Clinical reassessment of human embryo ploidy status between cleavage and blastocyst stage by Next Generation Sequencing
Clinical reassessment of human embryo ploidy status between cleavage and blastocyst stage by Next Generation Sequencing
Alberto Liñ aÂn 0 1
Barbara Lawrenz 1
Ibrahim El Khatib 0 1
Asina Bayram 0 1
Ana Arnanz 0 1
Carmen Rubio 1
Rupali Chopra 1
Human M. Fatemi 1
0 IVF Laboratory, IVIRMA Middle-East Fertility Clinic, Abu Dhabi, United Arab Emirates, 2 IVF department, IVIRMA Middle East Fertility Clinic, Abu Dhabi, United Arab Emirates, 3 Obstetrical Department, WomenÂs University Hospital Tuebingen , Tuebingen, Germany, 4 Igenomix, Valencia, Spain, 5 Igenomix, Dubai, UAE
1 Editor: Yang Yu, Peking University Third Hospital , CHINA
One of the most important limitations of genetic testing in preimplantation embryos is embryonic mosaicism, especially when performed on D3 with only a single blastomere evaluated. Previous publications, using Array-Comparative Genomic Hybridization (a-CGH) to compare day 3 (D3) biopsies versus trophectoderm biopsies for the analysis of aneuploid embryos, showed similar high concordance rates per embryo diagnosis for D3 biopsies and trophectoderm biopsies. Next generation sequencing (NGS) was introduced lately as a new technique for preimplantation genetic testing for aneuploidies (PGT-A). Using this technique, this retrospective descriptive study evaluated the degree of the concordance of the diagnosis between preimplantation human cleavage stage (D3) and blastocyst stage (D5) embryos. Double biopsies on D3 and D5 were performed on 118 embryos, reaching blastocyst stage on D5 and had not been selected for transfer. As the fertilization law of the United Arab Emirates does not allow embryo freezing, also surplus euploid embryos after D 3 biopsy were included. Analysis of the NGS results from D3 and D5 embryo biopsies showed a total concordance rate per embryo diagnosis of 85.6% for euploid and aneuploid embryos. The concordance rates per embryo chromosomal pattern for embryo diagnosed as aneuploid at both biopsy stages was 82.2%. However, the status regarding the affected chromosomes was not identical on D3 and D5. Hence, the total concordance rate between D3 biopsy and D5 biopsy was limited to 67.8%. This current study clearly demonstrated that the concordance rates between D3 and D5 biopsies in aneuploid and euploid embryos are lower than previously reported.
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported by Igenomix
who provided support in the form of salaries for
authors [C.R. and R.C.], but did not have any
additional role in the study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. The commercial affiliation of C.R.
and R.C. did not play any role in the study. The
Infertility is a common condition today [
] and exacerbated by the fact that couples tend to
postpone parenthood, leading to an increasing age in couples attempting to conceive [
the first successful IVF treatment with the birth of Louise Joy Brown in 1978, assisted
specific roles of these authors are articulated in the
`author contributions' section.
Competing interests: The authors have the
following interests. This study was supported by
Igenomix, the employer of Carmen Rubio and
Rupali Chopra. Both co-authors participated in the
design and in the blinded analysis of the samples,
however the final data collection and statistical
analysis of the results, as well as the decision to
publish was taken by A.L., B.L and H.F. There are
no patents, products in development or marketed
products to declare. This does not alter the
authors' adherence to all the PLOS ONE policies on
sharing data and materials, as detailed online in the
guide for authors.
reproductive technologies (ART) have improved the chances of infertile couples to achieve a
Despite the improvements in ART, success rates are still limited [
]. One of the most
crucial factors determining success of ART-treatment is the female age, due to increasing
aneuploidy rates in oocytes in women of advanced age . In addition to female age, also male age
has recently been identified as a determinant of delivery rates after ART [
], probably due to
factors such as age-dependent increase of sperm DNA-damage [
]. Other factors, being
relevant for aneuploidy in human spermatozoa are the severity of male factor infertility, abnormal
karyotype of the male partner and it seems that also lifestyle factors like smoking, alcohol
consum and exposure to pesticides might contribute to aneuploidy in human spermatozoa [
Nevertheless, the detection of chromosomal abnormality rates up to 62.9% in couples with a
mean female age of 32.33 years suggest that these aneuploidies are not exclusively attributed to
advanced maternal age [
The most important cause for implantation failure is chromosomal abnormality in the
]. Standard morphological criteria and development pattern cannot predict the
ploidy status of embryos  and the only reliable method for aneuploidy detection requires
biopsy of the embryo and subsequent analysis of the chromosomal status.
