Double versus single blastocyst biopsy and vitrification in PGT cycles: protocol for a systematic review and meta-analysis of clinical and neonatal outcomes.

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Alessandra Vireque, Vasileios Stolakis, Thalita S. Berteli, Maria C. Bertero, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5253174/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Apr, 2025 Read the published version in Systematic Reviews → Version 1 posted 5 You are reading this latest preprint version Abstract Background The number of re-biopsied blastocysts is widely increasing in PGT cycles and concerns regarding retesting, which involve 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, using PubMed/Medline, EMBASE, Cochrane Central Register of Controlled Trials, Scopus, Web of Science and Google Scholar to identify all relevant RCTs, cohort and case-control studies published until December 2024. The participants will include women undergoing preimplantation genetic testing and single euploid 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), low birthweight (LBW; birthweight < 2500g), preterm birth (gestation 4000g), 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 Additionally, manual search will target other unpublished reports and supplementary data.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 and will be used to assess the quality of the included studies. Studies that receive a score equal to or greater than 7 on the NOS will be considered high 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 I 2 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 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 technologies (ART) and prevent the transmission of genetic disorders to the offspring by eliminating embryos affected by a single gene mutation or mutations (PGT-M), structural rearrangements of chromosomes (PGT-SR), aneuploidy (PGT-A) as well as polygenic risk (PGT-P) [ 1 , 2 ]. The 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 with large amount of TE 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 or following vitrification and the safety of double vitrification or even single vitrification 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 or if a patient with untested vitrified embryos decides to undergo PGT. The 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 ]. The number of blastocysts rebiopsied is widely increasing and concerns regarding rebiopsy and retesting (double biopsy and double vitrification-warming) have been raised [ 2 , 4 , 15 – 19 ]. In a study designed to isolate the effect of repeated TE biopsy, by controlling for embryo exposure to double vitrification-warming, Sekhon and colleagues observed a 15% decrease in implantation rate in the double TE biopsy group [ 20 ]. 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 trophectoderm subsequently forms the placenta, it is proposed that multicellular TE biopsy is associated with adverse obstetrical or neonatal outcomes after single frozen-warmed blastocyst transfer [ 1 , 21 ]. Regarding repeated biopsy, contradictory findings have been reported with respect to blastocyst retest and obstetric and neonatal outcomes [ 12 , 22 – 24 ]. Recent studies have extracted DNA from blastocoel fluid and from the conditioned blastocyst culture medium in order to explore the clinical application of a noninvasive genetic screening [ 25 – 28 ]. However, current published data is not adequate in order to establish its application in clinical practice [ 29 ]. As with any assisted reproductive technology, blastocyst rebiopsy continue 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 risk of transferring single gene disorders to the offspring [ 2 , 15 ]. There is great interest across the board in more evidence that could provide patients and IVF providers with reliable data about the risks of retesting embryos. The present study therefore aims to collect and analyze existing data in order to provide a 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. Methods/Design Research aim The objective of this systematic review is to assess and synthesize pieces of evidence on the live birth and perinatal outcomes of singleton euploid blastocysts transferred 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 on IVF and neonatal outcomes of women undergoing euploid FET cycles compared to an embryo biopsied and vitrified-warmed once? Protocol and registration The study protocol was registered with PROSPERO (identifier CRD42024498955 - https://www.crd.york.ac.uk/PROSPERO/ ) and has been reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) [ 30 ]. Study eligibility criteria The selection criteria will be described according to Patients, Intervention, Comparison and Outcomes (PICO) statements as previously stated. We will include only RCT, cohort and case-control studies that compare the clinical outcomes between blastocysts biopsied and vitrified once and blastocyst retesting (biopsied and vitrified twice). Setting Single center and multicenter studies conducted in private fertility clinics and university-affiliated infertility practice addressing homologous and heterologous 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. 