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Systematic Reviews
Double versussingle blastocyst biopsy
andvitrication inpreimplantation genetic
testing (PGT) cycles: protocol forasystematic
review andmeta-analysis ofclinical
andneonatal outcomes
Alessandra A. Vireque1* , Vasileios Stolakis1, Thalita S. Berteli1, Maria C. Bertero1 and Jason Kofinas1
Abstract
Background The number of re-biopsied blastocysts is widely increasing in IVF cycles and concerns regarding retest-
ing, which involves double biopsy and vitrification-warming, have been raised. The re-biopsy intervention seems
to significantly reduce the pregnancy potential of a blastocyst but the evidence is still restricted to retrospective
observational studies reporting a low number of cycles with re-biopsied embryos. Additionally, the neonatal out-
comes after the transfer of re-biopsied and re-vitrified embryos are poorly documented to date.
Methods A systematic review will be conducted, using PubMed/Medline, EMBASE, Cochrane Central Register
of Controlled Trials, Scopus, Web of Science, and Google Scholar to identify all relevant randomized control trials
(RCTs), cohort and case–control studies published until December 2024. The participants will include women under-
going preimplantation genetic testing and single euploid frozen embryo transfer (FET) cycles. The primary outcomes
are live birth rate (LBR) and singleton birthweight, whereas secondary outcomes are post-warming embryo survival
rate, clinical pregnancy (fetal heart pregnancies at 4.5 weeks), miscarriage rate (loss of pregnancy before the 20th
week, and stillbirth), preterm birth (PB) rate, small-for-gestational age (SGA, < − 1.28 SDS (standard deviation score)),
large-for-gestational age (LGA, > + 1.28 SDS), low birthweight (LBW; birthweight < 2500 g), preterm birth (gesta-
tion < 37 weeks), macrosomia (birthweight > 4000 g), pre-eclampsia, eclampsia, perinatal death, and major congenital
malformations. Eligible studies will be selected according to pre-specified inclusion and exclusion criteria. Addition-
ally, manual search will target other unpublished reports and supplementary data. At least two independent review-
ers will be responsible for article screening, data extraction and bias assessment of eligible studies. A third reviewer
will resolve any disagreements. The Newcastle–Ottawa scale (NOS) will be used to assess the quality of the included
studies. Studies that receive a score of 7 or higher on the NOS will be considered to have high methodological quality.
The extracted data will be pooled and a meta-analysis will be performed. To carry out the data synthesis, a random
effects meta-analysis will be conducted using the RevMan software. Heterogeneity will be evaluated by Cochran’s Q
test and the I2 statistics and the strength of evidence will be rated with reference to GRADE. The review and meta-
analysis will be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses
(PRISMA) guidelines.
*Correspondence:
Alessandra A. Vireque
Avireque@kofinas.org
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Page 2 of 7
Virequeetal. Systematic Reviews (2025) 14:93
Discussion The findings of this systematic review will be important to clinicians, embryologists, patients, and assisted
reproductive service providers regarding the decision-making on retesting embryos for PGT in FET cycles.
Systematic review registration PROSPERO CRD42024498955.
Keywords Genetic testing/methods, PGT, Biopsy/adverse effects, Trophectoderm biopsy, Re-biopsy, Rewarming/
adverse effects, Live birth rate, Birthweight, Neonatal outcomes, Systematic review
Background
Preimplantation genetic testing (PGT) can significantly
enhance the success rate of assisted reproductive tech-
nologies (ART) and prevent the transmission of genetic
disorders to offspring by eliminating embryos affected by
a single gene mutation or mutations (PGT-M), structural
rearrangements of chromosomes (PGT-SR), and ane-
uploidy (PGT-A) [1, 2]. e genetic analysis requires
a trophectoderm biopsy (TE) from the embryo prior
to transfer [3] and the current standard for sampling
involves a blastocyst biopsy on days 5, 6, or 7 that extracts
an average of five cells [4–7]. It was previously reported
that TE biopsies containing a large number of cells were
associated with a lower live birth rate, suggesting that TE
cell number reduction, may affect clinical outcomes [5].
