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Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis
Abstract
STUDY QUESTION
Compared to the ‘single biopsy + single vitrification’ approach, do ‘double biopsy + double vitrification’ or ‘single biopsy + double vitrification’ arrangements compromise subsequent clinical outcomes following euploidy blastocyst transfer?
SUMMARY ANSWER
Both ‘double biopsy + double vitrification’ and ‘single biopsy + double vitrification’ led to reduced live birth/ongoing pregnancy rates and clinical pregnancy rates.
WHAT IS KNOWN ALREADY?
It is not uncommon to receive inconclusive results following blastocyst biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Often these blastocysts are warmed for re-test after a second biopsy, experiencing ‘double biopsy + double vitrification’. Furthermore, to achieve better workflow, IVF laboratories may choose to routinely vitrify all blastocysts and schedule biopsy at a preferred timing, involving ‘single biopsy + double vitrification’. However, in the current literature, there is a lack of systematic evaluation of both arrangements regarding their potential clinical risks in reference to the most common ‘single biopsy + single vitrification’ approach.
STUDY DESIGN, SIZE, DURATION
A systematic review and meta-analysis were performed, with the protocol registered in PROSPERO (CRD42023469143). A search in PUBMED, EMBASE, and the Cochrane Library for relevant studies was carried out on 30 August 2023, using the keywords ‘biopsy’ and ‘vitrification’ and associated variations respectively. Only studies involving frozen transfers of PGT-A tested euploid blastocysts were included, with those involving PGT-M or PGT-SR excluded.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Study groups included blastocysts having undergone ‘double biopsy + double vitrification’ or ‘single biopsy + double vitrification’, with a ‘single biopsy + single vitrification’ group used as control. The primary outcome was clinical pregnancy, while secondary outcomes included live birth/ongoing pregnancy, miscarriage, and post-warming survival rates. Random effects meta-analysis was performed with risk ratios (RR) and 95% CIs were used to present outcome comparisons.
MAIN RESULTS AND THE ROLE OF CHANCE
A total of 607 records were identified through the initial search and nine studies (six full articles and three abstracts) were eventually included. Compared to ‘single biopsy + single vitrification’, ‘double biopsy + double vitrification’ was associated with reduced clinical pregnancy rates (six studies, n = 18 754; RR = 0.80, 95% CI = 0.71–0.89; I2 = 0%) and live birth/ongoing pregnancy rates (seven studies, n = 20 964; RR = 0.72, 95% CI = 0.63–0.82; I2 = 0%). However, no significant changes were seen in miscarriage rates (seven studies, n = 22 332; RR = 1.40, 95% CI = 0.92–2.11; I2 = 53%) and post-warming survival rates (three studies, n = 13 562; RR = 1.00, 95% CI = 0.99–1.01; I2 = 0%) following ‘double biopsy + double vitrification’. Furthermore, ‘single biopsy + double vitrification’ was also linked with decreased clinical pregnancy rates (six studies, n = 13 284; RR = 0.84, 95% CI = 0.76–0.92; I2 = 39%) and live birth/ongoing pregnancy rates (seven studies, n = 16 800; RR = 0.79, 95% CI = 0.69–0.91; I2 = 70%), and increased miscarriage rates (five studies, n = 15 781; RR = 1.48, 95% CI = 1.31–1.67; I2 = 0%), but post-warming survival rates were not affected (three studies, n = 12 452; RR = 0.99, 95% CI = 0.97–1.01; I2 = 71%) by ‘single biopsy + double vitrification’.
LIMITATIONS, REASONS FOR CAUTION
All studies included in this meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies had accounted for potential confounding factors. Only one study reported neonatal outcomes.
WIDER IMPLICATIONS OF THE FINDINGS
Our data indicated adverse impacts of ‘double biopsy + double vitrification’ and ‘single biopsy + double vitrification’ on clinical outcomes following euploid blastocyst transfers. Patients should be carefully consulted about the risks when offered such approaches. The biopsy process should be carried out as carefully and competently as possible to minimize an inconclusive diagnosis.
STUDY FUNDING/COMPETING INTEREST(S)
R.W. is supported by a National Health and Medical Research Council Emerging Leadership Investigator Grant (2009767). There is no other external funding to report. All authors report no conflict of interest.
REGISTRATION NUMBER
CRD42023469143.
... The above-mentioned existing data on clinical outcomes with double-biopsied blastocysts have been meta-analyzed recently [23,86,87]. Based on three included studies, Cimadomo et al. [23] showed a similar LBR per euploid single embryo transfer (SET) and a similar MR per clinical pregnancy in the re-biopsied group (n = 86) compared to the single-biopsied group (n = 6896). ...
... Based on three included studies, Cimadomo et al. [23] showed a similar LBR per euploid single embryo transfer (SET) and a similar MR per clinical pregnancy in the re-biopsied group (n = 86) compared to the single-biopsied group (n = 6896). In contrast, enlarging the number of included studies, Bickendorf et al. [86] showed that 'double biopsy/double vitrification' (n = 385) resulted in reduced live birth rates (seven studies, RR = 0.72, 95% CI 0.63-0.82) compared to 'single biopsy/single vitrification' (n = 20,579). ...
... No significant changes were observed in miscarriage rates. To frame these findings, significant heterogeneity for multiple study outcomes needs to be acknowledged [86]. Potential confounding factors include inclusion criteria for biopsy (including poor-quality and/or delayed blastocysts or not), day of assisted hatching, protocols for both vitrification and biopsy (number of TE cells biopsied, pulling or flicking, overnight culture post-warming before second biopsy) and the skill level of biopsy operators. ...
