Non-invasive chromosome screening for embryo preimplantation using cell-free DNA : Reproductive and Developmental Medicine

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Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

He, Fang1; Yao, Ya-Xin2; Wang, Jing2; Zhao, Dun-Mei2; Wan, An-Qi2; Ren, Jun2,∗; Lei, Xi1,∗

Editor(s): Zhu, Yong-Qing

Author Information
Reproductive and Developmental Medicine 6(2):p 113-120, June 2022. | DOI: 10.1097/RD9.0000000000000023
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Abstract

Introduction

In vitro fertilization and embryo transfer (IVF-ET) is an effective procedure for treating infertility, which is an assisted reproductive technology, the patenets’ sperms and eggs fertilized in vitro conditions, then transfer the fertilized embryos back to the uterus. Each year millions of IVF-ET procedures were performed globally. However, the clinical outcomes of IVF treatments are often limited by low clinical pregnancy and high spontaneous abortion rates. In traditional IVF-ET practice, more than one embryo is often transferred per cycle of treatment, resulting in a high multiple pregnancy rate of ∼20% in IVF[1]. Multiple pregnancies increase chance of adverse pregnancy outcomes, which may hamper the health of both the mothers and the infants, so elective single ET (eSET) is the most effective way of reducing multiple pregnancy rate and has be increasing adopted internationally[2]. Therefore, how to select embryos with the best clinical outcome for eSET is the key to success in IVF-ET practice, mainly because of the lack of a systematic approach for evaluating the conceivability of the embryos. Presently, embryonic morphological assessment is still the most commonly used method for assessing embryo viability, but this method has limitations, such as the lack of quantifiable indicators and susceptibility to subjectivity from laboratory staff.

Chromosomal abnormalities commonly exist in early human embryos, and often cause embryo implantation failure and pregnancy loss IVF treatment, PGT for aneuploidies (PGT-A) can be used to identify embryos with chromosomal aneuploidy, thereby improving clinical outcomes in IVF patients[3–5]. Besides PGT-A, PGT for monogenic/single gene defects (PGT-M) and PGT for chromosomal structural rearrangements (PGT-SR) are included in PGT. PGT-M refers to testing for nuclear DNA pathogenic variant(s) causing monogenic disorders, with an autosomal dominant, autosomal recessive or X-linked transmission pattern, but also mitochondrial DNA (mtDNA) pathogenic variant(s). It also refers to exclusion testing and to human leukocyte antigen typing with or without concurrent testing for a monogenic disorder[6]. In case of familial rearrangements, PGT-SR provides an opportunity to identify chromosomally unbalanced progeny at the earliest stages of embryo development[7]. However, PGT relies heavily on invasive trophectoderm (TE) biopsy. The problem is that such biopsy procedure is often invasive and may hamper clinical outcomes as well as brings unknown health risks in long-term development of the embryos[8]. Also, embryo biopsy requires specialized equipment and extensive expertise in embryo treatment, which is difficult to standardize and very challenging to meet the demand of performing in every IVF-eSET treatment. Therefore, there is no doubt that an effective non-invasive chromosome screening approach is highly demanded to prioritize embryo for transfer in the clinical practice of IVF-eSET. Stigliani et al.[9] was the first to observe genome DNA contents in embryo culture medium, since then, multiple studies have been published of using culture medium or blastocoelic fluid for detection of chromosomal ploidy[10–13]. The current research focuses on the aneuploidy consistency between cell-free DNA (cfDNA) and embryos. However, the consistency comparison needs to consider many influencing factors, such as the definition of consistency, sampling methods, analysis methods, etc.

In this study, we aimed to comprehensively review the advancements and attempts at non-invasive PGT (niPGT)-A using fetal cfDNA and preliminarily summarize the research on embryonic cfDNA in non-invasive PGT-SR and PGT-M.

cfDNA research based on SCM

The discovery of cfDNA in SCM

Stigliani et al.[9] first demonstrated the presence of cfDNA in spent culture medium (SCM) in 2013. Subsequently, Hammond et al.[14] also detected mtDNA and gDNA in SCM. The presence of these genetic materials suggest that SCM can serve as a sampling source for early embryonic DNA, presenting a foundation for the development of a non-invasive method for PGT-A.

