817 RBMO VOLUME 44 ISSUE 5 202 2

REVIEW

Non-invasive preimplantation genetic testing

for aneuploidies: an update
BIOGRAPHY
Luis Navarro, PhD, has worked in the field of human genomics for over 13 years. During
his PhD he researched the genetics and epigenetics of neurodegenerative diseases.
Nowadays, he is part of the non-invasive research group at Igenomix, devoting his time to
the improvement of non-invasive tests for aneuploidy detection.

Luis Navarro-Sánchez1,*, Carmen García-Pascual1,2, Carmen Rubio1,2,

Carlos Simón1,2,3,4,5

KEY MESSAGE
Preimplantation embryos secrete cell-free DNA into culture medium, which can be used to identify
aneuploidies. Thus, cell-free DNA may provide a biomarker for prioritizing blastocysts for transfer in assisted
reproductive technology to improve implantation rates and reduce miscarriage rates and time to pregnancy.

ABSTRACT
Aneuploidy is common among preimplantation human embryos used in assisted reproductive technology. Because
abnormal chromosome number can negatively affect reproductive outcome, in-vitro-fertilized embryos routinely
undergo aneuploidy testing before transfer into the uterus. This testing typically involves an invasive trophectoderm
biopsy of a blastocyst-stage embryo. However, emerging evidence indicates that, during in-vitro development,
embryos secrete cell-free DNA into their culture medium; this phenomenon suggests the potential for an
alternative, non-invasive assay for aneuploidy. Embryonic cell-free DNA-based assays exhibit high concordance with
trophectoderm biopsies, inner cell mass and the whole blastocyst. Yet informativity and concordance rates may be
influenced by several factors: the culture day when the medium is collected, contamination with external and/or
cumulus cell DNA, and previous manipulation of the embryos. This review discusses non-invasive embryonic cell-
free DNA analysis as a biomarker to prioritize blastocysts for transfer to help increase implantation rates and reduce
miscarriage rates and time to achieve pregnancy. Ongoing research on the mechanisms underlying embryonic cell-
free DNA secretion and how this impacts its role as a biomarker of aneuploidy are also discussed.

KEY WORDS
1 Igenomix Headquarters, Paterna, Valencia, Spain
2 Igenomix Foundation, INCLIVA Health Research Institute, Valencia, Spain
3
Aneuploidy
Department of Obstetrics & Gynecology, University of Valencia, Valencia, Spain
4 Department of Obstetrics & Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA, Blastocyst
USA Cell-free DNA
5 Department of Obstetrics & Gynecology, Baylor College of Medicine, Houston TX, USA Culture medium
Non-invasive PGT for aneuploidies
© 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. E-mail address: luis.navarro@igenomix.com (L. Navarro-Sánchez). https://doi.org/10.1016/j.
rbmo.2022.01.012 1472-6483/© 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Declaration: L.N.-S., C.G.-P., C.R. and C.S. are employees of Igenomix and C.S. is the Chief of the Scientific Advisory
Board at Igenomix.
818 RBMO VOLUME 44 ISSUE 5 202 2
INTRODUCTION
A
neuploidy is common in
human embryos, with an
incidence ranging from 20%
to 80%, depending on female
age. About 10% of clinically recognized
pregnancies present trisomies or
monosomies, but for women in the later
child-bearing years, the incidence may
surpass 50%. Aneuploidy can result in
implantation failure and is a primary cause
of spontaneous abortion (Capalbo et al.,
2016; Franasiak et al., 2014; Rodrigo
et al., 2014; Rubio et al., 2020a) (FIGURE 1).
Conventional morphological analysis
does not prevent the transfer of
aneuploid embryos (Alfarawati et al.,
2011; Capalbo et al., 2014). During
assisted reproductive technology
(ART), some aneuploid embryos at
the blastocyst stage have optimal
morphological scores, meaning that these
embryos may be selected to undergo
transfer to the uterus. Depending on
the type of aneuploidy, some of these
embryos will not implant, others will
implant but will end in miscarriage,
and a few may result in a newborn with
aneuploidy. Thus, additional screening
measures are necessary to avoid the
transfer of aneuploid embryos in ART.
Another approach to embryo screening
is morphokinetics. Unfortunately, early
FIGURE 1 Incidence of different types of chromosomal abnormality in trophectoderm biopsies according to female age (Igenomix internal data).
Uniform aneuploidies: whole chromosome aneuploidies uniformly distributed in the biopsied cells. High degree mosaicism: whole chromosome
aneuploidies in 50–70% of the biopsied cells. Low degree mosaicism: whole chromosome aneuploidies in 30–50% of the biopsied cells. Segmental
aneuploidies: aneuploidies observed for a small segment of the chromosome (duplications/deletions >10 Mb).
morphokinetic parameters have not
shown the capacity to accurately predict
aneuploidy (Swain, 2013). Several studies
have not found a relationship between
morphokinetics and aneuploidy in
general (Rienzi et al., 2015; Yang et al.,
2014), while one study has identified
differences in morphokinetics only for
specific aneuploidies (Nogales et al.,
2017). In one report, compaction was
faster in aneuploid embryos compared
with euploid ones (Melzer et al., 2012),
and another determined that aneuploid
embryos initiated compaction later than
euploid embryos (Campbell et al., 2013).
Furthermore, the time to start blastulation,
the time to reach blastocyst expansion and
the time to blastocyst hatching significantly
differed between euploid and aneuploid
embryos, but selection according to
these parameters did not improve clinical
outcomes (Desai et al., 2016; Minasi
et al., 2016). Differences between groups
(euploid versus aneuploid) could result
from different conditions affecting embryo
dynamics, such as ovarian stimulation,
female age and culture conditions such
as temperature and pH (Zaninovic et al.,
2017).
Preimplantation genetic testing for
aneuploidies (PGT-A) provides a
robust alternative to these methods,
accurately identifying aneuploidies in
preimplantation embryos. Indeed, there
are several advantages to using PGT-A
within IVF programmes: aneuploidy
testing by this method can increase
pregnancy rates while also reducing
the time to conceive, the total number
of transfers required, the miscarriage
rate and the costs. These advantages
are especially relevant in cases with
advanced maternal age (Neal et al., 2018;
Somigliana et al., 2019). PGT-A can
be applied to different preimplantation
developmental stages, including the first
and second polar bodies, blastomeres
from cleavage-stage embryos on day 3,
and trophectoderm biopsies. However,
an invasive procedure, i.e. a biopsy,
is always required. Each stage has
specific diagnostic advantages as well as
critical limitations related to aneuploidy
genesis during both meiosis and the
preimplantation period of embryo
development. The most common PGT-A
approach requires a trophectoderm
biopsy of the blastocyst, followed by
the analysis of those 4–8 cells by next-
generation sequencing (NGS) (Fiorentino
et al., 2014; Friedenthal et al., 2018;
Huang et al., 2016; Rubio et al., 2020a).
More recently, a prioritization system has
been proposed combining PGT-A results
and morphology to transfer first those
blastocysts with the highest implantation
potential and ongoing pregnancy rates
(Viotti et al., 2021).
Blastocentesis has been proposed as
a less invasive strategy for embryo

