REstradiol and Antagonist Pretreatment Prior to Microdose Leuprolide in in Vitro Fertilization

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1 REstradiol and Antagonist Pretreatment Prior to Microdose Leuprolide in in Vitro Fertilization Does It Improve IVF Outcomes in Poor Responders as Compared to Oral Contraceptive Pill? FTThe Journal of Reproductive Medicine Alyaa Elassar, M.D., M.Sc., John Nulsen, M.D., Lawrence Engmann, M.D., and Claudio Benadiva, M.D., H.C.L.D. OBJECTIVE: To compare in vitro fertilization (IVF) outcomes in low responders stimulated with microdose leuprolide protocol (ML) following pretreatment with either oral contraceptive pill (OCP) or luteal estradiol (E 2 ) + GnRH antagonist (E 2 + antag) for follicular synchronization prior to controlled Dyielded superior IVF outcomes ovarian hyperstimulation (COH). STUDY DESIGN: This was a retrospective study of 130 women, who were poor responders, undergoing IVF with either OCP/ML or E 2 + antag/ml protocols. The main outcome measures were ongoing pregnancy rates, number of oocytes retrieved, and cancellation rate. RESULTS: Both groups were similar in baseline characteristics. There were no significant differences in gonadotropin requirement, cancellation rate, and number of embryos transferred. Ongoing pregnancy rates (40% vs. 15%) were significantly higher in the OCP/ML group. Trends toward greater number of oocytes retrieved (7.7± 3.4 vs. 5.9±4.2) and improved implantation rates (20% vs. 12%) were also noted, but these did not reach statistical significance. AOur findings do not indicate that E 2 +antag pretreatment CONCLUSION: E 2 +antag pretreatment does not appear to improve IVF outcomes in ML protocol when compared to the standard OCP in poor responders. Randomized trials with adequate power to study the optimal method of steroid pretreatments appear justified. (J Reprod Med 2015;60: ) Keywords: controlled ovarian hyperstimulation, estradiol, GnRH antagonist, leuprolide, lupron, luteal estradiol, luteal phase, oral contraceptives, ovarian stimulation, reproductive techniques, reproductive technologies. Despite recent advances in assisted reproductive technology (ART), management of poor responder patients remains controversial. 1 Poor response to controlled ovarian hyperstimulation (COH) generally implies a failure to achieve a certain number of oocytes or particular serum estradiol (E 2 ) level following adequate gonadotropin stimulation. This may result in high cycle cancellation and low pregnancy rates. Several stimulation protocols have From the Center for Advanced Reproductive Services, University of Connecticut Health Center, and the Department of Obstetrics and Gynecology, University of Connecticut, Farmington; and New York City IVF, Manhattan, New York. Address correspondence to: Alyaa Elassar, M.D., 400 East 56th Street, Suite #1, New York, NY (alyaa.elassar@gmail.com), and reprint requests to: Claudio Benadiva, M.D., Center for Advanced Reproductive Services, Dowling South Building, 263 Farmington Avenue, Farmington, CT (benadiva@up.uchc.edu). Financial Disclosure: The author has no connection to any companies or products mentioned in this article /15/ /$18.00/0 Journal of Reproductive Medicine, Inc. The Journal of Reproductive Medicine 1

2 TR2 The Journal of Reproductive Medicine DAFbeen employed to improve IVF outcomes in poor responders. These protocols aim at enhancing follicular recruitment with subsequent increase in the number of retrieved oocytes, mature oocytes, and embryos. However, no specific intervention can claim a consistent increase in ongoing pregnancy rates. Thus, the ideal COH protocol has yet to be defined. Various studies have supported the use of a microdose GnRH agonist (GnRH-a) flare protocol (ML) in this patient group, which demonstrated augmented ovarian response and clinical outcomes. 