TE biopsy is performed prior to or following vitrification
and the safety of double vitrification or even single vitrifi-
cation remains controversial [8–13].
In FET cycles, the combination of blastocyst biopsy and
vitrification involves a single vitrification–warming cycle.
A second biopsy can be performed whether the results of
the fresh biopsy are inconclusive, and a second vitrifica-
tion round will be requiredif a patient with untested vit-
rified embryos decides to undergo PGT. e main causes
of failure of PGT diagnosis are DNA amplification failure,
data inconsistency, and non-concurrent results. Under
these conditions, clinicians and patients face the dilemma
of whether to transfer these “unscreened” embryos or
to perform re-biopsy to obtain a PGT result. According
to ESHRE PGT Consortium data, the rate of ‘no result’
embryos is estimated at 11% for PGT-M and 7% for PGT-
SR whereas PGT-A fails to yield a diagnostic result in
0.86–3.8% of embryo biopsies [14].
As PGT has evolved in the setting of assisted repro-
ductive technologies, an increasing number of embryos
with undetermined results, yet potentially transferable,
have emerged. erefore, concerns regarding rebiopsy
and retesting (double biopsy and double vitrification-
warming) have been raised [2, 4, 15–19]. Approximately
2–6% of PGT embryos will require a second round of
biopsy and vitrification [20], and a portion of these
embryos will be transferred based on patient prefer-
ences. In this scenario, double biopsy and vitrification
have been less investigated compared to single proce-
dures (standard PGT), and no randomized controlled
trials have been conducted on blastocyst rebiopsy and
revitrification. e first report of blastocyst rebiopsy was
published in 2017 [21], and to date, most small-sample
observational studies on the association between blas-
tocyst rebiopsy and pregnancy outcomes have reported
an increased risk compared to single biopsy [6, 12, 17,
19]. In a study designed to isolate the effect of repeated
TE biopsies, by controlling embryo exposure to double
vitrification-warming, Sekhon and colleagues observed
a 15% decrease in implantation rate in the double TE
biopsy group [22]. Similarly, Zhuo and colleagues found
that rebiopsied euploid embryos exhibit significantly
lower odds of implantation and pregnancy compared to
single-biopsied euploid embryos [16]. Since trophecto-
derm subsequently forms the placenta, it is proposed that
multicellular TE biopsy is associated with adverse obstet-
rical or neonatal outcomes after a single frozen-warmed
blastocyst transfer [1, 23–28]. Regarding repeated biop-
sies, obstetrical and neonatal outcomes have been under-
reported to date and vary between studies [12, 19, 20, 22,
29, 30]. is lack of evidence creates uncertainty and lim-
its the guidance clinicians can provide to patients consid-
ering PGT testing for their previously biopsied embryos
[17]. Recent studies have extracted DNA from blasto-
coel fluid and from the conditioned blastocyst culture
medium in order to explore the clinical application of a
noninvasive genetic screening [31–34]. However, current
published data is not adequate in order to establish its
application in clinical practice [35].
As with any assisted reproductive technology, blas-
tocyst rebiopsy continues to evolve in FET cycles as a
strategy to increase the number of embryos available
for transfer, to optimize reproductive outcomes for the
patient, and to limit the risk of transferring single gene
disorders to offspring [2, 15]. ere is great interest
across the board in more evidence that could provide
patients and InVitro Fertilization (IVF) providers with
reliable data about the risks of retesting embryos. e
present study therefore aims to collect and analyze exist-
ing data in order to provide a comprehensive systematic
review of IVF and neonatal outcomes from pregnancies
conceived after retesting (an extra round of blastocyst
biopsy and vitrification) compared to those derived from
a single biopsy and vitrification in euploid FET cycles.
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Page 3 of 7
Virequeetal. Systematic Reviews (2025) 14:93
Methods/design
Research aim
e objective of this systematic review is to assess and
synthesize pieces of evidence on the live birth and peri-
natal outcomes of singleton euploid blastocysts trans-
ferred after undergoing a second round of biopsy and
vitrification-warming in comparison to embryos biopsied
and vitrified-warmed once.
