Day 5 versus day 3 embryo biopsy for preimplantation genetic testing for monogenic/single gene defects

Abstract

Background

Assisted reproductive technology (ART) has allowed couples with a family history of a monogenic genetic disease, or a disease‐carrying gene, to reduce the chance of them having a child with the genetic disorder. This is achieved by genetically testing the embryos using an advanced process called preimplantation genetic testing for monogenic or single gene disorders (PGT‐M), such as Huntington's disease or cystic fibrosis. This current terminology (PGT‐M) has replaced the formerly‐known preimplantation genetic diagnosis (PGD). During PGT‐M, one or more embryo cells are biopsied and analysed for genetic or chromosomal anomalies before transferring the embryos to the endometrial cavity. Biopsy for PGT‐M can be performed at day 3 of cleavage‐stage embryo development when the embryo is at the six‐ to the eight‐cell stage, with either one or two blastomeres being removed for analysis. Biopsy for PGT‐M can also be performed on day 5 of the blastocyst stage of embryo development when the embryo has 80 to 100 cells, with five to six cells being removed for analysis. Day 5 biopsy has taken over from day 3 biopsy as the most widely‐used biopsy technique; however, there is a lack of summarised evidence from randomised controlled trials (RCTs) that assesses the effectiveness and safety of day 5 biopsy compared to day 3 biopsy. Since biopsy is an invasive process, whether it is carried out at day 3 or day 5 of embryo development may have different impacts on further development, implantation, pregnancy, live birth and perinatal outcomes.

Objectives

To assess the benefits and harms of day 5 embryo biopsy, in comparison to day 3 biopsy, in PGT‐M in women undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) cycles.

Search methods

We searched the following electronic bibliographic databases in December 2021 to identify relevant RCTs: the Cochrane Gynaecology and Fertility Group (CGFG) Specialised Trials Register; CENTRAL, MEDLINE, Embase and PsycINFO. We also handsearched grey literature, such as trial registers, relevant journals, reference lists, Google Scholar, and published conference abstracts.

Selection criteria

Eligible RCTs compared day 5 versus day 3 embryo biopsy for PGT‐M. 

Data collection and analysis

We used standard methodological procedures recommended by Cochrane. The primary review outcomes were live births and miscarriages. We calculated outcomes per woman/couple randomised and reported odds ratios (ORs) with 95% confidence intervals (CIs).

Main results

We included one RCT involving 20 women. The evidence was of very low certainty; the main limitations of the study were serious risk of bias due to lack of blinding of study personnel, and imprecision.

We are uncertain whether day 5 embryo biopsy compared to day 3 biopsy has an effect on live births (OR 1.50, 95% CI 0.26 to 8.82; 1 RCT, 20 women; very low‐certainty evidence). The evidence suggests that if the chance of live birth following day 3 biopsy was assumed to be 40%, then the chance with day 5 biopsy is between 15% and 85%. It is also uncertain whether day 5 embryo biopsy compared to day 3 biopsy has an effect on miscarriages (OR 1.00, 95% CI 0.05 to 18.57; 1 RCT, 20 women; very low‐certainty evidence). 

We are uncertain whether day 5 embryo biopsy compared to day 3 biopsy has an effect on other secondary outcome measures, including viable intrauterine pregnancies (OR 2.25, 95% CI 0.38 to 13.47; 1 RCT, 20 women; very low‐certainty evidence), ectopic pregnancies (OR 0.16, 95% CI 0.01 to 3.85; 1 RCT, 20 women; very low‐certainty evidence), stillbirths (OR not estimable as no events in either group; 1 RCT, 20 women; very low‐certainty evidence) or termination of pregnancies (OR 3.32, 95% CI 0.12 to 91.60; 1 RCT, 20 women; very low‐certainty evidence).

No studies reported on gestational age at birth, birthweight, neonatal mortality and major congenital anomaly.

Authors' conclusions

We are uncertain if there is a difference in live births and miscarriages, viable intrauterine pregnancies, ectopic pregnancies, stillbirths or termination of pregnancies between day 5 and day 3 embryo biopsy for PGT‐M. There was insufficient evidence to draw any conclusions regarding other adverse outcomes. The results should be interpreted with caution, as the evidence was of very low certainty due to limited studies, high risk of bias in the included study, and an overall low level of precision.

Plain language summary

Day 5 versus day 3 embryo biopsy for genetic testing for single gene disorders before embryo implantation

Background

Couples who have a personal or family history of a single‐gene genetic disease, such as cystic fibrosis, can now undergo assisted reproductive technology (ART) to reduce the chance of them having a child with that same genetic disorder. This is known as preimplantation genetic testing for single gene disorders (PGT‐M). During PGT‐M, cells from a fertilised embryo are removed (biopsied) and analysed for the genetic disorder before those embryos that do not have the disorder are transferred back into the woman's uterus. The embryo cells can be biopsied at day 3 or day 5 of embryo development, with day 5 biopsy currently being the most widely used technique. Whether the embryo is biopsied at day 3 or day 5 could have different impacts on further development and implantation of the embryo, as well pregnancy and perinatal outcomes.

Key results

We found one randomised controlled trial that compared day 5 and day 3 embryo biopsy for PGT‐M in a total of 20 women undergoing ART. This study did not provide enough evidence to show whether there is a difference in the chance of live birth or miscarriage; we are very uncertain about the results.

The evidence suggests that if the chance of live birth following day 3 embryo biopsy is assumed to be 40%, then the chance of live birth following day 5 biopsy is between 15% and 85%. 

Evidence on other pregnancy and perinatal outcomes was poorly reported and inconclusive.

Limitations of the evidence

We have very little confidence in the evidence because we only found one small study, and the people performing the trial knew whether the women had day 3 or day 5 biopsy. This means that the results should be interpreted cautiously, and further studies are needed to confirm findings.

How up to date is this evidence?

The evidence is current to December 2021.

Summary of findings

Summary of findings 1. Day 5 compared to day 3 embryo biopsy for preimplantation genetic testing for monogenic/single gene defects.

Day 5 compared to day 3 embryo biopsy for preimplantation genetic testing for monogenic/single gene defects
Patient or population: people having preimplantation genetic testing for monogenic/single gene defects  Setting: fertility clinic Intervention: day 5 embryo biopsy Comparison: day 3 embryo biopsy
Outcomes	Anticipated absolute effects* (95% CI)		Relative effect (95% CI)	№ of participants (studies)	Certainty of the evidence (GRADE)	Comments
	Risk with day 3 embryo biopsy	Risk with day 5 embryo biopsy
Live birth	Study population		OR 1.50 (0.26 to 8.82)	20 (1 RCT)	⊕⊝⊝⊝ Very low a,b
	40 per 100	50 per 100 (15 to 85)
Miscarriage	Study population		OR 1.00 (0.05 to 18.57)	20 (1 RCT)	⊕⊝⊝⊝ Very low a,b
	10 per 100 c	10 per 100 (1 to 67)
Viable intrauterine pregnancy	Study population		OR 2.25 (0.38 to 13.47)	20 (1 RCT)	⊕⊝⊝⊝ Very low a,b
	40 per 100	60 per 100 (20 to 90)
Ectopic pregnancy	Study population		OR 0.16 (0.01 to 3.85)	20 (1 RCT)	⊕⊝⊝⊝ Very low a,b
	20 per 100	4 per 100 (0 to 49)
Stillbirth	Study population		Not estimable	20 (1 RCT)	⊕⊝⊝⊝ Very low a,d
	0 per 100	0 per 100 (0 to 0)
Termination of pregnancy	Study population		OR 3.32 (0.12 to 91.60)	20 (1 RCT)	⊕⊝⊝⊝ Very low a,b
	0 per 100	0 per 100 (0 to 0) e
*The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI).  CI: confidence interval; OR: odds ratio; RCT: randomised controlled trial
GRADE Working Group grades of evidence High certainty: we are very confident that the true effect lies close to that of the estimate of the effect. Moderate certainty: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different. Low certainty: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect. Very low certainty: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect.

