MODULE 1: OVULATION INDUCTION IN NORMAL RESPONDERS, POOR RESPONDERS, AND HYPERRESPONDERS

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1 MODULE 1: OVULATION INDUCTION IN NORMAL RESPONDERS, POOR RESPONDERS, AND HYPERRESPONDERS

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3 Contents OVULATION INDUCTION IN NORMAL RESPONDERS, POOR RESPONDERS, AND HYPERRESPONDERS Ovulation Induction Going Back in History Aim of Ovulation Induction Physiology of Ovulation Induction Causes of Anovulation Diagnostic Hormonal Levels in Anovulatory Infertility What is Ovarian Reserve? Predictors of Ovarian Response/Reserve Comparison of AMH and AFC for Assessment of Ovarian Reserve Markers of High and Low Ovarian Response Limitations of AMH and AFC Efficacy of Combined Markers Summary Drugs Used for Ovulation Induction Clomiphene Citrate Tamoxifen Aromatase Inhibitors Gonadotropins Regimens of Gonadotropin Therapy Conventional Step-up Protocol Step-down Protocol Chronic Low-Dose Protocol Sequential Protocol Newer Gonadotropins Used for Ovulation Induction Corifollitropin Alfa Combination of Follitropin Alfa and Lutropin Alfa Recombinant LH Adjuncts to Ovulation Induction Gonadotrophin-Releasing Hormone Advantages and Regimes Monitoring Ovulation Induction Cycles Summary

4 Selection of Protocol Key to prevention of OHSS Management Strategies for Normal Responders Current Strategies in the Management of Poor Responders Current Management Strategies Controlled Ovarian Stimulation GnRH Agonists GnRH Antagonist Natural Cycle IVF Pretreatment Adjuvant Therapy Summary Current Strategies in the Management of Hyperresponders Relation Between AFC and Age Hyperresponse and Genetic Variation Optimal Treatment Approach in Hyperresponders Submaximal Ovarian Stimulation Clomiphene + FSH is an Option to Submaximally Stimulate Ovary Is a GnRH Antagonist Protocol Better in Patients With PCOS? Does GnRH-Agonist Trigger Avoid OHSS? Reduced Low Birth Rate With GnRH Triggering Summary Questionnaire References

5 OVULATION INDUCTION IN NORMAL RESPONDERS, POOR RESPONDERS, AND HYPERRESPONDERS Ovulation Induction Going Back in History Infertility and its associated emotional burden have been known since ancient times. Before recent advances in this field, there were not many treatment options available to help overcome this affliction. Ovulation induction, a pillar of infertility treatment, was just an abstract thought in the 1960s, and medical treatment was limited to psychological support and proper technique, timing, and frequency of intercourse. However, since the early 1960s, we have witnessed many amazing breakthroughs in infertility 1 treatment. The first successful induction of ovulation with human gonadotropins was evidenced in 1958, materializing in pregnancy evidenced in In 1961, the first successful application of clomiphene citrate (MRL-41) was documented by Greenblatt et al. A major step forward was the application of human pituitary gonadotropins in hypophysectomized patients to achieve pregnancy and application of urine from menopausal women to 1 enable ovulation induction in amenorrheic women. During the 1970s, the application of gonadotropin-releasing hormone in pulses, which were precisely spaced in time, proved to be successful in ovulation induction among women with hypogonadotropic amenorrhea of hypothalamic origin. The period between 1974 and 1977 was significant for further research in this field. During this period, many publications presented and discussed new evidence that demonstrated the efficacy of bromocriptine in reducing the circulating levels of prolactin and restoring the luteinizing hormone (LH) balance pulsatility, thereby helping 1 reestablish normal menstruation and ovulation. Aim of Ovulation Induction Ovulation induction is often the first-line approach for treating infertility, including cases of unexplained infertility, irregular menstrual cycles, and 2 4 anovulation. The aims of ovulation induction are as follows: To induce monofollicular development and ovulation in anovulatory infertile women To overcome natural follicular selection process to increase the number of oocytes available for fertilization To restore normal fertility by generating normo-ovulatory cycles To mimic physiology and induce single dominant follicle selection and ovulation To augment ovulation in unexplained infertility For controlled ovarian hyperstimulation (COH) in intrauterine insemination (IUI) and assisted reproductive treatment (ART) Physiology of Ovulation Induction Ovulation is the result of a well-ordered series of events primarily controlled by the hypothalamic-pituitary-ovary axis. The menstrual cycle is directed by complex functional interactions between the ovaries and the hypothalamus-pituitary system that control each other by means of positive or negative feedback mechanisms. The main factors responsible for the function of this axis are: firstly, the hypothalamic hormones, particularly the gonadotropin releasing hormone (GnRH); secondly, the pituitary hormones or gonadotropins (FSH and LH); and finally, the ovarian 5,6 steroid hormones (estradiol and progesterone) (Figure 1). Figure 1: Physiology of ovulation induction. Paracrine control Endocrine control Gonadotropin Independent Primary recruitment of primordial follicles independent of GT Gonadotropin Sensitive Small antral 2 5 mm Gonadotropin Dependent Dominant mm Ovulatory 20 mm Secondary Primordial Primary Secondary recruitment phase of folliculogenesis and selection of ovulatory follicles is GT dependent and can be modulated with use of exogenous hormones I Recruitment II Recruitment Selection Dominance >120 days 85 days 14 days 1

