System Inefficiency in ART:

The Variable of the Variables

Pasquale Patrizio, M.D., MBE, HCLD

Yale University Fertility Center

New Haven, CT-USA

Topics

• Embryo Wastage

• Oocyte Wastage

• The “Super Donors”

• Pregnancy and Multiple rates in 40 or older

• New Genetic tools to identify competent Oocytes

and Embryos:

-CCOGE projects (genomics-transcriptomics)

High Rates of Embryos Wastage in ART

[Kovalevsky G & Patrizio P Fertil Steril 2005(84):325-30]

• To determine the percentage of embryos produced in ART and transferred that do not produce a Live Birth

• Examined trends in SART/CDC statistics from 1995-2001 and 2002-2008

Embryo Wastage =

[ Embryos Transferred / Infants delivered x 100]

Summary Statistics for Fresh Non-donor

ART in the US Between 1995–2001

Year

Mean

Embryos

TransferredTransfers

Total Embryos

Transferred Deliveries

Infants

born

Embryos

Wasted, %

Transfers

Leading to

Deliveries, %

1995 3.9 31,794 123,997 7,939 11,419 90.8 25.0

1996 4.0 35,859 143,436 10,011 14,468 89.9 27.9

1997 3.0 41,270 123,810 12,302 17,854 85.6 29.8

1998 3.5 47,529 166,352 14,789 21,503 87.1 31.1

1997 3.2 51,149 163,677 16,175 23,001 85.9 31.6

2000 3.1 59,004 183,502 18,793 26,254 85.7 31.9

2001 3.1 65,363 201,710 21,813 30,451 84.9 33.4

Kovalevsky G, Patrizio P. Fertil Steril 2005;84:325-330.

High Rate of Embryo Wastage in

Women Aged 41 - 42

0

10

20

30

40

50

60

70

80

90

100

2001 2002 2003 2004 2005

ImplantationFailure (%)

- Bromer et al.RBM, 2009

Low Rate of Multiples Over Age 40Age ≤40 41 – 42 43 – 44 p-value

Cycles 3178 363 192

Transfers 2674 235 149

Cycles with no transfer (%) 15.9 35.3 22.4 < 0.001

No. embryos transferred 2.5 3.6 3.6 < 0.001

SAB (%) 18.7 35.1 35.0 < 0.001

LBR (%)/transfer 38.1 21.2 8.7 < 0.001

- % singleton 71 82 77 0.13

- % twin 26 16 23 0.16

- % triplet 3 2 0 1.0

Singleton LBR / transfer (%) 26.5 17.4 6.7 0.004

Twin LBR / transfer (%) 10.6 3.4 2.0 < 0.001

Triplet LBR / transfer (%) 1.0 0.4 0.0 0.62

Multiple pregnancy LBR /

cycle start (%)9.9 2.5 1.6 < 0.001

- Bromer et al., 2009 RBM

High Rates of Oocyte Wastage in ART

[Patrizio P & Sakkas D Fertil Steril (91): 1061-66, 2009]

• To determine the percentage of oocytes retrieved and fully utilized that do not produce a Live Birth

• Examined SART statistics @ YFC (2005-06)

Oocyte Wastage =

[ Oocytes Retrieved / Infants delivered x 100]

Oocyte to Baby Rate

Patrizio P, Sakkas D. Fertil Steril 2009;91:1061-1066.

N Oocyte E.T. Frozen

L.B.

Fresh

L.B.

Frozen

L.B.

Oocyte, %

E.T.

(IR), %

Donors 87 1705 193 372 80 37 6.8 22.0

<35 y 206 2917 546 295 105 20 4.3 15.6

35–37 y 116 1235 299 70 49 7 4.5 15.5

38–40 y 97 843 268 14 24 1 3.1 8.9

41–42 y 45 383 134 12 4 0 1.0 2.7

Total 464 5378 1247 391 182 28 4.0 13.0

0

2

4

6

8

10

12

14

Best Prognosis Donor Other Donor

<10 Ooctes

10-20 Oocytes

>20 Oocytes

*

Live

Bir

th P

er

Oo

cyte

(%)

LB/oocyte in superDonors vs.other

Donors [Martin et al.FS2010]

Summary Points 1About 20% Embryos Transferred result in a Live Birth (LB)

About 5-7% Oocytes collected and fully utilized Result in LB (< 37 y.o.)

In women > 40 y. only 1-2% of Fresh oocytes Result in LB

Huge Biological wastage with current SOP’s, NEED to identify competent Oocytes and

Embryos

In “Super Donors” ~ 10% oocytes result in LB

• Adverse effect of IVF conditions and culture media on Oocytesand Embryos

• Follicles rescued from atresia may contain abnormal oocytes

Only a small number of Oocytes and Embryos able to produce a LIVE BIRTH in some cycles/year (i.e are competent)

Older women (particularly 40 or >) have much fewer competent oocytes, in some cycles/year

Possible Explanations

How to Identify competent Oocytes

and Embryos?