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) The 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 4000 g. 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), preterm birth (gestation < 37 weeks), pre-eclampsia, eclampsia, perinatal death, and major congenital malformations. To eliminate the confounding factors resulting from multiple pregnancies, we only included single euploid embryo FET cycles if provided in the publications. Search strategy and literature search We have searched the following electronic bibliographic databases: PubMed (MEDLINE), Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Scopus, Google Scholar, and Web of Science (science and social science citation index) according to recommendations by [ 31 ] for biomedical systematic reviews. The search strategy was developed according to P-I-C components of PICO [ 32 ]. The search strategy drafted for PubMed/MEDLINE is shown in Additional File 1. The search terms were adapted for use with other bibliographic databases. Controlled vocabulary terms, text words and medical subject headers (MeSH) were searched. Search strategy peer review was performed by the authors through PRESS Checklist. Databases syntax and thesaurus was extensively reviewed as well as proximity operators, truncation, subject headings (function explode/noexp), search fields (ti,ab), limits and filters. We also considered alternative spellings for keywords and surveyed the grey literature for non-reported negative studies of other Internet resources, conference proceedings, and contact with experts. A systematic search on OpenGrey, medRxiv, ProQuest, Google and ClinicalTrials.gov was performed [ 33 ]. 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. The searches in databases and grey literature will be re-run immediately prior to analysis to ensure that the most current information is presented in the review. We will not be retrieving or including any unpublished data. Study screening and selection Titles and/or abstracts of studies retrieved using the search strategy and those from additional sources were screened independently by two review authors to identify studies that potentially meet the inclusion criteria outlined above. To make a decision, two members of the review team performed full-text screenings of these potentially eligible studies independently. Any disagreements between them over the eligibility of particular studies were resolved through discussion with a third reviewer. Data extraction Before starting data extraction, we piloted 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 identified and resolved through discussion with a third author when is necessary. Upon completion of the data extraction template, the reviewers will extract the data and reasons for exclusion will be listed. Data extracted will include demographic information, methodology, intervention details, and all reported patient-important outcomes. More detailed information will be extracted such as: last name of the first author; year of publication; study setting; study population and participant baseline characteristics; type of control used; study design; statistical 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 of bias assessment The 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 Comparability, and 3 points for Outcomes. Studies that receive a score of 7 or above on the NOS will be considered high quality [ 34 – 35 ]. Two authors will check quality assessment 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 outcome 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 [ 36 – 38 ]. 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 [ 36 ]. The I 2 statistic will be used to quantify heterogeneity. If feasible, i.e. where heterogeneity is low to moderate ( I 2 < 60%), a random-effects meta-analysis will then be used to pool these estimates in a forest plot [ 38 ]. Otherwise, the fixed-effects model will be used ( I 2 < 50%). Where information is missing to calculate a common effect metric, additional information will be requested by contacting the authors. The funnel 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 asymmetry to assess the leverage of the studies on the results [ 36 ]. Subgroup analysis will be performed according to maternal age and biopsy day (5, 6, or 7). The sources of heterogeneity will be explored and appropriated quantified to avoid compromising interpretability of the results of the meta-analysis. The strength of evidence will be rated with reference to GRADE. The Review Manager (RevMan Version 7.2.0. Software, available at https://revman.cochrane.org ) will be used for statistical analysis. Data management Search results from bibliometric databases were imported to the web-based software Covidence ( https://www.covidence.org/ ) and de-duplicated. Results from grey literature searching will be into Sciwheel (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 [ 39 ]. All data extracted will 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 [ 40 – 43 ]. Timeline for systematic 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 additional IVF, obstetric and/or neonatal risks compared with euploid embryos undergoing a single biopsy and vitrification [ 1 , 2 , 6 , 12 , 17 , 18 , 44 ]. Therefore, this systematic review and meta-analysis will assess and analyze the current clinical outcomes of blastocyst re-biopsy compared with single biopsy and vitrification in single euploid FET cycles. This study can contribute to clinicians' decision-making and assist providers in supporting patients by thoroughly weighing the risks and benefits of embryo re-biopsy. The strengths and limitations of the evidence will be considered, and findings will be discussed in context with related studies. The results of this SR will summarize 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 development 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 Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Study funding/Conflict of interest The authors report no financial or commercial conflicts of interest. 