2,3 This approach retains the advantage of standard flare protocols, primarily by utilizing the initial rise of endogenous gonadotropins induced by GnRH-a in the early follicular phase. Additionally, the low dose minimizes the suppressive effect of leuprolide downregulation on gonadotropin secretion. Oral contraceptive pill (OCP) pretreatment is widely used in IVF cycles, including microdose flare protocol. It attenuates corpus luteum rescue and an early follicular phase progesterone rise, which may have detrimental effects on the endometrium and oocyte quality. 3 It may be beneficial to synchronize follicular development and prevent premature luteinizing hormone (LH) surge. 4 Recently there has been some concern regarding the effects of OCP pretreatment on follicular growth, hormonal profiles, and cycle outcomes. 5,6 Two large randomized studies reported that OCP pretreatment does not actually improve oocyte yield and may even exert a deleterious effect on cycle outcome. 5,7 The potential benefit of luteal phase estradiol and GnRH antagonist pretreatment has been assessed recently in GnRH antagonist cycles. 8,9 This approach allows synchronization of early antral follicle growth in the luteal phase prior to COH with subsequent increase in oocyte yield and possible improvement in pregnancy rates. 8 This approach seemed to be promising because endogenous estrogen secretion is actually the main factor involved in the negative regulation of follicle-stimulating hormone (FSH) secretion during the luteal-follicular transition. Estrogen suppresses luteal FSH rise and may also increase sensitivity to gonadotropins, while the GnRH antagonist induces luteolysis and prevents FSH rise. 10,11 Luteal antagonist administration is associated with lower basal FSH and inhibin levels and a reduction in the antral follicle heterogeneity. 12 The aim of this study was to compare the efficacy of OCP versus luteal estradiol plus a GnRH antagonist for luteal phase follicular synchronization in poor responder patients undergoing microdose flare COH protocol prior to IVF. Materials and Methods Patients This was a retrospective study of 130 poor responder patients undergoing IVF between January 2010 and May The Institutional Review Board at the University of Connecticut approved the study. Patients were included if they had a prior ovarian stimulation cycle at a starting dose of gonadotropins of 300 IU or more, yielding <5 oocytes or a prior canceled cycle due to low follicular recruitment. Low follicular recruitment was defined as 3 follicles 15 mm in diameter, following 10 days of stimulation. Patients underwent COH using ML protocol, preceded by luteal synchronization utilizing either OCP or transdermal E 2 + GnRH antagonist. Each patient contributed a single cycle to this study, so there was no crossover between groups. Assignment of patients to either protocol was made at their physician s discretion. Stimulation Protocols Luteal E 2 Patch + GnRH Antagonist Microdose Group (N=40). A transdermal E 2 (Vivelle Dot 0.1 mg, Novartis, Miami, Florida) was initiated every other day starting on day 7 after the LH surge. On the second day after the application of the E 2 patch, patients began daily subcutaneous injections of 0.25 mg ganirelix acetate (Ganirelix, Organon Pharmaceuticals, Roseland, New Jersey) for 3 consecutive days. Patients presented on day 2 of their ensuing menses for measurement of FSH, LH, and E 2 levels and a baseline ultrasound (for antral follicle count and endometrial thickness). The last E 2 patch was removed and patients started subcutaneous injections of 40 µg leuprolide acetate twice daily. On cycle day 4 ovarian stimulation was started using a high dose of gonadotropins (average of IU), either recombinant FSH (Follistim [Organon USA] or Gonal-F [Serono, Randolph, Massachusetts]), hmg (Menopur [Ferring Pharmaceuticals, Parsippany, New Jersey]), or a combination of both, employing a step-down protocol. Both LA and gonadotropins were continued until the day of human chorionic gonadotropin (hcg) administration. OCP Microdose Group (N=90). Patients in this group received OCP (Desogen [Organon USA]) for 21 days starting on the third day of the preceding menstrual

