ALTERNATIVAS TCNICAS EN MORFOCINTICA. CUL ELIJO PARA MI CENTRO?

JOSE L DE PABLO QUIRN BILBAO

BUSCANDO EL EMBRIN QUE EMBARAZA

En Fecundacin in vitro se trata de seleccionar los mejores embriones para transferirlos al tero materno. Para ello, los embrilogos nos basamos en criterios morfolgicos, como el nmero, simetra, la fragmentacin y su distribucin, la multinucleacin,...para identicar los embriones con mayor potencial de implantacin.
Un porcentaje elevado de todos los embriones transferidos no logran implantar
La capacidad de identificar al embrin con mayor potencial de implantacin es fundamental Neuber et al. Human Reprod. 2003

Necesitamos una seleccin embrionaria ms precisa

Scott et al. Fertil Steril. 2008

SECCIN 1

Introduccin

Debemos mejorar el microambiente embrionario para mejorar la salud embrionaria. Debemos establecer unas condiciones ptimas de cultivo durante el desarrollo preimplantacional. Es uno de los objetivos principales de investigacin en Reproduccin Humana Asistida.

INTRODUCCIN

Clasificacin Morfolgica segn criterios de ASEBIR

INTRODUCCIN
Evolucin de la poltica de transferencia

Registro SEF 2011

INTRODUCCIN
Los McCaughey, Iowa 1997

Disminuir las tasas de embarazo mltiple manteniendo unas buenas tasas de embarazo

The Biggest belly EVER!!!

SECCIN 2

Time-Lapse Technology
Con los mtodos actuales de clasificacin perdemos la informacin previa y posterior al momento evaluado Evaluacin embrionaria: Horario ms Flexible comparado con un Incubador estndar NO se alteran las condiciones de cultivo

Primeros pasos
Human Reproduction vol.12 no.3 pp.532541, 1997

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography
Dianna Payne1, Sean P.Flaherty, Michael F.Barry and Colin D.Matthews
Payne Lemmen Mio

Primeros pasos
Mio Fertility Clinic 65th Annual Meeting of the American Society of Reproductive Medicine ( ASRM ) October 2009 Atlanta, Georgia

Primera vez que se grab el proceso de Fecundacin

Time-Lapse

EmbryoScope
Embryoscope

Cmara Incubacin

Pantalla Tctil

Placas de Cultivo

Time-Lapse

10

11

Time-Lapse

12

Time-Lapse

Desarrollo Embrionario

Desarrollo Embrionario

Embriones Calidad A

Mala Calidad Embrionaria

13

Time-Lapse

6 Microscopios por Incubador

14

Time-Lapse
WOW

Una gota de medio para todos los embriones

15

Time-Lapse

Primo Vision EVO

16

Time-Lapse
Reproductive BioMedicine Online (2010) 21, 533536

CASE REPORT

Pregnancy achieved by transfer of a single blastocyst selected by time-lapse monitoring

Csaba Pribenszky, Szabolcs Mtys, Pter Kovcs, Eszter Losonczi, Jnos Zdori, Gbor Vajta

17

Time-Lapse

18

Time-Lapse

Two years of research at Standford University using a large set of cryo-preserved 2PN Human Embryos

ARTICLES
2010 Nature America, Inc. All rights reserved.

Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage
Connie C Wong1,2,7, Kevin E Loewke13,6,7, Nancy L Bossert4, Barry Behr2, Christopher J De Jonge4, Thomas M Baer5 & Renee A Reijo Pera1,2
We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction. Little is known about the basic pathways and events of early human embryo development, including factors that would aid in predicting success or failure to develop. Consequently, to increase the chances of pregnancy through IVF, multiple embryos are often transferred to the uterus, despite the potential for well-documented adverse outcomes. Development of the human embryo begins with the fusion of sperm and egg, the epigenetic reprogramming of the gametic pronuclei and a series of cleavage divisions that culminate with activation of the embryonic genome by day 3 of development1. The embryo compacts to form a morula and subsequently a blastocyst, containing the outer trophectoderm and inner cell mass1. Although development of the human embryo shares many features with other species, there are also some notable differences, including unique gene-expression and epigenetic patterns and a protracted period of transcriptional silence through the first 3 d after fertilization19. In the mouse, by contrast, activation of the zygotic genome is initiated concurrent with the first cleavage division on day 1 (refs. 7,8). Human embryo development is also more fragile than that of many other species. Human fecundity rates are relatively low, largely due to pre- and post-implantation embryo loss10,11. In vitro, 5070% of IVF embryos fail to reach the blastocyst stage12,13. Most human embryo research has been based on a small number of samples generated under diverse experimental conditions1,1417. Studies that involve imaging have been limited to measurements of early development, such as pronuclear formation and fusion and time to first cleavage1821, and molecular profiling studies have generally required pooling of oocytes, embryos or blastomeres, which masks differences in gene expression between embryos or between single blastomeres within an embryo1517,22,23. Here we sought to overcome these limitations and to define critical pathways and events of human embryo development by correlating imaging profiles and molecular data throughout preimplantation development from the zygote to the blastocyst stage. We studied a large set of supernumerary IVF embryos that had been cryopreserved at the zygote stage 1218 h after fertilization (Fig. 1). The embryos appeared representative of the typical IVF population, as they were frozen at the two-pronucleate (2PN) stage and thus indiscriminately selected for cryopreservation relative to those selected for culture. This is in contrast to embryos cryopreserved at the 8-cell stage or later, which are not selected for transfer during fresh IVF cycles and may therefore be of lower quality. With this unique set of embryos, we carried out a large-scale study that correlated time-lapse image analysis and gene expression profiling to show that successful development to the blastocyst stage can be predicted by the 4-cell stage, before EGA. RESULTS Cytokinesis as an embryo quality marker A normal human zygote undergoes the first cleavage division early on day 2, at ~2427 h after fertilization1820,24 (Fig. 2a, embryo H in Supplementary Video 1). Subsequently, the embryo cleaves to a 4- and 8-cell embryo on days 2 and 3, respectively, before compacting

1Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University, Stanford, California, USA. 2Department of Obstetrics and Gynecology, School of Medicine, Stanford University, Stanford, California, USA. 3Department of Mechanical Engineering, Stanford University, Stanford, California, USA. 4Reproductive Medicine Center, University of Minnesota, Minneapolis, Minnesota, USA. 5Stanford Photonics Research Center, Department of Applied Physics, Stanford University, Stanford, California, USA. 6Present address: Auxogyn, Inc., Menlo Park, California, USA. 7These authors contributed equally to this work.

19

Time-Lapse

Day 3 Blastocyst Prediction

100
# # #

80 60 40 20 0

85% 84% 57% 82%

Day 3 Morphology (n=343) Eeva Development (n=292) Eeva Validation (n=941) Eeva Prospective Cases (n=349)!

Specicity

!"#$%&%%%'

Wong et al. Nature Biotechnology (2010)

20

Time-Lapse
EEVA

Algoritmo HIGH and LOW

21

Time-Lapse

22

SECCIN 3

Comparativa y Conclusiones

Cul elijo para mi Centro

???

Comparativa

Informacin facilitada por Diego Ezcurra (Head of Global Fertility at Merck Serono/EMD Serono)

24

Cul?

Clnicas Grandes, muchos ciclos

25

Cul?

Incubadores Convencionales

26

Conclusiones

Podemos mejorar nuestro conocimiento respecto al desarrollo embrionario basndonos en los diferentes tiempos de divisin e intervalos de los principales eventos embrionarios (MORFOCINTICA) y no solo en la morfologa. Identificar embriones con mayor potencial reproductivo Debemos de ser capaces de reducir el Embarazo Mltiple Adaptar los parmetros clave a nuestros laboratorios

27

Quirn-Bilbao-Reproduccion-Asistida @Hospital_Quiron @QuironBilbao @jldepablo

jose.depablo@quiron.es

28