Journal of Genetics and Genomics xxx (xxxx) xxx

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Journal of Genetics and Genomics

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Noninvasive preimplantation genetic testing in assisted reproductive

technology: current state and future perspectives
Jingyi Li a, 1, Yifeng Liu a, 1, Yuli Qian b, Dan Zhang a, b, *
a
Key Laboratory of Reproductive Genetics (Ministry of Education) and Department of Reproductive Endocrinology, Women's Hospital, Zhejiang University
School of Medicine, Hangzhou, Zhejiang 310006, PR China
b
Key Laboratory of Women's Reproductive Health of Zhejiang Province, Hangzhou, Zhejiang 310006, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Invasive genetic screening of pre-implantation embryos via biopsied trophectoderm (TE) cells has been
Received 15 June 2020 in use for more than 20 years, while its benefits in selecting euploid embryos remain controversial.
Received in revised form Recent advances in the ability to process embryonic cell-free DNA (cfDNA) from blastocoel fluid (BF) and
10 November 2020
spent culture media (SCM) of blastocysts in a manner similar to that of a biopsied TE sample provide a
Accepted 13 November 2020
Available online xxx
potential alternative holding great promise for obtaining cytogenetic information of the embryos
without intrusive biopsy of traditional biopsy-based pre-implantation genetic testing (PGT). Several
studies have reported even higher diagnostic accuracy in non-invasive PGT (ni-PGT) than conventional
Keywords:
Pre-implantation genetic testing
PGT. However, there are still several technical challenges to be overcome before ni-PGT can be accepted
Spent culture media as a reliable genomic information source of embryo. In this review, we have summarized the emergence
Blastocoel fluid and current state of ni-PGT, and discussed our own perspectives on their limitations and future prospect.
Cell-free DNA There is still long way to go before truly wide clinical application of ni-PGT.
Non-invasive PGT Copyright © 2021, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and
Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

Preimplantation genetic testing for aneuploidy (PGT-A), defined
as a genetic test on embryos by in vitro fertilization (IVF)-based
assisted reproductive technology (ART) treatment, has been
increasingly used to select euploid embryos and improve the suc-
cessful rate of pregnancy in ART. Despite its application in ART
clinics for more than 20 years, many issues of PGT-A remain un-
solved (Gleicher et al., 2018). First, the effectiveness of PGT-A is
currently under debate as several studies have shown that PGT-A
had no benefits in improving live birth rates in patients with
advanced age, repeated implantation failures (RIF), or recurrent
miscarriages (RM) (Verpoest et al., 2018; Munne et al., 2019). Sec-
ond, in cases of mosaic embryos, PGT-A based on a single specimen
taken from the trophectoderm (TE) of the embryo may lead to false-
positive interpretation and thus abandonment of healthy embryos,
rendering the clinical application of PGT-A to be a risky procedure
(Lawrenz et al., 2019; Popovic et al., 2019). In addition, the biopsy
procedures such as the number of cells biopsied and the method to
separate the specimen from the embryo have not been standard-
ized and vary between IVF laboratories. Most crucially, the poten-
tial effect of the invasive biopsy procedure on embryo viability and
* Corresponding author.
E-mail address: zhangdan@zju.edu.cn (D. Zhang).
1
These two authors contributed equally to this article.
even the offspring health remains uncertain. In animal studies, data
have suggested that embryonic biopsies might have effects on ad-
renal or neural tube development in the fetus, increase the risk of
liver associated insulin resistance, and affect body weight and
behavior in adult mice (Zeng et al., 2013; Sampino et al., 2014;
Zheng et al., 2018). In addition, Shaia et al. (2020) report that pre-
implantation genetic testing (PGT) might cause a sex ratio skewed
to male offspring in the IVF population. Therefore, doubts remain
about the repercussions of the invasive action of removing cells
during biopsy on the health of these children.
1. Emergence and current status of noninvasive PGT
Recently, several noninvasive PGT (ni-PGT) methods have
emerged and shown promising diagnostic potentials. Palini et al.