6 Causes of Anovulation The causes of anovulation have been described in Table 1. Table 1: Causes of anovulation Group I: Hypothalamic/ Weight loss, systemic illness, 5% pituitary failure Kallmann s syndrome Hypogonadotropic hypogonadism After an initial workup to determine the cause of anovulation and going through the required lifestyle changes, the timing of ovulation induction plays a critical role in determining outcomes. Commonly, treatment is initiated in the luteal phase to minimize the consequences of flare effects, which is usually seen in the first few days of treatment. Optimal timing is determined from the diameter of preovulatory follicles, their ultrasonographic appearance, and circulating estradiol-17 levels during spontaneous or induced cycles. Premature or delayed induction is detrimental to the follicle and can lead to unfortunate outcomes and 7,8 probable complications. Diagnostic Hormonal Levels in Anovulatory Infertility Diagnostic hormonal levels in anovulatory infertility have been described in Table 2. Table 2: Diagnostic hormonal levels in anovulatory infertility What is Ovarian Reserve? Hyperprolactinemia Hypopituitarism Group II: H/P dysfunction PCOS 90% Group III: Ovarian failure Premature ovarian failure (POF) 5% Resistant ovary syndrome (RIS) Hypothalamic: Underweight Hyperprolactinemia Ovarian failure/menopause Mid-cycle PCOS FSH, LH, E2, n FSH, LH, E2 FSH, LH, E2 FSH, LH, E2 FSH, LH, E2 /n FSH, /n LH, /n E2 The loss of oocytes is a continuous process that begins just after the establishment of the oocyte pool during fetal life. At approximately 20 weeks gestation, the ovaries in the female fetus contain 6 7 million oocytes, but this number decreases rapidly. In total, 1 2 million oocytes remain at birth. At menarche, a female has about 300,000 ovarian follicles, which reduce to about a 00 during menopause. With advancing maternal age, the number of egg cells that can be successfully employed for a possible pregnancy declines; thus, there is an inverse correlation between age and female fertility. Studies have suggested that the ovaries begin to go through an accelerated decline in fertility as early as 13 years prior to 9 11 menopause (Figure 2). Ovarian reserve is a term used to establish the competence of the ovary to provide oocytes that are capable of fertilization, resulting in a healthy and successful pregnancy. It includes the secondary, preantral, and antral ovarian follicle pool. Figure 2: Quantitative (solid line) and qualitative (dotted line) 11 decline of the ovarian follicle pool. Number of follicles Optimal Fertility 30 Age (years) The main goal of individualized treatment in in vitro fertilization (IVF) cycles is to facilitate a superior probability of success with a lower risk from ovarian stimulation. This principally includes stimulation of the ovary and suppression of the pituitary. The treatment protocols in IVF are based on the most accurate calculation of ovarian response in an attempt to prevent poor,12 and hyperresponse. Predictors of Ovarian Response/Reserve 20 Declining Fertility Ovarian reserve tests (ORTs) help to predict ovarian reserves, based on which the treatment plan is formulated. Hence, they should be easy to execute, reproducible, and should help distinguish between a normal, hyper and poor ovarian response. However, recent advances indicate that they are effective only in predicting the ovarian response to stimulation, but are not accurate predictors of pregnancy or its outcome. Biological (age), biochemical, biophysical, and histological tests fall under the category of ORTs. Ovarian reserve tests include concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, inhibin, anti- Mullerian hormone (AMH); ovarian volume; ovarian antral follicle count (AFC); and ovarian biopsy. Most frequently performed tests include AMH 13,14 and AFC, which have a good sensitivity and specificity apart from age. Inhibin B and E2 are produced by early antral follicles in response to FSH, having the classical feedback loop of pituitary gonadal axis. With the decline of the follicle pool, serum levels of inhibin B and E2 decrease, leading to a rise in serum FSH level. Because these factors are part of feedback system, their serum levels are not independent of each other, and hence, they have to be measured collectively. Separately, their levels are poor predictors of ovarian reserve, because their levels vary widely by 15 assay, laboratory, population, and reproductive aging. Comparison of AMH and AFC for Assessment of Ovarian Reserve Anti-Mullerian hormone produced by the pre-antral follicles witnesses its maximum expression in granulosa cells of secondary, pre-antral, and small antral follicles up to 6 mm in diameter. Thus, the level of AMH may 40 End of Fertility Irregular Cycles 50 Menopause 60 2

7 Figure 3: Predictors of ovarian response/reserve. Excessive response >14 oocytes at OPU Accompanied by increasing number of immature oocytes OHSS Ovarian Response Indicative of diminished ovarian reserve Low pregnancy rates Need for higher FSH dosage? Poor response <4 oocytes at OPU Normal Response 4 14 oocytes at OPU correspond to the population of these follicles. Moreover, AFC is thought to correspond to the quantitative facet of ovarian aging. To compare significance, role, and reliability of AMH versus AFC in the assessment of ovarian reserve, a study was conducted with 75 patients with polycystic ovaries (PCO) undergoing IVF and 75 non-pco patients. Both (AMH and AFC) were found to have similar significance in oocytes retrieved on ovum pick up (OPU) in PCO patients. Difference in correlation for AFC and AMH was minimally significant in non-pco and not significant in PCO patients. Practically, AFC was found to be sufficient in 12 terms of OPU (Table 3). Table 3: 12 Correlation between AFC and follicles and AMH Normal ovaries AFC >12 mm Polycystic ovaries AFC >12 mm AFC AFC >12 mm >12 mm AMH >12 mm AMH >12 mm AMH AMH >12 mm >12 mm AFC: Antral follicle count; AHM: Anti-Mullerian hormone. With the ovarian tests one can only predict response and not the number of oocytes retrieved. So far, AFC (follicles of 2 mm size), which quantifies the number of antral follicles in the ovary by ultrasonography on day 3 of menstrual cycle, best predicts the quantitative