L8: Which POSEIDON groups may benefit of LH supplementation? C. Alviggi (Italy)
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1 L8: Which POSEIDON groups may benefit of LH supplementation? C. Alviggi (Italy)
2 LH in Poseidon Groups Agenda LH in natural and stimulated cycles LH in Group 1 Poseidon: The concept of hypo-response LH in Group 2 Poseidon: The relevance of androgens LH in Group 3 and 4 Poseidon
3 LH levels LH levels (IU/L) LH LH-R Leading follicle FSH Theca interna 4 -A E 2 Paracrine network (EGF, IGF-1, Inhibin) GRANULOSA Aromatase activity Production and release of 4 -androgens Day
4 LH levels LH levels (IU/L) LH serum level (IU/L) LH LH-R Leading follicle FSH Theca interna 4 -A E 2 Paracrine network (EGF, IGF-1, Inhibin) GRANULOSA Aromatase activity Production and release of 4 -androgens , , , ,0 0,0 GnRH-a Long protocol GnRH antagonist Spontaneous Stimulation day Day
5 Role of LH during folliculogenesis Since early follicular phase Induction of androgens production in the theca cells FSH receptor induction in granulosa cells responsiveness (Weil et al., 1999) Act synergistically with IGF1 growth (Vendola et al., 1999) Increase in pre-antral and antral follicles recruitability (Vendola et al., 1998; 1999; Spinder et al., 1989) Since intermediate follicular phase Expression of LH receptors in the granulosa Jeppesen et al., JCEM, 2012 Sustain of FSH-dependent granulosa activities, including aromatase induction and growth factors release (IGF-1, EGF etc ) Regulation of final follicle/oocyte maturation Optimization of steroidogenesis
6 Role of LH during COS Questions to keep in mind when a clinical trial is (meta)analyzed When LH supplemetation was started? In which subgroups of women? Which dose has been used? In which analogs regimen? Before COS; 1 day of COS; Antagonist day; day 6-8 Normo-responder; poor responder; hypo/suboptimal responder 75 IU/day; 150 IU/day; 2:1 ratio Antagonist; Agonist
7 LH in Poseidon Groups Agenda LH in natural and stimulated cycles LH in Group 1 Poseidon: The concept of hypo-response LH in Group 2 Poseidon: The relevance of androgens LH in Group 3 and 4 Poseidon
8 Four Groups of Patient with Lower Prognosis GROUP 1 Young patients <35 years with adequate ovarian reserve parameters (AFC 5; AMH 1.2 ng/ml) and with an unexpected poor or suboptimal ovarian response Subgroup 1a: <4 oocytes* Subgroup 1b: 4-9 oocytes retrieved* *after standard ovarian stimulation GROUP 2 Older patients 35 years with adequateovarian reserve parameters (AFC 5; AMH 1.2 ng/ml) and with an unexpected poor or suboptimal ovarian response Subgroup 2a: <4 oocytes* Subgroup 2b: 4-9 oocytes retrieved* *after standard ovarian stimulation GROUP 3 GROUP 4 Young patients (<35 years) with poor ovarian reserve pre-stimulation parameters (AFC <5; AMH <1.2 ng/ml) Older patients ( 35 years) with poor ovarian reserve pre-stimulation parameters (AFC <5; AMH <1.2 ng/ml) Poseidon Group, Fertil Steril 2016
9 Four Groups of Patient with Lower Prognosis GROUP 1 Young patients <35 years with adequate ovarian reserve parameters (AFC 5; AMH 1.2 ng/ml) and with an unexpected poor or suboptimal ovarian response Subgroup 1a: <4 oocytes* Subgroup 1b: 4-9 oocytes retrieved* *after standard ovarian stimulation Poseidon Group, Fertil Steril 2016
10 The meaning of ovarian sensitivity to FSH Follicle Output RaTe (FORT) Day stimulation 5-8 ~30% What to do? Increasing FSH? Adding LH? Poor! Hypo-sensitivity to FSH (15% of women with good ovarian reserve) ~70% Genro et al., 2011 Good!
