Relevance of LH activity supplementation
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1 Relevance of LH activity supplementation in ovulation induction Franco Lisi Servizio di Fisiopatologia della Riproduzione Clinica Villa Europa Roma, Italia
2 Comprehension of the role of LH in follicular growing and selection and in multiple l follicular l stimulation for ART.. has been one of the most controversial topics in human physiology and in assisted reproduction during the last few years
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4 Physiologic functions of FSH Binds to receptors on granulosa cells Activation of Adenylate cyclase system Increase in synthesis of the system cytochrome P 450 aromatasi and 3βhidroxy-steroid dehydrogenase Increased conversion of androstenedione in estrogens (Hillier et al,1981; Hsueh et al,1989)
5 Physiologic function of LH Provides substratum of androgens for aromatasi in granulosa cells Continuation of follicular growth and maturation initiated by FSH LH preovulatory surge Formation and support of Corpus Luteum (FILICORI 1986; FINK, 1989 ; LE COTONNEC,1993)
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7 LH supplementation in multiple follicular stimulation for IVF ET in patients undergoing down regulation In patients downregulated treated with rfsh no need of supplementation with LH Devroey, P., Mannaerts, B., Sith, J., et al. (1994) Clinical outcome of a pilot study on recombinant human follicle stimulating hormone ( Org ) combined with various gonadotrophin-releasing g hormone agonists regimens. Hum. Reprod.,, 9, In patients downregulated treated with FSH HP no need of supplementation with LH Scott Sills, E. P.,.Levy, D., Moomjy, M., et al. (1999) A prospective randomized comparison of ovulation o induction using highly purified follicle-stimulating hormone o alone and with recombinant human luteinizing hormone in in-vitro fertilization. Human. Reprod., 14(9),
8 Need of LH in multiple follicular stimulation for ART It doesn t seem necessary in women with normal ovulatory characteristics High levels l of LH have been recognized detrimental t to fertilization and pregnancies rates (HULL,1994; KOUSTA,1996; AGRAWAL,1997;REGAN,1990; CONWAY,1989; BALEN, 1995; SHOHAM,1990; STRANGER and YOVICH1985; HOWLES,1986,1987)
9 More recently : need of LH Supplementation with LH in follicular stimulation for IVF/ET In patients treated with GnRH analogue, supplementing LH to FSH from the mediofollicular phase, was observed an increase in steroidogenesis, in eggs and embryo quality and increase in embryo implantation rate. (Filicori,1986, 1999,2001, 2001;Fleming,1998,2000.)
10 Recombinant LH ( Luveris ) SERONO research available since 1993 Same concentrations after administration of urinary, pituitary and recombinant LH Has been demonstrated that doesn t cause allergy and other negative effects Porchet,1994;Porchet,1995;Le Cotennec,1998 HULL,1994; AGRAWAL,1997, CHANDRASEKHER et al, 1994.
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12 Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study 12 patients (17 cycles) that needed >3000 IU of recombinant FSH (rfsh) in a previous follicular stimulation attempt for IVF, after down regulation for 3 weeks with triptoreline 0.1 mg s.c. since midluteal l phase of previous cycle (day 21st) (group A), underwent another attempt (total 12 cycle) using rfsh supplemented with rlh (75IU) since 7 day of FSH stimulation (group B). Average basal FSH of 12.2 miu/ml patients measured day 3 of cycle was Average patients age was 36.1 y Lisi et al Reproductive BioMedicine Online 2001
13 Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study Group A rfsh alone Group B rfsh+rlh P value Lisi et al, Reproductive BioMedicine Online 2001
14 Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study Conclusions Supplementation with rlh was useful in patients with high basal FSH ( average 12.2 miu/ml ) and that in a previous downregulated cycle of stimulation needed > di 3000 UI of rfsh, No difference was observed in number of M2 eggs per patient (3.76 versus 4.17) or embryo - number and morphology between group A and group B There was a significant increase in fertilization incidence (60.9 versus 86.0%; P = 0.006) and clinical pregnancies (1 versus 6; P = 0.022) Lisi et al, Reproductive BioMedicine Online 2001