selection. This process requires
aspiration of the blastocoel fluid (Palini
et al., 2013) at the expanded blastocyst
stage, but without removing embryonic
cells. Blastocentesis appears to be a
promising technique. However, the DNA
in the blastocoel fluid originates, at least
in part, from cells that are necrotic or
apoptotic, which could compromise
DNA quantity or integrity (Hammond
et al., 2016; Rule et al., 2018). Indeed,
there is a significant variation in
amplification rates and concordance
of blastocoel fluid with the respective
trophectoderm biopsy (56–82% and
37–97%, respectively) (Gianaroli et al.,
2014; Magli et al., 2016; Tobler et al.,
2015; Tšuiko et al., 2018). The variation
probably stems from heterogeneity in
technical expertise in blastocentesis
(Magli et al., 2016). Thus, despite its
potential clinical value (Magli et al.,
2019), the application of blastocentesis
is not widespread.
The abovementioned approaches,
however, require embryo manipulation.
The zona pellucida must be perforated
in all cases, usually by laser, to retrieve
cells or fluids from the embryo,
and such invasive procedures entail
technical and economical challenges.
The high degree of technical skills
required to obtain samples and the
cost associated with both equipment
and operator training remain challenges
to wider implementation. In addition,
the possibility of harming the embryo
is a constant concern for doctors and
patients, despite the fact that the risk
is minimal in the hands of experienced
biopsy operators (Capalbo et al., 2016;
Neal et al., 2018). Trophectoderm
biopsy, after proper training and
under constant monitoring of the
operators’ performance, is considered
the gold standard for PGT, especially
for monogenic diseases (PGT-M) and
translocations (PGT-SR). However, recent
studies have linked trophectoderm
biopsy with a significant increase in
pre-eclampsia (Zhang et al., 2019a)
and hypertensive disorders among
mothers (Makhijani et al., 2021). Thus,
there is growing interest in non-invasive
approaches to identify chromosomal
abnormalities in preimplantation
embryos.
Ideally, a non-invasive approach would
offer the benefits of invasive PGT-A –
improved live birth rate at first embryo
transfer, reduced rate of miscarriage
and multiple pregnancies, and shorter
time to pregnancy – while avoiding the
disadvantages of embryo manipulation
and biopsy. Here, the use of embryonic
cell-free DNA (cfDNA) is reviewed,
including the concordance studies
between spent blastocyst medium
(SBM) and other blastocyst samples, the
different methodological approaches,
the origin of this cfDNA and its potential
clinical application as a biomarker to
prioritize the transfer of blastocysts
with a higher likelihood of resulting in a
healthy baby.
EMBRYONIC cfDNA ANALYSIS
FOR NON-INVASIVE PGT-A
(niPGT-A): CONCORDANCE
STUDIES
In 2016, Shamonki and colleagues
first attempted to ascertain whether
the cfDNA released by an embryo to
the SBM could be used to determine
the embryonic chromosomal content
(Shamonki et al., 2016). This proof-of-
concept study demonstrated that SBM
enabled successful embryonic aneuploidy
detection similar to trophectoderm
biopsy. After amplification of SBM, DNA
was detected in most of the samples (55
out of 57), but only two SBM samples
provided sufficient quality to diagnose
aneuploidy by array comparative genomic
hybridization (aCGH). For these two
cases, the diagnosis was fully concordant
with the trophectoderm biopsy.
Later studies compared SBM results
with those of other types of sample
taken from the same embryo. Important
measures in these studies were the
informativity rate of SBM, defined as
the percentage of diagnosable SBM
samples, and the ploidy concordance
rate, or the overall agreement between
the SBM and the reference sample
considering them euploid or aneuploid.
Ploidy concordance includes both full
concordance (when the chromosomal
status for all the chromosomes in
both samples is the same) and partial
concordance (the chromosomal status
for some chromosomes might differ
between samples, but they are both
aneuploid). Other relevant parameters
were the false-positive rate, i.e. when
the SBM is aneuploid and the reference
sample is euploid, and the false-negative
rate, i.e. when the SBM is euploid and
the reference sample is aneuploid.
Different examples of the comparison of
NGS results are shown in FIGURE 2.
RBMO VOLUME 44 ISSUE 5 202 2 819
Trophectoderm biopsy was typically
chosen as the reference sample against
which to compare SBM. In these cases,
the informativity rate of SBM and the
ploidy concordance rate varied, ranging
from 69.0% to 97.6% and from 33.3% to
89.1%, respectively (Chen et al., 2021a;
Ho et al., 2018; Huang et al., 2019; Lledo
et al., 2020; Rubio et al., 2019, 2020b;
Shitara et al., 2021; Vera-Rodriguez
et al., 2018; Yeung et al., 2019; TABLE 1).
Only one study compared SBM with
polar body biopsy and obtained an 81.8%
informativity rate for SBM and a 72.2%
ploidy concordance rate (22.2% full and
50.0% partial) (Feichtinger et al., 2017;
TABLE 1).
The whole embryo was considered as
the gold standard for comparison in two
studies. Xu and colleagues (Xu et al.,
2016) used vitrified day 3 embryos that
were warmed and cultured until day 5. All
42 embryos provided informative results,
