Preimplantation genetic testing for aneuploidy (PGT-A), previously known as ‘preimplantation genetic screening,’ has emerged as a transformative approach to improve the effectiveness of assisted reproductive technology (ART), particularly for high-risk patients who are susceptible to embryonic chromosomal abnormalities, such as those with advanced maternal age (AMA) [1], recurrent implantation failure (RIF) [2], and recurrent pregnancy loss (RPL) [3]. Since its introduction over two decades ago, PGT-A has represented a pivotal advancement in assisted reproduction [4]. Importantly, PGT-A has shown superior effectiveness in analyzing human chromosomes compared with conventional cytogenetic analysis [5]. A recent study comparing PGT-A with morphology-based embryo assessments underscored its clinical importance; specifically, the ongoing pregnancy rate (OPR) following single frozen embryo transfer (FET) facilitated by PGT-A reaches approximately 50% in patients with good prognoses [6]. Several randomized controlled trials (RCTs) [7,8] and meta-analyses [9] support the advantages of using comprehensive chromosome screening technology on trophectoderm (TE) cells for PGT-A. Given its high success rate, PGT-A is increasingly being integrated into standard in vitro fertilization (IVF) protocols worldwide [10].

Traditional biospy procedures (biopsy at fresh blastocyst) involve a sequential process starting with egg retrieval and fertilization during a fresh IVF and/or intracytoplasmic sperm injection (ICSI) cycle. The embryos are cultured to the blastocyst stage, biopsied, then cryopreserved. Normal embryos are selected based on biopsy results and transferred in the subsequent FET cycle. In clinical practice, most patients opt for embryo biopsy and PGT-A before initiating their IVF cycles; thus, embryos undergo biopsy prior to cryopreservation. However, this method is constrained to blastocysts derived from fresh IVF and/or ICSI cycles, especially regarding the limited timeframe available for comprehensive molecular diagnosis. Therefore, increasing numbers of patients—including those with repeated implantation failures or miscarriages following previous embryo transfer (ET)—request biopsy and PGT-A after embryo cryopreservation. However, limited research has investigated the outcomes of blastocysts that were cryopreserved, thawed, and subsequently biopsied, and even fewer have compared these outcomes with those of biopsy at fresh blastocyst.

Previous studies have highlighted the importance of critical processes occurring during the preimplantation stage, such as embryonic genome activation, maintenance of genomic imprinting, and methylation reprogramming of non-imprinted genes [11,12]. Because the reduction of embryonic mass and the potential disruption of intercellular communication during biopsy or cryopreservation may compromise embryo viability [12], assessing the cumulative impact of these procedures on clinical outcomes is crucial. Therefore, this study aimed to compare aneuploidy rates and clinical outcomes between trophectoderm biopsy at fresh blastocyst followed by cryopreservation and biopsy at vitrified-warmed blastocyst followed by next day transfer without re-vitrification.

In total, 3,485 biopsied embryos were obtained from 844 cycles involving 844 patients. Among these, 2,469 embryos from 531 patients (mean age, 37.2 years; range, 22 to 45) were included in the biopsy-fresh group, while 1,016 embryos from 313 patients (mean age, 36.9 years; range, 28 to 46) were included in the biopsy-vitri group. Euploid embryos selected for ET were transferred to 375 patients in the biopsy-fresh group and 226 patients in the biopsy-vitri group. ET was not performed in the remaining 156 patients in the biopsy-fresh group and 87 patients in the biopsy-vitri group due to the absence of euploid embryos. The detailed demographic and clinical characteristics of all patients are summarized in Table 1.

The proportions of euploid embryos (27.7% vs. 28.6%, p=0.116) and aneuploid embryos (71.7% vs. 70.3%, p=0.097) identified via PGT-A were not significantly different between the biopsy-fresh and biopsy-vitri groups. In total, 417 and 252 euploid embryos were transferred in the biopsy-fresh and biopsy-vitri groups, respectively, corresponding to an average of 1.1±0.3 embryos per ET cycle in both groups. Notably, the proportion of good-quality euploid embryos transferred was significantly higher in the biopsy-fresh group than in the biopsy-vitri group (57.1% vs. 32.1%, p<0.001) (Table 2). However, no significant differences were observed in embryo status post-warming (prior to biopsy) compared to embryo status at the time of transfer in either group (Table 3).

Furthermore, the CPR was significantly higher in the biopsy-fresh group compared with the biopsy-vitri group (58.7% vs. 45.6%; odds ratio [OR], 1.695; 95% confidence interval [CI], 1.215 to 2.364; p=0.002). A similar trend was observed for the IR (45.6% vs. 32.1%; OR, 1.767; 95% CI, 1.274 to 2.451; p=0.002) and OPR/LBR per ET cycle (48.0% vs. 35.8%; OR, 1.652; 95% CI, 1.177 to 2.319; p=0.004). However, no significant difference was found in miscarriage rates between the two groups (18.2% vs. 21.4%; OR, 0.818; 95% CI, 0.457 to 1.465; p=0.501) (Table 4).