11 Initial inadequate response (hypo-sensitivity) to FSH has been defined in different ways Normo-ovulatory normogonadotrophic-young patients characterized by an initial slow (poor) response to r-hfsh (normal follicular cohort with no follicle >10 mm on day 8 of stimulation or stagnation between day 7-10) De Placido et al., Hum Reprod, 2001; 2005; Ferraretti et al., Fertil Steril, 2004 Normogondotrophic patients with initial ovarian response to r-hfsh referred to as steady response De Placido et al., Clin Endocrinol, 2004 Normogondotrophic patients characterized by a suboptimal ovarian response, as they do not fit with the classical criteria to define poor responders Alviggi et al., RBMOnline, 2009, 2011
12 Hystory of Hypo-responders Normogonadotrophic women with initial poor (slow) response to FSH The experimental grouping was formed on day 8-10 patients were randomized into two study groups LH supplementation Increase in FSH dose De Placido et al. 2001; 2004; 2005; Ferraretti et al., 2004
13 Hypo responders (initial poor [slow] response to FSH) Addition of of LH from day 7-10 is more efficient than increasing FSH in rescuing oocyte number/competence Increased FSH LH supplementation N of oocytes retrieved Increased FSH (n = 50) LH supplemenntation (n = 54) 5.87 ± ± 6.91 De Placido et al., Human Reproduction, Ferraretti et al., Fertility and Sterility, ± ± 4.3 De Placido et al., Human Reproduction, 2005 N of oocytes mature 4.7 ± ± 4.3 De Placido et al., Human Reproduction, 2005 Implantation rate (%) Ferraretti et al., Fertility and Sterility, De Placido et al., Human Reproduction, De Placido et al., Human Reproduction, 2001 Pregnancy rate (%) Ferraretti et al., Fertility and Sterility, De Placido et al., Human Reproduction, 2005 Only statistically significant differences have been reported
14 Hypo responders (initial poor [slow] response to FSH) Addition of recombinant r-hlh from day 8: 150 IU are more effective than 75 IU R-hLH 75 IU (n = 23) R-hLH 150 IU (n = 23) p N of oocytes retrieved 6.39 ± ± 2.16 <0.001 N of oocytes mature (%) <0.05 Implantation rate (%) / Pregnancy rate (%) / De Placido et al. Clinical Endocrinology, 2004
15 The meaning of hypo-response Day stimulation 5-8 What to do? ~30% LH Increasing FSH? Adding LH? In 15% of normogonadotrophic good prognosis women (normal AMH and AFC) an initial inadequate (poor) response to standard FSH doses is observed Analysis of 3095 cycles «Hypo-reponse»: >2500 IU of r-hfsh in women <35 years, with normal ovarian reserve, having starting dose GnRH-a long protocol: 19.2% Antagonist protocol: 14.8% Overall: 15.5% Esteves unpubished data
16 The meaning of hypo-response Day stimulation 5-8 What to do? ~30% LH Increasing FSH? Adding LH? In 15% of normogonadotrophic good prognosis women (normal AMH and AFC) an initial inadequate (poor) response to standard FSH doses is observed This phenomenon reflects an hypo-sensitivity of granulosa cells to standard FSH doses. For this reason it has been defined hypo-response Hypo-response is associated to higher FSH consumption, low FORT, unexpected poor response (i.e. <3 eggs retrieved) and lower PRs If hypo-response is identified early (i.e., day 5-8 of COS), r-hlh is effective in rescuing follicle/oocyte number (FORT) and embryo competence
17 Hypo-sensitivity to FSH is different from reduced ovarian reserve Poor responder (ESHRE, Bologna criteria) Hypo responder At least two of the following three features must be present: Advanced maternal age ( 40 years) or any other risk factor for POR (Turner syndrome, X-fragile mutations, hystory of chemotherapy etc.) Young, normogonadotrophic women, with normal ovarian reserve who show sub-optimal or unexpected poor response to exogenous FSH A previous poor ovarian response (POR) ( 3 oocytes with a conventional stimulation protocol) An abnormal ovarian reserve test (i.e., AFC 5 7 follicles or AMH ng/ml) These women, even when the ovarian response is normal (i.e., >5 eggs) tend to show an increase in the cumulative FSH dose (i.e. > IU) and in the stimulation length (hypo-sensitivity to FSH) Ferraretti et al. Hum Reprod; De Placido, et al. Hum Reprod 2001; Clin Endocrinol 2004; Hum Reprod 2005; Drugs Ferraretti, et al. Fertil Steril Kailasam, et al. Hum Reprod Alviggi, et al. RBMOnline 2006; RBMOnline 2009; Reprod Biol Endocrinol 2009; Devroey, et al. Hum Reprod Update 2009 (EVAR) Workshop Group 2008.