Studies of SCM in niPGT-A

In recent years, an increasing number of studies have been conducted to evaluate the feasibility of SCM-based niPGT-A approaches, and some of the results are encouraging[15]. The success rate of cfDNA amplification and detection is high, ranging from 77.3% to 100%[11,12,16,17]. Many studies have found that the PGT-A by TE biopsy or whole embryo of SCM has a high consistency rate[12,13,17,18]. Nevertheless, other research groups have reported relatively low consistency rates when compared with TE or whole embryo results[16,19,20]. The consistency to conventional PGT-A fluctuated widely, ranging from 33% to 100%. Thus, it is critical to understand the factors that can influence accuracy in niPGT-A.

In a clinical context, Xu et al.[10] used niPGT-A for the first time in patients with balanced translocations and subsequently obtained live births. Additionally, Rubio et al.[11] 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 two-fold (52.9% vs. 16.7%) higher than that in the latter group. Zero miscarriages were observed (0/9) when both the TE and SCM results indicated that the embryos were euploid. Moreover, a single-center clinical trial was conducted in 2019 using niPGT-A in patient groups with either repeated implant failures (≥3) or repeated miscarriages (≥3). The results of this trial showed 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[21]. While the scale of the above studies and clinical trials was small, cfDNA-based niPGT-A proved that, in principle, it could reduce miscarriage and improve the sustained pregnancy rate. Large-scale randomized and controlled clinical trials are needed to determine whether niPGT-A can be an effective method for evaluating embryo implantation potential.

Research of SCM in non-invasive PGT-M and PGT-SR

Assou et al.[22] used quantitative polymerase chain reaction (qPCR) for the first time to detect the target genes for Y chromosome in SCM in 2014, which is valuable for avoiding X-linked diseases in males. Galluzzi et al.[23] performed whole-genome amplification (WGA) on collected SCM from day (D) 3 and D5/6 embryos. The detection rate of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, which was associated to unexplained recurrent pregnancy loss, was 62.5% (5/8). Among the five SCM samples with successful amplification, only three TE results were available, and one paired TE and SCM result was consistent. In an α-thalassemia study, the amplification success rate of the target fragments was 88.62% using fluorescent gap PCR[24]. Liu et al.[25] used multiple annealing and looping-based amplification cycles (MALBAC) technology to perform WGA on SCM, combined with PCR and next-generation sequencing (NGS) methods, where the results were 80% successful; the genotype consistency reached 88% when the results were compared with whole embryos. Contrastingly, Capalbo et al.[26] showed that concordance between SCM and TE results was only 21%. In 2016, Xu et al.[10] performed non-invasive PGT-SR for the first time clinically, and of two patients with balanced translocations, one achieved a live birth. Jiao et al.[12] tested blastocyst fluid (BF) and SCM mixtures from 41 embryos donated by 22 couples, and the mixture results were 100% consistent with whole embryos. Overall, these studies suggest that SCM has the potential to be applied to non-invasive PGT-M and PGT-SR.

cfDNA research based on BF

The discovery of cfDNA in BF

Palini et al.[27] reported for the first time in 2013 that gDNA was identified in 90% (26/29) BF samples using qPCR, with an average amount of 9.9 pg. Furthermore, they confirmed that the sex of embryos could be determined by qPCR using BF. Subsequently, Zhang et al.[28] performed NGS of WG-amplified BF-DNA and compared the data with paired blastomere biopsy samples. The two DNA sources showed decent concordance in genomic coverage and pattern regions. Further analysis of the gene annotation results suggested that cfDNA in the BF contained sequences of the majority of genes, indicating that BF-derived cfDNA could be used to investigate and potentially treat monogenic diseases.

