Double versus single blastocyst biopsy and vitrification in preimplantation genetic testing (PGT) cycles: protocol for a systematic review and meta-analysis of clinical and neonatal outcomes

Background: The number of re-biopsied blastocysts is widely increasing in IVF cycles and concerns regarding retesting, 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 outcomes 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 undergoing 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 (gestation < 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. Additionally, manual search will target other unpublished reports and supplementary data. At least two independent reviewers 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. 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. © The Author(s) 2025.