Gloria Calderón PhD

IVI-Barcelona

gloria.calderon@ivi.es

www.ivi.es

Key Success Factors For a

Successful IVF Laboratory

Diagnosis and therapeutic guidance

Ovarian stimulation

Oocyte retrieval

IVF procedures

Embryo transfer

Luteal phase treatment

TEAMWORK

Factors Affecting the Success of ART

Laboratory improvements affects directly in the

ART programs results.

Program results are mostly affected by laboratory variations

Rackowsky et al. 11th World Congress on IVF and Human Reproductive Genetics. Sydney, Australia 1999.

Why?

1. Everybody must follow standard operating protocols

2. Pay attention to every single detail

3. Internal tests passed by laboratory staff every 6 months.

4. Daily monitoring of results

5. Stable culture system to control:

A. Temperature

B. pH

C. Osmolarity

Quality Control

Human resources

2000 cycles/year

6 embryologists 10 technicians

1035 cycles/year

13 (10) embryologists

800 cycles/year

5 embryologists 6 technicians

ENOUGH TIME FOR EVERY CYCLE Optimal ratio of laboratory staff to ensure high attention during the procedures

CONCENTRATION

Each sample must be handled individually.

RESPONSABILITY For the correct application of the protocols in the laboratory during ART procedures

Cohen et al., 1997 Hum Reprod 12:1742-1749

Air Quality Influence

Air Quality Influence

What might be used in the near future?

• Morphology assessment of human embryo

• Time-Lapse (embryo kinetics)

• Cryopreservation (Vitrification)

• PGS (on demand)

• Transfer at the Blastocyst stage routinely (SET)

• “Omics”

Number of blastomeres Symmetry

Multinucleation Degree of fragmentation

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting Alpha Scientists in Reproductive Medicine and

ESHRE Special Interest Group of Embryology; Human Reproduction 2011; doi: 10.1093/humrep/der037

What we are used to - Embryo Prediction Model:

Static Morphology

Number of

blastomeres

with visible

single

nucleus

Day 1

Day 2

Day 3

Day 4

Day 5

Fertilization Checks: 2PN Assessments – Z scoring

Early cleavage/ Morphologic evaluation

Genetic screen ( PGD); metabolic evaluation,

morphologic evaluation; extended culture decision

Signals of compaction

Selection blastocyst for transfer,

embryo cryopreservation, PGD

Oocyte morphology: Dimorphism: cytoplasm, zona, PB’s Day 0

Historic and Current Approaches

Post-Modern lab: Time-lapse Assessment

1 2 4 8

• cc1=t2: First cell cycle

• cc2=t3-t2: Second cell

cycle, duration of period

as 2 cells

• s2=t4-t3: Synchrony in

division from 3 to 4

blastomere embryo

Exact Timing, Developmental Events, Kinetics

Meseguer et al. Hum Reprod, 2011;10:2658.

Embryo Development until day 3 Stage

using EmbryoScope

Development Anomalies on day 2

PGS using Embryoscope

Embryo Development until Blastocyst Stage

using EmbryoScope

Cryopreservation in ART Advantages

• Great flexibility in infertility treatments

• Makes feasible postponement of embryo transfer (ET)

for future cycles (OHSS, other multiple reasons)

• Contributes to decrease the incidence of multiple

pregnancies (SET)

• Contributes to the overall success of a single

stimulation cycle by increasing the cumulative

pregnancy rate

Physiological

solution

Cryoprotectant

solution

Vitrification

solution

Before

cooling

During

cooling

In LN2

Ice seeding

Slow cooling

Rapid cooling

Slow Freezing Vitrification

Ultra rapid

cooling 0.3ºC/min 200,000ºC/min

Cryopreservation Techniques

Vitrification appears to be associated with significantly

higher post-thawing survival rates than slow freezing.

The Post-Modern Lab: Standard Cleavage

Stage/Blastocyst Vitrification

Loutradi et al. Fertil Steril 2008;90:186.