Studies of BF in niPGT-A

Recently, BF has been studied as a source for minimally invasive PGT-A. However, this method usually isolates a very small amount of approximately 0.01 μL BF[29,30]. In 2014, Gianaroli et al.[31] isolated BF from 51 blastocysts with an amplification success rate of 76.5%. The results of BF detected in 38 cases (38/39, 97.4%) were consistent with the ploidy of TE biopsy results. Magli et al.[29] improved the sampling and amplification of BF samples to a success rate of 82% (95/116). The ploidy consistency of the BF and TE samples was 97.1% (67/69). However, the results of other research groups have been less satisfactory.

Tobler et al.[32] obtained a WGA success rate of 63% (60/96) after thawing and culturing donated embryos to extract BF. The concordance between the BF karyotype and whole embryo was 48.3% (29/60) using array comparative genomic hybridization analysis, suggesting that BF may not be suitable for PGT-A. In 2018, Tšuiko et al.[30] performed chromosome analysis using NGS on 16 donated blastocysts, and the success rate of BF amplification was 87.5% (14/16). However, only 10 samples (10/16, 62.5%) passed sequencing and quality control for subsequent analysis. The results showed that only 40.0% (4/10) of the BF samples agreed completely with the TE or ICM results. Capalbo et al.[26] performed PGT-A on 23 BF samples and compared the results with those of the TE biopsy. Only eight cases (34.8%) were successfully amplified, and only three (37.5%) were consistent with the ploidy of the TE detection results.

In summary, studies assessing the reliability of BF-DNA as a template for PGT-A have yielded conflicting conclusions, with a 37.5% to 97.4% range of concordance with TE samples (Table 1). Differences in consistency levels between studies may be related to differences in embryo handling. Tsuiko and Tobler used frozen embryos donated by patients after treatment, and BF was absorbed after blastocyst resuscitation and expansion, with Tobler's study specifically suggesting that these embryos are not suitable for clinical use. Contrastingly, the BF samples of Gianaroli et al. were obtained from fresh culture cycles and underwent many procedures (polar body [PB] and blastomere biopsies and assisted incubation) that were not performed or relatively few of which were performed in other studies. Thus, the observed increased rates of amplification and concordance may be due to: (1) the superior quality of BF obtained from freshly cultured embryos over frozen embryos or (2) an increase in the DNA amount in the blastocyst cavity resulting from unintentional cell lysis or death during the procedure.

Table 1 - The concordance rates between BF and TE samples.
Study Fresh/frozen embryos Day of BF isolation Volume used of WGA WGA method WGA products detection, % (n) Concordance, % (n)
Gianaroli et al. [31] Fresh D5 1 μL SurePLEX 76.5% (39/51) 100.0% vs. WB (9/9)97.4% vs. TE (38/39)93.3% vs. PB (28/30)
Magli et al. [29] Fresh D5 0.01 μL SurePLEX 82% (95/116) 94.4% vs. WB (34/36)97.1% vs. TE (67/69)94.1% vs. PB (32/34)
Magli et al. [40] Fresh D5 The aspirated fluid was transferred into a 1-μL droplet of PBS SurePLEX 71% (182/256) 93.6% vs. TE (161/172)
Tšuiko et al. [30] Cryopreserved blastocysts D5 0.01 μL PicoPLEX 62.5% (10/16) 40.0% vs. ICM or TE (4/10)
Capalbo et al. [26] Fresh D5 The aspirated fluid was transferred into a 5-μL droplet of medium SurePLEX 34.8% (8/23) 37.5% vs. TE (3/8)
BF: Blastocyst fluid; PB: Polar body; TE: Trophectoderm; WGA: Whole-genome amplification.

Research of BF in non-invasive PGT-M and PGT-SR

Galluzzi et al.[23] used PicoPLEX for WGA of SCM, and the amplification success rate was 44.4% (4/9). The folic acid metabolism-related gene, MTHFR C677T, was successfully amplified in all four WGA products, and only one had a TE result, which was the same as that of the BF results. Zhang et al.[28] also used similar methods, with an amplification success rate of 72.7% (8/11); the amplification efficiency of hotspot mutation regions of ten randomly selected genes was 84.0% (42/50). In spinal muscular atrophy and phenylpropionate ketoneuria, a total of seven samples had 100% WGA success rates, and the sequences of six BF samples were consistent with their paired TE results[33]. However, in a study by Capalbo et al.[26], with a total of 69 pairs of BF and TE samples, the results were only 2.9% consistent. There are different consistencies of BF-result concordance with TE results in different studies, and further exploration is required.

