The Ideal Time for Embryo Transfer
Ramazan Mercan, MD Koç University
School of Medicine
Aydıner et al., Cur.Mol.Med. 2010
OOCYTE BLASTOCYST
IN VITRO CULTURE
PN Morphology
0 16-‐18 24-‐25 48 72 96-‐120 Early Cleavage
Day 2 embryo Day 3 embryo
Multinucleation Sperm
Balaban HR 2001
Isiklar&Balaban J Rep.Med 2002
Rjinders HR 1998, Shapiro FS 2000,Ragione R.B.Endocrinol 2007
Yakin&Balaban FS 2006
Quality
Balaban FS 2000, Gardner FS 2000
Embryo Selection
• Pyruvate-‐lactate, glucose, aminoacids, sHLA-‐
G, lepDn in culture media
• O2 consumpDon
• Omics (Metabolomics, Genomic-‐PGS, proteomics, transcriptomics)
• Polarized microscopy
• Time-‐lapse monitorizaDon
Blastocyst stage embryo transfer-PROS
• BeLer correlaDon between embryo
morphology and chromosomal structure
• BeLer embryo/endometrium synchrony
• Improved the odds of transferring a viable embryo
• Decreased uterine contracDons
• Decreased mulDple pregnancy rate
Blastocyst transfer-CONS
• Higher cancellaDon rate
• The risk of losing and viable embryo due to extended culture
• Decreased rate of freezing
• Increased cost of laboratory
• Risk of monozygous twin
• Altered sex raDo
• EpigeneDc changes
• Increased neonatal/perinatal morbidiy
Comparative Studies
D2/3 vs D5/6
• ProspecDve-‐randomize
• ≥ follikül
• Max no. of embryos transferred: 2
• D2/3: 80, D5/6: 64
• ImplantaDon rates: %21.1 (D5/6) vs %20.9
• CPR: %36.7 vs %32.5
• At least 1 good quality embryo
– %60 vs %37.5 (higher in blastocyst group)
– Hreinsson J. Eur J Obstet Gynecol Reprod Biol 2004
Single cleavage vs single blastocyst
• ProspecDve randomized
• 351 women <36 years
• 176 cleavage stage and 175 blastocyst transfer
• LBR:
• %32 vs %21.6
• MonozygoDc twinning
– 2 in the cleavage group
– Papanikolaou EG. N Eng J Med 2006
Single ET& BT: A prospective randomised trial
Zech et al.,F&S 2007
Patients ≤36, 1st./2nd. Attempt, ≥5 2PN
PR: 42.2% when ≥4G1, with 95.5% suitable for SBT,
PR: 27.8%when ≤3G1, with 88.5% suitable for SBT
Guerif et al., HR 2009
FETs: Higher (not.sig.) cryosurvival with cleavage embryos than with blastocysts higher no.of deliviries(not. sig..) with sET compared with the SBT
ProspecGve
Single blastocyst vs two cleavage
• RetrospecDve
• LBR
– %27.2 vs D2: %24.8 (NS)
• MulDple pregnancy rate
– Significantly lower in blastocyst group
– Zander-‐Fox Dl. Aust NZ J Obstet Gynaecol 2011;Oct:406-‐10
Cycles without top quality embryos
• 450 women, <36 years
• ProspecDve
• Single blastocyst vs two cleavage stage
• Embryo transfer rate
– %88 vs %100
• LBR: % 26.7 similar in both groups
• MulDple pregnancy: %3.3 vs %23.3
• Guerif F, Hum Reprod 2011 Apr
The Cochrane Library 2013, Issue 6
A clinic with 31%
LBR with ET, may have a LBR of 32-‐42%
with BT
*POR
*Unselected
*Unselected
*
*
*
Moderate
All studies used slow-‐freezing for blastocyst cryopreservaGon!!
BeYer survival, transfer and IR are expected in terms of competence and benchmarks.