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 critically revised the protocol. JK conceived the study, critically revised the manuscript 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. Acknowledgements Not applicable. Author details AV is a biologist and research associate scientist; VS and TB are senior embryologists and researchers; MB is a clinician and IVF laboratory director; JK is a clinician, director of IVF and Research. References Mao D, Xu J, Sun L. Impact of trophectoderm biopsy for preimplantation genetic testing on obstetric and neonatal outcomes: a meta-analysis. Am J Obstet Gynecol. 2024;230(2):199–e2125. Verpoest W. O-051 Refreezing and rebiopsy – are these worth it? Hum Reprod 2023;38(1). Kokkali G, Traeger-Synodinos J, Vrettou C, et al. Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study. Hum Reprod. 2007;22:1443–9. Guzman L, Nuñez D, López R, et al. The number of biopsied trophectoderm cells may affect pregnancy outcomes. J Assist Reprod Genet. 2019;36(1):145–51. 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Supplementary Files PRISMA2020checklistVirequeetal..docx Cite Share Download PDF Status: Published Journal Publication published 25 Apr, 2025 Read the published version in Systematic Reviews → Version 1 posted Editorial decision: Minor revision 22 Dec, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers invited by journal 06 Nov, 2024 Editor assigned by journal 06 Nov, 2024 First submitted to journal 12 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5253174","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374975345,"identity":"371d7206-a205-4a0e-beb9-3436d6ac64e0","order_by":0,"name":"Alessandra Vireque","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYJCCA2CSvQFIGFiQooUHRBlIkGKXRAKYJKyQv4HH8HBFhXViv+Tzqxt+FEgw8Ld3J+A3+wCPwcEzZ9ITZ87OKbvZA3SYxJmzG/Bbc4At4WBj2+HEDbdz0m7wALUYSOTi1yIP1vLvcOL+m2fSbv4hRovBAeYDBxsbgLZIsB+7TZQthoeBWhqOpRvPOJPDdlvGQIKHoF/kjjc2f2yosZbtbz/+7OabPzZy/O29BLzPDCd5DEAsHvzKUTWyPyBW9SgYBaNgFIwwAAApFktFGFmzRgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2597-531X","institution":"Kofinas Fertility Group","correspondingAuthor":true,"prefix":"","firstName":"Alessandra","middleName":"","lastName":"Vireque","suffix":""},{"id":374975346,"identity":"c76e84d2-2a4e-4573-b41e-c3b05e7481c4","order_by":1,"name":"Vasileios Stolakis","email":"","orcid":"","institution":"Kofinas Fertility Group","correspondingAuthor":false,"prefix":"","firstName":"Vasileios","middleName":"","lastName":"Stolakis","suffix":""},{"id":374975347,"identity":"23d6d9c0-7800-4c35-a2a1-21fe2ba9337f","order_by":2,"name":"Thalita S. Berteli","email":"","orcid":"","institution":"Kofinas Fertility Group","correspondingAuthor":false,"prefix":"","firstName":"Thalita","middleName":"S.","lastName":"Berteli","suffix":""},{"id":374975348,"identity":"57f5d86f-6784-49fd-ba82-bdbb158d35c3","order_by":3,"name":"Maria C. Bertero","email":"","orcid":"","institution":"Kofinas Fertility Group","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"C.","lastName":"Bertero","suffix":""},{"id":374975349,"identity":"d90b0e6a-19a3-4f82-b7cf-a867ca8d0513","order_by":4,"name":"Jason Kofinas","email":"","orcid":"","institution":"Kofinas Fertility Group","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Kofinas","suffix":""}],"badges":[],"createdAt":"2024-10-12 22:10:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5253174/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5253174/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13643-025-02846-8","type":"published","date":"2025-04-25T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81569527,"identity":"81282182-a889-47b7-a97f-491b9279b182","added_by":"auto","created_at":"2025-04-28 16:05:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":650158,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5253174/v1/ca852663-b7d9-4ad2-804b-3c5fb9353a9c.pdf"},{"id":69661370,"identity":"b31155fb-fc59-44d3-94f6-625e613f10c0","added_by":"auto","created_at":"2024-11-22 19:22:41","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":33118,"visible":true,"origin":"","legend":"","description":"","filename":"PRISMA2020checklistVirequeetal..docx","url":"https://assets-eu.researchsquare.com/files/rs-5253174/v1/1f651440a1832decc066cd68.docx"}],"financialInterests":"","formattedTitle":"Double versus single blastocyst biopsy and vitrification in PGT cycles: protocol for a systematic review and meta-analysis of clinical and neonatal outcomes.","fulltext":[{"header":"Background","content":"\u003cp\u003ePreimplantation