Growth Hormone Supplementation in the Luteal Phase Before Microdose GnRH Agonist Flare Protocol for In Vitro Fertilization

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1 GYNAECOLOGY Growth Hormone Supplementation in the Luteal Phase Before Microdose GnRH Agonist Flare Protocol for In Vitro Fertilization Caitlin Dunne, MD, 1 Ken Seethram, MD, 1,2 Jeffrey Roberts, MD 1,2 1 Division of Reproductive Endocrinology and Infertility, University of British Columbia, Vancouver BC 2 Pacific Centre for Reproductive Medicine, Vancouver BC Abstract Objective: Growth hormone (GH) acts in both early and late follicular development to stimulate the proliferation and differentiation of granulosa cells and to increase the production of estradiol in animal and human ovaries. Investigators have therefore explored GH supplementation to improve outcomes in women undergoing in vitro fertilization, with the greatest interest in women with diminished ovarian reserve. Recent meta-analyses indicate that GH supplementation can be beneficial for poor responders undergoing IVF. In most studies, GH has been given concomitantly with gonadotropins during the follicular phase; this may not be optimal, since follicular recruitment begins during the preceding luteal phase. We therefore wished to examine the effect of GH supplementation in the luteal phase before controlled ovarian stimulation (COH) with a microdose GnRH agonist flare (MDF) protocol in women undergoing in vitro fertilization. Methods: We performed a retrospective matched case control study of patients undergoing treatment at a private IVF facility between June 2012 and July Patients identified as poor responders to COH were offered adjuvant GH treatment as part of their ovarian stimulation regimen. The patients in the experimental group chose to take GH, 3.33 mg daily by subcutaneous injection for 14 days, before starting COH. All patients had an MDF stimulation protocol using 450 IU of follicle stimulating hormone (FSH) daily. Results: A total of 42 women were included in the study. There were 14 women in the experimental group (GH) and 28 controls (C) matched for age, BMI, and day 3 FSH level. There was no difference between the groups in clinical pregnancy rate (GH = 29%, C = 32%, P = 0.99), number of mature oocytes retrieved (GH = 2.5, C = 5.0, P = 0.13), cycle cancellation rate (GH = 21%, C = 14%, P = 0.88), duration of COH (GH = 10.1, C = 10.1, P = 0.93), or mean peak estradiol level (GH = 4174 pmol/l, C = 5105 pmol/l, P = 0.44). Key Words: Growth hormone, in vitro fertilization, microdose GnRH agonist flare, poor responder Competing Interests: None declared. Received on January 13, 2015 Accepted on March 16, 2015 Conclusion: The administration of growth hormone during the luteal phase before a microdose GnRH agonist flare protocol for in vitro fertilization did not improve outcomes in poor responder patients. Résumé Objectif : L hormone de croissance (GH) agit pendant le développement folliculaire tant précoce que tardif pour stimuler la prolifération et la différenciation des cellules de la granulosa, ainsi que pour accroître la production d estradiol par les ovaires chez l animal et l homme. Les chercheurs se sont donc penchés sur le recours à la supplémentation en GH pour améliorer les issues chez les femmes qui font appel à la fécondation in vitro, tout en portant une attention particulière aux femmes qui présentent une réserve ovarienne amoindrie. De récentes méta-analyses indiquent que la supplémentation en GH peut être bénéfique pour les femmes qui réagissent mal à la FIV. Dans la plupart des études, on administre de la GH de façon concomitante avec des gonadotrophines pendant la phase folliculaire; cette façon de faire pourrait ne pas être optimale, puisque le recrutement folliculaire débute au cours de la phase lutéale qui précède. Nous avons donc souhaité examiner l effet de la supplémentation en GH pendant la phase lutéale, avant la tenue d une stimulation ovarienne contrôlée (SOC) au moyen d un «protocole de poussée» faisant appel à une microdose d agoniste de la GnRH (MDF), chez des femmes qui font l objet d une fécondation in vitro. Méthodes : Nous avons mené une étude cas-témoins appariés rétrospective se penchant sur des patientes qui ont fait l objet d un traitement au sein d un