Genetic testing of the embryo has led to the first reported pregnancy in 1995 after the use of
fluorescence in situ hybridization technique (FISH) [
]. Newer technologies have displaced
the FISH-technique, considering that only a limited number of chromosomes could be
evaluated, missing approximately up to 1/3 of aneuploidies [
]. Nowadays analysis of the whole
chromosome complement is performed with different genetic platforms like metaphase
comparative genomic hybridization (mCGH), array comparative genomic hybridization (aCGH),
single nucleotide polymorphism (SNP) microarray, quantitative polymerase chain reaction
(qPCR), and most recently, Next Generation Sequencing (NGS) [17±21].
In general, embryos can be assessed by testing single blastomeres from cleavage stage
embryos or by analysis of several cells from the trophectoderm at blastocyst stage. Despite the
different approaches, embryonic mosaicism is an important limitation of embryo biopsy
] and it has been described extensively, that mosaicism is present and common during
preimplantation development . If aneuploidy is detected with PGT-A, the embryo is
deemed abnormal. Hence, due to mosaicism, the discarding of potentially viable, euploid
embryos might occur.
The aim of this descriptive study was to evaluate the extend of the concordance /
discordance between the diagnosis in cleavage stage and blastocyst stage embryos, which may occur
as a result of mosaicism or technical error. For that purpose, we re-evaluated the NGS findings
after cleavage stage embryo biopsies by re-analysing the same embryos with trophectoderm
biopsy. To the best of our knowledge this is the only study which could not only re-evaluate
the results from aneuploid embryos, hence also from euploid embryos. This was possible due
to the law of the United Arab Emirates (UAE) governing ART which does not permit embryo
] as a routine procedure. As a result of this law, surplus euploid embryos cannot be
cryopreserved for future transfer and as a result are discarded.
Material and methods
Embryos of couples with primary or secondary infertility requiring IVF/ICSI and additional
Preimplantation Genetic Testing (PGT) were included in this descriptive study. The age of the
female partner ranged between 20 to 46 years and the Body Mass Index of the female partner
between 19 and 30. Embryos of patients, in whom more than 5 fresh, mature oocytes were
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retrieved at oocyte collection or more than 4 embryos were available for biopsy on D3, were
This retrospective descriptive study was performed in IVIRMA Middle East Fertility Clinic,
Abu Dhabi, from August 2016 to January 2017. Biopsies of the embryos had been performed
on day 3 (D3) and day 5 (D5) for confirmation of the genetic results. The best euploid embryo
(s) after biopsy on D3 and assessment of embryo development and blastocyst score had been
chosen for transfer. Surplus euploid blastocysts, which had not been selected for transfer and
were not cryopreserved in accordance with UAE law had been included. The genetic
laboratory was blinded regarding the embryo identity.
Ovarian stimulation protocols. Ovarian stimulation was performed by standard
protocols, either Gonadotropin-Releasing-Hormone (GnRH)-agonist- or
GnRH-antagonist-protocols, using recFSH (recombinant Follicle-stimulating-hormone) or
human-MenopausalGonadotropin (HMG) as stimulation medication. The dosage of the stimulation medication
was chosen according to the ovarian reserve parameters [
]. Final oocyte maturation was
achieved by administration of either 10.000 IU of hCG, 0.3 mg of GnRH agonist (Triptorelin)
or dual trigger (hCG and GnRH-analogue), as soon as 3 follicles 17 mm were present.
Oocyte retrieval was carried out 36 hours after administration of final oocyte maturation.
Embryo processing and embryo biopsy. Fertilization was assessed about 17±20 h post
ICSI, and embryo development was recorded every 24 h until the day of embryo transfer.
