Noninvasive preimplantation genetic testing for aneuploidy using blastocyst spent culture medium may serve as a backup of trophectoderm biopsy in conventional preimplantation genetic testing
Preimplantation genetic testing for aneuploidy and for structural rearrangements (PGT-A/-SR) performed on trophectoderm (TE) cells is an established and widely adopted clinical approach for identifying aneuploidies or chromosomal structural rearrangements in embryos obtained by in vitro fertilization (IVF). The aneuploidy rate of embryos increases with advancing maternal age, For patients aged 35–42, the aneuploidy rate ranges from 34.5 to 75.1% . Studies reported PGT-A/-SR could improve the clinical outcomes of IVF patients via the selection and transfer of euploid embryos , debates persist regarding the safety and accuracy of the technique, as well as which groups of IVF patients would benefit most from this approach . Given the need to perform TE biopsy, the invasiveness of PGT-A/-SR remains a concern, and its application may be limited as follows: (1) the biopsy may potentially negatively impact on the quality and development of the embryo; (2) the biopsy fraction may not fully represent the genetic status of the whole embryo due to mosaicism; and (3) biopsy is a sophisticated procedure that, despite the need for an experienced embryologist, still carries a risk of operational failure. Stigliani et al.  first demonstrated the existence of fetal cell-free DNA (cfDNA) in spent culture medium (SCM), with 63% (205/326) of collected SCM samples containing genomic DNA. The Research by Hammond et al. further supported the detection of fetal DNA materials in SCM . These findings have laid a groundwork for utilizing SCM as a source of early embryonic DNA sampling for developing noninvasive preimplantation genetic testing (niPGT). The noninvasive chromosome screening (NICS) method was first developed in 2016 . In the NICS method, multiple annealing and looping-based amplification cycles (MALBACs) were used for whole-genome amplification (WGA) to analyze embryonic cfDNA in SCM. In that study, the researchers achieved a 100% amplification rate for 42 embryonic SCM samples collected for noninvasive preimplantation genetic testing for aneuploidy (niPGT-A), with 88.2% sensitivity and 84.0% specificity in detecting aneuploid embryos. Several other studies have been conducted to compare PGT using SCM versus TE biopsy , and showing variation in concordance rates (78.7–62.1%) as well as the sensitivity (94.5–81.6%) and specificity values (71.7–48.3%) due to the different concordant metrics applied. Researchers and clinicians have questioned whether PGT with SCM, compared to PGT using TE biopsy, is more advanced in its reliability in representing the whole genetic profile of the tested embryo and its sensitivity in identifying moderately low-level mosaicism. Additionally, it is known that as the embryo passes through the different stages of cell proliferation, the biopsy procedure itself can interfere with the accuracy of PGT. A 20–30% level of mosaicism can be detected via microarray-based and NGS-based PGT , and mosaic results are commonly reported in PGT. Data from most reproductive centers indicate that the frequency of detecting mosaic embryos is approximately 5–20%  and up to 40% across IVF centers . Moreover, an improperly performed biopsy technique may lead to an increased rate of mosaicism, resulting in embryo waste and cancellation of the transplantation cycle. An expert consensus has proposed that when the diagnosis of the first biopsy is unclear, a second biopsy (including cleavage stage embryos and blastocysts) can be considered; if the embryo is frozen after biopsy, a secondary biopsy after thawing may be considered . However, secondary biopsies can also significantly affect the developmental potential of the embryo. SCM is a noninvasive material for embryonic copy number variation (CNV) detection and analysis. Therefore, we hypothesize that SCM can be used as a supplemental source of genetic material when mosaic results or failed amplifications occur in conventional PGT assays. In the current study, we collected SCM samples from 149 blastocysts of 51 couples who underwent TE biopsy PGT-A/-SR assays as part of the ICSI–IVF process. The SCM samples were first kept at -80 °C during the waiting period for the PGT test results and subsequently examined by WGA and next-generation sequencing (NGS) for chromosome screening. The ploidy and chromosomal structure data were acquired using PGT from paired TE biopsies. Additionally, a small fraction of the blastocysts with aneuploidy or structural rearrangements as determined by TE results were donated for further validation by again sampling the TE and inner cell mass (ICM) cells of whole embryos. This new niPGT approach enables the use of SCM as a backup sample for validation when ambiguous or mosaic TE results are reported to avoid a harmful secondary biopsy of the embryo.   