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

Secondary Logo

Journal Logo

Review Article

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

Lei, Cai-Xia1, 2; Sun, Xiao-Xi3, 4, 5,*

Author Information
Reproductive and Developmental Medicine: March 2022 - Volume 6 - Issue 1 - p 51-56
doi: 10.1097/RD9.0000000000000002
  • Open

Abstract

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.

References

[1]. Zhang S Lei CX Sun XX, et al. Current status and recent advances in preimplantation genetic testing for structural rearrangements. Reprod Dev Med 2020; 4(1):1-6. doi: 10.4103/2096-2924.281855.
[2]. Lei CX Zhang S Sun HY, et al. Outcome of couples with reciprocal translocation carrier undergoing the first preimplantation genetic testing cycles. Reprod Dev Med 2018; 2(1):30-37. doi: 10.4103/2096-2924.232873.
[3]. Lei CX Ye JF Sui YL, et al. Retrospective cohort study of preimplantation genetic testing for aneuploidy with comprehensive chromosome screening versus nonpreimplantation genetic testing in normal karyotype, secondary infertility patients with recurrent pregnancy loss. Reprod Dev Med 2019; 3(4):205-212. doi: 10.4103/2096-2924.274544.
[4]. Liu XL Xu CM Huang HF. Application and challenge of preimplantation genetic testing in reproductive medicine. Reprod Dev Med 2019; 3 (3):129-132. doi: 10.4103/2096-2924.268163.
[5]. Strom CM Levin R Strom S, et al. Neonatal outcome of preimplantation genetic diagnosis by polar body removal: the first 109 infants. Pediatrics 2000; 106(4):650-653. doi: 10.1542/peds.106.4.650.
[6]. Keymolen K Goossens V De Rycke M, et al. Clinical outcome of preimplantation genetic diagnosis for cystic fibrosis: the Brussels' experience. Eur J Hum Genet 2007; 15(7):752-758. doi: 10.1038/sj.ejhg.5201834.
[7]. Nekkebroeck J Bonduelle M Desmyttere S, et al. Mental and psychomotor development of 2-year-old children born after preimplantation genetic diagnosis/screening. Hum Reprod 2008; 23(7):1560-1566. doi: 10.1093/humrep/den033.
[8]. Desmyttere S De Schepper J Nekkebroeck J, et al. Two-year auxological and medical outcome of singletons born after embryo biopsy applied in preimplantation genetic diagnosis or preimplantation genetic screening. Hum Reprod 2009; 24(2):470-476. doi: 10.1093/humrep/den402.
[9]. Beukers F van der Heide M Middelburg KJ, et al. Morphologic abnormalities in 2-year-old children born after in vitro fertilization/intracytoplasmic sperm injection with preimplantation genetic screening: follow-up of a randomized controlled trial. Fertil Steril 2013; 99(2):408-413. doi: 10.1016/j.fertnstert.2012.10.024.
[10]. Schendelaar P Middelburg KJ Bos AF, et al. The effect of preimplantation genetic screening on neurological, cognitive and behavioural development in 4-year-old children: follow-up of a RCT. Hum Reprod 2013; 28(6):1508-1518. doi: 10.1093/humrep/det073.
[11]. Middelburg KJ van der Heide M Houtzager B, et al. Mental, psychomotor, neurologic, and behavioral outcomes of 2-year-old children born after preimplantation genetic screening: follow-up of a randomized controlled trial. Fertil Steril 2011; 96(1):165-169. doi: 10.1016/j.fertnstert.2011.04.081.
[12]. Seggers J Haadsma ML Bastide-van Gemert S, et al. Blood pressure and anthropometrics of 4-y-old children born after preimplantation genetic screening: follow-up of a unique, moderately sized, randomized controlled trial. Pediatr Res 2013; 74(5):606-614. doi: 10.1038/pr.2013.137.
[13]. Palini S Galluzzi L De Stefani S, et al. Genomic DNA in human blastocoele fluid. Reprod Biomed Online 2013; 26(6):603-610. doi: 10.1016/j.rbmo.2013.02.012.
[14]. Stigliani S Anserini P Venturini PL, et al. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Hum Reprod 2013; 28(10):2652-2660. doi: 10.1093/humrep/det314.
[15]. Stigliani S Persico L Lagazio C, et al. Mitochondrial DNA in day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome. Mol Hum Reprod 2014; 20 (12):1238-1246. doi: 10.1093/molehr/gau086.