PICO—research question
How do rebiopsy and revitrification impact IVF and neo-
natal outcomes of women undergoing euploid FET cycles
compared to an embryo biopsied and vitrified-warmed
once?
Protocol andregistration
e study protocol was registered with PROSPERO
(identifier CRD42024498955—https:// www. crd. york. ac.
uk/ PROSP ERO/) and has been reported according to the
Preferred Reporting Items for Systematic Reviews and
Meta-Analyses Protocols (PRISMA-P) [36].
Study eligibility criteria
e selection criteria will be described according to
Patients, Intervention, Comparison and Outcomes
(PICO) statements as previously stated. We will include
only Randomized Controlled Trials (RCTs), cohort and
case–control studies that compare the clinical outcomes
between blastocysts biopsied and vitrified once and blas-
tocyst retesting (biopsied and vitrified twice).
Setting
Single center and multicenter studies conducted in pri-
vate fertility clinics and university-affiliated infertility
practices addressing homologous and heterologous single
embryo transfer (SET) cycles.
Participants
Inclusion: All transferred euploid embryos biopsied and
vitrified-warmed twice compared to euploid embryos
biopsied and vitrified once from women undergoing FET
cycles. All embryos undergoing trophectoderm biopsy on
day 5, 6, and 7 followed by vitrification and single embryo
transfers. Since cleavage-stage embryo biopsy, which
involved removing one or more cells (blastomeres), has
been replaced by trophectoderm (TE) biopsy at the blas-
tocyst stage in PGT cycles, studies on cleavage-stage
biopsied embryos will be excluded from this systematic
review. Instead, we will focus on the effect of double tro-
phectoderm biopsy (performed only on blastocyst-stage
embryos) on clinical outcomes, as its relevance and appli-
cability are closely linked to the current PGT workflow in
IVF clinics.
Exclusion: Blastomere biopsy performed on cleavage-
stage embryos (day 3) and embryos cryopreserved by the
slow freezing method will not be included.
Intervention
Re-biopsied and re-vitrified blastocysts from patients
undergoing single euploid FET cycles.
Comparator
Blastocysts biopsied and vitrified-warmed once from
patients undergoing single euploid FET cycles.
Main outcome(s)
e primary outcomes are live birth rate (LBR) and
singleton birthweight. Live birth will be assessed as
live births per embryo transferred. Birthweight will be
assessed at the time of delivery after 37–42 weeks of
gestation. Low birthweight was defined as a birthweight
of < 2500g, and macrosomia was defined as a birthweight
of > 4000g.
Secondary outcomes
Embryo survival, clinical pregnancy rate (calculated
as fetal heart pregnancies at 4.5 weeks per blastocyst
transfers in the selected studies), miscarriage (clinical
pregnancies that did not result in live births in the first
20 weeks of pregnancy, including stillbirths), preterm
birth (PB), small-for-gestational age (SGA, < − 1.28 SDS),
large-for-gestational age (LGA, > + 1.28 SDS), preterm
birth (gestation < 37 weeks), pre-eclampsia, eclampsia,
perinatal death, and major congenital malformations. To
eliminate the confounding factors resulting from multi-
ple pregnancies, we only included single euploid embryo
FET cycles if provided in the publications.
Search strategy andliterature search
We will search the following electronic bibliographic
databases: PubMed (MEDLINE), Embase, Cochrane
Central Register of Controlled Trials (CENTRAL), Sco-
pus, Google Scholar, and Web of Science (science and
social science citation index) according to expert rec-
ommendations [37] for biomedical systematic reviews.
e search strategy was developed according to P-I-C
components of PICO [38]. e search strategy devel-
oped for PubMed/MEDLINE is shown in Additional
File 1. e search terms were adapted for use with other
bibliographic databases. Controlled vocabulary terms,
text words and medical subject headers (MeSH) will be
searched. Search strategy peer review was performed by
the authors through PRESS Checklist. Databases syntax
and thesaurus were extensively reviewed as well as prox-
imity operators, truncation, subject headings (function
explode/noexp), search fields (ti,ab), limits, and filters.