Ethics Statement and study patients The ethics committee of the International Peace Maternity & Child Health Hospital (IPMCH) approved this study. Informed consent was obtained from all patients included in this study as required by the ethics committee of IPMCH. All experiments were performed following the relevant guidelines and regulations. SCM samples and corresponding TE biopsy samples from a total of 149 fresh blastocysts were analyzed in this study. These blastocysts were obtained from 51 couples undergoing intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) from June 2019 to May 2022 at the IPMCH. In all, 23 patients (or their husbands) were known to carry a chromosomal rearrangement, while the remaining 28 couples had a normal karyotype. Embryo culture ICSI was performed to fertilize mature metaphase II oocytes collected from patients. Prior to ICSI, the oocytes will be denudated. The fertilized oocytes were cultured in 25 µL media under oil and then placed into an incubator. The embryos were cultured sequentially, switching to blastocyst medium on Day (D) 3. The cumulus and corona radiata cells were removed on D4, and the embryo was transferred to 25 µL fresh medium and cultured for D5 or D6. Collection of Trophectoderm cells Blastocysts with a grade of ≥ 4BC were transferred to a biopsy dish containing 10 µL G-GAMATE (Vitrolife; Sweden) under the oil used for the biopsy on D5 or D6. Four to six trophectoderm cells were collected from each blastocyst and immediately transferred into DNase-free PCR tubes containing 4 µL of cell lysis buffer. The biopsied cells were stored at -80 °C until the next step. Collection of ICM cells To obtain ICM cells, the blastocyst was held with a holding pipette, and the ICM was positioned at the 3 o’clock position. A laser was used to create a hole in the zona pellucida at the 3 o’clock position. A biopsy pipette was inserted into the blastocyst, and the ICM was removed with gentle suction and transferred into a 10 µL drop of G-GAMATE (Vitrolife; Sweden). Subsequently, four to six cells were collected from each isolated ICM. The collected cells were transferred into cell lysis buffer and stored at -80 °C. Collection of spent embryo culture medium After the blastocysts were removed, up to 20 µL spent embryo culture medium was collected into DNase-free PCR tubes containing 5 µL cell lysis buffer (Xukang Medical Technology (suzhou) Co., Ltd). These fluid samples were stored at -80 °C until the next step . Blank medium samples were cultured under the same conditions and served as the negative controls. PGT for TE Samples using a microarray WGA and array comparative genomic hybridization (aCGH) amplification were performed, starting with the biopsied cells. The Sureplex kit (Rubicon; Bluegnome) was used according to the manufacturer’s instructions. Quality control of amplification products was performed using Qubit 3.0 and 1.5% agarose gel electrophoresis. A 24Sure microarray and Blue-Fuse Multi software (Illumina) were utilized to detect and visualize the blastocysts’ subchromosomal imbalances and euploidy/aneuploidy. Chromosomal abnormalities with a mosaicism extent of ≥ 30% and size ≥ 10 Mb were reported. PGT for SCM, TE and ICM samples using CNV-Seq ChromInst™ (Xukang Medical Technology (Suzhou) Co., Ltd) was used to conduct WGA and NGS library preparation of TE and ICM according to the manufacturer’s instructions. NICSInst™ (Xukang Medical Technology (Suzhou) Co., Ltd) was used or the amplification and library construction. Quality control of the NGS libraries was performed using Qubit 3.0 and 1.5% agarose gel electrophoresis. The indexed libraries were pooled in equal proportions and then sequenced with a NextSeq 550 (Illumina) to obtain approximately 2 million raw reads (single-end, 55 bp) for each library. The high-quality read numbers were counted along the whole genome with a bin size of 1 Mb and normalized by the GC content and a reference dataset. The circular binary segmentation algorithm was used to detect CNV segments. The data were then analyzed and visualized using ChromGo™ Analysis Software (Xukang Medical Technology (Suzhou) Co., Ltd.) with default parameters. If the result indicated mosaicism, the embryo was initially classified as “euploid” when the extent of mosaicism was below 30% and as “aneuploid” when the extent of mosaicism was above 70%, with a detection limit for segmental aneuploidy of ≥ 10 Mb; that is, 30% mosaicism was regarded as a negative result, and ≥ 30% mosaicism was regarded as a positive result.   