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 biopsy procedures (fresh biopsy) 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. Few studies have specifically investigated the outcomes of patients whose blastocysts were cryopreserved, thawed, and subsequently biopsied, and even fewer have compared such cases to those undergoing fresh biopsy.

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]. These processes are essential because the reduction in viable embryonic material and the disruption of intercellular communication during biopsy and cryopreservation may adversely affect the embryo. Such disruptions can negatively impact embryonic development and implantation potential, thereby influencing clinical outcomes [12]. Thus, this study aimed to evaluate the clinical impact of cryopreservation and biopsy timing by comparing outcomes between fresh and frozen-thawed embryo biopsy procedures.

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) underwent fresh biopsy, while 1,016 embryos from 313 patients (mean age, 36.9 years; range, 28 to 46) underwent frozen biopsy. Euploid embryos selected for ET were transferred to 375 patients in the fresh biopsy group and 226 patients in the frozen biopsy group. ET was not performed in the remaining 156 patients in the fresh biopsy group and 87 patients in the frozen biopsy 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 fresh and frozen biopsy groups. In total, 417 and 252 euploid embryos were transferred in the fresh and frozen biopsy 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 fresh biopsy group than in the frozen biopsy 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 fresh biopsy group compared with the frozen biopsy 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).

Frozen-thawed embryo biopsy 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 frozen-thawed biopsy is procedural efficiency. Our data demonstrate that biopsies of frozen-thawed embryos can be performed within 1 to 2 hours post-thawing, enabling FET within 24 hours if euploid embryos 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 thawing and biopsy. If aCGH or NGS analyses could be expedited further, same-day FET would become possible. In our experience, extending embryo culture by up to 24 hours does not negatively affect embryo 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 embryo viability post-thawing. Vitrification is widely employed globally, and we have substantial experience and expertise in this technique [17], making our protocol highly suitable.

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 embryo 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 embryos, as euploid embryos are sometimes of poor morphological quality. Our results support these concerns, demonstrating that the fresh biopsy group had a higher proportion of good-quality embryos compared to the frozen biopsy group. In general, embryo morphology and euploid status are critical factors in selecting embryos for transfer. While multiple embryo selection methods exist, morphological evaluation alone has limitations because even high-quality embryos may be aneuploid, particularly in older patients or those with a history of RIF or RPL.

Cryopreservation and biopsy procedures can adversely affect embryo developmental potential and reduce IRs [19,20]. One previous study suggested that ZP damage during embryo biopsy makes embryos 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 embryos, we observed no significant difference in embryo survival between fresh and frozen biopsy groups. Both groups exhibited survival rates comparable to non-biopsied embryos [24]. Thus, cryopreservation timing and biopsy procedures in PGT-A did not negatively impact clinical outcomes in this context.

Our study directly compared fresh and frozen biopsy procedures to evaluate the clinical implications of biopsy timing and cryopreservation methods. We found that fresh biopsy resulted in significantly higher CPR, IR, and OPR/LBR. However, miscarriage rates did not significantly differ between procedures, suggesting that biopsy timing rather than the procedure itself influences outcomes. This discrepancy can primarily be explained by the higher proportion of good-quality embryos in the fresh biopsy group. These findings are consistent with prior studies demonstrating that embryo morphology and euploidy are key predictors of successful implantation and ongoing pregnancy [25]. High-quality embryos consistently yield higher LBRs compared to poor-quality embryos [26]. Hence, embryo morphology remains a critical determinant of pregnancy outcomes. Consequently, embryo quality and euploid status should both be considered essential criteria for ET selection. If frozen biopsy protocols can be optimized to produce higher-quality embryos, 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 frozen biopsy group included fewer good-quality embryos because many were surplus embryos remaining after previous ET cycles. Future studies should minimize such bias by either including only cycles where all embryos are cryopreserved or comparing embryos of similar quality. Additionally, the proportion of hatching and hatched blastocysts was significantly higher in the frozen biopsy group; however, hatching status is reportedly not associated with IR, CPR, or LBR [27]. Notably, embryo statuses post-warming (prior to biopsy) and at transfer were not significantly different between groups. It is important to acknowledge that some embryos in the frozen biopsy group were biopsied under slightly different conditions than those in the fresh biopsy group, as certain embryos could not be biopsied while fully expanded. These variations in biopsy conditions could have influenced clinical outcomes. Therefore, prospective RCTs are necessary to validate and reinforce these findings.

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