(2013) reported the detection of embryo DNA by real-time PCR in
blastocoel fluid (BF), the fluid inside the blastocoel cavity of a hu-
man blastocyst-stage embryo. Furthermore, the possibility for
directly determining the sex of the embryo via BF was demon-
strated, suggesting the potential of screening embryos for X-linked
disorders using BF (Palini et al., 2013). Later, several studies also
supported BF as a promising noninvasive alternative method to
obtain information of chromosome aneuploidy of the embryo
(Gianaroli et al., 2014; Magli et al., 2016). Besides, cell-free DNAs

https://doi.org/10.1016/j.jgg.2020.11.007
1673-8527/Copyright © 2021, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and
Science Press. All rights reserved.

Please cite this article as: J. Li, Y. Liu, Y. Qian et al., Noninvasive preimplantation genetic testing in assisted reproductive technology: current state
and future perspectives, Journal of Genetics and Genomics, https://doi.org/10.1016/j.jgg.2020.11.007
J. Li, Y. Liu, Y. Qian et al.
(cfDNAs) were also identified in BF to be suitable for monogenic
disease screening (Zhang et al., 2016). Nevertheless, although the
aspiration of BF is less invasive than TE biopsy, it is still not
completely noninvasive (Gianaroli et al., 2014). The spent culture
medium (SCM) of an embryo is proposed to be used for PGT
because it is completely noninvasive to the embryo. In the last three
years, a growing body of literature has demonstrated the capability
to detect, extract, and amplify cfDNA from SCM at the cleavage and
blastocyst stages and has investigated the clinical application of
SCM for ni-PGT (Shamonki et al., 2016; Xu et al., 2016; Hammond
et al., 2017), although the biological mechanism for the release of
embryonic cfDNA into the SCM remains elusive. Notably, Xu et al.
(2016) reported a specificity of 84.0% and a sensitivity of 88.2% in
identifying aneuploidies in Day 3 or Day 5 SCM samples of donated
vitrified-warmed embryos by next-generation sequencing (NGS) of
24 chromosomes in total. They claimed a higher efficiency in the
selection of euploid embryos than in identifying aneuploid ones,
raising a new nonintrusive and elegant prospect for preimplanta-
tion genetic diagnosis. Such a ni-PGT approach would outperform
current strategies by eliminating a costly micromanipulation bi-
opsy procedure and avoiding any cell biopsied associated risks.
However, the concordance between SCM and embryo biopsy
sample or whole embryo was reported to be in a wide range from
30.4% to 99.7% (Table S1). Recently, Huang et al. (2019) reported
that the false-positive rate was significantly lower in ni-PGT (20%)
than in routine PGT-A (50%). On the other hand, there was a 100%
concordance between ni-PGT and total blastocyst screening for
diagnosing euploid. Similar advantages of ni-PGT were reported by
another study in the same year conducted by a group called NICS-
Brazil involving 12 assisted reproduction centers (Franco, 2019).
2. Limitations of ni-PGT and future potential solutions
Although cfDNA was clearly present in the SCM, its representa-
tiveness of the chromosome constitution of the corresponding em-
bryo is uncertain. Growing evidence has shown that massive
parental contamination arising from cumulus cells, sperm, and polar
bodies may be present in most SCM samples (Feichtinger et al., 2017;
Hammond et al., 2017; Vera-Rodriguez et al., 2018) (Fig. 1). As to
reducing extraneous DNA contamination risks of maternal origin
from cumulus cells or polar bodies, BF aspirated from inside the
embryos may have certain advantages. BF removal has already been
performed in many clinics as part of routine in blastocyst cryopres-
ervation to decrease ice crystal formation. Considering that the
collapsing caused by BF removal artificially expands blastocysts
before vitrification and thus may improve pregnancy outcome, BF
aspiration seems to be an appealing approach with minimum inva-
siveness to obtain embryo-derived DNAs. However, studies by
Hammond et al. (2017) suggest that BF may affect intercellular
communication between the developing embryo and its surround-
ings. Whether removal of the BF would alter the property of the
embryo remains unknown. Sperm attached to the zona pellucida
during conventional IVF may be the main source of contamination of
paternal origin. Removing all outer cumulus cells carefully and per-
forming intracytoplasmic injection of single sperm (ICSI) for all may
be an alternative approach to reduce extraneous DNA contamination.
However, it will result in an artificial increase of ICSI usage, which is
ethically controversial and not recommended. The evidence so far
suggests existing differences in general physical health, metabolic,
and reproductive endpoints between children conceived by ICSI and
spontaneous conception (Catford et al., 2018). Therefore, elevated
usage of ICSI may pose potential risks to the health of offsprings. In
addition, it is well known that the cross talk between oocytes and
surrounding granulosa cells is required for the development and
maturation of oocytes. Therefore, removal of outer granulosa cells
2
Journal of Genetics and Genomics xxx (xxxx) xxx
may affect the maturation and fertilization of the oocytes, as well as
the embryo development at the early stage.