The ethics committee of the International Peace Maternity & Child Health Hospital (IPMCH) approved this study. Informed consent was obtained from all patients included in this study as required by the ethics committee of IPMCH. All experiments were performed following the relevant guidelines and regulations. SCM samples and corresponding TE biopsy samples from a total of 149 fresh blastocysts were analyzed in this study. These blastocysts were obtained from 51 couples undergoing intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) from June 2019 to May 2022 at the IPMCH. In all, 23 patients (or their husbands) were known to carry a chromosomal rearrangement, while the remaining 28 couples had a normal karyotype.   ICSI was performed to fertilize mature metaphase II oocytes collected from patients. Prior to ICSI, the oocytes will be denudated. The fertilized oocytes were cultured in 25 µL media under oil and then placed into an incubator. The embryos were cultured sequentially, switching to blastocyst medium on Day (D) 3. The cumulus and corona radiata cells were removed on D4, and the embryo was transferred to 25 µL fresh medium and cultured for D5 or D6.   Blastocysts with a grade of ≥ 4BC were transferred to a biopsy dish containing 10 µL G-GAMATE (Vitrolife; Sweden) under the oil used for the biopsy on D5 or D6. Four to six trophectoderm cells were collected from each blastocyst and immediately transferred into DNase-free PCR tubes containing 4 µL of cell lysis buffer. The biopsied cells were stored at -80 °C until the next step.   To obtain ICM cells, the blastocyst was held with a holding pipette, and the ICM was positioned at the 3 o’clock position. A laser was used to create a hole in the zona pellucida at the 3 o’clock position. A biopsy pipette was inserted into the blastocyst, and the ICM was removed with gentle suction and transferred into a 10 µL drop of G-GAMATE (Vitrolife; Sweden). Subsequently, four to six cells were collected from each isolated ICM. The collected cells were transferred into cell lysis buffer and stored at -80 °C.   After the blastocysts were removed, up to 20 µL spent embryo culture medium was collected into DNase-free PCR tubes containing 5 µL cell lysis buffer (Xukang Medical Technology (suzhou) Co., Ltd). These fluid samples were stored at -80 °C until the next step . Blank medium samples were cultured under the same conditions and served as the negative controls.   WGA and array comparative genomic hybridization (aCGH) amplification were performed, starting with the biopsied cells. The Sureplex kit (Rubicon; Bluegnome) was used according to the manufacturer’s instructions. Quality control of amplification products was performed using Qubit 3.0 and 1.5% agarose gel electrophoresis. A 24Sure microarray and Blue-Fuse Multi software (Illumina) were utilized to detect and visualize the blastocysts’ subchromosomal imbalances and euploidy/aneuploidy. Chromosomal abnormalities with a mosaicism extent of ≥ 30% and size ≥ 10 Mb were reported.   ChromInst™ (Xukang Medical Technology (Suzhou) Co., Ltd) was used to conduct WGA and NGS library preparation of TE and ICM according to the manufacturer’s instructions. NICSInst™ (Xukang Medical Technology (Suzhou) Co., Ltd) was used or the amplification and library construction. Quality control of the NGS libraries was performed using Qubit 3.0 and 1.5% agarose gel electrophoresis. The indexed libraries were pooled in equal proportions and then sequenced with a NextSeq 550 (Illumina) to obtain approximately 2 million raw reads (single-end, 55 bp) for each library. The high-quality read numbers were counted along the whole genome with a bin size of 1 Mb and normalized by the GC content and a reference dataset. The circular binary segmentation algorithm was used to detect CNV segments. The data were then analyzed and visualized using ChromGo™ Analysis Software (Xukang Medical Technology (Suzhou) Co., Ltd.) with default parameters. If the result indicated mosaicism, the embryo was initially classified as “euploid” when the extent of mosaicism was below 30% and as “aneuploid” when the extent of mosaicism was above 70%, with a detection limit for segmental aneuploidy of ≥ 10 Mb; that is, 30% mosaicism was regarded as a negative result, and ≥ 30% mosaicism was regarded as a positive result.   