3 Rcycle. On the third day after discontinuing the OCP (designated cycle day 2), baseline evaluation including FSH, LH, E 2, and ultrasound examination was performed and leuprolide acetate 40 µg subcutaneously twice daily was commenced and continued until the day of hcg administration. Gonadotropin stimulation, IU of FSH and/or hmg began on the third day of leuprolide acetate administration (cycle day 4), and both were continued until the day of hcg injection. In both groups, hcg was administered when at least 3 lead follicles had attained or exceeded 17 mm mean diameter by transvaginal ultrasound. Oocytes were harvested by transvaginal ultrasound guided follicular puncture 35 hours after hcg administration. Conventional oocyte insemination or intracytoplasmic sperm injection (ICSI) was performed as indicated. All embryos were transferred on day 3. Luteal support was achieved in both groups by administering 50 mg IM progesterone daily starting the evening after oocyte retrieval and continuing until a negative pregnancy test or confirmed clinical pregnancy (presence of an intrauterine gestational sac with fetal cardiac activity). Primary outcome measure was the live birth rate per started cycle. Secondary outcome measures were cancellation rate, number of oocytes retrieved and transferable embryos, implantation, and clinical pregnancy rates. Statistical Analysis AFStatistical analysis was performed using the Statistical Package for the Social Sciences v.17.0 (SPSS Inc., Chicago, Illinois). Student s t test was used for comparison of continuous variables, while χ 2 or Fisher s exact test were used for categorical variables. P<0.05 was considered statistically significant. Data are presented as the mean ± standard deviation (SD). Results The baseline characteristics of the patients enrolled in this study are shown in Table I. There were no significant differences between the two groups in the mean age, body mass index (BMI), or random day 3 FSH and E 2 levels. Mean number of prior attempted cycles was 2.3±1.31 in the E 2 /microdose group versus 2.1±1.2 in the OCP/microdose group; p=0.4. Table II compares cycle characteristics and outcomes between the two groups. As expected, cycle day 2 E 2 levels were significantly higher (85.5±43.8 vs. 32.8±13.5 pg/ml) in the E 2 / microdose group, while FSH (4.6±2.5 vs. 4.1±3.5 miu/ml) and LH levels (2.9±2.4 vs. 2.2 ± 2.1 miu/ ml) were similarly suppressed in the E 2 /microdose and OCP/microdose groups, respectively. The two groups were also comparable in duration of ovarian stimulation, total dose of gonadotropins, and peak E 2 levels. The cancellation rate was higher in the E 2 /microdose group (50% vs. 40%), although this was not found to be significant. There was a trend toward increased number of retrieved oocytes (7.7±3.4 vs. 5.9±4.2) and number of mature oocytes (6.0±3.23 vs. 4.4±3.6) in the OCP/microdose group; however, the number of 2 pronuclei oocytes (2PN), number of embryos transferred, and implantation rate were similar between groups. The ongoing pregnancy rate per started cycle was significantly higher in the OCP/microdose group (24.4% [22/90] vs. 7.5% [3/40]; p<0.05). Discussion Poor response to COH connotes a quantitative re- Volume 60, Number 0-0/Month-Month TTable I Demographic and Clinical Characteristics Characteristic E 2 /antag/ml (n=40) OCP/ML (n=90) p Value Age (yrs) 39.8± ± BMI (kg/m 2 ) 25.7± ± RD3 FSH (miu/ml) 8.9± ± RD3 LH (miu/ml) 4.3± ± RD3 E 2 (pg/ml) 48.6± ± No. of prior IVF cycles 2.3± ± Baseline FSH (miu/ml) 4.6± ± Baseline LH (miu/ml) 2.9± ± Baseline E 2 (pg/ml) 85.5± ±13.5 <0.01 Values are expressed as mean ± standard deviation or rate (%). RD3 = random day 3, BMI = body mass index, E 2 = estradiol, antag = gonadotropin-releasing hormone antagonist, OCP = oral contraceptive pill, ML = microdose leuprolide, FSH = follicle-stimulating hormone, LH = luteinizing hormone.d

