Roberta Maggiulli

Embryo development of fresh ‘versus’ vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study

BACKGROUND A successful oocyte cryopreservation programme is of utmost importance where a limited number of oocytes can be inseminated per cycle, to overcome legal and ethical issues related to embryo storage, for oocyte donation programmes and for fertility preservation (especially for cancer patients). Vitrification has been recently proposed as an effective procedure for this purpose. METHODS In order to validate the effectiveness of oocyte vitrification a non-inferiority trial was started on sibling metaphase II (MII) oocytes. To demonstrate the non-inferiority based on an absolute difference of 17% in the fertilization rate per sibling oocyte, a minimum of 222 oocytes were required. After oocyte denudation, MII oocytes with normal morphology were randomly allocated to fresh ICSI insemination or to vitrification procedure. If pregnancy was not obtained a subsequent ICSI cycle was performed with warmed oocytes of the same cohort. In both groups, three oocytes were inseminated per cycle by ICSI procedure. Primary end-points were fertilization rates calculated per warmed and per injected oocytes. Secondary end-points were zygote and embryo morphology. RESULTS A total of 244 oocytes were involved in this study. Of the 120 fresh sibling oocytes inseminated, 100 were fertilized (83.3%). Survival rate of sibling vitrified oocytes was 96.8% (120/124 oocytes). Fertilization rate after ICSI was 76.6% (95/124) per warmed oocyte and 79.2% (95/120) per survived/inseminated oocyte. No statistical difference in fertilization rates was observed between the two groups when calculated per sibling oocytes (absolute difference −6.73%; OR: 0.65; 95% CI = 0.33–1.29; P = 0.20) and per inseminated oocyte (absolute difference −4.17%; OR: 0.76; 95% CI = 0.37–1.53; P = 0.50). Embryo development was also similar in both treatment groups up till Day 2. The percentage of excellent quality embryos was 52.0% (52/100) in the fresh group and 51.6% (49/95) in the vitrification group (absolute difference −0.43%; OR: 0.98; 95% CI = 0.53–1.79; P = 0.9). The mean age of the 40 patients included in this study was 35.5 ± 4.8 years (range 26–42). Fifteen clinical pregnancies were obtained in the vitrification cycles of 39 embryo transfers performed (37.5% per cycle, 38.5% per embryo transfer), with an implantation rate of 20.2% (19/94). Three spontaneous miscarriages occurred (20%). Twelve pregnancies are ongoing (30.0% per cycle, 30.8% per embryo transfer) beyond 12 weeks of gestation. CONCLUSIONS Our results indicate that oocyte vitrification procedure followed by ICSI is not inferior to fresh insemination procedure, with regard to fertilization and embryo developmental rates. Moreover, ongoing clinical pregnancy is compatible with this procedure, even with a restricted number of oocytes available for insemination. The promising clinical results obtained, in a population of infertile patients, need to be confirmed on a larger scale. Clinical Trials Registration number: iSRCTN60158641.

Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts

STUDY QUESTION Does conventional blastocyst morphological evaluation correlate with euploidy (as assessed by comprehensive chromosome screening (CCS) of trophectoderm (TE) biopsies) and implantation potential? SUMMARY ANSWER A moderate relation between blastocyst morphology and CCS data was observed but the ability to implant seems to be mainly determined by the chromosomal complement of preimplantation embryos rather than developmental and morphological parameters conventionally used for blastocyst evaluation. WHAT IS KNOWN ALREADY Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select euploid embryos for transfer. Understanding the role of morphology in blastocyst stage preimplantation genetic screening (PGS) cycles may help in further optimizing the cycle management and clinical outcomes. STUDY DESIGN, SIZE, DURATION This is a multicenter retrospective observational study performed between January 2009 and August 2013. The study includes the data analysis of 956 blastocysts with conclusive CCS results obtained from 213 patients following 223 PGS cycles. Single frozen embryo transfer (FET) cycles of 215 euploid blastocysts were performed where it was possible to track the implantation outcome of each embryo transferred. PARTICIPANTS/MATERIALS, SETTING, METHODS PGS was offered to infertile patients of advanced maternal age (>35 years) and/or with a history of unsuccessful IVF treatments (more than two failed IVF cycles) and/or previous spontaneous abortion (more than two spontaneous miscarriages). Prior to TE biopsy for CCS, blastocyst morphology was assessed and categorized in four groups (excellent, good, average and poor quality). The developmental rate of each embryo reaching the expanded blastocyst stage was defined according to the day of biopsy post-fertilization. Day 5 and Day 6 biopsied blastocysts were defined as faster and slower growing embryos, respectively. A novel blastocyst biopsy method, not requiring the opening of the zona pellucida at the cleavage stage of embryo development, was used. Linear regression models were used to test the relationship between blastocyst morphology and developmental rate CCS data and FET cycle outcomes of euploid blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE Among the embryological variables assessed (morphology and developmental rate), only blastocyst morphology was predictive of the CCS data. The euploidy rate was 56.4, 39.1, 42.8 and 25.5% in the excellent, good, average and poor blastocyst morphology groups, respectively. A diagnosis of complex aneuploidy was also associated with blastocyst morphology (P < 0.01) with 6.8, 15.2, 17.4 and 27.5% of excellent, good, average and poor quality embryos, respectively, showing multiple chromosome errors. Faster and slower growing embryos showed a similar aneuploidy rate. Regression logistic analysis showed that none of the parameters used for conventional blastocyst evaluation (morphology and developmental rate) was predictive of the implantation potential of euploid embryos. The implantation potential of euploid embryos was the same, despite different morphologies and developmental rates. LIMITATIONS, REASONS FOR CAUTION The study is limited by its retrospective nature. A higher sample size or a prospective randomized design could be used in future studies to corroborate the current findings. WIDER IMPLICATIONS OF THE FINDINGS This study provides knowledge for a better laboratory and clinical management of blastocyst stage PGS cycles suggesting that the commonly used parameters of blastocyst evaluation are not good enough indicators to improve the selection among euploid embryos. Accordingly, all poor morphology and slower growing expanded blastocysts should be biopsied and similarly considered for FET cycles. This knowledge will be of critical importance to achieve similar cumulative live birth rates in PGS programs compared with conventional IVF, avoiding the potential for exclusion of low quality but viable embryos from the biopsy and transfer procedures. Future research to identify non-invasive biomarkers of reproductive potential may further enhance selection among euploid blastocysts. STUDY FUNDING/COMPETING INTEREST(S) No funding was obtained for the study. All authors have no conflicts to declare. TRIAL REGISTRATION NUMBER None.

Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance

Abstract 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.

No evidence of association between blastocyst aneuploidy and morphokinetic assessment in a selected population of poor-prognosis patients: a longitudinal cohort study

Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening.

Reduction of multiple pregnancies in the advanced maternal age population after implementation of an elective single embryo transfer policy coupled with enhanced embryo selection: pre- and post-intervention study