SCM Storage Feasibility at -80 °C for niPGT-A To evaluate the feasibility of storing SCM samples at -80 °C, we first randomly collected 40 SCM samples from routine embryo culture. Half of each SCM (10 µL) sample was used for WGA, while the other half was stored at -80 °C for 3–6 months. Thirty-six out of 40 fresh SCM samples were successfully amplified and sequenced, with 19 being euploid and 17 being aneuploid. Subsequently, 36 pairs of frozen SCM sample were thawed after 3–6 months of storage at -80 °C and subjected to WGA and sequencing. All thirty-six frozen-thawed SCM samples were successfully amplified and showed 100% (36/36) concordance with the freshly amplified SCM samples (Fig. , Table ). These results indicated that SCM can be feasibly and routinely stored at -80 °C and used as a source for niPGT-A assays. Evaluation of Concordance Rates between niPGT-A and PGT in normal and abnormal karyotype groups A total of 51 families who underwent IVF cycles of PGT-A or PGT-SR were included in the study, with 149 embryos screened using PGT with TE biopsy first. Standard PGT was performed on biopsies from these embryos, and the euploid embryos were implanted. However, the SCM samples of these embryos were collected for niPGT-A analysis. The SCM samples were stored at -80 °C for approximately three to six months before performing the niPGT-A assay. The embryos were grouped based on their parental karyotype information: 28 normal karyotype families with 59 embryos and 23 abnormal karyotype families with 87 embryos (Table ). Eighteen TE biopsies were screened by microarray, while the other 128 were sequenced by NGS. To rule out the effect of the use of different testing platforms, we reviewed the concordance rates and the CNV profiles of the PGT results obtained by microarray and NGS within the normal karyotype group. In this study, we considered only the overall ploidy status, namely, euploid versus aneuploid, when calculating the concordance rate between the niPGT-A and PGT results. No significant difference in the concordance rate was observed between the microarray (72.2%) and NGS (75.6%) detection platforms (Table ). Among these 149 ICSI embryos, paired TE biopsy and SCM samples were collected, and 146 (98.0%, 146/149) SCM samples were successfully amplified and sequenced (Fig. ). A concordance rate of 78.1% (114/146) was achieved between the niPGT-A results and the TE biopsy PGT-A and PGT-SR results, with 81 true-positive samples, 33 true-negative samples, 12 false-positive samples (euploid according to PGT but aneuploid according to niPGT-A), and 20 false-negative samples (aneuploid according to PGT but euploid according to niPGT-A) (Table ). The check for sex concordance showed that 2.7% (4/146) of the results might have been affected by maternal DNA contamination. In total, 32 samples presented inconsistent CNV profiles between the PGT and niPGT-A results, of which 12 and 20 tested positive for niPGT-A and PGT, respectively (Table ). A large proportion of the PGT-positive (55.0%, 11/20) and niPGT-A-positive (91.7%, 11/12) blastocysts were found to have mosaic aneuploidy, reflecting potential false-positive and false-negative representations of the TE biopsy PGT results (Table ). Concordance between SCM and ICM To confirm the concordance of TE and SCM with the gold standard ICM, ten PGT-A/SR-positive but niPGT-A negative embryos were donated from those families who gave birth to healthy babies. Clinical outcomes were collected from 23 patients who underwent single blastocyst transfer with embryos that were determined to be euploid by TE-PGT. Among them, 19 patients had euploid results from both TE-PGT and niPGT, and 8 achieved live birth (42.1%, 8/19); 4 patients with euploid TE-PGT results but aneuploid niPGT results, only 1 achieved live birth (25%, 1/4). The CNV profiles of the TE, ICM and niPGT-A samples of the same