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Pushing the limits of detection: Investigation of cell-free DNA for aneuploidy screening in embryos

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Pushing the limits of detection: Investigation of cell-free DNA for aneuploidy screening in embryos

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Content 1
Title
Pushing the Limits of Detection: Investigation of Cell-free DNA for Aneuploidy Screening in
Embryos

Author
Jacqueline R Ho, MD

Department/Degree
Clinical, Biomedical, and Translational Investigations
Master of Science
University of Southern California
August 2018

2
Table of Contents Pages
Introduction/Background 3-4
Materials and Methods 4-8
Results 8-11
Discussion 11-15
Acknowledgements 15
Tables 16-18
Figures 19-21
References 22-25

3
Introduction/Background
Selecting the best embryos is crucial for improving patients’ chance of achieving a live
birth with in vitro fertilization (IVF). Blastocyst transfer and preimplantation genetic testing-
aneuploidy (PGT-A) have led to improved outcomes.(1-3) While transferring euploid embryos
improves implantation rates, this may be a less viable option for older women or those with
severely diminished ovarian reserve.(4,5) This patient population may produce fewer or no
blastocysts during an IVF cycle, with reported no blastulation cycle rates ranging between 7.6-
32%.(6,7) For these reasons, there is increasing interest in noninvasive methods for embryo
assessment. Well-studied tools include time-lapse morphokinetics and metabolomics of spent
embryo media (SEM). However, there is limited evidence that these approaches improve clinical
outcomes.(8-12)

Studies have investigated nucleic acids in SEM, and demonstrate different microRNA
and mitochondrial DNA profiles in embryos based on quality and implantation potential.(13-15)
The next logical application for SEM is the use of cell free DNA (cfDNA) for aneuploidy
screening. Recent studies have documented the ability to detect and sequence cfDNA in SEM
using cytogenic techniques such as array comparative genomic hybridization (aCGH) and
modern sequencing platforms such single nucleotide polymorphism (SNP) sequencing and next
generation sequencing (NGS).(16-18) While results have been encouraging, further studies are
needed to assess the validity of this tool on different sequencing and bioinformatics platforms
prior to clinical implementation. In this study, our primary aim was to investigate the accuracy of
cfDNA in SEM using an improved method for DNA capture followed by NGS. We also sought
to determine other factors that may influence accuracy of cfDNA, such as the timing of SEM
collection, timing of assisted hatching (AH), and morphologic grade of the embryos. We also
4
sought to establish a threshold concentration for which cfDNA could be detected and used to
accurately predict the chromosomal status of an embryo. We hypothesized that cfDNA would
have >90% concordance with whole embryos or trophectoderm (TE) biopsies. We also
hypothesized that AH would increase cfDNA concentration and the accuracy of sequenced
cfDNA for aneuploidy screening. We predicted that poor morphologic grade would be associated
with higher DNA shedding and higher accuracy of cfDNA for aneuploidy screening.

Materials and Methods
We conducted a prospective study comparing the accuracy of aneuploidy screening using
cfDNA in SEM as compared to TE biopsies and whole embryos using NGS. The study was
composed of a pilot portion with two separate arms using donated research embryos, as well as a
clinical portion using patient samples. IRB approval was obtained from the University of Southern
California (HS-15-00858).