aspect of ovarian reserve. However, it might be sometimes difficult for the patient to get ultrasound done on a specific day; additionally, it requires for the patient measurement of the AFC by additional transvaginal ultrasound examination during early follicular phase. Therefore, in search of a better, time-independent parameter, serum anti-mullerian hormone (AMH) has emerged as a 15 promising test to assess the ovarian reserve. Markers of High and Low Ovarian Response Markers of ovarian reserve, in particular, AMH and AFC, allow identification of women who are likely to show a high or low response to controlled ovarian stimulation (COS). In addition, an AFC value of >16 is used to predict a high response with a sensitivity of 89% and a specificity of 92%. Cut-off levels of AMH values for poor ovarian response vary between 0.7 and 1.3 ng/ml and cut-off values of AFC used for predicting poor 13 response are <7 (Figure 4). Figure 4: Prediction of response. Markers of high response Markers of low response Normal response AFC AMH Dynamic test CCCT, EFORT FSH, E2, AMH, Inhibin B, Day P4 AFC, Ovarian volume Dynamic tests CCCT, EFORT 13 Prediction of hyperresponse AFC Optimum cut-off value for AFC =>14 Sensitivity 82% Specificity 89% AMH Optimal cut-off 3.36 ng/ml Sensitivity of 90.5% (95% CI ) Specificity of 81.3% (95% CI ) 13 Prediction of poor response AMH Optimum cut-off value for AMH=0.99 ng/ml Post-test probability was highest at cut-off levels of 0.59 ng/ml AFC Optimum cut-off value for AFC but the Post-test probability was highest at cut-off levels of <8 Figure 5: Expected poor-, normal-, or hyperresponders. AFC (n) AMH (ng/ml) Ovarian reserve AFC: Antral follicle count; AMH: Anti-Mullerian hormone High Low 13 Expected high response Expected normal response Expected poor response 3

8 Limitations of AMH and AFC 12,13 AMH predicts extremes of response as compared to FSH 12,13 Significant negative interaction between age and AMH 12,13 AMH and age independent determinants of oocyte yield Efficacy of Combined Markers The efficacy of combined markers has been given in Table 4. Table 4: Efficacy of combined markers Markers vs. eggs collected/amp FSH Regression Significance co-efficient Day 3 FSH+E p<0.01 Day 3 FSH+E2+AFC 0.7 p<0.001 Day 3 FSH+E2+AFC+ Inhibin B 0.8 p<0.001 Day 3 FSH+E2+AFC+AMH 0.73 p<0.001 AFC+Inhibin B 0.78 p<0.001 AFC+Day 3 AMH 0.7 p<0.001 AFC+Inhibin B+Day 3 AMH 0.78 p<0.001 Summary Age alone does not reflect the reproductive potential or ovarian reserve of a woman Circulating AMH may accurately reflect the total developing follicular cohort which may or may not represent the total ovarian reserve Available tests reflect, directly or indirectly, the size of the antral (2 5 mm) follicle pool AFC and AMH are equally accurate predictors of high ovarian response to COH and allow us to identify the patients who are at increased risk of OHSS Relationship between AFC and AMH concentrations is more reliable than that observed with FSH, inhibin B, and estradiol on cycle day 3 Basal FSH should not be used as a screening tool but instead used to counsel patients appropriately regarding the realistic chance of conception and aiding determination of appropriate GT dose AMH best single marker and Day 3 FSH + E2 + AFC + Inhibin B are best combined markers to predict poor response to GT stimulation CCCT is superior with regard to identification of potential low responders, and EFORT is superior to identify hyperresponders but with a very high rate of false positive results Though conventional markers cannot predict exact response they allow us to counsel women regarding complications and poor prognosis In ART population, first-cycle IVF still remains the most informative test in terms of how women will respond to ovarian stimulation Drugs Used for Ovulation Induction Clomiphene Citrate Clomiphene citrate is the first treatment of choice in the management of infertility in normally estrogenized, anovulatory women, primarily in PCO patients. It helps displace endogenous estrogen from estrogen receptor sites leading to a favorable modification in pulsatile gonadotrophinreleasing hormone (GnRH) secretion. Studies have shown >50% increase in endogenous FSH and a significant increase in LH concentration with 14 clomiphene citrate. It is administered orally and the dose is calculated based on body weight. Most women (52%) show an ovulatory response 16 after treatment with 50 mg. Use of clomiphene citrate requires careful monitoring to achieve favorable results. Clomiphene citrate has shown successful ovulation in 60% 90% patients and pregnancy rates in about % 40%. However, the peripheral antiestrogenic effects on the endometrium and cervical mucus maybe the reason for its drawback in a few cases. The estimated rate of associated multiple pregnancies is about % and 20%. Sometimes, mild ovarian hyperstimulation syndrome (OHSS) is observed in 13% of cases treated with clomiphene. Other minor effects include visual disorders, flushes, nausea, and breast discomfort. Cases of ovarian cancer have also been reported; however, the association remains uncertain. Considering these reports, the United Kingdom Committee for Medical Safety recommends the use of clomiphene only for six 14,17,18 consecutive cycles. Tamoxifen Tamoxifen is an antiestrogenic compound that acts as an agonist on the estrogen receptors of the vaginal mucosa and endometrium. It is at par with clomiphene citrate in efficiency. Limited evidence shows 50% 90% ovulation rates and 30% 50% pregnancy rates with Tamoxifen. It has also demonstrated positive results in cases that have failed with clomiphene citrate. This is primarily attributed to the higher score of cervical mucus and better functioning of the corpus luteum with tamoxifen. The drug has shown to increase the risk of endometrial cancer; however, it is improbable with short-term use. Despite exhibiting its usefulness, further studies with 19,20 tamoxifen are warranted. Aromatase Inhibitors Aromatase inhibitors, a common regimen in the management of postmenopausal breast cancer, are a new group of drugs to join the arsenal of infertility treatment. Third-generation aromatase inhibitors, primarily anastrozole and letrozole, are used for ovulatory disorders and 21 superovulation. They inhibit androgen to estrogen conversion, reduce the negative feedback, and thus, increase pituitary gonadotropin output, leading to an increased ovarian function. With increased estradiol produced by the developing follicle, FSH levels decrease as the pituitary and hypothalamic estrogen receptors are not blocked and the feedback mechanisms are intact. Therefore, aromatase inhibitors may be best suited for restoration of 21 monofollicular growth. 4