blastocyst are shown in Table . As determined by the ICM results, seven out of ten blastocysts were euploid, while the other three had mosaic aneuploidy or chromosomal abnormalities. Among the SCM samples, seven were actually true negatives, and three were false negatives (Fig. ; Table ). The 2nd round of TE PGT reported two positive embryos (true positive), one of which (EM81) was pinpointed to the same chromosomal locations as the ICM result. Moreover, the 2nd round of PGT results revealed five euploid embryos (true negatives), as supported by both the ICM and SCM (niPGT-A) results, which were marked as false positives in the 1st round of TE PGT. Considering the above ICM and 2nd TE PGT corrections, the overall concordance rate between the PGT and niPGT-A results improved to 82.9% (Table ). In addition, the 2nd round of TE biopsy PGT revealed a 50% (5/10) true-negative rate and a 20% (2/10) true-positive rate, while the 1st round of TE biopsy PGT revealed a 70.0% (7/10) false-positive rate. Notably, only a 40.0% (4/10) concordance rate was achieved between the two rounds of PGT tests using TE samples. The disagreement might be justified by the possible mosaicism of the blastocysts and technical variations between the two rounds of TE samplings.   To evaluate the feasibility of storing SCM samples at -80 °C, we first randomly collected 40 SCM samples from routine embryo culture. Half of each SCM (10 µL) sample was used for WGA, while the other half was stored at -80 °C for 3–6 months. Thirty-six out of 40 fresh SCM samples were successfully amplified and sequenced, with 19 being euploid and 17 being aneuploid. Subsequently, 36 pairs of frozen SCM sample were thawed after 3–6 months of storage at -80 °C and subjected to WGA and sequencing. All thirty-six frozen-thawed SCM samples were successfully amplified and showed 100% (36/36) concordance with the freshly amplified SCM samples (Fig. , Table ). These results indicated that SCM can be feasibly and routinely stored at -80 °C and used as a source for niPGT-A assays.   A total of 51 families who underwent IVF cycles of PGT-A or PGT-SR were included in the study, with 149 embryos screened using PGT with TE biopsy first. Standard PGT was performed on biopsies from these embryos, and the euploid embryos were implanted. However, the SCM samples of these embryos were collected for niPGT-A analysis. The SCM samples were stored at -80 °C for approximately three to six months before performing the niPGT-A assay. The embryos were grouped based on their parental karyotype information: 28 normal karyotype families with 59 embryos and 23 abnormal karyotype families with 87 embryos (Table ). Eighteen TE biopsies were screened by microarray, while the other 128 were sequenced by NGS. To rule out the effect of the use of different testing platforms, we reviewed the concordance rates and the CNV profiles of the PGT results obtained by microarray and NGS within the normal karyotype group. In this study, we considered only the overall ploidy status, namely, euploid versus aneuploid, when calculating the concordance rate between the niPGT-A and PGT results. No significant difference in the concordance rate was observed between the microarray (72.2%) and NGS (75.6%) detection platforms (Table ). Among these 149 ICSI embryos, paired TE biopsy and SCM samples were collected, and 146 (98.0%, 146/149) SCM samples were successfully amplified and sequenced (Fig. ). A concordance rate of 78.1% (114/146) was achieved between the niPGT-A results and the TE biopsy PGT-A and PGT-SR results, with 81 true-positive samples, 33 true-negative samples, 12 false-positive samples (euploid according to PGT but aneuploid according to niPGT-A), and 20 false-negative samples (aneuploid according to PGT but euploid according to niPGT-A) (Table ). The check for sex concordance showed that 2.7% (4/146) of the results might have been affected by maternal DNA contamination. In total, 32 samples presented inconsistent CNV profiles between the PGT and niPGT-A results, of which 12 and 20 tested positive for niPGT-A and PGT, respectively (Table ). A large proportion of the PGT-positive (55.0%, 11/20) and niPGT-A-positive (91.7%, 11/12) blastocysts were found to have mosaic aneuploidy, reflecting potential false-positive and false-negative representations of the TE biopsy PGT results (Table ).   