genetic testing (PGT) can significantly enhance the success rate of assisted reproductive technologies (ART) and prevent the transmission of genetic disorders to the offspring by eliminating embryos affected by a single gene mutation or mutations (PGT-M), structural rearrangements of chromosomes (PGT-SR), aneuploidy (PGT-A) as well as polygenic risk (PGT-P) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The genetic analysis requires a trophectoderm biopsy (TE) from the embryo prior to transfer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and the current standard for sampling involves a blastocyst biopsy on days 5, 6, or 7 that extracts an average of five cells [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It was previously reported that TE biopsies with large amount of TE cells were associated with a lower live birth rate, suggesting that TE cell number reduction, may affect clinical outcomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TE biopsy is performed prior or following vitrification and the safety of double vitrification or even single vitrification remains controversial [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn FET cycles, the combination of blastocyst biopsy and vitrification involves a single vitrification\u0026ndash;warming cycle. A second biopsy can be performed whether the results of the fresh biopsy are inconclusive or if a patient with untested vitrified embryos decides to undergo PGT. The 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 \u0026ldquo;unscreened\u0026rdquo; embryos or to perform re-biopsy to obtain a PGT result. According to ESHRE PGT Consortium data, the rate of \u0026lsquo;no result\u0026rsquo; 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% \u0026minus;\u0026thinsp;3.8% of embryo biopsies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe number of blastocysts rebiopsied is widely increasing and concerns regarding rebiopsy and retesting (double biopsy and double vitrification-warming) have been raised [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In a study designed to isolate the effect of repeated TE biopsy, by controlling for embryo exposure to double vitrification-warming, Sekhon and colleagues observed a 15% decrease in implantation rate in the double TE biopsy group [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similarly, Zhuo and colleagues found that rebiopsied euploid embryos exhibit significantly lower odds of implantation and pregnancy compared to single-biopsied euploid embryos [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Since trophectoderm subsequently forms the placenta, it is proposed that multicellular TE biopsy is associated with adverse obstetrical or neonatal outcomes after single frozen-warmed blastocyst transfer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Regarding repeated biopsy, contradictory findings have been reported with respect to blastocyst retest and obstetric and neonatal outcomes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recent studies have extracted DNA from blastocoel fluid and from the conditioned blastocyst culture medium in order to explore the clinical application of a noninvasive genetic screening [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, current published data is not adequate in order to establish its application in clinical practice [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs with any assisted reproductive technology, blastocyst rebiopsy continue 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 risk of transferring single gene disorders to the offspring [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. There is great interest across the board in more evidence that could provide patients and IVF providers with reliable data about the risks of retesting embryos. The present study therefore aims to collect and analyze existing data in order to provide a 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.\u003c/p\u003e"},{"header":"Methods/Design","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eResearch aim\u003c/h2\u003e \u003cp\u003eThe objective of this systematic review is to assess and synthesize pieces of evidence on the live birth and perinatal outcomes of singleton euploid blastocysts transferred after undergoing a second round of biopsy and vitrification-warming in comparison to embryos biopsied and vitrified-warmed once.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003ePICO \u0026ndash; Research question\u003c/strong\u003e \u003cp\u003eHow do rebiopsy and revitrification impact on IVF and neonatal outcomes of women undergoing euploid FET cycles compared to an embryo biopsied and vitrified-warmed once?\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtocol and registration\u003c/h3\u003e\n\u003cp\u003eThe study protocol was registered with PROSPERO (identifier CRD42024498955 - \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.crd.york.ac.uk/PROSPERO/\u003c/span\u003e\u003cspan address=\"https://www.crd.york.ac.uk/PROSPERO/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and has been reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStudy eligibility criteria\u003c/h3\u003e\n\u003cp\u003eThe selection criteria will be described according to Patients, Intervention, Comparison and Outcomes (PICO) statements as previously stated. We will include only RCT, cohort and case-control studies that compare the clinical outcomes between blastocysts biopsied and vitrified once and blastocyst retesting (biopsied and vitrified twice).