Trophectoderm (TE) biopsy is at present the most widely used procedure for preimplantation genetic testing (PGT). At the blastocyst stage, more TE cells (five to seven) can be obtained for genetic analysis. While removing TE cells and not touching the inner cell mass (ICM), the procedure is less invasive. Due to a natural selection happening between day 3 and day 5, 6 or 7 of human embryo development, fewer embryos will have to be biopsied and tested. An additional benefit, especially in view of aneuploidy testing (PGT-A), is the lower level of mosaicism present at the blastocyst stage. The biopsy procedure involves two steps: laser-assisted zona pellucida (ZP) opening and the excision of five to eight TE cells from the blastocyst with or without additional laser energy. Different protocols have emerged over time with variations regarding the technique, the exact moment of ZP opening, and the method of cell removal. The ‘pulling’ method involves laser excision, whereas the ‘flicking’ method represents a mechanical approach with or without laser assistance. Embryo developmental speed reaching the full/expanded or hatching/hatched blastocyst stage dictates the timing of the procedure, mostly on day 5 post-insemination, and to a lesser extent on day 6 or even on day 7. The inclusion of lesser quality or delayed blastocysts may impact the quality of the TE sample as well as the clinical outcome. Intracytoplasmic sperm injection (ICSI) is still the preferred method of fertilization for PGT-M (monogenic disorders) and PGT-SR (structural rearrangements). However, conventional in vitro fertilization (IVF) seems feasible for PGT-A (aneuploidy testing). In the absence of a (conclusive) genetic result, the re-biopsy of cryopreserved blastocysts is possible, however, with reduced clinical outcomes. So far, neonatal outcome post-TE biopsy has so far been reassuringly documented.
... However, to date, there is a lack of robust evidence regarding the benefits of PGT-A on the remaining cryopreserved embryos from this patient population [9,10]. Additionally, the potential impact of biopsy and two rounds of vitrification on implantation of rewarmed euploid blastocysts should be carefully balanced against the necessity of obtaining PGT-A results [11]. ...
Research question
Does preimplantation genetic testing for aneuploidy (PGT-A) on cryopreserved unbiopsied blastocysts improve pregnancy outcomes for women with previous IVF-related pregnancy loss?
Methods
This retrospective observational study included women who underwent vitrified blastocyst warming procedures, with or without trophectoderm biopsy for PGT-A, between January 2016 and June 2023. Participants had experienced two or more clinical pregnancy losses, with at least one loss following in vitro fertilization (IVF). The primary outcome was the cumulative live birth/ongoing pregnancy rate, analyzed using generalized estimating equations (GEE) with confounding adjustments.
Results
The cohort included 146 women, comprising 72 who intended to pursue PGT-A on thawed blastocysts (274 blastocysts) and 74 who proceeded directly to frozen embryo transfer (FET) without prior PGT-A (107 blastocysts). Fourteen women in the PGT-A group had no euploid embryos available for transfer. Among these, two patients had no warmed blastocysts suitable for testing, and twelve had all aneuploid embryoid. The cumulative live birth/ongoing pregnancy rate was significantly lower in the PGT-A group compared to the non-PGT-A group (34.7% [25/72] vs. 52.7% [39/74], adjusted odds ratio [AOR] 0.51, 95% confidence interval [CI]: 0.26–0.99, P = 0.048). Secondary outcomes, including live birth and pregnancy loss rates after initial FET, were comparable between the two groups. Among tested blastocysts, 58 (82.9%) had at least one euploid embryo, resulting in a euploidy rate of 48.6% (125/257).
Conclusions
PGT-A on cryopreserved unbiopsied blastocysts reduces cumulative live birth/ongoing pregnancy rates and could not improve pregnancy outcomes following the initial FET cycle in women with a history of IVF pregnancy loss.
... Our findings are consistent with a two recent meta-analyses that highlights the detrimental effects of both DVDB and DVSB on clinical outcomes [67,68]. ...
This study evaluates the effects of double cryopreservation and re-biopsy on embryo viability and clinical outcomes. Studies of interest were selected from an initial cohort of 1027 potentially relevant records retrieved. PubMed was systematically searched for peer-reviewed original papers identified by keywords and medical subject heading terms. Moreover, we elaborated the evidence tables for double cryopreservation and re-biopsy separately. Data were systematically extracted, focusing on live birth, survival, clinical pregnancy, and miscarriage rates. For each study, we identified absolute numbers (numerator and denominator) related to clinical outcomes. Finally, for each outcome, we calculated the percentage change between the control and study groups. Among studies on double cryopreservation, 13 out of 22 reported no effect on clinical outcomes, suggesting contradictory results. Similarly, findings on re-biopsy were controversial, with seven out of 12 studies showing negative effects on survival and clinical outcomes, while five reported no impact. In our analysis of the evidence tables, we observed a reduction in live birth rates of 22.2% and 39.3% in blastocysts undergoing double vitrification and re-biopsy, respectively. These findings suggest that repeated micromanipulations can impair embryo competence. Therefore, double cryopreservation and re-biopsy should be limited in the selected cases without other options by consulting patients about the possible harmful effects.
Background
Preimplantation genetic testing (PGT) is widely used in assisted reproduction to assess the genetic status of embryos. However, increasing evidence suggests that the trophectoderm (TE) may not fully reflect the genetic status of the inner cell mass (ICM), raising controversy about the accuracy of TE biopsy. Research in recent years has focused on cell-free DNA (cfDNA) found in blastocoel fluid (BF) and spent culture medium (SCM), as these may contain genetic information from both the TE and ICM. Therefore, further research and validation are essential to determine the reliability and clinical applicability of these diagnostic methods in PGT.
Methods
Relevant studies published between January 2000 and August 2024 were identified through PubMed and Web of Science (WOS). Risk assessment and publication bias were evaluated using QUADAS-2 and Deek’s test. Diagnostic meta-analysis was performed using a bivariate model to combine sensitivity and specificity, with results visualized through forest plots and summary receiver operating characteristic (SROC) curves.
Results
Out of 6,407 initially screened records, 36 studies involving 4,230 embryos were included. TE biopsy was identified as the best method for diagnosing the genetic status of embryos (sensitivity: 0.839; specificity: 0.791, AUC: 0.878), while SCM had slightly lower accuracy (sensitivity: 0.874; specificity: 0.719, AUC: 0.869). The effectiveness of BF (AUC: 0.656) was significantly lower than that of TE biopsy and SCM. Despite this, TE biopsy has not yet achieved ideal diagnostic performance. However, TE biopsies demonstrate a high level of accuracy in diagnosing PGT-SR (AUC: 0.957). Additionally, multiple TE biopsies (AUC: 0.966) or TE biopsies combined with SCM (AUC: 0.927) can enhance the diagnostic efficiency of PGT.