The cellular origins of embryonic DNAs disseminated into the
surrounding BF or SCM are still unknown. Some reports hypothe-
size that the cfDNA in BF is released by both apoptotic inner cell
mass and TE cells during normal embryo development (Hardy,
1997; Huang et al., 2019). However, Chi et al. (2011) reported that
cell necrosis may also be a possible source of long DNA fragments in
BF and SCM. If mosaic embryos discharge their aneuploid cells into
the BF or SCM during development as part of a self-correction
mechanism, it may lead to a potential mismatch between the
ploidy status of the SCM or BF-derived cfDNAs and the corre-
sponding embryo, which has succeeded in eliminating aneuploid
cells (Fig. S1). Aneuploidy embryos are known to arise from
segregation errors in both meiosis and mitosis. Aneuploidy em-
bryos due to meiotic errors are unlikely to be self-corrected. It is
accepted in general that the age-dependent elevated incidence of
spontaneous abortion and infertility is due to the increased
occurrence of oocytes with meiotic errors (Nagaoka et al., 2012).
Considering the prevailing meiotic error etiology in the main group
of patients undergoing PGT and the relatively low yield of mitotic
error correction, it seems that embryonic mosaicism may not be a
substantial confounding factor for ni-PGT. Vera-Rodriguez et al.
(2018) reported that the contribution of cell shedding mecha-
nisms to cfDNA was comparable between aneuploid and euploid
embryos, suggesting that self-correction for eliminating aneu-
ploidies may not be the main source of cfDNA in humans. Another
question is whether the DNA is truly ‘cell free’ or actually mixed
with shedding of some embryonic cells. However, studies remain
conflicting regarding whether the cfDNA obtained from BF or SCM
can truly represent the genetic composition of the embryo in
general. Future research focusing on the mechanism of cfDNA
production is warranted to address these critical questions.
No consensus yet regarding the most appropriate time for SCM
collection. Xu et al. (2016) reported the success in amplifying cfDNA
in all examined samples of exchanged culture media on Day 3 of
preimplantation development. Lagalla et al. (2017) reported a
higher accuracy of aneuploidy testing in SCM in contact with Day
4e5 embryos compared with Day 3e5 embryos, perhaps due to an
increase in the ratio of embryonic-to-maternal DNA from the
exponential rise of embryonic cells during blastulation. Other
studies proposed a continuous medium system until blastulation to
increase the cumulative cfDNA yield from embryos (Feichtinger
et al., 2017; Hammond et al., 2017). However, given the low suc-
cessful rate for embryo development into the blastocyst stage, the
requirement of collecting culture media until Day 5 may lead to
natural elimination of a portion of embryos with normal ploidy.
Furthermore, artificial increase in in vitro culture time of the em-
bryo may also pose potential detrimental effects on embryo
development and long-time health of the offsprings. It remains to
be elucidated which culture medium system and which stage of
embryo development for sampling are the safest and at the best
balancing point between elevating amplification rates and reducing
extraneous DNA contamination.
A technically reliable procedure qualified for successful DNA
extraction and amplification also remains to be determined. It has
been reported that failure rates of amplifying BF-derived DNAs are
disappointingly higher than those of TE cells, which was generally
accepted as 2% (Handyside, 2016). Some studies suggested trans-
ferring samples into cold PCR tubes and centrifuging them
promptly to improve DNA yield and thus the following amplifica-
tion or molecular analysis (Magli et al., 2016). With higher genome
coverage and a lower allele dropout ratio, multiple annealing and
looping-based amplification cycles (MALBAC) has been proposed as
the preferred amplification procedure in analyzing cfDNAs (Zong
J. Li, Y. Liu, Y. Qian et al.
Fig. 1. The procedure of noninvasive preimplantation and possible origins of parental contamination. BF, SCM from Day 3 or Day 5 embryos are the most reliable source of in-
formation on the embryonic genome; sperm, cumulus granulosa cells and polar bodies are the main source of parental contamination. BF, blastocoel fluid; SCM, spent culture
medium.
et al., 2012; Xu et al., 2016). The future of ni-PGT may closely rely on
the development of novel strategies of genome-wide massively
parallel sequencing (MPS) with high uniformity and fidelity.