^aDowngraded one level for risk of bias: high risk of performance bias due to lack of blinding of study personnel.
^bDowngraded two levels for imprecision: results based on one study, small sample size, wide CI that crosses the line of no effect
^cIn one twin pregnancy, one foetus was lost at 9 weeks.
^dDowngraded two levels for imprecision: results based on one study, small sample size, no events in either arm.
^eOne triplet pregnancy was reduced to twins.

Background

Assisted reproductive technology (ART) has enabled the possibility of allowing couples with a family history of a genetic disease, or a disease‐carrying gene, to have preimplantation genetic diagnosis (PGD) to reduce the chance of them having a child with the genetic disorder (Handyside 1989; Cimadomo 2016). The PGD is a type of preimplantation genetic testing (PGT) where DNA from oocytes (polar bodies) or embryos (cleavage stage or blastocyst) are analysed for human leukocyte antigen typing or for determining genetic abnormalities (Zegers‐Hochschild 2017). With the updated International Glossary on Infertility and Fertility Care, PGD was renamed preimplantation genetic testing for monogenic/single gene defects (PGT‐M) (Zegers‐Hochschild 2017). Along with PGT‐M, PGT is also used to test for aneuploidies (PGT‐A) and for chromosomal structural rearrangements (PGT‐SR) (Zegers‐Hochschild 2017). The PGT‐M and PGT‐A are used for different purposes; PGT‐M is used for people with genetic disease or a disease‐carrying gene, and PGT‐A is used for people with repeated implantation failure, recurrent miscarriage, or advanced maternal age (Jing 2016). While PGT‐A aims to improve the success of in‐vitro fertilisation (IVF) treatment in infertile couples, PGT‐M aims to prevent the birth of children affected with certain genetic diseases. The review is related to PGT‐M only.

Description of the condition

In 1990, Handyside and colleagues reported the first pregnancies using PGT‐M in two couples known to be at risk of transmitting adrenoleukodystrophy and X‐linked intellectual disability (Handyside 1990). In 1992, after PGT‐M testing for cystic fibrosis, the first birth following PGT‐M was reported (Handyside 1992). Since then, PGT‐M has been used for a range of single gene disorders, including Huntington's disease, cystic fibrosis, neurofibromatosis, hereditary breast cancer type 1, myotonic dystrophy type 1, beta‐thalassaemia, Fragile X syndrome, Marfan syndrome, Duchenne muscular dystrophy (Demko 2010; Lee 2018; van Montfoort 2021), and more recently, inherited eye disease and ocular cancer (Yahalom 2018). The Human Fertilisation and Embryology Authority (HFEA) publishes a list of conditions for which PGT‐M is currently approved or awaiting consideration (HFEA 2021).

Description of the intervention

Embryo biopsy is a key procedure in PGT‐M. In a biopsy procedure, one or more embryo cells are removed and analysed for genetic or chromosomal anomalies before transferring the embryos to the endometrial cavity. Biopsy for PGT‐M is usually performed at day 3 of cleavage‐stage embryo development, when the embryo is at the six‐ to eight‐cell stage (Harper 2010). The zona pellucida is opened with a near infra‐red laser, or by mechanical or chemical means, and either one or two blastomeres are removed for analysis (Braude 2002). From 1996 onwards, with transition to blastocyst culture occurring within ART, embryo biopsy at day 5 (the blastocyst stage) was introduced. In day 5 biopsy, generally five to six trophectoderm cells are removed from a typically 80‐ to 100‐cell blastocyst through micromanipulation (Henman 2005; McArthur 2005). In this systematic review, we plan to compare day 3 biopsy (cleavage‐stage biopsy) and day 5 biopsy (blastocyst‐stage biopsy).

Following day 5 or day 3 biopsy, the removed blastomere or trophectoderm cells are analysed using several different genetic tests. The most common PGT‐M test is polymerase chain reaction which represents 82% of PGT‐M tests in Europe in 2016 and 2017 (van Montfoort 2021). Single nucleotide polymorphism microarrays and whole genome amplification are used in small percentage of PGT‐M tests (van Montfoort 2021). 

According to the test results, the unaffected embryos are then selected for transfers. In 2016 and 2017, the European Society of Human Reproduction and Embryology (ESHRE) reported that day 3 biopsy accounted for 78% while day 5 biopsy accounted for 19% of all embryo biopsies (van Montfoort 2021). The ESHRE PGT‐M Consortium reported overall clinical pregnancy rates of 35% per embryo transferred, and a pregnancy with at least one live birth rate of 23% per embryo transferred (van Montfoort 2021).

How the intervention might work

It has been suggested that the removal of multiple cells on day 5 of embryo development, rather than a single cell on day 3, could potentially lead to improved sensitivity of genetic diagnosis, less biomass depletion of embryos, and better outcomes (de Boer 2004). The removal of one to two blastomeres for testing on day 3 decreases the mass of a human embryo by 12.5% to 25% (Cohen 2007). Whilst there is evidence of a capacity within a human embryo to both tolerate and overcome the possible resultant damage, it is likely that embryos that would otherwise progress to implantation (and result in the birth of a live, healthy infant) will be lost as a consequence of the impact of biopsy conducted at this stage of embryo development (Scott 2013). Artley and colleagues reported a high rate of spontaneous cleavage arrest at the four‐ to eight‐cell stage of human embryo development, and noted that the timing was attuned to the activation of the embryonic genome (Artley 1992). It may therefore be suggested that biopsy of human embryos via removal of blastomeres on day 3 of development refocuses resources that would otherwise be used by an embryo, in suitable in vivo or in vitro environments, for progress to the blastocyst stage and beyond.

In contrast, because day 5 biopsy generally involves the removal of five to six trophectoderm cells from an embryo that has typically reached cell numbers of 80 to 100 (McArthur 2005), a smaller proportion of embryo cell mass is removed, and it yields a sample of cells that does not comprise the inner cell mass. Since the embryo had already reached the blastocyst stage for possible implantation, it needs only to recover to the point of hatching or, if compromised, the regaining of implantation potential (McArthur 2005).

Along with embryo biopsy, other procedures in ART, such as IVF or intracytoplasmic sperm injection (ICSI), and transfer of fresh or frozen embryos, are also associated with implantation, pregnancy and live births (Glujovsky 2022; Wang 2010). ICSI is the method most often used for fertilisation; it accounted for 98% of PGT‐M fertilisations in Europe in 2016 and 2017 (van Montfoort 2021). In general, fresh embryos are transferred in day 3 biopsy, and frozen embryos are transferred in day 5 biopsy (Jing 2016).