Alpha consensus meeGng on cryo KPIs and benchmarks. RBM Online 2012
Cochrane-2012
• LBR (D2/3 vs D5/6)
– %31 vs %32-‐42, OR: 1.40 (4RCT)
• CPR
– %38.6 vs %41.6, OR: 1.14 (NS) (23 RCT)
• Miscarriage rate
– OR: 1.13 (NS) (13 RCT)
• CumulaDve pregnancy rate
– %56.8 vs %46.3, OR: 1.58 (4RCT)
• Embryo freezing
– OR: 2.28 (11RCT)
• CancellaDon rate
– %3.4 vs %8.9, OR: 0.35 (16 RCT)
Clinical outcome of fresh and vitrified-‐warmed blastocyst and cleavage-‐stage embryo transfers in ethnic Chinese ART
paGents
• <35 years (D5 vs D3)
– CPR: 41.07% vs 47.08%, p>0.05
– ImplantaDon rate: (31.8% vs 31.2%, p>0.05 – CPR with VET: %56.8
– ImplantaDon rate of VET : %47 – CumulaDve pregnancy rate:
• Blastocyst: %70.1, D3: %51.8, p<0.05
– CumulaDve mulDple pregnancy rate: Same
• >35 yaş (D5 vs D3)
– CPR: 33.33% vs 42.31%, p>0.05
– J Ovarian Res 2012
D3 group (n = 46) D5 group (n = 58) StaDsDcal significance
PaDents with no ongoing pregnancy in fresh IVF with vitrified embryos
65.7 % (23/35) 54.4 % (18/33) p = 0.347
PaDents going through VET 87 % (20/23) 88.8 % (16/18) p = 0.369
No. VET transfers/paDent 1.1 ± 0.6 (22/20) 1.5 ± 0.9 (24/16) p = 0.236
No. Embryos transferred/cycle 1.7 ± 0.47 1.33 ± 0.48 p = 0.02
Embryo survival rate 78.8 % (37/47) 82.1 % (32/39) p = 0.7
Clinical pregnancy rate/ VET cycle 50 % (11/22) 41.6 % (10/24) p = 0.571
ImplantaDon rate 43.2 % (16/37) 34.4 % (11/32) p = 0.452
Miscarriage rate 18.1 % (2/11) 20 % (2/10) p = 0.916
MulDple pregnancy rate 45.4 % (5/11) 10 % (1/10) p = 0.072
Ongoing pregnancy rate/paDent
in VET 45 % (9/20) 50 % (8/16) p = 1.000
CumulaDve ongoing pregnancy
rate/ paDent (fresh + VET) 43.4 % (20/46) 56.8 % (33/58) p = 0.174
Pregnancy outcome per patient in VET and cumulative pregnancy rate after fresh embryo transfer and VET
Fernandez-‐Shaw et al. J Assist Reprod Genet 2015
Cleavage: 46 pts, blastocyst: 58
Patients with poor prognosis
Weissman et al., RBM Online 2008
Prospective quasi-randomized, mixed general IVF population, No sig.dif. clinical parameters
Cancellation rate: 12/70: 17.1% ***
Weissman et al., RBM Online 2008
J Assist Reprod Genet. 2014 Mar;31(3):269-‐74. doi: 10.1007/s10815-‐013-‐0146-‐3. Epub 2013 Dec 19.
Comparison of the transfer of equal numbers of blastocysts versus cleavage-‐stage embryos a_er repeated failure of in vitro ferGlizaGon cycles.
Karacan M1, Ulug M, Arvas A, Cebi Z, Berberoglugil M, Batukan M, Camlıbel T.
METHODS:
RetrospecDve analysis of 238 couples (with previous implantaDon
failures) had equal number (two) of cleavage-‐stage embryos (n = 143) or blastocysts (n = 95) transferred in the same IVF center.
RESULTS:
The clinical pregnancy rates and live-‐birth rates were similar in the
cleavage-‐stage embryo transfer group and the blastocyst group (35.6%
vs. 40% and 32.1% vs. 35.7%; p > 0.05, respecDvely). Miscarriage rates (9.8% vs. 10.5%) and mulDple pregnancy rates (15.6% vs. 23.6%) did not differ. Although implantaDon rate was higher with blastocyst transfer than that with day 3 transfer, it did not reach to a staDsDcal significance (24.7% and 19%, respecDvely, p > 0.05).
High Estradiol Levels
• ProspecDve-‐randomized
• 200 pts, E2>3000 pg/ml, ≥4 high quality embryos
• Clinical pregnancy rates: %41 vs %59 (D3 vs D5)
• Ongoing pregnancy rates: %35 vs %52
• Elgindy EA, Reprod Biomed Online 2011 Dec:789-‐98
Comparative study of pregnancy outcomes between day 3 embryo transfer and day 5 blastocyst
transfer in patients with progesterone elevation
• RetrospecDve
• 2868 cycles
• D3 embryo transfer (n = 2345)
– CPR: 55.4% vs 46.7% (normal progesterone vs high progesterone)
– Ectopic pregnancy rates: 2.8% versus 7.9%
• D5 embryo transfer (n = 523)
– Similar clinical and ectopic pregnancy rates in both groups
– J Int Med Rs, 2013
Cleavage-‐stage biopsy significantly impairs human
embryonic implantaGon potenGal while blastocyst biopsy does not: a randomized and paired clinical trial
0 10 20 30 40 50 60
D3biyopsi + D3 biyopsi -‐ D5 biyopsi + D5 biyopsi -‐
PGS/PGD’de D5 tercih edilmeli
Metaanalysis
• Sex raDo
– OR: 1.29 (4 studies)
– Chang Hj. FerDl Steril, 2009
• MonozygoDc twin
– OR: 3.08 (9 studies)
• Luke et al., 2014
Demographics&Etiology : no stat.dif. Except mean age(29.9Bvs 30.8 E.) Treatment charecteristics: no.stat.dif. Except no.of COCs (12.3B vs. 10.4E)
Papanikolaou et al.,F&S 2010
**Delivery of 24 healthy babies reported
MZT: Hardening of ZP, disorder in the cell adhesion process secondary to the culture media, Costa HR 2001, Milki F&S 2003)
ICSI causing splitting of ICM through the artifical gap (Tarlatzis F&S 2002).