18 R-hLH versus in r-hfsh dose in POOR RESPONDERS Statistically significant increase in ongoing PR Ongoing PR per woman randomized ALL STUDIES PERFORMED IN HYPO-RESPONDERS!!! Favours r-hfsh Favours r-hfsh + r-hlh Mochtar MH, Cochrane Database, 2007, Issue 2 No difference in LH endogenous levels during stimulation
19 R-hLH versus in r-hfsh dose in POOR RESPONDERS Statistically significant increase in ongoing PR R-h (LH+FSH) vs r-hfsh: Meta-analysis 6443 patients Normal responders MANY STUDIES PERFORMED IN HYPO-RESPONDERS!!! Relative increase 30% in poor responders Poor responders Lehert et al., 2014
20 Four Groups of Patient with Lower Prognosis GROUP 1 Young patients <35 years with adequate At least two of the following three features must be present: ovarian reserve parameters (AFC 5; AMH 1.2 ng/ml) and with an unexpected poor or suboptimal ovarian response Advanced maternal age ( 40 years) or any other risk factor for POR (Turner syndrome, X-fragile mutations, hystory of chemotherapy etc.) Subgroup1a: A previous <4 poor oocytes* ovarian response (POR) ( 3 oocytes with Subgroup1b: a conventional 4-9 oocytes stimulation retrieved* protocol) *after standard ovarian stimulation An abnormal ovarian reserve test (i.e., AFC 5 7 follicles or AMH ng/ml) Hypo responder Young, normogonadotrophic women, with normal ovarian reserve who show sub-optimal or unexpected poor response to exogenous FSH These women, even when the ovarian response is normal (i.e., >5 eggs) tend to show an increase in the cumulative FSH dose (i.e. > IU) and in the stimulation length (hypo-sensitivity to FSH) WHY? ~30% Adding r-hlh and increasing r-hfsh are effective in improving FORT (De Placido et al., 2004; Ferraretti et al., 2004)
21 FSH (IU/l) FSH ampoules (n) FSH consumption is higher in carriers of FSH-R Ser680 and v-betalh variants FSH receptor Ser 680 genotype and ovarian response to FSH V-betaLH genotype and ovarian response to FSH * * * 11.6 %] v-lh carriers found 10.2 % heterozygotes 1.4% homozygotes Alviggi et al., RBMOnline 2009; Alviggi and Humaidan RB and E, n=46 n=72 n= n=46 n=72 n= Asn/Asn Asn/Ser * p < 0.05 Ser/Ser 0 * Asn/Asn Asn/Ser p < 0.05 Ser/Ser Perez Mayorga, et al. 2000; Sudo, et al. 2002; Choi, et al. 2004; Falconer, et al Greb, et al. JCEM, 2005; Gromoll & Simoni TEM 2005.
22 Frequency of FSH-R Ser680 is higher in patients selected as hypo-responders Background: 17 hyporesponder young patients who required a cumulative dose of recombinant FSH (rfsh) >2500 UI were compared with a control group which was randomly selected among patients who required a cumulative dose of rfsh <2500 UI (group B). Alviggi et al., Reprod sci, 2016
23 Multicentric study on the impact of Gonadotrophins and their receptors on COS Carlo Alviggi - Principal investigator Giuseppe De Placido, Pasquale De Rosa, Simona Alfano Dipartimento di Neuroscienze, Scienze Riproduttive ed Odontostomatologiche, U.O.C. di Chirurgia Ostetrica e Ginecologica, Laparotomica e Centro per lo Studio e la Terapia della Sterilità ed Infertilità di Coppia, Università degli Studi di Napoli Federico II, Italy; Ilpo Huhtaniemi - Department of Reproductive Biology, Hammersmith Campus, Imperial College London, United Kingdom; Kim Pettersson Department of Biotechnology, University of Turku, Finland; Peter Humaidan - Helle Olesen Elbæk The Fertility Clinic, Skive Regional Hospital, Skive, Denmark; Enrico Papaleo Unità Operativa di Ginecologia e Ostetricia Dimer, Centro Scienze della natalità, Istituto scientifico Universitario San Raffaele, Milan.