15 Use of rlh in a Group of unselected IVF patients ( 453 cycles ) Evaluation of LH supplementation in a group of IVF patients undergoing follicular stimulation with rfsh after down regulation All patients started with 150 UI/die of rfsh. In group A (122 cycle ) rfsh plus rlh 75 UI per day since 7 day of stimulation and eventually adjusting dose of rfsh: in group B (331 cases) treated with rfsh alone adjusting dose if necessary No difference in endocrinological and embriological parameters measured Implantation rate of 14.2 (group A) and of 9.8 (group B) ( p = 0.055) showed a general positive trend. Lisi et al 2002 Reproductive Biomedecine Online
16 Basal and peripheral levels of gonadotrophins and ovarian steroids for Group A and B Group A (n=122) (rfsh and rlh) (mean + SEM ) Group B (n=331) (rfsh ) (Mean + SEM) Basal FSH (miu/ml) NS Basal LH (miu/ml) NS Basal oestradiol ( pmol/ml) NS Basal prolactin (nmol/l) NS Down-reg LH (miu/ml) NS Down-reg FSH (miu/ml) NS Down-reg oestradiol (pmol/l) NS Maternal age NS Lisi et al: Use of recombinant LH in a group of unselected IVF patients. RBMOnline 2002
17 Outcome data following treatment of group A(rFSH+ rlh) and group B (rfsh) patients Group A (n=122) (rlh and rlh ) Mean + SEM Group B ( n=331) (rfsh) ( mean + SEM) No. Of days of stimulation Total FSH (IU) Terminal oestradiol (pmol/l) Mean. N oocytes per case Proportion of M 2 oocytes Oestradiol:M 2 ratio Proportion of M 2 oocytes fertilized Proportion of 2PN zygotes Proportion of grade 1 embryos Proportion of grade 1+2 embryos Proportion cleaved ( 2PN) Mean no. Embryos transferred Implantation rate (%) 41/228 (14.2) 74/753 (9.8) a Biochemical pregnancy (%) 36/122 (29.5) 83/331 (25.1) Deliveries (%) 30/122 (24.6) 62/331 (18.7%) a P 0,055 Lisi et al: Use of recombinant LH in a group of unselected IVF patients. RBMOnline 2002
18 Implantation rate in group of patients A(rFSH+ rlh) and B (rfsh) when LH at down regulation was <1.0mUI/ml or basal FSH was > 10mUI/ml or administration of FSH was > of 2500UI Down reg LH <1 Mui/ml Basal FSH >10 mui/ml FSH >2500 UI Groups of Group A Group B Group A Group B Group A Group B patients (rfsh+rlh) ( FSH alone ) (rfsh+rlh) ( FSH alone (rfsh+rlh) ( FSH alone Implantation rate 4/26 (15.4%) 0/38 a 2/21(9.5%) 0/19 5/7 (71,4%) 1/14 b (7,1%) Ap < 0,005 Bp < 0,001 Lisi et al: Use of recombinant LH in a group of unselected IVF patients. RBMOnline 2002
19 Use of recombinant LH in a Group of unselected IVF patients Conclusions (1) This study on 453 cycles demonstrates that adding rlh in unselected patients undergoing down regulation and multiple follicular stimulation with rfsh doesn t modify endocrinological and embriological parameters rlh overimposion showed a general but not significant increase in implantation rate ( p= 0,055)
20 Use of recombinant LH in a Group of unselected IVF patients Conclusions (2) Gives significantly better results if: The patient is deeply downregulated in terms of LH concentration ( <1UI/L) If an elevated amount of rfsh ( >2500 UI) is necessary to complete stimulation If basal FSH of patients is >10 UI/L
21 What is optimal amount of rlh to administrate? A research on immature mices showed that maximum effect is obtained with an activity of LH from 5% to 50% of FSH activity. Results with an LH activity from 5% to 50% remain constant (ANDERSEN et al, 1999) The European Recombinant Human LH Study Group studied efficacy of rlh in substaining follicular recruitment and growing induced by rfsh, showing that a range of IU rlh per day is well tolerated and non immunogenetic The European Recombinant Human LH Study Group 1998
22 Evaluation of two doses of recombinant luteinizing hormone supplementation in an unselected group of women undergoing follicular stimulation for in vitro fertilization To evaluate the efficacy of two doses of recombinant (r)lh, 75 IU (recommended) or 37.5 IU, for follicular stimulation and outcomes in a randomized cohort of IVF patients. Pituitary desensitization was achieved with triptorelin (0.1 mg SC), and gonadotropin stimulation was performed with either rfsh alone (group A) or in combination with rlh in one of two doses: 37.5 IU (group B) or 75 IU (group C), daily. All patients were started on a daily dose of 150 IU rfsh. If addition or a reduction in stimulation was required, this occurred by modulating the amount of rfsh only; With rlh supplementation there was a significant increase in the incidence of implantation (9% for rfsh only [group A] vs. 11% and 16% with 37.5 IU rlh and 75.0 IU rlh [groups B and C], respectively) and clinical pregnancy (19% vs. 23% and 31%) (P.01 and P.04, respectively), Lisi et al.fertility and Sterility Vol. 83, No. 2, February 2005
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24 ap.01 (No CP significantly higher in 75 IU rlh group than in FSH-only ygroup).