The clinical pregnancy rate has doubled with the introduction of vitrification

Tulandi, 2008; Cao et al., 2009; Smith et al., 2010

Efficiency in donation program not compromised with vitrification (RCT)

Cobo et al., 2007; 2011; Nagy et al., 2007

Prospective randomized study with own sibling oocytes demonstrates the lab

efficiency of the technique (RCT)

Rienzi et al., 2010

Cumulative ongoing pregnancy rate with oocyte vitrification without embryo

selection in a standard infertility program is comparable to what is obtained

with embryo cryopreservation

Ubaldi et al., 2010; Schoolcraft et al., 2009;

Chian et al., 2008; Kim et al., 2010;

Garcia et al., 2011

The Post-Modern Lab: Standard

Oocyte Vitrification

Fresh oocytes 37ºC

Vitrified oocytes

Donors oocytes

2 hours After OPU

Warming 1 hour after +

ICSI 2 hours after warming

Oocyte Vitrification: Clinical Evidence

Cobo et al. Fertil Steril 2011;96:277.

IVF Lab Will Resolve PGS Controversy

• Mastenbroek et al. N Engl J Med. 2007;357:9-17.

– 408 women 35-41 years: 206 PGS (434 IVF cycles), 202 Control (402 IVF cycles).

– Ongoing pregnancy rate: PGS 25% vs Control 37%.

– Live-birth rate: PGS 24% vs Control 35%.

– PGS significantly reduced the rates of ongoing pregnancies (viable intrauterine

pregnancy after 12 weeks of gestation) and live births after IVF in women of advanced maternal age.

• Hardarson et al. Hum Reprod. 2008;23:2806-2812.

– 118 women ≥38 years: 56 PGS, 53 Control (Halted accrual at interim analysis)

– Clinical pregnancy rate: PGS 8.9% vs Control 24.5% (P=0.039).

– Concluded study provided evidence against using PGS for AMA patients when performing IVF.

• Schoolcraft et al. Fertil Steril. 2009;92:157-62.

– 62 women: 32 PGS, mean age 38.3 years, 30 Control, mean age 38.2 years.

– Spontaneous abortion rate: PGS 25.9% vs Control 32.3%

– Delivery rate: PGS 78% vs Control 67.7%

– In infertile AMA patients PGS did not significantly improve outcome parameters, but a trend toward a decrease in spontaneous abortion rate with higher delivery rate was observed.

PGS: RCT Advanced Maternal age: 41-44 yrs

(2009- 2011)

Inclusion criteria:

• Age range: 41-44 years

• Number MII oocytes: ≥6 MII in a

single fresh cycle or in two cycles

(fresh+vitrified)

• Without chromosomal

abnormalities in previous

pregnancies

• < 2 previous miscarriages

• < 3 previous IVF/ICSI cycles

232 patients

informed

25 rejected

90 patients

Blastocyst

24 did not

meet inclusion criteria

Rubio et al., Poster ESHRE 2012

93 patients

PGS

183 patients

enrolled

Blastocyst PGS P-value

No. of cycles 90 93

Mean Age (SD) 41.7 (0.9) 41.8 (0.9) ----

No. of transfers (%) 73 (82.9) 70 (75.3) ----

% Abnormal embryos ---- 69.7 ----

Mean embryos transferred

(SD) 2.0 (0.8) 1.6 (0.6) ----

Ongoing PR/transfer (%) 14/74 (18.9) 30/70 (42.8) p=0.0021

OR 3.214, CI [1.518-6.806]

Ongoing PR/retrieval (%) 14/90 (15.5) 30/93 (32.3) p=0.0099

OR 2.585, CI [1.262-5.295]

Ongoing implantation rate

(%) 20/152 (13.1) 40/114 (35.1)

P<0.0001

OR 3.568, CI [1.943-6.551]

*Two-sides Fisher’s test

RCT Advanced Maternal Age 41-44 yrs

(2009-2011)

