Impact of cryopreservation and biopsy procedure timing on clinical outcomes in high-risk patients
Article information
Abstract
Objective
This study aimed to determine the optimal timing of cryopreservation and biopsy procedures in preimplantation genetic testing for aneuploidy (PGT-A) by comparing clinical outcomes between fresh embryo biopsy (fresh biopsy) and frozen-thawed embryo biopsy (frozen biopsy) procedures in high-risk patients.
Methods
This retrospective study included 844 patients undergoing 844 cycles conducted from August 2019 to December 2023. PGT-A was performed via trophectoderm biopsy using array comparative genomic hybridization and next-generation sequencing for comprehensive 24-chromosome screening. Patients were divided into two groups based on biopsy timing: fresh embryo biopsy (531 patients) and frozen-thawed embryo biopsy (313 patients).
Results
The clinical pregnancy rate was significantly higher in the fresh biopsy group compared to 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). Furthermore, the fresh biopsy group showed higher implantation rates (45.6% vs. 32.1%; OR, 1.767; 95% CI, 1.274 to 2.451; p=0.002), ongoing pregnancy or live birth rates per cycle (48.0% vs. 35.8%; OR, 1.652; 95% CI, 1.177 to 2.319; p=0.004), and rates of good-quality blastocysts (57.1% vs. 32.1%, p<0.001) compared with the frozen biopsy group. Miscarriage rates did not differ significantly between the groups (18.2% vs. 21.4%; OR, 0.818; 95% CI, 0.457 to 1.465; p=0.501).
Conclusion
Fresh biopsy demonstrated superior clinical outcomes compared with frozen biopsy, likely due to better embryo quality. Both fresh and frozen biopsies remain viable options for PGT-A, with frozen biopsy serving as a practical alternative. Embryo quality and euploid status continue to be critical considerations for embryo transfer selection.
Introduction
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.
Methods
1. Study population
This retrospective cohort study analyzed data from a single center involving 844 patients who underwent treatment (844 cycles) at Maria S IVF Center between August 2019 and December 2023. All included patients underwent PGT-A combined with FET, and were categorized into two groups based on biopsy timing: fresh biopsy (n=531) and frozen biopsy (n=313). During the study period, our center transitioned from array comparative genomic hybridization (aCGH) (n=95, 11.3%) to next-generation sequencing (NGS) (n=749, 88.7%) for PGT-A. Screening was limited to high-risk patients, including those with AMA (≥38 years), RIF (≥3 failed ETs), and/or RPL (≥2 miscarriages). Patients who underwent oocyte donation, had monogenic diseases, or had abnormal karyotypes were excluded.
Demographic information collected included patient age and body mass index at the time of oocyte retrieval, as well as the number of embryos biopsied. PGT-A results were categorized into euploid, aneuploid, or no-result embryos. Clinical outcomes assessed were clinical pregnancy, implantation, ongoing pregnancy/live birth, and miscarriage. This study was approved by the Institutional Review Board (IRB) of Maria Fertility Hospital (IRB reference number: 2021-005). The requirement for written informed consent was waived due to the retrospective nature of this cohort study.
2. Ovarian stimulation and oocyte retrieval
Ovarian stimulation involved protocols using a combination of long and short gonadotropin-releasing hormone agonists (Decapeptyl, Ferring Pharmaceuticals; or Lorelin Depot, Dongkook Pharm) or antagonists (Cetrotide, Merck-Serono; or Orgalutran, Organon), along with human menopausal gonadotropin (IVF-M HP, LG Chem; or Menopur, Ferring Pharmaceuticals). Gonadotropin doses were individually adjusted based on the follicular response observed using transvaginal ultrasonography. When one or two leading follicles reached a mean diameter of ≥18 or ≥17 mm, respectively, a subcutaneous injection of 250 µg recombinant human chorionic gonadotropin (hCG) (Ovidrel, Merck-Serono) was administered. Oocyte retrieval occurred 35 to 36 hours after hCG injection, followed by fertilization through IVF and/or ICSI.