24 Non-pharmacologic Interventional Study Objectives Assess the effects of polymorphisms, evaluated by sequencing the entire genes of FSH, FSH-R, LH and LH-R, on ovary response to recombinant gonadotropin treatment according to clinical practice in normo-gonadotropic women undergoing IVF/ICSI cycles Inclusion criteria Eumenorrhea-normogonadotrophism Age 20 and <35 years BMI 20 and 27 kg/m2 Basal FSH 10 IU/l Key primary endpoint Efficacy endpoint will be the cumulative gonadotrophins dose/mature oocyte retrieved Key secondary endpoint(s): Estradiol levels on the days 1, 5, 8 of stimulation and on the day of hcg Percentage of mature oocytes; fertilisation rate; number of embryos transferred Implantation rate Pregnancy rate per cycle and per transfer Clinical Pregnancy rate for started cycle and per transfer
25 SNPs investigated FSHR Thr/Ala Ala and 680Ser associated with reduced COH outcome and elevated FSH administration (Perez Mayorga et al., 2000; Sudo et al., 2002; de Castro et al., 2003, 2004; Choi et al., 2004; Asn/Ser680 Behre et al., 2005; Jun et al., 2006; Loutradis et al., 2006; Livshyts et al., 2009). 680Ser associated with lower clinical pregnancy (Choi et al., 2004; Jun et al., 2006). -29 (G/A) Women require highest amount of exogenous FSH for ovulation induction (Achrekar et al., 2009b). LHCGR 18insLeuGln Associated with increased receptor activity (Piersma et al., 2006, 2007; Simoni et al., 2008b) and their possible effects in steroid-hormone-related diseases have been suggested (Powell Asn291Ser et al., 2003; Piersma et al., 2007) Asn312Ser FSHB 2623 (T/C) Phenotypic effects on male reproductive characteristics, but its possible effects in the female have not yet been investigated -211 (G/T) LHB Trp8Arg More frequent among hypo-responders to rfsh (Alviggi et al., 2009a), and in women with ovarian resistance to rfsh (Alviggi et al., 2009b) Ala-3Gly Biochemical studies showed an association between SNP rs A/A genotype low levels of LH and higher fasting glucose levels in PCOS women (Liu et al., 2012)
26 Preliminary results LHCGR 312 Heterozigotic patients (T/C) needed less FSH for each mature oocyte recruited, than homozygotic women (C/C and T/T) (-96 IU, CI 95% IU) Basal estradiol levels and number of fertilized and mature oocytes were lower in homozygotic carriers of LHCGR 291 (T/T) than in heterozygotic C/T (p = and p = 0.05 respectively) - Number of oocytes with more than 10 mm of diameter recruited was significantly lower in homozigotic T/T than heterozygotic C/T (p = 0.035). - Similarly number of fertilized and mature oocytes were significantly lower in homozigotic T/T than heterozygotic C/T (p = 0.050). - Number of inseminated and fertilized oocytes were lower in homozigotic T/T patients than heterozigotic C/T (p = and p = 0.001, respectively) The presence of allele C on both FSH-29 and LHCGR 291 caused an increased ratio between cumulative r-fsh dose and total number of both oocytes and mature oocytes (RR: 5.47, CI 95 % : , p<0.001). Polymorphism of FSHR 680 seems to be involved in the same effect Analysis performed by Dr Daniele Santi University of Modena and Reggio Emilia
27 Four Groups of Patient with Lower Prognosis GROUP 1 Young patients <35 years with adequate At least two of the following three features must be present: ovarian reserve parameters (AFC 5; AMH 1.2 ng/ml) and with an unexpected poor or suboptimal ovarian response Advanced maternal age ( 40 years) or any other risk factor for POR (Turner syndrome, X-fragile mutations, hystory of chemotherapy etc.) Subgroup1a: A previous <4 poor oocytes* ovarian response (POR) ( 3 oocytes with Subgroup1b: a conventional 4-9 oocytes stimulation retrieved* protocol) *after standard ovarian stimulation An abnormal ovarian reserve test (i.e., AFC 5 7 follicles or AMH ng/ml) Hypo responder Young, normogonadotrophic women, with normal ovarian reserve who show sub-optimal or unexpected poor response to exogenous FSH These women, even when the ovarian response is normal (i.e., >5 eggs) tend to show an increase in the cumulative FSH dose (i.e. > IU) and in the stimulation length (hypo-sensitivity to FSH) WHY? ~30% Hypo-response may reflect polymorphisms of Gn and their receptors Possible pharmacogenomic approach (Behre et al., 2005; Alviggi et al., )