Pilot Study
For our pilot study, previously cryopreserved embryos donated to research were used. All
embryos were from prior oocyte donation cycles, previously fertilized using intracytoplasmic
sperm injection (ICSI) and subsequently cryopreserved at the zygote stage using the slow-freeze
technique with 1.5M propylene glycol (PROH) and 0.1M sucrose (Irvine Scientific, Santa Ana,
CA). Embryos were thawed using Irvine scientific embryo thaw kit (Santa Ana, CA) by serial
dilutions of cryoprotectant. After the thaw, embryos were placed in labeled wells corresponding
to their study ID. All embryos were cultured in 25 µl of continuous single culture media (CSC,
Irvine Scientific, Santa Ana, CA) using VitroLife Micro-Droplet (Sweden) dish, overlayed with
5
LiteOil (Life Global) at 37°C, with 5% O2 and 8% CO2. They were removed from the incubator
on days 3 and 5 for morphologic grading and for collection of SEM and TE biopsy.
We investigated factors that may potentially influence the concentration and accuracy of
cfDNA, including timing of SEM collection and AH. In the first arm, AH was performed on day
3, after which 5 µl of SEM was collected on both days 3 and 5. Blastocysts then underwent TE
biopsy on day 5. In the second arm, we collected 5 µl of SEM on days 3 and 5, prior to any AH.
Embryos then underwent AH on day 5 after SEM collection, followed by TE biopsy. All
corresponding whole embryos were saved for sequencing. All samples (days 3 and 5 SEM, TE
biopsies, and corresponding whole embryos) were placed in RNAse- and DNAse- free PCR
tubes and stored at -30°C until ready for analysis. Negative controls were culture media placed in
empty wells with the same incubation parameters. Pipette tips were changed between sample
collections to avoid contamination. For all TE biopsies, the Lykos laser system (Hamilton
Thorne, Beverly, MA) was used to remove approximately 5 cells. (Figure 1 for Experimental
Diagram)
Clinical Study Design
For the clinical arm, we prospectively recruited patients planning to undergo PGT-A as a
part of their IVF cycle from March through August of 2017. All oocytes underwent stripping of
all visible cumulus cells using hyaluronidase prior to ICSI. Stripped mature oocytes were then
fertilized using ICSI and cultured per clinical protocol in continuous single culture media (as
described above). All blastocysts underwent AH on day 5 and TE biopsy on days 5 or 6. Five µl
of SEM was collected from wells after embryos were removed for AH and biopsy. All samples
(SEM and TE biopsies) were placed in RNAse- and DNAse- free PCR tubes, and stored at -
30°C. All pilot and clinical samples were shipped on dry ice to a genetic laboratory for testing.
6
Morphologic Grading
On day 3 of embryo development, a cleavage stage morphologic score was assigned based
on a three point grading system using features including cell number, fragmentation, symmetry,
and shape.(19) At the blastocyst stage, morphologic score was based on expansion stage, quality
of inner cell mass (ICM), and quality of TE.(20)
Chromosomal analysis
TE biopsies and whole embryos were subject to cell lysis followed by whole genome
amplification (WGA) using PicoPlex (Rubicon, Inc). During the WGA process, DNA was
randomly amplified. Days 3 and 5 SEM samples underwent WGA in a similar fashion, however,
SEM underwent 20 cycles instead of the standard 14 cycles in order to ensure enough DNA
would be amplified. WGA products were quantified using Qubit dsDNA HS Assay (Life
technologies) and 300ng of whole genome DNA were subject to fragmentation, and adaptor
ligation. The pool of libraries was quantified, and 26pM was used for emulsion PCR for template
enrichment on Ion sphere using the OT2 200 Kit (Life Technologies). The enriched library was
then further purified to remove non-templated spheres, using Ion One Touch™ ES Enrichment.
Samples were individually barcoded, then mixed with a specific complexity to allow at least
200,000 reads per sample. For the library, approximately 40 samples were loaded onto each Ion
530 chip with a chip capacity of over 12 million reads and a chip loading of 80%. The final
library was sequenced using the Ion S5 Sequencer (Life Technologies). Each sample had an
average of 150,000 to 200,000 reads and approximately 200 bp size per amplicon, totaling 30 to
40 million bp per sample. All reads were filtered for polyclonals and aligned to the human
genome database using Torrent Suite™ Software for Sequencing Data Analysis. Quality reads
were scored for aneuploidy using Ion Reporter™ 5.0 software (Thermo Fisher Scientific). Each
7
sample had a unique study ID, thus investigators performing NGS on SEM were blinded which
arm of the study design samples derived from and were also blinded to the results of referent
samples (whole embryos or TE biopsies).
Statistical analysis
Concordance rates were calculated for aneuploidy and sex between the following groups:
day 3 SEM and corresponding whole embryos, day 5 SEM and whole embryos (for pilot
samples), day 5 SEM and TE biopsies, and TE biopsies and whole embryos. Chi-square,
Pearson’s exact, and Kruskall-Wallis were used to test for association between concordance and
blastulation status, AH (yes vs. no), ploidy (euploid vs. aneuploid), embryo morphology
(fair/good vs. poor), and fragmentation index (high >20%, medium 10-20%, and low <10%).
Day 3 morphology was used with statistical tests using day 3 cfDNA, and day 5 morphology
with statistical tests involving day 5 cfDNA, Sensitivity, specificity, positive predictive value
(PPV), and negative predictive values (NPV) were calculated for detection of aneuploidy and
sex. Given that previous data on cell-free fetal DNA (cffDNA) showed an increased ability to
distinguish aneuploid cases with increasing sequencing depth, we also performed a logistic
regression to test the relationship between sequence depths of days 3 and 5 cfDNA and
predictive value for determining ploidy status.(21) Receiver-operating curves were created and
the area under the receiver-operating curve (AUC) was determined. The AUC measures how
well a test can distinguish between normal and abnormal groups. A value of 1 represents a
perfect test and 0.5 represents inability to distinguish between groups. P-values <0.05 using a
two-tailed alpha were considered significant. STATA 13 (Statacorp, College Station, TX, USA)
was used for analyses. Samples yielding results with an indeterminate read for ploidy or sex were
re-sequenced. Samples with any final indeterminate read were excluded from our analysis.
8