Embryos were cultured in Quinn's Advantage Sequential medium, (SAGE, Målov, Denmark),
using Trigas incubators (6%CO2, 5%O2).
D3 embryo biopsy was only performed in embryos with five or more nucleated blastomeres
and less than 25% fragmentation degree. Embryos were placed on a droplet containing Ca2
+/Mg2+-free medium (G-PGD, Vitrolife, GoÈteborg, Sweden/LifeGlobal, Guilford, CT), the
zona pellucida was perforated by pulses of laser (OCTAX, Herborn, Germany) and one
blastomere was withdrawn from each embryo and placed in 0.2-mL PCR tubes containing 2 μL PBS.
For blastomere washing and handling, 1% polyvinylpyrrolidone (PVP) was used.
For trophectoderm biopsy, embryos were biopsied using Quinn's Advantage Medium with
HEPES, (SAGE, Målov, Denmark) supplemented with HSA, (Vitrolife, GoÈteborg, Sweden).
Three to five laser pulses were used to cut the trophectoderm cells inside the aspiration pipette
and then trophectoderm biopsies were placed in 0.2-ml PCR tubes containing 2 μL PBS.
Determination of aneuploidy by NGS. The herein used NGS platform has been validated
in previous studies [
] and is commercially available in the market. This platform has been
used to analyse blastomere biopsies and trophectoderm biopsies, (Resproseq,
A whole genome amplification (WGA) protocol was performed in all individual samples.
(PicoPlex technology by Rubicon Genomics,Inc; Ann Arbor,Michigan, USA.). After WGA,
library preparation consisted on the incorporation of individual barcodes for the amplified
DNA of each embryo. After Isothermal amplification and enrichment, sequencing was
performed in a 316 or 318 chip using the PGM sequencing machine (Life-Thermofisher, USA).
For sequencing analysis and data interpretation Ion Reporter software was employed.
Embryos were diagnosed as euploid, aneuploid or chaotic abnormal. In case of a result
indicating mosaicism, the embryo was classified as ªeuploidº when the extend of mosaicism was
below 30% and as ªaneuploidº when the extend of mosaicism was above 30%. Chaotic embryos
were defined as those showing a complex pattern of aneuploidies, involving more than six
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Concordance/Discordance rates. Discrepancy is described as a lack of agreement
between two or more facts, i.e. aneuploid on D3 and euploid on D5 or vice versa. The genetic
definition of discordance determines the degree of dissimilarity between the chromosomes
Herein we have included as discrepant results not only the euploid embryos on D3 that
were aneuploid on D5 and vice versa, hence also the aneuploid embryos on D3, that were still
aneuploid on D5, but with different chromosomes involved in the aneuploidy. For the
calculations of the ªfalse positiveº and ªfalse negativeº rates, we have chosen the blastocyst stage
biopsy as the reference, considering the higher technical and biological robustness of
trophectoderm biopy, the decreased incidence of procedural errors and lower impact of mosaicism on
the molecular analysis with day 5 trophectoderm biopsy [
Calculation of the concordance / discordance rates. Two types of concordance rates had
been calculated: the concordance rate per analysed chromosome, where the total number of
chromosomes was considered, independently whether the embryos were euploid or aneuploid
(24 chromosomes per embryo) and the concordance rate per embryo diagnosis, where
discrepancies in the embryo diagnosis (euploid or aneuploid) were considered. Concordance /
discordance rates have been calculated as previously described by Vera-Rodriguez et al. [
For the statistical analysis of the Confidence Intervall (CI), the 95% Confidence Interval
calculator for proportions has been applied.
Ethical approval. Written informed consent was obtained from the couples, whose
embryos were undergoing biopsies on day 3 and day 5. The study was approved by the Ethic
Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics
In this study, initial data of 134 embryos from 45 couples were analysed. The mean age of the
female partners was 33.4 years, ranging from 20 to 46 years, and the mean number of biopsied
embryos per couple was 4.4, with a range from 1 to 9. The indications to perform genetic
testing on the embryos are summarized in Table 1.