To confirm the concordance of TE and SCM with the gold standard ICM, ten PGT-A/SR-positive but niPGT-A negative embryos were donated from those families who gave birth to healthy babies. Clinical outcomes were collected from 23 patients who underwent single blastocyst transfer with embryos that were determined to be euploid by TE-PGT. Among them, 19 patients had euploid results from both TE-PGT and niPGT, and 8 achieved live birth (42.1%, 8/19); 4 patients with euploid TE-PGT results but aneuploid niPGT results, only 1 achieved live birth (25%, 1/4). The CNV profiles of the TE, ICM and niPGT-A samples of the same blastocyst are shown in Table . As determined by the ICM results, seven out of ten blastocysts were euploid, while the other three had mosaic aneuploidy or chromosomal abnormalities. Among the SCM samples, seven were actually true negatives, and three were false negatives (Fig. ; Table ). The 2nd round of TE PGT reported two positive embryos (true positive), one of which (EM81) was pinpointed to the same chromosomal locations as the ICM result. Moreover, the 2nd round of PGT results revealed five euploid embryos (true negatives), as supported by both the ICM and SCM (niPGT-A) results, which were marked as false positives in the 1st round of TE PGT. Considering the above ICM and 2nd TE PGT corrections, the overall concordance rate between the PGT and niPGT-A results improved to 82.9% (Table ). In addition, the 2nd round of TE biopsy PGT revealed a 50% (5/10) true-negative rate and a 20% (2/10) true-positive rate, while the 1st round of TE biopsy PGT revealed a 70.0% (7/10) false-positive rate. Notably, only a 40.0% (4/10) concordance rate was achieved between the two rounds of PGT tests using TE samples. The disagreement might be justified by the possible mosaicism of the blastocysts and technical variations between the two rounds of TE samplings.   In this study, we demonstrated that storage of SCM samples at -80 °C for 3 to 6 months did not affect the amplification success rate or the accuracy of CNV detection. In the future, in cases where the results of PGT-A are mosaic, multiple abnormalities, or not available (N/A), niPGT via precollected SCM could be an alternative method to confirm chromosome ploidy. If the patients in the PGT-A cycle fail to achieve a successful transplant on the first attempt and there are no euploid embryos to be transplanted, SCM could be used as a backup sample for reassessing embryonic CNVs. This would allow embryologists to avoid performing unnecessary second biopsies or experiencing embryo loss due to inaccurate PGT-A results. However, the safety and clinical effect of niPGT still require the nonselection study and a subsequent randomized controlled trial to support. The SCM sampling method was adjusted (D4–D5/D6) according to the conventional SCM collection period (D3–D5). Additionally, removing the culture medium from the early blastocyst stage (D3–D4) can further reduce the risk of maternal DNA contamination. One may be concerned that removing the medium would lead to the loss of cfDNA from the previous D3–D4 culture; indeed, the majority of niPGT-A studies using SCM extracted between D3–D5 achieved remarkably high amplification rates of 89–100% . Nevertheless, SCM collected between D4–D5/6/7 and D4–D6/D7 also yielded equivalent detection rates of 95% and 92%, respectively , which is further supported by our amplification rate of 98% (146/149). More importantly, this extra step would help reduce the maternal contamination rate, according to the data in Huang et al.’s study . In this study, only 2.7% (4/146) of samples were suspected of having maternal contamination, suggesting that the above sampling method indeed helped reduce the percentage of maternal DNA present in the collected SCM as a result of the elimination of granulosa cell DNA input after the medium change on D4, thereby minimizing the inconsistency in interpreting the results. As shown in the results, a higher concordance rate (88.5% versus 74.6%) between niPGT and PGT was achieved in the patient group with chromosome rearrangements compared to the group with normal karyotypes (Table ). This is primarily due to the higher proportion of positive embryos present among patients with abnormal karyotypes than in patients with normal karyotypes. Researchers have used different ways to calculate consistency in recent years. (1) Clinical consistency refers to the test results impacting clinical decisions, as some researchers suppose that