Total:587
Natural conception:0.42%
Result(s): No differences were found between the incidence of MZT in cycles that did vs.
did not use micromanipulaGon techniques. In addiGon, the length of embryo culGvaGon or type of culGvaGon media used did not affect the results. Estradiol levels and
implantaGon rates were significantly higher in group A. The incidence of MZT in families in group A was significantly higher than that in groups B and C.
F&S 2015
Neonatal Outcome
Kallen et al., F&S 2010
Kallen et al., F&S 2010
Neonatal Outcome
Congenital anomalies Preterm delivery
Blastocyst stage transfer
Kallen B. FerDl Steril 2010
Cleavage stage: 12.562 Blastocyst transfer: 1311
Clinics performing only blastocyt transfer Preterm Labor, Low birth weight, Low Apgar score, and respiratory problems
<32 weeks
<37 weeks
Congenital abnormaliDes OR: 1.29 (2 studies)
Preterm delivery OR: 1.32
Dar S. Hum Reprod Update 2014
Blastocyt Stage Transfer
Metaanalysis: 4 observaGonal study
Large for gestational age
• Zhu et al., 2014
• Maheswari and BhaLacharya., 2013
• Makinen et al., 2013
AJOG 2016
Possible etiologic factors for differences in neonatal outcome
• Extended culture of embryos in blastocyst group-‐epigeneDc effects
• Higher percentage of good prognosis paDents in blastocyst group and impaired placentaDon due to higher estradiol levels
• Higher preterm delivery and similar small for gestaDonal age rates suggest that increased perinatal mortality is primarily due to
embryonic factors in blastocyst group
Maheshwari F&S 2013
Conclusions
• Blastocyst stage transfer shortens the Dme to pregnancy
• Decreased mulDple pregnancy rate
• Although cumulaDve pregnancy rate is higher in D2/3 transfers, there is a need for further studies
• Neonatal outcome seems to be beLer in D2/3 transfer
• Does vitrificaDon increase the cumulaDve pregnancy rate in blastocyst group?
• CumulaDve pregnancy rate may even be
higher in high responders when D2/3 transfer performed
• Blastocyst transfer seems to be beLer in selected group of paDents:
– Good prognosis
– High estradiol levels?
– High progesterone levels?
– Male factor?
All-freeze protocol
Figure 2 SWOT analysis of a freeze-‐all strategy. OHSS, ovarian hypersGmulaGon syndrome.
Advantages of all-freeze
• Decreased risk of OHSS
• The need for PGS/PGD
• BeLer endometrial/embryo synchrony
• Increased endometrial recepDvity
• Decreased ectopic pregnancy rate
• Fang et al., 2015
• BeLer obstetrical and neonatal outcomes
• Pelkonen et al., 2010; Sazonova et al., 2012; Wennerholm et al., 2013; Ishihara et al., 2014
• The incidences of LBW and preterm birth of singleton FET pregnancies are similar to natural concepDons
• Pinborg et al., 2013
• A posiDve impact not only on implantaDon, but also on placentaDon and fetal growth
• Pinborg, 2012
• Scheduling of oocyte retrievals becomes easier
• The endocrine profile and, mainly, high
progesterone levels (.1.5 ng/ml) at the end of the follicular phase also become much less important
• Hormonal cycle monitoring becomes less crucial
• The avoidance of oocyte retrievals during weekend
• The possibility to iniDate ovarian sDmulaDon on any given day of the menstrual cycle
• The freeze-‐all protocol could also allow for a different approach to prevent premature LH surges, namely the use of oral
medroxyprogesterone acetate (MPA) instead of injectable GnRH analogs
Disadvantages
• Based on a few small and heterogeneous RCTs restricted mostly to high responders
– Aflatoonian et al., 2010; Shapiro et al., 2011a, 2011b
• OHSS is not completely avoided
Metaanalysis
• Higher implantaDon, clinical and ongoing pregnancy rate by performing FET
• Roque et al., 2013
