Biopsy droplets provide genetic signals for potential PGT-SR applications

Genetic material recovered from the liquid left behind during blastocyst biopsy can support copy-number analysis in patients undergoing preimplantation genetic testing for structural rearrangements, according to a single-center study of 218 blastocysts. After the biopsy, washing and temporary storage droplets from each embryo were combined and subjected to whole-genome amplification, library preparation and next-generation sequencing. Of the 218 biopsy-related droplet samples, 151, or 69.27%, contained enough amplified DNA to meet the predefined sequencing threshold. Interpretable copy-number variation profiles were obtained from 142 sequenced samples, giving a 94.04% detection success rate among those meeting that threshold. Across all collected droplets, the informative yield was 65.14%.

The study, conducted at the Center of Reproductive Medicine at Fujian Maternity and Child Health Hospital in Fuzhou, China, consecutively enrolled 62 couples undergoing 67 PGT-SR cycles during 2024. Four additional blastocysts from three patients in 2025 were included after their initial biopsy tests failed. The cohort comprised 42 cases involving balanced reciprocal translocations, 13 Robertsonian translocations and seven other structural abnormalities, including inversions, microdeletions, microduplications and complex rearrangements. Of 218 blastocysts available for biopsy, 211 biopsy-cell samples produced valid copy-number results initially, while seven failed initially but yielded results after a second biopsy. The researchers described the work as exploratory and hypothesis-generating rather than a demonstration that droplet testing can replace established embryo biopsy.

Biopsy-related droplets are operational by-products of trophectoderm biopsy. During the procedure, blastocoelic fluid and small amounts of embryonic cellular material can enter the surrounding biopsy, washing and temporary storage droplets. Because these droplets are already created when a clinician obtains trophectoderm cells for conventional testing, pooling them could add genetic information without requiring a separate embryo-derived fluid collection. In this study, the three droplets were combined into samples of approximately 15 microliters. Genomic DNA was amplified with the multiple annealing and looping-based amplification method, copy-number sequencing libraries were purified, and their concentrations were measured with a fluorometric assay. Sequencing was performed in paired-end mode, with at least two million reads per sample, and copy-number profiles were generated against the GRCh37/hg19 reference genome.

The researchers required a library concentration of at least 0.5 nanograms per microliter for droplet sequencing, a lower threshold than the 5 nanograms per microliter used for biopsy-cell libraries. Of the samples reaching the droplet threshold, 118 had concentrations of at least 1.0 nanogram per microliter and 33 had concentrations between 0.5 and 1.0. The researchers obtained interpretable copy-number profiles from 97.46% of the higher-concentration group and 81.82% of the lower-concentration group. Blank droplet controls produced no interpretable profiles, while trisomy 21 fibroblast controls were detected as expected. The analysis used a lower reporting threshold for deletions and duplications in droplets—10 megabases rather than 4 megabases for biopsy cells—because droplet-derived DNA was expected to be less abundant and more fragmented. Mosaic findings required an abnormal-segment proportion of at least 30%.

Among the 142 droplets with interpretable results, 87.32% agreed with biopsy findings on whether the embryo was euploid or non-euploid. Forty cases, or 40.85%, had identical copy-number profiles, while 55 cases, or 38.73%, shared at least one abnormality but also had discordant findings. The remaining 29 cases, representing 20.42%, had entirely different copy-number profiles, although 11 of these still agreed at the broader level of euploid versus non-euploid. The analysis therefore distinguished exact or overlapping chromosomal findings from ploidy-level agreement, an important qualification because a shared classification can conceal differences in individual chromosomes or segments.

Concordance was weaker when the analysis focused on the 89 blastocysts in which conventional PGT-SR detected abnormalities related to a parental structural rearrangement. Complete or partial agreement between the droplets and biopsy cells occurred in 69 cases, or 77.53%, while ploidy agreement was 83.15%. Droplet testing identified abnormalities involving the same chromosome affected by the rearrangement in 79.78% of this subgroup. Eighteen cases lacked corresponding rearrangement-related findings in the droplets: nine were below the sequencing threshold, three produced non-informative profiles after sequencing, and six contained abnormalities unrelated to the structural rearrangement. These results indicate that droplet analysis can recover rearrangement-associated information, but its lower yield and differing resolution prevent it from serving as a definitive replacement for haplotype-, breakpoint- or linkage-based PGT-SR testing.