but only 85.7% had ploidy concordance
across sample types (66.7% full and
19.0% partial). Yin and co-workers
worked with 75 warmed day 5/6 embryos,
which had already had trophectoderm
biopsy performed on them and which
had been cultured for 24 h (Yin et al.,
2021). Although the informativity rate was
78.7%, the ploidy concordance rate was
89.8% (32.2% full and 57.6% partial). Four
other groups (Chen et al., 2021a; Ho
et al., 2018; Huang et al., 2019; Shitara
et al., 2021) also compared some of
the SBM, not only with trophectoderm,
but also with whole embryos (33, 48, 16
and 256, respectively), obtaining high
informativity rates for the SBM (97.6%,
92.3%, 95% and 96.6%, respectively)
but disparate ploidy concordance
rates: 45.5%, 93.8%, 93.8% and 78.1%,
respectively (TABLE 2).
Several studies assessed the combination
of SBM plus blastocoel fluid. This
minimally invasive PGT-A approach
collapses the blastocyst and releases
its contents into the medium. In
addition to this mandatory artificial
shrinkage, the various studies conducted
extra embryo manipulations such as
trophectoderm biopsy, vitrification and/
or assisted hatching. In these cases,
for the same embryo, NGS results
from blastocoel fluid plus SBM were
compared with PGT-A results from
trophectoderm biopsy and whole
blastocysts. The number of samples
analysed in all cases was low and
the results were heterogeneous: the
820 RBMO VOLUME 44 ISSUE 5 202 2
FIGURE 2 Comparison of next-generation sequencing results in trophectoderm biopsies (TE) and spent blastocyst medium (SBM): full
concordance, partial concordance, false-negative (aneuploid TE–euploid SBM) and false-positive (euploid TE–aneuploid SBM).
informativity rate of SBM ranged from
87.5% to 100%, whereas the ploidy
concordance rates between the two
types of sample (SBM + blastocoel
fluid and trophectoderm or whole
blastocysts) were variable, ranging from
76.3% to 100% and from 70.4% to
100%, respectively. Although minimally
invasive, this approach did not provide
an advantage: no benefit was observed
for collapsing the blastocyst to retrieve
blastocoel fluid, as the use of blastocoel
fluid in combination with SBM did not
provide better results than the analysis
of the medium itself (Chen et al., 2020;
Jiao et al., 2019; Kuznyetsov et al., 2018;
2020; Li et al., 2018; Li et al., 2021;
Sialakouma et al., 2021; Zhang et al.,
2019b) (TABLE 3).
Discrepancies in the reported results
probably resulted from contamination
and/or from the different methodologies
applied to culture (drop culture
volume and time in culture), embryo
manipulation (assisted hatching,
biopsy, vitrification) or DNA analysis
(amplification and sequencing methods,
diagnosis algorithm). The significant
heterogeneity of procedures limits the
translation of this non-invasive approach
to the clinic. Indeed, despite being
non-invasive, most study protocols
require extra embryo manipulations
not feasible in the daily routine of an
embryology laboratory. In addition, when
performing niPGT-A, and due to the
low concentration of cfDNA released
to the culture medium, factors such as
decreasing the drop volume, prolonging
the time in culture and diminishing the
contamination (external or from maternal
cumulus cells) need to be adapted.
Therefore, before including the non-
invasive approach in their routine, each
laboratory needs to perform a validation
study to implement the required
modifications in the embryo culture
protocol and confirm that embryo
viability is not compromised and that
concordance rates are high, without the
presence of contamination.
Several niPGT-A options are currently
commercially available. In a report from
Hanson and colleagues (Hanson et al.,
2021), the high drop culture volume
(30 µl), the lack of measures to reduce
contamination, and chaotic SBM results
that could be related to the degradation
of DNA or storage conditions rather
than the actual embryo chromosomal
content could have contributed to the
controversial results observed. These
observations support a need to adapt
the IVF protocol and perform validation
before conducting niPGT-A. Results
indicated a high percentage of non-
informative media (37.3%) as well as a
poor correlation between trophectoderm
biopsy and SBM (63.5%, calculated as
ploidy concordance). Despite the poor
performance, when the results were
stratified by day of embryo development,
the tendency already observed by
Yeung and co-workers and Rubio and
collaborators (the longer the time in
culture, the better the results) was
confirmed: informativity and concordance
rates were higher for day 6 embryos
(Rubio et al., 2019; Yeung et al., 2019).
ORIGIN OF cfDNA
The discovery of embryonic cfDNA in
SBM prompted further research into its
origin. The compartment(s) from where
this DNA originates remains unclear;
both trophectoderm and inner cell
mass (ICM) are potential sources, but
there are challenges in examining the
mechanisms underlying cfDNA secretion
into the medium. Apoptosis during
preimplantation development (especially
the late preimplantation stages) may play
a role: the rapid transformation of the
embryo results in not only cell gain, but
also cell loss, even in genetically healthy
embryos (Hardy et al., 2001). Because
 RBMO VOLUME 44 ISSUE 5 202 2 821