établissement privé de FIV entre juin 2012 et juillet Les patientes identifiées comme réagissant mal à la SOC se sont vu offrir un traitement adjuvant à la GH dans le cadre de leur schéma thérapeutique de stimulation ovarienne. Les patientes du groupe expérimental ont choisi de recevoir de la GH, à raison de 3,33 mg par jour sous forme d injection sous-cutanée pendant 14 jours, avant le début de la SOC. Toutes les patientes ont fait l objet d un protocole de stimulation MDF faisant appel à 450 UI d hormone folliculostimulante (FSH) par jour. Résultats : Au total, 42 femmes ont participé à l étude. Le groupe expérimental (GH) comptait 14 femmes et le groupe témoin (C) comptait 28 femmes appariées en fonction de l âge, de l IMC et du taux de FSH au jour 3. Aucune différence n a été constatée entre les groupes en matière de taux de grossesse clinique (GH = 29 %, C = 32 %, P = 0,99), de nombre d ovocytes matures récupérés 810 SEPTEMBER JOGC SEPTEMBRE 2015

2 Growth Hormone Supplementation in the Luteal Phase Before Microdose GnRH Agonist Flare Protocol for In Vitro Fertilization (GH = 2,5, C = 5,0, P = 0,13), de taux d annulation de cycle (GH = 21 %, C = 14 %, P = 0,88), de durée de la SOC (GH = 10,1, C = 10,1, P = 0,93) ou de niveau moyen du pic d estradiol (GH = pmol/l, C = pmol/l, P = 0,44). Conclusion : L administration d hormone de croissance pendant la phase lutéale, avant la mise en œuvre d un «protocole de poussée» faisant appel à une microdose d agoniste de la GnRH aux fins de la fécondation in vitro, n a pas permis d améliorer les issues obtenues par les patientes «réagissant mal» à la SOC. J Obstet Gynaecol Can 2015;37(9): INTRODUCTION In women undergoing in vitro fertilization, controlled ovarian stimulation is used to promote a multi-follicular response and thereby maximize each woman s odds of generating at least one developmentally competent oocyte. Growth hormone, a pituitary peptide, stimulates proliferation and differentiation of granulosa cells, 1,2 and increases the production of estradiol in animal and human ovaries. 1,3 5 It is believed to activate granulosa cells directly by binding to the growth hormone receptor, and indirectly by stimulating the production of insulin-like growth factor I in ovarian or other tissues. 6 8 Both human and animal studies have suggested a role for GH in folliculogenesis. In rhesus macaque ovaries, GHR is expressed in the cumulus-oocyte-complex; administration of recombinant human GH increases cumulus expansion and development of embryos to the nine- to 16-cell stage, consistent with a stimulating effect of GH on oocyte maturation. 9,10 In humans, GHR mrna and protein are highly expressed in granulosa cells, ovarian stromal cells, and oocytes. 11,12 GHR expression in the corpus luteum may indicate a role for GH in the luteal phase. 13,14 Intrafollicular GH levels have been demonstrated to vary between human follicles, and higher GH concentrations have been associated with normal embryo morphology and vigorous cell growth during assisted reproduction procedures. 15 Together, these findings suggest that GH participates in follicular growth and development of oocytes and early embryos through several distinct cell types and at multiple steps. Investigators have therefore explored the potential for GH supplementation to improve outcomes in women ABBREVIATIONS COH controlled ovarian stimulation FSH follicle stimulating hormone GH growth hormone GHR growth hormone receptor POR poor ovarian response undergoing IVF, most commonly in those with a history of poor response to ovarian stimulation ( poor responders ). 2 In this study we assessed the effects of treatment with growth hormone in the luteal phase before COH with a microdose GnRH agonist flare (MDF) protocol in women undergoing in vitro fertilization. METHODS We performed a retrospective, matched case control study of patients undergoing in vitro fertilization at a private IVF facility between June 2012 and July Patients identified as poor responders to COH were offered adjuvant GH treatment as part of their ovarian stimulation regimen. All women who received GH (the experimental group) met the Bologna classification for poor ovarian response, having had two prior episodes of POR or at least two of the following: advanced maternal age or risk factors for POR, previous poor response to COH, or abnormal ovarian reserve testing. 