Middle East Lecture Tour, 2012 Use of LH in IVF and IUIDifferences between rec-hLH and LH Activity in HMG Preparations Sandro Esteves, MD, PhD Director, ANDROFERT Center for Male Reproduction Campinas, BRAZIL
What is in it for me? Role of LH in Reproductive Cycles LH window concept To Whom to Give LH Supplementation Recent Advances in Injectable Gonadotropin Preparations Rec-LH Products Differences between rec-hLH and LH Activity in HMG PreparationsEsteves, 2
Level ofevidence Individualization of Patient Treatment Lecture Structure Points I Consider Highly Relevant in Clinical Practice; Arguments Supported by Studies with High Level of Evidence. Level Type of evidence 1a Obtained from meta-analysis of randomised trials 1b Obtained from at least one randomised trial 2a Obtained from one well-designed controlled study without randomisation 2b Obtained from at least one other type of well-designed quasi- experimental study 3 Obtained from well-designed non-experimental studies (comparative and correlation studies, case series) 4 Obtained from expert committee reports or opinions or clinical experience of respected authoritiesEsteves, 3 Modified from Sackett et al. Oxford Centre for EBM Levels of Evidence (2009)
Use of LH in IVF and IUI Differences between rec-hLH and LH Activity in HMG Preparations Review this Lecture at: http://www.androfert.com.br/reviewEsteves, 4
What is in it for me? Role of LH in Reproductive Cycles 2 To Whom to Give LH Supplementation 3 Recent Advances in Injectable Gonadotropin Preparations Rec-LH Products Differences between rec-hLH and LH Activity in HMG PreparationsEsteves, 5
• Mild Stimulation (low dose rec-hFSH + GnRH ant.): Promotion of Steroidogenesis • 5 oocytes (TCs) early FP retrieved; • IR = 31% • Adequate estrogen production • Uterine/endometrial changes • Conventional Stimulation : Stimulation of final Follicular Maturation (GCs) late FP • 10 oocytes retrieved; • IR = 29% Verberg et al.Esteves, 6Esteves, 6 Alviggi et al.Hum Reprod Update 2009; 15: 5–12. Reprod Biomed Online 2006;12:221.
Evidence for LH threshold (1) Rec-hLH suppementation (UI): 0 25 75 225 3000 Serum Estradiol Levels 2500 225 2000 (pmol/L) 1500 75 1000 500 25 0 0 Day 1 Day 5 Day 10 hCG Day of StimulationEsteves, 8 The European Recombinant Human LH Study Group, JCEM 1998; 83:1507
Evidence for LH threshold (2) 0 25 75 225 rLH Endometrial Thickeness (mm) 75 8 Injected rLH LH Cmax 225 dose (UI) 6 75 UI 0.5 – 1.35 UI/L 4 25 2 0 0 Day 1 Day 5 Day 10 hCG Day of StimulationEsteves, 9 The European Recombinant Human LH Study Group, JCEM 1998; 83:1507
• Suppression of GC proliferation High • • Mild Stimulation Follicular atresia (non-dominant follicles) dose rec-hFSH + (low • Premature luteinization GnRH ant.): • Oocyte development compromised • 5 oocytes CEILING retrieved; Normal • IR = 31% • Normal androgen and estrogen biosynthesis • Normal follicular growth and development • Normal oocyte maturation THRESHOLD • Conventional Stimulation : Low • Insufficient androgen (and estrogen) synthesis • 10 oocytes • Follicular growth and maturation impaired retrieved; • Inadequate endometrial proliferation • IR = 29% Verberg et al.Esteves, 10Esteves, Balasch J, Fábreques F. Curr Opin Obstet Gynecol 2002, 14:265. Hum Reprod Update 2009; 15: 5–12.