Results
For the pilot study, 45 research embryos previously cryopreserved at the zygote staged
were thawed from three different patients. 41/45 (91.1%) embryos survived the thaw, and 23/41
(56%) became blastocysts. At day 3, cfDNA was detectable in 40/41 (97.6%) of SEM samples,
and only 16/41 (39%) samples generated sufficient sequence reads to obtain accurate
chromosome copy status. On day 5, cfDNA was detectable in 40/41 (97.6%) of day 5 SEM
samples, and 33/41 (80.4%) generated reads. Median day 3 cfDNA concentration was
significantly higher on day 3 (111ng/µl; interquartile range 96.6-120 ng/µl than on day 5
(105ng/µl; IQ range 41.4-117ng/µl), p<0.01. From our clinical arm, the median age at oocyte
retrieval was 37 (range 33-43) years. 20/20 (100%) day 5 SEM samples were sequenced, with
the median cfDNA concentration being 88.7 ng/µl (84.3-92.7ng/µl). The lowest concentrations
of cfDNA leading to accurate detection of ploidy were 76.2 ng/µl on day 3 and 63.2 ng/µl on day
5. (Table 3 for Demographics)
The concordance rate between TE biopsies and whole embryos samples for ploidy and
sex was 25/27 (93%) and 26/27 (96.3%), showing a high concordance as previously
established.(21) Concordance between day 3 cfDNA and corresponding whole embryos was 9/16
(56.3%) for ploidy and 13/16 (81.3%) for sex. Concordance for day 5 cfDNA media and whole
embryos had a concordance rate of 15/33 (45.5%) and 26/33 (78.8%) for ploidy and sex. 33/41
of research (80.5%) embryos generated sequence reads, with 20/33 (60.6%) of those progressing
to the blastocyst stage and 13/33 (39.4%) arresting prior to the blastocyst stage. Concordance for
ploidy in day 5 SEM and whole embryos was significantly higher in the blastocyst vs. arrested
embryo group 13/20 (65%) vs. 2/13 (18.2%), p=0.005. However, concordance was not different
9
for sex between blastocysts and arrested embryos 17/20 (85%) vs. 9/13 (69%) p=0.28. For all
blastocysts, including research (n=20) and clinical samples (n=20), concordance for ploidy and
sex between day 5 cfDNA and TE biopsies was 26/40 (65%) and 28/40 (70%). Of blastocysts,
there were 8/19 (42.1%) false positive and 3/21 (14.3%) false negative calls for aneuploidy from
day 5 cfDNA. In determining embryo sex, male embryos were more likely to be misdiagnosed
than females by cfDNA. There were 26 female embryos and 27 male embryos (as determined
from sequencing the whole embryo or TE biopsy). However, from day 5 cfDNA, 17/27 (63%)
of males were misdiagnosed as females whereas 0/26 (0%) of females were misdiagnosed as
males (p=0.001). (Table 1 for concordance rates, Table 4 for sequences, Supplemental Figures 1
and 2 for visual analysis of examples concordant and discordant cfDNA and whole embryo/TE
biopsy sequences)
Sensitivity, specificity, PPV and NPV were calculated for detection of aneuploidy and
sex using cfDNA. The reference for day 3 cfDNA was the corresponding whole embryo read and
for day 5 cfDNA was the corresponding TE biopsy read. Day 5 cfDNA had a sensitivity of 0.8,
specificity of 0.61, PPV of 0.47, and NPV of 0.88 for aneuploidy detection, and overall
performed better than day 3 cfDNA. (See Table 2) The odds ratio for determining ploidy based
on sequence depth using day 3 cfDNA was 0.97 (95% CI 0.95, 0.99), p=0.02. The AUC was
0.82, suggesting that sequence depth discriminated between euploid and aneuploid embryos,
with our data showing that aneuploid embryos had a higher number of reads on day 3. The odds
ratio for 5 cfDNA was 0.99 (95% CI 0.98, 1.0), p=0.12. The AUC was 0.6, indicating that day 5
cfDNA sequencing depth serves as a poor screening tool for distinguishing euploid and
aneuploid embryos. While increasing sequencing depth has been shown to provide better
discrimination for targeted trisomies 21 and 18 aneuploidy screening using cffDNA in maternal
10
serum, this is not true of general aneuploidy screening using embryonic cfDNA in SEM.(21)
(Figure 2 for ROC Curves)
AH was not associated with a difference in cfDNA concentrations, either on day 3
(median cfDNA was 112 vs. 110 ng/µl for AH vs. no AH, p=0.83) or on day 5 (median cfDNA
was 89.2 vs. 106 ng/µl for AH vs. no AH, p=0.17). Concordance rates were for ploidy and sex
were not significantly different between AH and no AH groups for day 3 and day 5. (See Table
1). When comparing euploid vs. aneuploid embryos, cfDNA concentration from day 3 SEM was
higher in aneuploid vs. euploid embryos (115.58 vs. 102.83 ng/µl, p=0.08). The number of
sequence reads was higher in aneuploid embryos vs. euploid embryos from day 3 cfDNA as well
(113,388 vs. 70,030, p=0.008). This relationship was not significant for cfDNA concentration
(84.6 vs. 85.8 ng/µl, p=0.9) or number sequencing reads (157,036 vs. 139,500, p=0.13) from day
5 SEM.
Morphology and fragmentation was not associated with cfDNA concentration or with
concordance rates. Embryos with low, moderate, and high fragmentation, had median
concentrations of 114, 96.2, and 112 ng/µl (p=0.13) on day 3, and 86.2, 92.8, and 103 ng/µl
(p=0.36) on day 5. Good/fair vs. poor embryos had similar cfDNA concentrations on day 3 (110
vs. 120 ng/µl, p=0.2) and day 5 (89.8 vs. 86 ng/µl, p=0.7). Concordance rates for ploidy were not
significantly different good/fair vs. poor morphology embryos using day 5 cfDNA (19/28, 67.9%
vs. 7/12, 58.3%), p=0.56. There were no poor-quality embryos yielding cfDNA on day 3. When
comparing low/medium and high fragmentation embryos, there was no difference in
concordance for ploidy using day 3 cfDNA [3/5 (60%) vs. 6/11 (54.5%), p=0.84. Concordance
was similar in low/medium vs. high fragmentation embryos in day 5 cfDNA [18/29 (62%) vs.
8/11 (72.7%), p=0.53].
11