Out of the 134 embryos analysed on D3, 6 embryos with ªcomplex abnormalº result and 5
embryos that failed to produce a result due to amplification failure (no DNA was detected
after cleavage stage biopsy) were excluded from the study. Out of these 6 embryos with
complex abnormal findings on cleavage stage, 3 were euploid after blastocyst biopsy. After
blastocyst stage biopsy, five embryos were excluded due to amplification failure. Finally a total of 118
Number of couples
Number of embryos
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embryos from 42 couples were included and biopsies on D3 and D5 were performed. Table 2
summarizes the data of the analysed embryos.
Concordance / Discordance rates
The overall concordance rate per analysed chromosome (24 chromosomes x 118 embryos)
was 98.1% (2779/2832 analysed chromosomes). For whole-chromosome aneuploidy, the
concordance rate was 98.7% (2795/2832) and for segmental aneuploidies was 99.4% (2816/2832).
The concordance rate for whole-chromosome aneuploidies per embryo diagnosis (euploid/
aneuploid) was 91.5% (108/118) and 94.1% (111/118) for segmental aneuploidies, leading to a
total concordance rate per embryo diagnosis of 85.6% (101/118). The data are summarized in
Per embryo diagnosis, 85.6% (95%CI: 79.26±91.93%) of the embryos were concordant, i.e.
they had the same diagnosis (euploid or aneuploid) at cleavage and trophectoderm biopsy.
However, 17 (14.4%; 95%CI: 8.07±20.74%) discrepancies per embryo diagnosis were found.
Table 4 summarizes the data.
The false positive rate per embryo diagnosis, i.e. embryos, which were initially diagnosed as
aneuploid on D3 were diagnosed as euploid embryos on D5 and hence transferable, was 13.6%
(16 out of 118 embryos). Out of these 16 embryos, 7.6% (9/118) were whole-chromosome
Concordance rate per embyo diagnosis (euploid /
(Percentage and number of embryos)
Concordance rates for segmental and whole chromosome aneuploidies per
(Percentage and number of chromosomes)
95% CI: 99.16±99.71%
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discrepancies and 5.9% (7/118) were segmental aneuploidies. False negative rate per embryo
diagnosis, i.e. embryos being diagnosed as euploid on D3 and being diagnosed as aneuploid
on D5, was 0.85% (1 out of 118 embryos, see Table 2).
Concordance rate for aneuploid embryos
The concordance rate for the chromosomal pattern in aneuploid D3 / aneuploid D5 embryos
was 82.2% (97/118) (95%CI: 75.30±89.10%), representing embryos aneuploid at both biopsy
stages. However, the chromosomal status regarding the affected chromosomes was not
identical on D3 and D5. 14 embryos had whole chromosomes discrepancies and 7 embryos had
segmental discrepancies. The discrepant chromosomes in both embryo stages are represented in
Out of 70 aneuploid embryos on D3, 16 (22.9%; 95% CI: 13.02±32.69%) showed a euploid
status after blastocyst biopsy. Fifty-four embryos were aneuploid after biopsy on D3 and D5.
In depth analysis of these 54 embryos 33 (61.1%; 95% CI: 48.11±74.11%) were found to be
concordant for all the aneuploid chromosomes. Twenty-one embryos (38.9%; 95% CI: 25.89±
51.89%) had discrepant chromosomes with different kinds of aneuploidy on D3 and/or D5.
Concordance rate for euploid embryos
The analysis of D3 euploid embryos showed that 47 out of 48 (97.9%; 95%CI: 93.88±101.96%)
were diagnosed as euploid on D5 as well. The embryo, which was not concordant, showed an
aneuploid chromosomal constitution (-12±13 +21 ♀) on D 5.
Overall concordance rate
The total rate of discrepancies was 32.2% (95% CI: 23.77±40.63%), composed of 13.6% (False
positive) + 0.85% (False negative) +17.8% (aneuploid D3-aneuploid D5, with different
aneuploidies either on D3 or D5). Hence the total concordance rate between D3 biopsy and D5
biopsy was limited to only 67.8%.
