2012 - Cleavage Stage Versus Blastocyst Stage Embryo Transfer in Assisted Reproductive Technology

Cleavage stage versus blastocyst stage embryo transfer in
assisted reproductive technology (Review)

Glujovsky D, Blake D, Bardach A, Farquhar C
This is a reprint of a Cochrane review, prepared and maintained by The Cochrane Collaboration and published in The Cochrane Library
2013, Issue 6
http://www.thecochranelibrary.com
Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology (Review)
Copyright 2013 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.
T A B L E O F C O N T E N T S
1 HEADER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 PLAIN LANGUAGE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 OBJECTIVES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Figure 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Figure 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Figure 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Figure 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Figure 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Figure 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Figure 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
16 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19 AUTHORS CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19 ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25 CHARACTERISTICS OF STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66 DATA AND ANALYSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Analysis 1.1. Comparison 1 Live birth rate, Outcome 1 Live birth per couple. . . . . . . . . . . . . . . 69
Analysis 1.2. Comparison 1 Live birth rate, Outcome 2 Live birth per couple: grouped by number of embryos transferred. 71
Analysis 1.3. Comparison 1 Live birth rate, Outcome 3 Live birth rate per couple: grouped by prognosis. . . . . 72
Analysis 1.4. Comparison 1 Live birth rate, Outcome 4 Live birth rate: grouped by day of randomisation. . . . . 73
Analysis 2.1. Comparison 2 Clinical pregnancy rate, Outcome 1 clinical pregnancy rate per couple. . . . . . . 75
Analysis 2.2. Comparison 2 Clinical pregnancy rate, Outcome 2 clinical pregnancy rate per couple: grouped by number of
embryos transferred. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Analysis 2.3. Comparison 2 Clinical pregnancy rate, Outcome 3 clinical pregnancy rate per couple: grouped by prognosis. 78
Analysis 2.4. Comparison 2 Clinical pregnancy rate, Outcome 4 clinical pregnancy rate per couple: grouped by day of
randomisation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Analysis 3.1. Comparison 3 Cumulative pregnancy rate, Outcome 1 cumulative pregnancy rate from fresh and frozen
transfers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Analysis 4.1. Comparison 4 Multiple-pregnancy rate, Outcome 1 multiple-pregnancy rate per couple. . . . . . 83
Analysis 4.2. Comparison 4 Multiple-pregnancy rate, Outcome 2 multiple-pregnancy rate per couple: grouped by number
of embryo transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Analysis 4.3. Comparison 4 Multiple-pregnancy rate, Outcome 3 multiple-pregnancy rate per couple: grouped by
prognosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Analysis 4.4. Comparison 4 Multiple-pregnancy rate, Outcome 4 high order pregnancies (more than 2 gestational sacs) per
couple. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Analysis 4.5. Comparison 4 Multiple-pregnancy rate, Outcome 5 high order pregnancy: grouped by number of embryos
transferred. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Analysis 4.6. Comparison 4 Multiple-pregnancy rate, Outcome 6 high order pregnancies: grouped by prognosis. . . 89
Analysis 4.7. Comparison 4 Multiple-pregnancy rate, Outcome 7 multiple-pregnancy rate per pregnancy. . . . . 90
Analysis 4.8. Comparison 4 Multiple-pregnancy rate, Outcome 8 high order pregnancies per total pregnancies. . . 91
Analysis 5.1. Comparison 5 Miscarriage rate, Outcome 1 miscarriage rate per couple. . . . . . . . . . . . 92
Analysis 5.2. Comparison 5 Miscarriage rate, Outcome 2 miscarriage rate per pregnancy. . . . . . . . . . . 93
Analysis 6.1. Comparison 6 Embryo freezing rate, Outcome 1 embryo freezing per couple. . . . . . . . . . 94
Analysis 6.2. Comparison 6 Embryo freezing rate, Outcome 2 Embyro freezing per couple: grouped by number of embryos
transferred. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Analysis 6.3. Comparison 6 Embryo freezing rate, Outcome 3 Embryo freezing per couple: grouped by prognostic
factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
i Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology (Review)
Analysis 7.1. Comparison 7 Failure to transfer embryos rate per couple, Outcome 1 Failure to transfer any embryos per
couple. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
couple: grouped by prognostic factors. . . . . . . . . . . . . . . . . . . . . . . . . . 98
couple: grouped by number of embryos transferred. . . . . . . . . . . . . . . . . . . . . . 100
101 ADDITIONAL TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
104 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
107 WHATS NEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
108 HISTORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
109 CONTRIBUTIONS OF AUTHORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
109 DECLARATIONS OF INTEREST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
109 SOURCES OF SUPPORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110 DIFFERENCES BETWEEN PROTOCOL AND REVIEW . . . . . . . . . . . . . . . . . . . . .
110 NOTES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110 INDEX TERMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology (Review)
[Intervention Review]
Cleavage stage versus blastocyst stage embryo transfer in
assisted reproductive technology
Demin Glujovsky
1
, Debbie Blake
2
, Ariel Bardach
3
, Cindy Farquhar
2
1
Reproductive Medicine, CEGYR (Centro de Estudios en Ginecologia y Reproduccin), Buenos Aires, Argentina.
2
Obstetrics and
Gynaecology, University of Auckland, Auckland, New Zealand.
3
Argentine Cochrane Centre IECS, Institute for Clinical Effectiveness
and Health Policy, Southern American Branch of the Iberoamerican Cochrane Centre, Buenos Aires, Argentina
Contact address: Demin Glujovsky, Reproductive Medicine, CEGYR (Centro de Estudios en Ginecologia y Reproduccin), Viamonte
1432 Buenos Aires, Argentina. demian.glujovsky@gmail.com.
Editorial group: Cochrane Menstrual Disorders and Subfertility Group.
Publication status and date: Edited (no change to conclusions), published in Issue 6, 2013.
Review content assessed as up-to-date: 21 February 2012.
Citation: Glujovsky D, Blake D, Bardach A, Farquhar C. Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive
technology. Cochrane Database of Systematic Reviews 2012, Issue 7. Art. No.: CD002118. DOI: 10.1002/14651858.CD002118.pub4.
A B S T R A C T
Background
Advances in cell culture media have led to a shift in in vitro fertilization (IVF) practice fromearly cleavage embryo transfer to blastocyst
stage transfer. The rationale for blastocyst culture is to improve both uterine and embryonic synchronicity and enable self selection of
viable embryos thus resulting in higher implantation rates.
Objectives
To determine if blastocyst stage (Day 5 to 6) embryo transfers (ETs) improve live birth rate and other associated outcomes compared
with cleavage stage (Day 2 to 3) ETs.
Search methods
Cochrane Menstrual Disorders and Subfertility Group Specialised Register of controlled trials, Cochrane Central Register of Controlled
Trials (CENTRAL) (The Cochrane Library), MEDLINE, EMBASE and Bio extracts. The last search date was 21 February 2012.
Selection criteria
Trials were included if they were randomised and compared the effectiveness of early cleavage versus blastocyst stage transfers.
Data collection and analysis
Of the 50 trials that were identied, 23 randomised controlled trials (RCTs) met the inclusion criteria and were reviewed (ve newstudies
were added in this update). The primary outcome was rate of live birth. Secondary outcomes were rates per couple of clinical pregnancy,
cumulative clinical pregnancy, multiple pregnancy, high order pregnancy, miscarriage, failure to transfer embryos and cryopreservation.
Quality assessment, data extraction and meta-analysis were performed following Cochrane guidelines.
Main results
Twelve RCTs reported live birth rates and there was evidence of a signicant difference in live birth rate per couple favouring blastocyst
culture (1510 women, Peto OR 1.40, 95% CI 1.13 to 1.74) (Day 2 to 3: 31%; Day 5 to 6: 38.8%, I
2
= 40%). This means that for a
1 Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology (Review)
typical rate of 31% in clinics that use early cleavage stage cycles, the rate of live births would increase to 32% to 42% if clinics used
blastocyst transfer.
There was no difference in clinical pregnancy rate between early cleavage and blastocyst transfer in the 23 RCTs (Peto OR 1.14, 95%
CI 0.99 to 1.32) (Day 2 to 3: 38.6%; Day 5 to 6: 41.6%) and no difference in miscarriage rate (13 RCTs, Peto OR 1.18, 95% CI 0.86
to 1.60). The four RCTs that reported cumulative pregnancy rates (266 women, Peto OR 1.58, 95% CI 1.11 to 2.25) (Day 2 to 3:
56.8%; Day 5 to 6: 46.3%) signicantly favoured early cleavage. Embryo freezing rates (11 RCTs, 1729 women, Peto OR 2.88, 95%
CI 2.35 to 3.51) and failure to transfer embryos (16 RCTs, 2459 women, OR 0.35, 95% CI 0.24 to 0.51) (Day 2 to 3: 3.4%; Day 5
to 6: 8.9%) favoured cleavage stage transfer.
Authors conclusions
This review provides evidence that there is a small signicant difference in live birth rates in favour of blastocyst transfer (Day 5 to 6)
compared to cleavage stage transfer (Day 2 to 3). However, cumulative clinical pregnancy rates from cleavage stage (derived from fresh
and thawcycles) resulted in higher clinical pregnancy rates than fromblastocyst cycles. The most likely explanation for this is the higher
rates of frozen embryos and lower failure to transfer rates per couple obtained from cleavage stage protocols. Future RCTs should report
miscarriage, live birth and cumulative live birth rates to enable ART consumers and service providers to make well informed decisions
on the best treatment option available.
P L A I N L A N G U A G E S U M M A R Y
Cleavage stage versus blastocyst stage embryo transfer in assisted conception
Embryos from assisted reproductive technologies (in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), thawed embryo
cycles) are commonly transferred into the womans uterus at either the early cleavage stage (Day 2 to 3 after egg collection) or blastocyst
stage (Day 5 to 6 after egg collection). The current thinking is that transferring embryos at the blastocyst stage is the most biologically
correct stage for embryos to be in the uterus as earlier stages are naturally in the fallopian tube, and longer culture in the laboratory
may give the scientist greater ability to select the best quality embryo(s) for transfer.
This review of 25 studies did not nd a difference in the chance of becoming pregnant between early cleavage and blastocyst stage
embryo transfer. Disappointingly, only half of the included studies reported miscarriage or live birth rates. These 12 studies showed a
small improvement in live birth rate per couple for blastocyst transfers. This would mean that for a typical rate of 31% in clinics that
use early cleavage stage cycles, the rate would increase to 32% to 42% live births if clinics used blastocyst transfer. In the 13 studies that
reported miscarriage rate, there was no difference between early cleavage and blastocyst stage transfers. Interestingly, in the four studies
that reported cumulative pregnancy rates (after both fresh and frozen thaw embryo transfers) there was an increase in those women
who had cleavage stage compared with blastocyst stage transfer.
Apart from a lowered cumulative pregnancy rate in women who had blastocyst transfer, other disadvantages included a lower rate of
excess embryos available for freezing per couple and a higher chance that there were no embryos that survived up to the stage of transfer.
For couples who obtain a high number of good quality embryos (high prognosis), however, the chance that there are no embryos for
transfer in blastocyst cycles is no different from cleavage stage transfers. These two factors may explain why there is a greater chance of
cumulative pregnancy in early cleavage stage than blastocyst stage cycles, where couples have received embryos for transfer in the initial
ovarian stimulated cycle, had excess embryos frozen, and then received thawed embryos in subsequent natural or controlled cycles.
B A C K G R O U N D
Description of the condition
Assisted reproductive technologies (ART) such as in vitro fertiliza-
tion (IVF), intracytoplasmic sperm injection (ICSI), and embryo
freezing are considered benecial for many couples who are un-
likely to conceive without treatment and for whom less invasive
forms of treatment have failed or are unlikely to be effective. The
edgling era of IVF, from1980 to the mid 1990s, was characterised
by relatively static successful pregnancy rates of around 20%. The
past decade however has given rise to advances in ovarian stimula-
tion, cell culture, embryo transfer and cryopreservationtechniques
that have culminated in signicant overall improvements in suc-
cessful pregnancies. This is evident in the annual statistical reports
from different areas of the globe. One such report, for example,
has demonstrated a doubling of the pregnancy rate per embryo
transfer cycle from 1994 to 2003 despite a decrease in the mean
number of embryos transferred (Waters 2006).
Description of the intervention
IVF involves the use of hormones to stimulate the ovaries to pro-
duce many eggs (oocytes), followed by egg collection (oocyte re-
trieval), addition or injection of sperm, fertilization, embryo cul-
ture and, lastly, the return of a few selected embryos to the uterus
(embryo transfer (ET)). Embryos have been conventionally trans-
ferred on either Day 2 or 3 when the embryos were two to eight
cells, or cleavage stage, because the uterus was thought to pro-
vide the best environment for the survival of the embryo (Laverge
2001). The question of optimal timing for embryo transfer arises
when examining the differences between the IVF procedures and
what happens naturally in vivo. Day 2 is the earliest time at which
morphological grading of the embryos is possible, allowing selec-
tion of the best embryos for transfer. Embryo morphology, along
with other factors, is thought to be highly indicative of pregnancy
outcome (De Placido 2002). Early replacement in the uterus may
be advantageous for the embryos by limiting the time spent in the
in vitro environment of the embryology laboratory.
Over the past decade there has been a steady shift in practice to
transfer of embryos on Day 5 or 6 when the embryos are blas-
tocysts, the 64-cell stage. With the introduction of a variety of
commercial preparations of sequential media in the late 1990s,
the ART service sector witnessed an explosion of worldwide in-
terest in blastocyst culture, with most clinics conducting research
into its application in their own setting. As a result, a substantial
volume of publications followed. These included conicting trials
and debates about the merits and drawbacks of extended culture.
A lack of strong consensus about the best practice for blastocyst
culture has not been aided by the fact that many of the trials were
not prospectively randomised or were underpowered. The need
for an evidence-based approach using meta-analysis of small trials
was, therefore, required to assist in deciphering the overall effect
of blastocyst culture to help identify patient subsets and practices
that might best benet from this approach.
How the intervention might work
Blastocyst culture is not novel; indeed the very rst report of an
IVF pregnancy was from a transferred blastocyst (Edwards 1995).
Despite this, cleavage stage transfer was adopted as standard global
practice early in the history of IVF for two reasons: the low devel-
opmental rate of embryos cultured past this stage; and the observa-
tion that unlike other primates, human embryos have an unusual
propensity to survive when replaced prematurely into the uterus
(Marston 1977). However, as knowledge of embryo metabolic re-
quirements expanded so did the range of more advanced culture
media (Scholtes 1996) and co-culture techniques (Menezo 1990;
Van Blerkom 1993; Yeung 1992). The most dramatic was the
understanding that the in vitro environment in which an early
cleavage stage embryo grows best is different from that for a blas-
tocyst. This led to the evolution of stage-specic (or sequential)
media (G1/G2) by Gardner in 1998 (Gardner 1998b); embryos
are transferred on Day 3 from a medium containing low concen-
trations of glucose and one or more amino acids to a medium
containing higher concentrations of glucose and a wider range of
amino acids (Gardner 1996). At this stage, the embryo undergoes
cell compaction and genomic activation so that the embryo is no
longer under the control of transcripts and RNA messages of ma-
ternal origin (Braude 1998). With the application of stage-specic
media, there have been reports of blastocyst development and im-
plantation rates as high as 60% to 65% (Schoolcraft 2001).
There are two central arguments why blastocyst culture has pur-
ported advantages over traditional cleavage stage transfer. Firstly, it
has long been recognised that it is physiologically premature to ex-
pose early-stage embryos to the uterine environment, particularly
one that has been subjected to superovulation and thus high lev-
els of estrogen (Valbuena 2001). In vivo, embryos travel through
the fallopian tubes and do not reach the uterus before the morula
(16 cell compacted) stage (Croxatto 1972), which equates to at
least Day 4 of in vitro culture. The uterus provides a different nu-
tritional environment from the oviduct; therefore it is postulated
that this may cause stress on the embryo and result in reduced
implantation potential (Gardner 1996). There is also evidence of
a signicant reduction in uterine pulsatility at the time when blas-
tocysts are transferred and therefore less chance that embryos can
be expelled (Fanchin 2001).
The second argument for blastocyst culture is their innately higher
implantation potential compared with early cleavage embryos. As
a consequence of self selection, it is postulated that only the most
viable embryos are expected to develop into blastocysts. It is widely
acknowledged that the morphological criteria used for selection
of the best embryos on Day 2 to 3 are limited. Many published
studies that debate the correlation of morphological features with
pregnancy rates can be found in the literature (Palmstierna 1998;
Puissant 1987; Roseboom 1995; Scott 2000; Sjoblom 2006; Steer
1992). It is now understood that a disturbingly large proportion
of morphologically normal Day 3 embryos are chromosomally ab-
normal, thus contributing to the 80% to 90% rate of implanta-
tion failure post-transfer that is observed in cleavage stage proto-
cols (Magli 1998). While the transfer of Day 5 blastocysts cannot
ensure the absence of chromosomal abnormality (Magli 2000),
Staessen 2004 have demonstrated that, at least in women older
than 36 years, the incidence can be reduced from 59% in Day 3
embryos to 35% in Day 5 blastocysts.
Arguments against blastocyst culture are largely related to this pro-
cess of self selection. Couples undergoing blastocyst culture are
expected to have a higher incidence of being cancelled due to failed
embryo development (Marek 1999) and of having fewer embryos
cryopreserved (frozen) (Tsirigotis 1998). Overall utilisation rates
have previously been described as the total number of embryos
transferred plus the embryos thawed divided by the number of fer-
tilized eggs. While this approach presents information about the
comparative number of pregnancy opportunities that each treat-
ment approach can provide a couple, it does not take into account
the implantationpotential for fresh and thawed embryos. Analter-
native efcacy formula was developed by Schoolcraft (Schoolcraft
2001) that does take this into account. Using the formula (mean
number of embryos transferred multiplied by implantation rate)
plus (mean number of embryos cryopreserved multiplied by im-
plantation rate) minus (1 minus cancellation rate), this group of
researchers were able to demonstrate a 19% greater efciency in
blastocyst culture compared with early cleavage stage transfers.
Disappointingly, such a utilisation and efciency analysis is not
possible in the majority of RCTs due to the lack of thaw cycle
outcomes within a reasonable time frame for trials.
There is also the question of how scientists can be so certain that
any given Day 3 embryo has the ability to become a viable blas-
tocyst in vivo but not in vitro. Based on the very wide range of
blastulation rates reported, there is evidence that not all clinical
andlaboratory environments are equal, despite identical sequential
media being used. This is an obvious compounding factor when
performing a meta-analysis. Variables such as number of incuba-
tors, gas mix, culture ware quality control (Gardner 2003b) and
the superovulation regimen (Bukulmez 2007; Schoolcraft 2001)
have all been reported to have an impact on blastocyst culture out-
comes. For this reason there may be an argument for introducing
a minimum Day 2 to 3 implantation rate (that is approximately
20%) for trial inclusion criteria, but this may differ depending on
the overall patient prognosis for each trial (for example Devreker
2000).
Other negative outcomes reported to be associated with blastocyst
culture include a higher incidence of monozygotic twinning and
altered sex ratio in favour of males (Menezo 1999). Monozygotic
twinning is frequently reported at above 1% in ART cycles (Sills
2000), while the background rate of monozygotic twins in spon-
taneous conceptions is in the order of 1 in 330. This twinning is
associated with miscarriage, serious structural congenital anoma-
lies, growth discrepancy and twin to twin transfusion syndrome.
Extended culture of an embryo has been implicated as one of the
interventions associated with anincrease inmonozygotic twinning
(Behr 2000; Cohen 1990; De Felici 1982; Jain 2004), but a recent
report suggests that improvements in cell culture techniques over
time can result in a signicant decrease in its incidence (Moayeri
2007). Similarly, as the underlying mechanisms that lead to an
altered sex ratio are elucidated, whether it be media constituents
or simply the morphological selection criteria (Luna 2007), the
imbalance may also be rectied.
Why it is important to do this review
Blastocyst culture has evolved against a backdrop of changing reg-
ulatory and community pressures. Until relatively recently it was
widely accepted that in order to achieve acceptable pregnancy
rates, several embryos were required to be replaced in the uterus
(Edwards 1983). However, pressure on the ART service sector to
assist in reducing the multiple-birth rate and high order birth rates
(more than two fetal sacs) over the past decade has seena steady de-
cline in the number of embryos transferred. Single embryo trans-
fers for selected patient groups are now considered standard prac-
tice in many clinics throughout the world (Hamberger 2005). The
importance of selecting the single most viable embryo for transfer
has intensied the search to improve the assessment of the quality
of embryos. Performing blastocyst culture may offer one of those
mechanisms (Gardner 2004; Milki 2004).
Advocates of blastocyst culture are condent that only the most
viable embryos will survive the extended culture to Day 5 to 6.
This would result in a higher probability of implantation and re-
quire fewer embryos to be transferred, thereby lowering the costly
multiple-birth rate (Gardner 1998b; Jones 1999). Critics of the
approach express concern at the increased incidence of women
failing to have embryos available for transfer (Marek 1999), al-
though the day of patient recruitment into the blastocyst program
is crucial to this argument. It is important to be aware that clinic
policies may differ on the minimum criteria for blastocyst culture
and the day on which this decision is made (for example number
of follicles, fertilized eggs, 8-cell embryos on Day 3) (Milki 1999).
It is also yet to be claried if there are patient groups for whom
blastocyst culture is disadvantageous. Most importantly, does blas-
tocyst culture achieve the primary aim of providing the subfertile
couple with a normal, healthy baby? Methods for identication
of viable blastocysts are indeed a popular research focus involving
a range of approaches which include polar-body and blastomere
genetic analysis (using microarrays or complete genomic hybridi-
sation) and metabolomic analysis of culture media (Jones 2008;
Nel-Themaat 2011).
The aimof this reviewis to determine whether the number of days
between oocyte retrieval and embryo transfer (that is the number
of days the embryos are grown in vitro) has any effect on the
success of ART treatment and in particular the live birth rate, the
most important outcome for couples undergoing treatment and
for service providers.
O B J E C T I V E S
Todetermine if blastocyst stage (Day 5to6) embryotransfers (ETs)
improve live birth rate and other associated outcomes compared
with cleavage stage (Day 2 to 3) ETs.
M E T H O D S
Criteria for considering studies for this review
Types of studies
All randomisedcontrolledtrials (RCTs) comparing early-stage em-
bryo transfers (Day 2 to 3) with blastocyst stage transfers (Day 5
to 6) were considered. Quasi-randomised controlled trials (trials
that stated they used random allocation but allocation was, for
example, the day of the week, which is not truly random) were
excluded and withdrawn from the previous versions of the review.
Types of participants
Inclusion criteria
Couples undergoing in vitro fertilization (IVF) or intracytoplas-
mic sperm injection (ICSI) for therapeutic reasons or for oocyte
donation within all patient prognosis groups.
Patient prognosis groups (patient subsets or populations) is a
term used to describe the categories that couples are assigned to
based on several factors such as their age, type of infertility, ovarian
response tothe superovulationdrugs andnumber of previous failed
attempts. See the subgroup analysis section in the Methods of the
review below for the categories.
Exclusion criteria
Couples whose IVF or ICSI cycle, or both, involved in vitro ma-
tured oocytes or pre-implantationdiagnosis. Couples where frozen
cycle results were shown but no data were available from the fresh
cycle.
Types of interventions
Inclusion criteria
Single and sequential media culture methods for IVF and ICSI
where the embryos were grown for between 2 and 6 days in vitro
prior to embryo transfer and where Day 2 to 3 transfers were
compared with Day 5 to 6 transfers.
Exclusion criteria
Use of co-culture methods as an intervention. Day 2 to 3 transfers
versus Day 5 to 6 transfers in frozen cycles where no data were
available from the fresh cycle.
Types of outcome measures
Primary outcomes
Live birth rate per couple (number of live births per couple)
Secondary outcomes
Clinical pregnancy rate per couple: number of couples
achieving a clinical pregnancy (dened by the demonstration of
fetal heart activity on ultrasound scan)
Cumulative pregnancy rate per couple (fresh and frozen
thaw embryo transfer)
Multiple-pregnancy rate per couple: number of multiple
pregnancies per couple
High order multiple-pregnancy rate per couple: three or
more fetal heartbeats per couple
Miscarriage rate: number of occurrences per couple and per
pregnant woman
Embryo freezing rate: number of couples that had embryos
frozen per couple
Failure to have any embryo transfer rate: percentage of
couples that did not have an embryo transfer
Additional outcomes not appropriate for statistical pooling
Data per cycle or per embryo transfer (ET) or per ovum pick up
(OPU) were not able to be pooled (Vail 2003). However, due to
the frequency that this form of data is reported in the literature
they have been entered into the Table of comparisons for the
following outcomes:
i) live births per OPU and ET;
ii) clinical pregnancy rate per OPU and ET;
iii) implantation rate, the number of fetal sacs divided by the
number of embryos transferred.
Search methods for identication of studies
All reports that described (or might have described) randomised
controlled trials comparing early-stage embryo transfer and blas-
tocyst stage transfer in the treatment of subfertility, using IVF or
ICSI, were obtained using the search strategy developed by the
Menstrual Disorders and Subfertility Group.
We searched the Cochrane Menstrual Disorders and Subfertil-
ity Group Specialised Register of Controlled Trials, the Cochrane
Central Register of Controlled Trials (CENTRAL) (The Cochrane
Library), MEDLINE (1966 to Feb 2012), EMBASE (1980 to Feb
2012) and Bio extracts using the Cochrane highly sensitive search
strategy and the following keywords: blastocyst/embryo or embryo
transfer/cleavage stage, ovum/culture media or embryo culture/
sequential culture/co-culture. See Appendix 1.
The MEDLINE search was combined with the Cochrane highly
sensitive search strategy for identifying randomised trials, which
appears in the Cochrane Handbook for Systematic Reviews of Inter-
ventions (Version 5.0.2, Chapter 6, 6.4.11).
The EMBASE search was combined with trial lters developed by
the Scottish Intercollegiate Guidelines Network (SIGN) (http://
www.sign.ac.uk/mehodology/lters.html#random). There was no
language restriction in these searches.
Electronic searches
Menstrual Disorders and Subfertility Group Specialised Register
(up to Feb 2012)
Ovid Cochrane Central Register of Controlled Trials (CEN-
TRAL) (up to Feb 2012)
Ovid MEDLINE(R) In-Process & Other Non-Indexed
Citations, Ovid MEDLINE(R) Daily and Ovid MEDLINE(R)
(up to Feb 2012)
Ovid EMBASE (01.01.10 to Feb 2012); EMBASE was only
searched one year back as the UKCC has handsearched EMBASE
to this point and these trials are already in CENTRAL
Ovid PsycINFO.
Searching other resources
The National Research Register (NRR), a register of ongoing and
recently completed research projects funded by or of interest to
the United Kingdoms National Health Service; entries from the
Medical Research Council Clinical Trials Register; and details on
reviews in progress that are collected by the NHS Centre for Re-
views and Dissemination were searched. The Clinical Trials reg-
ister (clinicaltrials.gov), a registry of both federally and privately
funded US and other clinical trials, was also searched.
The search was performed on titles, abstracts and keywords of the
listed articles. The citation lists of relevant publications, review
articles, and included studies were also searched. Relevant confer-
ence abstracts were handsearched.
A search for new trials is conducted bi-annually and the review
updated as and when new trials to be incorporated are found.
Data collection and analysis
Selection of studies
Three review authors (DG, CF and AB) performed the selection
of trials for inclusion in the review after employing the search
strategy described previously. This information was presented in a
Characteristics of included studies table and provides a context for
assessing the reliability of results. Excluded articles were detailed
in the Characteristics of excluded studies.
Data extraction and management
Three review authors (DG, CF and AB) independently extracted
data from eligible studies using a data extraction form designed
and pilot-tested by the authors. Any disagreements were resolved
by discussion. Data extracted included study characteristics and
outcome data. Where studies had multiple publications, the main
trial report was used as the reference and additional details derived
fromsecondary papers. We corresponded with study investigators
for further data, as required.
Assessment of risk of bias in included studies
Informationwas independently extractedonmethodological qual-
ity and outcome data by three review authors (DG, CF, AB) using
forms designed according to Cochrane guidelines. Another co-
author (CF) was available to resolve any discrepancies. Additional
information on trial methodology or actual original trial data were
sought from the principal author of trials that appeared to meet
eligibility criteria but were unclear in aspects of methodology, or
where the data were in a form unsuitable for meta-analysis. Re-
minder correspondence was sent when a reply was not received
within three weeks.
Measures of treatment effect
For dichotomous data (for example clinical pregnancy rate), results
for each study were expressed as odds ratios (OR) with 95% con-
dence intervals and combined for meta-analysis with RevMan
software using the Peto modied Mantel-Haenzel method.
Unit of analysis issues
The primary analysis was per woman randomised. Multiple live
births (for example twins or triplets) were counted as one live birth
event.
Data per cycle or per embryo transfer (ET) or per ovum pick up
(OPU) were not able to be pooled (Vail 2003). However, due to
the frequency that this form of data is reported in the literature
they have been entered into the Table of comparisons for the
following outcomes:
i) live births per OPU and ET;
ii) clinical pregnancy rate per OPU and ET;
iii) implantation rate, the number of fetal sacs divided by the
We used the number of women randomised as the denominator
even if the authors did not.
Dealing with missing data
Where possible, the data were analysed using an intention-to-treat
analysis.
Assessment of heterogeneity
We considered whether the clinical and methodological charac-
teristics of the included studies were sufciently similar for meta-
analysis to provide a clinically meaningful summary. We assessed
statistical heterogeneity by the measure of the I
2
statistic. An I
2
measurement greater than 50% was taken to indicate substantial
heterogeneity. When we detected substantial heterogeneity, we ex-
plored possible explanations in sensitivity analyses. We took sta-
tistical heterogeneity into account when interpreting the results.
Heterogeneity between the results of different studies was exam-
ined by inspecting the scatter of data points, the overlap in their
condence intervals and more formally by checking the results of
the Chi
2
tests. A priori, it was planned to look at the possible con-
tribution of differences in trial design to the heterogeneity identi-
ed. Where possible, the outcomes were pooled statistically.
Assessment of reporting biases
In viewof the difculty of detecting and correcting for publication
bias and other reporting biases, the authors aimed to minimise
their potential impact by ensuring a comprehensive search for
eligible studies and by being alert for duplication of data.
Data synthesis
Statistical analyses were performed in accordance with the
Cochrane Handbook for Systematic Reviews of Interventions (Higgins
2011).
Data were pooled for meta-analysis with RevMan software using
the Peto modied Mantel-Haenzel method. The data were entered
on the graphs so that for positive outcomes (for example preg-
nancy) points to the right of the line of no effect, and in negative
outcomes (for example miscarriage) points to the left of the line
of no effect.
Subgroup analysis and investigation of heterogeneity
The following subgroup analyses were planned.
Studies where the policy for the number of embryos