Conclusion
The findings of this study suggest that TE biopsy has yet to achieve optimal diagnostic accuracy, which may result in a significant number of missed embryo diagnoses and misdiagnoses. Our results confirm that SCM has the potential to serve as a supplementary test. Employing multiple biopsies or combining TE with SCM may enhance diagnostic efficiency and yield optimal results.
Objectives
This systematic review and meta-analysis aimed to evaluate the impact of double vitrification/thawing (DVT) versus single vitrification/thawing (SVT) on key embryonic and neonatal outcomes.
Data extraction
Information sources included systematic search in PubMed, Scopus, and Cochrane databases up to September 7, 2024. Data from each qualifying study were extracted by two reviewers using a standardized electronic data gathering form.
Data Analysis
Mantel-Haenszel odds ratio (MHOR) and mean difference (MD) with 95% confidence intervals (CI) were calculated using both fixed and random-effects models. Subgroup analyses were based on biopsy status, number of biopsy rounds, extended culture between rounds of vitrification, and embryo transfer strategy.
Results
A total of 35 studies involving 46,749 embryo transfer cycles were included. After excluding studies that used slow freezing, 28 studies were included in the meta-analyses. The findings indicated that DVT is associated with significant reductions in cryosurvival rates (MHOR: 0.4; CI: 0.3 to 0.8; P < 0.01), biochemical pregnancy (MHOR: 0.7; CI: 0.6 to 0.8; P < 0.01), clinical pregnancy (MHOR: 0.7; CI: 0.5 to 0.8; P < 0.01), and live birth rates (MHOR: 0.6; CI: 0.5 to 0.7; P < 0.01). Additionally, there was a significant increase in the miscarriage rate (MHOR: 1.4; CI: 1.2 to 1.7; P < 0.01). No significant differences were found in neonatal outcomes.
Conclusion
Poor-quality evidence suggests that the transfer of double-vitrified embryos might be associated with significantly lower rates of cryosurvival, pregnancy, and live births; however, it does not appear to affect neonatal outcomes such as birth weight and gestational age at birth. Given the small sample size in some subgroups, the high risk of selection, confounding and missing data biases, and the high level of heterogeneity for some outcomes, these findings should be interpreted cautiously.
Graphical Abstract
BACKGROUND
To prevent the transfer of embryos affected by monogenic conditions and/or chromosomal defects, preimplantation genetic testing (PGT) requires trophectoderm biopsy and cryopreservation. In 2–6% of biopsies, the diagnosis may be inconclusive due to DNA amplification failure or low-quality results. In these cases, a round of re-warming, re-biopsy, and re-cryopreservation is required to obtain a genetic diagnosis. In other cases, when the IVF centre starts providing PGT and/or when the patients develop an indication because of multiple failures, miscarriages or the birth of an affected child after IVF, cryopreserved untested embryos may be warmed, biopsied, and then re-vitrified. However, it is still unclear whether multiple manipulations may reduce reproductive outcomes after PGT.
OBJECTIVE AND RATIONALE
This study aimed at conducting a systematic review to investigate the available evidence on the safety of double biopsy and/or double cryopreservation–warming and provide recommendations in this regard. We performed meta-analyses of the differences in the reproductive outcomes (live birth per embryo transfer [LBR per ET], clinical pregnancy rate per ET [CPR per ET], and miscarriage rate per clinical pregnancy [MR per CP]) in double cryopreservation and single biopsy (CBC) or double biopsy and double cryopreservation (BCBC) flows vs the control single biopsy and single cryopreservation (BC) flow. Cryo-survival rates before ET and gestational and perinatal outcomes were also reported.
SEARCH METHODS
PRISMA guidelines were followed to gather all available information from the literature (PubMed, Scopus, and Embase). We used Medical Subject Headings (MeSH) terms and a list of specific keywords relevant for the study question. We searched for original studies in humans, published in peer-reviewed journals in English up to April 2024. Four independent authors assessed the articles for inclusion. One included paper was retrieved from another source.
OUTCOMES
A total of 4219 records were identified, and 10 studies were included in the meta-analysis. Certainty of evidence level ranged from low to moderate. Both the CBC and BCBC groups showed reduced reproductive outcomes compared to the control (BC). Specifically, live birth rates per embryo transfer were lower in the CBC group (OR: 0.56, 95% CI: 0.38–0.81, I2 = 58%; six studies) and the BCBC group (OR: 0.51, 95% CI: 0.34–0.77, I2 = 24%; six studies). CPR per ET were also lower in the CBC group (OR: 0.68, 95% CI: 0.51–0.92, I2 = 57%; seven studies) and the BCBC group (OR: 0.60, 95% CI: 0.46–0.78, I2 = 0%; seven studies). Additionally, MR per CPs were higher in both the CBC group (OR: 1.68, 95% CI: 1.02–2.77, I2 = 50%; seven studies) and the BCBC group (OR: 2.08, 95% CI: 1.13–3.83, I2 = 28%; seven studies). Cryo-survival as well as gestational and perinatal outcomes were within the expected norms in the studies reporting them.
WIDER IMPLICATIONS
Improved genetic technologies, standardization of laboratory protocols, operators’ proficiency with biopsy and cryopreservation, and continuous monitoring of the performance are essential to minimize inconclusive diagnoses and the putative impact of additional embryo manipulations. Although poorer reproductive outcomes might result from double biopsy and/or double cryopreservations, these practices may still be worthwhile to avoid transferring affected/aneuploid blastocysts. Therefore, the risks must be weighed against the potential benefits for each specific couple.
REGISTRATION NUMBER
PROSPERO (ID: CRD42024503678)
Purpose
To investigate whether embryo rebiopsy increases the yield of in vitro fertilization (IVF) cycles.
Methods
Retrospective study including 18,028 blastocysts submitted for trophectoderm biopsy and preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2021 in a private IVF center. Out of the 517 embryos categorized as inconclusive, 400 survived intact to the warming procedure, re-expanded, and were suitable for rebiopsy. Of them, 71 rebiopsied blastocysts were transferred. Factors affecting the probability of obtaining an undiagnosed blastocyst and clinical outcomes from blastocysts biopsied once and twice were investigated.
