LH vs HCG como suplemento de la actividad LH durante la estimulación ovárica. B. Coroleu, C. Dosouto, M. Alvarez , y PN. Barri. Departamento de Obstetricia, Ginecología y Reproducción del Hospital Universitario Quirón Dexeus

INTRODUCCIÓN In our IVF programme, we detected a significant decrease of PR from 38 years onwards, which is in agreement with what has been reported by other authors that have found that between 37 and 38 years the fertility decline accelerates (Faddy). 2

INTRODUCCIÓN Fase Folicular Precoz: Sintesis de Andrógenos In our IVF programme, we detected a significant decrease of PR from 38 years onwards, which is in agreement with what has been reported by other authors that have found that between 37 and 38 years the fertility decline accelerates (Faddy). Fase Folicular Precoz: Sintesis de Andrógenos Fase Folicular Tardía: Síntesis de estradiol Estimulación crecimiento folicular Maduración ovocitaria 3

VENTANA TERAPEUTICA DE LH Atresia folicular (folículos no dominantes) Supresión de proliferación de células de la granulosa Luteinización prematura (folículos pre-ovulatorios) Desarrollo ovocitario comprometido/baja calidad ovocitaria Baja tasa de fecundación, baja tasa de implantación Tasa aumentada de abortos Limite superior de LH VENTANA TERAPEUTICA DE LH LH “celling” <5-10 UI/L Nivel óptimo: para desarrollo folicular, los niveles deben estar por encima del nivel umbral pero por debajo del limite superior (o “celling”) In our IVF programme, we detected a significant decrease of PR from 38 years onwards, which is in agreement with what has been reported by other authors that have found that between 37 and 38 years the fertility decline accelerates (Faddy). Umbral de LH >1.2 UI/L Crecimiento folicular comprometido Maduración ovocitaria comprometida Sintesis de andrógenos y estrógenos inadecuada Limite inferior de LH Polo A y Espinós JJ. ”Cuadernos de Medicina Reproductiva, 2014 4

COMPOSICIÓN PEPTIDICA DE LAS GONADOTROPINAS Professor Claus Yding Andersen

CHARACTERISTICS OF THE GONADOTROPINS FSH LH hCG No. of sugar residues 4 3 8 Initial half life 3-4 hours 20 min ? 12 hours Chromosome localisation of the gene for the -chain 6q21.1-23. Chromosome localisation of the gene for -chain 11 19q13.3 No. of copies of the gene 1 However, it is interesting to know that the main benefit of freezing embryos was not the frozen embryo transfer itself but the fresh one, that is to say, freezing embryos increased the probability of selecting the best embryo for transfer in the fresh cycle. In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest. Professor Claus Yding Andersen

mRNA EXPRESSION LEVELS OF LHR, FSHR, CYP19A1 and AR IN GRANULOSA CELLS FROM HUMAN FOLLICLES In our IVF programme, we detected a significant decrease of PR from 38 years onwards, which is in agreement with what has been reported by other authors that have found that between 37 and 38 years the fertility decline accelerates (Faddy). Jeppesen J et al., JCEM, 2012. 7

hCG ES CONSIDERADA COMO UN SUPER AGONISTA DE LA LH Our aim was to analyze cumulative live birth rates (fresh and frozen) in each age group and to build a predictive model of expected cumulative live birth according to age and No. of oocytes retrieved. 8

ROL BIOLÓGICO DE LA hCG HORMONA ESENCIAL EN EL EMBARAZO (Implantación, embarazo y desarrollo placentario) Primera señal desde el trofoblasto hacia la madre. Estimulación de Progesterona por el cuerpo luteo. Unión al receptor LH/CG receptor (LH agonista) Angiogénesis Quiescencia miometrial Tolerancia inmunológica local Formación del sincitiotrofoblasto In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest.   Tsampalas et al, 2010, review

HCG EXPRESSION DURING NORMAL PREGNANCY In vivo In vitro hCG Secretion n=5 n=14 n=3 5000 10000 15000 20000 25000 30000 1er T 2nd T Terme *** hCG Serum concentrations   trimesters Frendo et al, 2001 Cortesía: Prof. Thierry Fournier

hCG Y SUS DIFERENTES TIPOS hCG incluye 5 moléculas diferentes, que comparten una secuencia de aminoácidos idéntica pero presentan gran variedad en la estructura y sus funciones biológicas. hCG es la glicoproteina más glicosilada (los oligosacáridos le confieren hasta el 28-39% del peso molecular). Es una molécula muy acídica con un pH de 3.5 que es repelido por la membrana basal glomerular. Tipos de hCG: hCG ordinaria hCG sulfatada hCG hiperglicosilada Subunidad beta de hCG Subunidad beta de hCG hiperglicosilada Cada una de estas moléculas tienen patrones de glicosilación diferentes