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Page 4 of 7
Virequeetal. Systematic Reviews (2025) 14:93
We also considered alternative spellings for keywords
and surveyed the grey literature for non-reported nega-
tive studies of other Internet resources, conference pro-
ceedings, and contact with experts. A systematic search
on OpenGrey, medRxiv, ProQuest, Google, and Clini-
calTrials.gov will be performed [39]. For completeness,
we will check the reference lists of all eligible studies and
review articles to assess additional references. If there are
errors or corrections of studies included with a complete
text, we will report the date on which they occurred. e
searches in databases and grey literature will be re-run
immediately prior to analysis to ensure that the most cur-
rent information is presented in the review. We will not
be retrieving or including any unpublished data.
Study screening andselection
Titles and/or abstracts of studies retrieved using the
search strategy and those from additional sources will be
screened independently by two review authors to iden-
tify studies that potentially meet the inclusion criteria
outlined above. To make a decision, two members of the
review team will perform full-text screenings of these
potentially eligible studies independently. Any disagree-
ments between them over the eligibility of particular
studies will be resolved through discussion with a third
reviewer.
Data extraction
Before starting data extraction, wewill pilot the process
to ensure reliability in the interpretation and use of the
inclusion criteria. Two unblinded review authors will
extract data independently, discrepancies will be identi-
fied and resolved through discussion with a third author
when is necessary. Upon completion of the data extrac-
tion template, the reviewers will extract the data and
reasons for exclusion will be listed. Data extracted will
include demographic information, methodology, inter-
vention details, and all reported patient-important out-
comes. More detailed information will be extracted such
as: last name of the first author; year of publication; study
setting; study population and participant baseline char-
acteristics; type of control used; study design; statisti-
cal methods implemented and main results (e.g., odds
ratios), relative risks; information for the assessment
of the risk of bias. Categorical data will be extracted as
a frequency from the number of events observed at the
endpoint (n, N, and CI) whereas continuous data will be
assessed as mean ± SD or median, IQ.
Risk ofbias assessment
e Newcastle–Ottawa scale (NOS) will be used to
assess the quality of the included articles. Attributing
one point to each answer marked with an asterisk
below scores the NOS quality instrument. Possible total
points are 4 points for Selection, 2 points for Compara-
bility, and 3 points for Outcomes. Studies that receive a
score of 7 or above on the NOS will be considered high
quality [40, 41]. e Cochrane Risk of Bias 2 (RoB 2)
tool will be used to assess quality of potential RCTs.
Whenever possible, grey literature will be evaluated
using the same standards as traditional studies. As part
of our critical appraisal approach, we will apply the
AACODS checklist for the domains authority, preci-
sion, coverage, objectivity, date, and significance, where
relevant [42, 43]. Two authors will check quality assess-
ment independently, and any disagreements solved by a
third reviewer until a consensus is reached.
Data synthesis
We will provide a narrative synthesis of the findings
from the included studies, structured around the type
of intervention, baseline characteristics, type of out-
come and intervention content. Where studies have
used the same type of intervention and comparator,
with the same outcome measure, a meta-analysis will
be performed [44–46]. Where most of the studies are
retrospective cohort studies, dichotomous outcomes
will be pooled to determine the odds ratio (OR) or risk
ratio (RR) with 95% confidence intervals (CIs). Data
from the continuous outcomes will be pooled using the
mean difference (MD) will be calculated between the
groups to determine the effect size [44]. e I2 statis-
tic will be used to quantify heterogeneity. A random-
effects model will then be used to pool the estimates in
a forest plot [46]. Where information is missing to cal-
culate a common effect metric, additional information
will be requested by contacting the authors. e fun-
nel plot will be used to assess potential publication bias
following the Cochrane recommendations on testing
for funnel plot asymmetry. Sensitivity analysis will be
performed for the outcomes with funnel plot asymme-
try to assess the leverage of the studies on the results
[44]. Potential heterogeneity sources will be examined
through subgroup analysis [47]. When sample size per-
mits, data will be grouped by maternal age or embryo
development stage at rebiopsy (day 5/6 embryos). e
sources of heterogeneity will be explored and appropri-
ated quantified to avoid compromising interpretabil-
ity of the results of the meta-analysis. e strength of
evidence will be rated with reference to GRADE. e
Review Manager (RevMan Version 7.2.0. Software,
available at https:// revman. cochr ane. org) will be used
for statistical analysis.