TABLE 1 SUMMARY OF STUDIES COMPARING RESULTS BETWEEN SBM AND TE OR PB BIOPSIES

Authors No. Informa- Concordance False False Drop Embryo Time in WGA PGT-A
of tive media TE–SBMa % positives negatives volume manipula- culture method technique
SBM % (n/N) (n/N) % (n/N) % (n/N) (µl)a tion
Shamonki 57 96.5 (55/57) Ploidy (all full): 33.3 – – 15 (15) AH on D3 D3–D5/6 Repli-G (Qia- aCGH
et al. (2016) (2/6)c gen) (Agilent Tech-
nologies)
Feichtinger 22 81.8 (18/22) Ploidy: 72.2 (13/18) 5.6 (1/18) 22.2 (4/18) 25 (5) PB biopsy, AH D0–D5/6 SurePlex aCGH (Illu-
et al. (2017)d Full: 22.2 (4/18) on D3 (Illumina) mina)
Partial: 50.0 (9/18)
Vera- 56 91.1 (51/56) Ploidy: 33.3 (17/51) – 66.7 (34/51) 25 (20) AH on D3 D3–D5 SurePlex (Illu- NGS (Thermo
Rodríguez Full: 17.6 (9/51) mina) + Re- Fisher)
et al. (2018) Partial: 15.7 (8/51) proSeq (Ther-
mo Fisher)
Ho et al. 41 97.6 (40/41) Ploidy: 65.0 (26/40) – – 25 (5) AH on D3 D1–D5 PicoPLEX NGS (Thermo
(2018) versus no AH (Rubicon) Fisher)
Huang et al. 52 92.3 (48/52) Ploidy: 89.1 (41/46)e 2.2 (1/46) 8.7 (4/46) 15 (3.5) AH on D3, D5–D6 MALBAC NGS (Illu-
(2019) Full: 65.2 (30/46) TE biopsy D6–D7 (Yikon) mina)
Partial: 23.9 (11/46) plus vitrifica- 24 h culture
tion on D5/6 after thawing
Yeung et al. 168 69.0 (116/168) Ploidy: 73.3 (85/116) 12.9 (15/116) 13.8 (16/116) 30 (3) AH on D3 D3–D5 SurePlex NGS (Illu-
(2019) D5: 55.6 Full: 23.3 (27/116) D5: 12 (6/50) D5: 12 (6/50) D3–D6 (Illumina) mina)
(50/90) Partial: 50.0 (58/116) D6: 13.6 D6: 15.2
D6: 84.6 D5: Ploidy 76 (38/50) (9/66) (10/66)
(66/78) D6: Ploidy 71.2
(47/66)
Rubio et al. 115 93.9 (108/115) Ploidy: 78.7 (85/108) 13.9 (15/108) 2.8 (3/108) 10 (10) None D4–D5 ReproSeq NGS (Thermo
(2019)f D5: 81.8 Full: 63.9 (69/108) D5: 29.6 D5: 3.7 (1/27) D4–D6/7 (Thermo Fisher)
(27/33) Partial: 14.8 (16/108) (8/27) D6/7: 2.5 Fisher)
D6/7: 98.8 D5: Ploidy 63 (17/27)g D6/7: 8.6 (2/81)
(81/82) D6/7: Ploidy 84 (7/81)
(68/81)
Rubio et al. 1301 85.2 Ploidy: 78.2 12.4 (137/1108) 8.3 (92/1108) 10 (10) None D4–D6/7 ReproSeq NGS (Thermo
(2020b)f (1108/1301) (866/1108) (Thermo Fisher)
Full: 67.7 (750/1108) Fisher)
Partial: 10.5 (116/1108)
Lledo et al. 92 92.4 (85/92) Ploidy: 74.7 (62/83) 12.0 or 15.7 13.3 or 12.0 20 (7.5 each AH on D3 D3–D5/6 MALBAC NGS (Illu-
(2020)h or 72.3 (60/83)e (10/83 or (11/83 or method) (Yikon) or mina)
13/83) 10/83) SurePlex
(Illumina)
Shitara et al. 20 95 (19/20) Ploidy: 88.9 (16/18)e 5.6 (1/18) 5.6 (1/18) – Vitrified D5/6 24 h for D5 SurePlex NGS (Illu-
(2021) Full: 66.7 (12/18) embryos 3 h for D6 (Illumina) mina)
Partial: 22.2 (4/18) Zona pelluci- blastocysts
da removed
Hanson et al. 166 62.7 (104/166) Ploidy: 63.5 (66/104) 26.9 (28/104) 8.7 (9/104) 30 (–) AH on D3 D5: 24–48 h MALBAC NGS (Illu-
(2021)f D3/4–D5: 17.6 D3/4–D5: Ploidy (and D3/4–D5: D3/4–D5: D6: 48–72 h (Yikon) mina)
(6/34) full) 50.0 (3/6) 33.3 (2/6) 16.7 (1/6) D7: 72–96 h
D3/4–D6/7: D3/4–D6/7: Ploidy D3/4–D6/7: D3/4–D6/7:
74.2 (98/132) 64.3 (63/98) 26.5 (26/98) 8.2 (8/98)4
Full: 30.6 (30/98)
Partial: 33.7 (33/98)
Chen et al. 265 96.6 (256/265)Ploidy: 74.2 (190/256)i 14.5 (37/256)i 11.3 (29/256)i – (20–25) None D3–D5/6 MALBAC NGS (Illu-
(2021a) (Yikon) mina)
a Ploidy concordance is the overall agreement between the SBM and the reference sample, considering them euploid or aneuploid. Ploidy concordance includes both full
concordance (when the chromosomal status for all the chromosomes in both samples is the same) and partial concordance (the chromosomal status for some chromo-
somes might differ between samples, but they are both aneuploid).
bDrop volume is shown for culture (and analysis).
c Despite having 55 informative SBM, aCGH was only performed in the six samples with the highest DNA concentration.
d SBM was compared with TE biopsy in all studies except in this one, where SBM was compared with PB biopsy.
e It was not possible to analyse the concordance of all the informative SBM because some of their respective TE biopsies did not provide a diagnosis.
f In these studies, there was a third type of discordance, called sex mismatch, i.e. SBM–TE pairs with concordant ploidy for autosomes but a different sex. More precisely,
values were 4.6% (5/108; Rubio et al., 2019), 1.2% (13/1108; Rubio et al., 2020b) and 1% (1/104; Hanson et al., 2021).
g On day 5, full concordance was 40.7% (11/27) and partial concordance was 22.2% (6/27). On day 6/7, full concordance was 71.6% (58/81) and partial concordance was
12.3% (10/81).
h Two different WGA methods were compared. The results for both are detailed: first for MALBAC and second for SurePlex.
i When not considering the multiple abnormal chromosomes (i.e. five or more abnormalities), for the remaining 229 samples the values of ploidy concordance, false positives
and false negatives changed to 78.6% (180/229), 10.9% (25/229) and 10.5% (24/229), respectively.
aCGH, array comparative genomic hybridization; AH, assisted hatching; D, day; NGS, next-generation sequencing; PB, polar body; PGT-A, preimplantation genetic testing
for aneuploidies; SBM, spent blastocyst medium; TE, trophectoderm; WGA, whole genome amplification.
82 2 RBMO VOLUME 44 ISSUE 5 202 2