4 R4 The Journal of Reproductive Medicine Table II COH Response E 2 /antag/ml (n=40) OCP/ML (n=90) p Value Stimulation days 9.9± ± Total gonadotropins (IU) 6,161±1,459 5,727±1, Peak E 2 (pg/ml) 1,353±992 1,553± Cancellation rate (%) 50 (20/40) 40 (36/90) 0.29 Values are expressed as mean ± standard deviation or rate (%). E 2 = estradiol, antag = gonadotropin-releasing hormone antagonist, OCP = oral contraceptive pill, ML = microdose leuprolide. Tduction of follicular response and thus a lower number of oocytes retrieved. It represents a considerable portion of the infertility population, with a prevalence ranging from 9 24%. 13 Due to the variable sensitivity of follicles to FSH, the FSH rise during the late luteal phase may lead to advanced growth of the few, more sensitive follicles 14 and ultimately result in a smaller follicular cohort available for recruitment, and hence a lower number of retrieved oocytes. This incoordinate growth is particularly evident in patients with diminished ovarian reserve who already have elevation in their baseline FSH levels. Luteal synchronization of early antral follicle growth using different regimens to increase oocyte yield has been proposed by many investigators. 15,16 In this study we investigated whether E 2 +antagonist pretreatment improves IVF outcomes over the standard OCP pretreatment in low responders receiving microdose-flare protocol. We found that the number of oocytes retrieved in the OCP group was not statistically significant but may be clinically significant (approximately +2 oocytes). Ongoing pregnancy rates were significantly higher in the OCP group. There was a trend towards increased AFimplantation and lower cancellation rates in the OCP group, but these did not reach statistical significance. After the initial reports of the use of microdose regimens for COH, 17 several studies adapting this protocol for poor responders appeared in the literature. 2,3 Pretreatment with a low-dose OCP is still the most commonly employed. The benefit of OCP pretreatment in flare protocols have been challenged in multiple studies 18,19 ; either no differences in IVF outcomes 18 or higher cancellation and lower clinical pregnancies were observed. 19 Luteal administration of either estradiol or GnRH antagonist prior to an antagonist cycle was initially proposed by deziegler et al. 15 Their series of studies noted an attenuation of follicular size discrepancies on day 2 of the pretreated cycle, translating into improved outcomes. These initial observations prompted several investigators 8,20,21,23 to use this protocol in low responders, with yield of variable results. Dragisic et al, 8 in a crossover study, reported that the use of luteal E 2 +antagonist yielded a lower cancellation rate and a higher number of retrieved oocytes and embryos transferred. Table III IVF Outcomes Outcome E 2 /antag/ml (n=40) OCP/ML (n=90) p Value No. of oocytes retrieved 5.9± ± No. of mature oocytes 4.4± ± No. of 2PN 3.6± ± Fertilization rate No. of embryos transferred 2.7± ± Implantation rate (%) 11.3 (6/53) 17.3 (26/150) 0.3 Clinical PR/started cycle (%) 17.5 (7/40) 28.9 (26/90) 0.17 Clinical PR/ET (%) 35.0 (7/20) 48.2 (26/54) 0.31 Ongoing PR/started cycle (%) 7.5 (3/40) 24.4 (22/90) 0.02 Ongoing PR/ET (%) 15.0 (3/20) 40.7 (22/54) 0.04 Pregnancy loss rate (%) 57.1 (4/7) 15.4 (4/26) 0.04 Values are expressed as mean ± standard deviation or rate (%). 2PN = 2 pronuclei, ET = embryo transfer, PR = pregnancy rate, E 2 = estradiol, antag = gonadotropin-releasing hormone antagonist, OCP = oral contraceptive pill, ML = microdose leuprolide.d

5 TRVolume 60, Number 0-0/Month-Month DOur group, in a series of studies, 20,21 compared low-responder patients who underwent luteal E 2 + antag before an antagonist protocol to those who were stimulated with OCP/microdose GnRH agonist. The number of oocytes, available embryos, and cancellation and pregnancy rates were comparable in both groups. Shastri et al 22 reproduced similar results in a cohort group of young poor responders. There was no difference in oocyte number; however, patients with luteal E 2 +antagonist pretreatment had a greater number and better quality of embryos transferred, with a trend toward improved implantation