Factors influencing niPGT

Sampling method

Culture system and sampling time

Currently, there are two common types of commercial embryo culture systems: sequential culture and single culture systems. Sequential culture medium is developed according to the needs of embryos for nutrients at different stages before implantation and the natural environment in the reproductive tract, mainly including fertilization, cleavage, and blastocyst culture medium. Single culture medium combines the nutrients needed during embryonic development, and the embryos choose the nutrients to take in. In culture, the quality of DNA in SCM degrades as a result of cfDNA degradation. Therefore, the amount of time and opportunity for cfDNA to degrade can be minimized by changing the media. Additionally, maternal DNA contamination introduced by residual cumulus cells can also be reduced or eliminated to a certain extent by changing the media. The results of a few studies that used a single culture system[25,34,35] and sequential culture medium[10,19,36] are listed in Table 2. Importantly, Rubio et al.[37] showed no significant differences in the influence of different culture systems on consistency when performing the same sampling process.

Table 2 - The concordance rates in different culture systems.
Study Media system Frozen: yes or no Timing of collection and embryo manipulation Volume for WGA Amplification method WGA products detection, % (n) DNA analysis Concordance rate, % (n)
Feichtinger et al. [34] Single medium No D1–D5/6 (fresh): embryos were cultured in single 25-μL droplets using a single step medium from fertilization until day 5/6. 5 μL SurePLEX 81.8% (18/22) aCGH 72.2% vs. PB (13/18)
Liu et al. [25] Single medium No D1–D5 (fresh): embryos were cultured in single 30-μL droplets using a single step medium from fertilization until day 5/6. 30 μL MALBAC 90.9% (80/88) NGS 64.5% vs. TE (20/31)
Ho et al. [35] Single medium Yes D1 (freeze-thaw) ∼D3: warmed 2PNs embryos were cultured in 25-μL medium until D3. 5 μL PicoPLEX 39.0% (16/41) NGS 56.3% vs. WB (9/16)
D1 (freeze-thaw) ∼D5: warmed 2PNs embryos were cultured in 25-μL medium until D5. 5 μL 80.4% (33/41) 65.0% vs. TE (26/40)
Vera-Rodriguez et al. [36] Sequential medium No D3–D5 (fresh): on day 3, each embryo was washed and moved to an individual 25-μL drop and cultured to blastocyst stage. 20 μL SurePLEX 91.1% (51/56) NGS 33.0% vs. TE (17/51)
Xu et al. [10] Sequential medium Yes D3 (freeze-thaw) ∼D5: warmed D3 embryos were placed in 30-μL droplets and cultured to blastocyst stage (D5) 5–20 μL MALBAC 100.0% (42/42) NGS 85.7% vs. TE (36/42)
Shamonki et al. [19] Sequential medium No D3–D5/D6 (fresh): on day 3, each embryo was washed and moved to an individual 15-μL drop and cultured to blastocyst stage. Repli-G 96.5% (55/57)
Rubio et al. [11] Single medium No D4–D6/D7 (fresh): on day 4, each embryo was washed and moved to an individual 10-μL drop and cultured to blastocyst stage (D6/7). 8–10 μL IonReproseq PGS Kit 94.8% (109/115) NGS 84.0% vs. TE (68/81)
D4–D5 (fresh): on day 4, each embryo was washed and moved to an individual 10-μL drop and cultured to blastocyst stage (D5). 63.0% vs. TE (17/27)
Rubio et al. [37] Sequential medium or Single medium No D4–D6/D7 (fresh): on day 4, each embryo was washed and moved to an individual 10-μL drop for at least 40 hours in culture. 10 μL IonReproseq PGS Kit 92.0% (1,197/1,301) NGS 78.2% (866/1108)
Huang et al. [43] Single medium Yes D5/D6 embryo (freeze-thaw): the thawed blastocysts were cultured for 24 hours in 15 μL medium, then the medium was collected. 3.5 μL MALBAC 92.3% (48/52) NGS 93.8% vs. WB (45/48)
Jiao et al. [12] Yes D5/D6 embryo (freeze-thaw): The thawed blastocysts were cultured in 12 μL medium for 15 hours; laser collapse to mix BF and SCM. 10 μL Improved MALBAC (MICS-Inst) 100.0% (41/41) NGS 90.5% vs. WB (19/21)
Li et al. [18] Yes D5/D6 embryo (freeze-thaw): The thawed blastocysts were cultured in 15 μL medium for 14–18 hours; laser collapse to mix BF and SCM. 10 μL Improved MALBAC (MICS-Inst) 97.6% (40/41) NGS 87.2% vs. WB (34/39)
Chen et al. [13] Sequential medium No D3–D5/D6 (fresh): on day 3, each embryo was washed and moved to an individual 30-μL drop and cultured to blastocyst stage. 20–25 μL MALBAC 100.0% (256/256) NGS 78.1% vs. WB (200/256)
aCGH: array-comparative genomic hybridisation; BF: Blastocyst fluid; MALBAC: Multiple annealing and looping-based amplification cycle; NGS: Next-generation sequencing; SCM: Spent culture medium; TE: Trophectoderm; WGA: Whole-genome amplification.