\u003c/p\u003e\n\u003ch3\u003eSetting\u003c/h3\u003e\n\u003cp\u003eSingle center and multicenter studies conducted in private fertility clinics and university-affiliated infertility practice addressing homologous and heterologous SET cycles.\u003c/p\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eInclusion: 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.\u003c/p\u003e \u003cp\u003eExclusion: Blastomere biopsy performed on cleavage-stage embryos (Day 3) and embryos cryopreserved by the slow freezing method will not be included.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntervention\u003c/h2\u003e \u003cp\u003eRe-biopsied and re-vitrified blastocysts from patients undergoing single euploid FET cycles.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparator\u003c/h3\u003e\n\u003cp\u003eBlastocysts biopsied and vitrified-warmed once from patients undergoing single euploid FET cycles.\u003c/p\u003e\n\u003ch3\u003eMain outcome(s)\u003c/h3\u003e\n\u003cp\u003eThe 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\u0026ndash;42 weeks of gestation. Low birthweight was defined as a birthweight of \u0026lt;\u0026thinsp;2500 g, and macrosomia was defined as a birthweight of \u0026gt;\u0026thinsp;4000 g.\u003c/p\u003e \u003cp\u003eSecondary outcomes\u003c/p\u003e \u003cp\u003eEmbryo 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, \u0026lt;-1.28 SDS), large-for-gestational age (LGA, \u0026gt;+1.28 SDS), preterm birth (gestation\u0026thinsp;\u0026lt;\u0026thinsp;37 weeks), pre-eclampsia, eclampsia, perinatal death, and major congenital malformations. To eliminate the confounding factors resulting from multiple pregnancies, we only included single euploid embryo FET cycles if provided in the publications.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSearch strategy and literature search\u003c/h2\u003e \u003cp\u003eWe have searched the following electronic bibliographic databases: PubMed (MEDLINE), Embase, Cochrane Central Register of Controlled Trials (CENTRAL), Scopus, Google Scholar, and Web of Science (science and social science citation index) according to recommendations by [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] for biomedical systematic reviews. The search strategy was developed according to P-I-C components of PICO [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The search strategy drafted for PubMed/MEDLINE is shown in Additional File 1. The search terms were adapted for use with other bibliographic databases. Controlled vocabulary terms, text words and medical subject headers (MeSH) were searched. Search strategy peer review was performed by the authors through PRESS Checklist. Databases syntax and thesaurus was extensively reviewed as well as proximity operators, truncation, subject headings (function explode/noexp), search fields (ti,ab), limits and filters. We also considered alternative spellings for keywords and surveyed the grey literature for non-reported negative studies of other Internet resources, conference proceedings, and contact with experts. A systematic search on OpenGrey, medRxiv, ProQuest, Google and ClinicalTrials.gov was performed [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. 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. The searches in databases and grey literature will be re-run immediately prior to analysis to ensure that the most current information is presented in the review. We will not be retrieving or including any unpublished data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStudy screening and selection\u003c/h2\u003e \u003cp\u003eTitles and/or abstracts of studies retrieved using the search strategy and those from additional sources were screened independently by two review authors to identify studies that potentially meet the inclusion criteria outlined above. To make a decision, two members of the review team performed full-text screenings of these potentially eligible studies independently. Any disagreements between them over the eligibility of particular studies were resolved through discussion with a third reviewer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eData extraction\u003c/h2\u003e \u003cp\u003eBefore starting data extraction, we piloted 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 identified and resolved through discussion with a third author when is necessary. Upon completion of the data extraction template, the reviewers will extract the data and reasons for exclusion will be listed. Data extracted will include demographic information, methodology, intervention details, and all reported patient-important outcomes. More detailed information will be extracted such as: last name of the first author; year of publication; study setting; study population and participant baseline characteristics; type of control used; study design; statistical 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\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median, IQ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRisk of bias assessment\u003c/h2\u003e \u003cp\u003eThe 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 Comparability, and 3 points for Outcomes. Studies that receive a score of 7 or above on the NOS will be considered high quality [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Two authors will check quality assessment independently, and any disagreements solved by a third reviewer until a consensus is reached.