Why it is important to do this review

Having a baby with a serious genetic disease can be a great source of distress for many families. Through PGT‐M, couples are empowered to select embryos with a high likelihood of developing into a healthy baby, by deselecting embryos afflicted with genetic disease variants (Demko 2010; Haude 2017). PGT‐M can help couples avoid later difficult decisions, including whether to terminate or continue with an affected pregnancy (Haude 2017; Klitzman 2018). However, regarding the biopsy procedure itself, concerns have been raised that the removal of cells in both day 3 and day 5 biopsy may result in a detrimental effect on the embryo by arresting its development. Firstly, there is the possibility that biopsy may negatively impact on pregnancy rates and perinatal outcomes. Secondly, there is a greater risk of amplification failure following day 3 biopsy, since a single cell with amplified DNA is examined, leading to higher false positive and false negative error rates (Cimadomo 2016; Wells 2000). As a result, euploid or non‐affected embryos are sometimes not transferred.

Not every embryo would reach the blastocyst stage of development; waiting for day 5 biopsy may result in no PGT‐M test or no transfer (Dahdouh 2015; Papanikolaou 2008). Following day 5 biopsy, embryos typically need to be cryopreserved and then thawed (Demko 2010), which precludes the transfer of a fresh embryo (De Rycke 2017). Furthermore, not all embryos can survive the freezing and thawing process (Taylor 2014). The two different biopsies of PGT‐M (day 3 biopsy versus day 5 biopsy) might have quite different likelihoods of a successful outcome. The removal of multiple cells by day 5 biopsy, rather than a single cell by day 3 biopsy, could potentially lead to improved sensitivity of genetic diagnosis and less biomass depletion of embryos, and therefore better treatment outcomes (de Boer 2004).

Along with embryo biopsy, other procedures such as IVF or ICSI, and transfer of fresh or frozen embryos can influence further development, implantation, pregnancy, live birth and perinatal outcomes. Cohen and colleagues suggested the removal of blastomeres at day 3 biopsy results in loss of cells during an embryo thawing process, and likely reduces the potential of the embryo development (Cohen 2007). In contrast, Magli and colleagues reported that for the purpose of PGT‐M, removing blastomeres on day 3 of embryo development does not affect the ongoing viability of an embryo (Magli 2004). It is important to eliminate the possibility of affecting the outcome through the biopsy stage (Demko 2010; Munne 2017; Wells 2000). However, there is a lack of summarised evidence from randomised controlled trials that evaluates the difference in treatment outcomes between day 3 and day 5 biopsy. This review compares the rates of embryo transfer, pregnancy and live birth between day 3 and day 5 biopsy for PGT‐M.

Objectives

To assess the benefits and harms of day 5 embryo biopsy, in comparison to day 3 biopsy, in PGT‐M in women undergoing in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) cycles.

Methods

Criteria for considering studies for this review

Types of studies

Published and unpublished randomised controlled trials (RCTs) were eligible for inclusion, irrespective of publication status, language or location. We excluded pseudo‐RCTs from the review. Cross‐over trials were eligible, but only data from the first phase, as the cross‐over is not a valid design in this context.

Types of participants

We included women/couples undergoing embryo biopsy following assisted reproductive technology (ART).

We excluded women/couples undergoing oocytes or zygotes biopsy (or both), mixed polar body and embryo biopsy, and embryo biopsy for PGT‐A or PGT‐SR.

Types of interventions

We compared day 5 embryo biopsy versus day 3 embryo biopsy in preimplantation genetic testing for monogenic/single gene defects (PGT‐M).

Types of outcome measures

We calculated the primary and secondary outcomes per woman randomised. We have preferably used the definitions provided in the core outcomes set (Duffy 2020), but studies reporting relevant outcomes with a different definition (i.e. live birth after 22 weeks) were still eligible.

Primary outcomes

1. Live birth, defined as the delivery of a foetus after 20 completed weeks of gestational age; which, after separation, shows signs of life such as a heart beat, umbilical cord pulsation, or definite movement of voluntary muscles.

2. Miscarriage, defined as the spontaneous loss of a clinical intra‐uterine pregnancy prior to 20 completed weeks of gestational age.

Secondary outcomes

1. Viable intrauterine pregnancy confirmed by ultrasound: at least one foetus with a discernable heartbeat on ultrasound.

2. Ectopic pregnancy: a pregnancy outside the uterine cavity, confirmed by ultrasound, surgical visualisation, or histopathology.

3. Stillbirth: we planned to subgroup these events according to the type of adverse event reported. Stillbirth is defined as the death of a foetus prior to the complete expulsion or extraction from its mother after 20 completed weeks of gestational age. The death is determined by the fact that, after such separation, the foetus does not breathe or show any other evidence of life, such as heartbeat, umbilical cord pulsation, or definite movement of voluntary muscles. It includes deaths occurring during labour.

4. Termination of pregnancy: intentional loss of an intrauterine pregnancy.

5. Gestational age at birth: calculated by the best obstetric estimate. 

6. Birthweight: collected within 24 hours of birth and assessed using a calibrated electronic scale with 10‐g resolution.

7. Neonatal mortality: death of a live born baby within 28 days of birth.

8. Major congenital anomaly: structural or functional disorders that occur during intrauterine life and can be identified prenatally, at birth or later in life.

Search methods for identification of studies

We searched for all published and unpublished RCTs of embryo biopsy for PGT‐M, without language restrictions and in consultation with the Cochrane Gynaecology and Fertility Group (CGF) Information Specialist.

Electronic searches

We searched the following electronic databases, trial registers and websites, from their inception to 14 December 2021:

1. The CGF Specialised Register, searched 14 December 2021, ProCite platform (Appendix 1);

2. CENTRAL (now including output from two trial registers and CINAHL) via The Cochrane Central Register of Studies Online (CRSO), searched 14 December 2021, web platform (Appendix 2);

3. MEDLINE, OVID platform, searched from 1946 to 14 December 2021 (Appendix 3);

4. Embase, OVID platform, searched from 1980 to 14 December 2021 (Appendix 4);

5. PsycINFO, OVID platform, searched from 1806 to 14 December 2021 (Appendix 5).

The MEDLINE search was combined with the Cochrane highly sensitive search strategy for identifying randomised trials, which appears in Chapter 4 of the Cochrane Handbook of Systematic Reviews of Interventions (Version 6.2 chapter 4, 4.4.7; 4.S1).

Other electronic sources of trials that were searched included the following:

1. LILACS and other Spanish/Portuguese databases, via the Virtual Health Library Regional Portal (VHL) (bvsalud.org/en/), searched 14 December 2021, web platform;

2. Google Scholar (for recent trials not yet indexed in the major databases) (scholar.google.com), searched 14 December 2021, web platform;

3. Epistemonikos database (www.epistemonikos.org/), a multilingual database of health evidence, searched 14 December 2021, web platform.

4. ClinicalTrials.govt (clinicaltrials.gov/); trial register of ongoing clinical trials, searched 24 March 2022, web platform;

5. International Clinical Trials Registry Platform (ICTRP) (www.who.int/clinical-trials-registry-platform), trial register of ongoing clinical trials, searched 24 March 2022, web platform.