28 LH in Poseidon Groups Agenda LH in natural and stimulated cycles LH in Group 1 Poseidon: The concept of hypo-response LH in Group 2 Poseidon: The relevance of androgens LH in Group 3 and 4 Poseidon
29 Four Groups of Patient with Lower Prognosis AGING Reduced androgens production FSH receptor induction in granulosa cells responsiveness (Weil et al., 1999) Act synergistically with IGF1 growth (Vendola et al., 1999) Increase in pre-antral and antral follicles recruitability (Vendola et al., 1998; 1999; Spinder et al., 1989) GROUP 2 Older patients 35 years with adequateovarian reserve parameters (AFC 5; AMH 1.2 ng/ml) and with an unexpected poor or suboptimal aovarian response Subgroup 2a: <4 oocytes* Subgroup 2b: 4-9 oocytes retrieved* *after standard ovarian stimulation LH can be effective in increasing oocyte/embryo quantity and competence (Bosch et al., 2011)
30 LH improves IR in advanced reproductive age in women with normal ovarian reserve (Group 2 Poseidon) R-hLH - IVF outcome according to age: retrospective-subgroup analysis from RCT <35 years + LH 35 years + LH <35 years - LH 35 years - LH Oocytes (no) PN (% per MII) ET no (%) 81 (85.3) 19 (90.5) 86 (88.7) 17 (94.4) Pos. HCG (% per ET) 41/81 (50.6) 7/19 (36.8) 40/86 (46.5) A 4/17 (23.5) A IR % B 31.2 G 13.3 B, G Clin. PR (% per cycle) 35/95 (36.8) 7/21 (33.3) 31/97 (32.0) 4/18 (22.2) A: p <0.05 ( 2 test) B: p <0.05 (Fishers Exact test) G: p <0.05 (Fishers Exact test) Humaidan et al., RBMOnline 2004
31 LH improves IR in advanced reproductive age in women with normal ovarian reserve (Group 2 Poseidon) <36 years old 36 to 39 years old rfsh 225 IU rfsh + rlh 150UI + 75 IU rfsh 300 IU rfsh + rlh 225UI + 75 IU Bosch et al., Fertility and Sterility, 2011
32 LH improves IR in advanced reproductive age in women with normal ovarian reserve (Group 2 Poseidon) No significant differences between the two stimulation protocols in terms of implantation, pregnancy, and ongoing pregnancy rates in patients aged <36 years old The implantation rate was significantly better in those patients given rfsh + rlh in the 36 to 39 years old age group. Clinical and ongoing pregnancy rates were also better in this group, but not statistically significant. Bosch et al., Fertility and Sterility, 2011
33 LH improves IR in advanced reproductive age in women with normal ovarian reserve (Group 2 Poseidon) rlh in advanced reproductive age Meta-analysis Clinical pregnancy (Hill et al., Fertil Steril 2012)
34 LH in Poseidon Groups Agenda LH in natural and stimulated cycles LH in Group 1 Poseidon: The concept of hypo-response LH in Group 2 Poseidon: The relevance of androgens LH in Group 3 and 4 Poseidon
35 Four Groups of Patient with Lower Prognosis ~70% If ovarian reserve is low and FORT is high no gonadotrophin can compensate!!! Most of Bologna patients patients fall in these groups!!! Waiting for ESPART subgroups analyses Duo-Stim can be an option GROUP 3 Young patients (<35 years) with poor ovarian reserve pre-stimulation parameters (AFC <5; AMH <1.2 ng/ml) GROUP 4 Older patients ( 35 years) with poor ovarian reserve pre-stimulation parameters (AFC <5; AMH <1.2 ng/ml) Poseidon Group, Fertil Steril 2016
36 Conclusions The concept of «low prognosis» should be developed considering new categories of abnormal ovarian response (i.e. hypo- and sub-optimal responders) and «ovarian quality» (age related aneuploidies) Hypo-response is different from POR This difference is crucial for right interpretation of RCT results There is evidence that r-hlh is effective in hypo-responders (Group 1 Poseidon) There is evidece (Level 1) that rlh improves implantation rate in women aged with good ovarian reserve (consistent with Groups 2 Poseidon) The efficacy of rlh in low ovarian reserve (Groups 3-4 Poseidon) is under evaluation
37 G. De Placido Acknowledgment I. Strina A. Conforti P. De Rosa S. Picarelli R. Vallone C. Buonfantino L. Avino I. Nuzzo F. Donnarumma S. Alfano J.A. Gilder D. Finizio University of Naples Federico II Reproductive Medicine IVF Unit Fertunina Special thank: Poseidon guys Excemed
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