9 Failure to conceive within 6 CC/Letrozole/Tamoxifen-induced ovulatory cycles is an indication to expand diagnostic evaluation to exclude other infertility factors or to change treatment strategy if evaluation is already complete. Letrozole has a short half-life, and no adverse impact is anticipated in the luteal phase if it is administered in the early follicular phase. Women with PCO resistant to clomiphene citrate have shown superior ovulation rate with letrozole. Letrozole displays an ovulation rate, live-birth rate, and miscarriage rate comparable to those of clomiphene citrate with a 21 significantly thicker endometrium. Gonadotropins Gonadotropins, which mainly include FSH and LH, are hormones synthesized by the anterior pituitary gland, and they play a crucial role in the process of ovulation. Urinary or recombinant gonadotropins are commonly used for ovulation induction, usually after other less complicated and costly methods have failed. However, premature ovarian failure does not respond to gonadotropins. It primarily causes an FSH upsurge, which fuels the proliferation of granulosa cells and follicular growth. Luteinizing hormone stimulates the production of androgen, which is converted to estrogen by granulosa cells. Although the primary objective is to promote the development of a single mature follicle, it is common to expect multiple pregnancies. Other adverse outcomes include premature 22,23 delivery and OHSS. Regimens of Gonadotropin Therapy Conventional Step-up Protocol The protocol maintains FSH at minimum levels to ensure employment of multiple follicles. It begins with IU of human menopausal gonadotropin (hmg) or FSH on day 2 or 3 of the cycle and continued for 5 7 days. The dose is increased by IU for another 5 7 days if the follicular and estradiol response is inadequate. If required, the dose is further augmented by 37.5 IU and continued in such doses until the preferred outcome is achieved (Figure 6). The protocol is beneficial for patients who show hyperstimulation with a conventional low-dose regimen; however, the prolonged duration of treatment can be taxing. Higher complication rates have been reported with the use of the 24,25 conventional step-up protocol. Figure 6: Conventional step-up protocol. 2,24 Step-down Protocol The step-down protocol tries to replicate the normal physiological negative feedback of FSH. It begins with 150 IU on day 2 or 3 of menses, which is continued for 2 or 3 days, and reduced to 75 IU for another 3 days, after which the patient undergoes follicular monitoring and serum estradiol measurement. The dose is decreased in two steps if follicles > mm are observed. The last dose is then continued till the day of human chorionic gonadotropin (hcg) injection (Figure 7). Although this protocol is intended to reduce the incidence of OHSS, the long half-life of gonadotropins makes it difficult to judge the proper dosage for 2,24 maintenance of a lead follicle without the risk of OHSS. Figure 7: Step-down protocol. Starting dose to IU/D Scan d 4 5 Follicle >9 mm HCG: Human chorionic gonadotropin. 2,24 Decrease by 37.5 IU Chronic Low-Dose Protocol Scan d 8 Decrease by 37.5 IU hcg 5000 IU leading follicle 16 mm The chronic low-dose protocol aims at administration of low-dose FSH till its threshold is reached and continued until satisfactory outcomes are achieved. In case of inadequate response, the dose in increased 50% every 7 9 days until there is indication of follicular growth (Figure 8). However, this protocol maybe impractical for clinical use considering the low cycle of induction. Most studies have shown poor outcomes; however, it has a 2,26 lower risk of OHSS and multiple pregnancies. Figure 8: Chronic low-dose protocol. Starting dose Scan d IU/d HCG: Human chorionic gonadotropin. Scan d 14 2 Increase dose by 50% Scan d 21 hcg 5000 IU leading follicle 16 mm Increase dose by 50% FSH threshold IU Increases of 75 IU FSH every 5 7 days when needed Days FSH: Follicle-stimulating hormone IU IU Sequential Protocol The sequential protocol causes dependence of FSH in leading follicle, which gradually decreases as the follicle grows. The negative feedback of the increasing estradiol on FSH levels prevents atresia in the leading follicle. It begins with an initial dose of gonadotropins 37.5 IU for the first 14 days, followed by an increased dose of 50% every 7 days till the dominant follicle is 14 mm, after which the dose is decreased by 50% 2 (Figure 9). 5

