Culture conditions and its impact on cell lineages and ......dpc 0.5 dpc 0.5 dpc 3.5 dpc 4.5 dpc 2.5...

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Culture conditions and its impact

on cell lineages and birth weight

SASREG Fertility expert forum 2019

07.-08. September 2019

SASREG 07.-08.09.2019 | PD Dr. rer. nat. Verena Nordhoff | culture conditions

Centre of Reproductive Medicine and Andrology

University Hospital Münster

Dr. rer. nat. Verena NordhoffAssistant Professor

Reproduktionsbiologin (AGRBM)

Senior Clinical Embryologist (ESHRE)

2SASREG 07.-08.09.2019 | PD Dr. rer. nat. Verena Nordhoff | culture conditions

I have no conflict of interest with respect to

the presentation.

The views expressed in this presentation are

those of the presenter and do not necessarily

reflect the views of Merck. This presentation

is for medical education purposes only.

Preimplantation development

- an important step in early life


Winslow 2001

https://embryology.med.unsw.edu.au

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Important changes occur during these steps

(modified from Sakkas and Vassalli 1993)


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Morphology and the creation of three lineages

Adapted from Zernicka-Goetz et al. 2009.

In preparation: Ph.D. thesis Tim Pock, CeRA



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Models for cell lineage allocation

adapted from Xenopoulos et al. Results Probl Cell Diff 2012

Fgf

signalling


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TS (Trophoblast)

ES (ICM, Epiblast Epi)

XEN (prim. Endoderm, Amnion)

Which cell lineage forms

which part of the embryo?


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Boiani lab


What is known?

➢ Influence on birth weight after ART1

➢ No difference on birth weight or length2

➢ Gene expression profile different in viable embryos

compared with non-viable embryos in various species3

➢ ART media have influence on pregnancy rates and birth

weights in humans and in mice4

Evaluate the interplay of

➢ intrinsic factors i.e. genetic predisposition or ‘nature’

➢ extrinsic factors, i.e. environment or ‘nurture’

1 Dumoulin et al. 2010, Nelissen et al. 2012, Kleijkers et al. 2016, Zandstra et al. 2015, 20182 Lin et al. 2013, de Vos et al. 2014, Vergouw et al. 2014, Lemmen et al. 20143 El-Sayed et al. 2006, Parks et al. 2011, Ghanem et al. 2011; Held et al. 2012, Jones et al. 20084 Dumoulin et al. 2010, Schwarzer et al. 2012, Hemkemeyer et al. 2014, Casser et al. 2017


Pre-implantation

development

mouse

human


Winslow 2001

www.devbio.biology.gatech.edu

Winslow 2001

https://embryology.med.unsw.edu.au

Schwarzer C, Esteves TC, Araúzo-Bravo MJ, Le Gac S, Nordhoff V, Schlatt S, Boiani M. HR 2012

Hemkemeyer SA, Schwarzer C, Boiani M, Ehmcke J, Le Gac S, Schlatt S, Nordhoff V. HR 2014

Schulte K, Ehmcke J, Schlatt S, Boiani M, Nordhoff V. Theriog. 2015

in vivohuman

culture media

(+ embryo transfer)

In vitro

ET

Experimental design


Preimplantation culture results in different

blastocyst and fetal rates

Schwarzer C, Esteves TC,

Araúzo-Bravo MJ, Le Gac S,

Nordhoff V, Schlatt S, Boiani M.

HR 2012

Boiani lab


Proportion of TE and ICM are changed

G FICM

TE

Schwarzer C, Esteves TC, Araúzo-Bravo MJ, Le Gac S, Nordhoff V, Schlatt S, Boiani M. HR 2012

Schulte K, Ehmcke J, Schlatt S, Boiani M, Nordhoff V. Theriog. 2015


But – do these changes remain after

implantation?

No difference in

weight or length

Normal skeleton

formation

Cartilage in blue

(AlcianBlue), ossified bone in

red (AlizarinRed).

Transparent preparation

Hemkemeyer SA, Schwarzer C, Boiani M, Ehmcke J, Le Gac S, Schlatt S, Nordhoff V. Human Reproduction 2014

ET in vitro

G F


Is there a special factor in the

culture medium that has an

influence on cell lineages?


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Eatsmarter.de

Eatsmarter.de

60.8 ±4.0 TE cells♂61.9 ±4.8 TE cells♀

45.8 ±3.1 TE cells♂38.6 ±3.6 TE cells♀

0.2mM

28mM

74%

56%

No change in body weight regardless

of glucose content

Bermejo-Alvarez et al. Mol Rep & Dev 2012

Impact of glucose on trophectoderm

Survival

to term


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Saenz-de-Juano et al., MHR 2016

Influence of hormonal stimulation

and in vitro culture

in vitro

follicle culture

Superovulation

in vitro culture

Mating and

in vitro culture

Mating

No culture


MEST, a maternal Imprint

– paternal and maternal Allel

X

SASREG 07.-08.09.2019 | PD Dr. rer. nat. Verena Nordhoff | culture conditions Saenz-de-Juano et al., MHR 2016

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Wale and Gardner HRU 2015

Impact of extrinsic stress on the embryo


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Similar but not identical

Pixabay.com


Monozygotic twins and the concept of totipotency

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adapted from chili-chins.de


The concept of totipotency

Totipotency is

the capability of a single cell to produce a

fertile adult organism

- when placed in a supportible environment

(Edwards et al. 1997, Condic et al. 2004)

Casser E, Israel S, Witten A, Schulte K, Schlatt S, Nordhoff V, Boiani M. Sci Rep 2017


Cell lineage allocation of twin embryos

Trophectoderm Primitive Epiblast

Endoderm


Casser E, Israel S, Witten A, Schulte K, Schlatt S, Nordhoff V, Boiani M. Sci Rep 2017

Concordant and discordant development

Only 34% concordant pairs!Casser E, Israel S, Witten A, Schulte K, Schlatt S, Nordhoff V, Boiani M. Sci Rep 2017


Concordant and discordant development

after embryo transfer

Casser E, Wdowik S, Israel S, Witten A, Schlatt S, Nordhoff V, Boiani M. Mol Hum Reprod accepted


Boiani lab

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Fetus and Placenta


Just submitted: Bachelor thesis by Lynn Ogoniak

Influence of growth factors in culture medium:

→ no change in placenta area or height

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Transgenerational effects?

Calle et al. Theriog 2015


3131

Münster

Muenster.de

Stadt-Muenster.de

Muensterland.de

WN.de

http://repro.klinikum.uni-muenster.de

Email: [email protected]

Thank your very much for your attention!

IMFUKM Münster

(NO 111212)

SCHL 394/9

NO 413/3-3

(NWO (NL) no. 63-258)

Funding

Ellen Casser, Michele Boiani, MPI Münster

Anika Witten, Human Genetics, UKM

Katharina Schulte, Jens Ehmke, Animal Facility, UKM

Séverine le Gac, MESA+, University Enschede, NL

Anke Cordes, Kinderwunschzentrum, UKM

CollaborationsStefan Schlatt

Sabine Kliesch

Jörg Gromoll

Sandra Laurentino

IVF-Lab

Friederike Macke

Elena Plester

Nordhoff-Lab

Tim Pock

Lynn Ogoniak

http://www.dfg.de/index.jsp

Culture conditions and its impact on cell lineages and ......dpc 0.5 dpc 0.5 dpc 3.5 dpc 4.5 dpc 2.5...

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