Clinical outcomes from a small subset of transferred embryos suggested that droplet results might add information to embryo prioritization. The investigators analyzed 37 blastocysts classified as euploid by trophectoderm biopsy that were subsequently transferred. Seven had euploid droplet profiles, seven had non-euploid profiles, and 23 had undetectable or non-informative results. Clinical pregnancy occurred in six of seven embryos in the euploid-droplet group, or 85.71%, and in two of seven in the non-euploid-droplet group, or 28.57%. Twenty of the 23 embryos with undetectable or non-informative droplet results, or 86.96%, achieved clinical pregnancy. The reported clinical-pregnancy measure included ongoing pregnancies, miscarriages and live births. The group sizes were very small, the design was observational, and the analysis was restricted to embryos already classified as euploid by biopsy, so the outcome differences cannot establish that droplet testing caused the pregnancies or reliably predicted embryo viability.

Among 12 live births in the transferred cohort, prenatal chromosomal microarray analysis of amniotic fluid found no significant fetal chromosomal abnormalities. The study also examined whether droplets collected during an initial failed biopsy could help inform the decision to perform another biopsy. Initial droplet samples were interpretable for six of seven blastocysts that later underwent a second biopsy. Five were non-euploid in the droplet analysis and were also non-euploid on second biopsy, while one was euploid in both. Overall, the droplet and second-biopsy classifications agreed for six of seven embryos, or 85.71%. None of the droplets produced during the second procedure reached the sequencing threshold. These observations suggest that retaining material from the first biopsy may provide supplementary evidence when a conventional test fails, although the sample was too small to determine whether such testing should replace repeat biopsy.

Several biological and technical constraints limit interpretation. Droplet DNA may include blastocoelic fluid, occasional embryo cells and genetic material of uncertain or non-embryonic origin. Its low concentration and fragmentation can increase amplification noise, destabilize copy-number profiles and make small structural abnormalities difficult to resolve. The timing of collection may also matter: five of nine droplets below the sequencing threshold in the rearrangement subgroup were collected more than one day after biopsy. Droplet concentrations were associated with blastocyst morphology, trophectoderm grade and biopsy-based ploidy. Samples from embryos classified as non-euploid had higher mean library concentrations than those from euploid embryos, while poorer morphological grades were also associated with higher concentrations. The authors said these associations might reflect differences in cellular stress, apoptosis or fragmentation, but the biological origins of embryo-associated DNA remain incompletely understood. Null results therefore cannot be treated as evidence that an embryo is euploid, and discordant results may reflect sampling or technical differences rather than true embryonic mosaicism.

The findings support storing biopsy-related droplets for selected retrospective analysis rather than testing every embryo routinely. Possible applications include adding context to embryos classified as euploid, examining possible mosaicism missed by a localized trophectoderm sample, investigating euploid-embryo transfer failures and informing—but not automatically determining—the need for repeat biopsy. The researchers proposed that a euploid or undetectable droplet result may be supportive when prioritizing an embryo, whereas a non-euploid result may be less supportive; a non-euploid finding after repeated euploid transfer failures could also point toward an embryonic contribution, while a euploid result may prompt consideration of other factors. These interpretations remain provisional. The study was conducted at one center, involved a PGT-SR population, and relied on several small subgroups. Further optimization of collection, amplification, contamination control and analysis, together with larger independent cohorts, is required before biopsy-related droplet testing can be used as a clinically validated adjunct to conventional PGT-SR.

Subject of Research: Genetic analysis of biopsy-related droplets as an adjunct to PGT-SR

Article Title: Genetic analysis of biopsy-related droplets and its potential applications in PGT-SR patients

Article References: Huang, Z., Chen, Y., Cai, X., Zhu, S., Wang, J., Xie, R., Shi, H., Yu, A., Zheng, B., & Sun, Y. (2026). Genetic analysis of biopsy-related droplets and its potential applications in PGT-SR patients. Frontiers in Endocrinology, 17, Article 1946046. https://doi.org/10.3389/fendo.2026.1946046

Image Credits: AI Generated

DOI: 10.3389/fendo.2026.1946046

Keywords: biopsy-related droplets, preimplantation genetic testing, structural rearrangements, copy-number variation, blastocyst biopsy, next-generation sequencing, embryo mosaicism, repeat biopsy, Genetic, analysis, biopsy-related, droplets