Both FSH and LH are essential for normal estradiol biosynthesis. 75 UI recLH is sufficient to promote optimal follicular and endometrial growth as well as androgen production in most HH patients. Evidence suggests that in reproductive cycles optimal follicular development occurs within an ‘LH window’, above a certain ‘LH threshold’ and below an ‘LH ceiling’ (1.2 to ? UI/L).Esteves, 11
What is in it for me? Role of LH in Reproductive Cycles To Whom to Give LH Supplementation Recent Advances in Injectable Gonadotropin Preparations Rec-LH Products Differences between rec-hLH and LH Activity in HMG PreparationsEsteves, 12
Central Paradigm Maximize Minimize beneficial effects complications of treatment and risks High-quality Cycle cancellation, oocyte yield OHSS, multiple pregnancy Fauser BC et al: Predictors of ovarian response: progress towards individualized treatment in ovulationEsteves, 13 induction and ovarian stimulation. Hum Reprod Update 2008;14:1-14.
Factors Determining Response to Ovarian Stimulation Demographics and anthropometrics (Age, BMI, Race) Genetic profile Cause of Infertility Years of Infertility Health status Nutritional statusEsteves, 14
Level 1a Female Age Negative Duration of infertility Predictors Basal FSH Type of infertility All reflecting Indication ovarian reserve Fertilization method Number of oocytes retrieved Positive Number of embryos transferred Predictor Embryo qualityEsteves, 15 van Loendersloot et al. Hum Reprod Update 2010; 16: 577–589.
Normal • ~80% normogonadotropic women undergoing Ovarian Stimulation1-3 • 15-20% of NG women have less sensitive ovaries • Older patients (≥35 years)4 Low • Poor responders5 • Slow/Hypo-responders6 • Deeply suppressed endogenous LH levels (endometriosis)7 1. Alviggi et al. Reprod Biomed Online 2006;12:221; 2. Tarlatzis et al. Hum Reprod 2006;21:90; 3. Esteves et al. Reprod Biol Endocrinol 2009;7:111; 4. Marrs et al. Reprod Biomed Online 2004;8:175;5. Mochtar MH, Cochrane Database, 2007; 6. Alviggi, et al.Esteves, 16 RBMOnline 2009; 7. De Placido et al. Clin Endocrinol (Oxf) 2004;60:637;
Up to 45% Infertility Patients • Older patients (≥35 years) aged 35 or Less Sensitive Ovaries • Poor responders above • Slow/Hypo-responders • Deeply suppressed endogenous LH (endometriosis) Poor Responders* Hypo/Slow Responders At least 2 of the following: Normal markers of ovarian reserve Advanced maternal age (≥40 years) Hypo-responders: Previous POR (≤3 oocytes with a d1-d7: normal initial follicullar recruitment conventional stimulation protocol) using fixed starting dose of FSH; d7- Abnormal ovarian reserve test (AFC<5; d10: plateau on follicullar growth AMH <1.1) despite continuing same FSH dosage Or: Slow responders: 2 episodes of POR after maximal High doses of FSH (>3,000UI) to promote stimulation follicular growth; May indicate genetic polymorphisms of LH and/or FSH receptor Marrs et al. Reprod Biomed Online 2004;8:175 De Placido et al. Clin Endocrinol (Oxf) 2004;60:637; Ferraretti et al. Fertil Steril. 2004; 82:1521-6;Esteves, 17 Mochtar MH, Cochrane Database, 2007; Alviggi, et al. RBMOnline 2012
