Stimulation protocols in ART.pptx
- 1. Dr. Deepeka. T. S. Fellow, Reproductive Medicine. CIMAR, Kochi. STIMULATION PROTOCOLS IN ART
- 2. Contents • The concept of ovarian stimulation • GnRH agonist – molecule and mechanism of action • Route and the dose of administration • Protocols using GnRH agonists • GnRH antagonist- molecule and mechanism of action • Route and the dose of administration • Protocols using GnRH antagonists • Difference between agonist and antagonist protocol • Summary
- 3. The concept of ovarian stimulation • Ovarian stimulation for assisted reproductive technology(ART) cycle aims to provide multiple pre-ovulatory follicles for oocyte collection. • The components of a conventional ART cycle- 1. Induction of multi-follicular growth with exogenous gonadotropins. 2. Prevention of endogenous leutinizing hormone (LH) surge by using Gonadotropin releasing hormone(GnRH) analogs. 3. inducing endogenous LH surge or mimicking it with exogenous human chorionic gonadotropin(hCG) for oocyte maturation. • This concept is known as “CONTROLLED OVARIAN STIMULATION”
- 5. Gonadotropin releasing hormone(GnRH) • GnRH is produced and released from a group of interconnected neurons • - arcuate nucleus(medical basal hypothalamus) and preoptic nucleus(ventral hypothalamus). • Released in a pulsatile fashion into the complex network(portal system) of pituitary. • GnRH was first isolated , charecterised and synthesised independently in 1971 by Andrew Schally and Roger Guilleman. • Single chain peptide, decapeptide(containing 10 amino acids), similar to several other brain peptides. • Structure is common to all mammals and its action is similar in both males and females.
- 6. • Inter-pulsatility frequency ranges from • 71 minutes in the late follicular phase and • 216 minutes in the late luteal phase. • Acute administration of GnRH agonistic analogs increases gonadtropin secretion(flare up effect- 2-3 days). • Prolonged administration leads to down regulation GnRH receptors. • Usually requires 7-14 days to achieve a state of pituitary supression. • Mechanism – pituitary receptor desensitization(down-regulation)
- 7. GnRH agonistic analogues • The agonist- bound receptor is internalised via receptor mediated endocytosis(with kinetics determined by the potency of the analog). • Subsequently undergoes dissociation, followed by degradation of the ligand and partial recycling of the receptors. • Native GnRH – short plasma half life , rapidly inactived by enzymatic cleavage. • Analogs with longer half-lives and higher receptor activities were created by structural change at the position of enzymatic breakdown of GnRH.
- 9. • The original goal for the development of GnRH agonistic analogs – TREATMENT OF ANOVULATION. • The potential for reversibly inducing a state of hypogonadotropic hypogonadism allowed the introduction of GnRH agonists in ART. • FIRST REPORT- buserelin and gonadotropin for ovarian stimulation for in vitro fertilization in 1984. • The major advantage – efficient abolition of spontaneous LH surge.
- 10. Routes of GnRH agonist administration • Intramuscular or subcutaneous depot injection • Subcutaneous daily administration • Intranasal
- 11. Comparing depot and daily s/c injection • A meta-analysis comparing depot and daily injections concluded that there is no clear difference in pregnancy rate. • Use of depot analogs is associated with increased gonadotropin requirements and longer stimulation periods. • Suppression of pituitary and ovarian function appears to be continued till 8 weeks after the injection. • Several authors claim a normal outcome of pregnancy following inadvertent administration of GnRH agonist during early pregnancy. • Lahat et al. reported a high incidence of attention deficit hyperactivity disorder in long-term follow -up of children.
- 12. • Though depot preparation seems attractive for its ease of administration for the patient, it is not cost effective. • One exception – prolonged use of GnRH agonist before IVF embryo transfer in patients with severe endometriosis is associated with higher ongoing pregnancy rates.
- 13. Intra nasal GnRH agonist…. • Nasal sprays are administered in six divided doses between 6 am and 11 pm. • It ensures uniform dosage through the day, mimicking the pulsatile release of GnRH. • The doses are reduced to two times a day once the gonadotropin injections are initiated. • The absorption flucuate inter and intra-individually, giving an unpredictable desensitization, but sufficient to prevent premature LH surge.
- 14. Dosage of GnRH agonist • The depot preparation of leuprolide of 3.75 mg is given intramuscularly as a single dose in the luteal phase of the previous cycle. • The down regulation lasts for atleast 4 weeks(28 days). • Daily subcutaneous dose leupride of 1 mg from day 21 of previous cycle followed by half the dose 0.5 mg from day of gonadotropin stimulation until hCG trigger.