• “New” PGS methodologies

- aCGH

- SNP array

• Cumulus Cells, Oocyte, Gene Expression

(the CCOGE projects)

• Proteomics and ? Metabolomics

- Spent culture media of Oocytes and Embryos

- Follicular fluid

CCOGE Projects

• Gene expression profiling of human oocytes

at different maturational stages and after in

vitro maturation (oocyte transcriptome)

• Aneuploidy and oocyte gene expression

• Cumulus cells gene expression and oocyte

aneuploidy

Wells D, Patrizio P. Am J Obstet Gynecol 2008;198:455 e451-459.Fragouli E et al. MHR 2010

CCOGE-Summary View of the Projects

14

Follicular fluid/medium

Proteomic/Metabolomic

Cumulus cells

Oocyte

Polar

body

Patrizio P and al.. RBMOnline 2007;15:346-35; Wells & Patrizio AJOG 2008; Fragouli et al MHR 2010.

GV MI MIIIVM-MII

Oocyte stage

Gene expression in Oocytes

• However, some immature features persist

• Expression profile of IVM-MII similar to in vivo MII

• up to 18,300 genes expressed in oocytes

• ~70% of genes shared between different oocyte stages

GV

IVM MII

MII

Venn Diagram

6,638 genes expressed in every oocyte tested

Biological

process

Total

H.Sapiens

genes

Number of genes up-regulated

in IVM-MII versus in vivo MII

Number of genes up-regulated

in GV versus in vivo MII

# Expected +/- P-value # Expected +/- P-value

Signal

transduction3406 24 43 - 2.14x10

-263 122 - 1.02x10

-8

Nucleic acid

metabolism3343 69 42 + 6.17x10-4 175 120 + 5.60x10-6

Cell surface

receptor 1638 9 21 - 4.17x10-1 23 59 - 6.67x10-6

Protein

metabolism and

modification

3040 62 38 + 2.55x10-3 159 109 + 2.48x10-5

tRNA

metabolism42 6 1 + 2.64x10-3 10 2 + 5.88x10-4

Pre-mRNA

processing291 12 4 + 5.47x10-1 26 10 + 4.44x10-3

mRNA slicing 214 10 3 + 8.80x10-2 20 8 + 2.68x10-2

Nuclear maturation achieved

Appropriate mRNA levels of most genes for meiosis, chromosome segregation, etc

Abnormal expression of storage and homeostatic proteins

Gene Expression in IVM Oocytes

Cytoplasmic maturation deficient

GV-like expression of genes for mRNA processing, protein synthesis, protein modification

Ferritins (iron storage)- FTL, FTHL17

Calcium ions (ATP2B4) and growth factor homeostasis (IGFPB5)

Wells and Patrizio,AJOG 2008

Aneuploidy and Oocyte Gene Expression

• Results (n=30 polar bodies)– 6 normal oocytes

– 6 abnormal oocytes

– 4 aneuploid, 1 double aneuploidy, 1 structural abnormality

• 48 genes with highly significant differences in expression between the 2 groups (P<0.01)

• A further 261 genes with smaller statistical expression differences (P<0.05)

Altered genes include several involved withSpindle dynamics (? explanation for aneuploidy); Chromatin structure; Cell cycle regulation and

specific Metabolic pathways

Wells D, Patrizio P. Am J Obstet Gynecol 2008;198: e451-459. Fragouli E. et al MHR 2010

To Reduce IVF inefficiency

• PGS for every embryo(aCGH and SNP)

• Microarray to assess oocytes by gene profiling(CCOGE and PB)

• Continuous Embryo Imaging• ? Markers embryo quality (proteomics,

metabolomics in spent culture media of oocytes and embryos)

[Selection of Competent Oocytes and Embryos]

Non-invasive imaging of human embryos before embryonic

genome activation predicts development to the blastocyst stage

[C.C Wong et al. Nature Biotechnology, Oct.2010 e-pub]

First cytokinesis: 14.3 ± 6.0 min

Time between first and second mitosis: 11.1 ± 2.2 h

Time between second and third mitosis: 1.0 ± 1.6 h

Predicting blastocyst stage (sensitivity and specificity of

94% and 93%), if embryos having a first cytokinesis

of 0–33 min, a time between first and second mitoses

of 7.8–14.3 h and a time between second and third

mitoses of 0–5.8 h

Conclusions and Future Directions

• The human oocyte is the most important

variable for the success of IVF

• To better understand genetic pathways for

oocyte competence (Cumulus cells screening)

• Better embryo-lab. conditions

• PGS for every embryo (aCGH or SNP)

• Blastocyst transfer for all

• Accept the “baby rate” (per oocyte or

embryo) as the proof of principle

The Ultimate Goal of ART:

A Single, Healthy and

Happy Baby

Thank You!!!