replaced was equal in both Day 2 to 3 and Day 5 to 6 groups
versus studies where fewer Day 5 to 6 than Day 2 to 3 embryos
were replaced.
Studies that actively selected for good prognosis participants
(for example four or more zygotes, rst two cycles, more than 10
follicles, young population, no male-factor individuals) versus
participants with poor prognostic factors (for example previous
failed ART cycles or poor response to ovulation stimulation)
versus studies with unselected participants.
Studies that randomised at the start of the cycle (that is
prior to ovarian stimulation) were compared with the days
immediately prior to and post OPU (that is day of nal
ultrasound scan and prior to human chorionic gonadotropin
(HCG) trigger up to and including the day of fertilization check,
when numbers of oocytes are anticipated).
Sensitivity analysis
The following sensitivity analyses were planned: studies that used
concealment of allocation, studies that reported the randomisation
method and the day of randomisation.
R E S U L T S
Description of studies
Results of the search
Two hundred and ninety-six trials have been identied as pro-
viding data comparing early cleavage stage and blastocyst stage
embryo transfer outcomes. Thirty-ve studies were potentially
eligible and were retrieved in full text. In this update ve new
studies were added (Brugnon 2010; Elgindy 2011; Fisch 2007;
Pantos 2004; Ten 2011) and in total there were 23 studies
that met our inclusion criteria. Thirteen studies were excluded.
See the study tables: Characteristics of included studies and Ex-
cluded studies. Replies were received from 11 contact authors
(Bungum 2003; Frattarelli 2003; Hreinsson 2004; Karaki 2002;
Levitas 2004; Levron 2002; Livingstone 2002, Papanikolaou
2005; Papanikolaou 2006; Plachot 1999; Rienzi 2002) who pro-
vided information regarding methodology and outcome data.
Included studies
Study design and setting
Twenty-three parallel-design randomised controlled trials were in-
cluded in this review. One of the studies had been published or
presented onseparate dates and both sets of data appear inthe table
Characteristics of included studies within single entries. Motta
1998 A & B were two conference abstracts presenting different
aspects of data from the same trial. The review consists of a total
of 3823 couples. The size of trials ranged from 20 (Fisch 2007) to
460 couples (Kolibianakis 2004) including both Day 2 to 3 and
Day 5 to 6 groups.
The majority of trials were carried out in less than six months,
except for the two largest studies. All studies were reported to have
beenperformed at single private or university-based clinics. Twelve
countries were represented in the included studies with Belgium
being the most prolic, with seven studies. The countries rep-
resented were: Australia (Livingstone 2002), Belgium (Devreker
2000; Emiliani 2003; Kolibianakis 2004; Papanikolaou 2005;
Papanikolaou 2006; Van der Auwera 2002), Brazil (Motta 1998 A
& B), Denmark (Bungum 2003), Egypt (Elgindy 2011), France
(Brugnon 2010), Greece (Pantos 2004), Israel (Coskun 2000;
Levitas 2004; Levron 2002), Italy (Rienzi 2002; Schillaci 2002),
Jordan (Karaki 2002), Spain (Ten 2011), Sweden (Hreinsson
2004), and USA (Fisch 2007; Frattarelli 2003; Gardner 1998).
Participants
Patient selection criteria comprised three main groups: unselected
patients (Emiliani 2003; Karaki 2002; Kolibianakis 2004; Motta
1998 A & B; Pantos 2004; Schillaci 2002; Van der Auwera
2002); good prognostic factors where participants were posi-
tively selected, that is those that would be expected to do well
with blastocyst culture (Brugnon 2010; Bungum 2003; Coskun
2000; Elgindy 2011; Fisch 2007; Frattarelli 2003; Gardner 1998;
Hreinsson 2004; Levron 2002; Livingstone 2002; Papanikolaou
2005; Papanikolaou 2006; Rienzi 2002; Ten 2011); and poor
prognostic factors, where couples were selected who had experi-
enced multiple failures with conventional treatment or had a poor
response to ovulation induction (Devreker 2000; Levitas 2004).
Most studies recruited women aged less than 40 years of age with
the exception of Gardner 1998 which had no age limit. The mean
age across all the studies varied from 29 years to 34 years.
Interventions
Eighteen trials used sequential media, of which 11 used Vitrolife
G1/G2 while the remaining media were combinations of brands
or made in-house. Three did not state the media used (Table 1 in
additional tables).
Freezing of embryos in both experimental groups was reported
in 13 of the 23 included trials (Brugnon 2010; Bungum 2003;
Gardner 1998; Hreinsson 2004; Karaki 2002; Kolibianakis 2004;
Levron 2002; Motta 1998 A & B; Pantos 2004; Papanikolaou
2005; Papanikolaou 2006; Rienzi 2002; Van der Auwera 2002).
Coskun 2000 reported no provision for Day 5 freezing. Levitas
2004 stated that most of the remaining embryos were not suitable
for freezing. Other interventions, such as assisted hatching, were
either not provided or not reported on for the majority of trials.
Gardner 1998 was the only trial that practised assisted hatching,
but only for the Day 3 ET group.
All the studies compared Day 2 or 3 embryo transfers versus blas-
tocyst stage. For the Day 2 to 3 transfer groups most transfers
were on Day 3, with the exception of four trials (Devreker 2000;
Emiliani 2003; Motta 1998 A & B; Van der Auwera 2002) and
Levitas 2004 had a policy of Day 2 or 3.
The trials that provided details on the ovarian stimulation regimen
mostly reported using a similar gonadotropin-releasing hormone
)GnRH) pituitary down-regulation
protocol prior to HMG and follicle stimulating hormone (FSH)
administration. However, the three most recent trials (Kolibianakis
2004; Papanikolaou 2005;
Papanikolaou 2006) all used a GnRH antagonist to varying de-
grees.
The number of embryos transfered
Outcomes
12/23 studies reported live birth
4/23 studies reported cumulative pregnancy rate
23/23 studies reported clinical pregnancy rate
16/23 studies reported multiple pregnancy rate
14/23 studies reported miscarriage rate
11/23 studies reported embryo freezing rate
16/23 studies reported failure to transfer embryos rate
Excluded studies
Eleven studies were excluded from the review, for the following
reasons:
2/11 fresh embryo transfers on day 5/6 were not the main
intervention;
3/11 used co-culture;
3/11 have duplicate data;
4/11 were not truly randomised studies.
Risk of bias in included studies
See Figure 1, Figure 2.
Figure 1. Risk of bias graph: review authors judgements about each risk of bias item presented as
percentages across all included studies.
Figure 2. Risk of bias summary: review authors judgements about each risk of bias item for each included
study.
Attempts were made to obtainadditional informationregarding all
aspects of randomisation, blinding, power analysis and intention
to treat from all trial authors.
Sequence generation method: 11 studies used computer generated
randomisation (Brugnon 2010; Elgindy 2011; Emiliani 2003;
Frattarelli 2003; Gardner 1998; Kolibianakis 2004; Levitas 2004;
Livingstone 2002; Papanikolaou 2005; Papanikolaou 2006; Rienzi
2002), six used sealed envelopes (Bungum 2003; Coskun 2000;
Hreinsson 2004; Karaki 2002; Levron 2002; Van der Auwera
2002) and the remaining studies did not state their method of
randomisation (Devreker 2000; Fisch 2007; Motta 1998 A & B;
Pantos 2004; Schillaci 2002; Ten 2011).
Allocation
In nine studies the method of concealing allocation was sealed en-
velopes (Bungum 2003; Coskun 2000; Elgindy 2011; Hreinsson
2004; Karaki 2002; Levitas 2004; Levron 2002; Livingstone 2002;
Van der Auwera 2002). Frattarelli 2003 stated that the allocation
was concealed although no details were provided. In the remaining
studies the method was unknown or a high risk method was used.
Blinding
The length of culture and the day of embryo transfer was different
for each of the experimental groups making it impossible to blind
whichgroupa participant was infor the doctor, scientist, nurse and
participant. There was no evidence to suggest that the statistician
in any trial was blinded to the assignment status.
Incomplete outcome data
Seventeen studies stated that they performed an intention-to-treat
analysis (Bungum 2003; Coskun 2000; Devreker 2000; Elgindy
2011; Frattarelli 2003; Gardner 1998; Hreinsson 2004; Karaki
2002; Kolibianakis 2004; Levitas 2004; Levron 2002; Motta 1998
A & B; Papanikolaou 2005; Papanikolaou 2006; Rienzi 2002;
Schillaci 2002; Ten 2011). The latter trial also performed an in-
terim analysis and was terminated after 50% of the intended pa-
tients were enrolled due to a signicant difference being detected.
Identication of participants failing to have an embryo transfer
was not stated or unclear in some trials. Coskun 2000 implied
that a 100% embryo transfer rate was achieved in both Day 2
to 3 and Day 5 to 6 groups, which is unexpectedly high and is
possibly explained by transferring embryos of a lesser stage when
blastocysts were not available. Where the number of couples and
the number of ETs were different, the number of couples was
used as the denominator even when exclusions took place post-
randomisation, assuming no pregnancies occurred. For example
Frattarelli 2003 excluded eight couples, including four for embryo
quality. These eight couples were able to be added to the denom-
inator and, therefore, an intention-to-treat analysis was possible.
Emiliani 2003 excluded 10 women because of protocol violations.
Van der Auwera 2002 excluded seven women post-randomisation
as three couples randomised to Day 2 requested blastocyst transfer
and four couples requested Day 2 transfer. These numbers were
added to the denominator, assuming they did not conceive. For
the remaining studies the Day 5 to 6 embryo transfer rate ranged
from 71% to 96%.
Selective reporting
No evidence was found of selective reporting in the included stud-
ies except from Fisch 2007, who published only preliminary data.
However only 12 of the 23 studies reported live birth rate.
Other potential sources of bias
No evidence of other potential sources of bias were found in the
included studies.
Effects of interventions
Cleavage stage versus blastocyst stage
1 Live birth per couple
Evidence of a signicant difference was detected between the two
treatment groups for live birth rate per couple favouring blastocyst
culture (12 RCTs, 1510 women, Peto odds ratio (OR) 1.40, 95%
CI 1.12 to 1.74) (Day 2 to 3: 31.2% versus Day 5 to 6: 38.8%).
Moderate heterogeneity was detected and the I
2
was 40% (see
Figure 3).
Figure 3. Forest plot of comparison: 1 Live birth rate, outcome: 1.1 Live birth per couple.
There was no difference betweenDay 2 to 3 and Day 5 to 6 transfer
based on prognosis. Day 2 to 3 of randomisation was associated
withbetter outcomes for Day 5to6transfers (2RCTs, 364women,
OR 2.17, 95% CI 1.42 to 3.33, I
2
= 0%). Sensitivity analysis,
including only those studies with low risk of bias by allocation
concealment, did not affect the conclusions for live birth rates
(OR 1.19, 95% CI 0.74 to 1.93).
2 Clinical pregnancy rate per couple
Evidence of a signicant difference was not detected between the
two treatment groups for clinical pregnancy rate per couple (23
RCTs, Peto OR 1.14, 95% CI 0.99 to 1.32) (Day 2 to 3: 38.6%
versus Day 5 to 6: 41.6%). Heterogeneity was detected and the
I
2
was 47% (Figure 4). Separate analyses did not show any dif-
ferences when single or equal embryo transfers were performed,
where more than one embryo was transferred or according to the
patients prognosis or timing of randomisation. Sensitivity analy-
sis including only those studies with low risk of bias by allocation
concealment did not affect the conclusions on clinical pregnancy
rates (OR 1.07, 95% CI 0.70 to 1.64).
Figure 4. Forest plot of comparison: 2 Clinical pregnancy rate, outcome: 2.1 clinical pregnancy rate per
couple.
3 Cumulative clinical pregnancy rate per couple
Evidence of a signicant difference was detected between the two
treatment groups for cumulative clinical pregnancy rate per couple
in favour of Day 2 to 3 transfers (4 RCTs, 266 women, Peto OR
1.58, 95% CI 1.11 to 2.25) (Day 2 to 3: 56.8% versus Day 5 to
6: 46.3%). Moderate heterogeneity was detected and the I
2
was
47% (see Figure 5).
Figure 5. Forest plot of comparison: 3 Cumulative pregnancy rate, outcome: 3.1 cumulative pregnancy rate
from fresh and frozen transfers.
4 Multiple-pregnancy rate
There was no evidence of a difference in multiple pregnancy rate
per couple between the two treatment groups (16 RCTs, Peto OR
0.92, 95% CI 0.71 to 1.19). There was minimal heterogeneity
detected and the I
2
was 27%. Separate analyses by embryo trans-
fer policy or prognosis did not suggest any subgroup differences
in the relative effect of blastocyst versus cleavage stage embryo
transfer. Sensitivity analysis excluding the studies which did not
report concealment of allocation did not affect the signicance of
multiple pregnancy rates (OR 0.95, 95% CI 0.65 to 1.39).
There was no evidence of a difference in high order pregnancy rate
per couple between the two treatment groups in 12 RCTs (OR
0.44, 95% CI 0.15 to 1.33). There was no heterogeneity detected
and the I
2
was 0%. Separate analyses by embryo transfer policy
did not suggest any subgroup differences in the relative effect of
blastocyst versus cleavage stage embryo transfer.
Seven RCTs reported if any of the multiple pregnancies were
monozygotic; there was one set in Day 2 to 3 transfer (Frattarelli
2003), two sets in Day 2 to 3 transfers (Papanikolaou 2006) and
one in Day 5 to 6 transfer (Levitas 2004).
5 Miscarriage rate
There was no evidence of a difference inmiscarriage rate per couple
between the two groups (14 RCTs, Peto OR 1.14, 95% CI 0.84
to 1.55). There was no heterogeneity detected and the I
2
was 0%.
6 Embryo freezing rate
Rates of embryo freezing per couple showed a signicant increase
for the Day 2 to 3 transfers compared to Day 5 to 6 (11 RCTs,
1729 women, Peto OR 2.88, 95% CI 2.35 to 3.51). Separate
analyses by embryo transfer policy and prognosis also showed a
signicant difference in favour of more embryos frozen with early
cleavage stage transfers. There was, however, signicant hetero-
geneity detected in all subgroups, with I
2
values of greater than
76%. Four trials reported cumulative pregnancy rates following
the transfer of fresh and frozen embryos (Brugnon 2010; Emiliani
2003; Rienzi 2002; Van der Auwera 2002), which also favoured
Day 2 or 3 (4 RCTs, OR 1.56, 95% CI 1.10 to 2.22).
7 Failure to transfer any embryos
Failure to transfer any embryos per couple was signicantly lower
in the Day 2 to 3 group (16 RCTs, 2459 women, OR 0.35, 95%
CI 0.24 to 0.51) (Day 2 to 3: 3.4% versus Day 5 to 6: 8.9%).
Heterogeneity was detected and the I
2
was 26%. This nding was
also true for subgroups analysed by number of embryos transferred
policy and all prognosis groups, except for high prognosis patients
where there was no signicant difference.
8 Other data
Blastocyst rates
Reported inTable 2 of the Additional tables, blastocyst rates (Day
5 to 6 transfer only) ranged from 28% (Coskun 2000) to 60.3%
(Schillaci 2002).
Implantation data
For Day 2 to 3 transfer, the implantation rate varied from 3% to
43.9%, and for Day 5 to 6 the implantation rate varied from4.2%
to 55.8% (see Table 2).
Publication bias
Only clinical pregnancy rate had 10 or more studies and a funnel
plot was generated, which suggested that there was no publication
bias (see Figure 6; Figure 7).
Figure 6. Funnel plot of comparison: 1 Live birth rate, outcome: 1.1 Live birth per couple.
Figure 7. Funnel plot of comparison: 2 Clinical pregnancy rate, outcome: 2.1 clinical pregnancy rate per
couple.
D I S C U S S I O N
Summary of main results
In the 12 RCTs that reported live birth rates, there was evidence
of a small but signicant difference in live birth rate per couple
favouring blastocyst culture and no difference in miscarriage rate.
Overall there was no difference in clinical pregnancy rate between
early cleavage and blastocyst transfer in the 23 RCTs. However,
cumulative clinical pregnancy rates from cleavage stage embryos
(derived from fresh and thaw cycles) resulted in higher clinical
pregnancy rates than fromblastocyst cycles. Embryo freezing rates
and failure to transfer embryos signicantly favoured early cleav-
age stage transfers. No evidence of a difference was found between
blastocyst and cleavage stage transfers for rates of miscarriage, mul-
tiple pregnancies, and high order multiples.
Overall completeness and applicability of
evidence
Implantation rates
Extended culture provides an opportunity to select those embryos
that have proven ability to survive and develop to an advanced
stage in vitro, with subsequent implantation success in vivo. The
transfer of Day 5 to 6 embryos also offers the opportunity to re-
place embryos into a uterine environment that is possibly more
synchronised than at Day 2 to 3. For these reasons, blastocyst cul-
ture is expected to result in higher implantation rates (number of
fetal sacs observed divided by the number of of embryos trans-
ferred). In this review most included trials reported higher rates
for blastocysts while the remaining (with three exceptions that
reported a decrease) reported no difference between the groups.
Pooling of implantation data could not be included in the meta-
analysis as this would not generate valid estimates or condence
intervals due to the unit of analysis used (Vail 2003). Nevertheless,
it is interesting to observe that the higher implantation potential
of blastocysts in this reviewdid translate into higher live birth rates
per couple randomised. This is despite a signicantly higher fail-
ure to transfer any embryos in the Day 5 to 6 group. The increased
rates of failure to transfer with Day 5 to 6 is largely the result of
patients whose embryos had arrested development prior to the day
of embryo transfer. Indeed, many of the studies that transferred
fewer blastocysts than cleavage stage did so out of a lack of op-
tions rather than by policy. Another point of consideration is the
widely variable policy for minimal quality of embryos for transfer
that may have existed amongst trials. Some trials accepted transfer
of developmentally delayed embryos on Day 5 to 6, whilst other
trials were more selective and denied transfer of embryos that were
less than a late morula or early blastocyst.
Blastulation rates and media
Blastocyst formation rates may also inuence the pregnancy rate
per embryo transfer for each trial. They ranged from 28% in the
Coskun 2000 trial to 60% in the Schillaci 2002 trial. Of the in-
cluded trials that provided information on the media used, all used
sequential media for the culture of blastocysts. However, while
the majority reported using various versions of Vitrolife G1/G2,
others used a combination of different brands or made the media
in-house. This highlights the possibility that different brands and
formulations are likely to inuence the blastulation rates and sub-
sequent outcomes.
Viability
In this review couples having blastocyst culture were three times
more likely to have a cycle cancelled prior to embryo transfer. Some
advocate that it is better for patients to learn that their embryos
failed to develop by Day 5 than go through with a transfer on Day
2 to 3 with embryos that had a low potential of success. There has,
however, been little research into the emotional status of couples
given such choices (Borg 2000). Before introducing blastocyst cul-
ture, clinics should have an established success rate with Day 2 to
3 embryos and a good understandng of the culture conditions and
protocols required for extended culture. One unintended conse-
quence of adopting a strict blastocyst culture policy that selects
the most viable embryos may be that the slow cleaving embryo
on Day 3 may not have had a chance of pregnancy if replaced
into the uterus early rather than be subjected to extended culture
(Racowsky 2000). Studies exploring what key indicators can be
detected for selecting which patient group might obtain the most
benet from blastocyst culture include the number of eight-cell
embryos on Day 3 (Racowsky 2000), number of pro-nuclear em-
bryos on Day 1, the pro-nuclear grading prole (Scott 2000) and
the number of early cleaving embryos. Certainly the Papanikolaou
2005 trial has clearly demonstrated that it is possible to obtain
zero cancellation rates and signicantly higher live birth rates with
criteria of four good quality embryos on Day 3 (in women under
38 years of age).
Number of embryos transferred and multiple pregnancy
Perhaps one of the greatest difculties indrawing conclusions from
published blastocyst trials is the variable embryo transfer policies
between the two experimental groups (Table 3). In this meta-anal-
ysis, signicantly fewer embryos were transferred in the Day 5 to
6 group than in the Day 2 to 3 group. There are two primary
reasons for this difference. Firstly, many clinics worried about the
high incidence of multiple pregnancy with blastocysts will have a
policy to transfer no more than two Day 5 to 6 embryos. Some
clinics state that by employing blastocyst culture they have been
able to reduce the multiple-pregnancy rate whilst maintaining the
pregnancy rate. In this review many of the studies were still trans-
ferring two to three embryos. When a subgroup analysis was per-
formed for trials where equal numbers of embryos were transferred
(including single embryo transfers (SETs)), the clinical pregnancy
rate remained unchanged. It could be argued that this is the most
valid comparison because trials with a greater number of cleav-
age stage embryos being transferred are probably advantaged in-
appropriately. Regardless of the embryo transfer policy, for many
patients there is simply a lack of choice as only one, if any, em-
bryo reaches the blastocyst stage. Three studies in this updated re-
view had a policy for single blastocyst transfer, although only one
reported live birth rate. Remarkably the policy of single embryo
transfer in one study reduced the multiple-pregnancy rate for blas-
tocyst transfer from 43% to zero (Papanikolaou 2006). Equally
remarkable is that the two more recent studies with SET did not
report multiple pregnancy rates (Brugnon 2010; Fisch 2007).
Miscarriage and monozygotic twinning
Miscarriage rates are a critical factor when evaluating a new mode
of treatment and they obviously impact on treatment efciency
and live birth outcomes. Yet only just over half of the included
trials provided these data. Theoretically, the rate of miscarriage
might be expected to be lowest with the transfer of highly se-
lected embryos that are transferred into a synchronous uterine
environment, such as in blastocyst culture. However, the results
to date reveal little change from the earlier reviews that showed
no evidence of a difference in miscarriage rates for couples ran-
domised (13 RCTs, Peto OR 1.18, 95% CI 0.86 to 1.60). Only
seven of the included trials reported on the presence or absence
of monozygotic twinning so this analysis remains underpowered
to comment meaningfully on monozygotic twin rates. A total of
three sets of monozygotic twins were reported, two with Day 2 to
3 embryo transfers and, reassuringly, only one set of monozygotic
twins from blastocyst transfer. Estimations of monozygotic twin
rates in ART are thought to be underestimated, with up to one
third being missed without genetic testing.
Embryo freezing
Overall this review found a signicant decrease in the number of
embryos frozen in the Day 5 to 6 group and an increase in cumu-
lative pregnancy rates (fresh and frozen cycle transfers) in women
with Day 2 to 3 transfers. A total of four trials reported data on
pregnancies following transfer of frozen embryos in both groups
(Brugnon 2010; Emiliani 2003; Rienzi 2002; Van der Auwera
2002). Van der Auwera 2002 used their trial data and results from
subsequent thaw cycles after one year to predict a cumulative live
birth rate that was almost identical in both groups (38% versus
39%). That is, the added benet of a higher cryopreservation rate
in the Day 2 to 3 group cancelled out the higher implantation rates
of the fresh Day 5 to 6 transfers. Similarly, Rienzi 2002 reported
no difference in cryo-augmented pregnancy rates when at least
one thaw cycle was carried out in the Day 2 to 3 group. Emiliani
2003, on the other hand, reported signicantly higher cumulative
pregnancy rates in the Day 2 to 3 group, presumably correlating to
the much lower cryo-survival rate they reported in their blastocyst
group (Day 2 to 3: 46% versus Day 5 to 6: 27%).The number
of embryos frozen is an important consideration when assessing
the effectiveness of a treatment as it offers the patient an addi-
tional opportunity to achieve a pregnancy. When considering an
alteration in treatment procedure from Day 2 to 3 to Day 5 to
6, the benets of possible higher implantation rates are weighed
up against the disadvantages of not only higher failure to transfer
but also lower cryopreservation rates. However, freezing protocols
for early cleavage and blastocyst stage embryos are different and
the effectiveness of the latter has yet to be widely accepted, par-
ticularly in embryos that have been cultured in sequential media.
Reports of improved blastocyst freezing techniques such as vitri-
cation may have a positive impact on the cumulative success rates
of future blastocyst RCTs (Edgar 2012; Gardner 2003a; Iwayama
2011; Zhu 2011).
Quality of the evidence
The overall risk of bias in this review is moderate. Blinding is
not possible with this intervention. More than two thirds of the
included studies have complete reporting for adequate sequence
generation but only one third reported their allocation conceal-
ment method. On the other hand, live birth rate was reported in
12 trials and also cumulative pregnancy rate after frozen embryo
transfer data was reported in four trials.
Other concerns with the quality of evidence that were identied
during the reviewprocess include the time of randomisation. Stud-
ies show that women with a high oocyte yield and good quality
eight-cell embryos on Day 3 are more likely to have blastocysts
by Day 5 to 6 compared with poor responders and those with
no eight-cell embryos by Day 3. This rationale is supported in
this review, where no difference was found in the rate of failure
to transfer embryos for the subgroup analysis of good prognosis
patients (2.4% for Day 2 to 3 versus 3.5% for Day 5 to 6), but if
randomisation was on Day 3 then Day 5 to 6 transfers resulted in
higher pregnancy rates suggesting that an assessment of suitability
for inclusion in the trial may have inuenced the nal outcomes.
The outcomes for trials randomising on Day 2 to 3 are in con-
trast to those that randomised couples prior to the start of the
treatment cycle, at a time when neither the number of oocytes re-
trieved nor fertilized, nor the number of eight-cell embryos, could
be anticipated. In some respects the trials can be divided into those
that investigated whether outright adoption of blastocyst culture
is superior to standard cleavage stage transfers (that is unselected
patient populations) or whether blastocyst culture can be incorpo-
rated into a clinical setting for enhancement of success in specic
patient subgroups (that is poor or high prognosis patients).
Perhaps one of the greatest difculties indrawing conclusions from
the trials in this review is the variable embryo transfer policies
between the two experimental groups. In this review, signicantly
fewer embryos were transferred in the Day 5 to 6 group than in the
Day 2to3group. There are twoprimary reasons for this difference.
Firstly, there is a reduced survival rate of Day 5 to 6 blastocysts and
secondly, many clinics that are worried about the high incidence of
multiple pregnancy with blastocysts will have a policy to transfer
no more than two Day 5 to 6 embryos. Some clinics state that
by employing blastocyst culture they have been able to reduce the
multiple pregnancy rate whilst maintaining the pregnancy rate.
In this review many of the studies were still transferring two to
three embryos. Regardless of the embryo transfer policy, for many
patients there is simply a lack of choice as only one, if any, embryo
reaches the blastocyst stage.
Potential biases in the review process
As far as possible the protocol methodology has been adhered to
in order to limit any potential biases. However, one new outcome
was added, cumulative clinical pregnancy rates (derived fromfresh
and thawcycles). This outcome reects the policy of fresh embryo
transfer andfreezing remaining embryos for transfer later andmore
correctly reects modern IVF practice than a single cycle transfer.
The issue of publication bias is important in systematic reviews as
it may result in incorrect conclusions being reached. For example,
it might be expected that the pressure for clinics to obtain high
implantation rates with blastocyst culture could lead to a bias in
publication towards those that do achieve this. The funnel plot for
clinical pregnancy rate however demonstrated that the studies were
distributed evenly across the graph, suggesting that publication
bias is not present.
Agreements and disagreements with other
studies or reviews
This review is in agreement with a systematic review published
by Papanikolaou (Papanikolaou 2008), which reported that cycles
where equal number of embryos were transferred had higher live
birth rates in blastocyst transfers than in cleavage stage embryos.
Our reivew also includes cumulative clinical pregnancy rate. No
other systematic reviews were found.
A U T H O R S C O N C L U S I O N S
Implications for practice
This review of the best available evidence based on data from ran-
domised controlled trials suggests that the margin of benet be-
tween cleavage stage and blastocyst transfer is unclear. Blastocyst
transfer appears to be a good option for the high prognosis sub-
groups where larger quantities of high quality embryos are present.
However, the freezing rate and failure to transfer rate tell a dif-
ferent story. The women who undergo cleavage stage transfer and
freeze the remaining embryos have higher cumulative pregnancy
rates.
Implications for research
Although this review provides evidence that there is a signicant
difference in live birth rates in favour of blastocyst transfer com-
pared to cleavage stage transfer cycles, cumulative clinical preg-
nancy rates from cleavage stage (derived from fresh and thawed
cycles) transfer result in higher clinical pregnancy rates than from
blastocyst cycles. The most likely reason for this is the higher rates
of frozenembryos obtained fromcleavage stage protocols. Another
reason could be differences in the cryopreservation efciencies of
cleavage and blastocyst stage techniques. Further studies of blas-
tocysts cycles must report miscarriage, live birth and cumulative
live birth rates to enable consumers and service providers to make
well informed decisions on the best treatment option.
Based on the results of this review, the following recommendations
are made to ensure valuable data are produced:
1. adherence to CONSORT recommendations for RCTs, espe-
cially methods of concealment (Begg 1996);
2. research into best patient selection and inclusion criteria;
3. same media composition and brand for both groups up to the
Day 2 to 3 stage;
4. explicit prespecied embryo transfer policies for both groups;
5. long-term follow-up reports of cumulative live birth rates (in-
cluding embryo thaws) presented as a survival analysis;
6. research into improved blastocyst cryopreservation techniques;
7. application of blastocyst culture for single embryo transfer;
8. reporting of miscarriage, live birth and cumulative pregnancy
rates.
A C K N O W L E D G E M E N T S
The authors acknowledge the helpful comments of those who
refereed previous versions of this review, in particular Mr Andy
Vail and Dr Gayle Jones. Thanks to Dr Plachot, Dr Huisman,
Dr Utsunomiya, Dr Hreinsson, Dr Rienzi, Dr Levron, Dr Levi-
tas, Dr Bungum, Dr Papanikolaou, Dr Karaki, Dr Frattarelli, Dr
Brugnon and Dr Vanderzwalmen for supplying additional infor-
mation. Thanks to the librarian Daniel Comand. Finally, special
thanks to the highly supportive team at the Cochrane ofce in
Auckland. Dr Neil Johnson was a review author for the previous
versionof this reviewand made a signicant contributionto the in-
terpretation of results and performed some data extraction. David
Olive, for the initial review, commented on drafts of the protocol
and review. Michelle Proctor, for the initial review, was involved in
selecting trials for inclusion, performed independent data extrac-
tion and quality assessment of the included trials, contributed to
discussion and interpretation of results. Quirine Lamberts, for the
2005 update, checked the data and study information extracted.
R E F E R E N C E S
References to studies included in this review
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randomized study. Reproductive Biomedicine Online April
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Devreker 2000 {published data only}
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Gardner D K, Schoolcraft W B, Wagley L, Schlenker
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blastocyst culture and transfer in in-vitro fertilization.
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Karaki 2002 {published data only}
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Ibrahim MH. Blastocyst culture and transfer: a step toward
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Kolibianakis EM, Zilopoulos K, Verpoest W, Camus M,
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Levitas 2004 {published data only}
Levitas E, Lunenfeld E, Hackmon-Ram R, Sonin Y, Har-
Vardi I, Potashnik G. A prospective, randomized study
comparing blastocyst versus 48-72 h embryo transfer in
women failed to conceive three or more in-vitro fertilization
treatment cycles. Abstracts from the 57th Annual Meeting
of ASRM. 2001.
Levitas E, Lunenfeld E, Har-Vardi I, Albotiano S, Sonin Y,
Hackmon-Ram R, et al.Blastocyst-stage embryo transfer
in patients who failed to conceive in three or more day 2-
3 embryo transfer cycles: a prospective, randomized study.
Levitas E, Lunenfeld E, Shoham-Vardi I, Hackmon-Ram R,
Albotiano S, Sonin Y, et al.Blastocyst stage versus 48-72h
embryo transfer in women who failed to conceive on three
or more IVF treatment cycles: a prospective, randomized
study. ESHRE Conference. Bologna, 2000:O021.
Levron 2002 {published data only}
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J. A prospective randomized study comparing day 3 with
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77:13001.
Livingstone 2002 {published and unpublished data}
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Serani P. Blastocyst vs. cleaving embryo transfer: a
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T, Tzigounis V. Comparison of embryo transfer on day 2,
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Papanikolaou 2005 {published data only}
Papanikolaou EG, Dhaeseleer E, Verheycn G, Van de Velde
H, Camus M, Van Steirteghem A, Devroey P, Tournaye H.
Live birth rate is signicantly higher after blastocyst transfer
than after cleavage-stage transfer when at least four embryos
are available on day 3 of culture. A randomized prospective
study. Human Reproduction 2005;20(11):3198203.
Papanikolaou 2006 {published data only}
Papnikolaou EG, Camus M, Kolibianakis EM, Landuyt LV,
Steirteghem AV, Devroey P. In vitro fertilization with single
blastocyst-stage versus cleavage-stage embryos. The New
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Rienzi 2002 {published data only}
Rienzi l, Ubaldi F, Iacobelli M, Ferrero S, Minasi MG,