Additionally, SCM collected at different time points during embryo culture may result in varying test results. When comparing the SCM collected on D3 to D5 and D4 to D5, Lane et al.[38] found that the accuracy was higher in later samples, with >95% ploidy consistency and 100% consistency of the sex chromosome. The primary explanation for these observations may be that the number of embryonic cells increases exponentially with development and that the concentration of cfDNA surges dramatically at later stages of development. Rubio et al.[11] transferred embryos into new culture drops at D4 and collected SCM on D5 to D7. The SCM consistency of D4 to D6/7 was significantly higher than that of D4 to D5 (84.0% vs. 63.0%), and the level of maternal contamination was also reduced. A multicenter clinical study with a large sample size, conducted by the same group, compared SCM and TE samples using 1301 embryos, which achieved an average of 78.2% (866/1108) concordance using samples collected from D4 to D6/7[37].

In the latest article published by Huang et al.[39] in 2021, the collection of SCM was standardized, and two sampling methods were provided according to clinical practices. Compared with other sampling methods, the two sampling methods in this study had a higher consistency of detection results (84%, 81.6% vs. 77.2%), and the maternal contamination rate was greatly reduced (5%, 6.8% vs. 13.8%; Table 3).

Table 3 - The concordance between NICS and PGT-A in different options.
Concordance rate % (n) False negative rate % (n) False positive rate % (n) Maternal contamination rate % (n)
Option 1 (D4–D6/D7) 84.0% (68/81) 2.5% (2/81) 8.5% (7/81) 5.0% (4/81)
Option 2 (D5–D6/D7) 81.6% (59/73) 1.3% (1/73) 16.1% (12/73) 6.8% (5/73)
Other option (D3–D5/D6) 77.2% (78/101) 7.9% (8/101) 14.8% (15/101) 13.8% (14/101)
NICS: Non-invasive chromosome screening; PGT-A: Preimplantation genetic testing for aneuploidies.

BF volume and minimally invasive procedures

According to the results of previous studies, BF-DNA constitutes a high proportion of DNA amplification failure. The percentage of BF samples that successfully underwent WGA and produced detectable levels of DNA ranged from 34.8% to 82.0%[26,28,32,40]. The difficulty in the successful amplification of BF-DNA lies in the small amount of fluid obtained from the cystic cavity, as the BF volume reported in various studies ranged from 0.01 μL to 1 μL[29,31,40]. The volume difference can significantly impact the concentration of BF-DNA, which may have a negative effect on the efficiency of subsequent amplification. Therefore, BF-DNA may be less suitable for clinical application as an alternative genetic source[14]. In 2019, Magli et al.[40] showed a significantly higher WGA success rate of BF in aneuploid blastocysts (n = 150, 81%) than in euploid blastocysts (n = 32, 45%). SCM samples had a higher DNA content and detection success rate than BF samples[41]. Given that the total amount of DNA in BF is relatively low, combining BF and culture medium may increase the amount of cfDNA, thereby improving the amplification success rate and consistency of detection[42].