10 Figure 9: Sequential protocol. Starting dose Scan d IU/day HCG: Human chorionic gonadotropin. Scan d 14 2 Increase dose by 50% Scan d 21 Follicle =14 mm Increase dose by 50% hcg 5000 IU leading follicle 16 mm Decrease dose by 50% Newer Gonadotropins Used for Ovulation Induction Corifollitropin Alfa Corifollitropin alfa is the first long-acting recombinant FSH that has been approved by the European Commission for COS. It functions similar to natural FSH, with a longer half-life. Seven daily injections of 150 UI recombinant follicular stimulating hormone (rfsh) are replaced with a single initial 0 or 150 g dose of corifollitropin alfa with no difference in safety or efficacy. Corifollitropin alfa does not show antibody formation, is well tolerated with subcutaneous administration, and the OHSS risk is similar to rfsh. Although not well-established, it must be avoided in patients with high AFC (>20) and in those with a history of 27,28 hyperresponse. Combination of Follitropin Alfa and Lutropin Alfa Combination of follitropin alfa and lutropin alfa allows administration of both gonadotrophins in a convenient single injection and is indicated for 29 severe endogenous FSH and LH deficiencies. Table 5: Adjuncts to ovulation induction Adjunct 31 GnRH analogs 32 Estrogen/progesterone 32 Growth hormone 31 Pulsatile GnRH 32 Dexamethasone 32 hcg/gnrh antagonist 32 Dopamine agonist 32 Metformin Application An LH surrogate to induce ovulation in PCOS Used as a pretreatment that helps reduce the amount of gonadotropin and thus improves synchronization of the follicular cohort instead of agonist alone Beneficial in poor responders Minimal application in diabetes Rarely used in PCOS Efficiency maybe improved when used as a pretreatment drug Beneficial in elevated androgen levels Highly effective adjunct to clomiphene citrate Minimal application in diabetes May be used as an alternative to hmg for the same dose of pure FSH when addition of LH activity is desired In high-risk OHSS patients, decreases hemoconcentration, ascites, and vascular permeability Decreases OHSS risk in PCOS patients GnRH: Gonadotrophin-releasing hormone; HCG: Human chorionic gonadotropin; PCOS: Polycystic ovarian syndrome; FSH: Follicular stimulating hormone; LH: Luteinizing hormone; OHSS: Ovarian hyperstimulation syndrome. Figure : Mechanism of action of GnRH agonist (A) and 33 antagonist (B). A GnRH Agonist Recombinant LH The physicochemical, immunological, and biological activities of rlh are comparable with those of pituitary LH. Recombinant LH may help in 30 situations where there is an inadequate response to FSH. Adjuncts to Ovulation Induction At times, adjuvant drugs are required along with ovulation induction to optimize ovulation. A few of them are listed in Table 5. Gonadotrophin-Releasing Hormone Gonadotrophin-releasing hormone (GnRH) plays a vital role in controlling the ovarian cycle in women. It helps adapt the molecular structure, and the analogues are synthesized with agonistic or antagonistic effects on the 33 gonadotrophic cells of the anterior pituitary gland. B Increased secretion of LH/FSH GnRH Antagonist After an initial stimulatory effect (flare-up), GnRH agonists lead to desensitization of the gonadotrophic cells and a reduction in the number of gonadotropin-releasing hormone receptors (GnRH-R) on the cell membrane (downregulation), thereby reducing the release of FSH and LH, which in turn leads to inhibition of androgen and estrogen production. In contrast, the GnRH antagonists produce an instant effect by competitive 33 blockade of GnRH-R (Figure ). Receptor blocked no micro-aggregation No effect immediate decrease of LH (FSH) GnRH: Gonadotropin-releasing hormone; FSH: Follicular stimulating hormone; LH: Luteinizing hormone. 6

11 Advantages and Regimes The advantages and regimes of GnRH have been listed in Table 6. Table 6: Advantages and regimes of GnRH agonists and antagonists Advantages GnRH agonist Fewer cycles cancelled Eliminates LH surge Controls basal LH secretion Oocyte recovery programmed Enhances intrafollicular growth and recovery of better quality oocytes Widens the window of uterine implantation thus increasing pregnancy rates Regimes Long or desensitization protocol Short protocol Long GnRH1a Microdose flare protocol Similar to the short protocol with reduced agonist dose Stopping gonadotropin-releasing hormone agonist protocol Started in the luteal phase 1 week before the expected start of menses, and stopped at the initiation of gonadotropin therapy. However, it is not popular due to the erratic response. Ultrashort protocol FSH Short flare GnRH1a Ultrashort GnRHa FSH Ovulation Ovulation Luteal support Luteal support Luteal support GnRH antagonist For prevention of premature LH surge Immediately suppress GTs by blocking GnRh receptor, restricting treatment only to those days when LH surge likely to occur Mechanism of action dependent on equilibrium between endogenous GnRh and dose of applied antagonist Lower GT consumption Lower risk of OHSS Opportunity to give GnRH agonist for trigger Reduces the burden of treatment Multiple-dose protocol (Lubeck protocol) Amp. hmg Gonadotropins Menses Single-dose regimen (French protocol) Gonadotropin GnRH antagonist GnRH antagonist hcg E2 >00 pg/ml follicle 20 mm OPU hcg OPU ET ET FSH Ovulation Monitoring Ovulation Induction Cycles Monitoring ovulation induction cycles helps in choosing the most suitable protocol to obtain the best possible outcome, prevent complications, determine responses to treatment, and narrow down the optimal dose and length of therapy. It also helps mitigate the risk of OHSS, predict high risk of multiple pregnancies, or inadequate ovarian 35,36 response (Figure 11). Serial transvaginal (TVS) ultrasound: It is carried out on day 2 to assess ovaries and look for AFC and rule out cysts. Serial transvaginal ultrasound is done from day 9; every alternate day till the leading 35 follicle reaches 18 mm or more in size. Ultrasound provides information on: Follicular growth Timing of ovulation Presence of cyst Ovarian morphology/reserve/blood flow Endometrial thickness/morphology/texture/blood flow Feasibility of oocyte retrieval Cancellation of ovarian stimulation cycles Definite Indication Poor follicular growth E2 levels < 0 pg/ml on day 5 6 Possible Indication Adnexal cyst secondary to GnRh agonist Endogenous LH surge Steady decline in E2 levels E2 < 250 pg/ml per mature follicle on day of hcg Serial serum estradiol E2 levels: They correlate closely with the stage of development of the dominant follicle. A value of more than pg/ml indicates sufficient dose of gonadotropins. Values occasionally depending on endometrial thickness and number, and size of follicles. 7