STUDY QUESTION Is an elective single-embryo transfer (eSET) policy an efficient approach for women aged >35 years when embryo selection is enhanced via blastocyst culture and preimplantation genetic screening (PGS)? SUMMARY ANSWER Elective SET coupled with enhanced embryo selection using PGS in women older than 35 years reduced the multiple pregnancy rates while maintaining the cumulative success rate of the IVF programme. WHAT IS KNOWN ALREADY Multiple pregnancies mean an increased risk of premature birth and perinatal death and occur mainly in older patients when multiple embryos are transferred to increase the chance of pregnancy. A SET policy is usually recommended in cases of good prognosis patients, but no general consensus has been reached for SET application in the advanced maternal age (AMA) population, defined as women older than 35 years. Our objective was to evaluate the results in terms of efficacy, efficiency and safety of an eSET policy coupled with increased application of blastocyst culture and PGS for this population of patients in our IVF programme. STUDY DESIGN, SIZE, DURATION In January 2013, a multidisciplinary intervention involving optimization of embryo selection procedure and introduction of an eSET policy in an AMA population of women was implemented. This is a retrospective 4-year (January 2010–December 2013) pre- and post-intervention analysis, including 1161 and 499 patients in the pre- and post-intervention period, respectively. The primary outcome measures were the cumulative delivery rate (DR) per oocyte retrieval cycle and multiple DR. PARTICIPANTS/MATERIALS, SETTING, METHODS Surplus oocytes and/or embryos were vitrified during the entire study period. In the post-intervention period, all couples with good quality embryos and less than two previous implantation failures were offered eSET. Embryo selection was enhanced by blastocyst culture and PGS (blastocyst stage biopsy and 24-chromosomal screening). Elective SET was also applied in cryopreservation cycles. MAIN RESULTS AND THE ROLE OF CHANCE Patient and cycle characteristics were similar in the pre- and post-intervention groups [mean (SD) female age: 39.6 ± 2.1 and 39.4 ± 2.2 years; range 36–44] as assessed by logistic regression. A total of 1609 versus 574 oocyte retrievals, 937 versus 350 embryo warming and 138 versus 27 oocyte warming cycles were performed in the pre- and post-intervention periods, respectively, resulting in 1854 and 508 embryo transfers, respectively. In the post-intervention period, 289 cycles were blastocyst stage with (n = 182) or without PGS (n = 107). A mean (SD) number of 2.9 ± 1.1 (range 1–4) and 1.4 ± 0.8 (range 1–3) embryos were transferred pre- and post-intervention, respectively (P < 0.01) and similar cumulative clinical pregnancy rates per transfer and per cycle were obtained: 26.8, 30.9% and 29.7, 26.3%, respectively. The total DR per oocyte retrieval cycle (21.0 and 20.4% pre- and post-intervention, respectively) defined as efficacy was not affected by the intervention [odds ratio (OR) = 0.8, 95% confidence interval (CI) = 0.7–1.1; P = 0.23]. However, a significantly increased live birth rate per transferred embryo (defined as efficiency) was observed in the post-intervention group 17.0 versus 10.6% (P < 0.01). Multiple DRs decreased from 21.0 in the preintervention to 6.8% in the post-intervention group (OR = 0.3. 95% CI = 0.1–0.7; P < 0.01). LIMITATIONS, REASONS FOR CAUTION In this study, the suitability of SET was assessed in individual women on the basis of both clinical and embryological prognostic factors and was not standardized. For the described eSET strategy coupled with an enhanced embryo selection policy, an optimized culture system, cryopreservation and aneuploidy screening programme is necessary. WIDER IMPLICATIONS OF THE FINDINGS Owing to the increased maternal morbidity and perinatal complications related to multiple pregnancies, it is recommended to extend the eSET policy to the AMA population. As shown in this study, enhanced embryo selection procedures might allow a reduction in the number of embryos transferred and the number of transfers to be performed without affecting the total efficacy of the treatment but increasing efficiency and safety. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER None.

Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies

STUDY QUESTION Is blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners? SUMMARY ANSWER The blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment. WHAT IS KNOWN ALREADY Recently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes. STUDY DESIGN, SIZE, DURATION This longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET). PARTICIPANTS/MATERIALS, SETTING, METHODS Seven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical outcomes following FEET cycles were stratified by biopsy operator and compared. Cellularity of the biopsy sample was also correlated with clinical outcomes. MAIN RESULTS AND THE ROLE OF CHANCE The seven practitioners performed 2586 biopsies, five in centre IVF-1 and one in each of the other two IVF centres (IVF-2 and IVF-3). Overall, 2437 out of 2586 (94.2%) blastocyst biopsies resulted in a conclusive diagnosis, 119 (4.6%) showed a nonconcurrent result and 30 (1.2%) failed to amplify, suggesting the absence of TE cells in the test tube or presence of degenerated/lysed cells only. Among the samples producing a conclusive diagnosis, a mean concurrence value of 0.253 (95% CI = 0.250–0.257) was observed. Logistic regression analysis adjusted for confounding factors showed no differences in the diagnosis rate and in the concurrence of the genetic analysis between different biopsy practitioners. An overall mean number of 7.32 cells (95% CI = 6.82–7.81; range 2–15) were predicted from all biopsies. Higher cellularity was significantly associated with a better quality of the CCS diagnosis (P < 0.01) and with the conclusive diagnosis rate, with nonconcurrent samples showing significantly lower numbers of cells (2.1; 95% CI=1.5–2.7) compared with samples resulting in a conclusive diagnosis (mean cells number 7.5; 95% CI = 7.1–7.9, P < 0.01). However, no differences were recorded between different biopsy practitioners regarding cellularity of the biopsy. Finally, logistic analysis showed no impact of the biopsy practitioners on the observed ongoing rates of implantation, biochemical pregnancy loss and miscarriage after the FEET cycles. LIMITATIONS, REASONS FOR CAUTION These data come from a restricted set of laboratories where all of the embryologists received identical training and use identical equipment. A single TE biopsy method and CCS technology was used and these data particularly apply to PGS programmes using blastocyst biopsy without zona opening at the cleavage stage and using qPCR-based CCS. To make firm conclusions on the potential impact of biopsy on biochemical pregnancy loss and miscarriages according to practitioner and biopsy cellularity, a larger sample size is needed. WIDER IMPLICATIONS OF THE FINDINGS We reported a very high consistency and reproducibility of the blastocyst biopsy approach coupled with qPCR-based CSS for both genetic and clinical outcomes across different practitioners working in different IVF centres when appropriate training is provided and when the same laboratory setting is used. These data are important considering the trend towards the use of blastocyst biopsy worldwide for PGD/PGS applications. STUDY FUNDING/COMPETING INTEREST(S) None.

The worldwide frozen embryo reservoir: methodologies to achieve optimal results

Cryopreservation of the human embryo has been successfully achieved at the zygote (day 1), cleavage (day 2/3), and blastocyst (day 5) stages; however, each stage presents specific advantages and disadvantages. During the past decades, two major methods have been applied: slow freezing (equilibrium procedure) and vitrification (nonequilibrium procedure). The overwhelming majority of published data prove that the latest vitrification methods induce less cellular trauma and are a more effective cryopreservation technique of human embryos than any other versions of slow freezing. For this reason, fragmented and slow‐cleaving embryos that normally would not be recommended may be revaluated for cryopreservation by using the vitrification method. Furthermore, if laser‐assisted necrotic blastomere removal is associated with the slow‐freezing/thawing procedure, good clinical results can be obtained. Finally, the most proper embryo cleavage stage at which to perform cryopreservation has to be assessed according to clinical indications and laboratory experience.

Failure mode and effects analysis of witnessing protocols for ensuring traceability during IVF

Traceability of cells during IVF is a fundamental aspect of treatment, and involves witnessing protocols. Failure mode and effects analysis (FMEA) is a method of identifying real or potential breakdowns in processes, and allows strategies to mitigate risks to be developed. To examine the risks associated with witnessing protocols, an FMEA was carried out in a busy IVF centre, before and after implementation of an electronic witnessing system (EWS). A multidisciplinary team was formed and moderated by human factors specialists. Possible causes of failures, and their potential effects, were identified and risk priority number (RPN) for each failure calculated. A second FMEA analysis was carried out after implementation of an EWS. The IVF team identified seven main process phases, 19 associated process steps and 32 possible failure modes. The highest RPN was 30, confirming the relatively low risk that mismatches may occur in IVF when a manual witnessing system is used. The introduction of the EWS allowed a reduction in the moderate-risk failure mode by two-thirds (highest RPN = 10). In our experience, FMEA is effective in supporting multidisciplinary IVF groups to understand the witnessing process, identifying critical steps and planning changes in practice to enable safety to be enhanced.