However, blastocyst puncturing for the obtainment of BF is still a minimally invasive procedure as an intracytoplasmic sperm injection (ICSI) needle is inserted into the blastocyst cavity to extract the fluid. In the presence of amplifiable cfDNA in BF, the low consistency of the test results with the results of TE biopsy or other gold standards suggests that technical variability exists. This may be as a result of the accidental acquirement of loose cells trapped in the lumen or cell material that has been shed. Thus, further optimization is required before BF-DNA can be utilized for PGT.

Methods of WGA

Different WGA techniques include multiple displacement amplification (MDA), MALBAC, and Sureplex/Picoplex.

Several niPGT-A studies have used MALBAC to amplify cfDNA in SCM with success rates of 90.9%[25], 92.3%[43], 97.5%[44], and 100%[10], and studies that used PicoPlex/SurePlex have exhibited success rates of 97.0%[35], 81.8%[34], and 89.3%[16]. Lledo et al.[45] compared Veriseq (Illumina®) and non-invasive chromosome screening (Non-invasive chromosome screening, Yikon®), both of which achieved 92.4% (85/92) amplification success rates, indicating that there was no significant difference between the two amplification techniques. A study using MDA in SCM showed an amplification success rate of 97%; however, only 2% of the amplified samples produced reliable PGT-A results[19]. The cfDNA of SCM is dominated by short fragments of length 160 to 220 bp[28], this would especially affect the MDA-based method, which requires longer DNA fragments to achieve optimal amplification.

Gold standard for niPGT evaluation

When evaluating the accuracy of SCM or BF detection, individual research groups may use different gold standards. At present, TE biopsy is the standard clinical practice for PGT-A, and is, therefore, often used as the gold standard. However, owing to the presence of mosaic embryos, researchers have questioned whether a small number of TE biopsy cells can, in fact, accurately represent the ploidy of the ICM.

Consequently, the D5 whole embryo may be the most appropriate gold standard for comparison with cfDNA when considering the accuracy of the selected comparison criteria. Several studies have reported the use of the whole embryo as the gold standard. In these studies, SCM collected from thawed blastocysts was assessed and overall consistency with whole embryo (WB) samples was achieved at a range of 90.5% to 96.4%[12,43,46]. Despite the fact that WB samples may be the most appropriate representation of the genetic status of the entire embryonic genome, it can be challenging to obtain donated WB samples.

Additionally, the state of the embryos (fresh or thawed), setting of mosaic thresholds, and determination of concordance are also factors that can affect detection accuracy.

Different fertilization methods may not be relevant

Intracytoplasmic sperm injection is usually required in patients undergoing PGT. American Society for Reproductive Medicine (ASRM) and European Society for Human Reproduction and Embryology (ESHRE) recommend ICSI for all couples undergoing PGT, including couples with non-male factor infertility. The main purpose of ICSI is to ensure single sperm fertilization to eliminate potential contamination by sperm attached to the zona pellucida and to prevent the presence of uncondensed sperm within blastomeres or cumulus cells[47,48].

Deng et al.[49] showed no significant differences in aneuploidy and mosaicism rates between IVF-and ICSI-fertilized embryos during split insemination cycles. De Munck et al.[50] used oocytes from 30 patients with normal ovarian function; half of the sibling oocytes were allocated to IVF, and the other half were allocated to ICSI. The euploid rate, blastulation rate, and total number of blastocysts were not different between the two fertilization methods. These results indicate that IVF can also be used for fertilization if sperm contamination is ruled out during PGT testing.