and ongoing pregnancy rates. Their findings suggest that luteal E 2 +antag may be a viable treatment option for poor responders. More recently, Cédrin-Durnerin et al 23 challenged the effect of adding luteal estradiol to an antagonistgonadotropin regimen in 472 patients. Women allocated to estrogen pretreatment required higher FSH doses and longer duration of stimulation, with no significant improvement in any of the IVF outcomes. Of note, the inclusion of solely normoresponders and in a setting of antagonist protocol hinders the applicability to the general population of true poor responders. To our knowledge the current study is the first to investigate the use of luteal E 2 patch + GnRH antagonist in microdose protocol in poor responder patients. Our results did not show improvement in IVF cycle outcomes when E 2 +antag was compared to the standard OCP for follicular coordination before start of microdose lupron. Patients in the OCP/ microdose group showed a trend towards greater number of retrieved and mature oocytes and a significantly higher ongoing pregnancy rate. The mean total dose of gonadotropins and the cancellation rate were higher in the E 2 +antag pretreatment group; this did not reach statistical significance. Patients in both groups demonstrated similar levels of FSH and LH suppression on cycle day 2, thus a higher level of gonadotroph suppression cannot explain the superior results achieved with OCP pretreatment. There is indirect evidence that OCP pretreatment achieved better follicular cohort synchronization as reflected by more retrieved oocytes, and subsequently improved pregnancy rates. Our findings were in agreement with those of Cédrin-Durnerin et al. 24 The study assessed the effects of 3 different steroid pretreatments on follicular growth and on hormonal profiles and favored OCP and progestogen pretreatment in antagonist protocol. OCP and progestogen were found to be AFmore suppressive than natural estrogen pretreatment. Their data suggest that the stronger the negative FSH feedback exerted by the steroid, the more effective the homogenization of the cohort. That study, however, excluded poor responders and patients with low ovarian reserve, and the stronger homogenization achieved by OCP was not reflected into a higher oocyte yield or improved pregnancy rate. Interestingly, those findings were refuted by a recent Cochrane review 25 that revealed that OCP pretreatment resulted in fewer clinical pregnancies, while estrogen-pretreated cycles yielded more retrieved oocytes; both required a higher amount of gonadotropins therapy than controls in antagonist cycles. Additionally, there has been some concern regarding the profound suppression of LH activity and possibly increased pregnancy loss after OCP-pretreated IVF cycles. 5,6 This observation was noted in GnRH antagonist protocols and was subsequently challenged by a recent large metaanalysis. 26 In the current study OCP was not associated with increased pregnancy loss; in fact, the miscarriage rate was found to be significantly lower than that in the E 2 +antag pretreated group. One limitation of this study was enrollment of patients into treatment groups according to physician preference. The 2 groups, however, were similar in age, BMI, and ovarian reserve. All patients included in the study were allocated to one treatment protocol: their first stimulation cycle at our center with either protocol. Thus, no patient crossover occurred between groups. Our findings in this study do not indicate that E 2 +antag pretreatment yielded superior IVF outcomes as compared to OCP pretreatment in poor responder patients undergoing microdose/ flare protocol. Due to the retrospective nature of our study, these findings should be validated by prospective randomized trials with adequate power to study the optimal method of steroid pretreatments and verify these findings. References 1. Kligman I, Z Rosenwaks: Differentiating clinical profiles: Predicting good responders, poor responders, and hyperresponders. Fertil Steril 2001;76: Scott RT, Navot D: Enhancement of ovarian responsiveness with microdoses of gonadotropin-releasing hormone agonist during ovulation induction for in vitro fertilization. Fertil Steril 1994;61: Schoolcraft WB, Schlenker T, Gee M, et al: Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose follicle stimulating