SCHEMATIC REPRESENTATION OF THE FIVE DIFFERENT TYPES OF HCG Our aim was to analyze cumulative live birth rates (fresh and frozen) in each age group and to build a predictive model of expected cumulative live birth according to age and No. of oocytes retrieved. Cole LA, Clim. Chem Acta 2012;413:48

Syncytio-trophoblast PROPERTIES OF 5 INDEPENDENT VARIANTS OF HCG. Parameter hCG Sulfated hCG H-hCG hCGβ H-hCGβ Synthesis Syncytio-trophoblast Gonadotrope Cytotropho blast Malignant cells Mode of action Endocrine Endo/autocrine autocrine Site of action LHR LHR/TGFβ anta TGFβ anta Total Mw 37.180 36.150 42.800 23.300 27.600 Peptide Mw 26.200 Sugar Mw 10.980 9.950 16.600 7.300 11.600 % sugar 30% 28% 39% 31% 42% pI (principal peak) 3.5 NK 3.2 Metabolic Clearance Rate 36 h 20 h 0.7 h N-linked sugars 4 2 O-linked sugars In our IVF programme, we detected a significant decrease of PR from 38 years onwards, which is in agreement with what has been reported by other authors that have found that between 37 and 38 years the fertility decline accelerates (Faddy). Cole LA, Clim. Chem Acta 2012;413:48 13

HCG-H: A MARKER OF EARLY TROPHOBLAST INVASION hCG-H ng/ml hCG ng/ml 50 100 150 200 250 300 350 400 450 9 10 11 12 13 14 15 16 17 18 19 2000 4000 6000 8000 10000 12000 Week of amenorhea hCG-H and hCG in serum Cortesía: Prof. Thierry Fournier Guibourdenche et al, 2010

hCG hiperGLICOSILADA La hCG hiperglicosilada se une y antagoniza los receptores de TGFB en las células de citotrofoblasto. Esta isoforma de la hCG (H-hCG)también actúa como hCG normal, ejerciendo acciones endocrinas. El déficit de H-hCG causa implantaciones incompletas del blastocisto, embarazos bioquímicos y abortos. La H-hCG no se une a los receptores hepáticos y por tanto continua siendo activa. Las isoformas acídicas del hCG (H-hCG) poseen una vida media más larga que las menos acídicas (igual que para la FSH).

CONSIDERACIONES CLINICAS USO DEL EFECTO LH/hCG 16

Balasch J. et al, Hum Reprod.,1995. 17

hCG EN LA ESTIMULACIÓN OVARICA Checa MA. et al, Fertil Steril,2012.

In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest.   Bosch E. et al. Hum Reprod., 2008

hMG VS. FSHr+LHr / FIV (Agonistas P. largo) A: hMG B: Pergoveris Para emitir un juicio ético debemos RAZONAR para valorar si algo se considera correcto o incorrecto. Vamos a razonar a partir de los datos que tenemos y de nuestro conocimiento sobre el tema a debatir. Pacchiarotti A. et al, Fertil Steril,2010. 20

hMG VS. FSHr+LHr / FIV (Agonistas P. largo) HMG-HP FSHr+LHr P Nº Folic. (día hCG) 10.1±2.5 13.5±2.9 <0.05 Nº Ovocitos 7.3±3.1 9.8±3.3 <0.01 MII 6.0±3.2 7.3±2.8 0.08 % Pac. Con Cong. 39% 71% Tasa de Emb./ciclo 48.4% NS In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest.   Fabregues F. et al, Gynecol Endocrinol, 2013.

TASA DE EMBARAZO EN PACIENTES TRATATAS CON FSHr + LHr o FSHr INDIVIDUALIZACIÓN TIPO DE GONADOTROPINAS TASA DE EMBARAZO EN PACIENTES TRATATAS CON FSHr + LHr o FSHr Recombinant Luteinizing Hormone (rLH) for controlled ovarian hyperstimulation in assisted reproductive cycles. Cochrane Database Syst Rev. 2007 Apr 18;(2):CD005070.Mochtar et al. 14 RCT involving 2612 women comparing COH with rFSH or rFSH/rLH in IVF/ICSI . 11 trials-2396 women used a GnRH agonist . There was no statistical difference in clinical pregnancy rates reported in seven trials OR 1.15, 95% CI 0.91 to 1.4; 3 trials used a GnRH antagonist. no statistical difference in clinical pregnancy rates (one trial: OR 0.79, 95% CI 0.26 to 2.43) comparing both groups. The pooled pregnancy estimates of trials including only poor responders showed significant increase in pregnancy rate, in favour of co-administrating rLH (three trials: OR 1.85, 95% CI 1.10 to 3.11). Bosdou et al, 2012: aumento no significativo de la tasa de embarazo del 6% Impact of luteinizing hormone administration on gonadotropin-releasing hormone antagonist cycles: an age-adjusted analysis. Fertil Steril. 2011;95(3):1031-6..Bosch et al RCT performed in two age subgroups. Recombinant (r) FSH versus rFSH + rLH GnRH antagonists administration was compared. Up to 35 years old (n = 380) and aged 36 to 39 years (n = 340), undergoing their first or second IVF cycle. rLH administration significantly increased the implantation rate in patients aged 36 to 39 years[26.7% versus 18.6%, OR 1.56 (95% CI 1.04-2.33)]. A clinically relevant better ongoing pregnancy rate per started cycle was observed, although the difference was not statistically significant. Patients younger than 36 years do not obtain any benefit from rLH administration. Bosdou et al, Hum Reprod Update 2012. rLH ↑ tasa de implantación en 36-39 años Bosch E. et al; Fertil Steril 2011 22