Results
The overall diagnostic rate was 97.1%, with 517 blastocysts receiving inconclusive reports. Several blastocyst and laboratory features, such as the day of the biopsy, the stage of development, and the biopsy methodology, were related to the risk of obtaining an inconclusive diagnosis after PGT-A. A successful diagnosis was obtained in 384 of the rebiopsied blastocysts, 238 of which were chromosomally transferable. A total of 71 rebiopsied blastocysts were transferred, resulting in 32 clinical pregnancies [(clinical pregnancy rate (CPR)=45.1%], 16 miscarriages [(miscarriage rate (MR)=41%], and, until September 2020, 12 live births [(live birth rate (LBR)=23.1%]. A significantly lower LBR and higher MR were obtained after transferring rebiopsied blastocysts compared to those biopsied once.
Conclusion
Although an extra round of biopsy and vitrification may cause a detrimental effect on embryo viability, re-analyzing the test-failure blastocysts contributes to increasing the number of euploid blastocysts available for transfer and the LBR.
Objective
To identify whether the transfer of blastocysts that have been vitrified, thawed, biopsied, revitrified, and subsequently rethawed affects clinical outcome and neonatal outcome.
Methods
A retrospective study was conducted in a single assisted reproduction technology center from September 2016 to March 2021. Women undergoing single frozen euploid blastocysts transfer were stratified into two groups based on number of vitrification‐thawing cycles: single vitrification coupled with single biopsy (group A, n = 177) and double vitrification coupled with single biopsy (group B, n = 30). Pregnancy and perinatal outcomes of the two groups were compared.
Results
Clinical pregnancy rates were similar between the two groups. Group B was associated with a lower chance of live birth when compared with group A by different multivariable analysis models (model 1: odds ratio, 0.42 [95% confidence interval, 0.18–0.97], P = 0.041; model 2: odds ratio, 0.38 [95% confidence interval, 0.16–0.92], P = 0.033). No major obstetrical complication was reported in the two groups and only one malformation live birth was reported in group A.
Conclusion
The procedure of double vitrification‐warming cycles, coupled with single biopsy, increases pregnancy loss and ultimately diminishes live birth but does not affect perinatal outcome. Future studies with a larger sample size would help to validate the results.
Background:
Preimplantation genetic testing for aneuploidy (PGT-A) with Next Generation Sequencing technology is a low-cost and powerful technology for the analysis of embryo quality. However, PGT-A requires freezing of embryos, suggesting that previously cryopreserved embryos cannot be tested. Here, we test whether use of the vitrification technique permits the refreezing of embryos, thus permitting PGT-A testing of cryopreserved embryos.
Methods:
The results are a retrospective analysis of cases performed at Create fertility between 2016 and 2017. Results obtained after traditional PGT-A are compared with results after the thaw biopsy and refreeze (TBR) procedure. A total of 220 patients were treated with PGT-A and 54 patients with the TBR procedure.
Results:
Maternal ages were not significantly different between the groups. The proportion of PGT-A normal embryos was not significantly different between the two groups. A clinical pregnancy rate of 61.5% was achieved with the PGT-A group and 52.4% with the TBR group. These results were not statistically significant. The efficiency of the thaw, biopsy and refreeze technique was not significantly different to that of fresh cycles for rates of survival, results obtained and aneuploidy incidence. Clinical pregnancy rates are not significantly different after the biopsy of fresh and previously cryopreserved embryos.
Conclusion:
The data shows that the TBR procedure has an equivalent success rate to that of classical PGT-A procedures.
Purpose
To describe diagnostic results following re-biopsy of blastocysts with inconclusive results on preimplantation genetic screening for aneuploidy (PGT-A) and to evaluate the reproductive potential of re-biopsied blastocysts.
Methods
This retrospective cohort study included all trophectoderm biopsies submitted for PGT-A by a large in vitro fertilization center to a single genetics laboratory from June 2016 to October 2018. PGT-A was performed using next-generation sequencing (NGS). No-result blastocysts that underwent re-biopsy were subsequently classified as euploid, aneuploid, mosaic/segmental, or no-result. Ongoing pregnancy and clinical loss rates were assessed following transfer of re-biopsied blastocysts. Logistic regressions were conducted to account for age and blastocyst morphology.
Results
Of the trophectoderm biopsies submitted for PGT-A, 635/25,199 (2.5%) were categorized as no-result. Those that underwent re-biopsy (n = 250) had a 95.2% diagnostic rate with 140 (56.0%) receiving euploid diagnoses. Thirty-six re-biopsied blastocysts deemed euploid were subsequently transferred, resulting in 18 (50.0%) ongoing pregnancies and 5 (13.9%) clinical losses. After adjusting for age and blastocyst morphology, there remained a lower ongoing pregnancy rate and a trend towards higher clinical loss rate following transfer of a re-biopsied blastocyst. When compared to blastocysts that underwent the same number of vitrification-warming cycles but only one biopsy, there were no differences in outcomes.
Conclusions
Failure to obtain an analytical result does not change the probability that a given blastocyst is euploid. Pregnancy outcomes following transfer of re-biopsied blastocysts are favorable, but further data must be accrued for an adequately powered comparison with outcomes after transfer of blastocysts biopsied once.
The world's first in vitro fertilization (IVF) baby was born in July 1978 in the UK. Since then, more than 7 million infants have been born worldwide as a result of IVF. Preimplantation genetic diagnosis (PGD) was introduced in the late 1980s for couples at risk of transmitting a genetic abnormality to their children. From the mid-1990s, this technology has been employed as an embryo selection tool for patients undergoing IVF and has been known as preimplantation genetic screening (PGS). The aim of this practice has been to identify and select euploid embryos for transfer, in order to increase efficacy of IVF cycle, ensure higher implantation rates or at least decreased time to pregnancy. In the early days, fluorescent in situ hybridization (FISH) technology was used for genetic analysis. New advancements in both biopsy and cytogenetic have made possible the improvement of PGD and PGT-A analysis. Currently, a variety of technologies have been implemented to individuate euploid embryos to be preferentially transferred in IVF treatments. The purpose of this review is to clarify the differences between PGD and PGT-A, and to discuss current indications and requirements for embryo biopsy and genetic methodologies used.