TABLE 2 SUMMARY OF STUDIES COMPARING RESULTS BETWEEN SBM AND WB

Authors No. of Informative Concordance False False Drop Embryo Time in WGA PGT-A
SBM media% WB–SBMa % positives negatives volumeb (µL) manipula- culture method technique
(n/N) (n/N) % (n/N) % (n/N) tion
Xu et al. 42 100 (42/42) Ploidy: 85.7 (36/42) 9.5 (4/42) 4.8 (2/42) 30 Vitrification D3–D5 MALBAC NGS (Illu-
(2016) Full: 66.7 (28/42) (5 to 20) on D3 (Yikon) mina)
Partial: 19.0 (8/42)
Ho et al. 41 97.6 (40/41) Ploidy: 45.5 (15/33)c– – 25 (5) AH on D3 D1–D5 PicoPLEX NGS (Thermo
(2018) versus no AH (Rubicon) Fisher)
Huang et al. 52 92.3 (48/52) Ploidy: 93.8 (45/48) 6.3 (3/48) – 15 (3.5) AH on D3, TE D5–D6 MALBAC NGS (Illu-
(2019) Full: 85.4 (41/48) biopsy D6–D7 (Yikon) mina)
Partial: 8.3 (4/48) plus vitrifica- Cultured for
tion on D5/6 24 h after
warming
Rubio et al. 81 90.1 (73/81) Ploidy: 84.4 6.3 (4/64) 9.4 (6/64) 10 (10) None D4–D6/7 ReproSeq NGS (Thermo
(2020b)d (54/64)c (Thermo Fisher)
Full: 68.8 (44/64) Fisher)
Partial: 15.6 (10/64)
Yin et al. 75 78.7 (59/75) Ploidy: 89.8 (53/59) 10.2 (6/59) – 25 (25) Biopsy on Cultured for MALBAC NGS (Illu-
(2021) Full: 32.2 (19/59) D5/6 and 24 h after (Yikon) mina)
Partial: 57.6 (34/59) vitrification warming
Shitara 20 95 (19/20) Ploidy: 93.8 (15/16)c – 6.3 (1/16) – Vitrified D5/6 24 h for D5 SurePlex NGS (Illu-
et al. (2021) Full: 56.3 (9/16) Zona pellucida 3 h for D6 (Illumina) mina)
Partial: 37.5 (6/16) removed blastocysts
Chen et al. 265 96.6 (256/265) Ploidy: 78.1 16.8 (43/256)3 5.1 (13/256)e – (20–25) None D3–D5/6 MALBAC NGS (Illu-
(2021a) (200/256) (Yikon) mina)
a Ploidy concordance is the overall agreement between the SBM and the reference sample considering them euploid or aneuploid. Ploidy concordance includes both full
concordance (when the chromosomal status for all the chromosomes in both samples is the same) and partial concordance (the chromosomal status for some chromo-
somes might differ between samples, but they are both aneuploid).
b Drop volume is shown for culture (and analysis).
c It was not possible to analyse the concordance of all informative SBM because some of their respective WB/ICM did not provide a diagnosis.
d SBM was compared with WB in all studies except in this one, where SBM was compared with ICM.
e When not considering the multiple abnormal chromosomes (i.e. five or more abnormalities), for the remaining 229 samples, the values of ploidy concordance, false posi-
tives and false negatives changed to 83.0% (190/229), 12.2% (28/229) and 4.8% (11/229), respectively.
AH, assisted hatching; D, day; NGS, next-generation sequencing; PGT-A, preimplantation genetic testing for aneuploidies; SBM, spent blastocyst medium; WB, whole blasto-
cyst; WGA, whole genome amplification.

aneuploid cells may be more prone
to apoptosis, cfDNA may show more
aneuploidies than trophectoderm biopsy.
DNA obtained via blastocentesis from the
blastocoel fluid of expanded blastocysts
showed higher amplification failure in
trophectoderm-euploid blastocysts than
in trophectoderm-aneuploid blastocysts;
this finding suggests that trophectoderm-
aneuploid blastocysts may have a higher
cfDNA concentration as a result of
embryonic mechanisms to correct for
aneuploidy (Magli et al., 2019).
Yet other findings do not support the
apoptotic origin of embryonic cfDNA
secreted to the external compartment,
the perivitelline space and the culture
medium. cfDNA quantity in the culture
drops of aneuploid blastocysts was similar
to that observed in euploid blastocysts
before and after whole genome
amplification (WGA; Vera-Rodriguez
et al., 2018) and the concordance rate
(both full and ploidy) of SBM and ICM
biopsies was similar to that between
the trophectoderm and ICM from the
same blastocysts (Rubio et al., 2020b).
These findings suggest that cfDNA
released to the SBM may originate from
both embryo compartments: ICM and
trophectoderm.
Another source of DNA in the culture
media could be the polar bodies.
Polar body DNA, primarily from the
second polar body, may be present at
the blastocyst stage in approximately
one-third of blastocysts. However,
severe polar body contamination was as
low as 4% (Chen et al., 2021b). In the
current authors’ experience, the risk
of contamination with DNA from the
polar body increases when medium is
collected on day 5 compared with day 6.
Furthermore, 35% of non-contaminated
samples had complementary aneuploidy,
suggesting a polar body origin or
mosaicism (Vera-Rodriguez et al., 2018).
Another study using vitrified-warmed
embryos and analysing whole
chromosome copy number per sample
found that ploidy concordance was
equivalent between niPGT-A and
trophectoderm biopsy, niPGT-A and
whole blastocysts, and trophectoderm
biopsy and whole blastocysts (Kuznyetsov
et al., 2018). A second study by the
same authors showed that embryonic
cfDNA could be successfully amplified
in both good- and bad-quality embryos,
suggesting that expelled cfDNA might
reflect general embryo ploidy status.
To determine the origin of embryonic
cfDNA, they obtained similar cfDNA
quantities from embryos with different
morphological grades and similar sizes of
amplified DNA fragments, suggesting that
apoptosis and necrosis were not likely
to be the only mechanisms driving DNA
release (Kuznyetsov et al., 2020).
Kobayashi and colleagues observed
cell-free mitochondrial DNA (mtDNA) in
SBM and investigated its relationship with
morphokinetics. The abundance of cell-
free mtDNA in SBM was linked with the
duration of expansion and was greater
when increased blastocyst collapse was
observed. There were no differences
 RBMO VOLUME 44 ISSUE 5 202 2 823