3. Assessment of fertilization, embryo culture, and blastocyst biopsy (fresh biopsy)
Fertilization was assessed 17 to 18 hours after insemination based on the observation of two distinct pronuclei and two polar bodies. Zygotes were cultured individually in 25-μL drops of Sydney IVF cleavage and blastocyst medium (Cook) or Sage single-step medium (Origio) in an EmbryoScope time-lapse incubator (Vitrolife) at 37 °C, with a humidified gas mixture of 6% CO2, 5% O2, and 90% N₂. Embryo quality was evaluated from days 2 to 7 using EmbryoViewer external image analysis software (Unisense FertiliTech), and blastocysts were graded according to the scoring system of Gardner et al. [13]. The biopsy process involved stabilizing the blastocyst using a holding pipette, creating a small hole in the zona pellucida (ZP) with a ZILOS-tk™ laser system (Hamilton Thorn Ltd.), and inserting a 21-μm polished biopsy pipette (TPC, CooperSurgical Inc.) through the opening. Subsequently, 5 to 10 TE cells were aspirated into the biopsy pipette and separated from the blastocyst using the recently described ‘new laser and flicking biopsy method’ [14]. The biopsied cells were rinsed four to five times, transferred into 0.2-mL polymerase chain reaction tubes containing 2.5 μL phosphate-buffered saline, and stored at −20 °C until further analysis via aCGH or NGS. All blastocysts were cryopreserved for subsequent FET cycles.
4. Blastocyst vitrification, thawing, and biopsy (frozen biopsy)
Blastocyst vitrification and thawing were performed as previously described [15]. Frozen-thawed embryo biopsy was conducted 1 to 2 hours after thawing, once blastocysts had re-expanded and both the inner cell mass and TE were clearly visible. The biopsy utilized the ‘new laser and flicking biopsy method’ [14]. All biopsied blastocysts were cultured without re-freezing and prepared for transfer the following day.
5. Testing for aneuploidy
Cells obtained from biopsy were analyzed using commercial genetic testing platforms appropriate for the employed method (aCGH by MGmed; NGS by GenoBro or Igenomix Korea). Complete genome amplification was performed, and biopsied cells were analyzed either using aCGH with the Illumina 24 sure+ array (Illumina Inc.) or NGS with a synthesis sequencer (Thermo Fisher Scientific).
6. Uterine preparation and embryo transfer
Endometrial preparation and ET procedures were conducted as previously described [15]. For patients in the fresh biopsy group, a FET cycle was initiated after confirmation of at least one euploid embryo. For patients in the frozen biopsy group, ET was performed within 24 hours after identifying at least one euploid embryo.
All embryos were transferred in natural or hormonally prepared cycles, in compliance with Korean Ministry of Health and Welfare guidelines. If multiple euploid embryos were available, one or two of the highest-quality embryos were selected; if only one euploid embryo was available, it was transferred regardless of quality.
7. Clinical outcome measures
Clinical outcomes evaluated in this study included clinical pregnancy rate (CPR), implantation rate (IR), OPR/live birth rate (LBR), and miscarriage rate. Clinical pregnancy was defined as a serum quantitative hCG >100 mIU/mL at 2 weeks post-retrieval, accompanied by the presence of a gestational sac on transvaginal ultrasound at 6 to 7 weeks of gestation. The IR was calculated by dividing the number of gestational sacs identified on ultrasound by the number of embryos transferred. OPR/LBR was defined as the birth of a neonate at or after 20 weeks of gestation. Miscarriage was defined as the loss of an intrauterine pregnancy after ultrasound identification of a gestational sac but before 20 weeks of gestation. CPR, OPR/LBR, and miscarriage rates were calculated per ET cycle, whereas the IR was calculated per euploid ET.
8. Statistical analysis
Continuous variables are presented as mean±standard deviation, and comparisons between groups were conducted using the independent-sample Student t-test. Categorical data are reported as frequencies with percentages, and group comparisons were performed using the chi-square test or the Fisher exact test. Statistical analysis was conducted using SPSS ver. 12.0 (SPSS Inc.), and p-values <0.05 were considered statistically significant.
Results
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.
Demographic characteristics of patients who underwent PGT-A, comparing the fresh biopsy group and the frozen biopsy group
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).
Comparison of euploidy and aneuploidy rates in PGT-A cycles between the fresh biopsy group and the frozen biopsy group
Comparison of embryo grades at the post-warming and ET stages in ET cycles
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).
Comparison of the clinical outcomes in PGT-A cycles between the fresh biopsy group and the frozen biopsy group
Discussion
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.
Notes
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
The authors extend special thanks to their IVF laboratory colleagues and the clinicians at Maria S Fertility Hospital for their dedicated efforts throughout the study. This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.
Author contributions
Conceptualization: JWK, CYH. Methodology: JWK, SJ, JK, JA. Formal analysis: JWK. Data curation: JWK. Project administration: CYH, JHL. Writing-original draft: JWK. Writing-review & editing: JWK. Approval of final manuscript: JWK, SJ, JK, JA, CYH, JHL.