Background:
Successful cryopreservation of oocytes and embryos is essential not only to maximize the safety and efficacy of ovarian stimulation cycles in an IVF treatment, but also to enable fertility preservation. Two cryopreservation methods are routinely used: slow-freezing or vitrification. Slow-freezing allows for freezing to occur at a sufficiently slow rate to permit adequate cellular dehydration while minimizing intracellular ice formation. Vitrification allows the solidification of the cell(s) and of the extracellular milieu into a glass-like state without the formation of ice.
Objective and rationale:
The objective of our study was to provide a systematic review and meta-analysis of clinical outcomes following slow-freezing/thawing versus vitrification/warming of oocytes and embryos and to inform the development of World Health Organization guidance on the most effective cryopreservation method.
Search methods:
A Medline search was performed from 1966 to 1 August 2016 using the following search terms: (Oocyte(s) [tiab] OR (Pronuclear[tiab] OR Embryo[tiab] OR Blastocyst[tiab]) AND (vitrification[tiab] OR freezing[tiab] OR freeze[tiab]) AND (pregnancy[tiab] OR birth[tiab] OR clinical[tiab]). Queries were limited to those involving humans. RCTs and cohort studies that were published in full-length were considered eligible. Each reference was reviewed for relevance and only primary evidence and relevant articles from the bibliographies of included articles were considered. References were included if they reported cryosurvival rate, clinical pregnancy rate (CPR), live-birth rate (LBR) or delivery rate for slow-frozen or vitrified human oocytes or embryos. A meta-analysis was performed using a random effects model to calculate relative risk ratios (RR) and 95% CI.
Outcomes:
One RCT study comparing slow-freezing versus vitrification of oocytes was included. Vitrification was associated with increased ongoing CPR per cycle (RR = 2.81, 95% CI: 1.05-7.51; P = 0.039; 48 and 30 cycles, respectively, per transfer (RR = 1.81, 95% CI 0.71-4.67; P = 0.214; 47 and 19 transfers) and per warmed/thawed oocyte (RR = 1.14, 95% CI: 1.02-1.28; P = 0.018; 260 and 238 oocytes). One RCT comparing vitrification versus fresh oocytes was analysed. In vitrification and fresh cycles, respectively, no evidence for a difference in ongoing CPR per randomized woman (RR = 1.03, 95% CI: 0.87-1.21; P = 0.744, 300 women in each group), per cycle (RR = 1.01, 95% CI: 0.86-1.18; P = 0.934; 267 versus 259 cycles) and per oocyte utilized (RR = 1.02, 95% CI: 0.82-1.26; P = 0.873; 3286 versus 3185 oocytes) was reported. Findings were consistent with relevant cohort studies. Of the seven RCTs on embryo cryopreservation identified, three met the inclusion criteria (638 warming/thawing cycles at cleavage and blastocyst stage), none of which involved pronuclear-stage embryos. A higher CPR per cycle was noted with embryo vitrification compared with slow-freezing, though this was of borderline statistical significance (RR = 1.89, 95% CI: 1.00-3.59; P = 0.051; three RCTs; I2 = 71.9%). LBR per cycle was reported by one RCT performed with cleavage-stage embryos and was higher for vitrification (RR = 2.28; 95% CI: 1.17-4.44; P = 0.016; 216 cycles; one RCT). A secondary analysis was performed focusing on embryo cryosurvival rate. Pooled data from seven RCTs (3615 embryos) revealed a significant improvement in embryo cryosurvival following vitrification as compared with slow-freezing (RR = 1.59, 95% CI: 1.30-1.93; P < 0.001; I2 = 93%).
Wider implications:
Data from available RCTs suggest that vitrification/warming is superior to slow-freezing/thawing with regard to clinical outcomes (low quality of the evidence) and cryosurvival rates (moderate quality of the evidence) for oocytes, cleavage-stage embryos and blastocysts. The results were confirmed by cohort studies. The improvements obtained with the introduction of vitrification have several important clinical implications in ART. Based on this evidence, in particular regarding cryosurvival rates, laboratories that continue to use slow-freezing should consider transitioning to the use of vitrification for cryopreservation.
STUDY QUESTION Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible
approach across different biopsy practitioners?
STUDY QUESTION Can next-generation sequencing (NGS) techniques be used reliably for comprehensive aneuploidy screening of human embryos
from patients undergoing IVF treatments, with the purpose of identifying and selecting chromosomally normal embryos for transfer?
Previous work has identified 6 important areas to consider when evaluating validity and bias in studies of prognostic factors: participation, attrition, prognostic factor measurement, confounding measurement and account, outcome measurement, and analysis and reporting. This article describes the Quality In Prognosis Studies tool, which includes questions related to these areas that can inform judgments of risk of bias in prognostic research.A working group comprising epidemiologists, statisticians, and clinicians developed the tool as they considered prognosis studies of low back pain. Forty-three groups reviewing studies addressing prognosis in other topic areas used the tool and provided feedback. Most reviewers (74%) reported that reaching consensus on judgments was easy. Median completion time per study was 20 minutes; interrater agreement (κ statistic) reported by 9 review teams varied from 0.56 to 0.82 (median, 0.75). Some reviewers reported challenges making judgments across prompting items, which were addressed by providing comprehensive guidance and examples. The refined Quality In Prognosis Studies tool may be useful to assess the risk of bias in studies of prognostic factors.