- 16. • Proper dose-finding studies for the use of GnRH in ART are still urgently needed.
- 17. Protocols using GnRH AGONISTS
- 18. Agonist protocols • Long protocol • Short/flare up protocol • Microdose flare protocol • Stop GnRH-a protocol • Ultra-short protocol • Ultra-long protocol
- 19. Long protocol- basics • The concept- Controlled ovarian hyperstimulation • Pituitary downregulation followed by stimulation of the ovary with optimum dose of exogenous gonadotropin in controlled manner aiming multifollicular development.
- 20. • The drug GnRH-a is started in the mid-luteal phase (preferably day 21), when E2 and progesterone start declining and FSH for the next menstual cycle starts rising. • In terms of gonadotropin supression and numbers of retrieved oocytes, the mid-luteal phase of the preceding cycle is the optimal moment for the initial of the GnRH agonist, in comparison to the follicular , early , or late luteal phase. • The first effect is the surge of FSH and LH, which lasts for about 2-3 days when the pituitary storage is depleted.
- 21. • The pituitary is now devoid of receptors and further daily administration of the agonist keeps the pituitary at “0 level” incapable of responding to GnRH • Continuous supression leads to decline of FSH, LH and therefore E2. • Optimum supression – 15 days after start of downregulation or with the onset of next menstruaion, whichever is earlier.
- 22. Criteria for downregulation 1. E2 <10 pg/ml 2. LH < 2 mIU/ml 3. P4 < 1.2 ng/ml. • Of these, E2 and progesterone are more important that LH. • If satisfactory level of E2 has been achieved and LH is still high, stimulation can be started. • If E2 is still high and satisfactory level of LH achieved, repeat estimation after 1 week.
- 23. • If P4 > 1.8 ng/ml, cycle has to be cancelled. • If P4 between 1.2 and 1.8 ng/ml, initiation of stimulation/cancellation has to be decided • Age of the patient • Ovarian reserve • Other factors like fibroid , endometriosis etc
- 24. Starting dose of hMG/FSH for ovarian stimulation FIRST CYCLE STIMULATION Parameters Dose Age <37 yrs 150 IU Age 37- 39 yrs 225 IU Age > 40 yrs 300 IU PCOS 100-200 IU BMI > 30 kg/m2 Increased by 75 IU Severe endometriosis Increased by 75 IU REPEATED CYCLE STIMULATION Parameters Dose Previous normal responders (= or > 5 oocytes) 150 IU Previous history of OHSS 75-100 IU Previous history of POOR RESPONSE(= or < 3 oocytes) 300-450 IU
- 25. Monitoring for response • There many be 3 types of response 1. Hyper response 2. Normal response 3. Poor response • Criteria for identification of different types of response serial folliculometry from D6/D7 after initiation of stimulation E2 as and when necessary Increment of endometrial thickness Folliculometry – significant and most practical marker of ovarian response.
- 26. • Ideally there should be gradual synchronous increment of follicular diameter with appearance of dominant and co-dominant follicles(>=14 mm diameter) between D9 and D13. • If around D11, there are more than 11 intermediate follicles of approximately 11 mm – presumed that patient is heading for hyperstimulation. • When at least 3 synchronously developing follicles reach a diameter of 17 and 20 mm, hCG trigger(10000 IU) should be administered , followed by oocyte retrieval 34-36 hours thereafter.
- 27. Monitoring…. I. E2 monitoring- when follicular response is too poor (<3) or too rapid( >10 follicles) observed on D6/D7 scan. • If E2 < 100 pg/ml- gonadotropin dose should be increased. • If E2 > 500 pg/ml - gonadotropin dose should be decreased. II. Estimation of endometrial thickness • ET should increase by 0.5 to 1 mm daily and should reach between 7 and 12.5 mm on the day of HCG trigger. • If < 7 mm- OPU should be performed, but embryos not to be transferred in the index cycle, but cryopreserved for transfer in subsequent cycle.