Martinez F, et al.Day 3 embryo transfer with combined
evaluation at the pronuclear and cleavage stages compares
favourably with day 5 blastocyst transfer. Human
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Schillaci 2002 {published data only}
Schillaci R, Castelli A, Vassiliadis A, Venezia R, Sciacca

GM, Perino A, Cittadini E. Blastocyst stage versus versus
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Ten 2011 {published data only}
Ten J, Carracedo M A, Guerrero J, Rodriguez-Arnedo A,
Llacer J, Bernabeu R. Day 3 or day 5 embryo transfer?
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Guerin 1991 {published data only}
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Sexualite 1991;19(7-8):6358.
Levitas 2001 {published data only}
Levitas E, Lunenfeld E, Hackmon Ram R, Sonin Y,
Har Vardi I, Potashnik G. A prospective, randomized
study comparing blastocyst stage versus 48-72hr embryo
transferin women failed to conceive three or more in-vitro
fertilization treatment cycles. Fertility and Sterility 2001;
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Levron 2001 {published data only}
Levron J, Bider D, Shulman A, Rabinovichi Y, Seidman D,
Dor J. A randomized prospective study on blastocyst versus
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1(3):4.
Loup 2009 {published data only}
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Papanikolaou 2005a {published data only}
Papanikolaou E G Sr, Verheyen G, Camus M, Van
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rate is signicantly higher with day 5 embryo transfer than
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[PUBMED: 21315339]
Indicates the major publication for the study

