Character of cell-free genomic DNA in embryo culture medium and the prospect of its clinical application in preimplantation genetic testing

Abstract

There is increasing evidence that cell-free DNA (cfDNA) in spent culture media (SCM) can be amplified for genetic testing. Therefore, this paper reviews the characteristics of cfDNA, including its fragment size, amount, origin, as well as some factors affecting the success rate of its amplification, together to provide researchers with a more comprehensive perspective on embryonic cfDNA. The origin of cfDNA in SCM is complicated and poses challenges to the interpretation of genetic test results. Advanced molecular techniques should distinguish between embryonic and contaminated DNA to maximize the success rate of amplification and analysis. Recent data showed that the type of culture medium, assisted hatching or not, the type of amplification kit, and fresh or thawed embryos were not related to the success rate of amplification, but the length of culture time might affect the success rate. The longer culture time, the more cfDNA is available in the SCM. Then we focused on the concordance between trophectoderm (TE), inner cell mass, whole embryo, and embryonic cfDNA. Despite successful amplification, the concordance between TE and embryonic cfDNA was low. In summary, non-invasive genetic testing using SCM could represent a major advance in future single embryo selection, however, contamination and timing for media collection are key factors affecting the results, and current noninvasive cfDNA testing should not be directly applied to clinical practice. Further research is needed to improve the methods used for testing techniques and genetic analysis to achieve greater accuracy and trace its origins before it can be used in the clinics.

Introduction

Assisted reproductive technology has been at the forefront of technological innovation since its inception^[1], but compared to these advances, there is currently no particularly effective and reliable single embryo selection method. At present, embryo morphology scoring and preimplantation genetic testing (PGT) are mainly used as the main methods of single embryo selection, while genetic testing currently requires cell biopsy^[2,3], which is traumatic and invasive. There are 3 main biopsy methods^[4]: (1) Polar body biopsy, the first polar body is obtained during egg extraction and the second polar body is obtained after fertilization; (2) Blastomere biopsy, 1 or 2 blastomeres are biopsied when the embryo divides into 6 to 8 blastomeres at cleavage stage; (3) Trophectoderm (TE) biopsy, 3 to 10 TE cells are obtained from blastocysts on 5 or 6 days after fertilization. Although most studies have revealed no observable detrimental effects on implantation rate, pregnancy rate, neonatal outcome and the first 2 to 4 years of development of children born after biopsy procedure^[5-10], some studies have observed fine motor dysfunction, muscle tone dysregulation, and paramedical care more frequently present after biopsy^[11,12]. And sparse of adult data meant the traumatic procedure of PGT need long-term follow up and investigation. Therefore, it is important to develop non-invasive method for single embryo selection. Genomic DNA (gDNA) and mitochondrial DNA (mtDNA) are found in the secretome of human embryos^[13,14], indicating spent culture medium (SCM) might be an ideal material for genetic testing like circulating cell-free DNA (cfDNA) in the plasm of pregnant women. Till now, even though many studies have discussed the applications of cfDNA in clinical use to replace invasive PGT, cfDNA in the culture medium^[15-33] or cfDNA in the blastocele fluid^[34-39], there are conflicting views based on the idea whether invasive PGT should be replaced completely by non-invasive PGT. Here, we review current application of non-invasive PGT using cfDNA in SCM in reproductive medicine.