Searching other resources

We searched the following conference abstracts:

1. European Society of Human Reproduction and Embryology (ESHRE) Annual Meeting (2021), Abstracts of the Scientific Oral and Poster Sessions, Program Supplement;

2. American Society for Reproductive Medicine (ASRM) in Fertility and Sterility Annual Meeting (2021), Abstracts of Scientific Oral and Poster Sessions, Program Supplement.

We handsearched reference lists of relevant trials and systematic reviews retrieved by the search and contacted experts in the field, to obtain any additional trials.

Data collection and analysis

Selection of studies

We used Covidence software for selection of studies (Covidence). Two review authors (TV and MG) conducted an initial screen of titles and abstracts retrieved by the search, and then retrieved the full texts of all potentially eligible studies. The same two authors (TV and MG) independently examined these full‐text articles for compliance with the inclusion criteria, and selected studies eligible for inclusion in the review. We recorded the reason(s) for excluding any study following a review of the full text. Disagreements regarding study eligibility were resolved by discussion or by a third author (LP). The selection process was documented in a PRISMA flow chart.

We constructed 'Characteristics of included studies' tables for those trials considered suitable for inclusion (Characteristics of included studies). The 'Characteristics of excluded studies' table lists the excluded studies with reasons for exclusion (Characteristics of excluded studies). 

Data extraction and management

Independently, two review authors (TV and MG) extracted data from the eligible studies including study characteristics and outcome data. TV also contacted the study authors for further data on the methods and results, where required. Any disagreements were resolved by discussion or by a third author (LP). Where studies had multiple publications, we collated multiple reports of the same under a single study ID with multiple references.

Assessment of risk of bias in included studies

Independently, two review authors (TV and MG) used the Cochrane Risk of Bias 1.0 tool to assess the risk of bias in each study, for the following domains (Higgins 2017): selection bias (random sequence generation and allocation concealment); performance bias (blinding of participants and personnel); detection bias (blinding of outcome assessors); attrition bias (incomplete outcome data); reporting bias (selective reporting); and other bias. We resolved disagreements by discussion, or by consultation with a third review author (LP). We described all judgements fully and presented the conclusions in the risk of bias table, and planned to incorporate them into the interpretation of the review findings by means of sensitivity analysis.

Measures of treatment effect

We reported odds ratios (ORs) with 95% confidence intervals (CIs), using the numbers of events in the day 5 biopsy and day 3 biopsy groups. 

For dichotomous outcomes, we used the numbers of events in the two groups (day 5 versus day 3 embryo biopsy) to calculate the Mantel‐Haenszel odds ratio. For continuous outcomes, we planned to calculate the mean difference (MD) between the two groups, provided the outcomes were measured in the same way across different trials.  We planned to reverse the direction of effect of individual studies, if required, to ensure consistency across trials. For all outcomes, we presented the 95% confidence intervals.

Unit of analysis issues

The primary analysis was per randomised woman.  If outcomes were reported in postrandomisation subgroups, such as per transfer cycle and per live birth, we planned to briefly summarise them in an additional table and not meta‐analyse them. However, we analysed 'per initiated cycle' data when the trial provided data for only one cycle per woman. We counted multiple live births (twins or triplets) as one live‐birth event. We planned to include only first‐phase data from cross‐over trials.

Dealing with missing data

We contacted the study authors to obtain missing data in order to perform analyses on an intention‐to‐treat basis. In the case of unobtainable data on live birth, we planned to conduct imputation using ongoing pregnancy data, where available. We assumed that live births had not occurred in women without a reported outcome. For secondary outcomes, we analysed only the available data.

We planned to conduct sensitivity analyses for the primary outcomes, with and without imputation.

Assessment of heterogeneity

We planned to consider whether the clinical and methodological characteristics of the included studies were sufficiently similar for meta‐analysis to provide a clinically meaningful summary. We also planned to assess statistical heterogeneity using the I² statistic and interpret a value greater than 50% as indicative of substantial heterogeneity (Higgins 2021).

Assessment of reporting biases

In view of the difficulty of detecting and correcting for publication bias and other reporting biases, we aimed to minimise their potential impact by ensuring we performed a comprehensive search for eligible studies, and by being alert for duplication of data. If there had been 10 or more studies in an analysis, we planned to use a funnel plot to explore the possibility of small‐study effects (the tendency for estimates of the intervention effect to be more beneficial in smaller studies) (Higgins 2021).

Data synthesis

We planned to restrict primary analyses of all review outcomes to studies at low risk of bias. 

We performed analysis using RevMan Web software (RevMan Web 2022). For binary (or dichotomous) outcomes, we expressed results for each study as an OR with 95% CI, and planned to combine them for meta‐analyses using a fixed‐effect model (Higgins 2021). For continuous outcome data, we planned to combine results for meta‐analyses using the mean differences (MDs) with 95% CIs. An increase in the odds of a particular outcome, which may be beneficial (e.g. live birth) or detrimental (e.g. adverse effects), are displayed graphically in the meta‐analysis to the right of the centre‐line, and a decrease in the odds of an outcome to the left of the centre‐line.

Subgroup analysis and investigation of heterogeneity

Where data were available, we planned to conduct subgroup analyses for the primary effectiveness outcome (live births) to investigate the efficacy of day 5 biopsy, depending on the following factors.

1. Transfer of fresh versus transfer of frozen‐thawed embryo

2. Fertilisation procedure (IVF versus ICSI)

3. Time at randomisation (before oocyte pick‐up (OPU) versus after OPU and before biopsy)

4. Ovarian stimulation protocol (agonist or antagonist)

In addition to visual inspection of the subgroup differences, we planned to use a significance test to determine the differences in effect estimates between two or more subgroups when the subgroups are independent (Higgins 2021). We planned to take any statistical heterogeneity into account when interpreting the results, especially if there was any variation in the direction of effect.

Sensitivity analysis

We planned to perform sensitivity analyses to examine the stability and robustness of the results for the primary outcomes (live birth and miscarriage) regarding the eligibility and analysis. These analyses would have included consideration of whether the review conclusions would have differed when accounting for the following factors.

1. Risk of bias (we planned to repeat the analysis including all studies, regardless of risk of bias)

2. Random effects (we planned to repeat the analysis using a random‐effects model rather than a fixed‐effect model)

3. Imputation of outcomes (we planned to repeat the analysis restricting it to studies where imputation strategies had not been implemented)

4. Relative risk (we planned to repeat the analysis using relative risk rather than odds ratio)

Summary of findings and assessment of the certainty of the evidence

We prepared a summary of findings table according to Cochrane methods, using GRADEpro GDT (GRADEpro GDT). This table evaluated the overall quality of the body of evidence for the main review outcomes (live birth, miscarriage, viable intrauterine pregnancy, ectopic pregnancy, stillbirth and termination of pregnancy), using GRADE criteria. Two independent review authors were involved in the GRADE assessment, which is based on: study limitations (risk of bias), consistency of effect, imprecision, indirectness, and publication bias (Schünemann 2011). We justified, documented, and incorporated into the reporting of results our judgements about evidence quality (high, moderate, low or very low) for each outcome. At least two review authors (TV and MG) made the GRADE judgements, working independently, and resolved disagreements by discussion. The main comparison in the summary of findings table compared the use of day 5 biopsy versus day 3 embryo biopsy.

Results

Description of studies

Results of the search

The search retrieved 1139 articles, of which we retrieved 28 articles in full text. Only one study met our inclusion criteria, and we excluded 27 articles. We did not identify any ongoing studies. See study tables for further details: Characteristics of included studies and Characteristics of excluded studies. Details of the screening and selection process are shown in the PRISMA study flow diagram (Figure 1).