[16]. Rodgaard T Heegaard PM Callesen H. Non-invasive assessment of invitro embryo quality to improve transfer success. Reprod Biomed Online 2015; 31(5):585-592. doi: 10.1016/j.rbmo.2015.08.003.
[17]. Capalbo A Ubaldi FM Cimadomo D, et al. MicroRNAs in spent blastocyst culture medium are derived from trophectoderm cells and can be explored for human embryo reproductive competence assessment. Fertil Steril 2016; 105(1):225-235. e221-223. doi: 10.1016/j.fertnstert.2015.09.014.
[18]. Hammond ER Shelling AN Cree LM. Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use. Hum Reprod 2016; 31(8):1653-1661. doi: 10.1093/humrep/dew132.
[19]. Shamonki MI Jin H Haimowitz Z, et al. Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertil Steril 2016; 106(6):1312-1318. doi: 10.1016/j.fertnstert.2016.07.1112.
[20]. Xu J Fang R Chen L, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. Proc Natl Acad Sci U S A 2016; 113(42):11907-11912. doi: 10.1073/pnas.1613294113.
[21]. Hammond ER McGillivray BC Wicker SM, et al. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified. Fertil Steril 2017; 107(1):220-228. e225. doi: 10.1016/j.fertnstert.2016.10.015.
[22]. Liu W Liu J Du H, et al. Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium. Ann Med 2017; 49(4):319-328. doi: 10.1080/07853890.2016.1254816.
[23]. Capalbo A Romanelli V Patassini C, et al. Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions. Fertil Steril 2018; 110 (5):870.e5-879.e5. doi: 10.1016/j.fertnstert.2018.05.031.
[24]. Farra C Choucair F Awwad J. Non-invasive pre-implantation genetic testing of human embryos: an emerging concept. Hum Reprod 2018; 33 (12):2162-2167. doi: 10.1093/humrep/dey314.
[25]. Ho JR Arrach N Rhodes-Long K, et al. Pushing the limits of detection: investigation of cell-free DNA for aneuploidy screening in embryos. Fertil Steril 2018; 110(3):467-475. e462. doi: 10.1016/j.fertnstert.2018.03.036.
[26]. Li P Song Z Yao Y, et al. Preimplantation genetic screening with spent culture medium/blastocoel fluid for in vitro fertilization. Sci Rep 2018; 8 (1):9275. doi: 10.1038/s41598-018-27367-4.
[27]. Huang L Bogale B Tang Y, et al. Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy. Proc Natl Acad Sci U S A 2019; 116(28):14105-14112. doi: 10.1073/pnas.1907472116.
[28]. Jiao J Shi B Sagnelli M, et al. Minimally invasive preimplantation genetic testing using blastocyst culture medium. Hum Reprod 2019; 34 (7):1369-1379. doi: 10.1093/humrep/dez075.
[29]. Rubio C Rienzi L Navarro-Sanchez L, et al. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications. Fertil Steril 2019; 112(3):510-519. doi: 10.1016/j.fertnstert.2019.04.038.
[30]. Sanchez-Ribas I Diaz-Gimeno P Quinonero A, et al. NGS analysis of human embryo culture media reveals mirnas of extra embryonic origin. Reprod Sci 2019; 26(2):214-222. doi: 10.1177/1933719118766252.
[31]. Kuznyetsov V Madjunkova S Abramov R, et al. Minimally invasive cell-free human embryo aneuploidy testing (miPGT-A) utilizing combined spent embryo culture medium and blastocoel fluid - towards development of a clinical assay. Sci Rep 2020; 10(1):7244. doi: 10.1038/s41598-020-64335-3.
[32]. Galluzzi L Palini S Stefani S, et al. Extracellular embryo genomic DNA and its potential for genotyping applications. Future Sci OA 2015; 1(4): FSO62. doi: 10.4155/fso.15.62.
[33]. Vera-Rodriguez M Diez-Juan A Jimenez-Almazan J, et al. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod 2018; 33 (4):745-756. doi: 10.1093/humrep/dey028.
[34]. Gianaroli L Magli MC Pomante A, et al. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril 2014; 102(6):1692.e6-1699.e6. doi: 10.1016/j.fertnstert.2014.08.021.
[35]. Herrera C Morikawa MI Castex CB, et al. Blastocele fluid from in vitroand in vivo-produced equine embryos contains nuclear DNA. Theriogenology 2015; 83(3):415-420. doi: 10.1016/j.theriogenology.2014.10.006.