5 The experimental group comprised women who elected to have GH treatment before COH; they received daily subcutaneous injections of 3.33 mg of GH (Saizen; EMD Serono, Mississauga, ON) for 14 days before beginning injections of FSH (Figure 1). The control group subjects were women undergoing contemporaneous IVF cycles who were not receiving GH; they were matched for age, day 3 FSH levels, and BMI. All patients had controlled ovarian stimulation using a microdose GnRH agonist flare protocol; this involved days of a combined oral contraceptive preparation, followed three days later by twice daily subcutaneous injections of 40 μg of leuprolide acetate (Lupron; Abbvie Corp., Saint-Laurent, QC) and daily subcutaneous injections of 450 IU of recombinant FSH (r-fsh) beginning on the third day of withdrawal bleeding. All subjects had undergone routine screening for thyroid dysfunction, glucose intolerance (if indicated), uterine abnormalities, and respiratory or cardiovascular dysfunction before beginning in vitro fertilization treatment. Ovarian follicular growth was monitored by serial transvaginal ultrasound and serum estradiol measurement. When two or more follicles reached a mean of 17 mm in diameter, IU of human chorionic gonadotropic was administered subcutaneously to trigger final oocyte maturation. Transvaginal oocyte retrieval was performed 36 hours after human chorionic gonadotropic administration, and standard fertilization in vitro or intracytoplasmic sperm injection was performed as appropriate. Luteal phase support was given in the form of micronized vaginal progesterone. Clinical pregnancy was SEPTEMBER JOGC SEPTEMBRE

3 GYNAECOLOGY Figure 1. Controlled ovarian stimulation protocol OCP start OCP stop FSH 450 IU hcg Oocyte retrieval Embryo transfer GnRH agonist start 40 μg BID 21 d 3 d 2 d 36 hr 3 5 d GH x 14 days, stop All subjects used a microdose GnRH agonist flare protocal. The experimental group also used growth hormone (3.33 mg daily) for 14 days prior to the start of FSH. defined as the visualization of an intrauterine gestational sac on transvaginal ultrasound at seven weeks gestational age. Demographic data including age, BMI, and day 3 FSH levels were compared between the two groups using Welch s t tests. Clinical pregnancy rates were compared using a chisquare test of two proportions with a continuity correction. All secondary outcome binary variables were analyzed using chi-square tests of two-proportions with continuity corrections. Count data were compared using Poisson regression and continuous variables were compared with Welch s t tests. Statistical analyses were carried out using SPSS (IBM Corp., Armonk, NY). Ethics approval for the study was provided by the University of British Columbia s Clinical Research Ethics Board. RESULTS Baseline characteristics were not significantly different between the two groups (Table 1). The mean age of study subjects was 39.5 years in the experimental (GH) group and 40 years in the control (C) group, and there was no difference between the groups in mean BMI (GH = 24.1 kg/m 2, C = 25.9 kg/m 2, P = 0.3), day 3 FSH level (GH = 10.0 IU/L, C = 8.2 IU/L, P = 0.07), or use of ICSI (GH = 43%, C = 36%, P = 0.91). None of the patients experienced adverse effects attributable to the use of GH, and none discontinued GH treatment prematurely. The addition of GH in the luteal phase before COH was not associated with any improvement in IVF cycle outcomes (Table 2). The clinical pregnancy rate was similar between groups (GH = 29%, C = 32%, P = 0.99) (Figure 2), as were the mean number of mature oocytes retrieved (GH = 2.5, C = 5.0, P = 0.13) and mean number of embryos available for cryopreservation (GH = 0, C = 0). Mean peak estradiol level (GH = 4174, C = 5105 pmol/l, P = 0.44), mean number of follicles 14 mm (GH = 3.5, C = 4, P = 0.94) and mean number of days of COH (GH = 10.1, C = 10.1, P = 0.93) were also not significantly different between groups. Cycle cancellation rates were similar at 21% in the experimental group and 14% in the controls (P = 0.88). DISCUSSION This case control study did not identify any benefit from administration of growth hormone in the luteal phase before an MDF protocol for IVF treatment in women classified as poor responders. To our knowledge, no other studies have examined the use of luteal phase GH supplementation exclusively with an MDF protocol. Kucuk et al. conducted a randomized placebo-controlled trial of luteal phase GH supplementation in conjunction with a long luteal GnRH agonist protocol, but did not find a statistically significant increase in the clinical pregnancy rate with GH supplementation. 9 They did, however, report a significant 812 SEPTEMBER JOGC SEPTEMBRE 2015