The size, amount, and origin of cfDNA in the SCM

Just like maternal serum cfDNA comes from apoptotic or necrotic cells of maternal cells and the placental cells, and from free DNA that is spontaneously secreted by these cells in pregnant women^[40], cfDNA in culture medium is also thought to be released by apoptotic or necrotic embryonic cells or maternal cumulus cells during embryo development or may be secreted from these cells. It has been hypothesized that fragments appeared during early embryo development are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, and early studies have demonstrated both gDNA and mtDNA are detectable in the secretome and the DNA profiles are significantly associated with fragmentation feature^[14]. By analyzing SCM collecting after embryo transfer or cryopreservation on Day 2 (n = 166) or Day 3 (n = 634), Stigliani et al.^[14] demonstrated that the amounts of total dsDNA in SCM were from 0.9 to 5 ng with the average 3 ng, and the gDNA size from 100 to 1,000 base pairs with the mean size 400 base pairs. They firstly purified cfDNA, and then quantified cfDNA using microfluidic electropherograms and realtime PCR quantification methods to get these results, which is like that of circulating cfDNA originating from apoptotic placental trophoblast cells^[41]. This is the only paper analyzing cfDNA size in the SCM, and another paper analyzed size of cfDNA in blastocoele fluid before whole genome amplification (WGA) by calculating the density of sequencing reads following next generation sequencing (NGS) after removing the contribution of adaptors, and they found size distribution ranging from 160 to 220 bp and 300 to 400 bp^[39]. One paper analyzed size of SCM cfDNA after WGA and found the mean size of the DNA amplified from medium samples was approximately 400 bp^[22]. We can infer that cfDNA fragments in SCM include fragments less than 200 bp and fragments greater than 250 bp. In a review from Jiang and Lo, they have written: “Plasma DNA fragments less than 200 bp would likely be derived from apoptosis whereas fragments greater than 250 bp would more likely be associated with other cell death phenomena^[42].” This suggests that embryo development period is complicated, and the origin of cfDNA in the SCM is not all related to cell apoptosis. More research is needed to elucidate the production of cfDNA in early embryo culture.

Many articles reported amount of cfDNA in SCM. Galluzzi et al.^[32] evaluated the amount of gDNA in SCM from Day 3 (n = 32) and Day 5/6 (n = 54), and they found 80 ± 70 pg (range 3-257 pg; median 58 pg) and 99 ± 113 pg (range 2.1-633 pg; median 67 pg) of gDNA were present in the medium on Day 3 and Day 5/6, respectively. After amplification, Shamonki et al.^[19] demonstrated DNA ranging from 2 to 642 ng/μL were detected in the products amplified using φ 29 polymerase. These results suggest that cfDNA in culture medium is sufficient for a variety of genetic tests, since the total amount of gDNA we need for singlecell sequencing is only the amount of gDNA in a single nucleus, that is, 6 to 9 pg. However, it is the proportion of embryonic gDNA in each SCM sample that determines the reliability of the final genetic test results, since the origin of cfDNA in SCM is complicated.

Origin of cfDNA detected in SCM should mostly reflect the genetic constitution of discarded cells diverging from the growing embryo genetic status, but the ingredients of culture medium and maternal contamination may also be a part of it. Hammond et al.^[21] found that there was consistently a very low level of DNA contamination in the G5 series and continuous singleculture media controls that had not been exposed to embryos, and thought it originated from the protein supplement. Human serum albumin with a high binding affinity for DNA is the major protein supplement in the culture medium, which will make it challenging to perform genetic testing and interpretation. Low levels of mtDNA were detected in media controls, the base medium and protein supplement components of the three commercial culture media^[21]. And significantly more mtDNA copies present in the SCM compared with the control media, suggesting that the proportion of gDNA in the SCM should be considered when testing. Another main contamination is from the maternal cumulus cells. Vera-Rodriguez et al. calculated the fraction of embryonic DNA in each SCM with the lowest percentage of 0% and the maximum percentage of 100% (median 8%) according to the haplotypes of the maternal DNA and embryonic DNA^[33]. All these results indicate that origin of cfDNA in culture medium is complicated, including not only embryonic gDNA and mtDNA, but also medium background DNA and maternal DNA contamination, and it is challenging to interpret genetic testing results from SCM amplification products. Recently, Chen et al. performed whole genome DNA methylation sequencing for SCM cfDNA, and the results demonstrated that SCM cfDNA was derived from blastocysts, cumulus cells and even polar bodies^[43]. Their work suggests that maternal DNA contamination should be considered first when performing SCM cfDNA tests, and that DNA methylation sequencing can accurately detect chromosomal aneuploidy in SCM and distinguish between low false negative rates and sex inconsistence rates in SCM samples after integrating origin analysis.