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+6, +16, -18, +22, ♂
+21, +22, ♂
+3, +4, +6, -22, ♀
+7, +10, +16, +18, +20, ♀
-1q, +8, +16, ♂
+1q, +7p, ♂
+8, -19, ♀
-7q, -22, ♂
-2q, -13, ♀
-9, +14, -15, ♀
+15, +16, +19, -20, X0
To the best of our knowledge, this is the first study re-evaluating not only aneuploid, but also
euploid embryos with double biopsy on D3 and D5 using NGS technology. This is unique due
to the law governing ART in the United Arab Emirates, which does not permit the
cryopreservation of surplus embryos routinely.
Contrary to previous publications, which reported higher concordance rates between
blastomere and trophectoderm biopsies with aCGH technology [
], the current descriptive study,
which includes 118 embryos from 42 couples revealed a total concordance rate per embryo
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diagnosis (euploid/aneuploid) of 85.6%. This concordance rate of 85.6% represents embryos
with the same diagnosis following both cleavage stage and trophectoderm biopsy. In 14.4% (17
embryos) discrepancies per embryo diagnosis (euploid/aneuploid) were detected. However,
when the discrepancies between the diagnosis of the aneuploid embryos were considered
(different chromosomes involved in the aneuploidy between D 3 and D 5 biopsy), the total
discrepancy rate was as high as 32.2%.
The false positive rate per embryo diagnosis, meaning that embryos, which were initially
diagnosed as aneuploid on D3 were finally diagnosed as euploid embryos on D5, was 13.6%.
This finding underlines the decrease of aneuploidy from cleavage to blastocyst stage embryo,
which is in agreement with the data from Fragouli et al. [
], who reported an aneuploidy rate
of 83% in cleavage stage embryos and 58% in blastocyst stage embryos. However, Fragouli
et al. [
] compared the aneuploidy rate, detected with microarray comparative genomic
hybridisation, in embryos biopsied at cleavage stage and embryos at blastocyst stage, which
were derived from different couples and did not re-biopsy the same embryo, as performed in
the herein presented data.
The biological explanation for the discrepancies in this study might be potential mitotic
errors in the division of the blastomeres during the embryo development, giving rise to mosaic
embryos with euploid and aneuploid blastomeres [
]. Contrary to meiotic errors, mitotic
errors are not consistent with maternal age, showing only weak propensities for specific
chromosomes, and often affect many chromosomes simultaneously [
]. However, also
amplification artefacts from single cell genetic technologies on D3 biopsies could be the underlying
cause for discrepancies, leading to a high percentage of false positive rate per embryo diagnosis
]. A previous study by Capalbo et al [
] identified a sensitivity of 86.4% and a specificity of
95% with D3 biopsy with the use of aCGH technique. Compared to aCGH-technigue, NGS
technique allows identification of embryos with chromosomal mosaicism and segmental
aneuploidy more precisely than the PGS/aCGH platform [
] and this fact may account partly for
the lower concordance rates observed in our study, compared to the previous aCGH studies
The topic of discrepant results of genetic testing between cleavage and blastocyst stage
embryos with a decline in the aneuploidy rate towards blastocyst stage has been addressed by
several studies. The main limitation of the first publications, which described inconsistent
results between cleavage and blastocyst stages, is the use of FISH (Fluorescent in situ
hybridization)±technique [38±40]. Later on, other studies confirmed the discrepant results by the use of
different techniques [32,41±44] and those findings are in agreement with our results.
The following mechanisms have been suggested for the reduced rate of aneuploidy in
blastocyst stage embryos: the occurrence of cell arrest or apoptosis of the aneuploid blastomeres,
the active correction or self-correction of the aneuploidy and the allocation of
diploid/aneuploid blastomeres to embryonic or extra-embryonic tissues. Moreover, the possibility of
primary misdiagnosis has to be considered [
]. Cell arrest is initiated after the time of
embryonic genome activation and seems to prevent chromosomal abnormal blastomeres from
further development . Additionally, apoptosis of aneuploidy blastomeres could also be
initiated by weaker mitotic checkpoints [
]. Finally the activation of apoptosis of aneuploid
blastomeres in the blastocyst would result in embryos with a higher proportion of euploid cells
]. Self-correction has been observed in uniparental disomy, however the exact mechanism
is not known. Theoretically, mechanism like anaphase lagging or non-disjunction, which
might cause mitotic errors, might be also able to correct them [
]. The finding of aneuploid
cells only in the placenta and not in the embryo is the basis of the theory of ªpreferential
allocationº, assuming that aneuploid blastomeres are allocated to the trophectoderm where the
detrimental impact of the aneuploidy is lower [
]. However, presence of a similar degree of
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mosaicism in the inner cell mass when compared to trophectoderm, does not support the idea
of preferential allocation of aneuploid blastomeres.