1.

Figure 1. Study flow diagram.

Included studies

Study design and setting

We included only one RCT in this review. This was a study conducted at the Centre for Human Reproduction, Genesis Hospital, Athens, and the Laboratory of Medical Genetics, Athens University, Greece. 

Participants

The study included 20 couples where both the female and male partners were beta‐thalassaemia carriers.  

Interventions

The study compared day 3 with day 5 embryo biopsy and transfer for preimplantation genetic diagnosis of beta‐thalassaemia.

Outcomes

The study reported live birth, miscarriage, viable intrauterine pregnancy, ectopic pregnancy, termination of pregnancy, and beta‐thalassaemia status. 

Excluded studies

We excluded 27 full‐text articles from the review, for the following reasons:

• 26/27 did not compare day 3 versus day 5 embryo biopsy for PGT‐M

• 1/27 was not an RCT

Risk of bias in included studies

Allocation

Sequence generation

We rated the study at low risk of selection bias related to sequence generation, as they used random number tables for block randomisation (Figure 2; Figure 3). 

2.

Risk of bias summary: review authors' judgements about each risk of bias item for each included study.

3.

Risk of bias graph: review authors' judgements about each risk of bias item presented as percentages across all included studies.

Allocation concealment

We rated the study at low risk of bias related to allocation concealment, as they used sequentially numbered, sealed envelopes (Figure 2; Figure 3).

Blinding

Blinding of participants and personnel (performance)

The participants in the study were initially blinded but were then informed of their allocation on the day of embryo transfer. We did not consider that blinding of participants was likely to influence findings for any of the review outcomes. Personnel were not blinded either, and we thought that this could have influenced how procedures were performed, and therefore the outcomes. We therefore rated the study at high risk of performance bias (Figure 2; Figure 3).

Blinding of outcome assessors (detection)

We did not consider blinding of outcome assessors likely to influence the primary review outcomes (live birth and miscarriage). Blinding might influence outcomes for other observer‐measured outcomes, such as birthweight, but the study did not report relevant data for this outcome. We therefore rated the study at low risk of detection bias (Figure 2; Figure 3).

Incomplete outcome data

We rated the study at low risk of attrition bias because they analysed all of the women randomised (Figure 2; Figure 3).

Selective reporting

We rated this study as at unclear risk of reporting bias because they did not prespecify their outcomes (Figure 2; Figure 3). 

Other potential sources of bias

The study acknowledged Hamilton Thorne Biosciences Inc. Beverly, MA, USA for donating the ZILOS‐tk laser optical system, so we deemed the risk of bias to be unclear (Figure 2; Figure 3).

Effects of interventions

See: Table 1

Day 5 compared to day 3 embryo biopsy for PGT‐M

We identified only one study (Kokkali 2007), which reported live birth, miscarriage, viable intrauterine pregnancy, ectopic pregnancy, stillbirth, and termination of pregnancy data.

Primary outcomes

Live birth

There were five events reported in the 10 women randomised to the day 5 arm, and four events in the 10 women randomised to the day 3 control arm. We are uncertain whether there is any difference in live births between the groups (OR 1.50, 95% CI 0.26 to 8.82, 1 RCT, 20 women; very low‐certainty evidence; Analysis 1.1; Figure 4). 

1.1. Analysis.

Comparison 1: Day 5 versus day 3 embryo biopsy, Outcome 1: Live birth

4.

Forest plot of comparison: 1 Day 5 versus day 3 embryo biopsy, outcome: 1.1 Live birth.

Miscarriage

There was one event reported in the 10 women randomised to the day 5 arm and one event in the 10 women randomised to the day 3 control arm (one foetus was lost at nine weeks in one of the twin pregnancies). We are uncertain whether there is any difference in miscarriages between the groups (OR 1.00, 95 % CI 0.05 to 18.57, 1 RCT, 20 women; very low‐certainty evidence; Analysis 1.2; Figure 5).

1.2. Analysis.

Comparison 1: Day 5 versus day 3 embryo biopsy, Outcome 2: Miscarriage

5.

Forest plot of comparison: 1 Day 5 versus day 3 embryo biopsy, outcome: 1.2 Miscarriage.

Secondary outcomes

Viable intrauterine pregnancy

There were six events reported in the 10 women randomised to the day 5 arm and four events in the 10 women randomised to the day 3 control arm. We are uncertain whether there is any difference in viable intrauterine pregnancies between the groups (OR 2.25, 95% CI 0.38 to 13.47, 1 RCT, 20 women; very low‐certainty evidence; Analysis 1.3).

1.3. Analysis.

Comparison 1: Day 5 versus day 3 embryo biopsy, Outcome 3: Viable intrauterine pregnancy

Ectopic pregnancy

There were no events reported in the 10 women randomised to the day 5 arm and two events in the 10 women randomised to the day 3 control arm. We are uncertain whether there is any difference in ectopic pregnancies between the groups (OR 0.16, 95% CI 0.01 to 3.85, 1 RCT, 20 women; very low‐certainty evidence; Analysis 1.4).

1.4. Analysis.

Comparison 1: Day 5 versus day 3 embryo biopsy, Outcome 4: Ectopic pregnancy

Stillbirth

There were no events reported in either of the two study arms (OR not estimable, 1 RCT, 20 women; very low‐certainty evidence; Analysis 1.5).

1.5. Analysis.

Comparison 1: Day 5 versus day 3 embryo biopsy, Outcome 5: Stillbirth

Termination of pregnancy

There was one event in the 10 women randomised to the day 5 arm (one triplet pregnancy was selectively reduced to twins) and no events in the day 3 control arm. We are uncertain whether there is any difference in termination of pregnancy between the groups (OR 3.32, 95 % CI 0.12 to 91.60, 1 RCT, 20 women; very low‐certainty evidence; Analysis 1.6).

1.6. Analysis.

Comparison 1: Day 5 versus day 3 embryo biopsy, Outcome 6: Termination of pregnancy

Gestational age at birth

Kokkali 2007 reported mean gestational age at birth for all full‐term deliveries; however, these data were not usable as the calculation excluded gestational age at birth for preterm deliveries. 

Birthweight

The included study did not report birthweight data.

Neonatal mortality

The included study did not report neonatal mortality data.

Major congenital anomaly

The included study did not report major congenital anomaly data.

Sensitivity analyses

We could not perform sensitivity analysis on risk of bias and imputation strategies due to lack of data.

The sensitivity analyses using a random‐effects model and relative risk did not lead to change in results (data not shown). 

Discussion

Summary of main results

This review compared the clinical outcomes for day 5 (blastocyst stage) versus day 3 (cleavage stage) embryo biopsy for PGT‐M. We found one RCT that compared day 5 versus day 3 biopsy for preimplantation genetic diagnosis of beta‐thalassaemia. The aim of this comparison was to assess the potential advantages of performing the embryo biopsy for PGT‐M at day 5 to increase live births and prevent miscarriages.