TABLE 3 SUMMARY OF STUDIES COMPARING RESULTS BETWEEN SBM PLUS BF AND TE BIOPSIES OR WB

Authors No. Inform- Con- False False Con- False False Drop Embryo Time in WGA PGT-A
of ative cordance posi- nega- cordance posi- nega- volume manipu- culture method tech-
SBM media TE-SB- tives % tives % WB-SB- tives % tives % (µl)b lation nique
% (n/N) M+BFa (n/N) (n/N) M+BF % (n/N) (n/N)
% (n/N) (n/N)
Kuznyetsov 47 100 (47/47) Ploidy: 88.4 2.3 (1/43) 9.3 (4/43) Ploidy: 89.3 7.1 (2/28) 3.6 (1/28) 25 (25) 28 vitrified; D4–D5/6 SurePlex NGS
et al. (2018) (38/43)c (25/28)c 24 with (Illumina) (Illumina)
Full: 69.8 Full: 78.6 previous
(30/43) (22/28) TE biopsy;
Partial: 18.6 Partial: 10.7 all laser
(8/43) (3/28) collapse
Li et al. 40 97.5 Ploidy: 76.3 13.2 (5/38) 10.5 (4/38) Ploidy: 78.9 15.8 (6/38) 5.3 (2/38) 25 (25) Small D3–D5 MALBAC NGS
(2018) (39/40) (29/38)c (30/38)c breech in (Yikon) (Illumina)
Full: 47.4 Full: 55.3 the zona
(18/38) (21/38) pellucida
Partial: 28.9 Partial: 23.7
(11/38) (9/38)
Jiao et al. 62 98.4 Ploidy: 98.3 – 1.7 Ploidy: 96.7 3.3 (2/61) – 12 (10) AH, 14 h cul- MALBAC NGS
(2019) (61/62) (58/59)c (1/59) (59/61) vitrification ture after (Yikon) (Illumina)
Full: 88.1 Full: 88.5 D5/6; warming
(52/59) (54/61) artificial
Partial: 10.2 Partial: 8.2 shrinkage
(6/59) (5/61)
Zhang 32 87.5 (28/32)– – – Ploidy: 70.4 14.8 (4/27) 7.4 (2/27) Approx- Vitrifica- D4–D5 MALBAC NGS
et al. (19/27)c imately tion D2/3; D5–D6 (Yikon) (Illumina)
(2019b)d Full: 66.7 20–30 (ap- artificial
(18/27) proximatelyshrinkage
Partial: 3.7 20–30)
(1/27)
Kuznyetsov 102 88.2 Ploidy: 88.9 3.3 (3/90) 7.8 (7/90) – – – 25 (5) AH on D4; D4–D6 SurePlex NGS
et al. (90/102) (80/90) artificial (Illumina) (Illumina)
(2020) Full: 78.9 shrinkage
(71/90)
Partial: 10.0
(9/90)
Chen et al. 26 100 (26/26)Ploidy: 100 – – Ploidy: 100 – – 15 (10) TE biopsy 15 h cul- MALBAC NGS
(2020) (26/26)e (26/26)f D5/6, vit- ture after (Yikon) (Illumina)
Full: 76.9 Full:80.8 rification; warming
(20/26) (21/26) artificial
Partial: 23.1 Partial: 19.2 shrinkage
(6/26) (5/26)
Li et al. 41 95.1 (39/41) Ploidy: 76.3 7.9 (3/38) 15.8 (6/38) Ploidy: 87.2 10.3 (4/39) 2.6 (1/39) 15 (10) TE biopsy 14–18 h MALBAC NGS
(2021)g (29/38)c,e (34/39) D5/6, culture (Yikon) (Illumina)
Full: 68.4 Full: 84.6 vitrifica- after
(26/38) (33/39) tion; laser warming
Partial: 7.9 Partial: 2.6 collapse
(3/38) (1/39)
Sialakouma 40 100 Ploidy: 81.8 3.0 (1/33) 15.2 (5/33) Ploidy (all – – 10 (–) Artificial D3–D5/6 SurePlex NGS
et al. (2021) (40/40) (27/33)h full): 100 shrinkage (Illumina) (Thermo
Full: 63.6 (4/4)i Fisher)
(21/33)
Partial: 18.2
(6/33)
a Ploidy concordance is the overall agreement between the SBM+BF and the reference sample considering them euploid or aneuploid. Ploidy concordance includes both
full concordance (when the chromosomal status for all the chromosomes in both samples is the same) and partial concordance (the chromosomal status for some chromo-
somes might differ between samples, but they are both aneuploid).
b Drop volume is shown for culture (and analysis).
c It was not possible to analyse the concordance of all informative SBM+BF because some of their respective TE/WB did not provide a diagnosis.
d In this study, there was a third type of discordance, called sex mismatch, in two cases (7.4%), i.e. SBM+BF–WB pairs with concordant ploidy for autosomes but a different
sex.
e TE rebiopsies were used to compare SBM+BF with.
f In this study, SBM+BF was compared with ICM, not WB.
g A mosaicism threshold of 50% was considered.
h Although all 40 media amplified, diagnosis could only be obtained for 33 of them due to low quality.
i It was possible to analyse the chromosomal content for only four of the embryos.
AH, assisted hatching; BF, blastocoel fluid; D, day; NGS, next-generation sequencing; PGT-A, preimplantation genetic testing for aneuploidies; SBM, spent blastocyst medi-
um; TE, trophectoderm; WB, whole blastocyst; WGA, whole genome amplification.
824 RBMO VOLUME 44 ISSUE 5 202 2
between IVF and intracytoplasmic sperm
injection (ICSI). Furthermore, less
(although not significant) cell-free mtDNA
was observed in SBM from blastocysts
that implanted successfully (Kobayashi
et al., 2020a). Interestingly, cell-free
mtDNA was also found in SBM from
porcine embryos, and the quantity of
cell-free mtDNA was affected by embryo
ploidy (Kobayashi et al., 2020b).
Recent studies assessing genome-wide
DNA methylation and sequencing in
SBM detected cfDNA from blastocysts,
cumulus cells and polar bodies in the
culture drop. Interestingly, epiblast and
trophectoderm samples differed in
their methylation profiles, and one-
third of the cfDNA was aligned with
trophectoderm and two-thirds with the
epiblast, suggesting that embryonic
cfDNA can be derived from both ICM
and trophectoderm. Application of an
algorithm to discriminate the percentage
of contamination with cumulus
and polar bodies revealed higher
concordance when there was lower DNA
contamination from outside sources.
Higher embryonic cfDNA quantity was
observed in day 6 embryos, with lower
cumulus cell contamination, but similar
polar body cfDNA compared with day
5. Therefore, the analysis of day 6 SBM
may provide more reliable results (Chen
et al., 2021b).
In summary, there is growing evidence to
support an embryonic origin of cfDNA