Occasionally, clinical scenarios arise where embryos, previously cryostored and warmed, need to be recryopreserved. The outcome of 30 such transfer cycles from 25 women where embryos were recryopreserved is detailed. In 16 cases, embryos were initially cryopreserved by slow freezing and in 14 cases by vitrification. The cryopreservation stages were the pronuclear stage (n = 16), day-3 cleavage stage (n = 12), blastocyst (n = 1) and oocytes (n = 1). All recryopreservation was by Cryotop-based vitrification. From this mixed source, 30/31 twice-cryopreserved embryos survived warming and were transferred, resulting in 13 pregnancies, 11 deliveries with normal gestational age and birthweight, one pre-term birth at 33 weeks and two miscarriages. There were no malformations reported for the live births. Recryopreservation using vitrification by CryoTop has been used in a variety of clinical scenarios to preserve surplus cryopreserved embryos. The current study, although limited in numbers, resulted in high survival rates, clinical pregnancy rates similar to once-cryopreserved embryos and healthy live births independently of the initial stage and cryopreservation method. The technique may increasingly be applicable to elective single-embryo transfer and blastocyst transfer to maximize the pregnancy rate while minimizing the number of cryopreserved embryo transfers.
To evaluate the effects of sequential, repetitive freezing on their in-vitro development, mouse embryos at the eight- to 16-cell stage were subjected to one of five treatments. They were (i) cultured as unfrozen controls, (ii) frozen once and cultured, (iii) subjected to two consecutive freeze-thaw cycles, (iv) frozen and thawed, and then cultured for 18-30 h before being frozen a second time, and (v) frozen three times in succession without being cultured. To assess their functional survival after freezing and thawing, all embryos were cultured in vitro to the hatched blastocyst stage in Whitten's medium. In one experiment, hatched embryos that developed after one, two or three cycles of freezing and thawing were stained with Hoechst 33342 to determine their mean cell number. More embryos of the culture control group and the once-frozen group developed into hatching blastocysts than those of the refrozen groups. There was no difference in the second post-thaw rate of in-vitro development for embryos refrozen with the culture-refreeze or direct-refreeze procedure. Furthermore, there was no difference among in-vitro development rates for embryos frozen two or three times. However, among those embryos subjected to repeated cycles of freezing and thawing that did not survive, there was a considerable amount of damage to their zonae pellucidae. Furthermore, frozen mouse embryos had fewer cells per embryo at the time of hatching than the unfrozen embryos. Nevertheless, these results demonstrate that mouse embryos can survive even three successive freeze-thaw cycles yet still be capable of in-vitro development.
Importance:
There is a lack of consensus and reliable evidence regarding the impact of re-cryopreservation technique on human embryos, particularly with respect to embryo viability and in vitro fertilization (IVF) outcomes.
Objective:
To investigate the impact of re-cryopreservation on embryo viability and IVF outcomes by comparing to single cryopreservation.
Data sources:
Databases PubMed, Embase, Cochrane Library and Scopus were searched until 10 October 2022.
Study selection and synthesis:
All the comparative studies comparing embryonic outcomes and IVF outcomes between repeated and single cryopreservation of embryos were included. The random-effect and fixed-effect meta-analysis model were used to pool the odds ratio (OR) and the corresponding 95% confidence intervals (CIs). Subgroup analysis was performed based on different methods of cryopreservation and different time of embryo cryopreservation/transfer.
Main outcomes:
Outcomes referring to embryo survival, IVF outcomes (including clinical pregnancy rate, embryo implantation rate, miscarriage rate and live birth rate) and neonatal outcomes (including low birth weight rate and preterm birth rate) were evaluated.
Results:
Fourteen studies were eligible for the present meta-analysis, involving 4525 embryo transfer cycles in total (3270 cycles with single cryopreservation (control group) and 1255 with re-cryopreservation (experimental group)). A decreased embryo survival rate (OR, 0.51; 95% CI, 0.27-0.96; P = 0.04) and clinical pregnancy rate (OR, 0.47; 95% CI, 0.23-0.96; P = 0.04) were found in embryos re-cryopreserved by slow freezing. The live birth rate of re-vitrified embryos was also notably affected (OR, 0.60; 95% CI, 0.38-0.94; P = 0.02). Overall, re-cryopreservation resulted in a decreased live birth rate (OR, 0.67; 95% CI, 0.50-0.90; P = 0.007) and an increased miscarriage rate (OR, 1.52; 95% CI, 1.16-1.98; P = 0.003) compared with single cryopreservation. No significant difference was found in neonatal outcomes. When embryos were cryopreserved and transferred at the blastocyst stage, both the embryo implantation rate (OR, 0.59; 95% CI, 0.39-0.89; P = 0.01) and live birth rate (OR, 0.60; 95% CI, 0.37-0.96; P = 0.03) were significantly different between two groups.
Conclusion and relevance:
The present meta-analysis suggested re-cryopreservation, compared to single cryopreservation, can lead to impaired embryo viability and lower rate of IVF success, with no affected neonatal outcomes. Clinicians and embryologists should retain a cautious attitude towards re-cryopreservation strategies.
The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement, published in 2009, was designed to help systematic reviewers transparently report why the review was done, what the authors did, and what they found. Over the past decade, advances in systematic review methodology and terminology have necessitated an update to the guideline. The PRISMA 2020 statement replaces the 2009 statement and includes new reporting guidance that reflects advances in methods to identify, select, appraise, and synthesise studies. The structure and presentation of the items have been modified to facilitate implementation. In this article, we present the PRISMA 2020 27-item checklist, an expanded checklist that details reporting recommendations for each item, the PRISMA 2020 abstract checklist, and the revised flow diagrams for original and updated reviews.
STUDY QUESTION
Does trophectoderm biopsy for preimplantation genetic testing (PGT) increase the risk of obstetric or perinatal complications in frozen–thawed embryo transfer (FET) cycles?
SUMMARY ANSWER
Trophectoderm biopsy may increase the risk of hypertensive disorders of pregnancy (HDP) in pregnancies following FET cycles.
WHAT IS KNOWN ALREADY
Trophectoderm biopsy has replaced blastomere biopsy as the standard of care to procure cells for PGT analysis. Recently, there has been concern that trophectoderm biopsy may adversely impact obstetric and perinatal outcomes. Previous studies examining this question are limited by use of inappropriate control groups, small sample size or reporting on data that no longer reflects current IVF practice.