C H A R A C T E R I S T I C S O F S T U D I E S
Characteristics of included studies [ordered by study ID]
Brugnon 2010
Methods Randomisation: computer generated random list
Blinded: no
Size: 52 D2 55 D5-6
Single centre: Clermont Ferrand Universit dAuvergne, Biologie de la reproduction
CECOS EA 975, Clermont Ferrand, France
Participants Criteria: If more than5oocytes were retrievedand3topquality embryos (34blastomeres,
< 20% fragmentation without multinuclear blastomeres) were observed at day 2, the
couples were included in the study. Cause/duration: NA. Previous Treatment:NA. Fert
rate: NA. Blast rate: NA
Interventions Ov Stim: NS Luteal support: NS Media: Sequential commercial brand Culture method:
NS AHA: N
SET
Outcomes Cumulative pregnancy rate (D2 versus D5/6) 51.9 versus 49.1%. Clinical pregnancy
rate per ET (D2 versus D5/6): 46.2% versus 41.8%
Notes
Risk of bias
Bias Authors judgement Support for judgement
Random sequence generation (selection
bias)
Low risk Computer generated random list
Allocation concealment (selection bias) Unclear risk Not stated
Blinding (performance bias and detection
bias)
All outcomes
High risk Not blinded
Incomplete outcome data (attrition bias)
All outcomes
Unclear risk Not stated
Selective reporting (reporting bias) Low risk
Other bias Unclear risk Not stated
Bungum 2003
Methods Randomisation: sealed envelope on day3
Blinded: no
Size 118 separate women
D3 57 D5 61
No dropouts
Single centre: Denmark
Power calc: Yes retrospective
Therapeutic
Participants Criteria: D3 3 or more 8-cell embryos <20% fragmentation, <40, BMI<30, FSH<12
Age: D3 31.3 D5 31.2
Cause/duration: NS
Previous Treatment:
rst or second cycle:
D3 84% D5 88%
ICSI: D3 51% D5 64%
(lower blast rate in ICSI than IVF)
FSH: D3 6.5 D5 6.5
#eggs: D3 12.84.4
D5 13.55.3
Fert rate: D3 60.2%
D5 60.7%
Blast rate: 55.2%
ET policy: 2 embryos both groups
#ET: D3 2.0 D5 1.96
(2 patients D5 had 1 only due to lack of blasts)
Pregnancy determination: HCG + US 7 weeks
Interventions Ov Stim: GnRHa long + rFSH + HCG 35hr
Luteal support: prog vaginal
Media: G1/G2 Vitrolife
Culture method: microdrops low oxygen incubator
AHA: NS
Outcomes Primary
a) LB: NS
b) preg/OPU/ET/woman:
D3 63.2% (36/57)
D5 52.5% (32/61)
c) multiple rate:
D3 41.6% (15/36)
D5 40.6% (13/32)
Secondary
a) Imp: D3 43.9% (50/114)
D5 36.7% (44/120)
b) miscarriage pre 12 weeks: D3 15% 6/36
D5 29.2% 12/32
c) embryo freeze:
D3 95% (3.42.4)
Bungum 2003 (Continued)
D5 59% (1.31.6)
d) embryo utilisation: NS
e) ET rate: 100% both
f ) high order rate: nil
g) monozygotic twin: NS
Notes Prognosis: good
3 or more 8C D3
Lower blast rate in ICSI than IVF
Outcome no advantage of Blast culture for this selected group
Letter sent and reply received
Risk of bias
bias)
Low risk Sealed envelope
bias)
All outcomes
All outcomes
Low risk No dropouts or exclusions
Other bias Low risk
Coskun 2000
Methods Randomisation: enrolled consecutively sealed envelope until day of fert check
Blinded: no
Size: 201 separate women randomised and analysed:
D3 101 D5 100
Single centre: Uni clinic in Israel
Power calc: NS
Therapeutic
Participants Criteria: 4 or more zygotes
Age: D3 30.7 D5 30.4
Primary/Secondary: NS
Cause/duration - similar except for Unexp:
D3 15% D5 6%
Previous Treatment: NS
Coskun 2000 (Continued)
ICSI: D3 67% D5 64%
FSH: NS
Criteria: 4 or more fert eggs
#eggs: NS
Fert rate: D3 68%, D5 67%
Blast rate: 28% 197/703 (77% patients had at least 1) (lower in male infertility patients
26%)
ET policy: 2 for both D3 and D5
But women with at least 6 attempts had up to 3 embryos transferred
#ET: D3 2.3 (0.6), D5 2.2 (0.5)
Pregnancy determination: HCG + ultrasound
Interventions Ov Stim: GnRH down regulation hMG + 36hr trigger
Luteal support: prog IM
Media: Sequential Medicult to D3 and G1/G2 for D5
Culture method: NS
AHA: NS
Outcomes Primary
a) LB (ongoing) per OPU and ET: D3 33/101
D5 35/100
b) preg OPU: D3 39% (39/101)
D5 39% (39/100)
b) preg/ET: D3 39% (39/101), D5 39% (39/100)
c) multiple preg:
D3 13/39 33%
D5 15/39 38%
Secondary
a) Imp: D3 21% (50/235)
D5 24% (52/218) not sig
b) miscarriage: D3 5/39
D5 3/39
c) embryo freeze: No D5 freezing
e) embryo transfer rate:
D3 101/101 100%
D5 100/100 100%
e) cancellation rate:
D3 0%, D5 0%
f ) high order: D3 1/39
D5 0/39
g) monozygotic twins: NS
4 or more zygotes
Young women
Good ET policy
Mixture of media brands
100% ET rate therefore all stages transferred
Coskun 2000 (Continued)
Low blast rate
High implantation rate considering
No dropouts unusual
Risk of bias
bias)
Allocation concealment (selection bias) Low risk Sealed envelopes
bias)
All outcomes
All outcomes
Other bias High risk Reported no provision for Day 5 freezing
Devreker 2000
Methods Randomisation:
randomly allocated
Size: 23 separate women
D2 12
D5 11
Single unit: Uni afliated
Belgium
Power calc: NS
Therapeutic
Participants Criteria: <40y, >4 previous cycles
Age: no diff
Cause/duration: no diff
Previous treatment: similar in both groups
ICSI: NS
FSH: NS
#eggs: NS
Fert rate: NS
Blast rate: NS
ET policy: max 3 for both groups
#ET: D3 2.83 (34/12)
D5 1.73 (19/11)
Devreker 2000 (Continued)
Pregnancy determination: NS
Interventions Ov Stim: NS
Luteal support: NS
Media: Sequential commercial brand
Culture method: NS
AHA: NS
Outcomes Primary
a) LB: D3 8.3% (1/12)
D5 27.3% (3/11)
b) preg/OPU: NS
b) preg/ET: D3 8.3% (1/12)
D5 36.4% (4/11)
Secondary
a) Imp: D3 3% (1/34)
D5 42% (8/19)
b) miscarriage: D1 0%
D5 20% (1/4)
c) embryo freeze: NS
e) ET rate:100% both
f ) multiple rate: NS
g) high order rate: NS
h) b) monozygotic twin: NS
Notes Prognosis: poor
Abstract only
Letter sent regarding randomisation
Risk of bias
bias)
bias)
All outcomes
All outcomes
Selective reporting (reporting bias) Unclear risk Not stated
Elgindy 2011
Methods Randomisation: computer generated block randomisation
Allocation: identical, sequentially numbered, dark-sealed envelopes
D3 100
D5 100
Single unit: Uni afliated
Egypt
Power calc: 95 women would be required to be able to reject the null hypothesis that
the success
rates are equal with a probability (power) of 0.8 and type-I error probability of 0.05
Participants Criteria: <35y, with regular cycles, serumday-3 FSH concentration <9.5 IU/l and antral
follicle count >6. At least at least four good-quality embryos on day 3.
Age: no diff
Duration: no diff
FSH: NS
#eggs: NS
Fert rate: NS
#ET: D3 2.8
D5 1.96
Interventions Ov Stim: long luteal-phase GnRH agonist down regulation protocol and both recom-
binant FSH and human menopausal gonadotrophin
Media: Sequential commercial brand
Culture method: NS
AHA: N
Outcomes LB: D3 35% (35/100)
D5 52% (52/100)
Notes
Risk of bias
bias)
Low risk Prospective randomised controlled trial
Allocation concealment (selection bias) Low risk
bias)
All outcomes
Unclear risk Open label
All outcomes
Low risk
Elgindy 2011 (Continued)
Other bias Low risk
Emiliani 2003
Methods Randomisation: list with permuted blocks for each new cycle
Blinded: NS
Size: 171 separate women. But reported on 193 cycles
D2 94 cycles
89 women
D5 99 cycles
82 women
18 dropouts
Single centre: Uni afliated
Belgium
Power calc: Yes
Therapeutic
Participants Criteria: <39y, 3 or less previous cycles, 4 or more 2PN on day1
Age: D3 313
D5 324
Cause/duration: NS
Previous treatment:
D2 1.7+/-0.9
D5 2.0+/- 1.0
ICSI: 67% both groups
FSH:NS
#eggs: D2 11.94.4
D5 12.04.9
Fert rate: D2 67.2%
D5 67.5%
Blast rate: 48.3%
ET policy: D2 2-3 depending on age and embryo quality
D5 2 embryos
#ET: D2 2.10.4
D5 1.90.3
Interventions Ov Stim: GnRHa, hMG, HCG 36h
Luteal support:
NS
Media: in-house sequential media
Culture method: 30ul microdrops low oxygen
AHA: no
Outcomes Primary
a) LB/OPU:
D2 44.1% (41/94)
D5 33.3% (33/99)
Emiliani 2003 (Continued)
LB/woman
D2 46.1% (41/89)
D5 40.2% (33/82)
Note that 5% D2 and 17% D5 were from same women
LB/Per ET
D2 44.1% (41/93)
D5 37.1% (33/89)
b) preg/OPU:
D2 44.2% (46/94)
D5 39.4% (39/99)
Preg/randomised/cycle
D2 44.2% (46/104)
D5 36.4% (39/107)
b) preg/ET
D2 44.1% (46/93)
D5 37.1% (39/89)
c) multiple rate per preg:
D2 22% (9/46)
D5 36% (12/39)
Secondary
a) imp: D2 29% (57/197)
D5 30% (50/168)
b) miscarriage:
D2 8.5% (4/46)
D5 15.4% (6/39)
c) embryo freeze:
D2 73% cycles (4.3+/-2.4)
D5 54% cycles (3.3+/-2.3)
D2 1.1% D5 10% (lack of blasts)
f ) high order rate: NS
Cumulative delivery rate including thaws to date were: D2 50% D5 36.4%
g) monozygotic twin: nil
Notes Prognosis: mixed unselected
Outcome: discontinued blast culture
Gives cumulative fresh thaw rates.
Not all different women - per cycle data only
Risk of bias
bias)
Low risk Randomisation list with permuted blocks
for each new cycle
Emiliani 2003 (Continued)
bias)
All outcomes
All outcomes
High risk 10 women were subsequently excluded for
protocol violations
Other bias Low risk
Fisch 2007
Methods Randomisation: N/A
Blinded: N/A
Size: 20 D3 8 D5 12
No dropouts
Single centre: Private practice. USA
Power calc: these are preliminary results and no ulterior study has been published (4
years later)
Participants Criteria: D3 3 or more 8-cell embryos <20% fragmentation, <40, BMI<30, FSH<12
Age: D3 31.3 D5 31.2 Cause/duration: NS Previous Treatment:rst or second cycle:D3
84% D5 88% ICSI: D3 51% D5 64%(lower blast rate in ICSI than IVF) FSH: D3
6.5 D5 6.5 #eggs: D3 12.8+/-4.4D5 13.5+/-5.3 Fert rate: D3 60.2%D5 60.7% Blast
rate: 55.2% ET policy: 2 embryos both groups #ET: D3 2.0 D5 1.96(2 patients D5 had
1 only due to lack of blasts) Pregnancy determination: HCG + US 7 weeks. Inclusion
criteria: Women <41, with <=2 prior fresh cycles with at least one embryo on day 3
GES R70 and sHLA-G OD: 0.148-0.210
Interventions n/a
Outcomes Pregnancy rate: 92% versus 50%
Notes
Risk of bias
bias)
bias)
All outcomes
Fisch 2007 (Continued)
All outcomes
Low risk No dropouts
Selective reporting (reporting bias) High risk Preliminary data
Frattarelli 2003
Methods Randomisation: computer generated randomisation table. Allocation concealed until
intervention assigned. Randomisation day of OPU.
Blinded: no
Size: 57 separate women randomised, 8 withdrawals, 5 from day 3, and 3 from day 5
which were added to number of womenrandomised inorder to achieve anITT, including
4 who did not meet embryo criteria. Assumed no pregnancy for the withdrawals after
randomisation.
(D3 28 D5 29)
Single centre: Army Medical Centre
Hawaii
Power calc: performed but not included - needed 68 women per group
Participants Criteria: <39y, 3 or less previous cycles, 4 or more 2PN on day 1
Age: D3 313
D5 324
Cause/duration: NS
Previous treatment:
D2 1.70.9
D5 2.01.0
FSH:NS
#eggs: D2 11.94.4
D5 12.04.9
Fert rate: D2 67.2%
D5 67.5%
Blast rate: 48.3%
ET policy: D2 2-3 depending on age and embryo quality
D5 2 embryos
#ET: D2 2.10.4
D5 1.90.3
Criteria: 6 or more high grade embryos day3, less than 35y, FSH <12mIU/ml, >10 or
more follicles day of HCG.
Age D3 31.0 D5 30.2
Cause/duration: NS
Previous Treatment: 1st
ICSI: 5x in each group
#eggs:D3 17 D5 20
Frattarelli 2003 (Continued)
Blast rate: not calculated
ET policy:
D3 3-4 embryos
D5 2-3 embryos
# ET: D3 2.96(0.5)
D5 2.04(0.2)
Pregnancy determination: US 6 weeks, ongoing US 12 weeks
Luteal support: NS
Media: sequential for both
Culture method: NS
AHA: NS
Outcomes Primary
a) LB per OPU/ET/woman
D3 34.8% (8/23)
D5 57.7% (15/26)
b) preg OPU/ET/ woman
D3 43.5% (10/23)
D5 69.2% (18/26)
c) multiple preg (initial):
D3 70% (7/10)
D5 27.7% (5/18)
Secondary
a) Imp: D3 26.1% (18/69)
D5 43.4% (23/53)
b) miscarriage
D3 8.7% (2/23)
D5 11.5% (3/26)
D3 5/28 D5 3/29 (not due to lack of blastocysts)
f ) high order: D3 1/10 quad
D5 0/18
number of triplets NS
g) monozygotic twins: 1x D3
6 or greater high grade embryos D3, 1st cycle, young, high numbers of oocytes
No dropouts due to lack of blasts
High blast imp rate
Risk of bias
Frattarelli 2003 (Continued)
bias)
Low risk Computer generated randomisation table
Allocation concealment (selection bias) Unclear risk Allocation was concealed but method not
given
bias)
All outcomes
All outcomes
Low risk Intention-to-treat analysis was applied
Other bias Low risk
Gardner 1998
Methods Randomisation: computer generated on day 8 prior to OPU
Blinding: NS
Inclusion decided on day of trigger
Size: 92 separate women randomised no cancellations: day 3 47 cycles day 5 45 cycles
excluded patients: NS
single private clinic. USA.
Time: 5 months 97/98
Power calculation: NS
Intention to treat: needs clarication
Therapeutic
Participants Criteria: all ages and >10 follicles on day of trigger
Age: D3 34.5 D5 33.6
Primary/secondary: NS
Cause: no signicant difference
Previous treatment: D3 0.21 D5 0.62 cycles (signicantly different)
ICSI: D3 34%, D5 33%
FSH: comparative details NS
Criteria : >10 follicles on day of trigger
# eggs retrieved: NS
Fertilization rate: NS
#PN embryos: D2 512.3,
D5 522.0
Blast rate 46.5% (85% patients had 2+ blasts)
ET policy: changed for day 5 mid study from 2-3. day NS
# ET: D3 3.7, D5 2.2 (4% had no ET)
Gardner 1998 (Continued)
Luteal support: NS
Media: sequential for both
Culture method: NS
AHA: NS
AMS F10 15% FCS, D5 G1 /G2
Method: tubes in humidicrib
AH for nearly all DOv Stim: Lupron D21. hMG dose? hCG 2 follicles >18mm OPU
35hrs trigger
Luteal: steroids and tetracyclin for 4 days post OPU. Progesterone IM from 2 days post
OPU
Culture media: Sequential D3 H3 - nil for D5
Outcomes Primary
a) live birth: NS
b) preg/OPU:
D3 31/47 66%,
D5 32/45 71%
b) preg/ET:
D3 66% (31/47),
D5 74% (32/43)
c) mult preg: D3 - NS
D5 46%
Secondary
a)imp: D3 (64/174) 37%, D5 (53/95) 55.4% p<0.01
b) miscarriage: NS
b) MZ twinning: NS
c) embryo fz rate: D3 30% (14/47) patients, 3.0 embryos
D5 64% (29/45) patients, 3.2 embryos
d) embryo utilisation rate: NS
e) embryo transfer rate: D3 -NS but must be (47/47) 100%, D5 (43/45) 95.5%
f ) high order: D3 NS, D5 15.5%
g) monozygotic NS
Notes Prognosis: good <1 previous cycle and >10 follicles
Different media used for each arm of study
Excluded patients not mentioned
Number of ET policy change partway through -?unblinded interim analysis
Risk of bias
bias)
Low risk Computer generated random list
Gardner 1998 (Continued)
bias)
All outcomes
All outcomes
Unclear risk Need more information
Other bias High risk No age limit
Hreinsson 2004
Methods Randomisation: NS
Allocation: sealed envelope on day prior to HCG
Blinded: no
D2/3 80
day5/6 64
no dropouts
Single centre: Sweden
Power calc: Yes but not achieved
Study stopped early due to change of national guidelines
Therapeutic
Participants Criteria: 6 follicles day prior to hCG
Age: D3 33.1y
D5 32.1y
Cause/duration: similar
Previous cycles: NS
ICSI: D3 42 % D5 45%
FSH: NS
# eggs: D3 14.6 D5 14.9
Fert rate: D3 54%
D5 61%
Blast rate: 33%
ET policy: 1-2 at start of study but then only 1 at end of study
#ET D3 1.8 D5 1.9
HCG: NS
Interventions Ov Stim: long-course GnRHa or short centrorelix
hMG: rFSH, HCG 36hr
Luteal supp: NS
Media: Vitrolife mixture of IVF/CCM and G1/G2 sequential
Culture method: microdrops 6% CO2
AHA: not done
Hreinsson 2004 (Continued)
Outcomes Primary
a) LB: NS
b) preg/OPU
D3 25/80 (31.3%)
D5 22/64 (34.4%)
b) preg/ET
D3 25/77 (32.5%)
D5 22/60 (36.7%)
c)multiple rate:
D3 4/25 (16%)
D5 2/22 (9%)
Secondary
a) Imp: D3 29 /139
D5 24/114 (21.1%)
b) miscarriage pre 12 weeks: D3 2/25 (8%)
D5 3/22 (13.6%)
c) embryo freeze:
D3 34/80 (42.5%)
D5 15/64 (23.4%)
e) ET cancellation:
D3 3/80 (4%)
D5 4/64 (6%)
f ) high order rate: nil
Notes Prognosis: good excluded poor responders.
Mixture of media types and ET policy change over course of study
Outcome no advantage of blast culture
Risk of bias
bias)
Allocation concealment (selection bias) Low risk Sealed envelope
bias)
All outcomes
All outcomes
Hreinsson 2004 (Continued)
Other bias Low risk
Karaki 2002
Methods Randomisation: sealed envelope on D1 fert check
Blinded: no
Size: 162 separate women randomised and analysed
D3 82 D5 80
Single centre: Uni afliated. Jordan.
Power calc: NS
Therapeutic
Participants Criteria: 5 or more zygotes
Age: D3 29.2 D5 30.0
Primary /Secondary: NS
Cause: No sig diff
(Av: 51% Male factor, 9% tubal, 6% unexp)
Duration: D3 6.7 D5 6.8 Previous treatment:
D3 1.1 D5 0.9
ICSI: Yes but no effect found in outcome of imp or blast rate
FSH: D3 7.5 D5 7.8
# eggs: D3 13.4 (5.2)
D5 13.0 (3.2)
Fert rate: D3 56.7%
D5 63.8%
Blast rate:33% day5/6
ET policy: 1-2 embryos but if >35 and/or >2 previous cycles then up to 3 (same policy
for both groups)
# Et: D3 3.50.63
D5 2.01.0
Preg determination:
HCG for preg rate
US for viable preg rate
Interventions Ov Stim: GnRH long and short + rFSH and HP FSH + 35hr trigger
Luteal support: Prog V pessary
Media: D3 Medicult, D5 G1/G2 Vitrolife
Culture method: NS
AHA: NS
Outcomes Primary
a)LB: NS
b) preg/OPU/ET/woman (initial hCG):
D3 29% (24/82)
D5 35% (28/80)
Viable: D3 26% (21/82)
D5 29% (23/80)
c) multiple rate:
Karaki 2002 (Continued)
D3 47.6% (10/21)
D5 39.1% (9/23)
Secondary
a) Imp: D3 13% (37/291)
D5 26% (37/142)
b)miscarriage: info only until 7 weeks
D3 12.5% (3/24)
D5 17.9% (5/28)
c) embryo freeze:
D3 42% (35/82) 1.74+/-3.0
D5 28% (22/80) 0.65+/- 1.0
D3 nil (0/82)
D5 11% (9/80) due to lack of blasts
f ) high order rate:
D3 19% (4/21)
D5 4% (1/23)
Notes Prognosis: moderate, young women, moderately high oocyte numbers. Large difference
in embryo ET# between groups
Sent letter
Risk of bias
bias)
bias)
All outcomes
All outcomes
Other bias Low risk
Kolibianakis 2004
Methods Randomisation: computer generated list. Sequence not concealed. Prior to stimulation
Size: 460 different women
D3 234 D5 226
Power Calc: 5% diff in preg rate, baseline preg rate 30% need 1416 women in each
group - unrealistic so aimed for estimate
Single centre: Uni afliated
Belgium
Participants Criteria: <43y, no PGD or azoospermia
Age: D3 31.3y D5 31.5y
Cause: Similar in both groups (av: 65% male, 10% tubal, 16% unexp)
Duration: NS
Previous Treatment:
D3 0.7 D5 0.8
ICSI: 74% of all patients