Theca cells Increase in LH LH drive LH Granulosa Increase in cells FSH drive FSH Increasing the Number % Cycle Pregnancy Level Stimulation Dose of oocytes cancellation rates 1b FSH… retrieved Manzi et al, 1994 …is not associated with better Klinkert et al, 2004 IVF outcome Berkkanoglu & Ozgur, 2010Esteves, 18
Reduced oocyte quality Less Sensitive Ovaries Reduced Fertilization Rate Reduced Embryo Quality Increase Miscarriage Rates Westergaard et al., 2000; Esposito et al., 2001; Humaidan et al., 2002 Reduced Androgen Decreased Reduced ovarian LH receptor LH secretory numbers of paracrine poly- bioactivity capacity functional activity morphisms while reduced LH receptors imnuno- • Piltonen et al., reactivity Hurwitz & Alviggi et al., unchanged Santoro 2004 2006 2003 • Vihko et al. 1996 • Mitchell et al. 1995; Marama et al 1984Esteves, 19
Level LH Supplementation in Poor 1a Responders… Effect on Regimen Outcome Pregnancy Mochtar et al, 2007 r-hFSH+rLH vs. OR 1.85 3 RCT (N=310) OPR r-hFSH alone* (95% CI: 1.10; 3.11) Poor responders CPR RD: +6%, Bosdou et al, 2012 r-hFSH+rLH vs. (95% CI: -0.3; +13.0) 7 RCT (N= 603) r-hFSH alone* Poor responders LBR RD: +19% (only 1 RCT) (95% CI: +1.0; +36.0%) Hill et al, 2012 r-hFSH+rLH vs. 7 RCT (N=902) OR 1.37 r-hFSH alone CPR Women advanced (95% CI: 1.03; 1.83) age ≥35 yrs. *long GnRH-a protocol; OR=odds-ratio; RD=risk difference Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Bosdou JK et al,Esteves, 20 Hum Reprod Update 2012; 8(2):127-45. Hill MJ et al. Fertil Steril 2012; 97:1108-4.
Action of LH at the follicular level that increasesandrogen production for its later aromatization toestrogens in a dose dependent manner mayrestore the follicular milieu in these patients torecover oocyte quality and, therefore, embryoquality and implantation rates. Jamnongjit M et al. PNAS 2005;102:16257-16262
Level LH Supplementation in 1b Hypo/Slow Responders (1) • RCT 260 pts with “steady” response on COS D8 (E2 <180pg/mL; >6 follicles <10mm) • 3 groups: Mean No. oocytes retrieved IR (%) OPR (%) 40 32 22 18 14 10 9 11 6 FSH step-up (+150 UI) LH supplementation Normal Responders (+150 UI)Esteves, 22 De Placido et al. Hum Reprod. 2004; 20: 390-6.
Level 1b LH Supplementation in Hypo/Slow Responders (2) • RCT • 126 pts. follicular stagnation during d7-d10 COS • 4 groups: Mean No. oocytes retrieved LBR (%) 41 37 22 18 8 11 11 10 increase in r- increase in r- increase in r- controls hFSH dose hFSH dose + r- hFSH dose + LH (max. 450UI) hLH (75-150UI) supplementation supplementation with HMGEsteves, 23 Ferraretti et al. Fertil Steril. 2004; 82: 1521-6.