The cost-benefit ratio of ni-PGT should not to be ignored
either. Ni-PGT will result in additional costs. In particular, to
assess SCM cfDNA, embryos have to be cultured separately,
which may lead to an increase in extra requirement of culture
medium volume, number of Petri dishes, and use of incubators in
total per patient.
3. Future perspectives
Increasing infertility rate and desired pregnancies with
advanced maternal age are major drivers of PGT. It is estimated
that if a woman conceives at the age of 35e39, the chance of the
child suffering from aneuploidy is 1% and will increase to
approximately 3.5% by the age of 40e45. Moreover, chromosomal
rearrangements or aneuploidy themselves can lead to infertility or
repeated spontaneous abortions. PGT is expected by some groups
to become a regular feature in the future for patients undergoing
IVF at an advanced age. As described previously, ni-PGT is a
promising method that may avoid blastomere biopsy-related risks
and reduce the false-positive rate in preimplantation genetic
screening. As the demand for PGT increases, ni-PGT may become
the preferred procedure in selection of euploid embryos in ART in
3
Journal of Genetics and Genomics xxx (xxxx) xxx
future when the limitations and technical challenges discussed
are overcome.
The future of ni-PGT will closely depend on methods for maxi-
mally reducing maternal contamination. In noninvasive prenatal
screening, studies have shown that DNA fragments of fetal origin
differ in size and have different preferred ends as corresponding to
fragments of maternal origin (Chan et al., 2016). Thus, further well-
designed studies focusing on identifying differences between DNA
fragments from embryos and those from maternal admixture in
SCM should be addressed rigorously before ni-PGT can be used
clinically. Recently, healthy children born from ni-PGT selected
euploid blastocysts for couples carrying genetic alterations were
reported (Xu et al., 2016; Jiao et al., 2019). However, the validity of
ni-PGT in RIF, recurrent spontaneous abortion, advanced maternal
age, and so on has yet to be addressed. Randomized trials with ni-
PGT relevant to these populations should be conducted.
With regard to preimplantation genetic testing for monogenic/
single gene diseases (PGT-M) to detect gene variants in embryos,
most of the professional guidelines in the postnatal area relevant
for genetic/genomic testing are applicable to PGT. Zhang et al.
(2019) reported on the development and validation of a gene
panel in noninvasive prenatal sequencing for common dominant
monogenic diseases. Follow-up studies confirmed results of 100%
sensitivity and 100% specificity. This procedure provided promising
prospects for detecting a wide range of monogenic diseases
J. Li, Y. Liu, Y. Qian et al.
through cfDNA, complementing current aneuploidy screening for
ni-PGT.
In conclusion, although the existence of measurable cfDNA in
the BF and SCM from human embryos has been confirmed by many
studies, technical and ethical challenges remain to be overcome
before ni-PGT can be accepted as a reliable source of embryonic
genomic information source and as a routine clinical approach.
Poor embryonic genome representation by BF and SCM-derived
cfDNA, largely due to low efficiency in DNA yield, poor integrity
of nucleic acid, presence of parental contamination, and potential
embryo mosaicism, is the main current limitation. Standard pro-
cedures for sample collection and cfDNA amplification and for
modified NGS analysis are required to move ni-PGT to the clinic.
Furthermore, well-designed randomized trials with or without ni-
PGT are also required to evaluate the clinical benefits and cost-
benefit ratio of these procedures on pregnancy outcomes. After
rigorously overcoming such limitations, ni-PGT may become a very
promising approach someday to complement or even replace the
current procedure for TE biopsy-based PGT.
Acknowledgment
We thank professors Cynthia Casson Morton and Yiping Shen
from Harvard Medical School and professor Sharon YC Ruan
from Hong Kong Polytechnic University for revising the manu-
script. This work was supported by the National Key Research
and Development Program of China (2018YFC1005003), the
National Natural Science Foundation of China (81974224,
81771535), the Natural Science Foundation of Zhejiang Province
(LZ18H040001, LQ19H040007), Zhejiang Provincial Key Medical
Technology Program (WKJ-ZJ-1826), and Zhejiang University
Education Foundation Global Partnership Fund. The authors
declared no competing interests.
Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jgg.2020.11.007.
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