FSH: D3 10.4 D5 10.4
#eggs: D3 12.1 D5 12.0
Fert rate: D3 61.3%
D5 63.4%
Blast rate: 50.7%2.4
ET policy:1-2 embryos for both groups
#ET: D3 1.90.1
D5 1.80.1
Pregnancy determination: ongoing pregnancy beyond 12 weeks
Interventions Ov Stim: initially GnRHa long protocol with HMG, replaced by GnRHantagonist with
rFSH
hCG 36h
Luteal support:
vag micronised prog until 7 week of gestation
Meida: Sequential G1/G2 Vitrolife
Culture method: NS
AHA: no
Outcomes Primary
a) LB: NS
b) preg/randomised couple:
D3 32.1% (75/234)
D5 33.2% (75/226)
preg/OPU:
D3 33.1% (75/227)
D5 33.2% (75/215)
b) preg/ET
D3 34.4% (75/218)
D5 39.5% (75/190)
c) multiple rate:
D3 26.7% (20/75)
D5 20% (15/75)
Secondary
Kolibianakis 2004 (Continued)
a) imp: ongoing
D3 24.52.5% (96/234)
D5 26.62.7% (94/226)
b) miscarriage: (rst 12 weeks)
D3 21.9% (21/96)
D5 20.2% (19/94)
c) embryo freeze:
D3 61.5% (144/234)
D5 50.4% (114/226)
D2 6.8% D5 15.9%
(from randomisation to ET) day5 9.1% due to lack of blasts.
f ) high order rate:
D3 nil D5 nil
Notes Prognosis: mixed unselected
Risk of bias
bias)
Low risk Computer generated list
Allocation concealment (selection bias) High risk The sequence of randomisation was not
concealed
bias)
All outcomes
High risk Not stated
All outcomes
Other bias Low risk
Levitas 2004
Methods Randomisation: computer generated random number table, blinded sealed envelopes
prior to treatment.
Size: 54 different women were randomised and analysed:
D2/3 31
day5/6/7 23
Setting: single Uni clinic in Israel
Time: NS
Therapeutic
Participants Criteria: min 3 failed IVF cycles, <37, adequate ovarian response
Age: D2/3 31.2
day5 29.1 no stat diff
Duration:
D2/3 7.0y
D5 7.1 y
Previous cycles:
D2/3 4.3 D5 4.9
Cause: D2/3 62% Male, 33% tubal
D5 79% Male, 21% tubal
ICSI: D2/3 62% D5 51%
# eggs: D2/3 12.8+/-5.3
D5 13.0+/-5.1
Fert rate: D2/3 53.9% D5 66.1%
Blast rate: 43% (65/151)
ET policy:
D2/3 was 3-4 embryos
D5 was 2-3 embryos
# embryo ET:
D2/3 3.40.7
D5 1.90.4
Preg determination: hCG + US
Interventions Ov Stim: GnRHa long and short +hMG doses adjusted daily, + hCG 36-38hr
Luteal: hCG x5, or Prog +less hCG
Media:
D2/3 P1 Irvine (23x) and Cook (8x)
D5 Sequential G1/G2 Vitrolife
Culture system: NS
AHA: NS
Outcomes Primary
a) Live birth:
D2/3 9.7% (3/31)
D5 13.0% (3/23)
b) preg/OPU:
D2/3 12.9% (4/31),
D5 21.7% (5/23)
b) preg/ET:
D2/3 13.8% (4/29),
D5 29.4% (5/17)
Levitas 2004 (Continued)
c) Multiple preg:
D2/3 75% (3/4),
D5 40% (2/5)
Secondary
a) Imp: D2/3 (6/100) 6.0% D5 (7/33) 21.2%
b) miscarriage: D2/3 (1/4)
D5 (2/5)
c) embryo freeze rate:
D3 22.6% (7/31)
D5 13.0% (3/23)
D2 2/31 (6.4%), embryo arrest at 2PN
D5 6/23 (26%) lack of blasts
f ) high order:
D2/3 nil
D5 1x trip (monozygotic twin)
g) monozygotic twinning: 1 in D5
Notes Prognosis: both good and poor
Note: young women with large numbers of failed cycles
Uneven number in each group
Similar gures to the 2001 abstract
letter sent and reply received
Risk of bias
bias)
Low risk Computer generated randomisation list
bias)
All outcomes
Unclear risk Not clear which of participants or outcome
assessors were blinded
All outcomes
Other bias Low risk
Levron 2002
Methods Randomisation: sealed envelope performed on day1 post OPU
Blinded: Yes
Size: 90 different women
D3 44 D5 46
Single centre: Uni clinc
Israel
Power calc: No
Participants Criteria: <38y, <5 prev IVF,
>5 or more oocytes D1
Age: D3 31.5 D5 30.9
Cause/Duration: NS
Previous Treatment: <5
ICSI: D3 59.1%
D5 51.2%
FSH: NS
# eggs: D3 16.3
D5 15.2
Fert rate: D3 60.1%
D5 62.0%
Blast rate: 34.2% (3 patients no blasts 6.5%)
ET policy: max 3 for both D3 and D5
#ET: D3 3.1 (0.6)
D5 2.3 (0.8)
Luteal support: NS
Media: sequential Cook
Culture method: NS
AHA: NS
Outcomes Primary
a) LB per woman:
D3 34% (15/44)
D5 17.4% (8/46)
b) Preg/OPU/woman
D3 45.5% (20/44)
D5 17.4 (8/46)
b) Preg/Et
D3 45% (20/44)
D5 18.6% (8/43)
c) multiple preg
D3 40% (8/20)
D5 50% (4/8)
Secondary
a) Imp: D3 38.7 % (55/137)
D5 20.2% (20/99)
b) miscarriage: NS
Levron 2002 (Continued)
c) embryo freeze:
D3 56.8% (25/44)
D5 26.1% (12/46)
d) cancellation rate
D3 0%
D5 6.5% (due to lack of blasts available)
f ) High order
D3 3/20 pregs
D5 1/8 pregs
Notes Prognosis: moderate
Young women
Moderately high numbers of oocytes
Clarication letter sent
Same ET policy
Risk of bias
bias)
Unclear risk Method of randomisation unknown
bias)
All outcomes
Unclear risk Blinded, unclear who was blinded
All outcomes
High risk 20 women were excluded post randomisa-
tionas they did not have a transfer, not clear
from which group they came
Other bias Low risk
Livingstone 2002
Methods Randomisation: sealed envelope
Participants Size: 79 recruited
59 separate women analysed
20 failed criteria or various reasons - outcomes given where possible
D3 29 D5 30
Single Centre: Uni afliated Australia
Power Calc: Yes for reduction of twins
Livingstone 2002 (Continued)
Intention to treat: not done (those with no ET were excluded)
Prognosis: moderate
Young women
Moderately high numbers of oocytes
Clarication letter sent
Criteria: <38y, 3 or less previous cycles
Age: D3 31.6 D5 33.5
Cause: Similar in both
Duration: D3 3.8y D5 4.1y
Previous treatment: NS
ICSI: yes but no details
FSH: NS
ET policy: day2/3 2x embryos, day5 1x embryo
#ET D3 2.0 D5 1.0
Preg determination: NS
Interventions Ov Stim: GnRH long+ rFSH + hCG 36 hr trigger
Luteal support: hCG x2 or prog pessaries
Media: Syd IVF sequential
Culture method:
microdrops under oil in Minc incubator
AHA: NS
Outcomes Primary
a) LB/woman: D3 37.9% (11/29)
D5 46.7% (14/30)
b) preg/woman
D3 51.7% (15/29)
D5 50% (15/30)
c) multiple rate:
D3 13.8% (4/15)
D5 0% (0/15)
Secondary
a) Imp: D3 37.9% (22/58)
D5 56.2% (18/32)
b) miscarriage or ectopic:
D3 20% (3/15)
D5 7% (1/15)
c) embryo freeze: performed but no details given
e) cancellation rate: overall 26% (but not due lack of blasts - other various reasons - not
included in stats)
f ) high order rate: Nil for both groups
Notes Prognosis: high
Data from Thesis 2002 and not abstract 2001
Low fert rate
Livingstone 2002 (Continued)
Aim to reduce twinning
Risk of bias
bias)
Low risk Computer generated
bias)
All outcomes
Unclear risk Not possible to blind
All outcomes
Unclear risk 20 recruited but not randomised
Selective reporting (reporting bias) Low risk Live birth and multiple pregnancy rate reported
Other bias Unclear risk Single blastocyst transfer (Day 5) but double embryo
transfer (Day 3)
Motta 1998 A & B
Methods Randomisation:randomly assigned
Blinding: NS
Size: 83 women in 116 cycles randomly assigned and analysed: D2 58 and D5 58
Setting: Uni clinic private in Brazil
Time: NS
Power analysis: NS
Therapeutic
Participants Criteria: unselected
Age overall: 33.8 6.6y
? All ICSI
Baseline and population characteristics:NS
#eggs: D3 12.9, D5 11.7
Fert rate: D3 7.8, D5 9.1
# PN embryos: D3 452, D5 528
Cycles with no fert: 2/116
Cycles with no blasts: 6
ET policy: D3 3-5 embryos
D5 1-3 embryos
#embryos ET: D3 4.6,
D5 2.3
Preg determination: ultrasound
Motta 1998 A & B (Continued)
Interventions Ov Stim: down reg with gonadotropin 150-450
luteal: NS
Media: sequential media P1/Irvines Blast
Culture method: NS
AH: NS
Outcomes Primary
a) Live birth: NS
b) preg/ET: D3 (21/57) 36.8%
D5 (21/52) 40.4%
b) preg/OPU: D3 (21/58) 36.2%
D5 (21/58) 36.2%
c) Multi preg: D3 10/21 57%,
D5 3/21 14%
Secondary
a) Imp: D3 (51/262) 19.4% D5 (36/120) 30.1%, P<0.01
b) miscarriage: NS
c) emb Fz D3 5.1 /cycle 74%/pat (45/58)
D5 1.4/cycle 26% pat (15/58)
d) embryo utilisation rate: NS
e) ET Rate: D2 (57/58) 98.3%
D5 (52/58) 89.6%
e) cancellation rate: D2 1.7% (1/58), D5 10.4% (6/58)
f ) high order: NS
g) monozygotic: NS
Notes Prognosis: moderate to good. No letter sent
Risk of bias
bias)
bias)
All outcomes
All outcomes
Pantos 2004
Methods Randomisation: not stated
Allocation: not stated
Blinded: no
Size: 243, D2 81, D3 81, D5 81
No dropouts Single centre: Greece
Participants <41 years olf. less than 4 previous unsuccessful ART attempts
Interventions Ov Stim: long or short protocol, using GnRH agonist and recombinant FSH; Luteal
support: NS Media: sequential media commercial AHA: N
Outcomes Total: Pregnancy rate: D2 46.9%, D3: 48.1%, D5 37%
Day 2 or 3: Pregnancy rate: 47.5%; Multiple PR: 29.9%; High Order Multiple PR: 3.
9%; Miscarriage Rate: 11.7%; Embryo Freezing rate: 48.8%
Day 5: Pregnancy rate: 37.0%; Multiple PR:33.3% ; High Order Multiple PR: 6.7% ;
Miscarriage Rate: 33.3% ; Embryo Freezing rate: 19.8%
Notes
Risk of bias
bias)
bias)
All outcomes
All outcomes
Low risk
Other bias Low risk
Papanikolaou 2005
Methods Randomisation: computer generated list
Randomised on day 3 following OPU
Concealment?
Blinded: no
Size:164 separate patients
D3 84 D5 80
Single centre
Belgium
Power calc: yes prospectively
Interim analysis terminated at halfway point
Participants Criteria: 4 good quality embryos on D3, <38y,
<4 cycles FSH <12 D3
Age: D3 29.6 D5 29.9
Cause: no difference
Prev treatment:
D3 1.3 D5 1.4
ICSI:D3 66.7% D5 67.5%
FSH: NS
# eggs:
D3 13.9 D5 15.1
Fert rate: D3 65%
D5 65%
Blast rate: NS
ET policy: 2 embryos
# embryos transferred:
D3 2.0 D5 1.97
(4 women D5 had 1 embryo transferred due to lack of availability)
Pregnancy determinant:
HCG + US 7 weeks
Interventions Ov Stim: 2 types
a) GnRH agonist
b) GnRH antagonist
rFSH/HCG
Luteal support:
prog vaginal
Media: G1/G2 Vitrolife
Culture method: NS
AHA: no
Outcomes Primary
a) LB/woman, OPU and ET
D3 27.4% (23/84)
D5 47.5% (38/80)
b) Preg/woman, OPU, ET
D3 32.1% (27/84)
D5 52% (42/80)
c) Multiple rate:
Papanikolaou 2005 (Continued)
D3 29.6% (8/27)
D5 42.9% (18/42)
Secondary:
a) Imp: D3 20.6% (35/170)
D5 37.3% (59/158)
b) miscarriage from positive hCG:
D3 34.3% (12/35)
D5 28.3% (15/53)
c) embryo freeze:
D3 36.3% 3.30.5
D5 23.5% 2.30.3
d) embryo utilisation
D3 58% (5.3/9.1)
D5 43.6% (4.27/9.8)
e) ET rate: 100% both groups
f ) High order rate: nil
g) monozygotic twin rate: NS
Notes Prognosis: high, 4 high qual embryos D3, young women
Letter sent regarding concealment
100% ET rate
Risk of bias
bias)
Unclear risk Computer generated list
Allocation concealment (selection bias) High risk No concealment
bias)
All outcomes
All outcomes
Other bias Low risk
Papanikolaou 2006
Methods Randomisation: computer generated list
Randomised prior to start of cycle
Concealment: no but used group A or B
Blinded: no
Size:351 separate patients
D3 176 D5 175
To OPU
D3 166 D5 163
To ET
D3 156 D5 149
Single centre
Belgium
Power calc: yes prospectively
Interim analysis: Yes - study terminated at halfway point.
Intention to treat: yes
Participants Criteria: single embryo transfer, <36y, <3 cycles FSH 12 on D3
Age: D3 30.4 D5 30.5
Cause: no difference
Prev treatment:
D3 7.4% D5 9.1%
ICSI:D3 63.2% D5 64.5%
FSH: NS
# eggs:
D3 12.5 D5 13.9
Fert rate: D3 60%
D5 58%
Blast rate: NS
ET policy: 1 embryo
# embryos transferred:
D3 1.0 D5 1.0
(Number patients where no embryos were available for ET:
D3 8 D5 11
Pregnancy determinant:
HCG + US 7 weeks
Interventions Ov Stim: GnRH antagonist
rFSH/HCG
Luteal support:
prog vaginal
Media: NS
Culture method: NS
AHA: no
Outcomes Primary
a) LB/woman
D3 21.6% (38/176)
D5 32.0% (56/175)
b) Preg/woman
Papanikolaou 2006 (Continued)
D3 23.3% (41/176)
D5 33.1% (58/175)
c) Multiple rate:
D3 5% (2/176)
D5 0% (0/175)
Secondary:
a) imp: D3 24.2% (38/156)
D5 38.9% (58/149)
b) miscarriage from positive hCG:
D3 35.6% (21/59)
D5 23.3% (17/73)
c) embryo freeze:
D3 ?% 4.24.1
D5 ?% 2.22.7
D3 65% (5.2/8.0)
D5 42.6%(3.2/7.5)
e) cancellation rate (from those those that had OPU:
D3 5.3% D5 8.6%
f ) High order rate: nil
g) monozygotic twin rate (clinical preg):
D3 2/41 D5 0
Notes Prognosis: high, young women
Letter sent regarding concealment, media and freezing
Risk of bias
bias)
Low risk Computer generation list
Allocation concealment (selection bias) High risk Not concealed
bias)
All outcomes
All outcomes
Low risk Intention-to-treat analysis was applied
Other bias Low risk
Rienzi 2002
Methods Randomisation: computer generated randomised list. Randomised on day 1 following
OPU
Blinded: No
Size: 98 separate women randomised and analysed
no dropouts
D3 48 D5 50
Single centre
Italy
Power calc: No
Therapeutic
Participants Criteria: <38y, ICSI only, 8 or more zygotes
Age: D3 31.63.1
D5 32.22.5
Cause/Duration: NS
FSH: NS
# eggs: D3 12.77.1
D5 13.15.2
Fert rate: D3 71.2%
D5 71.8%
Blast rate: 44.8% (211/470)
ET policy: Max 2 for both groups
#ET: D2 2.0 D5 2.0
Preg determination
hCG + US 8 weeks
LBR:yes
Cumulative including freezing: yes
Interventions Ov Stim: down reg with rFSH + hCG trigger 36h
Luteal support: NS
Media: G1/G2 Vitrolife both groups
Culture method: NS
AHA: NS
Outcomes Primary
Fresh transfer cycles only
a) LB/woman:
D3 50% (24/48)
D5 48% (24/50)
b) preg/OPU/ET: (clinical)
D3 56% (27/48)
D5 58% (29/50)
c) multiple rate/preg:
D3 25.9% (7/27)
D5 31.0 (9/29) from personal communication
Secondary
a) imp: D3 35% (34/96)
Rienzi 2002 (Continued)
D5 38% (38/100)
b)miscarriage:
D3 6.3% (3/48)
D5 10% (5/50)
c) embryo freeze:
D3 87.5% (42/48)
D5 36% (18/50)
d) embryo utilisation: cumulative LBR results including fresh and thawed:
D3 77% (37births/48 OPUs)
D5 52% (26births/50 OPUs)
e) ET rate
f ) high order rate
D3 nil
D5 nil
Notes Prognosis: Good
>8 zygotes
Conclude that embryo/PN score system as good as blast culture
Cumulative fresh thawed results
Risk of bias
bias)
Low risk Computer generated list
Allocation concealment (selection bias) Unclear risk Method of allocation not stated
bias)
All outcomes
All outcomes
Other bias Low risk
Schillaci 2002
Methods Randomisation: method NS randomly allocated on day1 fert check
Size 120 separate patients randomised and analysed no dropouts
D3 60 D5 60
Single centre
Italy
Power calc: NS
Therapeutic
Participants Criteria: 8 or more MII oocytes and 3 zygotes
Age: NS
Cause/Duration: NS
ICSI: yes but no details
FSH: NS
#eggs D3 9.0 D5 8.9
Fert rate: no diff
Blast rate: 60.3%
ET policy: 2-3 embryos for both groups except only 2 if expanded blasts for D5
#ET: D3 2.80.2
D5 1.80.4
Preg determination: NS
Luteal support: NS
Media: D3 IVF-20 D5 G1/G2 both Vitrolife
Culture method: NS
AHA: NS
Outcomes Primary
a) LB: NS
b) preg/OPU/ET/woman:
D3 38.3% (23/60)
D5 40.0%(24/60)
c) multiple rate: NS
Secondary
a) imp: D3 13.7% (23/168)
D5 23.6% (26/110)
b)miscarriage: NS
e) ET rate: nil cancellations
f ) high order rate: NS
Notes Prognosis: ? unclear ? moderate #eggs
Abstract only
?Error in No. of patients 51 or 60?
Schillaci 2002 (Continued)
Risk of bias
bias)
bias)
All outcomes
All outcomes
Unclear risk No dropouts or exclusions
Ten 2011
Methods Randomisation: method NS distributed randomly on day 3 fert check
55 separate patients randomised and analysed no drop outs
D3 27 D5 28
Single centre
Spain
Power calc: NS
Participants Criteria: at least one embryo type A and 2 type B
Age: NS
rst or second cycle
Cause/Duration: NS
IVf and ICSI
Interventions day 3 versus day 5
Outcomes clinical pregnancy rate but cumulative pregnancy rate later
number of frozen embryos
Notes abstract only
Risk of bias
bias)
Unclear risk Distributed randomly
Ten 2011 (Continued)
Allocation concealment (selection bias) Unclear risk Distributed randomly
bias)
All outcomes
All outcomes
Other bias Unclear risk It is an abstract
Van der Auwera 2002
Methods Randomisation: blind randomisation with sealed envelope at start of hormone stimula-
tion
Size: 136 separate patients randomised and 129 were analysed due to 7 dropouts
(3x D2 and 4xD5)
D2 63 D5 66
Single centre
Belgium
Power calc: performed but abandoned due to high multiple rate, difculty in recruitment
and high cancellation rate in D5
Therapeutic
Participants Criteria: unselected
Age: D2 31.73.3
D5 31.53.5
Primary/Secondary: no diff between groups
Duration: D2 3.32.0
D5 3.41.5
Cause: no diff (approx 50% male <10% tubal, slightly more unexp in D5)
Previous treatment:
D2 1.7 D5 1.7
ICSI: D2 19/63
D5 25/66
FSH: NS
#eggs: D2 10.75.4
D5 11.55.1
Fert rate: D2 51.4%
D5 55.7%
Blast rate: 44.7%
ET policy: max of two for both groups.
NOTE that D2 group had only 5x PN cultured on for transfer with remaining frozen.
While all D5 group were cultured to ET.
#ET: D2 1.860.28
D5 1.870.22
Van der Auwera 2002 (Continued)
Pregnancy determination: +ve HCG also clinical pregnancy (denition NS)
Luteal support: NS
Media: D3 IVF-20 D5 G1/G2 both Vitrolife
Culture method: NS
AHA: NS
Ov Stim: GnRH down reg+Humegon+HCG trigger 36h
Luteal support: HCG every 3 days or prog pessaries
Media: either Cook sequential or Vitrolife G1/G2 simultaneous trial
Culture method NS but used low O2
AHA: NS
Outcomes Primary
a) LB/OPU/woman:
D2 27% (17/63)
D536% (24/66)
LB/ET
D2 30% (17/57)
D5 50% (24/48)
b) preg/OPU/woman: (clinical only)
D2 32% (20/63)
D5 44% (29/66)
b) preg/ET
D3 35% (20/57)
D5 60% (2948)
c) multiple rate:
D2 45% (9/20)
D5 31% (9/29)
Secondary
a) imp: D2 27.4% (31/106)
D5 51.1% (41/90)
b) miscarriage:
per randomised:
D2 3/66
D5 5/70
D2 15% (3/20)
D5 17.2% (5/29)
c) embryo freeze: D2 56%
(but many 2PN) D5 39%
d) embryo utilisation:
D2 73.3% (252/344)
D5 42.1% (178/423)
D2 10% (6/63)
D5 27% (18/66) lack of blasts on day6
f ) high order rate: Nil
Van der Auwera 2002 (Continued)
Notes Prognosis: mixed - unselected
Aim to reach highest cryoaugmented preg rate
Smaller cohort of embryos to choose from in D2 group due to freezing on day1.
New policy patients with >5 zygotes go to D5 transfer with 79% preg rate
Risk of bias
bias)
Low risk Not stated
bias)
All outcomes
All outcomes
Low risk
Other bias Low risk
ET - embryo transfer
Blast - blastocyst
Fert - fertilization
OPU - oocyte pick up
AH - assisted hatching
# - number
NS - not stated
US - ultrasound
Fz - freeze
D3 - embryo transfer on day 3 post OPU (i.e early cleavage stage)
D5 - embryo transfer on day 5 post OPU (i.e. blastocyst stage)
Imp - implantation
Clin preg - clinical pregnancy
IM - intramuscular injection