STUDY DESIGN, SIZE, DURATION
This was a retrospective cohort study conducted at a single university-affiliated fertility center. A total of 756 patients who underwent FET with transfer of previously vitrified blastocysts that had either trophectoderm biopsy or were unbiopsied and resulted in a singleton live birth between 2013 and 2019 were included.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Obstetric and perinatal outcomes for patients aged 20–44 years who underwent FET with transfer of previously vitrified blastocysts that were either biopsied (n = 241) or unbiopsied (n = 515) were analyzed. Primary outcome was odds of placentation disorders including HDP and rate of fetal growth restriction (FGR). Binary logistic regression was performed to control for potential covariates.
MAIN RESULTS AND THE ROLE OF CHANCE
The biopsy group was significantly older, had fewer anovulatory patients, was more often nulliparous and had fewer embryos transferred compared to the unbiopsied group. After controlling for potential covariates, the probability of developing HDP was significantly higher in the biopsy group compared with unbiopsied group (adjusted odds ratio (aOR) 1.943, 95% CI 1.072–3.521; P = 0.029).There was no significant difference between groups in the probability of placenta previa or placenta accreta. There was also no significant difference in the rate of FGR (aOR 1.397; 95% CI, 0.815–2.395; P = 0.224) or the proportion of low (aOR 0.603; 95% CI, 0.336–1.084; P = 0.091) or very low (aOR 2.948; 95% CI, 0.613–14.177; P = 0.177) birthweight infants comparing biopsied to unbiopsied groups.
LIMITATIONS, REASON FOR CAUTION
This was a retrospective study performed at a single fertility center, which may limit the generalizability of our findings.
WIDER IMPLICATIONS OF THE FINDINGS
Trophectoderm biopsy may increase the risk of HDP in FET cycles, however, a prospective multicenter randomized trial should be performed to confirm these findings.
STUDY FUNDING/COMPETING INTEREST(S)
No specific funding was obtained for this study. The authors declare no conflict of interest.
TRIAL REGISTRATION NUMBER
NA.
Research Question
: Do multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively impact IVF outcomes?
Design
: Patients undergoing in vitro fertilization (IVF) with homologous single embryo transfer, and who underwent trophectoderm biopsy for PGT-A from 2013 through 2017, were divided into 3 groups based on degree of embryonic micromanipulation: once-biopsied, once-cryopreserved (Group BC, n = 2603), once-biopsied, twice-cryopreserved (Group CBC, n = 95) and twice-biopsied, twice-cryopreserved (Group BCBC, n = 15). The primary outcome was live birth; secondary outcomes included positive serum pregnancy test, clinical pregnancy, and miscarriage.
Results
: Group CBC had a significantly lower chance of live birth (aRR 0.57, 95% CI 0.41-0.79) and clinical pregnancy (aRR 0.67, 95% CI 0.53-0.85) compared to Group BC. Miscarriage rates were similar between Groups BC (6.6%) and CBC (8.4%).
Conclusions
: Multiple cryopreservation-warming cycles, coupled with blastocyst biopsy, negatively impact IVF outcomes. While PGT-A is thought to improve reproductive outcomes on a per transfer basis, caution must be exercised in counseling patients on the possibility of diminishing returns due to further embryonic micromanipulation after an embryo has been cryo-preserved.
The high incidence of chromosome aneuploidy in human gametes and embryos is a major cause of in vitro fertilization (IVF) failure and miscarriage. In order to improve live birth rates with single-embryo transfer, the use of preimplantation genetic testing for aneuploidy (PGT-A) has significantly increased. PGT encompasses methods that allow embryos to be tested for inherited conditions or screened for chromosomal abnormalities. However, PGT-A is a screening method and results can never be used to definitively predict the chromosomal status of the embryo and fetus. The objective of this manuscript is to review prenatal screening and diagnostic methods available in pregnancies conceived by IVF-PGT-A.
Study question:
Can a second round of biopsy, vitrification and chromosomal testing provide a valid diagnosis where the first attempt fails?
Summary answer:
The risk of inconclusive chromosomal-assessment after trophectoderm biopsy was 2.5% but a further biopsy and vitrification-warming appeared not to impair the competence of euploid blastocysts.
What is known already:
The increasing implementation of multicell trophectoderm biopsy has significantly reduced the risk of inconclusive diagnosis after preimplantation-genetic-testing (PGT). Yet, few reports have defined the variables that influence the risk of failure or described the technical and clinical outcomes after re-biopsy.
Study design, size, duration:
Retrospective multicenter study involving 8990 blastocyst biopsies conducted between April 2013 and September 2017 at six IVF centers but analyzed at a single genetic laboratory. A total of 206 blastocysts were successfully re-biopsied after warming and re-expansion, then re-vitrified. And 49 of these blastocysts were diagnosed euploid and used in single-embryo-transfers (SETs). Logistic regression analyses were conducted.
Participants/materials, setting, methods:
A total of 3244 PGT-for-aneuploidies (PGT-A) cycles with a freeze-all approach, vitrification and qPCR-based analysis were performed by 2687 consenting couples. DNA amplification failure (AF) or non-concurrent data resulted in inconclusive diagnoses. In case of DNA amplification, the cellularity of the biopsy was estimated according to a previously validated method. Euploid SETs were performed. Clinical pregnancy, miscarriage, live birth rates (LBR) and perinatal outcomes were monitored.
Main results and the role of chance:
Overall, 2.5% of trophectoderm biopsies resulted in an inconclusive diagnosis (N = 228/8990). Specifically, 2% (N = 176/8990) resulted in AF and 0.5% (N = 52/8990) in non-concurrent results. The only parameters significantly associated with inconclusive diagnoses were the IVF center and the embryo age (days) at biopsy. Among samples with successful amplification, the number of cells in the biopsy and the day of biopsy were critical to limit non-concurrent results. In total, 213 blastocysts with an inconclusive diagnosis were warmed for re-analysis and the survival rate was 96.7% (N = 206/213). The euploidy rate in blastocysts biopsied twice was 51.9% (N = 107/206) and the euploid embryos were re-vitrified. Overall, 49 euploid embryos were warmed for replacement and all survived. The LBR after SET was 38.8% (N = 19/49). No minor/major obstetrical/perinatal complication was reported.