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData synthesis\u003c/h2\u003e \u003cp\u003eWe will provide a narrative synthesis of the findings from the included studies, structured around the type of intervention, baseline characteristics, type of outcome 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 [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The \u003cem\u003eI\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e statistic will be used to quantify heterogeneity. If feasible, i.e. where heterogeneity is low to moderate (\u003cem\u003eI\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;60%), a random-effects meta-analysis will then be used to pool these estimates in a forest plot [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Otherwise, the fixed-effects model will be used (\u003cem\u003eI\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;50%). Where information is missing to calculate a common effect metric, additional information will be requested by contacting the authors. The funnel 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 asymmetry to assess the leverage of the studies on the results [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Subgroup analysis will be performed according to maternal age and biopsy day (5, 6, or 7). The sources of heterogeneity will be explored and appropriated quantified to avoid compromising interpretability of the results of the meta-analysis. The strength of evidence will be rated with reference to GRADE. The Review Manager (RevMan Version 7.2.0. Software, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://revman.cochrane.org\u003c/span\u003e\u003cspan address=\"https://revman.cochrane.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) will be used for statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eData management\u003c/h2\u003e \u003cp\u003eSearch results from bibliometric databases were imported to the web-based software Covidence (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.covidence.org/\u003c/span\u003e\u003cspan address=\"https://www.covidence.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and de-duplicated. Results from grey literature searching will be into Sciwheel (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 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. All data extracted will exported to RevMan (ReviewManager, Cochrane) for quantitative analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eReporting\u003c/h2\u003e \u003cp\u003eTo 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 [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTimeline for systematic review\u003c/h2\u003e \u003cp\u003eData extraction started in September 2024 and will be completed by December 2024. A draft manuscript will be completed by January 2025.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePotential 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 additional IVF, obstetric and/or neonatal risks compared with euploid embryos undergoing a single biopsy and vitrification [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, this systematic review and meta-analysis will assess and analyze the current clinical outcomes of blastocyst re-biopsy compared with single biopsy and vitrification in single euploid FET cycles. This study can contribute to clinicians' decision-making and assist providers in supporting patients by thoroughly weighing the risks and benefits of embryo re-biopsy. The strengths and limitations of the evidence will be considered, and findings will be discussed in context with related studies. The results of this SR will summarize 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 development of additional guidelines on PGT practice.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProtocol amendments\u003c/h2\u003e \u003cp\u003eAny 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ein vitro fertilization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFET\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efrozen transfer cycles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epreimplantation genetic testing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGT-A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epreimplantation genetic testing for aneuploidy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGT-M\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epreimplantation genetic testing for monogenic/single genes defects\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGT-SR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epreimplantation genetic testing for structural rearrangements\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData sharing is not applicable to this article as no datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy funding/Conflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no financial or commercial conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors were involved in the conception and design of the protocol. AV developed the search strategy and drafted the protocol. VS, TB and MB critically revised the protocol. JK conceived the study, critically revised the manuscript 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAV is a biologist and research associate scientist; VS and TB are senior embryologists and researchers; MB is a clinician and IVF laboratory director; JK is a clinician, director of IVF and Research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMao D, Xu J, Sun L. 