For the correct interpretation of the results from genetic testing on D3 and D5, the
biological and technical limitations have to be considered [
]. Due to the highly variable rate of
mosaicism in cleavage-stage embryos [15,51±53]. D3 biopsies for PGT-A have been criticized
for not being the optimal and accurate technique for ploidy screening and trophectoderm
biopsy is considered to be the more reliable technique [
]. However, a recently published
study showed similar concordance rates with whole blastocyst results on D5, performed as
reanalysis on embryos with previous D3 biopsy [
Presence of mosaicism in the embryo might pose an important factor for the observed
discordance rate found in this study. Four different types of mosaic embryos have been described
at blastocyst stage, according to the cell lineage affected: Total mosaic, inner cell mass (ICM)
mosaic, trophectoderm (TE) mosaic and ICM/TE mosaic [
]. The different types of
mosaicism might make it impossible to detect some of the mosaic embryos with a trophectoderm
biopsy, especially as ICM/TE and ICM mosaicism will never be detectable with the use of a
trophectoderm biopsy. Furthermore, the diagnosis of TE mosaic embryos will differ, depending
on the biopsy location of the TE cells [
]. Therefore the factors mosaicism type and degree,
biopsy location, number of cells biopsied and quality of the biopsy sample are critical
regarding the discrepancies between blastocyst and cleavage stage embryos.
Contrary to cleavage stage biopsy, trophectoderm biopsies contain multiple cells and
therefore an increased number of copies of DNA material in the biopsy specimen. This provides
greater fidelity and may reduce the non-result rate [54±56]. Consequently, a lower rate for
mosaicism is expected with the approach of trophectoderm biopsy [
The predictive value of a TE biopsy to identify a mosaic embryo was evaluated by two
studies. Two to three biopsies in the same embryo were performed and high concordance
(95%100%) were found [56±57]. In those studies also ICM from the same embryos were analysed to
re-evaluate the concordance rate between ICM and TE. A discordant mosaicism rate of
approximately 3%-4% was shown and based on this findings, blastocyst biopsy was confirmed
as a valid method to diagnose blastocyst mosaicism accurately.
The limitations of this study are the retrospective and descriptive character and a possible
bias regarding the concordance/discordance rate due to the fact, that embryos, which had
been selected for transfer, did not undergo biopsy on day 5. The embryos had been selected
according to the results of the genetic testing, done on day 3 and in case of several euploid
embryos, according to morphology scoring. The importance of our findings might be scaled
down by the increasing use of blastocyst biopsies instead of cleavage stage embryo biopsies,
especially as cleavage stage biopsy can have an impact on future developmental and
implantation potential. Despite this circumstances the current study clearly demonstrated that the
concordance rates between D3 and D5 biopsies in aneuploid and euploid embryos are lower than
previously reported. Future studies, including inner cell mass biopsies are required to evaluate
the aetiology of the observed discrepancies between trophectoderm and blastomeres.
We thank Dr. Carol Coughlan for the linguistic revision and correction of the manuscript and
Nicolas Garrido, PhD, for the support of the statistical analysis.
Conceptualization: Alberto LiñaÂn, Barbara Lawrenz, Ibrahim El Khatib, Human M. Fatemi.
Investigation: Alberto LiñaÂn, Ibrahim El Khatib, Asina Bayram, Ana Arnanz.
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Methodology: Alberto LiñaÂn, Ibrahim El Khatib, Rupali Chopra.
Writing ± original draft: Alberto LiñaÂn, Barbara Lawrenz.
Writing ± review & editing: Carmen Rubio, Human M. Fatemi.
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