Ov Stim - ovarian stimulation regimen
FCS - fetal chord serum
G1/G2 sequential media from Vitrolife
emb - embryo
trans - transfer
years - years
Unex - unexplained
morula- embryonic stage prior to blastocysts (usually embryos with delayed development on day5)
rFSH - recombinant follicle stimulating hormone (fertility ovarian stimulation drug)
IU - international unit of drug administration
hCG - human chorionic gonadotropin (trigger injection that initiates ovulation and maturation of oocytes)
Blastocyte rate - number of blastocysts developed divided by number of 2PN embryos available
Characteristics of excluded studies [ordered by study ID]
Study Reason for exclusion
Bungum 2002 Used co-culture
Guerin 1991 Used co-culture
Levitas 2001 Duplicate of Levitas 2004
Levron 2001 Quasi Randomised
Loup 2009 Included transfer of embryos on two separate days within the same cycle
Menezo 1992 Used co-culture
Papanikolaou 2005a Duplicate data
Papanikolaou 2005b Duplicate data
Utsonomiya 2004a non-randomised study (sequentially numbered)
Vanderzwalmen 2006 non-randomised study - according to even or odd year of birth
Zech 2007 non-randomised study - according to even or odd year of birth
D A T A A N D A N A L Y S E S
Comparison 1. Live birth rate
Outcome or subgroup title
No. of
studies
No. of
participants
Statistical method Effect size
1 Live birth per couple 12 1510 Peto Odds Ratio (Peto, Fixed, 95% CI) 1.40 [1.13, 1.74]
2 Live birth per couple: grouped by
number of embryos transferred
12 Peto Odds Ratio (Peto, Fixed, 95% CI) Subtotals only
2.1 more cleavage stage than
blastocyst embyros transferred
6 483 Peto Odds Ratio (Peto, Fixed, 95% CI) 1.52 [1.03, 2.23]
2.2 single embryo transfer 2 458 Peto Odds Ratio (Peto, Fixed, 95% CI) 1.46 [0.98, 2.19]
2.3 equal number of embryos
transferred
3 Live birth rate per couple:
grouped by prognosis
12 1510 Odds Ratio (M-H, Random, 95% CI) 1.37 [1.01, 1.85]
3.1 good prognostic factors 8 1126 Odds Ratio (M-H, Random, 95% CI) 1.43 [0.99, 2.07]
3.2 poor prognostic factors 2 77 Odds Ratio (M-H, Random, 95% CI) 1.99 [0.49, 8.04]
3.3 unselected group 2 307 Odds Ratio (M-H, Random, 95% CI) 1.05 [0.56, 1.97]
4 Live birth rate: grouped by day
of randomisation
4.1 randomisation at start of
cycle
4.2 randomised on day of
OPU and day 1 after OPU
4.3 randomised Day 2 to 3
post OPU
4.4 day of randomisation
unstated
Comparison 2. Clinical pregnancy rate
No. of
studies
No. of
participants
1 clinical pregnancy rate per
couple
couple: grouped by number of
embryos transferred
23 Odds Ratio (M-H, Fixed, 95% CI) Subtotals only
2.1 equal numbers of ET 11 1854 Odds Ratio (M-H, Fixed, 95% CI) 1.19 [0.99, 1.44]
blastocyst embryos transfered
12 1387 Odds Ratio (M-H, Fixed, 95% CI) 1.07 [0.86, 1.33]
2.3 Single embryo transfer 3 478 Odds Ratio (M-H, Fixed, 95% CI) 1.24 [0.84, 1.82]
couple: grouped by prognosis
3.1 Good prognostic factors 14 1756 Odds Ratio (M-H, Random, 95% CI) 1.15 [0.83, 1.58]
3.2 Poor prognostic factors 2 77 Odds Ratio (M-H, Random, 95% CI) 2.59 [0.75, 8.92]
3.3 Unselected group 7 1408 Odds Ratio (M-H, Random, 95% CI) 1.01 [0.81, 1.25]
couple: grouped by day of
randomisation
4.1 Randomised start of cycle 7 1371 Odds Ratio (M-H, Fixed, 95% CI) 1.19 [0.95, 1.49]
4.2 Randomised on day of
OPU or day 1
4.3 Randomised on day 2 to 3 4 537 Odds Ratio (M-H, Fixed, 95% CI) 1.59 [1.13, 2.23]
4.4 Day of randomisation
unstated
Comparison 3. Cumulative pregnancy rate
No. of
studies
No. of
participants
1 cumulative pregnancy rate from
fresh and frozen transfers
Comparison 4. Multiple-pregnancy rate
No. of
studies
No. of
participants
1 multiple-pregnancy rate per
couple
couple: grouped by number of
embryo transfer
2.1 Equal number of embryos
transferred
2.2 More cleavage stage than
blastocyst embryos transferred
2.3 Single embryo transfer 1 351 Odds Ratio (M-H, Fixed, 95% CI) 0.20 [0.01, 4.17]
couple: grouped by prognosis
3.1 Good prognostic factors 11 1498 Odds Ratio (M-H, Fixed, 95% CI) 0.88 [0.63, 1.22]
3.2 Poor prognostic factors 1 54 Odds Ratio (M-H, Fixed, 95% CI) 0.89 [0.14, 5.81]
3.3 Unselected 4 929 Odds Ratio (M-H, Fixed, 95% CI) 0.96 [0.62, 1.47]
4 high order pregnancies (more
than 2 gestational sacs) per
couple
5 high order pregnancy: grouped
by number of embryos
transferred
5.1 Equal number of embryos
transferred
5.2 More cleavage stage than
6 high order pregnancies: grouped
by prognosis
6.1 Good prognostic factors 9 1385 Odds Ratio (M-H, Fixed, 95% CI) 0.29 [0.08, 1.06]
6.2 Poor prognostic factors 1 54 Odds Ratio (M-H, Fixed, 95% CI) 4.2 [0.16, 107.89]
6.3 Unselected 2 596 Odds Ratio (M-H, Fixed, 95% CI) 0.0 [0.0, 0.0]
pregnancy
14 Odds Ratio (M-H, Fixed, 95% CI) Totals not selected
8 high order pregnancies per total
pregnancies
12 Odds Ratio (M-H, Fixed, 95% CI) Totals not selected
Comparison 5. Miscarriage rate
No. of
studies
No. of
participants
1 miscarriage rate per couple 14 2127 Peto Odds Ratio (Peto, Fixed, 95% CI) 1.14 [0.84, 1.55]
2 miscarriage rate per pregnancy 14 Odds Ratio (M-H, Fixed, 95% CI) Totals not selected
Comparison 6. Embryo freezing rate
No. of
studies
No. of
participants
1 embryo freezing per couple 11 1729 Peto Odds Ratio (Peto, Fixed, 95% CI) 2.88 [2.35, 3.51]
2 Embyro freezing per couple:
grouped by number of embryos
transferred
transferred
3 Embryo freezing per couple:
grouped by prognostic factors
3.1 good prognostic factors 6 612 Odds Ratio (M-H, Random, 95% CI) 6.39 [3.12, 13.10]
3.2 poor prognostic factors 0 0 Odds Ratio (M-H, Random, 95% CI) 0.0 [0.0, 0.0]
3.3 unselected 4 874 Odds Ratio (M-H, Random, 95% CI) 2.60 [1.31, 5.16]
Comparison 7. Failure to transfer embryos rate per couple
No. of
studies
No. of
participants
per couple
per couple: grouped by
prognostic factors
2.1 good prognostic factors 9 1315 Odds Ratio (M-H, Fixed, 95% CI) 0.67 [0.35, 1.27]
2.2 poor prognostic factors 2 77 Odds Ratio (M-H, Fixed, 95% CI) 0.20 [0.04, 1.08]
2.3 unselected 5 1067 Odds Ratio (M-H, Fixed, 95% CI) 0.27 [0.17, 0.43]
per couple: grouped by number
of embryos transferred
transferred
blastocyst embryos tranferred
3.3 single embryo transfer 1 351 Odds Ratio (M-H, Fixed, 95% CI) 0.71 [0.28, 1.81]
Analysis 1.1. Comparison 1 Live birth rate, Outcome 1 Live birth per couple.
Review: Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology
Comparison: 1 Live birth rate
Outcome: 1 Live birth per couple
Study or subgroup Day 5/6 Day 2/3
Peto
Odds Ratio Weight
Peto
Odds Ratio
n/N n/N Peto,Fixed,95% CI Peto,Fixed,95% CI
Brugnon 2010 22/55 21/52 7.8 % 0.98 [ 0.46, 2.12 ]
Devreker 2000 3/11 1/12 1.0 % 3.53 [ 0.43, 29.14 ]
Elgindy 2011 52/100 35/100 14.8 % 1.99 [ 1.14, 3.48 ]
Emiliani 2003 33/82 41/89 12.6 % 0.79 [ 0.43, 1.44 ]
Frattarelli 2003 15/29 8/28 4.2 % 2.57 [ 0.90, 7.35 ]
Levitas 2004 3/23 3/31 1.6 % 1.40 [ 0.26, 7.65 ]
Levron 2002 8/46 15/44 5.2 % 0.42 [ 0.16, 1.08 ]
Livingstone 2002 14/30 11/29 4.4 % 1.42 [ 0.51, 3.96 ]
0.02 0.1 1 10 50
Favours day 2/3 Favours day 5/6
(Continued . . . )
(. . . Continued)
Peto
Odds Ratio Weight
Peto
Odds Ratio
Papanikolaou 2005 38/80 23/84 11.6 % 2.35 [ 1.25, 4.43 ]
Papanikolaou 2006 56/175 38/176 20.7 % 1.70 [ 1.06, 2.72 ]
Rienzi 2002 24/50 24/48 7.4 % 0.92 [ 0.42, 2.03 ]
Van der Auwera 2002 24/70 17/66 8.6 % 1.49 [ 0.72, 3.10 ]
Total (95% CI) 751 759 100.0 % 1.40 [ 1.13, 1.74 ]
Total events: 292 (Day 5/6), 237 (Day 2/3)
Heterogeneity: Chi
2
= 18.43, df = 11 (P = 0.07); I
2
=40%
Test for overall effect: Z = 3.07 (P = 0.0021)
Test for subgroup differences: Not applicable
0.02 0.1 1 10 50
Analysis 1.2. Comparison 1 Live birth rate, Outcome 2 Live birth per couple: grouped by number of
embryos transferred.
Outcome: 2 Live birth per couple: grouped by number of embryos transferred
Peto
Odds Ratio Weight
Peto
Odds Ratio
1 more cleavage stage than blastocyst embyros transferred
Devreker 2000 3/11 1/12 3.3 % 3.53 [ 0.43, 29.14 ]
Elgindy 2011 52/100 35/100 47.5 % 1.99 [ 1.14, 3.48 ]
Frattarelli 2003 15/29 8/28 13.4 % 2.57 [ 0.90, 7.35 ]
Levitas 2004 3/23 3/31 5.1 % 1.40 [ 0.26, 7.65 ]
Levron 2002 8/46 15/44 16.6 % 0.42 [ 0.16, 1.08 ]
Livingstone 2002 14/30 11/29 14.1 % 1.42 [ 0.51, 3.96 ]
Subtotal (95% CI) 239 244 100.0 % 1.52 [ 1.03, 2.23 ]
Heterogeneity: Chi
2
= 9.66, df = 5 (P = 0.09); I
2
=48%
2 single embryo transfer
Brugnon 2010 22/55 21/52 27.3 % 0.98 [ 0.46, 2.12 ]
Papanikolaou 2006 56/175 38/176 72.7 % 1.70 [ 1.06, 2.72 ]
Subtotal (95% CI) 230 228 100.0 % 1.46 [ 0.98, 2.19 ]
Heterogeneity: Chi
2
= 1.40, df = 1 (P = 0.24); I
2
=29%
3 equal number of embryos transferred
Brugnon 2010 22/55 21/52 11.3 % 0.98 [ 0.46, 2.12 ]
Emiliani 2003 33/82 41/89 18.4 % 0.79 [ 0.43, 1.44 ]
Papanikolaou 2005 38/80 23/84 16.8 % 2.35 [ 1.25, 4.43 ]
Papanikolaou 2006 56/175 38/176 30.1 % 1.70 [ 1.06, 2.72 ]
Rienzi 2002 24/50 24/48 10.8 % 0.92 [ 0.42, 2.03 ]
Van der Auwera 2002 24/70 17/66 12.6 % 1.49 [ 0.72, 3.10 ]
Subtotal (95% CI) 512 515 100.0 % 1.35 [ 1.04, 1.75 ]
Heterogeneity: Chi
2
= 8.52, df = 5 (P = 0.13); I
2
=41%
Test for subgroup differences: Chi
2
= 0.28, df = 2 (P = 0.87), I
2
=0.0%
0.05 0.2 1 5 20
Favoursday 2/3 Favours Day 5/6
Analysis 1.3. Comparison 1 Live birth rate, Outcome 3 Live birth rate per couple: grouped by prognosis.
Outcome: 3 Live birth rate per couple: grouped by prognosis
Study or subgroup Day 5/6 Day 2/3 Odds Ratio Weight Odds Ratio
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
1 good prognostic factors
Brugnon 2010 22/55 21/52 9.1 % 0.98 [ 0.45, 2.13 ]
Elgindy 2011 52/100 35/100 12.6 % 2.01 [ 1.14, 3.55 ]
Frattarelli 2003 15/29 8/28 5.7 % 2.68 [ 0.89, 8.02 ]
Levron 2002 8/46 15/44 6.6 % 0.41 [ 0.15, 1.09 ]
Livingstone 2002 14/30 11/29 6.1 % 1.43 [ 0.51, 4.04 ]
Papanikolaou 2005 38/80 23/84 11.0 % 2.40 [ 1.25, 4.60 ]
Papanikolaou 2006 56/175 38/176 14.4 % 1.71 [ 1.06, 2.76 ]
Rienzi 2002 24/50 24/48 8.8 % 0.92 [ 0.42, 2.04 ]
Subtotal (95% CI) 565 561 74.4 % 1.43 [ 0.99, 2.07 ]
Heterogeneity: Tau
2
= 0.13; Chi
2
= 13.71, df = 7 (P = 0.06); I
2
=49%
2 poor prognostic factors
Devreker 2000 3/11 1/12 1.4 % 4.13 [ 0.36, 47.30 ]
Levitas 2004 3/23 3/31 2.8 % 1.40 [ 0.26, 7.66 ]
Subtotal (95% CI) 34 43 4.2 % 1.99 [ 0.49, 8.04 ]
Heterogeneity: Tau
2
= 0.0; Chi
2
= 0.51, df = 1 (P = 0.48); I
2
=0.0%
3 unselected group
Emiliani 2003 33/82 41/89 11.8 % 0.79 [ 0.43, 1.45 ]
Van der Auwera 2002 24/70 17/66 9.6 % 1.50 [ 0.72, 3.15 ]
Subtotal (95% CI) 152 155 21.4 % 1.05 [ 0.56, 1.97 ]
Heterogeneity: Tau
2
= 0.09; Chi
2
= 1.75, df = 1 (P = 0.19); I
2
=43%
Total (95% CI) 751 759 100.0 % 1.37 [ 1.01, 1.85 ]
0.02 0.1 1 10 50
(Continued . . . )
(. . . Continued)
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
Heterogeneity: Tau
2
= 0.10; Chi
2
= 18.35, df = 11 (P = 0.07); I
2
=40%
2
= 1.01, df = 2 (P = 0.60), I
2
=0.0%
0.02 0.1 1 10 50
Analysis 1.4. Comparison 1 Live birth rate, Outcome 4 Live birth rate: grouped by day of randomisation.
Outcome: 4 Live birth rate: grouped by day of randomisation
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
1 randomisation at start of cycle
Brugnon 2010 22/55 21/52 9.1 % 0.98 [ 0.45, 2.13 ]
Emiliani 2003 33/82 41/89 11.8 % 0.79 [ 0.43, 1.45 ]
Levitas 2004 3/23 3/31 2.8 % 1.40 [ 0.26, 7.66 ]
Papanikolaou 2006 56/175 38/176 14.4 % 1.71 [ 1.06, 2.76 ]
Van der Auwera 2002 24/70 17/66 9.6 % 1.50 [ 0.72, 3.15 ]
Subtotal (95% CI) 405 414 47.7 % 1.25 [ 0.90, 1.73 ]
Heterogeneity: Tau
2
= 0.02; Chi
2
= 4.47, df = 4 (P = 0.35); I
2
=11%
2 randomised on day of OPU and day 1 after OPU
Frattarelli 2003 15/29 8/28 5.7 % 2.68 [ 0.89, 8.02 ]
Levron 2002 8/46 15/44 6.6 % 0.41 [ 0.15, 1.09 ]
Rienzi 2002 24/50 24/48 8.8 % 0.92 [ 0.42, 2.04 ]
0.02 0.1 1 10 50
(Continued . . . )
(. . . Continued)
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
Subtotal (95% CI) 125 120 21.1 % 0.97 [ 0.37, 2.58 ]
Heterogeneity: Tau
2
= 0.50; Chi
2
= 6.28, df = 2 (P = 0.04); I
2
=68%
3 randomised Day 2 to 3 post OPU
Elgindy 2011 52/100 35/100 12.6 % 2.01 [ 1.14, 3.55 ]
Papanikolaou 2005 38/80 23/84 11.0 % 2.40 [ 1.25, 4.60 ]
Subtotal (95% CI) 180 184 23.6 % 2.17 [ 1.42, 3.33 ]
Heterogeneity: Tau
2
= 0.0; Chi
2
= 0.16, df = 1 (P = 0.69); I
2
=0.0%
4 day of randomisation unstated
Devreker 2000 3/11 1/12 1.4 % 4.13 [ 0.36, 47.30 ]
Livingstone 2002 14/30 11/29 6.1 % 1.43 [ 0.51, 4.04 ]
Subtotal (95% CI) 41 41 7.6 % 1.68 [ 0.65, 4.38 ]
Heterogeneity: Tau
2
= 0.0; Chi
2
= 0.61, df = 1 (P = 0.43); I
2
=0.0%
Total (95% CI) 751 759 100.0 % 1.37 [ 1.01, 1.85 ]
Heterogeneity: Tau
2
= 0.10; Chi
2
= 18.35, df = 11 (P = 0.07); I
2
=40%
2
= 4.89, df = 3 (P = 0.18), I
2
=39%
0.02 0.1 1 10 50
Analysis 2.1. Comparison 2 Clinical pregnancy rate, Outcome 1 clinical pregnancy rate per couple.
Comparison: 2 Clinical pregnancy rate
Outcome: 1 clinical pregnancy rate per couple
Peto
Odds Ratio Weight
Peto
Odds Ratio
Brugnon 2010 23/55 24/52 3.6 % 0.84 [ 0.39, 1.80 ]
Bungum 2003 32/61 36/57 3.9 % 0.65 [ 0.31, 1.34 ]
Coskun 2000 39/100 39/101 6.5 % 1.02 [ 0.58, 1.79 ]
Devreker 2000 4/11 1/12 0.6 % 4.84 [ 0.69, 33.64 ]
Elgindy 2011 59/100 41/100 6.8 % 2.05 [ 1.18, 3.56 ]
Emiliani 2003 39/82 46/89 5.8 % 0.85 [ 0.47, 1.54 ]
Fisch 2007 4/8 11/12 0.5 % 0.12 [ 0.02, 0.91 ]
Frattarelli 2003 18/29 10/28 2.0 % 2.82 [ 1.01, 7.89 ]
Gardner 1998 32/45 31/47 2.7 % 1.27 [ 0.53, 3.04 ]
Hreinsson 2004 22/64 25/80 4.3 % 1.15 [ 0.57, 2.32 ]
Karaki 2002 28/80 24/82 4.8 % 1.30 [ 0.67, 2.51 ]
Kolibianakis 2004 75/226 75/234 13.7 % 1.05 [ 0.71, 1.55 ]
Levitas 2004 5/23 4/31 1.0 % 1.87 [ 0.45, 7.83 ]
Levron 2002 8/46 20/44 2.6 % 0.27 [ 0.11, 0.67 ]
Livingstone 2002 15/30 15/29 2.0 % 0.93 [ 0.34, 2.57 ]
Motta 1998 A % B 21/58 21/58 3.7 % 1.00 [ 0.47, 2.13 ]
Pantos 2004 30/81 77/162 7.2 % 0.65 [ 0.38, 1.12 ]
Papanikolaou 2005 42/80 27/84 5.4 % 2.29 [ 1.24, 4.26 ]
Papanikolaou 2006 58/175 41/176 9.6 % 1.62 [ 1.02, 2.58 ]
Rienzi 2002 29/50 27/48 3.3 % 1.07 [ 0.48, 2.38 ]
Schillaci 2002 24/60 23/60 3.9 % 1.07 [ 0.52, 2.22 ]
Ten 2011 17/28 14/27 1.9 % 1.42 [ 0.50, 4.10 ]
Van der Auwera 2002 29/70 20/66 4.3 % 1.61 [ 0.80, 3.24 ]
Total (95% CI) 1562 1679 100.0 % 1.14 [ 0.99, 1.32 ]
Heterogeneity: Chi
2
= 41.63, df = 22 (P = 0.01); I
2
=47%
0.1 0.2 0.5 1 2 5 10
Analysis 2.2. Comparison 2 Clinical pregnancy rate, Outcome 2 clinical pregnancy rate per couple: grouped
by number of embryos transferred.
Outcome: 2 clinical pregnancy rate per couple: grouped by number of embryos transferred
n/N n/N M-H,Fixed,95% CI M-H,Fixed,95% CI
1 equal numbers of ET
Brugnon 2010 23/55 24/52 7.4 % 0.84 [ 0.39, 1.80 ]
Bungum 2003 32/61 36/57 9.2 % 0.64 [ 0.31, 1.34 ]
Coskun 2000 39/100 39/101 12.3 % 1.02 [ 0.58, 1.79 ]
Fisch 2007 4/8 11/12 2.3 % 0.09 [ 0.01, 1.08 ]
Hreinsson 2004 22/64 25/80 7.6 % 1.15 [ 0.57, 2.32 ]
Kolibianakis 2004 75/226 75/234 25.5 % 1.05 [ 0.71, 1.56 ]
Papanikolaou 2005 42/80 27/84 6.5 % 2.33 [ 1.24, 4.40 ]
Papanikolaou 2006 58/175 41/176 14.2 % 1.63 [ 1.02, 2.61 ]
Rienzi 2002 29/50 27/48 6.0 % 1.07 [ 0.48, 2.39 ]
Ten 2011 17/28 14/27 2.9 % 1.44 [ 0.49, 4.18 ]
Van der Auwera 2002 29/70 20/66 6.2 % 1.63 [ 0.80, 3.30 ]
Subtotal (95% CI) 917 937 100.0 % 1.19 [ 0.99, 1.44 ]
Heterogeneity: Chi
2
= 15.31, df = 10 (P = 0.12); I
2
=35%
2 more cleavage stage than blastocyst embryos transfered
Devreker 2000 4/11 1/12 0.4 % 6.29 [ 0.58, 68.42 ]
Elgindy 2011 59/100 41/100 10.8 % 2.07 [ 1.18, 3.64 ]
Emiliani 2003 39/82 46/89 14.9 % 0.85 [ 0.47, 1.55 ]
Frattarelli 2003 18/29 10/28 2.5 % 2.95 [ 1.00, 8.65 ]
Gardner 1998 32/45 31/47 5.6 % 1.27 [ 0.53, 3.07 ]
Karaki 2002 28/80 24/82 9.9 % 1.30 [ 0.67, 2.52 ]
Levitas 2004 5/23 4/31 1.7 % 1.88 [ 0.44, 7.94 ]
0.2 0.5 1 2 5
(Continued . . . )
(. . . Continued)
Levron 2002 8/46 20/44 10.9 % 0.25 [ 0.10, 0.66 ]
Livingstone 2002 15/30 15/29 4.9 % 0.93 [ 0.34, 2.59 ]
Motta 1998 A % B 21/58 21/58 8.6 % 1.00 [ 0.47, 2.13 ]
Pantos 2004 30/81 77/162 20.8 % 0.65 [ 0.38, 1.12 ]
Schillaci 2002 24/60 23/60 8.9 % 1.07 [ 0.52, 2.23 ]
Subtotal (95% CI) 645 742 100.0 % 1.07 [ 0.86, 1.33 ]
Heterogeneity: Chi
2
= 24.30, df = 11 (P = 0.01); I
2
=55%
3 Single embryo transfer
Brugnon 2010 23/55 24/52 31.1 % 0.84 [ 0.39, 1.80 ]
Fisch 2007 4/8 11/12 9.5 % 0.09 [ 0.01, 1.08 ]
Papanikolaou 2006 58/175 41/176 59.3 % 1.63 [ 1.02, 2.61 ]
Subtotal (95% CI) 238 240 100.0 % 1.24 [ 0.84, 1.82 ]
Heterogeneity: Chi
2
= 6.61, df = 2 (P = 0.04); I
2
=70%
0.2 0.5 1 2 5
by prognosis.
Outcome: 3 clinical pregnancy rate per couple: grouped by prognosis
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
1 Good prognostic factors
Brugnon 2010 23/55 24/52 4.4 % 0.84 [ 0.39, 1.80 ]
Bungum 2003 32/61 36/57 4.6 % 0.64 [ 0.31, 1.34 ]
Coskun 2000 39/100 39/101 6.0 % 1.02 [ 0.58, 1.79 ]
Elgindy 2011 59/100 41/100 6.0 % 2.07 [ 1.18, 3.64 ]
Fisch 2007 4/8 11/12 0.7 % 0.09 [ 0.01, 1.08 ]
Frattarelli 2003 18/29 10/28 2.8 % 2.95 [ 1.00, 8.65 ]
Gardner 1998 32/45 31/47 3.7 % 1.27 [ 0.53, 3.07 ]
Hreinsson 2004 22/64 25/80 4.9 % 1.15 [ 0.57, 2.32 ]
Levron 2002 8/46 20/44 3.3 % 0.25 [ 0.10, 0.66 ]
Livingstone 2002 15/30 15/29 3.0 % 0.93 [ 0.34, 2.59 ]
Papanikolaou 2005 42/80 27/84 5.4 % 2.33 [ 1.24, 4.40 ]
Papanikolaou 2006 58/175 41/176 6.9 % 1.63 [ 1.02, 2.61 ]
Rienzi 2002 29/50 27/48 4.2 % 1.07 [ 0.48, 2.39 ]
Ten 2011 17/28 14/27 2.8 % 1.44 [ 0.49, 4.18 ]
Subtotal (95% CI) 871 885 58.8 % 1.15 [ 0.83, 1.58 ]
Heterogeneity: Tau
2
= 0.20; Chi
2
= 30.58, df = 13 (P = 0.004); I
2
=57%
2 Poor prognostic factors
Devreker 2000 4/11 1/12 0.7 % 6.29 [ 0.58, 68.42 ]
Levitas 2004 5/23 4/31 1.8 % 1.88 [ 0.44, 7.94 ]
Subtotal (95% CI) 34 43 2.5 % 2.59 [ 0.75, 8.92 ]
Heterogeneity: Tau
2
= 0.0; Chi
2
= 0.73, df = 1 (P = 0.39); I
2
=0.0%
3 Unselected group
0.1 0.2 0.5 1 2 5 10
(Continued . . . )
(. . . Continued)
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
Emiliani 2003 39/82 46/89 5.7 % 0.85 [ 0.47, 1.55 ]
Karaki 2002 28/80 24/82 5.2 % 1.30 [ 0.67, 2.52 ]
Kolibianakis 2004 75/226 75/234 7.8 % 1.05 [ 0.71, 1.56 ]
Motta 1998 A % B 21/58 21/58 4.5 % 1.00 [ 0.47, 2.13 ]