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The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021;372:n71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuarneri C, Reschini M, Pinna M, Perego L, Sanzani E, Somigliana E, Sorrentino U, Cassina M, Zuccarello D, Ciaffaglione M. The impact of a second embryo biopsy for preimplantation genetic testing for monogenic diseases (PGT-M) with inconclusive results on pregnancy potential: results from a matched case-control study. J Assist Reprod Genet. 2024;41:1173\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"systematic-reviews","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sysr","sideBox":"Learn more about [Systematic Reviews](http://systematicreviewsjournal.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/sysr/default.aspx","title":"Systematic Reviews","twitterHandle":"@MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Genetic Testing/methods, PGT, Biopsy/adverse effects, trophectoderm biopsy, re-biopsy, Rewarming/adverse effects, live birth rate, birthweight, neonatal outcomes, systematic review","lastPublishedDoi":"10.21203/rs.3.rs-5253174/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5253174/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe number of re-biopsied blastocysts is widely increasing in PGT cycles and concerns regarding retesting, which involve 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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA systematic review, using PubMed/Medline, EMBASE, Cochrane Central Register of Controlled Trials, Scopus, Web of Science and Google Scholar to identify all relevant RCTs, cohort and case-control studies published until December 2024. The participants will include women undergoing preimplantation genetic testing and single euploid 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, \u0026lt;-1.28 SDS), large-for-gestational age (LGA, \u0026gt;+1.28 SDS), low birthweight (LBW; birthweight\u0026thinsp;\u0026lt;\u0026thinsp;2500g), preterm birth (gestation\u0026thinsp;\u0026lt;\u0026thinsp;37 weeks), macrosomia (birthweight\u0026thinsp;\u0026gt;\u0026thinsp;4000g), 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 and will be used to assess the quality of the included studies. Studies that receive a score equal to or greater than 7 on the NOS will be considered high 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\u0026rsquo;s Q test and the \u003cem\u003eI\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e 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.\u003c/p\u003e\u003ch2\u003eDiscussion\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e\u003ch2\u003eSystematic review registration:\u003c/h2\u003e \u003cp\u003ePROSPERO CRD42024498955\u003c/p\u003e","manuscriptTitle":"Double versus single blastocyst biopsy and vitrification in PGT cycles: protocol for a systematic review and meta-analysis of clinical and neonatal outcomes.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-22 19:22:36","doi":"10.21203/rs.3.rs-5253174/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2024-12-22T23:51:38+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-11-20T07:20:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-06T17:45:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-06T07:04:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Systematic Reviews","date":"2024-10-12T18:10:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"systematic-reviews","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sysr","sideBox":"Learn more about [Systematic Reviews](http://systematicreviewsjournal.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/sysr/default.aspx","title":"Systematic Reviews","twitterHandle":"@MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b0fb58ec-4cc6-47d4-aed3-d01aceca34c6","owner":[],"postedDate":"November 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T15:59:02+00:00","versionOfRecord":{"articleIdentity":"rs-5253174","link":"https://doi.org/10.1186/s13643-025-02846-8","journal":{"identity":"systematic-reviews","isVorOnly":false,"title":"Systematic Reviews"},"publishedOn":"2025-04-25 15:57:01","publishedOnDateReadable":"April 25th, 2025"},"versionCreatedAt":"2024-11-22 19:22:36","video":"","vorDoi":"10.1186/s13643-025-02846-8","vorDoiUrl":"https://doi.org/10.1186/s13643-025-02846-8","workflowStages":[]},"version":"v1","identity":"rs-5253174","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5253174","identity":"rs-5253174","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}