The niPGT study by Rubio et al.[51], which included ICSI and IVF populations, showed similar sensitivity (80.9% vs. 87.9%) and specificity (78.6% vs. 69.9%). In 2020, ASRM proposed that PGT for ICSI of non-male factors should be limited to cases where contamination of extraneous sperm could affect the accuracy of test results[52]. In the future, both fertilization methods and chromosome screening could be achieved non-invasively, and non-invasive preimplantation chromosomal aneuploidy detection may be performed without the limitation of fertilization methods.

Problems with the application of cfDNA to PGT

The origin of cfDNA

At present, there are several opinions regarding the origin of cfDNA. It is believed that during embryonic development, cfDNA may be released into culture media through cell lysis, apoptosis, cell debris, or other mechanisms[14]. In apoptotic cells, DNA is cleaved to form DNA fragmentsoligomers of approximately 180 bp[53]. A study by Zhang et al.[28] shows that these fragments have two peaks. The first peak has a range of 160 to 220 bp, whereas the second is broader, ranging from 300 to 400 bp. Bolton et al.[54] found that apoptosis was frequently observed within the ICM and TE of euploid and aneuploid cells in a mouse model. The results also exhibited that the percentage of apoptotic cells in the ICM and TE was 41.4% and 3.3%, respectively, in aneuploid embryos and 19.5% and 0.6%, respectively, in euploid embryos. The study by Bolton et al.[54], and others like it, have demonstrated that if cfDNA mainly originates from apoptotic cells, it primarily arises from the ICM. Moreover, Victor et al.[55] reported that, compared with aneuploid embryos and mosaic embryos, the TE and ICM in euploid cells exhibit lower levels of cell proliferation and apoptosis.

Maternal contamination

The discordance that generally occurs in SCM results and control embryos is mainly attributable to the high percentage of maternal DNA in the SCM. Vera-Rodriguez et al.[36] analyzed the SCM and TE results from 56 samples. Among them, 17 embryos were detected as aneuploid or aneuploid males (XY) by TE, whereas all were detected as aneuploid females (XX) by SCM. When Feichtinger et al.[34] compared the consistency between SCM and the PB, the negative control (SCM of fertilization failure oocytes) was also successfully amplified, indicating that oocytes are unlikely to abandon their DNA in the culture media. Therefore, maternal contamination may originate from cumulus cells or other exogenous DNA sources. However, contamination caused by in vitro DNA degradation may be minimized by changing the solution of a sequential culture on D3 and delaying the sampling time. In sequential culture, if the granular cells are not removed adequately before ICSI, it is recommended to remove them again while changing the medium at D3.

To reduce human interference and subjectivity, a calculation method should be developed to eliminate maternal DNA for internal quality control. Hammond et al. performed a short tandem repeat analysis on abandoned SCM, but the amount of DNA in the SCM was too small to identify any DNA. Short tandem repeat analysis involves long-range amplification, and highly fragmented SCM-DNA is unsuitable for this detection. Vera-Rodriguez et al.[36] conducted SNP sequencing of three groups of samples (TE/follicular fluid-DNA/embryonic SCM-DNA) from 35 embryos and successfully quantified the proportion of maternal DNA contamination in SCM. This suggests that SNP detection is a useful technique to evaluate maternal contamination in SCM.

Additionally, considering that the fragment size of cfDNA may differ from that of maternal DNA contamination, and that cfDNA possesses cleavage characteristics at the restriction site, maternal DNA can be identified and excluded, and analysis can be restricted to DNA that conforms to embryonic DNA. This proposes the premise for a DNA amplification and database construction method that can maintain the characteristics of DNA fragments or restriction sites, so that the original cfDNA template can be amplified with high fidelity.