Level LH Supplementation in 1b OI and IUI LH levels 1.2 UI/L (WHO group I) Higher follicular development pts. receiving LH (67% vs 20%; p=0.02): Shoham et al., 2008. Similar follicular development HMG vs FSH+rLH; higher cumulative PR after 3 cycles in FSH+LH (56% vs 23%; p=0.01): Carone et al., 2012. WHO group II Clomiphene-resistant: fewer intermediate-sized follicles and OHSS in LH-supl. vs FSH group; similar ovulation rate (Plateau, 2006); Previous over-response: higher monofollicular development in LH group (32% vs 13%; p=0.04): Hughes et al., 2005; IUI: higher monofollicular development in LH group without intermediate-size (42% vs 11%; p=0.03); lower cycle cancellation due to risk OHSS (-7% difference): Segnella et al., 2011.Esteves, 24
What is the optimal LH supplementation protocol? Existing studies give us some clues but the optimal LH protocol has yet to be established How much LH should be used? Should the dose be fixed or flexible? At what stage of the cycle should LH be administered? Is LH needed in a GnRH antagonist Protocol? FSH LH 2:1? 1:1? Fixed? Mimic of natural LH levels?Esteves, 25
Level Is LH needed in a GnRH 1a antagonist Protocol? Unselected women undergoing COS; r-hFSH+r-hLH vs. r-hFSH alone in antagonist cycles Mochtar et al. Kolibianakis et al. Baruffi et al. 3 RCT (N=216) 2 RCT (N=176) 5 RCT (N= 434) Estradiol on WMD 571 - WMD 514 hCG day (pg/ml) (95% CI 259; 882) (95% CI 368; 660) No. retrieved WMD 0.50 WMD 0.41 - oocytes (95% CI -0.68; 1.68) (95% CI -0.44; 1.3) †OR 0.79 *OR 0.86 †OR 0.89 CPR†/LBR* (95% CI: 0.26; 2.43) (95% CI: 0.04; 1.85) (95% CI: 0.57; 1.39) WMD weight mean difference Mochtar MH et al. Cochrane Database Syst Rev. 2007;2:CD005070; Kolibianakis et al, Hum Reprod Update. 2007;13:445-52; Baruffi RL et al, Reprod Biomed Online. 2007;14:14-25.Esteves, 26
Level Is LH needed in a GnRH 1b antagonist Protocol? RCT; 292 NG women aged 36-39; Fixed (D6) antagonist COH protocol rFSH rFSH + rLH P= 0.027 68% 61% OR=1.49 OR=1.56 95% CI 0.93-2.38 95% CI 1.04-2.33 33% 25% 27% 19% %2PN Ongoing PR Implantation Yes, for women aged >35 yoEsteves, 27 Bosch et al. Fertil Steril. 2011; 95:1031-6.
Women with less sensitive ovaries (ovarian aging) have poorer IVF outcomes. Androgen secretory capacity decreases with ovarian ageing. Mechanisms include decreased number of functional LH receptors and ovarian paracrine activity resulting in reduced LH bioactivity. LH-r polymorphisms possibly involved in hypo- responders. LH supplementation to COS is an evidence-based strategy to maximize pregnancy results. 4 subgroups benefit of LH supplementation in COS: Poor responders Slow/hypo-responders Age >35 years Deeply suppressed endogenous LH levelsEsteves, 28
3 subgroups clearly benefit of LH supplementation in OI and IUI: WHO group I anovulation WHO group II clomiphene resistant WHO group II with previous over-response to OS Other potential indications include: Poor responders Slow/hypo-responders Age >35 years Deeply suppressed endogenous LH levelsEsteves, 29
What is in it for me? Role of LH in Reproductive Cycles To Whom to Give LH Supplementation Recent Advances in Injectable Gonadotropin Preparations Rec-LH Products Differences between rec-hLH and LH Activity in HMG PreparationsEsteves, 30
r-hFSH Long- FbM r-hFSH acting u-FSH HP +r-hLH r-hFSH; Pituitary r-hFSH FbM u-FSH FSH r-hLH u-hMG Puriity Horse and Safety, Quality, PMSG Specific Consistency and Patient Activity Convenience 1930s 1950 1962 1980 1993 1995 2000 2003 2007 2010 Intramuscular administration sc Injector pens sc, subcutaneous; FbM, filled by Mass; HP, highly-purifiedEsteves, 31 Adapted from Lunenfeld. Hum Reprod Update 2004;10:453–67
• Same injection device design for all gonadotropins; • Color-coded for differentiation; • Pre-filled, ready-to- use family of pens for fertility treatment.Esteves, 33
Conventional FbM: Novel Bioassay analitycal method High Protein content by Rat ovary mass weight variability gain Minimal batch-to- batch variability (1.6%) Urinary gonadotropins Follitropin beta Follitropin alfa and rec-hLH Bassett et al. Reprod Biomed Online 2005;10:169–177;Esteves, 34 Driebergen et al. Curr Med Res Opin 2003;19:41–46.