only euploid embryos would be considered for implantation. This means that clinical consistency is the foundation, aligns with the transplantation decision. (2) Full consistency can only be considered consistent when the test result is in line with the karyotype, especially for patients with known chromosomal abnormalities. (3) Partial consistency: Some researchers consider SCM and the gold standard to be two different types of samples. In this study, we calculated the clinical consistency, mainly considering that implantation is usually avoided in clinical practice when chromosomal abnormalities are identified within the embryos. Several studies have reported that some mosaic PGT-A embryos can also result in live births, indicating TE cells biopsied could not represent the entire embryos . The DNA in the spent medium likely originates from the ICM and TE, which undergo apoptosis during preimplantation development. Huang et al. reported niPGT-A has the potential to be superior to TE biopsy for PGT-A . The findings reported in this study agree with others on using a noninvasive approach (SCM) for human embryonic PGT. We conclude that embryonic cfDNA released between D4–D5/6 blastocyst cultures can be detected and that the whole genome can be amplified in almost all SCM samples. Further validation and testing are required to optimize the reporting threshold of mosaic chromosomal variations in niPGT-A readouts to maintain sensitivity and minimize the false-positive rate. Nevertheless, the noninvasiveness makes niPGT-A an informative supplement to PGT with TE biopsy and potentially a substitution in the near future. This study provides a noninvasive detection assay as a remedy for failed PGT. In the future, for patients of advanced maternal age or with low-quality embryos found in the early embryo development stage, the embryo culture media may be stored at -80 °C in advance. When an uncertain PGT-A result is obtained, especially for mosaic embryos, the reserved SCM samples can be used for CNV validation to avoid further damage from invasive TE biopsy and secondary vitrification. Li et al.  found that the SCM samples of embryos initially determined to be mosaic by PGT-A had a remarkably high amplification success rate of 97.6%, as well as an 87.2% ploidy consistency with the whole embryo, suggesting that SCM can be used for secondary validation of PGT outcomes. In addition, for patients who have not undergone PGT-A in the first IVF cycle with implantation failure or first-trimester miscarriage, simultaneously collecting SCM during embryo culture is also recommended. NiPGT-A results might provide further explanations, and rather than conventional PGT-A and a second cryopreservation, niPGT-A should be considered for the remaining cryopreserved embryos. Before clinical application of niPGT, the prognostics cohort study is still needed to evaluate the predictive values. If the predictive values are good, randomized controlled trial should be performed to assess the clinical efficiency. A recent study demonstrated that the application of the gentle WGA lysis process to biopsy cells prior to PGT was unable to amplify sperm DNA. Once the technique is improved to ensure that paternal cell interference is indeed neglectable, IVF insemination may be preferred to PGT for patients with a normal sperm number, motility, and morphology . The application of niPGT using SCM samples can be extended to IVF-inseminated embryos, avoiding the economic burden caused by ICSI insemination and allowing more people to potentially benefit. The study has several limitations. First, the WGA method is limited to NICSInst technique. It remains unclear whether other WGA methods can successfully amplify samples stored at -80 °C for six months. Further experimental design is required to validate this result. Second, the culture medium samples were not subjected to maternal contamination testing, which could potentially affect the CNV results. Third, the number of clinical outcomes collected is small. A wide range of factors could explain that difference observe in the two cohorts. Therefore, the larger nonselection studies and a randomized controlled trial are needed to confirm the clinical efficacy of niPGT.   In conclusion, this study demonstrated SCM stored at -80 °C for 6 months without affecting niPGT results based on NICSInst amplification.   Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2   