All findings for this comparison were uncertain because of the very low‐certainty evidence, due to limited studies and the high risk of bias and small sample size of the only included study. In summary, it is unclear if day 5 biopsy has an effect on the primary outcomes of live births or miscarriages, compared to day 3 biopsy. There is also insufficient evidence of a difference in secondary outcomes, including viable intrauterine pregnancies, ectopic pregnancies, stillbirths, and termination of pregnancy. No data were available for gestational age at birth, birthweight, neonatal mortality and major congenital anomaly.

Overall completeness and applicability of evidence

The comparison of day 5 versus day 3 embryo biopsy for PGT‐M included one study with 20 women (Kokkali 2007). The average age of women randomised to day 5 and day 3 embryo biopsy was 35 years and 36.8 years, respectively. It was a single centre study conducted at a private fertility clinic in Greece. The indication for PGT‐M in this study was to prevent a pregnancy affected with homozygous beta‐thalassaemia in parents who are known carriers of this disease. The older mean age of women included, the fact that it was conducted at a single centre, and the specific indication for PGT‐M, mean that the results of the study may not be applicable to younger women, to other settings, or to other monogenic defects. 

It is important to mention that day 3 embryo biopsy is the most widely used biopsy stage for PGT‐M at present (van Montfoort 2021). 

No studies reported data on gestational age at birth, birthweight, neonatal mortality, or major congenital abnormality. Although the included study reported gestational age at birth for all full‐term deliveries, these data were not usable in the analysis because they excluded gestational age at birth for preterm deliveries (Kokkali 2007). 

Quality of the evidence

Using the GRADE approach, we found the evidence for all outcomes comparing day 5 and day 3 embryo biopsy for PGT‐M to be of very low certainty due to high risk of bias and imprecision. We downgraded the evidence by one level due to high risk of performance bias and two levels due to the results being based on only a single study with a small sample size, wide confidence intervals, and the confidence intervals crossing the line of no effect. It was not possible to assess for inconsistency across studies or the risk of publication bias given that there was only one included study.

With regard to risk of bias assessment, we graded the study at low risk of selection and detection bias but high risk of performance bias. We were not confident that the lack of blinding of study personnel would not influence the outcomes. The risk of reporting bias was unclear because we could not find a published protocol and the authors did not prespecify their outcomes in the methods section. 

Potential biases in the review process

The original protocol for this review was published in 2018 by different authors. Before commencing the review, we updated the protocol and changed some of the outcomes to be more in line with the Cochrane core outcomes for infertility trials (CGF 2022). The protocol was updated before we started screening titles and abstracts in order to reduce the risk of introducing bias. 

We used a very broad and extensive search strategy, which included searching electronic databases, trial registers, conference proceedings, and handsearching reference lists of systematic reviews with the aim of identifying all studies for inclusion in this review. We also contacted the authors of the included study to gain as much information as possible about the study methods and information to assist with our risk of bias assessment. The authors were forthcoming with providing further information; however, they did not send us their raw data. 

Agreements and disagreements with other studies or reviews

We were unable to find any similar published reviews addressing the same or similar objectives.

McArthur 2008 performed a non‐randomised sequential study for people undergoing PGT‐M. They compared viable pregnancy rates, miscarriages, and live births for biopsies taken on day 3 versus biopsies taken on day 5 or 6. Their results aligned with our results in that there was insufficient evidence as to whether day 5 or 6 biopsy improves the mentioned outcomes.

The ESHRE acknowledges that day 5 has overtaken day 3 biopsy as the most widely used embryo biopsy technique as it provides more cells, is more likely to give a conclusive diagnosis, and is less sensitive to potential damage (ESHRE PGT Consortium 2020). However, the ESHRE Consortium data from 2016 to 2017 reported that day 5 biopsy is still less common than day 3 biopsy in PGT‐M, in comparison to other modalities (van Montfoort 2021). 

Authors' conclusions

Implications for practice.

There is insufficient evidence of a difference in live births, miscarriages, viable intrauterine pregnancies, ectopic pregnancies, stillbirths or termination of pregnancies between day 5 and day 3 embryo biopsy for preimplantation genetic testing (PGT) for monogenic/single gene defects (PGT‐M). Data were lacking on gestational age at birth, birthweight, neonatal mortality and major congenital anomaly. The results should be interpreted with caution, as the evidence was of very low certainty due to limited studies, high risk of bias, and low precision.

There are other potential considerations when deciding on timing of embryo biopsy for PGT. Day 5 biopsy may confer a diagnostic advantage over day 3 biopsy because it involves sampling a higher number of cells, and therefore DNA, for analysis (ESHRE PGT Consortium 2020). Day 5 biopsy may also be more cost‐effective since only embryos which have been able to develop to blastocyst stage are biopsied. Furthermore, day 5 biopsy allows PGT to be concurrently performed for other indications such as PGT for aneuploidies (PGT‐A) or for chromosomal structural rearrangements (PGT‐SR). A potential downside of day 5 biopsy is that cryopreservation following biopsy is mandatory. On the other hand, day 3 biopsy can be used in fresh embryo transfers or cryopreserved. In day 3 biopsy it is also possible to extend the embryo culture to blastocyst stage and rebiopsy if the initial genetic result was inconclusive (ESHRE PGT Consortium 2020).

Implications for research.

This review has highlighted the paucity of randomised controlled trials (RCTs) evaluating day 5 versus day 3 embryo biopsy for PGT‐M. Furthermore, there were no RCTs that evaluated gestational age at birth, birthweight, neonatal mortality, and major congenital anomaly outcomes between day 5 and day 3 embryo biopsy. Given the significance of these outcomes, new developments should be properly investigated before their routine clinical application. 

Future studies should ideally randomise women, rather than embryos, and use the correct unit of analysis (e.g. per woman). They should also include all relevant fertility outcomes, including live births and miscarriages. 

History

Protocol first published: Issue 12, 2018

Date	Event	Description
13 December 2021	New search has been performed	New search performed
8 December 2021	New search has been performed	Four new authors to revise the protocol published in 2018 and to do the entire review

Appendices

Appendix 1. Cochrane Gynaecology and Fertility specialised register search strategy

Searched 14 December 2021

PROCITE platform

Keywords CONTAINS "PGD" or "PGS" or "pre‐implantation genetic diagnosis" or "pre‐implantation genetic screening" or "preimplantation genetic analysis" or "preimplantation genetic diagnosis" or "preimplantation genetic screening" or "genetic analysis" or "genetic screening" or "genetic techniques" or "chromosomal abnormalities" or "chromosomes" or "aneuploidy" or "aneuploidy screening" or "comparative genomic hybridization" or "CGH" or "aCGH" or "array comparative genomic hybridization" or "genotyping" or "embryo aneuploidy" or "blastocyst biopsy" or "embryo biopsy" or "FISH" or Title CONTAINS "PGD "or "PGS" or "pre‐implantation genetic diagnosis" or "pre‐implantation genetic screening" or "preimplantation genetic analysis" or "preimplantation genetic diagnosis" or "preimplantation genetic screening" or "genetic analysis" or "genetic screening" or "chromosomes" or "aneuploidy" or "aneuploidy screening" or "comparative genomic hybridization" or "CGH" or "aCGH" or "array comparative genomic hybridization" or "genotyping" or "embryo biopsy" (283 records)

Appendix 2. CENTRAL via the Cochrane Register of Studies Online (CRSO) search strategy