- 28. Estimation of response(D6/D7 USG, D7/D8 E2) • 3-8 follicle(at least 3 synchronous) • 150-200 pg/ml NORMAL RESPONSE • >10 follicles(usually asynchronous ) • > 500 pg/ml HYPER RESPONSE • <3 follicles • < 150 pg/ml POOR RESPONSE
- 29. Possible etiological factors of hyper-response PCOS/hyperestrogenism- with exogenous gonadotropin augmentation of partially mature follicles. Vascular endothelial growth factor (VEGF) – abundance of VEGF in both ovaries – multifollicular development- OHSS. Hyperinsulinemia – insulin resistant PCOS are more prone for OHSS. Insulin acts as co-gonadotropin because of insulin receptor in hypothalamus. Mutated FSH gene on the granulosa cell membrane receptors
- 30. Possible etiological factors of hypo-response in PCOS • Obese PCOS- Leptin hormone in adipocytes – reduces the follicular sensitivity to gonadotropin both at follicular as well as hypothalamic pituitary level. • Androgen and prolactin antagonize the follicular response to gonadotropin. • Response can be improved by antagonizing androgen with metformin and also by supressing prolactin with cabergoline.
- 31. Advantages of long GnRH agonist protocol Prevents unscheduled LH surge Downregulated elevated LH in early follicular phase, ensuring proper estrogen environment during early follicular development. Contribution to planning ovum pick-up, since both initiation of gonadotropins after pituitary desensitization and the administration of hCG can be delayed without any detrimental effect on IVF outcome.
- 32. Prevention of premature leutinization of immature granulosa cells. A meta-analysis comparing ultra-short, short and long IVF protocols showed a higher number of oocytes retrieved and higher pregnancy rates in the long protocol. Low cancellation rates.
- 33. Drawbacks of long GnRH agonist protocol • Prolonged and complicated treatment procedure • Expensive • Multiple pregnancy • Ovarian hyperstimulation syndrome(OHSS) • Ovarian cyst formation
- 34. Formation of functional cysts • Ketz et al. observed both a poor stimulation outcome and a reduction in the pregnancy rates in a cycle with cyst formation. • Feldberg et al. contradicted it • Cyst formation can be reduced when pretreatment with an oral contraceptive. • Damario et al showed the beneficial effect of this strategy in high responder patients with respect to cancellation rates and pregnancy rates. • Another advantage of including oral contraceptive – coincidence of GnRH use and early pregnancy is prevented.
- 35. Short agonist protocol/Flare-up protocol • Since the use of long protocol in poor responders has been found to result in reduced ovarian responses to gonadotropin stimulation, short protocol has been proposed. • GnRH agonist(0.5 to 1 mg) is started at cycle day 2 , until day of hCG. • Gonadotropin treatment(150- 225 IU) is started on day after GnRH agonist(usually D3/D4)
- 36. • The immediate stimulatory action of the GnRH serves as the initial stimulus for follicular recruitment(flare-up) and improve ovarian response. • Adequate follicular maturation is on average reached in 12 days, which should allow enough time to prevent premature LH surge. • Folliculometry from D7/D8 – gonadotropin dose adjusment – estimation of response-hCG trigger.
- 37. Short agonist protocol Advantages • Flare- up action. • Less expensive. • Shorter duration. • Patient friendly. • Effective for poor responders. Drawbacks • Unscheduled LH surge-cycle cancellation • Cyst formation due to flare action- inteferes with subsequent gonadotropin stimulation
- 38. • Associated with lower oocyte quality and reduced ongoing pregnancy rates compared to the long protocol. • The protocol has been practically abandoned. • Nevertheless, short protocol has a role in poor responders.
- 39. Microdose flare protocol • Similar to short protocol, except the dose of agonist is reduced to a quarter of that in short protocol. • 50 microgram of leuprolide acetate.
- 41. BOLOGNA CRITERIA FOR POOR RESPONDERS
- 42. Early cessation protocol/Stop GnRH-a protocol • The mean desensitization phase with an agonist in the long protocol is about 3 weeks. • The agonist begun in the previous cycle in stopped a the initiation of gonadotropins, believing that supression outlasts the gonadotropin stimulation. • Increased gonadotropin requirement and cancellation rate. • Some reported otherwise-no of retreived oocytes were significantly higher and the amount of required gonadotropins was reduced. • No advantages in terms of pregnancy and implantation rates. • Currently available data do not favour an early cessation protocol , but this approach might have some beneficial effects in poor responders.
- 43. Ultra-short protocol • Modification of short agonist protocol • Agonist is given over a period of three days in the early follicular phase. • On 2 nd day of agonist administration, gonadotropin stimulation is started. • Advantage – patient comfort, useful in poor responders • Premature LH surge, low pregnancy rates.
- 44. Ultralong/continuous long agonist protocol • GnRH is continued during the luteal phase • In large prospective randomized study comparing this continuous long protocol versus standard long protocol , higher implantation and pregnancy rates were found in continuous-long protocol.