12 Figure 12: Why monitor ovulation induction cycles? Patient s initial parameters Baseline Scan TRO Ovarian or uterine pathology, AFC Baseline hormonal profile Ovarian reserve, FSH: LH ratio, androgen excess, thyroid profile, and hyperprolactinemia Choose appropriate stimulation regimen to prevent OHSS, multiple pregnancy, and predict response to ovarian stimulation Ovarian response to ovulation induction Confirmation of downregulation after GnRH agonist Determine response to drug Determine the dose and length of GT TX Determine optimal time for hcg administration Detect ovulation Time OR Identify-poor responders and women at risk of OHSS Completion of therapy Diagnose complications of OI 1. Premature Luteinization 2. LUF 3. Endogenous LH surge 4. Retention/Functional cyst Confirm pregnancy TRO multiple pregnancy TRO late onset OHSS The main complications of ovulation induction include OHSS, multiple 37 pregnancies, and spontaneous abortions. Summary New treatment modalities have revolutionized infertility treatment with good rates of successful pregnancies. Clomiphene citrate is the first treatment of choice in the management of infertility in estrogenized, anovulatory women; it is used primarily in PCO patients and has shown successful ovulation in 60% 90% patients and pregnancy rates in % 40% women. Tamoxifen is an antiestrogenic compound that acts as an agonist on the estrogen receptors of the vaginal mucosa and endometrium, its efficiency being at par with clomiphene citrate. Gonadotropins are hormones synthesized by the anterior pituitary gland; they mainly include FSH and LH. They play a crucial role in the process of ovulation and are usually used after other less complicated and costly methods have failed. Regimens of gonadotropin therapy used commonly include conventional step-up protocol, step-down protocol, chronic low-dose protocol, and sequential protocol. GnRH agonist regimens include long or desensitization protocol, short protocol, microdose flare protocol, stopping gonadotropin-releasing hormone agonist protocol, and ultrashort protocol. GnRH antagonist regimens include multiple-dose protocol (Lubeck protocol) and single-dose regimen (French protocol). Selection of Protocol In a clinical scenario, it is imperative to select the correct stimulation protocol. Individualized protocols are based on the following parameters of 22,28,38 ORT: COS and BMI Age FSH AMH AFC Alternative strategies for all categories of ovarian response, including in particular normal and poor responders, have been reported, with the optimal strategy still to be fully elucidated. Ovarian response categories dictate risk, and treatment strategies are designed to minimize risk while maximizing oocyte yield within each response category. Negligible response means that the conventional criteria for triggering (three follicles R17 mm) are unlikely to be achieved. For all antagonist cycles with an excessive response, an agonist trigger is adopted. The AMH measurements are for the AMH Gen II assay, and the values are in pmol/l. The suggested antral follicle count (AFC) thresholds are based on the correlation of AMH and AFC and associated response category literature, 22,28,38 depicted in Figure 13. Figure 13: Anti-Mullrerian hormone (AMH)-stratified 22,28,38 individualization of treatment. AFC AMH High 40 Response Normal Response 7 Reduced Response 2 1 Negligible AMH-tailored protocol has been displayed in Table 7. Table 7: AMH-tailored protocol AMH <2.2 pmol/l (0.3 ng/ml) AMH < pmol/l (<2.2 ng/ml) AMH pmol/l (2 4 ng/ml) AMH >28.6 pmol/l (>4 ng/ml) Conversion: 1 pmol/l=ng/ml Treatment strategy Antagonist Control Agonist Trigger Antagonist Control HCG Agonist Trigger Long Down-regulation Agonist Control Flare Agonist Flare Agonist/Oocyte Donation Exclude, counsel, offer alternative ART 300 IU GT GnRH antagonist 200 IU rfsh or 225 IU HMG GnRH agonist/antagonist 150 IU GT + GnRH antagonist Dosage is subject to change as per patient profile increases with BMI and age years 36 8

13 It is hence clear that the poor responders and hyperresponders have a different and individualized treatment approach, populated in Figure 14. Figure 14: Individualization of protocols. Follicular phase AMH AFC Age History Titrating dose Rec FSH or hmg GnRH a or A Protocol Patient friendly Effective & safe Day of oocyte trigger Normal response Rec hcg 250 mcg 5000 u hcg Rec hcg 125 Or 250 mcg 3500 u hcg >18 dominant follicles E2 >5000 pg/ml GnRHAa Triggering 1 mg SC Luteal phase set or DET Freeze surplus Proven LPS Signs off OHSS Freeze all embryos Proceed to day 5 Evaluate patient P4+ E hcg on day of OR for LPS Freeze all embryos Freeze half D 2/3 Culture rest to blastocyst set Freeze surplus OHSS Freeze all embryos set Freeze surplus set Freeze surplus Gonadotropin-releasing hormone agonist (GnRH-a) trigger has been used for the induction of final follicular maturation and ovulation with the aim of reducing the OHSS risk. Several studies have shown that the releases of endogenous follicular stimulating hormone (FSH) and LH after administration of GnRH agonist in in vitro fertilization (IVF) cycles are able to precede the final follicular maturation leading to removal of fertile oocyte with normal development of the embryo and ultimately pregnancy. Recent modifications of luteal phase after GnRH-a trigger make it possible to transfer embryo in the same cycle for many women at risk of OHSS and provide a good outcome. But based on the results of some studies, using GnRH-a trigger leads to defect luteal-phase resulting to reduce the implantation and clinical pregnancy rates and also increase in abortion in fresh embryo transfer cycles compared to routine IVF cycle with hcg triggering. The main clinical advantage of GnRH-a trigger is the potential to induce a rapid and reversible luteolysis and therefore decrease the risk of OHSS progression. On the other hand, this is concomitant with severe luteal phase defect resulting from a short period of the induced LH and FSH