Discussion
Age-associated aneuploidy contributes to decreased pregnancy rates and higher
miscarriage rates.(22,23) To obviate this problem, clinicians have incorporated PGT-A to
improve the selection of euploid embryos. TE biopsies from blastocysts have been shown to be
safer than cleavage stage blastomere biopsies, and do not have negative effects on implantation
rates based on a randomized control trial.(24,25) TE biopsies also provide more accurate reads
due to a higher number of cells sampled and possibly aneuploidy self-correction that occurs
during embryo development.(26,27) However, there is still debate regarding the efficiency of
PGT-A and whether it improves live birth rates across all age groups.(28-30) CfDNA has
emerged as a noninvasive strategy for aneuploidy screening. If optimized, it may be an option for
those that produce no or poor-quality blastocysts and may theoretically mitigate potential adverse
effects of TE biopsies on embryos, particularly in laboratories with less experience performing
them. Two studies involving aspiration of blastocoel fluid (BF) as a potential source of cfDNA
have yielded reasonable concordance rates (48-97%).(31,32) However, studies with BF have not
been replicated using modern sequencing platforms, and techniques for BF collection still
involve embryo manipulation. SEM has been more recently investigated, with one study showing
a high accuracy for ploidy screening with NGS (86%).(17) Despite this,

it is necessary to
replicate results in different centers with different sequencing and bioinformatics platforms to
assess generalizability of the previous findings. Additionally, it was unclear whether accurate
diagnoses of aneuploidy could be made earlier in the cleavage stage.
CfDNA can be derived from apoptotic or necrotic cells, or from release by actively
dividing cells.(33) CfDNA usually consists of fragmented segments averaging 70-200 bp in
12
length compared to genomic DNA, which has 3 million bp.(34) This brings into question
whether random fragments of cfDNA released by the embryo provide sufficient genomic
coverage. With TE biopsy an average of five cells is sampled, and after cell lysis and
amplification, the typical genomic coverage is around 72% with a depth of 30x reads.(35,36)
Using the Thermo Fisher Scientific platform for NGS, the coverage for TE biopsies is 50-60
million reads, which leads to about 2% coverage of the genome. We were able to achieve similar
numbers of reads for cfDNA in SEM. Despite the ability to reliably sequence cfDNA on day 5,
calculations for specificity and NPV at 0.61 and 0.88 are still not high enough to reassure
patients that a euploid embryo is being selected. CffDNA as a screening tool for trisomies 21, 18,
and 13, yields specificity values of 0.99, 0.9, and 1.0 and NPV of 1.0, 1.0, and 1.0.(37)
Meanwhile, PGT-A with TE biopsy using NGS yields a specificity of 0.99-1.0 and NPV of 1.0
for aneuploidy.(38,39) Xu et al. published rates of specificity and NPV at 0.84 and 0.91 using
cfDNA sequenced on an MALBEC-NGS platform.(17) These rates are higher than what we
found in our study, possibly due to differences in methodology. In their study, embryos were
previously vitrified on day 3 and thawed and grown to the blastocyst stage. In our study, research
embryos were previously cryopreserved and thawed at the zygote stage, while clinical embryos
were grown in continuous media after fertilization. This may potentially lead to detection of
cfDNA from residual cumulus cells. While it is theoretically possible that residual cumulus cells
not completely stripped may lead to maternal contamination, we attempted to minimize this
effect by removing all visible cumulus cells from oocytes prior to ICSI. Vera-Rodriguez et al,
reported a maternal contamination rate of 60.8% in cfDNA from SEM.(18) Their study reported
a lower concordance rate than Xu et al, with matching aneuploid calls at 30.4%.(18) While our
overall concordant rates for ploidy using day 5 cfDNA were 65%, if we exclude matching 46,XX
13
calls (due to an inability to account for maternal contamination in these samples), we have a
concordant aneuploid call rate of 8/30 (27%) between day 5 cfDNA and TE biopsies, similar to
the Vera-Rodriguez study. They also reported a sensitivity for Y chromosome detection of
around 29.6%, similar to our 34% sensitivity for Y chromosome detection using day 5
cfDNA.(18) The misdiagnosis of male embryos as female embryos may reflect a limited ability