USO DE LHr EN BAJA RESPONDEDORA Lehert Ph. et al. RB&Endoc., 2014

LHr vs. hMG: DONACIÓN DE OVOCITOS Es un fenómeno a nivel mundial Requena A. et al, Repro. Biol. and Endoc., 2014 24

LHr vs. hMG: DONACIÓN DE OVOCITOS En españa, la edad media del primer hijo se sitúa casi en los 32 años. Requena A. et al, Repro. Biol. and Endoc., 2014 25

LHr vs. hMG: DONACIÓN DE OVOCITOS rFSH+rLH HP-hMG+uFSH P Oocytes Retrieved 16.5±4.1 11.8±2.6 0.049 MII 11.8±3.7 9.5±1.8 NS % MII/Oocytes 71.2 80.6 0.003 Fertilization Rate 67.8 78.2 Top Q Embryos 3.0±0.5 3.6±0.6 Frozen Embryos 1.6±0.8 1.8±1.0 N of Transferred Embryos 1.6±0.4 1.8±0.3 The increased postponement of childbearing is mainly due to social, proffesional and economic reasons. This phenomenon leads to an increased level of childlessness couples and an increased use of ART, in fact in Europe in 2010 approximately 500.000 ART cycles were performed. Requena A. et al, Repro. Biol. and Endoc., 2014

FSH+LH vs. hMG EN INSEMINACIÓN ARTIFICIAL (>35 a.) The increased postponement of childbearing is mainly due to social, proffesional and economic reasons. This phenomenon leads to an increased level of childlessness couples and an increased use of ART, in fact in Europe in 2010 approximately 500.000 ART cycles were performed. Moro F. et al, Hum Reprod, 2015.

hMG-HP (Meriofert®) vs. hMG-HP (Menopur®): FIV Alviggi C. et al, Gynecol Endocrinol, 2013. 28

hMG-HP (Meriofert®) vs. hMG-HP (Menopur®): FIV Alviggi C. et al, Gynecol Endocrinol, 2013. 29

De Ziegler et al (No publicado) In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest.   De Ziegler et al (No publicado) 30

De Ziegler et al (No publicado) In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest.   De Ziegler et al (No publicado) 31

De Ziegler et al (No publicado) In order to counsell our patients we built a predictive model of CLBR according to age and number of oocytes retrieved. As the graph shows, the higher the number of oocytes retrieved, the higher the CLBR. Despite this fact, we can see that in 44 years and over a CLBR of 3% would never be reached irrespective of the number of oocytes retrieved. The main limitations of the study are: its retrospective nature and that freezing was done with slow freezing as it was our routine during that period This is the largest study analyzing CLBR (fresh cycle + subsequent FET) in women ≥38years. Cryopreservation significantly increased LBR in fresh but the extra benefit of the frozen cycles was limited. Women of ≥44years should be advised against doing an IVF with their own oocytes. Women in the other age groups should be counselled regarding CLBR according to their age and oocyte yield. Being able to predict ovarian response in advance is of crucial interest.   De Ziegler et al (No publicado) 32

CONCLUSIONES El efecto LH/HCG es importante en la foliculogénesis y maduración folicular. Existen diferentes formas de hCG. Las formas glicosiladas (procedentes de orina de mujer embaraza) presentan una vida media mas larga que la hCG hipofisaria y una dinámica de metabolización diferente. El uso de Gonadotropinas con contenido de hCG podrían tener un efecto positivo en la maduración final de los ovocitos El efecto LH/hCG tendría una acción positiva en perfiles de mujeres con edad >35 años. La hCG es fundamental para el proceso de implantación. El presente/futuro de la estimulación ovárica es encontrar para cada perfil de paciente la mejor gonadotrofina o combinación de las mismas. 33

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