Success rate of amplification and its influencing factors: the culture medium, incubation days, assisted hatching (AH), amplification kits, and fresh or thawed embryos

The culture medium might influence the results of cfDNA amplification because the medium contains not only cfDNA, but also some other supplements that may affect the amplification. The basic composition of any media is water, salts, bicarbonate, glucose, pyruvate, amino acids, vitamins, and added with proteins. Since the first study analyzing mtDNA in the culture medium, different commercial media have been reported in cfDNA studies, for instance, Sydney IVF Blastocyst medium from Cook Medical^[14,15], G-series medium from Vitrolife^{[18,19,26,32,44-46]}, CCM medium from Vitrolife^[33], Quinn's Advantage Protein Plus blastocyst medium from SAGE^[20,23], Continuous single culture medium from Irvine Scientific^[21,23,29], Sage 1-Step medium from Origio^[47], and Origio Sequential Blast media from Cooper Surgical (Table 1)^[48]. All these media were successfully amplified, indicating that the components in the media had a low influence on the success rate of amplification. However, contaminated DNA in the culture medium may have an impact on the results. One study found that there was consistently a very low level of DNA contamination which might originate from the protein supplement in negative media controls that had not been exposed to embryos, and the occurrence of contamination will make it challenging to perform genetic interpretation^[21]. According to the above analysis, purification of cfDNA in the medium before amplification can reduce the influence of salts in the medium, and application of advanced molecular techniques can distinguish embryonic DNA from contaminated DNA, thus maximizing the success rate of amplification and analysis.

The incubation days might influence the results of cfDNA amplification because programmed cell death plays a key role in mammalian development and the origin of cfDNA in the culture medium are mainly dependent on apoptosis^[49]. Some researchers believe that culture time may significantly affect the success rate of amplification. The success rate of cfDNA amplification in collected media was analyzed for different incubation days ranging from 1 to 3 days at cleavage stage, 4 to 5 days at morula stage, 5/6/7 days at blastocyst stage, and 24 hours after thawing (Table 1). Early studies by Stigliani et al.^[14,15] have shown that successful amplification rate reached 62.9% and 73.2% from samples collected 2 or 3 days after fertilization. Galluzzi et al.^[32] have reported comparable success rate in samples collected at Day 3 and Day 5/6. Hanson et al.^[48] compared success rate based on the day of media changeover and timing of blastocyst biopsy and concluded that a shorter total duration of exposure of an embryo to its surrounding culture media were associated with higher rates of DNA amplification failure. Of the samples undergoing media collection and TE biopsy after 1 day of exposure to the culture media, 96.2% (25/26) demonstrated DNA amplification failure; after 2 days of exposure, 42.9% (27/63) exhibited DNA amplification failure; after 3 days of exposure, the rate of amplification failure decreased to 15.2% (10/66); and after 4 days of exposure, none of the 11 samples exhibited DNA amplification failure. From the above data, we can see a trend that the amplification success rate of culture media collected at the blastocyst stage is higher than that of culture media collected at the cleavage stage, suggesting that there may be more cfDNA from embryos at the blastocyst stage.