Alfa Unit Beta unit Carboxyl terminal (biological action segment and receptor (determines half-life) affinity) LH 92 AA; 121 AA Absent; half life of 20’ hCG Identical to LH 144 AA Present; half-life of Higher receptor affinity 24h Purity FSH LH activity (LH content) activity (IU/vial) (IU/vial) Rec-hLH >99% 0 75 Rec-hLH + rec-hFSH >99% 150 75 hMG-HP Unknown* 75 75* *derives primarily from the hCG component, which preferentially is concentrated during the purification process and sometimes was added to achieve the desired amount of LH-like biological activity.Esteves, 35 ASRM Practice Committee. Fertil Steril. 2008; 90:S13-20.
Level 2a • Matched case-control study; • N=4,719 pts.; long GnRH-a protocol • 3 groups 35 30 P=0.02 Duration of 31 Stimulation (days) 25 26 25 Mean No. oocytes 20 retrieved 15 IR (%) 10 5 CPR per transfer (%) 0 2:1 r-hFSH+r- HMG rec-hFSH + hLH HMGEsteves, 36 Buhler KF, Fisher R. Gynecol Endocrinol 2011; 1-6.
Level 1a Lower expression of LH/hCG receptor gene as well as genes involved in in biosynthesis of cholesterol and steroids in granulosa cells in pts. treated with HMG preparations May reflect down-regulation of LH receptors, as shown in animals: Caused by a constant ligand exposure during the follicular phase due to longer half life and higher binding affinity of hCG to LHr May explain the observed lower progesterone levels: Caused by lower LH-induced cholesterol uptake, a decrease in the novo cholesterol synthesis and a decrease in steroid synthesis. Trinchard-Lugan I et al. Reprod Biomed Online 2002; 4:106-115; Menon KM et al. Biol ReprodEsteves, 37 2004; 70:861-866; Grondal ML et al. Fertil Steril 2009; 91: 1820-1830.
Recombinants vs Urinary products Recombinant LH preparations have 3 major differences compared to urinary products: Higher purity and specific activity (SC delivery in very small volumes)) Higher dose precision (FbM) LH activity in u-HMG is hCG dependent: hCG is concentrated during purification or added to achieve the desired amount of LH-like biological activity; hCG has higher half-life and biological activity compared to rec-hLH.Esteves, 38
Differences between rec-hLH and LH Activity in HMG Preparations Lower expression of LH receptor gene in pts. treated with HMG (LH-r down- regulation). Preparations used for COS are important for granulosa cell function and may influence the developmental competence of the oocyte and the function of corpus luteum.Esteves, 39
Use of LH in IVF and IUI Progesterone Issues Supplementary MaterialEsteves, 41
Steroidogenesis During Normal Follicular Phase and Following COS The expression of LH-R (GCs) is linked to the synthesis of progesterone during COS. Levels of LH-R and progesterone synthesis vary depending on the hormones used during the stimulation protocol.Esteves, 42 Steroidogenesis Consensus Meeting III, Copenhagen, Denmark, May 2011.
Steroidogenesis in COS Endogenous LH then results in higher levels of progesterone synthesis following treatment with FSH than hMG: higher levels of LHR expressed on granulosa cells and increased number of granulosa cells.Esteves, 43 Steroidogenesis Consensus Meeting III, Copenhagen, Denmark, May 2011.