Searched 14 December 2021

Web platform

#1 MESH DESCRIPTOR Chromosome Aberrations EXPLODE ALL TREES 552

#2 MESH DESCRIPTOR Aneuploidy EXPLODE ALL TREES 181

#3 MESH DESCRIPTOR Preimplantation Diagnosis EXPLODE ALL TREES 43

#4 (Preimplant* adj3 gene*):TI,AB,KY 271

#5 Preimplant* adj2 Diagnos*:TI,AB,KY 114

#6 aneuploid*:TI,AB,KY 489

#7 (PGS or PGD or PGT):TI,AB,KY 745

#8 (NGS or CGH* or aCGH):TI,AB,KY 727

#9 ((embryo* or blasto* or Trophectoderm or cleavage) adj3 biops*):TI,AB,KY 231

#10 chromosome*:TI,AB,KY 2463

#11 "generation sequenc*":TI,AB,KY 978

#12 MESH DESCRIPTOR In Situ Hybridization EXPLODE ALL TREES 298

#13 (In Situ Hybridization):TI,AB,KY 1089

#14 (In Situ Hybridisation):TI,AB,KY 111

#15 (Preimplant* adj2 screen*):TI,AB,KY 131

#16 (gene* adj2 screen*):TI,AB,KY 1074

#17 MESH DESCRIPTOR Comparative Genomic Hybridization EXPLODE ALL TREES 10

#18 (genom* adj2 hybridi?ation):TI,AB,KY 97

#19 (Preimplant* adj2 Diagnos*):TI,AB,KY 114

#20 #1 OR #2 OR #3 OR #4 OR #6 OR #7 OR #10 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 5262

#21 MESH DESCRIPTOR Blastocyst EXPLODE ALL TREES 182

#22 Blastocyst*:TI,AB,KY 1490

#23 (day 5 or day 6):TI,AB,KY 7844

#24 (day5 or day6):TI,AB,KY 84

#25 (day five or day six):TI,AB,KY 529

#26 #21 OR #22 OR #23 OR #24 OR #25 9524

#27 #20 AND #26 346

Appendix 3. MEDLINE search strategy

Searched from 1946 to 14 December 2021

OVID platform

1 exp chromosome aberrations/ or exp aneuploidy/ (157891)
2 exp Preimplantation Diagnosis/ (3502)
3 (Preimplant$ adj3 gene$).tw. (4214)
4 aneuploid$.tw. (21817)
5 ((embryo$ or blasto$ or Trophectoderm or cleavage) adj3 biops$).tw. (1559)
6 (PGS or PGD or PGT).tw. (13619)
7 chromosome$.tw. (294287)
8 exp in situ hybridization/ or exp in situ hybridization, fluorescence/ (95297)
9 In Situ Hybridi?ation.tw. (101714)
10 FISH.tw. (178702)
11 (Preimplant$ adj2 Diagnos$).tw. (2773)
12 (Preimplant$ adj2 screen$).tw. (540)
13 (gene$ adj2 screen$).tw. (27299)
14 exp Comparative Genomic Hybridization/ (6491)
15 (genom$ adj2 hybridi?ation).tw. (11272)
16 NGS.tw. (15170)
17 next generation sequenc*.tw. (41233)
18 CGH.tw. (6852)
19 aCGH.tw. (1842)
20 or/1‐19 (721522)
21 exp Blastocyst/ (28619)
22 (Blastocyst$ or blastomere$).tw. (27862)
23 (day 5 or day 6).tw. (37868)
24 (day5 or day6).tw. (46)
25 (day five or day six).tw. (2027)
26 or/21‐25 (82804)
27 randomized controlled trial.pt. (552223)
28 controlled clinical trial.pt. (94572)
29 randomized.ab. (543333)
30 randomised.ab. (108156)
31 placebo.tw. (229886)
32 clinical trials as topic.sh. (198253)
33 randomly.ab. (371579)
34 trial.ti. (252678)
35 (crossover or cross‐over or cross over).tw. (91505)
36 or/27‐35 (1485655)
37 exp animals/ not humans.sh. (4923906)
38 36 not 37 (1367643)
39 20 and 26 and 38 (231)

Appendix 4. Embase search strategy

Searched from 1946 to 14 December 2021

OVID platform

1 exp prenatal diagnosis/ (110295)
2 (prenatal adj3 diagnos$).tw. (33782)
3 exp chromosome aberration/ (187902)
4 (Preimplant$ adj3 gene$).tw. (6695)
5 ((embryo$ or blasto$ or Trophectoderm or cleavage) adj3 biops$).tw. (4035)
6 aneuploid$.tw. (29051)
7 (PGS or PGD or PGT).tw. (18962)
8 chromosome$.tw. (321136)
9 exp in situ hybridization/ or exp hybridization/ (341423)
10 In Situ Hybridi?ation.tw. (119424)
11 (Preimplant$ adj2 Diagnos$).tw. (4121)
12 (Preimplant$ adj2 screen$).tw. (1485)
13 (gene$ adj2 screen$).tw. (37088)
14 exp comparative genomic hybridization/ (19259)
15 next generation sequenc$*.tw. (67056)
16 (genom$ adj2 hybridi?ation).tw. (14778)
17 (CGH$ or NGS).tw. (46431)
18 aCGH.tw. (3966)
19 or/1‐18 (936602)
20 exp BLASTOCYST/ (29343)
21 (Blastocyst$ or blastomere$).tw. (36823)
22 (day 5 or day 6).tw. (58049)
23 (day5 or day6).tw. (458)
24 (day five or day six).tw. (3267)
25 or/20‐24 (99885)
26 19 and 25 (10487)
27 Clinical Trial/ (1010302)
28 Randomized Controlled Trial/ (682338)
29 exp randomization/ (92418)
30 Single Blind Procedure/ (44503)
31 Double Blind Procedure/ (187338)
32 Crossover Procedure/ (68751)
33 Placebo/ (360869)
34 Randomi?ed controlled trial$.tw. (271815)
35 Rct.tw. (44456)
36 random allocation.tw. (2239)
37 randomly.tw. (488409)
38 randomly allocated.tw. (39630)
39 allocated randomly.tw. (2711)
40 (allocated adj2 random).tw. (833)
41 Single blind$.tw. (27642)
42 Double blind$.tw. (218465)
43 ((treble or triple) adj blind$).tw. (1437)
44 placebo$.tw. (329252)
45 prospective study/ (729704)
46 or/27‐45 (2705391)
47 case study/ (82460)
48 case report.tw. (458556)
49 abstract report/ or letter/ (1171325)
50 or/47‐49 (1699829)
51 46 not 50 (2646128)
52 (exp animal/ or animal.hw. or nonhuman/) not (exp human/ or human cell/ or (human or humans).ti.) (6307291)
53 51 not 52 (2466509)
54 26 and 53 (840)

Appendix 5. PsycINFO search strategy

Searched from 1806 to 14 December 2021

OVID platform

1 exp Prenatal Diagnosis/ (743)
2 (Preimplant$ adj3 gene$).tw. (130)
3 exp Chromosome Disorders/ (10632)
4 aneuploid$.tw. (268)
5 (PGS or PGD or PGT).tw. (872)
6 chromosome$.tw. (8169)
7 Preimplant$ Diagnos$.tw. (9)
8 (genom$ adj2 hybridi?ation).tw. (238)
9 (Preimplant$ adj2 screen$).tw. (4)
10 next generation sequenc*.tw. (758)
11 NGS.tw. (272)
12 In Situ Hybridi?ation.tw. (3628)
13 (CGH or aCGH).tw. (189)
14 ((embryo$ or blasto$ or Trophectoderm or cleavage) adj3 biops$).tw. (7)
15 (gene$ adj2 screen$).tw. (1772)
16 or/1‐15 (24797)
17 exp Embryo/ (1821)
18 exp Infertility/ or exp Reproductive Technology/ (3828)
19 Blastocyst*.tw. (91)
20 (day 5 or day 6).tw. (2064)
21 (day5 or day6).tw. (2)
22 (day five or day six).tw. (205)
23 or/17‐22 (7841)
24 16 and 23 (225)
25 random.tw. (63532)
26 control.tw. (475418)
27 double‐blind.tw. (23977)
28 clinical trials/ (12000)
29 placebo/ (6135)
30 exp Treatment/ (1121927)
31 or/25‐30 (1544423)
32 24 and 31 (74)

Data and analyses

Comparison 1. Day 5 versus day 3 embryo biopsy.