4 Growth Hormone Supplementation in the Luteal Phase Before Microdose GnRH Agonist Flare Protocol for In Vitro Fertilization Table 1. Baseline characteristics Experimental Control Test result Variable n = 14 n = 28 P Age, years 39.5 (1.6) 40.0 (1.3) 0.58 BMI, kg/m (3.0) 25.9 (2.1) 0.30 Day 3 FSH, IU/L 10.0 (1.8) 8.2 (1.0) 0.07 Rate of ICSI, % 43 (6) 36 (10) 0.91 Table 2. Results Variable Experimental n = 14 Control n = 28 Test result P Clinical pregnancy rate 29 (4) 32 (9) 0.99 Number of mature oocytes (median) 2.5 (0 to 12) 5.0 (0 to 17) 0.13 Peak serum estradiol, pmol/l 4174 (2095) 5105 (1380) 0.44 Number of frozen embryos 0 (0 to 4) 0 (0 to 3) 0.60 Number of follicles 14 mm 3.5 (0 to 14) 4.0 (0 to 8) 0.94 Number of days of stimulation 10.1 (1.4) 10.1 (1.0) 0.93 Rate of ICSI, % 43 (6) 36 (10) 0.91 Cycle cancellation rate, % 21(3) 14 (4) 0.88 increase in the number of two pronuclei embryos in the GH group compared to the control group (4.4 ± 1.8 vs 1.5 ± 0.9; P < 0.001). Another randomized trial by Eftekhar et al. in 82 poor responders studied the effect of GH supplementation in the luteal phase followed by a GnRH antagonist protocol. 12 They also found greater numbers of oocytes in the GH group (6.1 ± 2.9 vs. 4.8 ± 2.4, P = 0.035) but, as in our study and that of Kucuk et al., 9 they did not find a significant increase in clinical pregnancy rate (GH group = 12.5%, control = 11.2%, P > 0.05). 12 To date there has been no comparison of clinical effects between early (luteal phase) and late (follicular phase) initiation of GH treatment in women undergoing IVF. Several small trials have demonstrated an increase in clinical pregnancy and live birth rates with the addition of GH to standard IVF protocols. Two meta-analyses have reviewed these clinical trials of GH usage in IVF. 2,16 All but one of the studies included in these meta-analyses involved initiation of GH treatment during the follicular phase, at the time of initiation of gonadotropin stimulation. 2,8,16 A 2009 Cochrane Collaboration review included 10 studies involving 440 patients, with a range of 14 to 61 subjects in each study. 2 The findings of this review did not support the routine use of GH in IVF protocols; however, the metaanalysis did find higher live birth rates and pregnancy rates in users of adjuvant GH in women who had been deemed to be poor responders (OR 3.28, 95% CI 1.74 to 6.2). In another meta-analysis, Kolibianakis et al. focused only on poor responders, including six of these same studies, for a total of 169 subjects. 16 These authors also found an increase in clinical pregnancy rate with the addition of GH (+16%, 95% CI +4% to +28%). 7,8,16 The authors of both metaanalyses were cautious in their conclusions, acknowledging that larger trials would be necessary to produce definitive conclusions. Therefore, while recent meta-analyses indicate that GH supplementation can have beneficial effects in poor responders undergoing IVF, the sample sizes in studies to date have been too small to confirm an overall benefit. 2,10,16 Inconsistency between studies may be related to the variable criteria used for defining poor ovarian response and the different stimulation protocols employed in IVF treatment cycles. Perhaps most crucially, the studies to date have varied with regard to the timing, dose, and duration of the adjuvant GH treatment. We theorized that the use of GH in the follicular phase might not have an optimal effect because follicular recruitment begins before this point. If earlier developmental events were constrained by limited endogenous GH, then initiating GH supplementation in the preceding luteal phase would be expected to lead to greater improvement of clinical outcomes. However, the administration of GH during the luteal phase in our study failed to produce a clinically apparent benefit. We cannot draw definitive conclusions about the benefit of GH in a microdose GnRH agonist SEPTEMBER JOGC SEPTEMBRE