peak. Besides, it particularly inhibits the secretion of vasoactive products, 39 especially VEGF, from the corpus luteum. hcg trigger has provided a simple method in the clinic for fresh embryo transfer. This conventional method has been modified by another trigger, GnRH-a, which unlike hcg, does not affect the early luteal phase. On the other hand, the GnRH-a trigger reduces LH levels through pituitary downregulation, so that the amount of LH is inadequate for continuing the function of the corpus luteum. The reduction of the activity of the corpus luteum caused to decrease the progesterone levels in luteal phase which is very low for optimal embryo implantation. Therefore, the use of GnRH-a trigger without accurate luteal phase support causes a decrease in pregnancy rate. The preliminary results of the administration of GnRH-a trigger for final oocyte maturation revealed unsatisfactory results so that the high rate of pregnancy loss was associated with a significant reduction 39 in OPR. Induction of follicular maturation and ovulation is primarily approached as the following: Urinary hcg ,000 IU IM Rec hcg g SC GnRha - 1 mg SC Key to Prevention of OHSS Experience with OI therapy and recognition of risk factors for OHSS Highly individualized OI regimens carefully monitored with USG and E2 Use of minimum dose and duration of GT therapy necessary to achieve the therapeutic goal Management Strategies for Normal Responders Ovulation induction in normal responders can be optimized by individualizing the stimulation protocols and dose of gonadotropins based on ovarian reserve tests and previous response to controlled ovarian 36 stimulation. A meta-analysis was conducted to recognize the optimal daily starting dose of rfsh. Factors that were considered were ovarian response, pregnancy chances, rate of cycle cancellation, and the incidence of OHSS. The study suggested that oocyte yield was greater with the use of >200 IU/day dose of rfsh; however, the pregnancy rates remained the same with lower doses as well. Use of 0 IU/day demonstrated a higher risk of insufficient response, and administration of 200 IU/day demonstrated a high risk of OHSS. The number of embryos available for cryopreservation was lowest with 0 IU/day, but similar with 150 and 200 IU/day. Thus, the meta-analysis concluded that the optimal daily rfsh stimulation dose is 150 IU/day in presumed normal responders. A customized approach may further help improve the risk benefit balance 37,38 while sustaining an adequate ovarian response. Current Strategies in the Management of Poor Responders The European Society of Human Reproduction and Embryology (ESHRE) consensus (Bologna criteria) defines poor response to ovarian stimulation as presence of at least two of the following three 40,41 features: Advanced maternal age ( 40 years) or any other risk factor for poor response A previous poor response ( 3 oocytes with a conventional stimulation protocol) An abnormal ovarian reserve test (AFC <5 7 follicles or AMH < ng/ml) The common causes or risk factors present in poor responders include advancing age, structural chromosomal aberrations or mutations or early menopause, primary ovarian insufficiency, pelvic infection, ovarian 9

14 endometriomas, ovarian surgery, and chemotherapy. One stimulated cycle is deemed essential for the diagnosis of poor ovarian response (POR). However, patients over 40 years of age with an abnormal ORT may be 41 classified as expected PORs. Introduction and approach to poor responders have been summarized in Figure 15. n=398) and Group B was treated with hmg (n = 450). The pregnancy rate in both groups was increasingly enhanced in coherence to the number of retrieved oocytes. When more than 8 oocytes were available, Group A had a significantly better outcome in terms of the pregnancy rate. Thus, rfsh + 44 rlh results in a significantly higher pregnancy rate than hmg (Figure 16). Figure 15: Poor responders. Causes Idiopathic Ovarian failure Antibodies to exogenous GTS Current Management Strategies Controlled Ovarian Stimulation Change in OI protocols and ART Modifications of the long GnRH-a protocol Use of GnRH agonist short protocol Use of GnRH antagonist protocol Natural or Mild cycle IVF Modifications of ovarian stimulation Use of LH in COS Use of adjuvants Intracytoplasmic sperm injection Day 2 better than day 3 or 5 ET A high initial dose of rfsh in the follicular phase is the standard protocol. This is expected to help enhance early follicular maturation and minimize atresia. However, one major drawback is the number of oocytes that are already recruited in the cycle. It is suggested that in a natural cycle, FSH begins to rise during the foregoing luteal phase, 12 days after the LH surge. This is contrary to the classical belief that follicular phase supports follicle growth and recruitment. The mid-luteal rise of FSH suggests its impending role in the luteal follicular transition of follicular 42 development. Also, addition of a low-dose hmg in an antagonist protocol has demonstrated a pregnancy rate of 28% in women <40 years of age. However, it requires further evidence to substantiate its use in 43 clinical practice. A study was conducted to evaluate the effectiveness of COS protocols with rfsh + rlh versus hmg. A total of 848 IVF patients classified as expected poor or normal responders according to AFC and were treated with basal (day 3) FSH. Group A was treated with rfsh + rlh (2:1 ratio, Abnormal response to controlled ovarian stimulation Premature luteinization Follicle <15 mm with echoes Correlates with high P4 levels in follicular phase Premature and suboptimal LH surge Progesterone production but no ovulation Oocyte maturation without follicular rupture Poor quality oocytes and embryos Endometrium out of phase Reduces implantation rate LUF Diagnosed when dominant follicle is still apparent 48 hours after administration of hcg or LH surge Size mm with internal echoes Perifollicular RI Endogenous surge Seen