld 1991). The last category shows a comparison of GnRH antagonist with GnRHa flareup protocol. This protocol was represented by two trials (Marci 2003; Malmusi 2005) with 44 participants in the antagonist group and 49 in the flare-up group. 17

18 Over-responders and polycystic ovarian syndrome patients Compared to the agonist long protocol, GnRH-antagonist protocol has been shown to reduce the incidence of OHSS in high responders, making it an attractive choice in these patients. However, variable data regarding the success rates with the two protocols prevents us from making any firm conclusions regarding the best protocol for these patients. 18

19 Starting dose for the standard patients varies and is in the range of IU/day. The variability of ovarian response to the same gonadotropin dose in standard patients is well documented and a possible result of the several factors that affect the response to controlled ovarian hyperstimulation (COH). Age, BMI, smoking status, history of endometriosis, the antral follicle count (AFC), ovarian volume, stromal blood flow and endocrine parameters like basal FSH levels, inhibin B and Anti-Müllerian hormone levels can affect the ovarian response to COH. As there are so many factors involved, selecting the optimal dose of gonadotropins for the first cycle is complicated. Each clinician will have his or her own formula to define the starting dose. Some kind of guide is presented here. In the recent years, an attempt has been made to use models combining the above factors to personalize the starting dose and hence optimize the ovarian response to COH. More recently, the CONSORT study (Olivennes et al., 2009) has reported that the use of fixed dose regimens calculated by computerized dosing algorithms based on basal FSH, BMI, age and AFC resulted in adequate oocyte yield and good pregnancy rates (overall, 34.2%). 19

20 There are 3 regimens of gonadotropin use for ovarian stimulation. In the fixed dose regimen, the gonadotropin dose is kept constant throughout the stimulation. If the optimal staring dose has been determined, this protocol is simple to follow and results in good outcomes. In the "step-down" regimen, a high starting dose of gonadotropins ( IU) is used for the first 2 days, followed by a dose reduction ( IU/day). This enables supra-physiological levels of FSH required for maximal multifollicular recruitment during the critical period in the early follicular phase and seems to result in greater synchrony of follicles. The higher dose during the first few days theoretically increases the risk of over-response and OHSS, but the following dose reduction balances this risk. In the "step-up" regimen, the starting dose of gonadotropin is low and is then gradually increased on cycle day 5 or later, depending on the response. This protocol can result in high FSH serum levels in the late follicular phase leading to an asynchronous development of multiple follicles, an undesirable outcome. Reference: Textbook of assisted reproductive techniques 2nd edition, Ed Gardner, Weissman, Howles, Shoham 20

21 There are several different types of gonadotropins that have been used in IVF cycles. 21

22 Cycle monitoring is an essential part of any IVF protocol as it can indicate over- or under-response, enabling dose adjustments to optimize response. Initially, cycle monitoring was comprised of daily ultrasound scans and hormonal tests. But with increasing clinical experience, cycle monitoring is becoming less cumbersome for patients as well as for the clinic staff. Each center will have its own protocol for monitoring ART cycles, but usually an ultrasound scan and measurement of serum estradiol level is done on day 5 or 6 of stimulation. Subsequent scans are done every 2-3 days or more frequently depending on the patient s response. A recent Cochrane review (Kwan et al., 2008) concluded on the basis of the existent material that there is no significant difference in pregnancy rates and live birth in cycles monitored using ultrasound and serum estradiol or those monitored by ultrasound alone. 22

23 OHSS is a rare but serious complication of ovarian stimulation. Therefore, one of the main aims of cycle monitoring is to prevent its occurrence. If ovarian over-response is recognized during cycle monitoring, the dose of gonadotropin is reduced or stopped (coasting). Frequent (even daily) monitoring is performed to continually assess the response. If the risk of OHSS seems to be very high hcg trigger is withheld and the cycle is abandoned. On the other hand, if the risk of OHSS does not seem to be very high, the HCG trigger is given and oocyte retrieval is performed. The patient is then observed for symptoms and signs of OHSS (bloatedness, nausea, vomiting, sharp increase in abdominal girth and weight). If no such symptoms and signs are identified, the embryo replacement is carried out. Otherwise, the embryos are cryopreserved for subsequent transfer in a thaw cycle. A randomized trial with a sufficiently large sample size to test the effects of different monitoring protocols on a rare outcome like OHSS is difficult. Nevertheless, the Cochrane review mentioned in the previous slide (Kwan et al., 2008) suggested that cycle monitoring using transvaginal ultrasound plus serum estradiol be retained as a precautionary good practice point. 23

24 The first monitoring visit is usually on day 5 or 6 of stimulation, when an ultrasound scan and estradiol level determination is done to assess the ovarian response. On this day, If the estradiol level is pmol/L continue with the same dose If the estradiol level is > 1500pmol/L decrease the dose If the estradiol level is < 500pmol/L consider increasing the dose (but anticipate less than ideal response) The next checkup depends on the ovarian response and is usually performed on day 8 or 9 of stimulation. Most experts believe that the best results are obtained in cycles where the dose of the gonadotropin is maintained rather than increased. Hence, estimating the correct starting dose is extremely important for optimal results. The effectiveness of dose increases, though widely practiced, does not seem to be based on the evidence. Several studies have shown that in patients receiving a step-up dosing of gonadotropin following a low response after 5 days of stimulation, the number of oocytes retrieved as well as the pregnancy rates remain significantly lower than in patients treated with a fixed dose. 24

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