5 Gynaecology Figure 2. Clinical pregnancy rate (%) Growth hormone (n = 14) Control (n = 28) flare protocol because of the case control design of our study and the inherent potential for bias when patients are permitted to choose a treatment rather than be randomly assigned. A larger, randomized trial to compare luteal versus follicular phase administration of GH in poor responders would be ideal to explore this concept further. CONCLUSION In this case-control study of women undergoing in vitro fertilization, the administration of growth hormone in poor responders during the luteal phase before a microdose GnRH agonist flare stimulation protocol did not improve clinical or laboratory outcomes. Since some previous studies have suggested improved clinical outcomes associated with administration of GH in the follicular phase of poor responders undergoing IVF, and some have also shown a benefit associated with use in the luteal phase, a larger randomized study of the effect of follicular versus luteal phase administration of GH in this setting is indicated. ACKNOWLEDGEMENTS We thank Arianne Albert, PhD and Kathryn Dewar, PhD of the Women s Health Research Institute (WHRI), Vancouver, BC, for their help with statistical analysis and study design. REFERENCES 1. Langhout DJ, Spicer LJ, Geisert RD. Development of a culture system for bovine granulosa cells: effects of growth hormone, estradiol, and gonadotropins on cell proliferation, steroidogenesis, and protein synthesis. J Anim Sci 1991;69(8): Duffy JM, Ahmad G, Mohiyiddeen L, Nardo LG, Watson A. Growth hormone for in vitro fertilization. Cochrane Database Syst Rev 2010;(1):CD Lanzone A, Fortini A, Fulghesu AM, Soranna L, Caruso A, Mancuso S. Growth hormone enhances estradiol production follicle-stimulating hormone-induced in the early stage of the follicular maturation. Fertil Steril 1996;66(6): Mason HD, Martikainen H, Beard RW, Anyaoku V, Franks S. Direct gonadotrophic effect of growth hormone on oestradiol production by human granulosa cells in vitro. J Endocrinol 1990;126(3):R Ferraretti AP, La Marca A, Fauser BCJM, Tarlatzis B, Nargund G, Gianaroli L. ESHRE consensus on the definition of poor response to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod 2011;26(7): EMD Serono. Saizen product monograph. Rockland (MA): EMD Serono; Adashi EY. The IGF family and folliculogenesis. J Reprod Immunol 1998;39(1 2): Hillier SG. Gonadotropic control of ovarian follicular growth and development. Mol Cell Endocrinol 2001;179(1 2): Kucuk T, Kozinoglu H, Kaba A. Growth hormone co-treatment within a GnRH agonist long protocol in patients with poor ovarian response: a prospective, randomized, clinical trial. J Assist Reprod Genet 2008;25(4): SEPTEMBER JOGC SEPTEMBRE 2015

6 Growth Hormone Supplementation in the Luteal Phase Before Microdose GnRH Agonist Flare Protocol for In Vitro Fertilization 10. de Prada JKN, VandeVoort CA. Growth hormone and in vitro maturation of rhesus macaque oocytes and subsequent embryo development. J Assist Reprod Genet 2008;25(4): Abir R, Garor R, Felz C, Nitke S, Krissi H, Fisch B. Growth hormone and its receptor in human ovaries from fetuses and adults. Fertil Steril 2008;90(4 Suppl): Eftekhar M, Aflatoonian A, Mohammadian F, Eftekhar T. Adjuvant growth hormone therapy in antagonist protocol in poor responders undergoing assisted reproductive technology. Arch Gynecol Obstet 2013;287(5): Ménézo YJ, Mouatassim el S, Chavrier M, Servy EJ, Nicolet B. Human oocytes and preimplantation embryos express mrna for growth hormone receptor. Zygote 2003;11(4): Tamura M, Sasano H, Suzuki T, Fukaya T, Watanabe T, Aoki H, et al. Immunohistochemical localization of growth hormone receptor in cyclic human ovaries. Hum Reprod 1994;9(12): Mendoza C, Ruiz-Requena E, Ortega E, Cremades N, Martinez F, Bernabeu R, et al. Follicular fluid markers of oocyte developmental potential. Hum Reprod 2002;17(4): Kolibianakis EM, Venetis CA, Diedrich K, Tarlatzis BC, Griesinger G. Addition of growth hormone to gonadotrophins in ovarian stimulation of poor responders treated by in-vitro fertilization: a systematic review and meta-analysis. Hum Reprod Update 2009;15(6): SEPTEMBER JOGC SEPTEMBRE