- 45. GnRH antagonist
- 46. GnRH antagonist – molecule • GnRH antagonists are analogs of GnRH, which have a direct inhibitory, reversible, supressive effect on gonadotropin secretion. • Antagonist molecule compete for and occupy pituitary GnRH receptors, the competely blocking the access of GnRH , thus inhibiting gonadotropin stimulation.
- 48. Comparing the mechanism of action of GnRH agonist and antagonist GnRH agonist GnRH antagonist Receptor downregulation Receptor blockage without activation Pituitary desensitization Competitive inhibition Initial flare-up Immediate and dose dependent suppression Slower reversibility Rapid reversibility
- 49. Synthesis and evolution of GnRH antagonist • Over the past 3 decades, thousands of GnRH analogs, both agonists and antagonists, have been synthesised. • The first generation of antagonistic analogs were hydrophilic, and contained replacement of amino acids which resulted in increased inhibitory activity. • However, histamine release also increased, resulting in anaphylactic reactions.
- 50. • Though both GnRH agonists and antagonists were available in the early 1980s for supression of endogenous LH surge, the use of GnRH agonist became universl through 1980s, 1990s and until early 2000s, charecterizing IVF throughout this period as the “GnRH agonist era”. • The third generation GnRH anatgonists introduced in early 2000s, lacked histamine release problems and was perceived by the scientific community as a great opportunity to simplify and optimise ovarian stimulation.
- 51. Third Generation GnRH antagonists • Cetrorelix • Ganirelix • ORG-30850 • Ramorelix • Nal-Glu • Antide • AzalineB • A-75998 • Abarelix
- 52. Dose and route of administration • Dosing schemes • Daily dosing scheme • Single-dose scheme (inhibits premature LH surge for 4 days) • On the basis of dose-finding study, the optimal dose for the daily dose GnRH antagonist scheme is 0.25 mg(both cetrorelix and ganirelix) and 3 mg for the single- dose scheme. • Two comparative trials comparing single-dose and daily dose GnRH agonist schemes shows no difference in the probability of clinical pregnancy. • Route - subcutaneous
- 53. Protocols with GnRH antagonist
- 54. GnRH antagonist protocol • Gonadotrophins started from D2/d3. • Antagonist is started on a fixed day ie 6 th day/after certain criteria , on which it is assumed that LH becomes imminent and continued till hCG trigger day. • hCG trigger of 10000 IU is given when atleast 3 follicles reach 17- 20 mm, followed by OPU 34- 36 hours.
- 55. Multiple dose protocol Single dose protocol - 3 mg s/c given on day6/day 7 of stimulation (TOPPED UP WITH 0.25 MG DAILY DOSE ) Flexible protocol- 0.25 mg s/c daily started when follicle size 12-14mm/E2 ≥200 till and on day of hCG Fixed protocol- 0.25mg s/c daily started from day 6 of stimulation till and on day of hCG
- 56. • Fixed protocol is simpler and requires less monitoring. • Flexible protocol might avoid unnecessary GnRH antagonist administration. • A stratified analysis of 4 RCTs comparing fixed and flexible protocol suggests no diffference in clinical pregnancy rates.
- 57. Programming the initiation of GnRH antagonist cycle • Initiation of gonadotropin stimulation is dependent on occurrence of menstruation. • There have been efforts to prevent the occurrence of oocyte retrieval on Sundays or on weekends. • Pretreatment with OCPs for 14-18 days before initiation of stimulation has been used. • However, this is associated with decreased probability of ongoing pregnancy. • Associated with increased duration of stimulation and increased gonadotropin consumption.
- 58. • Alternative way of programming – delaying the day of starting gonadotropin from day 2 to day 3 and/or postponing hCG administration by 1 day. • However postponing HCG administration for 2 or more days as soon as three or more follicles of ≥ 17mm ---- -associated with significantly decreased pregnancy rate in GnRH antagonist cylces.
- 59. Gonadotropin/FSH starting dose Based on • Age • BMI • AFC • AMH • Previous response to stimulation A starting dose of 150-200 IU is generally considered appropriate for a typical patient.
- 60. • Two studies performed to determine whether higher doses( 200-225 IU) would increase the probability of pregnancy. • Pregnancy rates are not increased by using higher than the standard dose. • Does not take into account the advantage provided by availability of surplus frozen embryos for later replacement in patients with high dose stimulations. • Increasing FSH dose at GnRH antagonist initiation did not show a beneficial effect on the probability of clinical pregnancy.
- 61. Elevated serum progesterone (> 1.5 ng/ml) at the initiation of stimulation(D2) is associated with lower pregnancy rate. Administration of GnRH antagonist for 3 consecutive days(from D2)- result in acceptable pregnancy rates.