25 Evaluation of two doses of recombinant luteinizing hormone supplementation in an unselected group of women undergoing follicular stimulation for in vitro fertilization Conclusions (1) With the supplementation with rlh there was a significant increase in the incidence of implantation (9% for rfsh alone [group A] vs. 11% e 16% with 37.5 IU rlh e 75.0 IU rlh [group B and C]. There was a significant increase in incidence of clinical pregnancy (19% (Group A) vs. 23% ( Group B) and 31% (Group C) (P.01 and P.04, respectively). 75IU of rlh gave better results than 37,5IU even if not in a significant way
26 Evaluation of two doses of recombinant luteinizing hormone supplementation in an unselected group of women undergoing follicular stimulation for in vitro fertilization Conclusions (2) There was a significant increase (P.01) in incidence of pregnancy when rlh was added in patients who had a basal FSH > 10 IU/L In a group of patients who underwent a deep downregulation, there wasn t a significant difference in percentage of clinical pregnancy with rlh supplementation (38%) compared with the group of patients treated only with rfsh (17%) but there was an obvious trend towards an improvement in the results.
27 Number of IU of rlh used for supplementation N. patients Basal FSH UI di rlh Implantation rate Laml Media 7,75IU/L75IU/L 75 (since 1st day) 16.6% 6% Lisi % Lisi Non selezionate % Lisi % De Placido <10 IU/L 75 versus % (75UI) 13.5% (150UI) Marrs % ( <35aa ) 21.7% ( >35 aa ) Ferraretti Non indicato % De Placido < 9IU/L ,1% Lisi Non selezionate 37,5 versus 75 11% (37,5 UI ) 16% (75 UI )
28 But why peripheral LH is so important for follicular growing and multiple follicular stimulation? The presence of circulating LH seems to be fondamental in completion of M1 and embryo competence
29 Current LH debate Requirement for LH - Oocyte/Embryo development Moor et al, 1985 FSH with LH stimulated oocyte protein synthesis Armstrong et al, 1991 human embryo development only went beyond D2 with FSH:LH/1:10 Zhang et al, 1993 Improved in vitro matured oocytes and cleavage with HMG Weston et. al, 1996 In Macaque LH improves embryo s tolerance to cryopreservation Cortvrindt et al 1998 Improvement in completion of M1 Anderiesz et al 2000 maturation to M2, embryo developmental competence Alviggi et al 2007 cumulus apoptosis in LH absence; rlh improves cumulus chromatin quality for oocyte maturation
30 Role of LH in completion of M1 COMPLETION OF MEIOSIS 1 IN MOUSE OOCYTES IN VITRO 76% 46% FSH FSH/LH 39% LH Addition of rlh to rfsh to culture medium did significantly improve (P < 0.05) the completion of the first meiotic division
31 rlh on Maturation of Human Oocytes and Embryo Development in vitro Anderiesz et al, 2000 Sig Developmental Competence and Expression of Oocyte Proteins Me ean % rfsh & rlh on OM & Embryo Development In Vitro No Hormones FSH FSH:LH/1:10 Day 2 Day 3 Day 4 Day 5
32 Study on cleavage and blastulation in human in presence or absence of LH in follicular stimulation Fishel et al paper in press
33 Preliminary examination of the data There is a minimum requirement for peripheral LH to ensure maximal embryo cleavage An adequate cleavage is presumably caused by the initial effect on oocyte competence Fishel et al in press
34 Proportion (%) of embryos reaching advanced cell stage % 30 LH <1.0; FSH only LH<1.0; rlh + FSH LH>1.0; FSH only 0 Day 2 Day 3 Day 4 Day 5 Day of Cleavage Fishel et al, in press
35 Summary Patients profoundly down-regulated have significantly reduced chance of pregnancy if remaining LH is depleted during Follicular Phase Numerous studies The result of profound down regulation could compromise oocytes leading to compromised embryo cleavage This study Lack of LH during maturation in vitro causes depleted protein synthesis Several studies The cause of compromised oocytes may also be erroneous downstream epigenetic pg (gene methylation) effects as a consequence of LH deprivation hypothesis Fishel et al, in press
36 Thank You!
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