to amplify and sequence the Y chromosome from low DNA concentrations. However, the similar
rate of discordant aneuploid calls and low sensitivity for Y chromosome detection suggest that
maternal contamination likely diminishes the ability to detect and sequence pure embryonic
DNA. An alternative explanation for misdiagnosis is detection of cfDNA from individual cells
that have undergone post-mitotic error or amplification error. Another factor to consider with
cfDNA is the inability to detect and characterize mosaicism, which carries a higher risk of
miscarriage, failed implantation, and lower pregnancy rates.(40,41) While we could not assess
for mosaicism in our study due to the nature of the DNA source, this remains to be characterized
in future studies.
Despite having higher concentrations of cfDNA on day 3, high quality cfDNA was more
likely to be successfully amplified in day 5 SEM. However, we were still able to achieve
accurate reads from some day 3 cfDNA samples. Interestingly, there was a significant difference
in sequence reads for aneuploid vs. euploid embryos on day 3, suggesting that at that stage,
abnormal cells may have higher rates of DNA shedding via cell degradation or active secretion
of DNA into the media, which leads to detection of cfDNA not reflective of the rest of the
embryo. In comparison to the cleavage stage, blastocyst stage embryos may have a higher
proportion of embryonic cfDNA reflective of actual ploidy status in SEM as dividing embryos
continue to secrete cfDNA while cfDNA released from abnormal cells is concurrently degraded.
14
AH is used prior to TE biopsy, but has also been utilized with the goal of improving
clinical pregnancy rates in poor prognosis patients.(42,43) We hypothesized that AH would
facilitate the release of cfDNA into the culture media. While AH is thought to be benign in the
human preimplantation embryo, temperatures during AH using the diode laser beam can reach
anywhere from 130° to 160° C.(44) A study on mouse embryos examining the effects of the
diode laser on DNA damage found higher DNA damage in AH vs. non AH embryos, and even
higher proportions when AH was performed earlier in embryonic development.(45) Any
potential DNA damage from laser AH could theoretically lead to cross-contamination with
actively secreted biological DNA. In contrast to our hypothesis, our results demonstrated slightly
higher cfDNA concentrations and concordance rates for embryos that did not undergo AH prior
to SEM collection. This, however, did not reach statistical significance, possibly due to a small
sample size.
Strengths of the study include the exploration of several factors influencing accuracy of
cfDNA including timing of AH and SEM collection as early as the cleavage stage. We were also
able to include TE biopsies and whole embryos as reference groups in our research cohort.
Limitations include a small sample size and inability to distinguish false aneuploid reads as
amplification errors vs. contamination from aneuploid cells undergoing apoptosis. Protocols for
cfDNA included additional 6 rounds of amplification, which could theoretically lead to a higher
error rate due to amplification bias.(46) However, bias is typically minimized during targeted
NGS, as there is high coverage for consensus sequences, which reduces the noise from random
errors.(47) We also did not follow patients to invasive prenatal diagnostic screening, which
would provide better information regarding the accuracy of cfDNA as a screening tool.
15
To our knowledge, this is the first study to sequence cfDNA from the cleavage stage and
determine whether AH influences the viability of cfDNA as a screening tool. CfDNA is
detectable and can be successfully sequenced at the cleavage and blastocyst stages, with a
minimum concentration of 63.2ng/µl leading to an accurate ploidy diagnosis. Additionally, we
found that DNA was more likely to be amplified and accurate on day 5. AH does not appear to
be necessary or helpful for detection and sequencing of cfDNA for PGT-A. While sequence
depth does not appear to influence accuracy of ploidy discrimination, enhancing embryonic
cfDNA isolation protocols may improve the purity of the sample and thus sensitivity and
specificity of sequence reads. In conclusion, cfDNA in SEM is not currently optimized for
aneuploidy screening in embryos, but with further improvement, remains a promising tool for
noninvasive PGT-A.