[36]. Skryabin NA Lebedev IN Artukhova VG, et al. Molecular karyotyping of cell-free DNA from blastocoele fluid as a basis for noninvasive preimplantation genetic screening of aneuploidy. Russ J Genet 2015; 51 (11):1123-1128. doi: 10.1134/S1022795415110150.
[37]. Tobler KJ Zhao Y Ross R, et al. Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a wholegenome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis. Fertil Steril 2015; 104(2):418-425. doi: 10.1016/j.fertnstert.2015.04.028.
[38]. Magli MC Pomante A Cafueri G, et al. Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? Fertil Steril 2016; 105(3):676-683. e675. doi: 10.1016/j.fertnstert.2015.11.018.
[39]. Zhang Y Li N Wang L, et al. Molecular analysis of DNA in blastocoele fluid using next-generation sequencing. J Assist Reprod Genet 2016; 33 (5):637-645. doi: 10.1007/s10815-016-0667-7.
[40]. Hu Z Chen H Long Y, et al. The main sources of circulating cell-free DNA: apoptosis, necrosis and active secretion. Crit Rev Oncol Hematol 2021; 157:103166. doi: 10.1016/j.critrevonc.2020.103166.
[41]. Taglauer ES Wilkins-Haug L Bianchi DW. Review: cell-free fetal DNA in the maternal circulation as an indication of placental health and disease. Placenta 2014; (Suppl 35):S64-S68. doi: 10.1016/j.placenta.2013.11.014.
[42]. Jiang P Lo YMD. The long and short of circulating cell-free DNA and the ins and outs of molecular diagnostics. Trends Genet 2016; 32(6):360-371. doi: 10.1016/j.tig.2016.03.009.
[43]. Chen Y Gao Y Jia J, et al. DNA methylome reveals cellular origin of cellfree DNA in spent medium of human preimplantation embryos. J Clin Invest 2021; 131(12):e146051. doi: 10.1172/JCI146051.
[44]. Wu H Ding C Shen X, et al. Medium-based noninvasive preimplantation genetic diagnosis for human alpha-thalassemias-SEA. Medicine 2015; 94(12):e669. doi: 10.1097/MD.0000000000000669.
[45]. Yeung QSY Zhang YX Chung JPW, et al. A prospective study of noninvasive preimplantation genetic testing for aneuploidies (NiPGT-A) using next-generation sequencing (NGS) on spent culture media (SCM). J Assist Reprod Genet 2019; 36(8):1609-1621. doi: 10.1007/s10815-019-01517-7.
[46]. Chen J Jia L Li T, et al. Diagnostic efficiency of blastocyst culture medium in noninvasive preimplantation genetic testing. Fertil Steril Rep 2021; 2(1):88-94. doi: 10.1016/j.xfre.2020.09.004.
[47]. Kuznyetsov V Madjunkova S Antes R, et al. Evaluation of a novel noninvasive preimplantation genetic screening approach. PLoS One 2018; 13 (5):e0197262. doi: 10.1371/journal.pone.0197262.
[48]. Hanson BM Tao X Hong KH, et al. Noninvasive preimplantation genetic testing for aneuploidy exhibits high rates of deoxyribonucleic acid amplification failure and poor correlation with results obtained using trophectoderm biopsy. Fertil Steril 2021; 115(6):1461-1470. doi: 10.1016/j.fertnstert.2021.01.028.
[49]. Ramos-Ibeas P Gimeno I Canon-Beltran K, et al. Senescence and apoptosis during in vitro embryo development in a bovine model. Front Cell Dev Biol 2020; 8:619902. doi: 10.3389/fcell.2020.619902.
[50]. Rubio C Navarro-Sanchez L Garcia-Pascual CM, et al. Multicenter prospective study of concordance between embryonic cell-free DNA and trophectoderm biopsies from 1301 human blastocysts. Am J Obstet Gynecol 2020; 223(5):751.e1-751.e13. doi: 10.1016/j.ajog.2020.04.035.
[51]. Rubio C Racowsky C Barad DH, et al. Noninvasive preimplantation genetic testing for aneuploidy in spent culture medium as a substitute for trophectoderm biopsy. Fertil Steril 2021; 115(4):841-849. doi: 10.1016/j.fertnstert.2021.02.045.
Keywords:

Cell-free DNA; Embryo culture medium; Genomic DNA; Non-invasive; Pre-implantation genetic testing

Copyright copyright 2022 Reproductive and Developmental Medicine, Published by the Wolters Kluwer Health, Inc.