Growth Factors as Therapy for the

oocyte or Embryo Bad Idea

Marius Meintjes

Frisco Institute for Reproductive Medicine Dallas, Texas, USA

mmeintjes@friscoinfertility.com

Introduction

Not making the case that growth factors are not needed

Growth factors should not be added to today’s culture media

How not to do it

Are Current Culture Conditions Devoid of Growth Factors?

Control HSA SSS

10 KDa

75 KDa

250 KDa

Protein and Growth Factors

Hormone/ Growth Factor

HSA Company 1

HSA Company 2

SSS Lot 1

SSS Lot 2

SSS Lot 3

FSH (mIU/mL) 8.2 6.5 4.0 3.9 4.0 hCG (mIU/mL) 51.4 28.0 18.0 10.9 10.7 Progesterone (ng/mL) - - 0.31 0.4 0.32 DHEA-S (μg/dL) 240.6 68.1 63.5 216.7 224.4 TSH (μIU/mL) 0.057 0.110 0.144 0.138 0.143 IGF-2 (ng/mL) - - 328 281 284 VEGF (pg/mL) - 17 30 27 23 Insulin (μIU/mL) - - 2.6 4.1 4.5

Common hormones and growth factors in commercial IVF protein supplements

Another Source of Growth Factors The Embryo

Embryo produces IL1, LIF, EGF, TGFB, IL6

(Paria & Dey, 1990)

Bovine Embryos Cultured in Groups (30/400 μl) or Individually (1/20μl).

Treatment Group Cleavage (%) Blastocysts (%) Hatching (%) 72 h Vitrification

Group Culture 92.1a 54.8a 96.9a

Individual Culture 86.0b 44.6b 73.5b

Group Culture and Laboratory Outcomes

(Pereira et al., Theriogenology, 2005) a,b Χ2; P<0.05

Species Author Number of Embryos/Drop

Embryo Density (μl/embryo)

Mouse Wiley et al., 1986 20 0.5

Mouse Canseco et al., 1992 5 2.0

Hamster Schini & Bavister, 1988 2 <0.5

Cow Nagao et al., 2008 50 0.5

Cow Donnay et al., 1997 20 1.0

Human Ebner et al., 2011 4 7.5

Group Culture and Optimal Embryo Density

When culturing 250 cow embryos in 50 μl (0.2 , μl/embryo), embryo development was compromised (Nagao et al., 2008)

Species Author Embryo Distance (μm)

Pig Stokes et al., 2005 80-160 (>640)

Cow Gopichandran & Leese, 1992 165 (>540)

Human Ebner et al., 2011 Physical touch > separation

Group Culture and Spacing

Variables to Consider when Optimizing Group Culture:

Embryo density Embryo spacing Quality of companion embryos Microdrop volume Culture medium Changing of medium Protein supplementation Type of dish

Culture Dynamics Group vs., Single Culture

  Time to evaluate

  Time to change out media

  Incubator recovery time

  Temperature control

  pH control

  Cost

Should ensure that the information gained justifies the damage done.

E.g., PGS/PGD

Are Growth Factors Needed during the Cleavage Stage?

Culture Dynamics Group Culture and Growth Factors: When culturing human embryos in groups between days 1 and 3, no benefit was seen (Spyropoulou et al., 1999) (Rebollar-Lazaro & Matson, 2010)

However, when culturing to the blastocyst stage, more usable blastocysts were obtained in women <35 years of age when cultured in groups (Rebollar-Lazaro & Matson, 2011)

30

40

50

60

70

Complex Simple

62.8

54.2

Liv

e B

irth

Rat

e (%

)

Protein Supplement

Live Birth Rate per Retrieval for the Complex and Simple Protein Supplementation Treatment Groups

Protein and Live Births

Live Birth Rate is Different. GLM; P<0.05 (Meintjes et al., Human Reproduction, 2009)

40

45

50

55

60

Low Growth Factor High Growth Factor

Mal

e / F

emal

e (%

)

Culture System 2

Male:Female Sex Ratio for Low- and High Growth Factor Protein Supplementation

Males Females

Protein and Sex Ratio

Sex ratio different χ2, P<0.09. (Meintjes et al., Fertil Steril, 2009)

20

30

40

50

60

70

Day 3 Day 5

*

Live

Birt

h R

ate

(%)

Day of Transfer

Protein Supplementation and Day of Transfer

Simple Complex

*Live Birth Rate is Different. GLM; P<0.02

Protein and Embryonic Genome

(Meintjes et al., Human Reproduction, 2009)

Why Can the Addition of Growth Factors be Bad?

Cultured in the presence of serum very bad •  Altered genetic imprinting igf2, H19, Grb 10Grb 7

(Khosla et al 2001)

•  Large offspring (Behboodi et al., 1995)

Operate at nano- and pico-level •  Dose difficult to measure/supplement •  Overdose in quantity or time may induce birth

defects •  Timing and temporal combinations almost

impossible

Why Can the Addition of Growth Factors be Bad?

Not growth factors, but cytokines •  Dynamic modulation •  T½ may be minutes •  Inhibition

Single factor can have diverse and/or opposite effects that can be dose dependent, presence/absence of others

No single cytokine is likely to affect embryo growth - EmbryoGen® not the way to do it.

GM-CSF (EmbryoGen®) •  1322 Patients (564 treatment, 585 control) •  Effect of GM-CSF affected by HSA

concentration in medium •  HSA 2 mg/ml – suboptimum control medium

performance. After 620 patients, increase to 5 mg/ml.

GM-CSF (EmbryoGen®) 2mg/ml HSA 5mg/ml HSA Overall

Control IR 12 weeks 16.7% 21.1% 18.7%

GM-CSF IR 12 weeks 23.5% 22.4% 23.0%a

Control Live Births 22.3% 26.2% 24.1%

GM-CSF Live Births 28.4% 29.5% 28.9%A

a,A, P<0.03

Ziebe et al., 2013, Fertil Steril 99(6):1600-1609

Why Current Cytokine Conditions May be Sufficient Look Elsewhere?

Studies in the human demonstrating rescue from suboptimum culture conditions e.g., ongoing implantation rates from 18.7% to 23.0% (Ziebe et al., 2013)

To demonstrate an effect, always in absence of- or low protein

Cross-over and back-up mechanisms – knock-outs rarely successful

Why Current Cytokine Conditions May be Sufficient Look Elsewhere?

Some expression may not have anything to do with embryo quality, but may be hatching, endometrium receptivity or synchronization – limited with freeze all.

The effect of aneuploidy – some report implantation rates >80%. Concentrate on selection (metabolomics, time lapse, PGS)

Addition of Growth Factors

Improved Clinical Outcome

Compromised Clinical Outcome

No Change

Long-Term Effects

Risk Benefit

?