Table 1 - Success rate of amplification of embryonic cell-free DNA and its influencing factors

Reference	Collection day	Culture medium	Assisted hatching	DNA extraction	WGA kit	Quantification of gDNA	CCS	Fresh/thawed embryos	Amplification success rate	PGT success rate	Concordance rate
Stigliani et al.[14]	Day 2 Day 3	Sydney IVF	NA	QIAamp c	GenomePlex	Quantitative PCR (qPCR)	aCGH	Fresh	62.9% (205/326)	NA	NA
Stigliani et al.[15]	Day 3	Sydney IVF	NA	QIAamp c	NA	qPCR	NA	Fresh	73.2% (512/699)	NA	NA
Galluzzi et al.[32]	Day 3	G-1 PLUS	NA	NA	PicoPlex	qPCR	NA	Fresh	93.7% (30/32)	NA	NA
	Day 5/6	G-2 PLUS	NA	PicoPlex	qPCR	NA	Fresh	94.4% (51/54)	NA	NA
Wu et al.[44]	Day 3-4 Day 4-5	G-2 v5	NA	NA	NA	qPCR	NA	Fresh	82.1% (339/413)	NA	NA
Shamonki et al.[19]	Day 3	G-2	Yes, Day 3, laser	NA	Repli-G	Qubit	aCGH	Fresh	96.5% (55/57)	100.0% (6/6)	NA
Xu et al.[20]	Day 5	Quinn's	NA	NA	MALBAC	NA	NGS	Fresh	100% (42/42)	100% (42/42)	Compared with WE: 84.0% (21/25)
Hammond et al.[21]	Day 3 Day 5 Day 6	G-1 PLUS G-2 PLUS CSC	NA	NucleoSpin	REPLI-g; GenomePlex	QuantStudio 3D Digital PCR	TaqMan	Fresh	NA	NA	NA
Liu et al.[22]	Day 5	NA	NA	NA	MALBAC	Agilent 2100 instrument	NGS	Fresh	90.9% (80/88)	NA	NA
Capalbo et al.[23]	Day 5	Quinn's CSC	NA	EXTþAMP DirectAMP; QIAamp m	SurePlex	NA	NGS	Fresh	89.7% (339/378)	84.1% (58/69)	Compared with TE: 20.8%
Ho et al.[25]	Day 3	CSC	Yes, Day 3, diode laser	NA	PicoPlex	Qubit dsDNA HS Assay	NGS	Fresh	97.6% (40/41)	39.0% (16/41)	Compared with WE: 56.3% (9/16)
	Day 5							Fresh	97.6% (40/41)	80.4% (33/41)	Compared with TE: 45.5% (15/33)
Kuznyetsov et al.[47]	24 hours Post-thaw	Sage 1-Step	NA	NA	SurePlex	Qubit 3.0 Fluorimeter	NGS	Thawed	100% (28/28)	100.0% (28/28)	Compared with TE: 87.5%; Compared with WE: 96.4%
	Day 5/6							Fresh	100% (19/19)	100.0% (19/19)	Compared with TE: 100%
Li et al.[26]	Day 5/6	G-2 PLUS	NA	NA	MALBAC	Nanodrop	NGS	Fresh	92.5% (37/40)	92.5% (37/40)	NA
Vera-Rodriguez et al.[33]	Day 5/6	CCM	Yes, Day 3	NA	SurePlex	qPCR	NGS	Fresh	91.1% (51/56)	91.1% (51/56)	Compared with TE: 33.3% (17/51)
Huang et al.[27]	24 hours post-thaw	NA	NA	NA	MALBAC	NA	NGS	Thawed	100% (52/52)	100.0% (52/52)	Compared with WE: 93.8% (45/48)
Jiao et al.[28]	Day 5/6	NA	NA	NA	MALBAC	NA	NGS	Thawed	100% (62/62)	100.0% (62/62)	Compared with WE: 90.24%
Rubio et al.[29]	Day 5/6/7	CSC	NA	NA	Ion Reproseq	NA	NGS	Fresh	94.8% (109/115)	94.8% (109/115)	Compared with TE: 78.7%
Yeung et al.[45]	Day 5/6	G-2	Yes, Day 3, laser	NA	SurePlex	NA	NGS	Fresh	89.3% (150/168)	77.3% (116/150)	Compared with TE: 62.1%
Kuznyetsov et al.[31]	Day 5/6	NA	NA	NA	SurePlex	Qubit	NGS	Fresh	100% (86/86)	90.7% (78/86)	Compared with TE: 97.8% (88/90)
Rubio et al.[50]	Day 6/7	NA	NO hatching	NA	Ion ReproSeq	Qubit	NGS	Fresh	NA	90.1% (73/81)	Compared with TE: 78.2% (866/1,108)
Chen et al.[46]	Day 5/6	G-2	NA	NA	MALBAC	NA	NGS	Thawed	100.0% (26/26)	100% (26/26)	Compared with ICM: 80.8% (21/26)
Hanson et al.[48]	Day 5/6/7	Origio	Yes, Day 3, laser	NA	MALBAC	NA	NGS	Fresh	62.7% (104/166)	62.7% (104/166)	Compared with TE: 59.6% (62/104)
ICM: Inner cell mass; TE: Trophectoderm; WE: Whole embryos.
Companies producing the blastocyst culture medium listed above: 1, Sydney IVF: Sydney IVF Cleavage/ Blastocyst medium (Cook Medical, Australia; Cook Medical, Bloomington, IN). 2, G-1 PLUS: G-1 PLUS medium, G-2 PLUS: G-2 PLUS medium, G-2 v5: G-2 v5 medium, G-2: G-2 medium, G-MOPS PLUS (Vitrolife, Goteborg, Sweden). 3, CCM: CCM medium (Vitrolife AB, Sweden). 4, Quinn's: Quinn's Advantage Cleavage Medium/Quinn's Advantage Protein Plus blastocyst medium (SAGE). 5, CSC: Continuous single culture medium (Irvine Scientific, Australia). 6, Sage 1-Step: Sage 1-Step medium with serum protein supplement (Origio, Denmark). 7, Origio: Origio Sequential Blast media (Cooper Surgical, Trumbull, CT).
DNA extraction kit: 1, QIAamp c: QIAamp Circulating Nucleic Acid kit, QIAamp m: QIAamp DNAMicro Kit (Qiagen, Hilden, Germany). 2, NucleoSpin: NucleoSpin Plasma XS kit (Macherey-Nagel). 3, EXTþAMP (DNA extraction before amplification). 4, DirectAMP (loci amplification without prior DNA extraction).
Whole genome amplification (WGA) kit: 1, GenomePlex: GenomePlex Single Cell WGA kit (Sigma-Aldrich, St. Louis, MO). 2, PicoPlex: PicoPlex WGA kit (Rubicon Genomics, MI). 3, Repli-G: Repli-G single cell kit (Qiagen). 4, MALBAC: Multiple annealing and looping-based amplification cycles method (Yikon Genomics, Shanghai, China). 5, Sureplex: Sureplex DNA amplification system (Illumina, CA). 6, Ion ReproSeq: Ion ReproSeq PGS Kit (ThermoFisher Scientific, MA).
Comprehensive chromosome testing (CCS). 1, aCGH: array comparative genomic hybridisation. 2, NGS: Next-generation sequencing. 3, TaqMan: TaqMan PreAmp system.

Assisted hatching (AH) might influence the results of cfDNA amplification. Sampling from the embryos relies on creating a hole in the zona pellucida to allow TE cells to herniate out. Many methods can be used to breach the zona, such as acid tyrodes and laser AH. Although AH is considered benign in human preimplantation embryos, temperatures can reach 130 to 160°C during AH using a diode laser beam, which may cause harm to the embryos. The purpose of AH is twofold: on Days 5/6, to allow extrusion of the TE to facilitate TE biopsy, and to allow embryonic cfDNA to enter the culture medium through opening created by AH. Ho et al. have conducted a study to determine whether AH influences the viability of cfDNA and found slightly higher cfDNA concentration and concordant rates for embryos that did not undergo AH with no significance^[25]. They observed cfDNA concentration decrease in AH groups either on Day 3 (median cfDNA was 112 vs. 110 ng/mL for AH vs. no AH, respectively; P = 0.83) or on Day 5 (median cfDNA was 89.2 vs. 106 ng/mL for AH vs. no AH; P = 0.17). Rubio et al. retrospectively analyzed results of 115 samples of embryonic cfDNA from embryos completely without AH conducted before biopsy, and they found successful amplification and interpretable NGS results were obtained for 94.8% of these samples, informativity was significantly increased on Day 6/7 compared with Day 5 sample collection (P = 0.0004)^[29]. These results indicate that AH has little effect on the success rate of cfDNA amplification, but more data are needed to determine whether AH reduces the success rate of cfDNA amplification.

Various amplification technologies have been used to amplify SCM samples. In the early study, cfDNA was concentrated and purified in SCM by using the QIAamp^® Circulating Nucleic Acid kit^[14,15]. However, considering that the DNA amount in the SCM is minimal, it may be better to perform WGA first and then other experiments. Later studies did amplify SCM first without exceptions using many WGA kit, including GenomePlex Single Cell WGA kit^[14], PicoPlex WGA kit^[25,32,45], SurePlex WGA kit^[38], Repli-G single cell kit^[19], TaqMan PreAmp system^[21], Ion ReproSeq PGS Kit^[29,33], and multiple annealing and loopingbased amplification^{[20,22,26-28]}. The success rate of amplification was shown in Table 1. There were differences among different amplification kits, ranging from 62.7% to 100.0%. In addition, not all successfully amplified products can obtain copy number variation results after microarray or sequencing, and the probability of successful analysis ranges from 39% to 100%. Thus, the failure rate of embryonic cfDNA amplification is high, and the probability of effective results of successful amplification products is also reduced. This greatly limits the clinical application of this technique.

The amount of cfDNA in the culture medium may be increased after frozen-thawed embryos because the apoptosis rate is higher than that of embryos in fresh culture, and the collected SCM may allow easier and more accurate detection after amplification. Many studies showed a satisfied amplification rate in the thawing blastocysts. Kuznyetsov et al.^[47] collected cfDNA from 28 frozen-thawed and 19 fresh culture samples, and all samples were successfully amplified and performed PGT. Huang et al.^[27] thawed 52 frozen donated blastocysts and collected each of their SCM after 24 hours culture and performed PGT by NGS. The success rate of amplification and PGT of these samples reached 100%. Similarly, Jiao et al.^[28] and Chen et al.^[46] performed PGT using 62 and 26 SCM samples post thawed embryos respectively, and the amplification rate all reached 100%. These studies provide the possibility for rapid genetic analysis of frozen-thawed embryo before transfer. The culture media collected overnight after frozen-thawed blastocyst culture could also be successfully amplified, indicating that sufficient cfDNA could be generated in the culture media for effective amplification after 24 hours of culture. This 24 hour is critical, for if we could collect enough amount cfDNA within 24 hours after frozen-thawed culture, then we could also collect enough amount during the fresh culture period before transfer, following rapid NGS testing to get genetic results, and fresh euploid embryo transfer can be achieved. More data are needed at the appropriate time point for culture collection.

The ploidy concordant rates of cfDNA in SCM and TE/inner cell mass (ICM)/whole embryos

Whether testing of cfDNA can replace testing of biopsied cells also depends on the accuracy of the technology. Many studies have compared the ploidy concordant rates of SCM and TE. Capalbo et al.^[23] analyzed concordance of SCM samples with biopsied samples and found 20.8% of samples produced haplotypes identical to the corresponding TE. Kuznyetsov et al.^[47] compared concordant rate per sample between cfDNA in SCM 24 hours post thaw and Day 5/6 SCM from freshly cultured embryos with the corresponding biopsied samples, finding 87.5% and 100.0%, respectively. Other studies showed that concordant rate between 50% to 80%^{[29,31,33,45,46,48]}. Rubio et al.^[50] evaluated 78.2% (866/1,108) concordance in a large multicenter prospective study of human blastocysts, and concluded that concordance exceeded 86% when all defined steps in the culture laboratory were controlled to minimize the impact of maternal and operator contamination. These studies showed that the concordance between SCM and TE results is still poor, and there is a high risk of direct clinical application of this technique.

Even results from biopsied samples have significant limitations, including the possibility of false positives and false negatives. Due to the possibility of mosaicism of the whole embryo, the genomic composition of inner cell mass (ICM) cells may be different from that of TE cells, or the genomic composition of different TE cells may be different, thus the biopsied samples represent only a small fraction of TE cells. Some researchers believe that the cfDNA in SCM is more representative of the whole embryo, not just the TE layer. By profiling whole embryos, Xu et al.^[20] identified 21 of 25 SCM samples showed concordance, and the remaining 4 were false positives. Ho et al.^[25] found that the ploidy concordances between cfDNA and corresponding whole embryos was 9/16(56.3%) on Day 3. In 2 studies, SCM ploidy results were compared with that of whole embryo sequencing, and the concordance rate were 93.8%^[27] and 90.2%^[28], respectively. Chen et al.^[46] compared the concordance of SCM and ICM and found that using SCM can satisfactorily predict the karyotype of ICM, which may be more reliable than TE biopsy of mosaic embryos.

Conclusions and perspectives

As a new biomolecule, cfDNA in SCM has great potential in PGT and is currently in the critical transition period of rapid development. There is increasing evidence that cfDNA in the culture media can be amplified to give acceptable results in genetic testing. Therefore, this paper reviews the characteristics of cfDNA, including its origin and fragment size, as well as some factors affecting the success rate of its amplification, together to provide researchers with a more comprehensive perspective on embryonic cfDNA. The origin of cfDNA in culture medium is complicated and poses challenges to the interpretation of genetic test results. Advanced molecular techniques should distinguish between embryonic and contaminated DNA to maximize the success rate of amplification and analysis. Recent data showed that the type of culture medium, AH or not, the type of amplification kit and fresh or thawed embryo were not related to the success rate of amplification, but the length of culture time might affect the success rate of amplification. The longer culture time, the more cfDNA is available in the culture medium. Subsequently, we focused on the concordance between TE, ICM, whole embryo and embryonic cfDNA. Despite successful cfDNA amplification, the concordance between TE and embryonic cfDNA was low. However, some studies suggested that the concordance between SCM and ICM or the whole embryo was satisfactory. Ideally, SCM testing would be tested on day 3, since it has been reported that SCM testing is already representative of the whole embryo at this time point^[25]. If the results can be returned within 24 to 48 hours, it is not difficult to achieve fresh euploid embryo transfer. Moreover, the greatest advantage of SCM testing is to assess the ploidy status of embryos without biopsy, which may make it more attractive and lead to greater potential global application. If it can be proven to be reliable, it can provide the benefits of invasive PGT without the technical burdens. In addition, it can be provided as a preferred model to detect cfDNA in SCM, which, combined with morphology, can select live blastocysts that are most likely to produce healthy babies. This model can facilitate blastocyst prioritization so that providers and couples can select the best blastocysts for the first embryo transfer and retain others for later transfers^[51].

In summary, non-invasive genetic testing using SCM could represent a major advance in future single embryo selection, however, contamination and timing for media collection are key factors affecting the results, and current non-invasive cfDNA testing should not be directly applied to clinical practice. Further research is needed to improve the methods used for testing techniques and genetic analysis to achieve greater accuracy and trace its origins before it can be used in the clinics.

Acknowledgments

None.

Author contributions

Conception and design of the review: C.L. and X.S.

Funding(s)

This review was supported by Shanghai Shen Kang Hospital Development Center Municipal Hospital New Frontier Technology Joint Project (SHDC12017105).

Conflicts of interest

All authors declare no conflict of interest.