Acknowledgements
This research was funded by a Vivere Scientific Advisory Board Research Grant as well as
Progenesis, Inc. I would like to thank my research mentors, Dr. Lynda McGinnis, Dr. Karine
Chung, Dr. Richard Paulson, Dr. Ali Ahmady, Dr. Sue Ingles, and Dr. Nabil Arrach.

16
Tables

Table 1. Concordance rates between cfDNA, trophectoderm biopsy, and whole embryos

Concordance for Ploidy Concordance for Sex
Pairs Total
Assisted
Hatching
No
Assisted
Hatching
P-
value
c
Total
Assisted
Hatching
No
Assisted
Hatching
P-
value
c
Day 3 Cell-Free
DNA vs. Whole
Embryo
a
(n=16)
9/16
(56.3%)
4/8
(50.0%)
5/8
(62.5%)
0.61
13/16
(81.3%)
5/8
(62.5%)
8/8
(100%)
0.06
Day 5 Cell-Free
DNA vs. Whole
Embryo
a
(n=33)
15/33
(45.5%)
5/16
(31.3%)
10/17
(58.8%)
0.11
26/33
(78.7%)
12/16
(75.0%)
14/17
(82.4%)
0.61
Day 5 Cell-Free
DNA vs.
Trophectoderm
Biopsy
b
(n=40)
26/40
(65.0 %)
16/28
(57.1%)
10/12
(83.3%)
0.16
28/40
(70.0%)
17/28
(60.7%)
11/12
(91.7%)
0.07
Day 5
Trophectoderm
Biopsy vs.
Whole Embryo
a

(n=27)
25/27
(92.6%)
12/14
(85.7%)
13/13
(100%)
0.22
26/27
(96.3%)
14/14
(100%)
12/13
(92.3%)
0.48
Values depicted as n (percentage),
a
Includes research embryos only,
b
Includes both research
embryos and clinical samples,
c
Chi-square analysis or Fisher’s Exact used to compare AH vs. no
AH groups, p<0.05 considered significant.

