We have here compared MDA and LBA as WGA methods for PGT-SR, with a particular focus on distinguishing between normal diploids and balanced reciprocal translocations. In CNV analysis, both of the MDA and LBA methods yielded consistent results, suggesting that these were stable approaches for conducting PGT-SR. Historically, the MDA method using φ29 DNA polymerase with random primers under isothermal conditions has been widely applied in WGA, but it has shown problems with its quantitative performance and is not generally used for CNV analysis. To overcome the limitations of the MDA, MALBAC or LBA were developed as an alternative WGA method for use in CNV analysis. In that system, synthesized DNA preferentially amplified in earlier cycles forms a loop structure that can avoid excess amplification in later cycles [9]. It is not unreasonable to expect therefore that LBA would perform better in quantitative analysis. However, our present results have indicated no problems in interpreting the results when MDA is used in CNV analysis, suggesting that advances in MDA amplification technology, such as fine tuning of reaction buffers or protocols, produced a performance for this technique that was comparable to LBA.

Both the MDA and LBA-amplified WGA products failed to detect copy number abnormalities at the proximal 22q11 region generated by unbalanced t(11;22) and t(8;22) translocations. The predicted size of the unbalanced region estimated by the breakpoint location is ~5 Mb, which may be above the threshold for detection by NGS-based CNV analysis [13]. However, this region includes many segmental duplications and low copy repeats that might hinder the quantitative analysis of the sequence reads [12]. This might potentially require careful attention in clinical practice since it could potentially lead to a misdiagnosis from PGT-SR. However, unbalanced translocations usually involve copy number abnormalities of two translocation-related chromosomes. Even when CNVs are hard to detect at the proximal 22q11 region, the size of the unbalanced region of the partner chromosome, 8q24.13-qter or 11q23-qter, is large enough to be detected by standard CNV analysis. Thus, it does not affect the sensitivity of the detection of unbalanced translocations in PGT-SR for t(11;22) and t(8;22).

It was notable that the MDA-amplified WGA DNA samples yielded a substantial amount of translocation-specific PCR products. The positive PCR findings in this regard correctly reflected the presence of each translocation derivative chromosome predicted by CNV analysis. In contrast, the LBA-amplified WGA DNA did not yield any translocation-specific PCR products. Although the LBA method is useful for detecting aneuploidy in PGT-A, and also partial aneuploidy in PGT-SR, it is generally considered unsuitable for use in PGT-M due to coverage bias and amplification product length issues [14]. The WGA product lengths from the LBA method are reported to be shorter than those obtained with the MDA method: 0.2–2.0 kb for LBA and ~10 kb for MDA [15]. Hence, the conventional MDA method is preferentially used for PGT-M since various type of pathogenic variants must be handled. LBA method has been optimized for quantitative analysis. To achieve a cost-effectiveness as a clinical use in PGT, low depth sequencing, usually ×0.01–0.1, is enough for CNV analysis of 1 Mb resolution in standard PGT-SR. However, insufficient genome coverage hinders analysis of each specific locus [11]. Further, translocation breakpoint region might be recalcitrant to amplification due to DNA secondary structure. Reduction of the sequencing cost and increase the read coverage might overcome this weakness of the LBA method. In this sense, it is not surprising that LBA WGA products did not work in the translocation-specific PCR and that MDA appears to be superior to LBA when CNV analysis and translocation-specific PCR are required for the same sample.

Our present methodology allowed us to distinguish balanced translocation from normal diploidy among four samples with putative normal findings by CNV analysis. One sample was finally determined to have a balanced translocation since both the der(11) and der(22) results were positive, and the others were determined as normal diploid since none of the PCR tests for derivative chromosomes were positive. Our data thus suggest that CNV analysis using WGA products amplified with the MDA method, combined with translocation-specific PCR, can successfully distinguish balanced translocation from normal diploidy. In addition, samples appearing to be aneuploid for translocation-related chromosomes in the CNV analysis occasionally showed positivity in both PCR tests for translocation-derivative chromosomes, suggesting that we could use these techniques also to distinguish 3:1 segregations and meiotic errors. Although resolution of the unbalanced translocation as well as accurate quantification of additional germline or somatic aneuploidy needs to be re-evaluated, MDA and translocation-specific PCR, which can distinguish balanced translocation from normal diploidy, has the potential to become standard practice in PGT-SR.

We tested the performance of WGA methods for translocation-specific PCR using recurrent constitutional t(11;22) and t(8;22) embryo samples. The rationale for this approach was that we did not need to set up translocation-specific PCR since these translocations share the same breakpoints located at the center of PATRRs among unrelated families. The breakpoints of the t(11;22) and t(8;22) were located within a few hundred base pair regions, allowing us to use same PCR system with the same primer to detect the translocation junctions. However, since the breakpoints of general reciprocal translocations distribute randomly, it is necessary to determine the translocation breakpoints individually when establishing a tailor-made translocation-specific PCR system. This necessarily requires time, money, and technical effort, and there are still challenges in generalizing the resulting data. Recent advances in long-read sequencing have enabled the detection of breakpoints for structural rearrangements and have spurred the establishment of junction-specific PCR for PGT [16, 17].

Finally, we found using these aforementioned techniques that it became possible to distinguish embryos with various balanced reciprocal translocations from those with normal diploidy using PGT-SR with translocation-specific PCR. Currently in Japan, PGT-SR is used for translocation carriers presenting with reproductive issues due to embryos with unbalanced translocations and thereby selection against embryos with unbalanced translocations or aneuploidy improve the pregnancy rate and reduce the miscarriage rate [18]. Techniques that can be used to detect balanced reciprocal translocations in euploid embryos are essentially prohibited by current Japan Society of Obstetrics and Gynecology (JSOG) regulations unless a severe clinical phenotype is predicted in relation to the balanced translocation carrier [19]. In Japan at present, the potential for future reproductive problems associated with a balanced translocation are not considered sufficient to exclude an embryo from transfer. However, in couples involving a translocation carrier, the psychological burden of potentially passing on reproductive problems to their offspring can be heavy. Balanced translocation carriers face complex psychological burdens characterized by mixed emotions and significant social challenges [20]. Many carriers grapple with the implications for their relationships, particularly with their partners, fearing potential blame, misunderstanding, or rejection. Additionally, carriers experience anxiety about their children’s futures, including concerns about whether to disclose their carrier status to siblings and how to navigate uncertainties surrounding future pregnancies. Therefore, we propose that considering this distinction using the present method is potentially a solution to alleviate psychological burdens when multiple euploid embryos are available for transfer. However, the current Japanese guidelines for PGT-SR address the presence or absence of balanced translocations should not be disclosed, which poses challenges for the clinical application of our methodology in Japan [19]. There might be a concern that the reduction of number of embryos available for transfer affects pregnancy or live birth rates if the balanced translocation embryos are deselected or deprioritized. However, when multiple transferable euploid embryos are available in PGT-SR, our own common experience is that they would like to transfer a normal euploid embryo without any translocations. Our current results demonstrate the technical feasibility of doing just that using a standard PGT-SR system, although we acknowledge that there remain some ethical issues relating to the clinical application of our methodology in Japan. Further discussions on the medical, ethical, legal, and social implications are warranted to establish appropriate indications for PGT-SR in embryo transfer.

In conclusion, we demonstrate a method for distinguishing between normal diploid embryos and those harboring balanced reciprocal translocations that combines an MDA method and translocation-specific PCR. Although some challenges remain in this regard, distinguishing normal diploidy from balanced reciprocal translocations may well become standard practice the future PGT-SR testing.