on USG as premature rupture of follicles at diameter <16 17 mm Compromised oocytes and embryo quality result of exposure to inappropriate LH levels Requires extensive endocrine monitoring Prevented with use of GnRH agonist or antagonist Functional cyst Presence of cyst at pre-stimulation baseline scan following GnRh agonist stimulation for downregulation Characterized by sharp edges and anechogenic contents Initial FSH surge when GnRh agonist is commenced triggers functional cyst formation Requires USG-guided aspiration before commencing OI Persistent/Retention cyst Presence of cyst at baseline scan follicle from previous cycle or persistent CL Figure 16: Pregnancy rate per embryo transfer (PR/ET) according to the number of retrieved oocytes (A) or according to the number of 44 mature (MII) oocytes (B). A PR/ET (%) rfsh+rlh hmg Number of oocytes Number of MII oocytes Pale columns correspond to patients who received rfsh + rlh/2:1 (Group A, n=398), dark columns correspond to patients who received hmg (Group B, n=450). *p=0.0038; **p= * B PR/ET (%) rfsh+rlh hmg ** 9

15 GnRH Agonists The long protocol is a well-defined, superior protocol with no adverse effects on cycle treatment. Moreover, it has clear effects on enhancing the embryo cleavage speed and the pregnancy rate, but with a higher rate of 45 multiple pregnancies. Several strategies have been suggested for preventing cycle cancellation in order to technically eradicate excessive ovarian suppression while capitalizing the initial stimulatory effect of GnRH-agonists on pituitary gonadotropin release. The overall clinical pregnancy rates when using flare protocols in POR ranged between 12% and 26.3%. Furthermore, it is suggested that the agonist-antagonist protocol is at par with the microdose protocol. However, some reports on microdose flare protocol have demonstrated conflicting results in cycle cancellation rate (CCR) and clinical pregnancy rates despite better cycle outcomes. A study was conducted to compare the microdose flare-up protocol to the ultrashort gonadotropin releasing hormone agonist flare combined with the fixed multidose GnRH antagonist protocol in 120 poor responders undergoing ovarian stimulation. There were no significant differences between groups in the number of used ampoules, duration of stimulation, number of retrieved oocytes, fertilization rate, and number of transferred embryos. Although not statistically significant, an inclination toward higher clinical pregnancy rate in the ultrashort agonist/antagonist protocol 49 was observed (Table 8). Table 8: Microdose flare-up regimen versus ultrashort GnRH 49 agonist combined with fixed GnRH antagonist GnRH Antagonist p value GnRH agonist/ antagonist Microdose flare-up No. of used gonadotropin ampoules ± ±6.93 Duration of stimulation (Days) ± ±1.61 No. of retrieved oocytes ± ±3.63 No. of transferred embryos ± ±2.41 Fertilization rate (%) (Per cycle) ±27 58±30 Clinical pregnancy rate (%) (Per cycle) % % GnRH-antagonists have been shown to have a lower probability of clinical pregnancy per treatment cycle compared with GnRH-agonists. Although GnRH-antagonists have several theoretical advantages over the GnRH- 50 agonists, evidence in multiple studies has been inconclusive. A meta-analysis concluded that GnRH-antagonist resulted in a statistically significantly lower duration of stimulation compared with GnRH-agonist, but there was no significant difference in the number of total oocytes retrieved or mature oocytes retrieved. Moreover, no significant difference 50 was found in the CCR or clinical pregnancy rate. Another study was conducted to compare the results of fixed versus flexible GnRH-antagonist protocols in 0 patients with PCOS. The study suggested that using flexible antagonist protocol favors better outcomes in terms of number of good-quality oocytes and embryo and cryopreservation. However, flexible protocol showed a pregnancy rate of 66.7% and fixed protocol showed a pregnancy rate of 0% 51 (p=0.013) (Table 9). Table 9: Comparison of COS outcomes in fixed/flexible groups Variable Group 1 Flexible (n=50) Group 2 Fixed (n=50) Days of ovulation induction (n) 9.6±2.3.5± Gonadotropin injections (n) 19.2± ± Cetrotide injections (n) 3.8± ± Oocytes (n) 14.75± ±3.3 <0.01 Oocytes in metaphase II (n) 11.3± ±2.5 <0.01 Germinal vesicle 1.8± ±1.2 NS Atretic 0.5± ±0.4 NS 2PN Fertilized oocyte 8.4± ±2.3 <0.01 Transferred embryo (n) 3± ±1.1 NS Frozen embryo (n) 9.2± ±2.4 <0.01 Embryo grading A 31 (77.5) 34 (73.9) NS B 8 (20.0) (21.7) NS C 1 (2.5) 2 (4.3) NS Data are presented as Mean ± SD, and Number (%) Endocrine consequences of GT - hcg include: Abnormal FSH/LH ratio Blockade of LH surge Hyperprolactinemia Premature luteinization Short luteal phase Follicular atresia/dyssynchrony Heterogenicity of estradiol response Natural Cycle IVF 51 p value Natural cycle IVF has regained popularity owing to the increased physical and psychological burden, high cost, and a risk of OHSS associated with conventional stimulated IVF. In addition, repeated natural cycles can be carried out in successive months. However, it is associated with a high risk of cancellation due to abnormal follicular maturation, premature ovulation of the single follicle, failed oocyte retrieval, or empty follicle at retrieval and 52 failed fertilization. A retrospective cohort study was conducted to determine if a modified natural cycle in IVF is an acceptable treatment option for an infertile couple. It demonstrated that the efficacy of a modified natural cycle was at par with conventional stimulated IVF and concluded that it is an acceptable treatment option for patients considering IVF, particularly for women 35 years old and for women 36 years old with a normal 52 ovarian response. Pretreatment GnRH-antagonist protocols begin stimulation on day 2 or 3, which makes it difficult to plan stimulation and laboratory activities. In such cases, pretreatment can play a vital role. Oral contraceptive pills (OCPs) and 11