Limitations, reasons for caution:
A single aneuploidy-testing method was adopted in this retrospective analysis. A more powered report of the clinical and obstetrical/perinatal outcomes after re-biopsied and re-vitrified blastocysts euploid SET requires a larger sample size.
Wider implications of the findings:
It is important to re-biopsy and re-vitrify undiagnosed blastocysts since healthy live births can result from them.
Study funding/competing interest(s):
None.
Trial registration number:
None.
The transition in biopsy timing from blastomere to trophectoderm biopsy has led to a remarkable decrease in the percentage of undiagnosed blastocysts. However, patients with few or no euploid blastocysts can be affected by this residual percentage of diagnosis failure. The aim of this study is to assess whether blastocyst rebiopsy and revitrification is an efficient and safe procedure to be applied in cases of no results after analysis. Fifty-three patients agreed to the warming of 61 blastocysts to perform a second biopsy and PGT-A by aCGH. Only 75.4% of the blastocysts survived, reexpanded, and could be rebiopsied. After the second biopsy and analysis, 95.6% of the blastocysts were successfully diagnosed with an euploidy rate of 65.9%. Eighteen euploid blastocysts were warmed and transferred to 18 patients with a 100% survival and reexpansion rate. Seven clinical pregnancies have been achieved with 4 live births, 1 ongoing pregnancy, and 2 miscarriages. Thus, although few transfers of rebiopsied and revitrified blastocysts have been performed till date, our preliminary results show that this approach is efficient and safe to be applied for undiagnosed blastocysts, as it ultimately allows the transfer of euploid blastocysts and good clinical outcomes.
Objective:
To assess the impact of multiple blastocyst biopsy and vitrification-warming procedures on clinical outcomes.
Design:
Retrospective study.
Setting:
Private fertility clinic.
Patient(s):
Preimplantation genetic diagnosis (PGD) patients undergoing comprehensive chromosome screening, including monogenic disorder and chromosome rearrangement cases.
Intervention(s):
Warming and transfer of euploid blastocysts biopsied and vitrified-warmed once (group 1 [G1, control]; n = 2,130), biopsied once but vitrified-warmed twice (group 2 [G2]; n = 34), or biopsied and vitrified-warmed twice (group 3 [G3]; n = 29).
Main outcome measure(s):
Thaw (for transfer) survival rate and clinical pregnancy rate (CPR).
Result(s):
The thaw survival rates were 98.4% for G1, 97.3% for G2, and 93.3% for G3, with once biopsied and vitrified-warmed embryos being significantly higher than twice biopsied and vitrified-warmed embryos (G1 vs. G3; P=.032). There was a slight reduction in CPR with an additional vitrification-warming (G1 54.3% vs. G2 47.1%) and larger reduction with an additional embryo biopsy (G2 47.1% vs. G3 31.0%), but neither difference was statistically significant. However, the combined effect of both additional biopsy and vitrification-warming resulted in a significantly reduced CPR (G1 54.3% vs. G3 31.0%; P=.013).
Conclusion(s):
This study indicates that blastocysts biopsied and vitrified-warmed twice have reduced clinical outcomes compared with blastocysts biopsied and vitrified-warmed once. PGD patients should be advised that performing a second biopsy and vitrification-warming in cases of failure to obtain a result from initial biopsy will reduce the chance of pregnancy. Patients with inherited disorders may elect to proceed with the second biopsy and vitrification to avoid transfer of embryos with the genetic condition.
To evaluate the clinical value of re-examining the test-failure blastocysts in preimplantation genetic diagnosis/screening cycles.
Retrospective study.
University-affiliated center.
Women with test-failure blastocysts cryopreserved in preimplantation genetic diagnosis/screening cycles.
Cryopreserved test-failure blastocysts were warmed and underwent a second round of biopsy, single nucleotide polymorphism microarray analysis, and vitrification, and the normal blastocysts were warmed again for ET.
The percentage of test-failure blastocysts for transfer, the implantation rate per transferred blastocyst, and the live birth rate.
A total of 106 test-failure blastocysts from 77 cycles were warmed for re-examination. A total of 73 blastocysts that completely expanded were considered to have survived the warming process and were successfully rebiopsied. After single nucleotide polymorphism array analysis, 70 blastocysts yielded whole genome amplification product, and 31 had normal chromosomes (44.3%). A total of 19 normal blastocysts were warmed for ET, of which 18 survived and were transferred. The clinical pregnancy rate (implantation rate) was 50.0% in 10 single blastocyst transfer cycles, and all the implanted blastocysts resulted in healthy live births.
Test-failure blastocysts that survived from the first warming procedure can tolerate a second round of biopsy, vitrification, and warming, have a high chance of having normal chromosomes, and are worth being re-examined.
Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
Over 200 recessive X chromosome-linked diseases, typically affecting only hemizygous males, have been identified. In many of these, prenatal diagnosis is possible by chorion villus sampling (CVS) or amniocentesis, followed by cytogenetic, biochemical or molecular analysis of the cells recovered from the conceptus. In others, the only alternative is to determine the sex of the fetus. If the fetus is affected by the defect or is male, abortion can be offered. Diagnosis of genetic defects in preimplantation embryos would allow those unaffected to be identified and transferred to the uterus. Here we report the first established pregnancies using this procedure, in two couples known to be at risk of transmitting adrenoleukodystrophy and X-linked mental retardation. Two female embryos were transferred after in vitro fertilization (IVF), biopsy of a single cell at the six- to eight-cell stage, and sexing by DNA amplification of a Y chromosome-specific repeat sequence. Both women are confirmed as carrying normal female twins.
Analysis of clinical outcome performing fresh or vitrified-warmed blastocyst transfer after trophectoderm biopsy in 307 Preimplantation Genetic Screening with array comparative genomic hybridization cycles
- A Colasante
- M Minasi
- A Ruberti
- V Casciani
- R Cotarelo
- S Bono
- A Nuccitelli
- A Biricik
- K Litwicka
- C. Mencacci
Does double biopsy and/or double vitrification of blastocyst impact treatment outcomes following preimplantation genetic testing for aneuploidy: a systematic review and meta-analysis?
- K Bickendorf
- F Qi
- K Peirce
- J Natalwala
- V Chapple
- Y. Liu