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Page 5 of 7
Virequeetal. Systematic Reviews (2025) 14:93
Data management
Search results from bibliometric databases were
imported to the web-based software Covidence
(https:// www. covid ence. org/) and de-duplicated.
Results from grey literature searching will be into Sci-
wheel (Sciwheel, Reference Manager and Generator,
Harvard, APA) and de-duplicated. All results from grey
literature and the second round of databases search will
be then imported into the Covidence for title/abstract
screening, full-text screening as well as data extraction
[48]. All data extracted will be exported to RevMan
(ReviewManager, Cochrane) for quantitative analysis.
Reporting
To allow for transparency and reproducibility of the
findings, the methods and results of this systematic
review and meta-analysis will be reported according to
the Preferred Reporting Items for Systematic Reviews
and Meta-Analysis (PRISMA) guidelines [49–52].
Timeline forsystematic review
Data extraction started in September 2024 and will be
completed by December 2024. A draft manuscript will
be completed by January 2025.
Discussion
Potential re-biopsy-related damage to the blastocyst and
the impact on live birth and neonatal outcomes are still
debatable. It is paramount to evaluate whether blastocyst
retesting (double biopsy and vitrification) poses addi-
tional IVF, obstetric and/or neonatal risks compared with
euploid embryos undergoing a single biopsy and vitrifi-
cation [1, 2, 6, 12, 17, 18, 53]. erefore, this systematic
review and meta-analysis will assess and analyze the cur-
rent clinical outcomes of blastocyst re-biopsy compared
with single biopsy and vitrification in single euploid FET
cycles. is study can contribute to clinicians’ decision-
making and assist providers in supporting patients by
thoroughly weighing the risks and benefits of embryo
re-biopsy. e strengths and limitations of the evidence
will be considered, and findings will be discussed in con-
text with related studies. e results of this SR will sum-
marize the existing evidence of the impact of embryo
retesting on clinical outcomes and help to identify gaps
in knowledge where further research is required. It is also
expected that the findings will be useful for the develop-
ment of additional guidelines on PGT practice.
Protocol amendments
Any amendment that is made to the protocol whilst
conducting the systematic review will be detailed
clearly in the published article and will be updated on
PROSPERO.
Abbreviations
IVF In vitro fertilization
FET Frozen transfer cycles
PGT Preimplantation genetic testing
PGT-A Preimplantation genetic testing for aneuploidy
PGT-M Preimplantation genetic testing for monogenic/single genes defects
PGT-SR Preimplantation genetic testing for structural rearrangements
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13643- 025- 02846-8.
Supplementary Material 1.
Acknowledgements
Not applicable.
Authors’ contributions
All authors were involved in the conception and design of the protocol. AV
developed the search strategy and drafted the protocol. VS, TB and MB criti-
cally revised the protocol. JK conceived the study, critically revised the manu-
script and gave final approval for the manuscript to be published, and agree
to be accountable and guarantor for all aspects of the review. All authors read,
provided feedback, and approved the final manuscript.
Data availability
Data sharing is not applicable to this article as no datasets were generated or
analyzed during the current study.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors report no financial or commercial conflicts of interest.
Author details
1 Kofinas Fertility Group 65, Broadway, New York, NY 10006, USA.
Received: 12 October 2024 Accepted: 2 April 2025
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Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Alessandra A. Vireque is a biologist and research associate scien-
tist; VS and TB are senior embryologists and researchers; MB is a clini-
cian and IVF laboratory director; JK is Chief Medical Office of Kofinas
Fertility Group and division director of RE/I at The Brooklyn Hospital
Center.
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