PGS Results IVI – Barcelona

Nb Oocytes (X ± DS) 1906 (9.5 ± 5.6)

Nb Biopsied E (%) 1358 (91.3)

Nb no informatives(%) 29 (2.2)

Nb abnormal (%) 879 (68.6)

% Transfers 64.2

E Transferred (X ± DS) 1.63 ± 0.8

% + BHCG 68.8

% Clinical pregnancy 64.3

% Implantation 54.2

% Miscarriage 17.2

% Twin pregnancies 33.3

% Triplets 1.9

Period: 06/2010-07/2011 Nb=242

X Age= 39.29 ± 3.7 ALL INDICATIONS

Calderón G. ALPHA 2012, London

Key factor for PGS success:

Highly trained team

Low O2 tension incubators

Stable culture system

Culture medium up to Day 5 (LifeGlobal ® Media)

Protein support (LifeGlobal ® LGPS)

Global Global TOTAL p Nb of cycles 50 42

X Age 35,48 ± 0,13 36,71 ± 0,6 NS (0,07)

X oocytes 9,44 ± 1,65 8,76 ± 1,31 NS

Nb cells D2 3,99 ± 0,97 3,81 ± 1,09 NS

% fragments D2 6,96 ± 3,68 6,69 ± 3,09 NS

Nb cells D3 7,96 ± 1,65 7,32 ± 1,67 p 0,02

% fragments D3 6,76 ± 3,88 6,48 ± 3,88 NS

Good Quality Embryos 55,16% (187/339) 53,11% (128/241) NS

Nb Transfers 48 40

X Embryos Transferred 2 ± 0,16 2,08 ± 0,18 NS

X Embryos Frozen 1,82 ± 0,86 1,07 ± 0,6 NS

Cancellation Rate 4% (2/50) 5% (2/42) NS

Implantation Rate 34% 31% NS

Pregnancy Rate 50% (24/48) 45% (18/40) NS

Multiple Preg Rate 29,17% (7/24) 33,33 (6/18) NS

Miscarriage Rate 4,17% (1/24) 5,55% (1/18) NS

IVI-BARCELONA Results: Prospective, Randomized Trial May-July 2011

‘OMICS’ Technologies

Genome

DNA

≈25,000 Genes

Transcriptome

RNA

≈1,000,000

Proteins

Proteome Metabolome

Proteome Metabolites

Chromosomes

≈2500

metabolites

Transcription Translation

23 pairs

Post-Modern Lab:

Future Directions in ‘Omics’

Assou et al. 2011 HRU.

• Development

comprehensive Omic database

• “Grand Unification”

of transcriptomic/

proteomic and

metabolomic

technologies

Single Embryo Transfer

The ideal situation to avoid multiple pregnancies

would be to perform SET routinely when:

1. Couple meets indication, basically woman’s age

2. Perfect culture system till blastocyst stage

3. Large cohort of good quality embryos

4. Extremely good cryopreservation program

5. Controlled randomized trials have demonstrated

cumulative pregnancy rates of SET+ FET are equal

to DET

Single Embryo Transfer

Conclusion: eSET is associated with decrease risk of PTB and

LBW compared with DET but higher risk of PTB when compared

with naturally conceived singletons.

Grady et al. Fertil Steril 2012;97:324-31

Single Embryo Transfer

Conclusion

The post-modern laboratory will have:

Improved techniques to evaluate embryos

- Time-lapsed morphology assessments with computerized

algorithms linked to clinical outcomes

Optimized cryopreservation (vitrification) techniques to

with higher survival, pregnancies and implantation rates

Understands value of PGS

Utilizes and unifies all data from ‘Omics’ to help more

accurate embryo selection

Allows for SET – maximizing child outcome

Unify criteria in quality control and quality assurance

Gracias por su atención

Thank you for your attention

IVI-Barcelona

Ronda General Mitre 14, 08017 Barcelona

Tel. +3493 206 3000 – Fax. +3493 205 34 28

ivibarcelona@ivi.es