Table 2. Characteristics of cfDNA as a screening test for aneuploidy and sex

Sensitivity Specificity
Positive
Predictive
Value
Negative
Predictive
Value
Day 3 cfDNA aneuploidy
detection (n=16)
0.33
(-0.2-0.87)
0.69
(0.44-0.94)
0.20
(-0.15-0.55)
0.82
(0.69-1.05)
Day 3 cfDNA sex detection
0.38
(0.12-0.65)
0.80
(0.55-1.05)
0.71
(0.38-1.05)
0.50
(0.26-0.75)
Day 5 cfDNA aneuploidy
detection (n=40)
0.80
(0.55-1.0)
0.61
(0.41-0.81)
0.47
(0.23-0.71)
0.88
(0.71-1.0)
Day 5 cfDNA sex detection
0.34
(0.15-0.54)
1.0
(1.0-1.0)
1.0
(1.0-1.0)
0.57
(0.41-0.71)
Trophectoderm biopsy
aneuploidy detection (n=27)
1.0
(1.0-1.0)
0.90
(0.78-1.03)
0.75
(0.45-1.05)
1.0
(1.0-1.0)
Trophectoderm biopsy sex
detection
1.0
(1.0-1.0)
0.94
(0.84-1.05)
0.88
(0.65-1.1)
1.0
(1.0-1.0)
Values depicted as point estimate (95% confidence interval)

17
Table 3. Demographics
Variable Values
Age at Retrieval (years) 31.8 (9.2)
Duration of Stimulation
(days)
10 (0.76)
Total Gonadotropin Dose
(IU)
2657.8 (734.3)
Number of Oocytes
Retrieved
34 (14.2)
Number of MIIs 26.3 (12.7)
Fertilization Type All 100% ICSI
Fertilization Rate 81.4% (16.3%)
Duration of Cryopreservation
for Research Embryos (days)
4253.6 (1247.7)
Aneuploidy Rate 18 (30.5%)
% Males 27 (46.6%)
Values depicted as means (std. deviation) or n (%)

Table 4. CfDNA Sequences

Matching Aneuploid Calls from Day 3 Media and Whole Embryo
Day 3 Media Day 5 Whole Embryo
49,X,+3,+8,+12+17 47,X,+3,+12,-11
49,XX,+4,+9,+20 46,XY,+9,-10
50,XX,+9,+10,+17,+20 47,XY,+9
50,XX,+2,+4,+8,+9,+10,+13,-17,-20 46,X,+13
False Negative Reads from Day 3 Media
Day 3 Media Day 5 Whole Embryo
46,XX 47,XX,+21 (Mosaic 21)
46,XX 46,XY,+20,-9
46,XX 48,XX,+7,+18
False Positive Reads from Day 3 Media
Day 3 Media Day 5 Whole Embryo
47,XX,+5 46,XX
55,XX,+3,+5,+6,+10,+10,+11,+12,+15,+19 46,XX
45,XX,-14 46,XX
47,XX,+9 46,XY
46,XXX,+3,+7,+15,-1,-11,-13,-20 46,XX
49,XX,+9,+10,+20 46,XX
47,XX,+10 46,XX
48,XX,+13,+15 46,XX
53,XX,+4,+9,+10,+13,+15,+17,+20 46,XX
55,XX,+2,+4,+8,+9,+10,+13,+15,+17,+20 46,XX
18
48,XX,+9,+10 46,XY
47,XX,+20 46,XX
46,X,+4 46,XX
48,XX,+9,+20 46,XX
49,XX,+9,+10,+17 46,XX
Matching Aneuploid Calls from Day 5 Media and Trophectoderm Biopsy
Day 5 Media Trophectoderm Biopsy
45,X 46,XXX,-1
49,XX,+14,+16,+18 47,XY,+9
45,XY,-16 45,XY,-16
49,XXY,+8,+21 47,XY,+21
46,XY,+3,-14 46,XY,+3,-14
45,X 43,XY,-4,-6,-14
45,X 45,XY,-3
46,X,+8 44,XX,-1,-13
False Negative Reads from Day 5 Media
Day 5 Media Trophectoderm Biopsy
46,XX 45,XY,-4
46,XX 47,XY,+22
46,XX 45,XY,+9,-10,-20
46,XX 47,XY,+9
46,XX 46,XY,+20,-9
46,XX 48,XX,+7,+18
False Positive Reads from Day 5 Media
Day 5 Media Trophectoderm Biopsy
44,XY,-9,-11 46,XY
47,XX,+4,+16,-1 46,XX
47,XX,+10 46,XY
50,XX,+2,+7,+10,+17 46,XX
44,XX,-8,-18 46,XX
45,XX,-18 46,XX
47,XX,+6 46, XX
48,XX,+8,+18 46, XX
45,X 46,XY
47,XY,+18 46,XY
45,XX,-2 46,XX
45,X 46,XY