Outcome or subgroup title	No. of studies	No. of participants	Statistical method	Effect size
1.1 Live birth	1		Odds Ratio (M‐H, Fixed, 95% CI)	Totals not selected
1.2 Miscarriage	1		Odds Ratio (M‐H, Fixed, 95% CI)	Totals not selected
1.3 Viable intrauterine pregnancy	1		Odds Ratio (M‐H, Fixed, 95% CI)	Totals not selected
1.4 Ectopic pregnancy	1		Odds Ratio (M‐H, Fixed, 95% CI)	Totals not selected
1.5 Stillbirth	1	20	Odds Ratio (M‐H, Fixed, 95% CI)	Not estimable
1.6 Termination of pregnancy	1		Odds Ratio (M‐H, Fixed, 95% CI)	Totals not selected

Characteristics of studies

Characteristics of included studies [ordered by study ID]

Kokkali 2007.

Study characteristics
Methods	Study design: single centre, single blinded, randomised trial Duration and location of trial: between January 2004 and May 2005, private infertility clinic, Athens, Greece Sample size calculation: a sample size with power calculation was not performed 20 women randomised
Participants	Inclusion criteria: both partners are beta‐thalassaemia carriers requesting PGT to avoid initiation of a pregnancy affected with homozygous beta‐thalassaemia Exclusion criteria: not specified
Interventions	Type of biopsy: cleavage (day 3) or blastocyst (day 5) stage biopsy Number and type of cells biopsied: one blastomere (day 3 biopsy) or four to five trophectoderm cells (day 5 biopsy) Embryo transfer: blastocyst (day 5‐6) stage transfer. Per woman, on average 2.1 day 5 embryos were transferred versus 3 day 3 embryos. Genetic analysis: real‐time PCR multiplexed with fluorescent microsatellite analysis
Outcomes	Cycles to embryo transfer HCG positive Ectopic pregnancy  Miscarriage Number of foetal sacs Implantation rate Viable pregnancies Premature delivery (25 weeks) Pregnancies to term Babies born Average week of full‐term delivery Reduction to twins Spontaneous abortion of one foetus
Notes	Treatment procedure: ICSI Cycles included: only the first cycle for each woman was included in the study Embryo transfer policy: not specified Funding: Hamilton Thorne Biosciences Inc. Beverly, MA, USA, donated the ZILOS‐tk laser optical system. No other funding reported. Note that some clarifications were obtained via personal communication with the lead author. No mention of clinical trial registration and ethical approval. We obtained additional information on allocation concealment, blinding, and results via personal communication.
Risk of bias
Bias	Authors' judgement	Support for judgement
Random sequence generation (selection bias)	Low risk	Quote: "randomisation in blocks with the use of random number tables"
Allocation concealment (selection bias)	Low risk	Personal correspondence: sequentially numbered and sealed envelopes
Blinding of participants and personnel (performance bias) All outcomes	High risk	Personal correspondence: participants were informed about their allocation on the day of embryo transfer; however, this was unlikely to influence the outcomes. Personnel were not blinded either, which could have influenced how procedures were performed and, therefore, the outcomes.
Blinding of outcome assessment (detection bias) All outcomes	Low risk	Personal correspondence: outcomes were measured by a midwife who was blinded. It is possible that the blinding could have been broken since the participants knew which intervention they were allocated by that stage. However, this is unlikely to have influenced outcome assessment.
Incomplete outcome data (attrition bias) All outcomes	Low risk	No missing outcome data
Selective reporting (reporting bias)	Unclear risk	Outcomes were not prespecified in the methods.
Other bias	Unclear risk	Quote: "Hamilton Thorne Biosciences Inc. Beverly, MA, USA, donated the ZILOS‐tk laser optical system".

HCG:human chorionic gonadotropin; ICSI: intracytoplasmic sperm injection; PCR: polymerase chain reaction; PGT: preimplantation genetic testing

Characteristics of excluded studies [ordered by study ID]

Study	Reason for exclusion
Alcaraz 2016	Inappropriate intervention
Balaban 2002	Inappropriate intervention
Chamayou 2015	Inappropriate intervention
Dahdouh 2015	Inappropriate study design (review)
Fakih 2015	Inappropriate intervention
Forman 2012	Inappropriate intervention
Gleicher 2012	Inappropriate study design (review)
Goossens 2009	Inappropriate intervention
Harper 2012	Inappropriate study design (review)
Insogna 2020	Inappropriate intervention
ISRCTN37972669	Inappropriate intervention
McArthur 2008	Inappropriate study design (not RCT)
NCT01332643	Inappropriate intervention
NCT01546350	Inappropriate intervention
NCT01571076	Inappropriate intervention
NCT01946945	Inappropriate intervention
NCT01950104	Inappropriate intervention
Reddy 2018	Inappropriate intervention
Scott Jr 2013	Inappropriate study design
Staessen 2004	Inappropriate intervention
Sui 2020	Inappropriate intervention
Ten 2011	Inappropriate intervention
Tisser 2017	Inappropriate intervention
Treff 2011	Inappropriate control
Verpoest 2017	Inappropriate intervention
Zech 2013	Inappropriate intervention
Zheng 2021	Inappropriate study design (review)

Differences between protocol and review

We implemented the core outcomes set for infertility research (Duffy 2020). 

We planned to restrict our main analysis to studies with low risk of bias and perform a sensitivity analysis with all studies. We added the sensitivity analyses 'random effects model' and 'relative risk', and we removed the sensitivity analysis 'publication type'. 

We added the following outcomes to the summary of findings table: ectopic pregnancy, stillbirth, and termination of pregnancy; and removed multiple births, number of transferable embryos and adverse events.

Contributions of authors

TV updated the protocol.

TV and MG were the two independent reviewers for title and abstract screening, and assessing full‐text reports.

LP resolved any disagreements.

TV and MG extracted data.

TV and MG independently assessed risk of bias and did the GRADE assessment.

RvE assisted with statistical analyses.

TV wrote the review.

All authors reviewed and approved the final draft of the review.

Sources of support

Internal sources

• The Cochrane Gynaecology and Fertlity editorial team, Other

External sources

• No sources of support provided

Declarations of interest

TV has no conflicts of interest to declare.

MG has no conflicts of interest to declare.

RvE has no conflicts of interest to declare.

LP has no conflicts of interest to declare.

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