1. Key Success Factors For a
Successful IVF Laboratory
Gloria Calderón PhD
IVI-Barcelona
gloria.calderon@ivi.es
www.ivi.es
2. Factors Affecting the Success of ART
Diagnosis and therapeutic guidance
Ovarian stimulation
Oocyte retrieval
IVF procedures
Embryo transfer
Luteal phase treatment
TEAMWORK
3. Why?
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.
4. Quality Control
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
6. 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
9. 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”
10. What we are used to - Embryo Prediction Model:
Static Morphology
Number of
blastomeres
with visible
Number of blastomeres Symmetry
single
nucleus
Degree of fragmentation Multinucleation
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
11. Historic and Current Approaches
Day 1
Day 2
Day 3
Day 4
Day 5
Oocyte morphology: Dimorphism: cytoplasm, zona, PB’s
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
Day 0
13. Exact Timing, Developmental Events, Kinetics
• 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
1 2 4 8
Meseguer et al. Hum Reprod, 2011;10:2658.
18. 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
19. Slow Freezing Vitrification
Physiological
solution
Cryoprotectant
solution
Vitrification
solution
Before
cooling
During
cooling
In LN2
Ice seeding
Slow cooling
Rapid cooling
Ultra rapid
0.3ºC/min cooling
200,000ºC/min
Cryopreservation Techniques
20. The Post-Modern Lab: Standard Cleavage
Stage/Blastocyst Vitrification
Vitrification appears to be associated with significantly
higher post-thawing survival rates than slow freezing.
Loutradi et al. Fertil Steril 2008;90:186.
21. 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
22. Fresh oocytes
37ºC
Donors oocytes
Vitrified oocytes
2 hours
After OPU
Warming
1 hour after +
ICSI
2 hours after warming
24. 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.
25. 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
26. RCT Advanced Maternal Age 41-44 yrs
(2009-2011)
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
27. 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
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)
Calderón G. ALPHA 2012, London
28. IVI-BARCELONA Results:
Prospective, Randomized Trial May-July 2011
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
30. Post-Modern Lab:
Future Directions in ‘Omics’
• Development
comprehensive
Omic database
• “Grand Unification”
of transcriptomic/
proteomic and
metabolomic
technologies
Assou et al. 2011 HRU.
31. 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
33. 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
34. 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
35. 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