6 TR6 The Journal of Reproductive Medicine Dhormone flare, growth hormone protocol. Fertil Steril 1997; 67: Gonan Y, Jacobson W, Casper RF: Gonadotropin suppression with oral contraceptives before in vitro fertilization. Fertil Steril 1990;53: Kolibianakis EM, Papanikolaou EG, Camus M, et al: Oral contraceptive pill pretreatment on ongoing pregnancy rates in patients stimulated with GnRH antagonists and recombinant FSH for IVF. A randomized controlled trial. Hum Reprod 2006;21: Meldrum DR, Scott RT Jr, Levy MJ, et al: Oral contraceptive pretreatment in women undergoing controlled ovarian stimulation in ganirelix acetate cycles may, for a subset of patients, be associated with low serum luteinizing hormone levels, reduced ovarian response to gonadotropins, and early pregnancy loss. Fertil Steril 2009;91: Rombauts L, Healy D, Norman RJ: A comparative randomized trial to assess the impact of oral contraceptive pretreatment on follicular growth and hormone profiles in GnRH antagonist-treated patients. Hum Reprod 2006;21: Dragisic KG, Davis OK, Fasouliotis SJ, et al: Use of a luteal estradiol patch and a gonadotropin-releasing hormone antagonist suppression protocol before gonadotropin stimulation for in vitro fertilization in poor responders. Fertil Steril 2005;84: Elassar A, Mann JS, Engmann L, et al: Luteal phase estradiol and antagonist protocol for controlled ovarian stimulation before in vitro fertilization in poor responders. Fertil Steril 2011;95: Fanchin R, Salomon L, Castelo-Branco A, et al: Luteal estradiol pre-treatment coordinates follicular growth during controlled ovarian hyperstimulation with GnRH antagonists. 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Semin Reprod Med 2005;23: de Ziegler D, Jääskeläinen AS, Brioschi PA, et al: Synchronization of endogenous and exogenous FSH stimuli in controlled ovarian hyperstimulation (COH). Hum Reprod 1998; AF13: Lindheim SR, Barad DH, Witt B, et al: Short-term gonadotropin suppression with oral contraceptives benefits poor responders prior to controlled ovarian hyperstimulation. J Assist Reprod Genet 1996;13: Brzyski RG, Muasher SJ, Droesch K, et al: Follicular atre sia associated with concurrent initiation of gonadotropinreleasing hormone agonist and follicle-stimulating hormone for oocyte recruitment. Fertil Steril 1988;50: al-mizyen E, Sabatini L, Lower AM, et al: Does pretreatment with progestogen or oral contraceptive pills in low responders followed by the GnRHa flare protocol improve the outcome of IVF-ET? 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Fertil Steril 2009;92: Shastri SM, Barbieri E, Kligman I, et al: Stimulation of the young poor responder: Comparison of the luteal estradiol/ gonadotropin-releasing hormone antagonist priming protocol versus oral contraceptive microdose leuprolide. Fertil Steril 2011;95: Cédrin-Durnerin I, Guivarc h-levêque A, Hugues JN: Pretreatment with estrogen does not affect IVF-ICSI cycle outcome compared with no pretreatment in GnRH antagonist protocol: A prospective randomized trial. Fertil Steril 2012; 97: Cédrin-Durnerin I, Bständig B, Parneix I, et al: Effects of oral contraceptive, synthetic progestogen or natural estrogen pre-treatments on the hormonal profile and the antral follicle cohort before GnRH antagonist protocol. Hum Reprod 2007; 22: Smulders B, van Oirschot SM, Farquhar C, et al: Oral contraceptive pill, progestogen or estrogen pre-treatment for ovarian stimulation protocols for women undergoing assisted reproductive techniques. Cochrane Database Syst Rev 2010 Jan 20;(1):CD Griesinger G, Shapiro DB, Kolibianakis EM, et al: No association between endogenous LH and pregnancy in a GnRH antagonist protocol: Part II, recombinant FSH. Reprod Biomed Online 2011;23: