*2.3.4 Standard of the mosaic threshold of embryo*

Embryonic mosaicism refers to the existence of two or more cell lines with different genotypes in the same embryo. Some studies suggest that abnormal cells in a mosaic embryo are gradually diminished, i.e., a certain degree of self-repair occurs during embryonic development [47–49]. Mosaic embryos, especially those with a low proportion, still have a decent success rate of pregnancy after being implanted [50, 51]. Therefore, there is a trend of accepting low-frequency mosaic embryos in clinical practice.

Chromosome ploidy of mosaic embryos can be quantified by the proportion of mosaics (M), and samples with results above a certain threshold of M are identified as aneuploidy embryos. Therefore, the proportion of false positives and false negatives in embryo chromosome detection relies on the threshold setting of M. We observed that different studies had different criteria for reporting mosaicism, ranging from 30% to 60%. Yeung et al. [34] set the minimum threshold as 30% and recommend against the implantation of mosaic embryos >30%. Bolton et al. [47] found that the elimination of aneuploidy cells in the ICM through apoptosis may result in an increase in aneuploidy fragments in the SCM. Thus, increasing the chimaerism threshold of SCM samples may reduce the false positive rate and improve the consistency rate. In the study of Jiao et al., samples with a mosaic ratio of less than 40% were defined as euploid, while samples with ≥40% were defined as aneuploid [25]. Rubio et al.'s study directly defined samples with a chimaerism ratio of <50% as euploidy and ≥ 50% as aneuploidy, and no mosaic embryos were reported [28]. Huang et al. observed that using 60% as the threshold for distinguishing aneuploidy from euploid embryos achieved a false negative rate of zero, significantly improving the results of non-invasive PGT-A [24]. This study suggests that appropriately increasing the aneuploidy threshold can minimize the mosaic false positives caused by NGS results to a certain extent; however, these adjustments would require validation on a much larger scale.

#### *2.3.5 Whole embryo samples are more suitable as the gold standard for niPGT evaluation*

Individual research groups may use different gold standards when evaluating the accuracy of SCM or BF detection results. At present, TE biopsy is the standard clinical practice for PGT-A; thus, TE biopsy is often used as the gold standard. However, due to the presence of chimaeric embryos, studies have questioned whether a couple of TE biopsy cells is able to accurately represent the ploidy of the inner cell masses. There was also a study performed to compare the ploidy results from cfDNA and polar body biopsy [36]. Although the polar body is naturally released from oocytes, which are minimally invasive, it only examines aneuploidy of maternal meiosis origin and is limited by its inability to directly evaluate the genetic status of the entire embryo [52].

Consequently, the D5 whole embryo for comparison with cfDNA as the gold standard may be the most appropriate when considering the accuracy of the given selected comparison criteria. Several studies have reported the use of the whole embryo as the gold standard. SCM collected from thawed blastocysts was assessed, which achieved overall consistency with the WB samples, ranging from 90.48% to 96.4% in all these studies [23–25]. Nevertheless, it can be challenging to obtain

donated WB, even though it may be the best representation of the genetic status of the entire embryonic genome.

#### *2.3.6 The determination for concordance*

The concordance between the SCM and the selected gold standard, e.g., conventional TE biopsy or whole embryo, can be analysed from several aspects, as shown in **Table 2**.

First, the statistics of the consistency rate can be grouped into the following three situations: (1) the negative consistency rate (euploidy vs. euploidy), that is, both SCM and gold standard are negative results reported as euploidy, which is also the most critical indicator in clinical practice. Generally, clinicians choose euploid embryos for transplantation, and the accuracy of this indicator often directly impacts the clinical outcome. (2) The positive consistency rate (aneuploidy vs. aneuploidy), that is, both SCM and gold standard are positive results reported as aneuploidy. Usually, clinicians would not choose such embryos for transplantation. Hence, the accuracy will directly affect the availability of embryos for transplantation and the cycle cancellation rate. (3) The overall consistency, which is a comprehensive evaluation including both euploidy and aneuploidy.

Second, the researchers likewise have different ways to calculate consistency. (1) Clinical consistency refers to the test results for the impact on the clinical decision, as some researchers suppose that only euploid embryos would be considered for implantation, which means clinical consistency is the foundation, which agrees with the transplant decision (as shown in **Figure 6**). (2) Full consistency: it can only be considered consistent when the test result is absolutely in line with the karyotype, especially for patients with known chromosomal abnormalities (as shown in **Figure 6**). (3) Partial consistency: Some researchers consider SCM and the gold standard to be two different types of samples. Thus, they should be considered as consistent if there is partial consistency with the karyotypes.

Third, the criteria for the reporting of aneuploidy are not uniform. Researchers usually consider whether to report fragmentation abnormalities or whole chromosome abnormalities and whether to report mosaic results and their reporting criteria.

Through the analysis of a series of related studies (see **Table 2**), we observed that although different investigators used different methods to calculate consistency, the consistency results were superior to other methods when only clinical consistency rates were considered.

In the studies of Huang et al. [24], Jiao et al. [25], Li et al. [35], Liu et al. [38], and Yeung et al. [34], when the karyotype was completely consistent or partially consistent, the results' coincidence rates to the gold standard were 83.3%, 76.2%, 50%, 64.5% and 62.1%, respectively. However, the consistency rates increased to 93.8%, 90.5%, 78.9%, 83.9% and 73.3%, respectively, when only clinical consistency was considered.

In addition, in the analysis of Kuznyetsov et al., if only chromosomal aneuploidy was considered, the clinical consistency rate was often better than the results of fragment abnormalities and consistent karyotypes. The clinical concordance of frozen embryos and fresh embryos was 96.4% and 100% without considering chimaerism and fragment abnormalities, respectively, but decreased to 78.5% and 73.7% when considering chimaerism and fragment abnormalities, respectively. In addition, only euploidy and aneuploidy were distinguished according to the chromosomal test results given, and the positive and negative consistency rates in each study were calculated.


#### *Non-Invasive Chromosome Screening for Embryo Preimplantation Using Cell-Free DNA DOI: http://dx.doi.org/10.5772/intechopen.98416*



 *rate.* *Non-Invasive Chromosome Screening for Embryo Preimplantation Using Cell-Free DNA DOI: http://dx.doi.org/10.5772/intechopen.98416*

#### **Figure 6.**

*Definition of concordance between SCM and the gold standard are at different levels in studies. Yeung et al. defines concordance at chromosome level, while Rubio et al. defines at the level of clinical concordance.*

In general, the negative consistency rate is better than the positive consistency rate in SCM, which may indicate high false positives in the niPGT-A test. On the other hand, it also shows that the negative result of this approach is more reliable. In summary, the definition of consistency has a direct impact on the final results of the assessment method. Considering the speciality of cfDNA presented in SCM, it seems insufficient to define consistency purely based on the perfect match of the CNV patterns. Chen et al. [27] proposed the embryos can be categorized or scored by the obtained niPGT-A results into three groups: (A) chromosomal normal, (B) chromosomal abnormal, and (C) multiple abnormal chromosomes or uncertain. The results showed group A predicts a normal embryo with 90.0% probability, while groups B and C predict 27.8% and 72.2% normal probability, respectively. Thus, it may be necessary to fully consider the conditions, such as mosaicism, ploidy status and test resolution, when establishing the analytical model.

It seems more appropriate to use whole blastocysts as the gold standard for evaluation of the accuracy of niPGTA. Apparently, the establishment of the model would require large embryos of SCM and WB paired samples, with standardized sample collection and data analysis procedures. Furthermore, the model can evaluate SCM according to the euploidy probability of embryos to obtain the priority or exclusion order of embryos for implantation to avoid the waste of embryos due to false-positive decisions led by conventional PGT-A.

#### **2.4 Clinical application of non-invasive PGT-A**

At present, several studies have applied non-invasive PGT-A to diverse patient groups to preliminarily evaluate the clinical manifestations of the technique. Xu et al. [31] used non-invasive PGT-A for the first time in balanced translocation patients and obtained five live births from seven couples. Rubio et al. [28] compared the clinical outcomes of two groups of patients, one with both TE biopsy and SCM results of euploidy and the other with TE biopsy negative and SCM results of aneuploidy. The transplant success rate of patients with both euploidy TE and SCM results was twofold (52.9% vs. 16.7%) higher than that of the latter group. Zero miscarriages were observed (0/9) when both the TE and SCM results indicated that the embryos were euploid. Moreover, Fang et al. [53] reported the results of a single-centre clinical trial in 2019 applying non-invasive PGT-A on patient groups with either repeated implant failures (≥3) or repeated miscarriages (≥3). The trial included 45 couples with 50 ART cycles, resulting in a biochemical pregnancy rate of 72% (36/50), a clinical pregnancy rate of 58% (29/50), and a spontaneous miscarriage rate of 10% (3/29), with a total of 27 babies successfully delivered. While the clinical trial scale of the above studies was small, the cfDNA-based niPGT-A proved in principle that it could reduce miscarriage and improve the ongoing pregnancy rate. Conversely, large-scale randomized controlled clinical trials are needed to conclude whether non-invasive PGT-A can be an effective way to evaluate embryo implantation potential.