detected in SBM that is not derived
solely from the necrosis and apoptosis
of embryo cells. Additional research is
needed to determine what mechanisms
are involved in releasing cfDNA to SBM.
METHODOLOGY AND
CHALLENGES TO
IMPLEMENTATION
When culturing embryos for niPGT-A and
for proper interpretation of NGS results
and diagnosis, it is important to consider
the following.
Can different embryo culture
protocols be applied?
In the development of niPGT-A
approaches, a broad variety of protocols
for embryo culture and various
strategies to analyse the cfDNA have
been used. Most methods involve
small culture volumes to increase the
cfDNA concentration. For conventional
incubators, different volumes have been
used: drops of 4 µl (Lane et al., 2017),
10 µl (Capalbo et al., 2018; Rubio et al.,
2019; 2020b; Sialakouma et al., 2021),
12 µl (Jiao et al., 2019), 15 µl (Huang
et al., 2019; Shamonki et al., 2016), 20 µl
(Lledo et al., 2020), 25 µl (Feichtinger
et al., 2017; Ho et al., 2018; Kuznyetsov
et al., 2018; 2020; Li et al., 2018) or
30 µl (Fang et al., 2019; Xu et al., 2016;
Yeung et al., 2019). For time-lapse
systems, 20–25 µl of culture media have
been used (Vera-Rodriguez et al., 2018).
Small volumes do not appear to affect
embryo development (Minasi et al.,
2015), and both single-step or sequential
culture systems can be used without
interfering with the results (Belandres
et al., 2019). Time lapse is suggested
only if the wells are not connected,
showing similar concordance rates as
culture with conventional incubators (see
FIGURE 3, unpublished data). Interestingly,
no significant differences have been
detected in concordance rates between
trophectoderm biopsies and SBM
analysis across clinics using different
brands of medium and incubator (Rubio
et al., 2020b). Therefore, equipment and
materials do not appear to influence the
results. Albumin percentage was also
determined to be free of DNA traces
that could interfere with medium-based
diagnostics (Hammond et al., 2016).
Regarding the fertilization techniques,
although ICSI is the most common, both
ICSI and IVF provide similar sensitivity
(87.9% versus 80.9%) and specificity
(69.9% versus 78.6%). Interestingly,
IVF is not linked to an increase in male
contamination from spermatozoa.
Therefore, both ICSI and IVF can be
safely applied as long as oocytes or
zygotes are carefully denuded before
ICSI or on day 1 (fertilization check) in
IVF respectively (Rubio et al., 2020b).
How long should the medium be in
contact with the embryo before its
collection?
Extending embryo culture until day 6
(versus day 5) can make a substantial
difference in concordance rates with
trophectoderm biopsy, ICM and whole
blastocysts (Huang et al., 2019; Rubio
et al., 2019; 2020b). Indeed, SBM in
contact with the embryo from day 3
to day 5 had higher non-informativity
rates and an increased presence of
degraded DNA (coming from residual
cells) that can interfere with the results
(Vera-Rodriguez et al., 2018). SBM from
embryos cultured until day 6 showed
higher informativity rates and higher
concordance rates (Rubio et al., 2019;
Yeung et al., 2019) (TABLE 1).
The main concern with extended embryo
culture is the impact on viability and
reproductive potential if embryos are
left in culture to day 6. Notably, several
studies showed that in PGT-A cases with
day 6 vitrification, clinical outcomes
were similar to those with day 5 embryos
(Kaye et al., 2017; Shear et al., 2020;
Tiegs et al., 2019; Viñals-Gonzalez
et al., 2019). Additionally, in a pilot study
using niPGT-A and culturing blastocysts
until day 6, pregnancy rates were high
and similar to those of PGT-A cases
with day 5 or day 6 vitrification (Franco
et al., 2021). Similar results have been
published from one centre participating
in the multicentre niPGT-A study from
Rubio and colleagues (Rubio et al.,
2020b; see also Ocali et al., 2021).
Is assisted hatching or previous
vitrification needed?
Embryos in culture release small amounts
of cfDNA to the media. In most studies
involving non-invasive testing protocols,
assisted hatching was performed to
increase the release of DNA and assure
enough embryonic cfDNA in the sample
(Feichtinger et al., 2017; Ho et al., 2018;
Huang et al., 2019; Jiao et al., 2019;
Kuznyetsov et al., 2020; Lledo et al.,
2020; Shamonki et al., 2016; Vera-
Rodriguez et al., 2018; Yeung et al.,
2019). The benefit of such a procedure
has not been confirmed (Ho et al.,
2018). Other manipulations that were
performed include vitrification (Chen
et al., 2020; Huang et al., 2019; Jiao
et al., 2019; Kuznyetsov et al., 2018;
Li et al., 2021; Shitara et al., 2021; Xu
et al., 2016; Yin et al., 2021; Zhang
et al., 2019b), collapsing of the embryo
and/or biopsy before media collection
(Chen et al., 2020; Feichtinger et al.,
2017; Huang et al., 2019; Jiao et al.,
2019; Kuznyetsov et al., 2018, 2020; Li
et al., 2021; Sialakouma et al., 2021;
Zhang et al., 2019b). Moreover, there
are publications where a completely
non-invasive protocol is performed
achieving ploidy concordance rates with
trophectoderm biopsy as high as 84%
(Rubio et al., 2019; 2020b).
How can contamination be minimized?
It is essential to handle media in
sterile conditions to prevent external
contamination with exogenous DNA,
assuring that only embryonic cfDNA is