Background cfDNA contamination of SCM

Some studies have suggested that another source of exogenous DNA contamination is the low level of background DNA in SCM. In 2017, Hammond et al.[56] detected low baseline levels of DNA in the base media and protein supplement components of three commercial culture media. They also detected a copy of nuclear DNA in the culture media with no previous contact with any embryos. Vera-Rodriguez et al.[36] detected DNA quantification using qPCR in 53 SCM and 17 control samples (culture medium with no previous contact with embryos). The average total amount of cfDNA in the blank control group was 1.4 pg, which means there is exogenous DNA contamination in the control group. In addition, Li et al.[44] found that the background DNA of the control culture did not interfere with the detection results, and all samples from the same embryo had the same sex chromosome diagnosis. The results indicated that although there was a low baseline of exogenous DNA in the control SCM, the effect could be ignored because the amount was negligible. Additionally, some commercial media use human serum albumin (HSA) to improve embryonic development. As HSA can adsorb DNA, the amount of HSA added should be controlled within a reasonable range. We suggest that the blank culture medium should be set as parallel culture at the same time as recovering blastocyst culture media for the quality control of SCM samples.

Future prospects

Currently, standard procedures are used for SCM sampling, amplification, and sequencing. With the accumulation of data, SCM can be used to develop detection methods for different populations. For low-risk populations with chromosomal abnormalities, the risk value of chromosomal abnormalities on high-risk chromosomes can be determined. For high-risk populations with chromosomal abnormalities, such as elderly individuals, the consistency between SCM test results and embryos is high, and the interference of false positives is relatively low. For this group, it is recommended to conduct whole-chromosome screening, determine the risk value of chromosomal abnormalities of each embryo, and determine the order of ET according to this risk value. For people with clear indications of PGT-A, we suggest that SCM samples be stored while biopsy samples are collected. If there is no result or the result of the biopsy cannot be used owing to sample loss or complications that occurred during the operation, SCM test results could provide remedial measures for detection failure. In addition, for IVF cycles with poor quality or a small number of embryos, the damage to embryos caused by biopsy operations should be minimized, and instead, the detection of chromosome aneuploidy by SCM should be considered under the premise of patient's informed consent.

Currently, exogenous contamination of embryos is a matter of concern. To improve the detection accuracy, DNA fragments from embryos should be identified by differentiating DNA fragment length or linkage analysis using a bioinformatics platform. Furthermore, studies have shown that the lysis conditions for WGA in biopsy cells (polar bodies, blastomeres, or TE cells) are mild enough to amplify sperm DNA, whereas single sperm DNA amplification requires strong lysis conditions, and IVF may also allow for PGT testing to be performed.

Concurrently, niPGT detection can provide a non-invasive option for aneuploidy detection and expand its comprehensive evaluation on the basis of morphology, while taking into account the DNA concentration, mosaic ratio, resolution, consistency with the gold standard, and other factors of embryonic SCM. The accumulation of clinical outcomes can also be combined with the clinical data of patients as an index to predict the clinical outcomes of embryos. A non-invasive artificial intelligence embryo evaluation model could be established, which would not only provide suggestions for clinicians on the order of embryo implantation, but also provide patients with the most suitable and economical detection scheme. This would ultimately alleviate possible stressful events during pregnancy and improve the overall success rate of IVF-ET.

Conclusion

We summarized the research and applications of SCM and BF in PGT and expounded both the factors that affect the detection results and current problems with the utilization of cfDNA in PGT. Non-invasive or minimally invasive PGT presents economic and operational advantages to genetic testing of embryos because it is practically harmless to the embryos and it waives the need for invasive biopsy. Currently, there are several studies on niPGT-A; however, more research is needed to evaluate the reliability of cfDNA in PGT. The advancement of reproductive technology will give rise to more studies focusing on the application of SCM or BF in non-invasive PGT-M and PGT-SR, which will ultimately benefit more patients.

Acknowledgments

None.

Author contributions

X.L., J.R., and F.H. designed the review; F.H. and Y.Y. wrote the manuscript; J.W., D.Z. and A.W. revised the manuscript and provided edits. All authors approved the final manuscript.

Funding(s)

This work was supported by the Science and Technology Major Projects of Birth Defect Collaborative Prevention in Hunan Province (No. 1019SK1010).

Conflicts of interest

All authors declare no conflict of interest.

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Keywords:

Aneuploid; Cell-free DNA; Non-invasive preimplantation genetic testing; Spent culture media

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