- 62. Elevated progesterone on the day of trigger – associated with sigificantly decreased pregnancy rate. It is worth considering freezing all embryos for transfer in subsequent cycles.
- 63. Trigerring of final oocyte maturation in GnRH agonist cycle • Although the incidence of SEVERE OHSS is significantly decreased, OHSS can still occur. • GnRH trigger is an option for high responders. • Mean LH and FSH rose over 4-12 hours, elevated for 24-34 hours after GnRH administration. • Approximately 6 days of elevated hCG after 5000 IU of hCG adminstration.
- 64. • GnRH agonist not only effectively induce final oocyte maturation, but also eliminates the incidence of severe OHSS in unsupported luteal phase. • However in patients using their own oocytes, if embryo transfer performed in the same cycle under standard luteal support, • GnRH triggering is associated with significantly decreased probability of pregnancy, due to alteration in the quality of ensuing luteal phase.
- 65. Difference Between Agonist And Antagonist Protocol Agonist protocol Antagonist protocol Prevention of LH surge Effective Effective Supression of pituitary Deep suppression of pituitary Avoids No of oocytes retrieved High Low Cycle cancellation rate Low high OHSS High low GnRH trigger Not possible possible Duration of treatment Long short Cost High low
- 66. Modified natural cycle • Paulson et al. in 1994 • Exogenous hormones or drugs are used when IVF is being performed during a spontaneous cycle with the aim of collecting ‘a naturally selected single oocyte’. • This includes • Use of hCG to induce final oocyte maturation. Luteal phase support may or may not be administered. • Use of GnRH antagonist to block the spontaneous LH surge with or without FSH or hMG as add-back therapy.
- 67. • Severe studies have compared MNC with conventional antagonist protocol. • An RCT concluded than Modified natural cycle provides • Comparable pregnancy rates, • Lower doses and shorter duration of gonadotropins • And hence safe, patient friendly and cost-effective treatment option in poor responders. • Patient should be counselled regarding the advantages and disadvantages. • Oocyte retrieval – 40-80 % • Embryo transfer- 50 % • Pregnancy and live birth – 0-20 %(prognosi better – younger patients).
- 68. Mild stimulation protocol • The term ‘‘mild-stimulation IVF’’ is denoted as • ‘‘a method when follicle stimulating hormone (FSH) or human menopausal gonadotropin (hMG) is administered at a lower dose and or for a shorter duration in a gonadotropin releasing hormone (GnRH)-antagonist co-treated cycle, or • when oral compounds, anti-estrogens or aromatase inhibitors (AIs) are used either alone or in combination with gonadotropins (Gn) with the aim of collecting fewer oocytes’’
- 70. Nargund. Mild IVF. Fertil Steril 2017. Advantages • Higher safety profile (OHSS,multiple pregnancy) • Higher birth weights • Higher patient satisfaction Disadvantages • Higher cycle cancellation rate • Fewer embryos for cryopreservation
- 72. Conclusion about mild stimulation protocol • Except for two RCTs (24, 25), all reported MSIVF to be equally successful in terms of pregnancy outcomes per ET. • The majority of RCTs that analyzed cumulative pregnancy (CPRs/LBRs) found no difference in these outcomes between the two approaches. • The current weight of evidence points to comparable LBRs between MS-IVF and C-IVF in good responders and a trend toward better outcomes in poor responders • Mild-stimulation IVF incorporating tamoxifen or aromatase inhibitors has secured a place in treatment of women with estrogen-sensitive malignancies (breast or endometrial). Nargund. Mild IVF. Fertil Steril 2017
- 73. To summarise……….. GnRH agonists are widely used in IVF to control endogenous LH surge and to achieve augmentation of multifollicular development. Disadvantages , such as necessity of luteal phase support, increased total gonadotropin dose per treatment cycle, and higher costs appear to be outweighed by the observed increase in ability to control the cycle, the higher yield of good quality oocytes and subsequently embryos. The long GnRH agonist protocols give the highest pregnancy rates in normal responders. Further research into finding the right dose and protocol could still improve the clinical benefits of the GnRH agonists.
- 74. GnRH antagonists represent a more physiological and patient- friendly way of ovarian stimulation with equivalent pregnancy rates compared to agonist protocols. GnRH antagonists are particularly advantageous in special groups of patients ie hyper-responders and poor-responders, where they are associated with fewer complications(OHSS) and acceptable pregnancy rates. Finally, GnRH agonists can be used to induce final maturation and ovulation as an alternative to hCG in non GnRH agonist co-treated ART cycles.