FIGURE 3 Ploidy concordance rate in conventional incubators and time-lapse systems (Igenomix internal data).
analysed. Contamination is primarily from
cumulus cells or from external sources
such as technicians, contaminated media,
working surfaces or materials (Leaver and
Wells, 2020; Rubio et al., 2019, 2020b).
Avoiding contamination is one of the
greatest challenges in niPGT-A (Chen
et al., 2021b; Ho et al., 2018; Rubio et al.,
2019, 2020b; Vera-Rodriguez et al.,
2018; Xu et al., 2016; Yeung et al., 2019).
Hence careful embryo manipulation is
critical to assuring accurate results based
on SBM.
Important steps to avoid or minimize
contamination with cumulus cells are
gentle denudation after IVF or before
ICSI, media changes and extra serial
washes of embryos before transfer to
the culture drop. Embryos also should
be handled individually using new
capillaries for each embryo to avoid
cross-contamination (Rubio et al., 2019,
2020b).
Do amplification methods, sequencing
platforms or bioinformatic analyses
impact results?
Once medium has been collected,
WGA is performed. Strategies such as
SurePlex (Illumina, Cambridge, UK),
modified MALBAC (Yikon Genomics,
Shanghai, China) and Reproseq
(ThermoFisher Scientific, MA, USA) are
the most common (TABLES 1–3). As the
concentration of cfDNA present in SBM
samples could be low, modified WGA
protocols have been developed to avoid
this potential limitation (Rubio et al.,
2019, 2020b). In addition, the initial
cell lysis/DNA extraction step during
WGA is unnecessary as cfDNA analysis
provides good results even when this
step is not included (Kuznyetsov et al.,
2020). The next step is the chromosomal
analysis of WGA products. aCGH was
the technique used for aneuploidy
detection in the first studies on niPGT-A
(Feichtinger et al., 2017; Shamonki et al.,
2016). However, NGS is now the most
common technique used to detect
aneuploidy, although with different
platforms. The NGS platform does not
affect the results (TABLES 1–3).
For the interpretation of NGS results,
customized algorithms are available.
They adapt the cut-off values for
chromosome aneuploidy calling
to obtain higher sensitivity and/
or specificity when comparing the
results with trophectoderm biopsies
or whole embryos. The results can
be influenced by differences in the
diagnostic parameters established by
each laboratory as shown by Huang
RBMO VOLUME 44 ISSUE 5 202 2 825
and colleagues and Li and co-workers
(Huang et al., 2019; Li et al., 2021). In
those studies, the ploidy concordance
between different embryonic samples
varied depending on the threshold
for mosaicism used, obtaining optimal
results for 60% and 50%, respectively.
Therefore, the appropriate threshold
must be set up to define a chromosome
as abnormal because the criteria applied
to trophectoderm biopsies and to SBM
samples can differ. Moreover, methods to
deduce the contribution of cumulus and
polar body DNA fractions in SBM could
improve results (Chen et al., 2021b).
CLINICAL RESULTS
Non-invasive aneuploidy testing using
SBM for cfDNA analysis, in combination
with morphology evaluation, would
significantly improve clinical outcomes
in IVF programmes. This approach could
be used to inform prioritization, whereby
blastocysts are ranked for their likelihood
to result in a healthy baby. Indeed,
the first embryo transfer could utilize
the top-ranked blastocysts, while the
remaining blastocysts could be stored for
future transfer by rank order.
The current authors’ group conducted
a study on 46 couples with a PGT-A
826 RBMO VOLUME 44 ISSUE 5 202 2
indication (mainly advanced maternal
age) whereby trophectoderm biopsy
results were used to guide single embryo
transfers (SETs) and clinical outcomes
were retrospectively calculated in
two different scenarios: when euploid
trophectoderm was concordant with
euploid SBM, and when euploid
trophectoderm was discordant with
aneuploid SBM. Ongoing implantation
rates were three-fold higher in euploid
trophectoderm/euploid SBM compared
with euploid trophectoderm/aneuploid
SBM (52.9% versus 16.7%, respectively),
although without reaching statistical
significance due to the small sample
size. Interestingly, SETs did not end in
miscarriage when trophectoderm biopsy
and SBM were euploid concordant
(Rubio et al., 2019).
Several groups have evaluated niPGT-A
with the transfer of euploid embryos,
based only on SBM diagnosis, showing
good results. In a pilot study with only
seven SETs in couples with a PGT-A
or PGT-SR indication, five pregnancies
were achieved, resulting in five livebirths
(Xu et al., 2016). In a second study, in
couples with recurrent spontaneous
abortion or repeated implantation failure,
50 transfers of euploid medium were
performed, resulting in a 58% clinical
pregnancy rate with 27 healthy babies
born (Fang et al., 2019). Similar results
were described in good prognosis
patients (less than 38 years of age) with
ongoing pregnancy rates comparable
to those of PGT-A (61.5%) and higher
than those of conventional IVF or ICSI
(48.5%) (Franco et al., 2021).
CONCLUSIONS:
CONSIDERATIONS IN THE
APPLICATION OF niPGT-A
Morphology and morphokinetics are
non-invasive embryo strategies, but their
ability to identify euploid embryos is not
well established. Conventional PGT-A
is the gold standard for aneuploidy
detection. However, this approach
requires a biopsy, which imparts
significant economic cost, owing to
the need for highly trained personnel
and specialized equipment. In addition,
although trophectoderm biopsy in the
hands of properly trained personnel is
related to a minimal risk to the embryo,
patients and doctors are concerned
about the harm that can be caused by
this invasive procedure. In contrast,
niPGT-A provides information about
blastocyst chromosome status using the cfDNA and assess the clinical benefits of
SBM in which the embryo is cultured, niPGT-A using metrics such as miscarriage
which is typically discarded. Furthermore, rates and live birth rates and including
niPGT-A would provide wider access to long-term follow-up.
embryo genetic analysis and circumvent
many legal and ethical considerations. Consequently, incorporation of
These features represent significant niPGT-A in clinical programmes relies
advantages over conventional PGT-A. on an analysis of embryonic cfDNA
as a biomarker to prioritize embryos
Another advantage of niPGT-A is that for transfer. It is important that the
it can be performed in any laboratory centres interested in this new strategy
without the need for extra equipment. evaluate how to include it in their daily
Moreover, consistent results are obtained routine, determine which patients may
with different types of incubator and benefit the most and understand the
culture medium (Rubio et al., 2020b). sensitivity and specificity of the niPGT-A
However, some changes are needed protocol. niPGT-A with the analysis of
in the daily routine of an embryology the embryonic cfDNA in the SBM is
laboratory since the amount of cfDNA a powerful biomarker, but currently it
present in the culture drop is low. To should not be considered a substitute
increase the concentration of sample for trophectoderm biopsy, since it has
DNA, it is necessary to extend the not yet reached the diagnostic test
amount of time in culture and to category. However, it might help to
reduce the volume of the drop, as prioritize for transfer the embryo with
well as to minimize external/maternal higher reproductive potential, better
contamination. Before introducing than morphology or morphokinetics.
niPGT-A into clinical practice, validation In this scenario, all embryos could be
experiments are needed. New users will considered for transfer according to their
need time to practise, work on the new priority order. This could be an attractive
protocol and obtain good results without option for those patients who want to
compromising the viability of embryos. increase their clinical outcomes without
niPGT-A leverages the cfDNA released by the need for an invasive trophectoderm
the embryo to the culture drop to enable biopsy, and for those patients who do not
DNA analysis. The origin of this cfDNA want to discard embryos.
in the media is confirmed as primarily
embryonic. The amount of cfDNA in
the SBM is independent of the sex and
chromosomal content of the embryo,
as it was the same for male and female
as well as for euploid and aneuploid
embryos (Shamonki et al., 2016; Vera-
Rodríguez et al., 2018). In addition, ploidy
concordance rates between SBM and
trophectoderm biopsy, ICM and whole
embryos can be higher than 90%.
Nevertheless, the precise origin of the
cfDNA is unknown. Therefore, although
the observations reviewed here indicate
that all types of embryo release DNA into
the culture medium during their growth,
the mechanisms involved are unclear.
Despite promising data, until the cellular
mechanisms involved in DNA release are
fully understood, niPGT-A will remain
under scrutiny as a reliable measure for
embryonic chromosomal status. It is
necessary to clarify how representative
the genetic material present in the culture
medium is of the embryo under study.
Further research, including randomized
and non-selection studies, as well as risk
assessment and cost-effectiveness analysis,
is needed to understand the origins of
 RBMO VOLUME 44 ISSUE 5 202 2 827

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Received 26 August 2021; received in revised form
19 January 2022; accepted 24 January 2022.