Biopsy at vitrified-warmed blastocyst may be considered in various clinical contexts, including RIF or miscarriage following previous ET, embryos cryopreserved without prior biopsy or PGT-A, a preference for single ET, or when considerable time has elapsed since the initial IVF cycle, preventing biopsy and PGT-A at that earlier stage. Among the notable advantages of biopsy at vitrified-warmed blastocyst is procedural efficiency. Our data demonstrate that biopsies of vitrified-warmed blastocysts can be performed within 1 to 2 hours post-thawing, enabling FET within 24 hours if euploid blastocysts are available. This expedited timeline is feasible since biopsy and analysis (aCGH or NGS) generally require 12 to 16 hours. Therefore, we typically schedule FET the day after warming and biopsy. If aCGH or NGS analyses could be expedited further, same-day FET would become possible. In our experience, extending blastocyst culture by up to 24 hours does not negatively affect blastocyst quality. Consistently, Mukaida et al. [16] demonstrated similar IRs when transferring blastocysts thawed after overnight incubation compared to those thawed the same day. Typically, selecting a biopsy site is easier once blastocysts fully expand, contract, and then re-expand approximately 2 to 3 hours post-thawing. However, this approach may delay FET. Thus, effective vitrification programs are essential to maintain blastocyst viability post-thawing. Vitrification is widely employed globally, and we have substantial experience and expertise in this technique [17] validate the clinical feasibility of our protocol.

Particularly when numerous embryos are cryopreserved, thawing all embryos solely to identify euploid embryos and subsequently re-freezing surplus embryos is unnecessary. Although some reports suggest that re-freezing blastocysts does not further compromise their quality or implantation potential [18], others indicate that re-freezing negatively impacts blastocyst viability and reduces IRs [19]. Given the uncertainty about the long-term effects of repeated freezing and thawing on human embryos, selective biopsy and analysis may be advisable to prevent multiple freeze-thaw cycles. However, the limitation of selectively thawing embryos is the possibility of identifying no euploid embryos suitable for transfer, potentially resulting in FET cancellation. Additionally, selecting a limited subset of embryos for biopsy may reduce the availability of good-quality euploid blastocysts, as euploid blastocysts are sometimes of poor morphological quality. Our results support these concerns, demonstrating that the biopsy-fresh group had a higher proportion of good-quality blastocysts compared to the biopsy-vitri group. In general, blastocyst morphology and euploid status are critical factors in selecting blastocysts for transfer. While multiple selection methods exist, morphological evaluation alone has limitations because even high-quality blastocysts may be aneuploid, particularly in older patients or those with a history of RIF or RPL.

Cryopreservation and biopsy procedures can adversely affect blastocyst developmental potential and reduce IRs [19,20]. One previous study suggested that ZP damage during blastocyst biopsy makes blastocysts more vulnerable to cryopreservation and thawing [21]. Conversely, Zech et al. [22] reported no detrimental effects on survival or further development post-vitrification. Additionally, a previous study comparing cryopreservation protocols found no significant difference in survival between biopsied and non-biopsied blastocysts [23]. In our study, using the ‘new laser and flicking biopsy method’ designed to minimize damage to the ZP and blastocysts, we observed no significant difference in blastocyst survival between biopsy-fresh and biopsy-vitri groups. Both groups exhibited survival rates comparable to non-biopsied blastocysts [24]. Thus, our optimized protocol—specifically performing biopsy at vitrified-warmed blastocysts followed by next day transfer without re-vitrification—does not negatively impact clinical outcomes, offering a viable and efficient strategy in PGT-A cycles.

Our study directly compared TE biopsy at fresh blastocyst with TE biopsy at vitrified-warmed blastocyst to evaluate the clinical implications of blastocyst status and cryopreservation protocols on PGT-A outcomes. We found that the biopsy-fresh group resulted in significantly higher CPR, IR, and OPR/LBR. However, miscarriage rates did not significantly differ between the groups, suggesting that blastocyst quality at the time of biopsy, rather than the procedure itself, primarily influences outcomes. This discrepancy can primarily be explained by the higher proportion of good-quality blastocysts in the biopsy-fresh group. These findings are consistent with prior studies demonstrating that blastocyst morphology and euploidy are key predictors of successful implantation and ongoing pregnancy [25]. High-quality blastocysts consistently yield higher LBRs compared to those of poor morphological quality [26]. Hence, blastocyst morphology remains a critical determinant of pregnancy outcomes. Consequently, blastocyst quality and euploid status should both be considered essential criteria for ET selection. If protocols for biopsy at vitrified-warmed blastocyst can be optimized to utilize higher-quality blastocysts, our findings suggest the biopsy procedure itself would not adversely impact clinical outcomes.

This retrospective study has several limitations. Although baseline clinical characteristics were similar between groups, the biopsy-vitri group included fewer good-quality blastocysts because many were surplus blastocysts remaining after previous ET cycles. Future studies should minimize such selection bias by either including only cycles where all blastocysts are cryopreserved or by comparing blastocysts of equivalent morphological quality. Additionally, while the proportion of hatching and hatched blastocysts was significantly higher in the biopsy-vitri group, hatching status is reportedly not associated with IR, CPR, or LBR [27]. Notably, blastocyst statuses post-warming (prior to biopsy) and at the time of transfer did not significantly differ between the groups. It is also important to acknowledge that some blastocysts in the biopsy-vitri group were biopsied under slightly different conditions than those in the biopsy-fresh group; specifically, certain blastocysts could not be biopsied in a fuly expanded state. These variations in biopsy conditions may have influenced clinical outcomes. Therefore, prospective RCTs are necessary to further validate and reinforce these findings.

In conclusion, our findings demonstrated that both biopsy at fresh blastocyst and biopsy at vitrified-warmed blastocyst are viable ART options for PGT-A. Euploid blastocysts biopsied post-warm can be successfully transferred within 24 hours without compromising ET outcomes, and aneuploidy rates do not increase after biopsy at vitrified-warmed blastocyst. Blastocyst quality and euploid status are critical determinants for successful ET. While biopsy at fresh blastocyst provides superior outcomes, likely due to better blastocyst quality, biopsy at vitrified-warmed blastocyst remains a practical alternative for patients with cryopreserved blastocysts. Ultimately, our findings suggest that the biopsy at vitrified-warmed blastocyst approach can be as effective as biopsy at fresh blastocyst, informing evidence-based clinical decisions in ART.