Failure mode and effects analysis of witnessing protocols for ensuring traceability during PGD/PGS cycles

Preimplantation genetic diagnosis and aneuploidy testing (PGD/PGS) use is constantly growing in IVF, and embryo/biopsy traceability during the additional laboratory procedures needed is pivotal. An electronic witnessing system (EWS), which showed a significant value in decreasing mismatch occurrence and increasing detection possibilities during standard care IVF, still does not guarantee the same level of efficiency during PGD/PGS cycles. Specifically, EWS cannot follow single embryos throughout the procedure. This is however critical when an unambiguous diagnosis corresponds to each embryo. Failure Mode and Effects Analysis (FMEA) is a proactive method generally adopted to define tools ensuring safety along a procedure. Due to the implementation of a large quantitative PCR (qPCR)-based blastocyst stage PGD/PGS programme in our centre, and to evaluate the potential procedural risks, a FMEA was performed in September 2014. Forty-four failure modes were identified, among which six were given a moderate risk priority number (>15) (RPN; product of estimated occurrence, severity and detection). Specific corrective measures were then introduced and implemented, and a second evaluation performed six months later. The meticulous and careful application of such measures allowed the risks to be decreased along the whole protocol, by reducing their estimated occurrence and/or increasing detection possibilities.

Abnormally fertilized oocytes can result in healthy live births: improved genetic technologies for preimplantation genetic testing can be used to rescue viable embryos in in vitro fertilization cycles

OBJECTIVE To test whether abnormally fertilized oocyte (AFO)-derived blastocysts are diploid and can be rescued for clinical use. DESIGN Longitudinal-cohort study from January 2015 to September 2016 involving IVF cycles with preimplantation genetic testing for aneuploidy (PGT-A). Ploidy assessment was incorporated whenever a blastocyst from a monopronuclear (1PN) or tripronuclear zygote (2PN + 1 smaller PN; 2.1 PN) was obtained. SETTING Private IVF clinics and genetics laboratories. PATIENT(S) A total of 556 women undergoing 719 PGT-A cycles. INTERVENTION(S) Conventional chromosome analysis was performed on trophectoderm biopsies by quantitative polymerase chain reaction. For AFO-derived blastocysts, ploidy assessment was performed on the same biopsy with the use of allele ratios for hetorozygous SNPs analyzed by means of next-generation sequencing (1:1 = diploid; 2:1 = triploid; loss of heterozygosity = haploid). Balanced-diploid 1PN- and 2.1PN-derived blastocysts were transferred in the absence of normally fertilized transferable embryos. MAIN OUTCOME MEASURE(S) Ploidy constitution and clinical value of AFO-derived blastocysts in IVF PGT-A cycles. RESULT(S) Of the 5,026 metaphase II oocytes injected, 5.2% and 0.7% showed 1PN and 2.1PN, respectively. AFOs showed compromised embryo development (P<.01). Twenty-seven AFO-derived blastocysts were analyzed for ploidy constitution. The 1PN-derived blastocysts were mostly diploid (n = 9/13; 69.2%), a few were haploid (n = 3/13; 23.1%), and one was triploid (n = 1/13; 7.7%). The 2.1PN-derived blastocysts were also mostly diploid (n = 12/14; 85.7%), and the remainder were triploid. Twenty-six PGT-A cycles resulted in one or more AFO-derived blastocysts (n = 26/719; 3.6%). Overall, eight additional balanced-diploid transferable embryos were obtained from AFOs. In three cycles, the only balanced-diploid blastocyst produced was from an AFO (n = 3/719; 0.4%). Three AFO-derived live births were achieved: one from a 1PN zygote and two from 2.1PN zygotes. CONCLUSION(S) Enhanced PGT-A technologies incorporating reliable ploidy assessment provide an effective tool to rescue AFO-derived blastocysts for clinical use.