19
Figures

Figure 1. Experimental Diagram for Pilot Study

Figure 2. ROC Curves for Day 3 and Day 5 Cell-free DNA Based on Sequencing Depth

(Left) The AUC for day 3 cfDNA using varying sequencing depth cutoffs was 0.82. This
indicates that sequencing depth does discriminate between euploid and aneuploid embryos, with
aneuploid embryos having higher amounts of DNA in SEM.

(Right) The AUC for cfDNA predictive values using varying sequencing depth cutoffs was 0.60.
This indicates that sequencing depth does not discriminate between euploid and aneuploid
embryos, and does not enhance day 5 cfDNA as a screening tool for aneuploidy.

20

Figure 3. Visual Analysis of Day 3 CfDNA and Whole Embryo Sequences

A) Concordant aneuploid calls between day 3 cfDNA and whole embryo
B) Discordant aneuploid calls between day 3 cfDNA and whole embryo (false positive)

21
Figure 4. Visual Analysis of Day 5 CfDNA and Trophectoderm Biopsy Sequences

A) Concordant aneuploid calls between day 5 cfDNA and trophectoderm biopsy
B) Discordant aneuploid calls between day 5 cfDNA and trophectoderm biopsy (false positive)

22
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Abstract (if available)

Abstract Objective: To determine the accuracy of cell-free DNA (cfDNA) in spent embryo media (SEM) for ploidy and sex detection at the cleavage and blastocyst stages. To determine if assisted hatching (AH) and morphologic grade influence cfDNA concentration and accuracy. ❧ Materials and Methods: This was a prospective cohort study at an academic fertility center. We included 41 previously cryopreserved zygote embryos donated for research and 9 patients undergoing IVF with preimplantation genetic testing for aneuploidy (PGT-A). In a donated embryo arm, SEM was collected on days 3 and 5, with half of embryos undergoing AH before and half after. In a clinical arm, SEM was collected on day 5 before trophectoderm biopsy. Samples underwent PGT-A with next generation sequencing. CfDNA results were compared with corresponding whole embryos and trophectoderm biopsies. We calculated concordance rates, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for ploidy and sex detection using cfDNA. ❧ Results: Of 141 samples, cfDNA was amplified in 39% and 80.4% of days 3 and 5 SEM. Concordance for ploidy and sex was 56.3% and 81.3% between day 3 cfDNA and whole embryos, and 65% and 70% between day 5 cfDNA and TE biopsies. Day 5 cfDNA sensitivity and specificity for aneuploidy were 0.8 and 0.61. PPV and NPV were 0.47 and 0.88. Timing of AH and morphology did not influence cfDNA concentration or accuracy. ❧ Conclusions: CfDNA is detectable on days 3 and 5, but more accurate on day 5. While our data suggest moderate concordance rates, PGT-A using cfDNA must be further optimized before clinical implementation.

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Creator Ho, Jacqueline Rowena (author)

Core Title Pushing the limits of detection: Investigation of cell-free DNA for aneuploidy screening in embryos

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Publication Date 07/17/2018

Defense Date 07/17/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cell-free DNA,embryo,genetic testing,OAI-PMH Harvest

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Language English

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Advisor Paulson, Richard (committee chair), Chung, Karine (committee member), McGinnis, Lynda (committee member)

Creator Email jackierho@gmail.com,jacquerh@usc.edu

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