Pantos 2004 30/81 77/162 6.2 % 0.65 [ 0.38, 1.12 ]
Schillaci 2002 24/60 23/60 4.6 % 1.07 [ 0.52, 2.23 ]
Van der Auwera 2002 29/70 20/66 4.8 % 1.63 [ 0.80, 3.30 ]
Subtotal (95% CI) 657 751 38.8 % 1.01 [ 0.81, 1.25 ]
Heterogeneity: Tau
2
= 0.0; Chi
2
= 5.21, df = 6 (P = 0.52); I
2
=0.0%
Total (95% CI) 1562 1679 100.0 % 1.13 [ 0.92, 1.40 ]
Heterogeneity: Tau
2
= 0.11; Chi
2
= 40.15, df = 22 (P = 0.01); I
2
=45%
2
= 2.44, df = 2 (P = 0.30), I
2
=18%
0.1 0.2 0.5 1 2 5 10
by day of randomisation.
Outcome: 4 clinical pregnancy rate per couple: grouped by day of randomisation
Study or subgroup Day 5/6 day 2/3 Odds Ratio Weight Odds Ratio
1 Randomised start of cycle
Brugnon 2010 23/55 24/52 4.1 % 0.84 [ 0.39, 1.80 ]
Emiliani 2003 39/82 46/89 6.6 % 0.85 [ 0.47, 1.55 ]
Gardner 1998 32/45 31/47 2.5 % 1.27 [ 0.53, 3.07 ]
Kolibianakis 2004 75/226 75/234 14.1 % 1.05 [ 0.71, 1.56 ]
Levitas 2004 5/23 4/31 0.8 % 1.88 [ 0.44, 7.94 ]
Papanikolaou 2006 58/175 41/176 7.8 % 1.63 [ 1.02, 2.61 ]
Van der Auwera 2002 29/70 20/66 3.5 % 1.63 [ 0.80, 3.30 ]
Subtotal (95% CI) 676 695 39.5 % 1.19 [ 0.95, 1.49 ]
Total events: 261 (Day 5/6), 241 (day 2/3)
Heterogeneity: Chi
2
= 5.29, df = 6 (P = 0.51); I
2
=0.0%
2 Randomised on day of OPU or day 1
Coskun 2000 39/100 39/101 6.8 % 1.02 [ 0.58, 1.79 ]
Fisch 2007 4/8 11/12 1.3 % 0.09 [ 0.01, 1.08 ]
Frattarelli 2003 18/29 10/28 1.1 % 2.95 [ 1.00, 8.65 ]
Hreinsson 2004 22/64 25/80 4.2 % 1.15 [ 0.57, 2.32 ]
Karaki 2002 28/80 24/82 4.4 % 1.30 [ 0.67, 2.52 ]
Levron 2002 8/46 20/44 4.8 % 0.25 [ 0.10, 0.66 ]
Rienzi 2002 29/50 27/48 3.3 % 1.07 [ 0.48, 2.39 ]
Schillaci 2002 24/60 23/60 4.0 % 1.07 [ 0.52, 2.23 ]
Subtotal (95% CI) 437 455 29.9 % 1.00 [ 0.76, 1.31 ]
Heterogeneity: Chi
2
= 16.10, df = 7 (P = 0.02); I
2
=57%
3 Randomised on day 2 to 3
Bungum 2003 32/61 36/57 5.1 % 0.64 [ 0.31, 1.34 ]
Elgindy 2011 59/100 41/100 4.8 % 2.07 [ 1.18, 3.64 ]
0.1 0.2 0.5 1 2 5 10
(Continued . . . )
(. . . Continued)
Study or subgroup Day 5/6 day 2/3 Odds Ratio Weight Odds Ratio
Papanikolaou 2005 42/80 27/84 3.6 % 2.33 [ 1.24, 4.40 ]
Ten 2011 17/28 14/27 1.6 % 1.44 [ 0.49, 4.18 ]
Subtotal (95% CI) 269 268 15.1 % 1.59 [ 1.13, 2.23 ]
Heterogeneity: Chi
2
= 8.08, df = 3 (P = 0.04); I
2
=63%
4 Day of randomisation unstated
Devreker 2000 4/11 1/12 0.2 % 6.29 [ 0.58, 68.42 ]
Livingstone 2002 15/30 15/29 2.2 % 0.93 [ 0.34, 2.59 ]
Motta 1998 A % B 21/58 21/58 3.8 % 1.00 [ 0.47, 2.13 ]
Pantos 2004 30/81 77/162 9.3 % 0.65 [ 0.38, 1.12 ]
Subtotal (95% CI) 180 261 15.5 % 0.84 [ 0.57, 1.25 ]
Heterogeneity: Chi
2
= 3.83, df = 3 (P = 0.28); I
2
=22%
Total (95% CI) 1562 1679 100.0 % 1.14 [ 0.99, 1.32 ]
Heterogeneity: Chi
2
= 40.15, df = 22 (P = 0.01); I
2
=45%
2
= 6.94, df = 3 (P = 0.07), I
2
=57%
0.1 0.2 0.5 1 2 5 10
Analysis 3.1. Comparison 3 Cumulative pregnancy rate, Outcome 1 cumulative pregnancy rate from fresh
and frozen transfers.
Comparison: 3 Cumulative pregnancy rate
Outcome: 1 cumulative pregnancy rate from fresh and frozen transfers
Peto Odds
Ratio(Non-event) Weight
Peto Odds
Ratio(Non-event)
Brugnon 2010 (1) 24/55 25/52 21.5 % 1.19 [ 0.56, 2.55 ]
Emiliani 2003 43/99 56/94 39.0 % 1.90 [ 1.08, 3.34 ]
Rienzi 2002 31/50 41/48 15.5 % 3.28 [ 1.35, 8.02 ]
Van der Auwera 2002 24/66 22/63 24.0 % 0.94 [ 0.46, 1.93 ]
Total (95% CI) 270 257 100.0 % 1.58 [ 1.11, 2.25 ]
Heterogeneity: Chi
2
= 5.54, df = 3 (P = 0.14); I
2
=46%
0.1 0.2 0.5 1 2 5 10
Favours Day 5/6 Favours 2/3
(1) Study had policy of single embryo transfer
Analysis 4.1. Comparison 4 Multiple-pregnancy rate, Outcome 1 multiple-pregnancy rate per couple.
Comparison: 4 Multiple-pregnancy rate
Outcome: 1 multiple-pregnancy rate per couple
Peto
Odds Ratio Weight
Peto
Odds Ratio
Bungum 2003 13/61 15/57 9.5 % 0.76 [ 0.33, 1.77 ]
Coskun 2000 15/100 13/101 10.6 % 1.19 [ 0.54, 2.65 ]
Elgindy 2011 12/59 8/41 6.9 % 1.05 [ 0.39, 2.84 ]
Emiliani 2003 12/82 8/89 7.8 % 1.72 [ 0.68, 4.37 ]
Frattarelli 2003 5/29 7/28 4.2 % 0.63 [ 0.18, 2.23 ]
Hreinsson 2004 2/64 4/80 2.5 % 0.63 [ 0.12, 3.23 ]
Karaki 2002 9/80 10/82 7.4 % 0.91 [ 0.35, 2.37 ]
Kolibianakis 2004 15/226 20/234 14.2 % 0.76 [ 0.38, 1.52 ]
Levitas 2004 2/23 3/31 2.0 % 0.89 [ 0.14, 5.63 ]
Levron 2002 4/46 8/44 4.6 % 0.44 [ 0.13, 1.49 ]
Livingstone 2002 0/30 4/29 1.7 % 0.12 [ 0.02, 0.88 ]
Motta 1998 A % B 3/58 10/58 5.1 % 0.30 [ 0.10, 0.95 ]
Papanikolaou 2005 18/80 8/84 9.7 % 2.63 [ 1.14, 6.06 ]
Papanikolaou 2006 0/175 2/176 0.9 % 0.14 [ 0.01, 2.17 ]
Rienzi 2002 9/50 7/48 5.9 % 1.28 [ 0.44, 3.72 ]
Van der Auwera 2002 9/70 9/66 6.9 % 0.93 [ 0.35, 2.51 ]
Total (95% CI) 1233 1248 100.0 % 0.92 [ 0.71, 1.19 ]
Heterogeneity: Chi
2
= 20.59, df = 15 (P = 0.15); I
2
=27%
0.1 0.2 0.5 1 2 5 10
Analysis 4.2. Comparison 4 Multiple-pregnancy rate, Outcome 2 multiple-pregnancy rate per couple:
grouped by number of embryo transfer.
Outcome: 2 multiple-pregnancy rate per couple: grouped by number of embryo transfer
1 Equal number of embryos transferred
Bungum 2003 13/61 15/57 18.1 % 0.76 [ 0.32, 1.77 ]
Coskun 2000 15/100 13/101 16.3 % 1.19 [ 0.54, 2.66 ]
Hreinsson 2004 2/64 4/80 5.1 % 0.61 [ 0.11, 3.46 ]
Kolibianakis 2004 15/226 20/234 27.2 % 0.76 [ 0.38, 1.53 ]
Papanikolaou 2005 18/80 8/84 9.0 % 2.76 [ 1.12, 6.77 ]
Papanikolaou 2006 0/175 2/176 3.7 % 0.20 [ 0.01, 4.17 ]
Rienzi 2002 9/50 7/48 8.7 % 1.29 [ 0.44, 3.78 ]
Van der Auwera 2002 9/70 9/66 12.0 % 0.93 [ 0.35, 2.52 ]
Subtotal (95% CI) 826 846 100.0 % 1.05 [ 0.75, 1.46 ]
Heterogeneity: Chi
2
= 7.64, df = 7 (P = 0.37); I
2
=8%
2 More cleavage stage than blastocyst embryos transferred
Elgindy 2011 12/59 8/41 14.3 % 1.05 [ 0.39, 2.86 ]
Emiliani 2003 12/82 8/89 12.5 % 1.74 [ 0.67, 4.49 ]
Frattarelli 2003 5/29 7/28 11.2 % 0.63 [ 0.17, 2.27 ]
Karaki 2002 9/80 10/82 16.7 % 0.91 [ 0.35, 2.38 ]
Levitas 2004 2/23 3/31 4.4 % 0.89 [ 0.14, 5.81 ]
Levron 2002 4/46 8/44 14.2 % 0.43 [ 0.12, 1.54 ]
Livingstone 2002 0/30 4/29 8.6 % 0.09 [ 0.00, 1.81 ]
Motta 1998 A % B 3/58 10/58 18.1 % 0.26 [ 0.07, 1.01 ]
Subtotal (95% CI) 407 402 100.0 % 0.75 [ 0.49, 1.13 ]
Heterogeneity: Chi
2
= 8.70, df = 7 (P = 0.27); I
2
=20%
3 Single embryo transfer
Papanikolaou 2006 0/175 2/176 100.0 % 0.20 [ 0.01, 4.17 ]
0.1 0.2 0.5 1 2 5 10
(Continued . . . )
(. . . Continued)
Subtotal (95% CI) 175 176 100.0 % 0.20 [ 0.01, 4.17 ]
Heterogeneity: not applicable
0.1 0.2 0.5 1 2 5 10
Analysis 4.3. Comparison 4 Multiple-pregnancy rate, Outcome 3 multiple-pregnancy rate per couple:
grouped by prognosis.
Outcome: 3 multiple-pregnancy rate per couple: grouped by prognosis
Bungum 2003 13/61 15/57 10.1 % 0.76 [ 0.32, 1.77 ]
Coskun 2000 15/100 13/101 9.1 % 1.19 [ 0.54, 2.66 ]
Elgindy 2011 12/59 8/41 6.2 % 1.05 [ 0.39, 2.86 ]
Frattarelli 2003 5/29 7/28 4.9 % 0.63 [ 0.17, 2.27 ]
Hreinsson 2004 2/64 4/80 2.9 % 0.61 [ 0.11, 3.46 ]
Levron 2002 4/46 8/44 6.2 % 0.43 [ 0.12, 1.54 ]
Livingstone 2002 0/30 4/29 3.7 % 0.09 [ 0.00, 1.81 ]
Motta 1998 A % B 3/58 10/58 7.9 % 0.26 [ 0.07, 1.01 ]
Papanikolaou 2005 18/80 8/84 5.0 % 2.76 [ 1.12, 6.77 ]
Papanikolaou 2006 0/175 2/176 2.1 % 0.20 [ 0.01, 4.17 ]
Rienzi 2002 9/50 7/48 4.8 % 1.29 [ 0.44, 3.78 ]
Subtotal (95% CI) 752 746 62.8 % 0.88 [ 0.63, 1.22 ]
0.1 0.2 0.5 1 2 5 10
(Continued . . . )
(. . . Continued)
Heterogeneity: Chi
2
= 15.38, df = 10 (P = 0.12); I
2
=35%
Levitas 2004 2/23 3/31 1.9 % 0.89 [ 0.14, 5.81 ]
Subtotal (95% CI) 23 31 1.9 % 0.89 [ 0.14, 5.81 ]
3 Unselected
Emiliani 2003 12/82 9/89 6.1 % 1.52 [ 0.61, 3.83 ]
Karaki 2002 9/80 10/82 7.3 % 0.91 [ 0.35, 2.38 ]
Kolibianakis 2004 15/226 20/234 15.2 % 0.76 [ 0.38, 1.53 ]
Van der Auwera 2002 9/70 9/66 6.7 % 0.93 [ 0.35, 2.52 ]
Subtotal (95% CI) 458 471 35.2 % 0.96 [ 0.62, 1.47 ]
Heterogeneity: Chi
2
= 1.41, df = 3 (P = 0.70); I
2
=0.0%
Total (95% CI) 1233 1248 100.0 % 0.91 [ 0.70, 1.18 ]
Heterogeneity: Chi
2
= 16.79, df = 15 (P = 0.33); I
2
=11%
2
= 0.09, df = 2 (P = 0.96), I
2
=0.0%
0.1 0.2 0.5 1 2 5 10
Analysis 4.4. Comparison 4 Multiple-pregnancy rate, Outcome 4 high order pregnancies (more than 2
gestational sacs) per couple.
Outcome: 4 high order pregnancies (more than 2 gestational sacs) per couple
Study or subgroup Day 5/6 Day 2/3 Odds Ratio Odds Ratio
Bungum 2003 0/61 0/57 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/100 1/101 0.33 [ 0.01, 8.28 ]
Frattarelli 2003 0/29 1/28 0.31 [ 0.01, 7.95 ]
Hreinsson 2004 0/64 0/80 0.0 [ 0.0, 0.0 ]
Karaki 2002 1/80 4/82 0.25 [ 0.03, 2.26 ]
Kolibianakis 2004 0/226 0/234 0.0 [ 0.0, 0.0 ]
Levitas 2004 1/23 0/31 4.20 [ 0.16, 107.89 ]
Levron 2002 1/46 3/44 0.30 [ 0.03, 3.04 ]
Papanikolaou 2005 0/80 0/84 0.0 [ 0.0, 0.0 ]
Papanikolaou 2006 0/175 0/176 0.0 [ 0.0, 0.0 ]
Rienzi 2002 0/50 0/48 0.0 [ 0.0, 0.0 ]
Van der Auwera 2002 0/70 0/66 0.0 [ 0.0, 0.0 ]
Total (95% CI) 1004 1031 0.44 [ 0.15, 1.33 ]
Heterogeneity: Chi
2
= 2.29, df = 4 (P = 0.68); I
2
=0.0%
0.1 0.2 0.5 1 2 5 10
Analysis 4.5. Comparison 4 Multiple-pregnancy rate, Outcome 5 high order pregnancy: grouped by number
of embryos transferred.
Outcome: 5 high order pregnancy: grouped by number of embryos transferred
1 Equal number of embryos transferred
Bungum 2003 0/61 0/57 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/100 1/101 0.33 [ 0.01, 8.28 ]
Hreinsson 2004 0/64 0/80 0.0 [ 0.0, 0.0 ]
Kolibianakis 2004 0/226 0/234 0.0 [ 0.0, 0.0 ]
Papanikolaou 2005 0/80 0/84 0.0 [ 0.0, 0.0 ]
Papanikolaou 2006 0/175 0/176 0.0 [ 0.0, 0.0 ]
Rienzi 2002 0/50 0/48 0.0 [ 0.0, 0.0 ]
Van der Auwera 2002 0/70 0/66 0.0 [ 0.0, 0.0 ]
Subtotal (95% CI) 826 846 0.33 [ 0.01, 8.28 ]
Heterogeneity: Chi
2
= 0.0, df = 0 (P = 1.00); I
2
=0.0%
2 More cleavage stage than blastocyst embryos transferred
Frattarelli 2003 0/29 1/28 0.31 [ 0.01, 7.95 ]
Karaki 2002 1/80 4/82 0.25 [ 0.03, 2.26 ]
Levitas 2004 1/23 0/31 4.20 [ 0.16, 107.89 ]
Levron 2002 1/46 3/44 0.30 [ 0.03, 3.04 ]
Subtotal (95% CI) 178 185 0.46 [ 0.14, 1.49 ]
Heterogeneity: Chi
2
= 2.27, df = 3 (P = 0.52); I
2
=0.0%
Total (95% CI) 1004 1031 0.44 [ 0.15, 1.33 ]
Heterogeneity: Chi
2
= 2.29, df = 4 (P = 0.68); I
2
=0.0%
2
= 0.03, df = 1 (P = 0.86), I
2
=0.0%
0.01 0.1 1 10 100
Analysis 4.6. Comparison 4 Multiple-pregnancy rate, Outcome 6 high order pregnancies: grouped by
prognosis.
Outcome: 6 high order pregnancies: grouped by prognosis
Bungum 2003 0/61 0/57 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/100 1/101 0.33 [ 0.01, 8.28 ]
Frattarelli 2003 0/29 1/28 0.31 [ 0.01, 7.95 ]
Hreinsson 2004 0/64 0/80 0.0 [ 0.0, 0.0 ]
Karaki 2002 1/80 4/82 0.25 [ 0.03, 2.26 ]
Levron 2002 1/46 3/44 0.30 [ 0.03, 3.04 ]
Papanikolaou 2005 0/80 0/84 0.0 [ 0.0, 0.0 ]
Papanikolaou 2006 0/175 0/176 0.0 [ 0.0, 0.0 ]
Rienzi 2002 0/50 0/48 0.0 [ 0.0, 0.0 ]
Subtotal (95% CI) 685 700 0.29 [ 0.08, 1.06 ]
Heterogeneity: Chi
2
= 0.03, df = 3 (P = 1.00); I
2
=0.0%
Levitas 2004 1/23 0/31 4.20 [ 0.16, 107.89 ]
Subtotal (95% CI) 23 31 4.20 [ 0.16, 107.89 ]
3 Unselected
Kolibianakis 2004 0/226 0/234 0.0 [ 0.0, 0.0 ]
Van der Auwera 2002 0/70 0/66 0.0 [ 0.0, 0.0 ]
Subtotal (95% CI) 296 300 0.0 [ 0.0, 0.0 ]
Heterogeneity: Chi
2
= 0.0, df = 0 (P<0.00001); I
2
=0.0%
Test for overall effect: Z = 0.0 (P < 0.00001)
Total (95% CI) 1004 1031 0.44 [ 0.15, 1.33 ]
Heterogeneity: Chi
2
= 2.29, df = 4 (P = 0.68); I
2
=0.0%
2
= 2.26, df = 1 (P = 0.13), I
2
=56%
0.1 0.2 0.5 1 2 5 10
Analysis 4.7. Comparison 4 Multiple-pregnancy rate, Outcome 7 multiple-pregnancy rate per pregnancy.
Outcome: 7 multiple-pregnancy rate per pregnancy
Bungum 2003 13/32 15/36 0.96 [ 0.36, 2.52 ]
Coskun 2000 15/39 13/39 1.25 [ 0.49, 3.16 ]
Elgindy 2011 12/59 8/41 1.05 [ 0.39, 2.86 ]
Emiliani 2003 12/39 9/46 1.83 [ 0.67, 4.95 ]
Hreinsson 2004 2/22 4/25 0.53 [ 0.09, 3.19 ]
Karaki 2002 9/23 10/21 0.71 [ 0.21, 2.34 ]
Kolibianakis 2004 15/75 20/75 0.69 [ 0.32, 1.47 ]
Levitas 2004 2/5 3/4 0.22 [ 0.01, 3.98 ]
Levron 2002 4/8 8/20 1.50 [ 0.29, 7.81 ]
Motta 1998 A % B 3/14 10/21 0.30 [ 0.06, 1.40 ]
Papanikolaou 2005 18/42 8/27 1.78 [ 0.64, 4.98 ]
Papanikolaou 2006 0/58 2/38 0.12 [ 0.01, 2.67 ]
Rienzi 2002 9/29 7/27 1.29 [ 0.40, 4.13 ]
Van der Auwera 2002 9/29 9/20 0.55 [ 0.17, 1.79 ]
0.1 0.2 0.5 1 2 5 10
Analysis 4.8. Comparison 4 Multiple-pregnancy rate, Outcome 8 high order pregnancies per total
pregnancies.
Outcome: 8 high order pregnancies per total pregnancies
Bungum 2003 0/32 0/36 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/38 1/38 0.32 [ 0.01, 8.22 ]
Frattarelli 2003 0/18 1/10 0.17 [ 0.01, 4.62 ]
Hreinsson 2004 0/22 0/25 0.0 [ 0.0, 0.0 ]
Karaki 2002 1/23 4/21 0.19 [ 0.02, 1.89 ]
Kolibianakis 2004 0/75 0/75 0.0 [ 0.0, 0.0 ]
Levitas 2004 1/5 0/4 3.00 [ 0.09, 95.17 ]
Levron 2002 1/8 3/20 0.81 [ 0.07, 9.18 ]
Papanikolaou 2005 0/42 0/27 0.0 [ 0.0, 0.0 ]
Papanikolaou 2006 0/58 0/38 0.0 [ 0.0, 0.0 ]
Rienzi 2002 0/29 0/27 0.0 [ 0.0, 0.0 ]
Van der Auwera 2002 0/29 0/20 0.0 [ 0.0, 0.0 ]
0.1 0.2 0.5 1 2 5 10
Analysis 5.1. Comparison 5 Miscarriage rate, Outcome 1 miscarriage rate per couple.
Comparison: 5 Miscarriage rate
Outcome: 1 miscarriage rate per couple
Peto
Odds Ratio Weight
Peto
Odds Ratio
Bungum 2003 12/61 6/57 9.3 % 2.02 [ 0.74, 5.48 ]
Coskun 2000 3/100 5/101 4.7 % 0.60 [ 0.15, 2.47 ]
Devreker 2000 3/11 0/12 1.7 % 9.97 [ 0.93, 107.33 ]
Elgindy 2011 4/59 4/41 4.4 % 0.67 [ 0.16, 2.89 ]
Frattarelli 2003 3/29 2/28 2.8 % 1.48 [ 0.24, 9.14 ]
Hreinsson 2004 3/64 2/80 2.9 % 1.91 [ 0.32, 11.44 ]
Karaki 2002 5/80 3/82 4.6 % 1.73 [ 0.42, 7.14 ]
Kolibianakis 2004 19/226 21/234 22.2 % 0.93 [ 0.49, 1.78 ]
Levitas 2004 2/23 1/31 1.7 % 2.78 [ 0.27, 28.68 ]
Livingstone 2002 1/30 3/29 2.3 % 0.34 [ 0.04, 2.52 ]
Papanikolaou 2005 15/80 12/84 13.7 % 1.38 [ 0.61, 3.14 ]
Papanikolaou 2006 17/175 21/176 20.6 % 0.80 [ 0.41, 1.56 ]
Rienzi 2002 5/50 3/48 4.5 % 1.64 [ 0.39, 6.92 ]
Van der Auwera 2002 5/70 3/66 4.6 % 1.59 [ 0.38, 6.62 ]
Total (95% CI) 1058 1069 100.0 % 1.14 [ 0.84, 1.55 ]
Heterogeneity: Chi
2
= 10.60, df = 13 (P = 0.64); I
2
=0.0%
0.1 0.2 0.5 1 2 5 10
Analysis 5.2. Comparison 5 Miscarriage rate, Outcome 2 miscarriage rate per pregnancy.
Comparison: 5 Miscarriage rate
Outcome: 2 miscarriage rate per pregnancy
Bungum 2003 12/32 3/36 6.60 [ 1.66, 26.28 ]
Coskun 2000 3/39 5/39 0.57 [ 0.13, 2.56 ]
Elgindy 2011 4/59 4/41 0.67 [ 0.16, 2.86 ]
Emiliani 2003 6/39 4/36 1.45 [ 0.38, 5.64 ]
Frattarelli 2003 3/18 2/10 0.80 [ 0.11, 5.82 ]
Hreinsson 2004 3/22 2/25 1.82 [ 0.27, 12.01 ]
Karaki 2002 5/28 3/24 1.52 [ 0.32, 7.16 ]
Kolibianakis 2004 19/94 21/96 0.90 [ 0.45, 1.82 ]
Levitas 2004 2/5 1/4 2.00 [ 0.11, 35.81 ]
Livingstone 2002 1/15 4/15 0.20 [ 0.02, 2.02 ]
Papanikolaou 2005 15/53 12/35 0.76 [ 0.30, 1.90 ]
Papanikolaou 2006 17/73 21/59 0.55 [ 0.26, 1.18 ]
Rienzi 2002 5/29 3/27 1.67 [ 0.36, 7.77 ]
Van der Auwera 2002 5/29 3/20 1.18 [ 0.25, 5.62 ]
0.1 0.2 0.5 1 2 5 10
Analysis 6.1. Comparison 6 Embryo freezing rate, Outcome 1 embryo freezing per couple.
Comparison: 6 Embryo freezing rate
Outcome: 1 embryo freezing per couple
Peto Odds
Ratio(Non-event) Weight
Peto Odds
Ratio(Non-event)
Brugnon 2010 42/55 51/52 3.2 % 6.63 [ 2.17, 20.30 ]
Bungum 2003 36/61 54/57 5.6 % 7.08 [ 3.04, 16.48 ]
Hreinsson 2004 15/64 34/80 8.4 % 2.32 [ 1.16, 4.64 ]
Karaki 2002 22/80 35/82 9.7 % 1.94 [ 1.02, 3.69 ]
Kolibianakis 2004 114/226 145/234 29.5 % 1.60 [ 1.10, 2.31 ]
Levron 2002 12/46 25/44 5.7 % 3.51 [ 1.52, 8.09 ]
Motta 1998 A % B 15/58 45/58 7.6 % 7.80 [ 3.77, 16.10 ]
Pantos 2004 16/81 79/162 13.4 % 3.37 [ 1.95, 5.81 ]
Rienzi 2002 18/50 42/48 6.1 % 8.56 [ 3.81, 19.22 ]
Ten 2011 20/28 26/27 2.0 % 5.95 [ 1.44, 24.53 ]
Van der Auwera 2002 26/70 35/66 8.8 % 1.89 [ 0.96, 3.71 ]
Total (95% CI) 819 910 100.0 % 2.88 [ 2.35, 3.51 ]
Heterogeneity: Chi
2
= 35.44, df = 10 (P = 0.00011); I
2
=72%
0.01 0.1 1 10 100
Favours day 5/6 Favours 2/3
Analysis 6.2. Comparison 6 Embryo freezing rate, Outcome 2 Embyro freezing per couple: grouped by
Outcome: 2 Embyro freezing per couple: grouped by number of embryos transferred
Odds
Ratio(Non-
event) Weight
Odds
Ratio(Non-
event)
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
Brugnon 2010 42/55 51/52 4.0 % 15.79 [ 1.98, 125.67 ]
Bungum 2003 36/61 54/57 7.2 % 12.50 [ 3.51, 44.49 ]
Hreinsson 2004 15/64 34/80 10.7 % 2.41 [ 1.17, 5.00 ]
Kolibianakis 2004 114/226 145/234 13.0 % 1.60 [ 1.10, 2.32 ]
Rienzi 2002 18/50 42/48 8.6 % 12.44 [ 4.43, 34.93 ]
Ten 2011 20/28 26/27 3.7 % 10.40 [ 1.20, 90.09 ]
Van der Auwera 2002 26/70 35/66 11.0 % 1.91 [ 0.96, 3.79 ]
Subtotal (95% CI) 554 564 58.1 % 4.35 [ 2.11, 8.97 ]
Heterogeneity: Tau
2
= 0.63; Chi
2
= 26.68, df = 6 (P = 0.00017); I
2
=78%
2 more cleavage stage than blastocyst embryos transferred
Karaki 2002 22/80 35/82 11.2 % 1.96 [ 1.02, 3.79 ]
Levron 2002 12/46 25/44 9.6 % 3.73 [ 1.53, 9.06 ]
Motta 1998 A % B 15/58 45/58 9.8 % 9.92 [ 4.23, 23.27 ]
Pantos 2004 16/81 79/162 11.4 % 3.87 [ 2.06, 7.24 ]
Subtotal (95% CI) 265 346 41.9 % 3.95 [ 2.09, 7.46 ]
Heterogeneity: Tau
2
= 0.27; Chi
2
= 8.74, df = 3 (P = 0.03); I
2
=66%
Total (95% CI) 819 910 100.0 % 4.06 [ 2.49, 6.60 ]
Heterogeneity: Tau
2
= 0.44; Chi
2
= 38.82, df = 10 (P = 0.00003); I
2
=74%
2
= 0.04, df = 1 (P = 0.84), I
2
=0.0%
0.1 0.2 0.5 1 2 5 10
Favours day 5/6 Favours 2/3
Analysis 6.3. Comparison 6 Embryo freezing rate, Outcome 3 Embryo freezing per couple: grouped by
prognostic factors.
Outcome: 3 Embryo freezing per couple: grouped by prognostic factors
Odds
Ratio(Non-
event) Weight
Odds
Ratio(Non-
event)
n/N n/N
M-
H,Random,95%
CI
M-
H,Random,95%
CI
Brugnon 2010 42/55 51/52 4.8 % 15.79 [ 1.98, 125.67 ]
Bungum 2003 36/61 54/57 8.3 % 12.50 [ 3.51, 44.49 ]
Hreinsson 2004 15/64 34/80 11.9 % 2.41 [ 1.17, 5.00 ]
Levron 2002 12/46 25/44 10.8 % 3.73 [ 1.53, 9.06 ]
Rienzi 2002 18/50 42/48 9.8 % 12.44 [ 4.43, 34.93 ]
Ten 2011 20/28 26/27 4.5 % 10.40 [ 1.20, 90.09 ]
Subtotal (95% CI) 304 308 50.2 % 6.39 [ 3.12, 13.10 ]
Heterogeneity: Tau
2
= 0.40; Chi
2
= 11.01, df = 5 (P = 0.05); I
2
=55%
Subtotal (95% CI) 0 0 0.0 % 0.0 [ 0.0, 0.0 ]
Test for overall effect: not applicable
3 unselected
Karaki 2002 22/80 35/82 12.4 % 1.96 [ 1.02, 3.79 ]