Level 1a Progesterone on the Day of hCG and Probability of Pregnancy in IVF Progesterone Elevation x No Progesterone Elevation Venetis et al, 2007 Kolibianakis et al, 2012 GnRH Agonists Antagonists Antagonists analogue n = 2,624 n = 109 n = 109 OR: 0.86 OR: 0.57 WMD: -9% CPR (95% CI: 0.59; 1.25) (95% CI: 0.09; 3.56) (95% CI: -17; -2) E2 levels on WMD: 413.06 WMD: 956 the day of (95% CI: 240.14; 585.99) (95% CI 248; 1664) hCG (pg/mL) Number of WMD: +2.96 WMD: 0.00 WMD: +2.9 retrieved (95% CI: +1.74; +4.18) (95% CI: -2.98; +2.99) (95% CI: +1.5; +4.4) oocytes heterogeneity of the studies included; arbitrary serum progesterone threshold values Venetis et al, Hum Reprod Update. 2007;13:343-55;Esteves, 44 Kolibianakis et al, Curr Pharm Biotechnol. 2012;13:464-70.
Level 2b Progesterone on the Day of hCG and Probability of Pregnancy in IVF Bosch et al. 2010 (N=4,032) OPR: inversely associated with serum P levels on the day of hCG irrespective of the GnRH analogue: CUT-OFF: 1.5 ng/mL Serum P levels ≤1.5 ng/ml: ↑OPR 31% versus 19.1%; P = 0.00006; OR: 0.53 (95% CI, 0.38 – 0.72) positively FSH dose associated No. oocytes with P levels Estradiol (day of hCG) (P < 0.0001 for all). Serum P levels: agonists: 0.84 ± 0.67 vs antagonists: 0.75 ± 0.66 (P = 0.0003)Esteves, 45 Bosch et al. Hum Reprod. 2010; 25(8):2092-100.
Level Progesterone on the Day of hCG 2b and Probability of Pregnancy in IVF Xu et al, 2012 (N=11,055 long agonist protocol) For fresh cycles, OPR inversely associated with serum P levels on hCG day FSH dose Positively No. oocytes associated with P levels ■ Fresh Estradiol (day of hCG) ■ FET Serum P Ovarian Number of threshold response oocytes (ng/mL) Poor ≤4 1.5 Intermediate 5-19 1.75 High ≥20 2.25 Xu et al. Fertil Steril 2012;97:1321–7.Esteves, 46
Progesterone on the Day of hCG and Probability of Pregnancy in IVF The rise in progesterone levels seen during COS for IVF/ICSI cycles cannot be explained by luteinization of granulosa cells Conversion FSH activity Granulosa of cholesterol to cell progesterone LH Conversion Teca bioactivity of progesterone cell to androgens FSH dose, number of follicles and rec-hFSH (x HMG): correlation to P increase on hCG day Bosch et al. Hum Reprod. 2010;25:2092-100;Esteves, 47 Xu et al. Fertil Steril 2012;97:1321–7; Smitz J et al. Hum Reprod 2007;22:676–87.
LevelsProgesterone on the Day of hCG 2b, 3 and Probability of Pregnancy in IVF Hofmann et al, Fertil Steril. 1993;60:675-9; Xu et al. Fertil Steril 2012;97:1321–7; Huang et al. Fertil Steril 2012; 98:664–70; Melo et al. Hum Reprod 2006; 21:1503–1507.Esteves, 48
Serum Progesterone and IVF OutcomeMost circulating P4 (95%) is produced in the intrafollicularcompartment by the granulosa cells;Intrafollicular P4 and Hydroxi-progesterone are terminalproducts and cannot be converted to estradiol by GCsunder the effect of LH/hCG activity contained in hMG, due tolack of expression of an hydrogenase and P450-17α neededfor this pathway;Higher serum Progesterone increments are related withmore follicles developed and more oocytes retrieved and it’seffect in pregnancy still controversial. Increments up to>7nmol/L seems not to affect clinical pregnancy rates.
Serum Progesterone and IVF Outcome Treatment with FSH results in higher levels of progesterone than treatment with hMG. A large number of developing follicles leads to increased levels of progesterone. The higher the level of LH present, the higher the level of progesterone. The effect of high progesterone levels at the time of hCG administration on pregnancy outcome is still controversial. Further detailed analyses are required to understand why, when and how much progesterone is detrimental for implantation rates.Esteves, 50