Kolibianakis 2004 114/226 145/234 14.1 % 1.60 [ 1.10, 2.32 ]
Motta 1998 A % B 15/58 45/58 11.1 % 9.92 [ 4.23, 23.27 ]
Van der Auwera 2002 26/70 35/66 12.2 % 1.91 [ 0.96, 3.79 ]
Subtotal (95% CI) 434 440 49.8 % 2.60 [ 1.31, 5.16 ]
Heterogeneity: Tau
2
= 0.38; Chi
2
= 14.93, df = 3 (P = 0.002); I
2
=80%
Total (95% CI) 738 748 100.0 % 4.17 [ 2.41, 7.21 ]
Heterogeneity: Tau
2
= 0.52; Chi
2
= 37.69, df = 9 (P = 0.00002); I
2
=76%
2
= 3.15, df = 1 (P = 0.08), I
2
=68%
0.1 0.2 0.5 1 2 5 10
Favours Day 5/6 Favours Day 2/3
Analysis 7.1. Comparison 7 Failure to transfer embryos rate per couple, Outcome 1 Failure to transfer any
embryos per couple.
Comparison: 7 Failure to transfer embryos rate per couple
Outcome: 1 Failure to transfer any embryos per couple
Odds
Ratio(Non-
event)
Odds
Ratio(Non-
event)
Gardner 1998 2/45 0/47 0.18 [ 0.01, 3.92 ]
Bungum 2003 0/61 0/57 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/100 0/101 0.0 [ 0.0, 0.0 ]
Devreker 2000 0/11 0/12 0.0 [ 0.0, 0.0 ]
Emiliani 2003 10/99 1/94 0.10 [ 0.01, 0.76 ]
Frattarelli 2003 3/29 5/28 1.88 [ 0.40, 8.77 ]
Hreinsson 2004 4/64 3/80 0.58 [ 0.13, 2.71 ]
Karaki 2002 9/80 0/82 0.05 [ 0.00, 0.80 ]
Kolibianakis 2004 36/226 16/234 0.39 [ 0.21, 0.72 ]
Levitas 2004 6/23 2/31 0.20 [ 0.04, 1.08 ]
Levron 2002 3/46 0/44 0.14 [ 0.01, 2.78 ]
Motta 1998 A % B 6/58 1/58 0.15 [ 0.02, 1.31 ]
Papanikolaou 2005 0/80 0/84 0.0 [ 0.0, 0.0 ]
Papanikolaou 2006 11/175 8/176 0.71 [ 0.28, 1.81 ]
Rienzi 2002 0/50 0/48 0.0 [ 0.0, 0.0 ]
Van der Auwera 2002 18/70 6/66 0.29 [ 0.11, 0.78 ]
Total (95% CI) 1217 1242 0.35 [ 0.24, 0.51 ]
Heterogeneity: Chi
2
= 12.46, df = 10 (P = 0.26); I
2
=20%
0.1 0.2 0.5 1 2 5 10
Favours Day 2/3 Favours Day 5/6
embryos per couple: grouped by prognostic factors.
Outcome: 2 Failure to transfer any embryos per couple: grouped by prognostic factors
Odds
Ratio(Non-
event)
Odds
Ratio(Non-
event)
Bungum 2003 0/61 0/57 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/100 0/101 0.0 [ 0.0, 0.0 ]
Frattarelli 2003 3/29 5/28 1.88 [ 0.40, 8.77 ]
Gardner 1998 2/45 0/47 0.18 [ 0.01, 3.92 ]
Hreinsson 2004 4/64 3/80 0.58 [ 0.13, 2.71 ]
Levron 2002 3/46 0/44 0.14 [ 0.01, 2.78 ]
Papanikolaou 2005 0/80 0/84 0.0 [ 0.0, 0.0 ]
Papanikolaou 2006 11/175 8/176 0.71 [ 0.28, 1.81 ]
Rienzi 2002 0/50 0/48 0.0 [ 0.0, 0.0 ]
Subtotal (95% CI) 650 665 0.67 [ 0.35, 1.27 ]
Heterogeneity: Chi
2
= 3.53, df = 4 (P = 0.47); I
2
=0.0%
Devreker 2000 0/11 0/12 0.0 [ 0.0, 0.0 ]
Levitas 2004 6/23 2/31 0.20 [ 0.04, 1.08 ]
Subtotal (95% CI) 34 43 0.20 [ 0.04, 1.08 ]
Heterogeneity: Chi
2
= 0.0, df = 0 (P = 1.00); I
2
=0.0%
3 unselected
Emiliani 2003 10/99 1/94 0.10 [ 0.01, 0.76 ]
Karaki 2002 9/80 0/82 0.05 [ 0.00, 0.80 ]
Kolibianakis 2004 36/226 16/234 0.39 [ 0.21, 0.72 ]
Motta 1998 A % B 6/58 1/58 0.15 [ 0.02, 1.31 ]
0.1 0.2 0.5 1 2 5 10
(Continued . . . )
(. . . Continued)
Odds
Ratio(Non-
event)
Odds
Ratio(Non-
event)
Van der Auwera 2002 18/70 6/66 0.29 [ 0.11, 0.78 ]
Subtotal (95% CI) 533 534 0.27 [ 0.17, 0.43 ]
Heterogeneity: Chi
2
= 4.07, df = 4 (P = 0.40); I
2
=2%
Total (95% CI) 1217 1242 0.35 [ 0.24, 0.51 ]
Heterogeneity: Chi
2
= 12.46, df = 10 (P = 0.26); I
2
=20%
2
= 5.57, df = 2 (P = 0.06), I
2
=64%
0.1 0.2 0.5 1 2 5 10
embryos per couple: grouped by number of embryos transferred.
Outcome: 3 Failure to transfer any embryos per couple: grouped by number of embryos transferred
Odds
Ratio(Non-
event)
Odds
Ratio(Non-
event)
Bungum 2003 0/61 0/57 0.0 [ 0.0, 0.0 ]
Coskun 2000 0/100 0/101 0.0 [ 0.0, 0.0 ]
Hreinsson 2004 4/64 3/80 0.58 [ 0.13, 2.71 ]
Kolibianakis 2004 36/226 16/234 0.39 [ 0.21, 0.72 ]
Papanikolaou 2005 0/80 0/84 0.0 [ 0.0, 0.0 ]
Rienzi 2002 0/50 0/48 0.0 [ 0.0, 0.0 ]
Van der Auwera 2002 18/70 6/66 0.29 [ 0.11, 0.78 ]
Subtotal (95% CI) 651 670 0.37 [ 0.23, 0.61 ]
Heterogeneity: Chi
2
= 0.60, df = 2 (P = 0.74); I
2
=0.0%
2 more cleavage stage than blastocyst embryos tranferred
Devreker 2000 0/11 0/12 0.0 [ 0.0, 0.0 ]
Emiliani 2003 10/99 1/94 0.10 [ 0.01, 0.76 ]
Frattarelli 2003 3/29 5/28 1.88 [ 0.40, 8.77 ]
Gardner 1998 2/45 0/47 0.18 [ 0.01, 3.92 ]
Karaki 2002 9/80 0/82 0.05 [ 0.00, 0.80 ]
Levitas 2004 6/23 2/31 0.20 [ 0.04, 1.08 ]
Levron 2002 3/46 0/44 0.14 [ 0.01, 2.78 ]
Motta 1998 A % B 6/58 1/58 0.15 [ 0.02, 1.31 ]
Subtotal (95% CI) 391 396 0.23 [ 0.11, 0.46 ]
Heterogeneity: Chi
2
= 9.44, df = 6 (P = 0.15); I
2
=36%
3 single embryo transfer
Papanikolaou 2006 11/175 8/176 0.71 [ 0.28, 1.81 ]
0.005 0.1 1 10 200
Favours day2/3 Favours day 5/6
(Continued . . . )
(. . . Continued)
Odds
Ratio(Non-
event)
Odds
Ratio(Non-
event)
Subtotal (95% CI) 175 176 0.71 [ 0.28, 1.81 ]
0.005 0.1 1 10 200
Favours day2/3 Favours day 5/6
A D D I T I O N A L T A B L E S
Table 1. Culture techniques of included studies
Trial Culture Tech Day 2/3 Culture Tech Day 5/6
Brugnon 2010 G series medium (Vitrolife, Sweden) G series medium (Vitrolife, Sweden)
Bungum 2003 Sequential G1 VItrolife Sequential G1/G2 Vitrolife
Coskun 2000 Sequential Medicult Sequential G1/G2 Vitrolife
Devreker 2000 NS NS
Elgindy 2011 NS NS
Emiliani 2003 In-house sequential (based on G1/G2) In-house sequential (based on G1/G2)
Fisch 2007
Frattarelli 2003 Sequential NS Sequential NS
Gardner 1998a Single Hams F10 In-house Sequential G1/G2 In-house
Hreinsson 2004 Vitro life IVF Sequential G1/G2 or CCM Vitrolife
Karaki 2002 Medicult Sequential G1/G2 Vitrolife
Kolibianakis 2004 Sequential G1 Vitrolife Sequential G1/G2 Vitrolife
Levitas 2004 NS Sequential - G1/G2 Vitrolife
Levron 2002 NS NS
Table 1. Culture techniques of included studies (Continued)
Livingstone 2002 Sequential - Sydney IVF Cook Sequential - Sydney IVF Cook
Motta 1998 Sequential - Irvines P1 Sequential - Irvines P1 then Blast media
Pantos 2004
Papanikolaou 2005 Sequential - Vitrolife G1/G2 GII or GIII Sequential - Vitrolife G1/G2 GII or GIII
Papanikolaou 2006 Assume Sequential - Vitrolife G1/G2 Assume Sequential - Vitrolife G1/G2
Rienzi 2002 Sequential G1 Vitrolife Sequential G1/G2 Vitrolife
Schillaci 2002 NS NS
Ten 2011 NS NS
Van der Auwera 2002 Sequential both Cook and Vitrolife Sequential both Cook and Vitrolife
Table 2. Blastocyst and implantation rate (in Day 5 to 6 transfers)
Study Blastocyst rate Implantation D2/3 Implantation D5/6 Other
Brugnon 2010 Not stated 24/52 46.2% 23/55 41.8%
Bungum 2003 55.2% 50/114 43.9% 44/120 36.7% 2/61 patients had only 1 blastocyst
Coskun 2000 28% 50/235 21.3% 52/218 23.9% 77% patients had at least 1 blastocyst
Devreker 2000 Not stated 1/34 2.9% 8/19 42.1%
Elgindy 2011 97% 71/197 36% 53/280 19%
Emiliani 2003 48% 57/197 28.9% 50/168 29.8%
Fisch 2007 Not stated 11/12 92% 4/8 50%
Frattarelli 2003 Not stated 18/69 26.1% 23/53 43.4%
Gardner 1998 46.5% 64/174 36.8% 53/95 55.8% 85% patients had at least 2 blastocysts
Hreinsson 2004 33% 29/139 20.9% 24/114 21.1% 2 morula replace (one implanted). 60% preg
rate when top quality blasts transferred
Karaki 2002 33% 37/291 12.7% 37/142 26.1% 9/80 cancelled due to lack of blastocysts (un-
selected)
Kolibiankis 2004 50.7% 96/234 41.0% 94/226 41.6%
Table 2. Blastocyst and implantation rate (in Day 5 to 6 transfers) (Continued)
Levitas 2004 43% 4/56 7.1% 10/24 4.2% Day 5-7 26% cancelled due to lack of blas-
tocysts (poor prog)
Levron 2002 34.2% 53/137 38.7% 20/99 20.2% 6.5% cancelled due to lack of blastocysts
(good prog)
Livingstone 2002 not stated
Motta 1998 Not stated 51/262 19.5% 36/120 30.0% 6/58 cycles cancelled D5 no blastocysts
Pantos 2004 44.6% 15.8% 15.8%
Papanikolaou 2005 Not stated 35/170 20.6% 59/158 37.3% 4/158 women had only 1 blast transferred
due to lack of availability and 1 had it on
request
Papanikolaou 2006 Not stated 38/156 24% 58/149 38.9% Number of patients with no embryos avail
D3: 8 and D5: 11
Rienzi 2002 44.8% 34/96 35.4% 38/100 38.0% Good prognosis
Schillaci 2002 60.3% 23/168 13.7% 26/110 23.6% Unselected population nil cancellations D5
Ten 2011 Not stated 21/54 38.9% 26/56 46.4% Good prognosis
Van der Auwera 44.7% 31/106 29.2% 41/90 45.6% 27% cancellation D5 (unselected popula-
tion)
Table 3. Mean number of embryos transferred
Study ID Day 2/3 Day 5/6
Brugnon 2010 1 1
Bungum 2003 2.00 1.97
Coskun 2000 2.3 2.2
Devreker 2000 2.83 1.73
Elgindy 2010 2.8 1.97
Emiliani 2003 2.1 1.9
Fisch 2007 1 1
Frattarelli 2003 2.96 2.04
Table 3. Mean number of embryos transferred (Continued)
Gardner 1998 3.7 2.2
Hreinsson J 1.8 1.9
Karaki 2002 3.5 2.0
Kolibiankis 2004 1.9 1.8
Levitas 2004 3.4 1.9
Levron 2002 3.1 2.3
Livingstone 2.0 1.0
Motta 1998 4.6 2.3
Pantos 2004 4 3.4
Papanikolaou 2005 2 1.97
Papanikolaou 2006 1 1
Rienzi 2002 2.0 2.0
Schillaci 2002 2.8 1.8
Ten 2011 2 2
Van der Auwera 2002 1.86 1.87
A P P E N D I C E S
Appendix 1. Search string
MEDLINE (12.11.2010)
1 exp embryo transfer/ or exp fertilization in vitro/ or exp sperm injections, intracytoplasmic/ or exp oocyte donation/ (28694)
2 embryo transfer$.tw. (6779)
3 in vitro fertili?ation.tw. (14235)
4 intracytoplasmic sperm injection$.tw. (3856)
5 (ivf or icsi).tw. (15044)
6 ET.tw. (134290)
7 or/1-6 (167154)
8 day 2.tw. (14522)
9 day3.tw. (13)
10 48$.tw. (378794)
11 72$.tw. (243895)
12 cleav$.tw. (127733)
13 pronuclear.tw. (1700)
14 day 3.tw. (18495)
15 day2.tw. (17)
16 (early adj3 embryo$).tw. (18909)
17 (day two or day three).tw. (1779)
18 exp Cleavage Stage, Ovum/ (1853)
19 or/8-18 (755874)
20 exp Blastocyst/ (18307)
21 Blastocyst$.tw. (13428)
22 (day 5 or day 6).tw. (23950)
23 (day5 or day6).tw. (8)
24 (day ve or day six).tw. (1035)
25 or/20-24 (49046)
26 7 and 19 and 25 (2829)
27 randomized controlled trial.pt. (306110)
28 controlled clinical trial.pt. (83388)
29 randomized.ab. (219168)
30 placebo.tw. (131763)
31 clinical trials as topic.sh. (153027)
32 randomly.ab. (162058)
33 trial.ti. (94444)
34 (crossover or cross-over or cross over).tw. (50486)
35 or/27-34 (743932)
36 exp animals/ not humans.sh. (3599763)
37 35 not 36 (687968)
38 26 and 37 (170)
EMBASE (12.11.2010)
1 exp embryo transfer/ (14920)
2 exp fertilization in vitro/ (30627)
3 exp intracytoplasmic sperm injection/ (8214)
4 exp oocyte donation/ (2258)
9 ET.tw. (237744)
10 or/1-9 (279043)
11 day 2.tw. (16218)
12 day3.tw. (69)
13 48$.tw. (423980)
14 72$.tw. (275907)
15 cleav$.tw. (129599)
17 day 3.tw. (20427)
18 day2.tw. (58)
21 exp oocyte cleavage/ (2345)
22 or/11-21 (831598)
23 exp BLASTOCYST/ (10629)
25 (day 5 or day 6).tw. (25626)
26 (day5 or day6).tw. (48)
28 or/23-27 (41680)
29 10 and 22 and 28 (2971)
30 Clinical Trial/ (815265)
31 Randomized Controlled Trial/ (284101)
32 exp randomization/ (52851)
33 Single Blind Procedure/ (13493)
34 Double Blind Procedure/ (99910)
35 Crossover Procedure/ (29559)
36 Placebo/ (170225)
37 Randomi?ed controlled trial$.tw. (57394)
38 Rct.tw. (6101)
39 random allocation.tw. (998)
40 randomly allocated.tw. (14840)
41 allocated randomly.tw. (1679)
42 (allocated adj2 random).tw. (679)
43 Single blind$.tw. (10503)
44 Double blind$.tw. (114194)
45 ((treble or triple) adj blind$).tw. (226)
46 placebo$.tw. (152155)
47 prospective study/ (158218)
48 or/30-47 (1097150)
49 case study/ (10547)
50 case report.tw. (193062)
51 abstract report/ or letter/ (759204)
52 or/49-51 (959233)
53 48 not 52 (1065274)
54 29 and 53 (311)
55 2010$.em. (1016089)
56 54 and 55 (50)
EBM Reviews - Cochrane Central Register of Controlled Trials (12.11.10)
1 exp embryo transfer/ or exp fertilization in vitro/ or exp sperm injections, intracytoplasmic/ or exp oocyte donation/ (1474)
6 ET.tw. (4143)
7 or/1-6 (6693)
8 day 2.tw. (2673)
9 day3.tw. (3)
10 48$.tw. (30640)
11 72$.tw. (18242)
12 cleav$.tw. (438)
14 day 3.tw. (3110)
15 day2.tw. (1)
18 exp Cleavage Stage, Ovum/ (50)
19 or/8-18 (51749)
20 exp Blastocyst/ (101)
22 (day 5 or day 6).tw. (3531)
25 or/20-24 (4175)
26 7 and 19 and 25 (154)
MDSG search strategy (15.11.10)
Keywords CONTAINS day 2orday 3or day 3 embryo transferorday 4 embryo transferor cleavage stageorcleavage trans-
ferorpronuclear morphologyorearly cleavage assessmentorearly cleavage mediumorearly cleavage status or Title CONTAINS
day 2orday 3or day 3 embryo transferorday 4 embryo transferor cleavage stageorcleavage transferorpronuclear morphol-
ogyorearly cleavage assessmentorearly cleavage mediumorearly cleavage status
AND
Keywords CONTAINS day 5 or day 5 transfer or day 6 transfer or Blastocyst or blastocyst culture technique or blastocyst
media or blastocyst stage or blastocyst transfer or Title CONTAINS day 5 or day 5 transfer or day 6 transfer or Blastocyst
or blastocyst culture technique or blastocyst media or blastocyst stage or blastocyst transfer
PsycINFO (12.11.10)
1 exp reproductive technology/ (1022)
6 ET.tw. (73167)
7 or/1-6 (74312)
8 day 2.tw. (1072)
9 day3.tw. (2)
10 48$.tw. (41932)
11 72$.tw. (25340)
12 cleav$.tw. (1318)
14 day 3.tw. (900)
15 day2.tw. (2)
18 exp embryo/ (990)
19 or/8-18 (69832)
21 (day 5 or day 6).tw. (1091)
24 or/20-23 (1206)
25 7 and 19 and 24 (7)
W H A T S N E W
Last assessed as up-to-date: 21 February 2012.
Date Event Description
28 May 2013 Amended Correction of author order
H I S T O R Y
Protocol rst published: Issue 2, 2000
Review rst published: Issue 2, 2002
Date Event Description
21 February 2012 New citation required but conclusions have not
changed
5 new studies have been added. There are no changes
to the conclusions reported in the 2007 update
21 February 2012 New search has been performed History of this review: the review was rst published
in The Cochrane Library in 2000 with 10 RCTs, and a
journal paper version was published in Human Repro-
ductionin2003withanadditional four RCTs. The au-
thors were Debbie Blake, Michelle Proctor, Neil John-
son and David Olive. There was no evidence for a dif-
ference in pregnancy rate and only one trial reported
live birth rates
In the 2005 update, seven RCTs from the 2000 re-
view were excluded (for quasi-randomisation or per
cycle data only or other study design problems) and
13 new RCTs were added. In addition the outcomes
in Metaview were recongured. Cindy Farquhar and
Quirine Lamberts assisted with the update and were
added to the authors. Some protocol changes were
made to the outcome measures and to the sensitivity
analysis (day of randomisation) and subgroup analyses
(prognosis). More included trials reported live birth
outcomes but there was still no evidence of a difference
in success rates
The 2007 update had two new trials added to bring
the total to 18. There was a new subcategory for sin-
gle embyro transfer. This update was performed by
Debbie Blake, Neil Johnson and Cindy Farquar. It re-
ported for the rst time a signicant difference in live
birth and pregnancy outcomes in favour of blastocyst
culture
In this 2012 update led by Demian Glujovsky with
the assistance of Cindy Farquhar and Debbie Blake,
ve new studies were added (Brugnon 2010; Elgindy
2011, Fisch 2007, Pantos 2004, Ten 2011).
(Continued)
29 August 2011 Amended Plain language summary corrected
17 November 2010 Amended New search strategies performed.
11 November 2010 Amended New author
23 July 2007 New citation required and conclusions have changed Substantive amendment
C O N T R I B U T I O N S O F A U T H O R S
Debbie Blake: for the initial review and updates to 2005, took the lead in writing the protocol and review, performed initial searches
of databases for trials, involved in selecting trials for inclusion, performed independent data extraction and quality assessment of the
included trials, was responsible for statistical analysis and interpretation of the data. Also contributed to the nal analysis and text of
the 2012 update.
Cindy Farquhar: for the 2005 update, added in the new studies, redesigned the table of comparisons and rewrote the results section
as well as edited the review. Also contributed to the 2007 and 2012 update with assistance in extraction and interpretation of the data
and writing in all sections.
Demin Glujovsky: for the 2012 update, took the lead in writing the update of the review, performed new searches of databases for
trials, involved in selecting trials for inclusion, performed independent data extraction and quality assessment of the included trials,
was responsible for statistical analysis and interpretation of the data in the update.
Ariel Bardach: for the 2012 update, involved in selecting trials for inclusion, performed independent data extraction and quality
assessment of the included trials.
D E C L A R A T I O N S O F I N T E R E S T
No authors have any conict of interest to declare.
S O U R C E S O F S U P P O R T
Internal sources
Cindy Farquhar, Not specied.
University of Auckland
Debbie Blake, Not specied.
Auckland University of Technology
External sources
No sources of support supplied
D I F F E R E N C E S B E T W E E N P R O T O C O L A N D R E V I E W
Addition of cumulative pregnancy rate to the outcomes.
N O T E S
Conict of interest added.
I N D E X T E R M S
Medical Subject Headings (MeSH)
Blastocyst; Cleavage Stage, Ovum [
transplantation]; Embryo Transfer [
methods]; Live Birth [epidemiology]; Pregnancy Outcome;

Pregnancy Rate; Pregnancy, Multiple; Randomized Controlled Trials as Topic
MeSH check words
Female; Humans; Pregnancy

2012 - Cleavage Stage Versus Blastocyst Stage Embryo Transfer in Assisted Reproductive Technology

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Cleavage stage versus blastocyst stage embryo transfer in

assisted reproductive technology (Review)

Karaki RZ, Samarraie SS, Younis NA, Lahloub TM,

Levron J, Shulman A, Bider D, Seidman D, Levin T, Dor

Rienzi l, Ubaldi F, Iacobelli M, Ferrero S, Minasi MG,

Schillaci R, Castelli A, Vassiliadis A, Venezia R, Sciacca

Van der Auwera I, Debrock S, Spiessens C, Afschrift H,

Indicates the major publication for the study

Blastocyst; Cleavage Stage, Ovum [

transplantation]; Embryo Transfer [

methods]; Live Birth [epidemiology]; Pregnancy Outcome;

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