A deep dive into the morphokinetics

and ploidy of low-quality blastocysts

Molly M. Quinn, M.D.,a Philip Marsh, M.S.,b Salustiano Ribeiro, M.S.,b Rhodel K. Simbulan, M.S.,b
and Mitchell P. Rosen, M.D.b
a
Department of Obstetrics and Gynecology, University of Southern California, Los Angeles, California; and b Department of
Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, California.

Objective: To describe morphokinetic parameters and ploidy among low-quality blastocysts not meeting the criteria for clinical use.
Design: Prospective cohort study.
Setting: Academic medical center.
Patient(s): Two hundred patients undergoing in vitro fertilization between February 2018 and November 2019.
Intervention(s): All embryos were cultured in a time-lapse incubator. All expanded blastocysts underwent preimplantation genetic
testing for aneuploidy using next-generation sequencing.
Main Outcome Measure(s): Static blastocyst morphology grading; morphokinetic parameters, including time to each cell division (2-
cell formation to 8-cell formation); time to morula formation; time to the start of blastulation; time to blastocyst formation; and
preimplantation genetic testing for aneuploidy results.
Result(s): A total of 1,306 embryos progressed to the expanded blastocyst stage; of these, 935 embryos met the criteria for clinical use
and were designated as high quality, whereas 371 embryos were graded as low quality and did not meet the criteria for use. In mor-
phokinetic evaluation, low-quality embryos developed more quickly to 5-cell formation (t5) 48.4 [42.4–48.7) vs 50.2 [46.3–50.1]
hours, but progressed more slowly thereafter with tM 91.5 [85.9–92.3] vs 88.3 [82.1–88.3] and tB 114.0 [106.4–113.9] vs 106.9
[101.3–107.4] hours. Among the low-quality embryos, 75.5% were aneuploid, 22.4% were euploid, and 2.2% had undetermined
chromosome copy number results. Morphokinetic parameters did not differ between the euploid and aneuploid low-quality embryos.
Conclusion(s): Morphokinetic analysis did not distinguish between euploid and aneuploid low-quality embryos. (Fertil Steril RepÒ
2022;3:231–6. Ó2022 by American Society for Reproductive Medicine.)
Key Words: IVF, embryo morphokinetics, aneuploidy, embryo quality, time-lapse imaging
Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfre-d-22-00075

I
n vitro fertilization has progressed cycle with an attendant delay in trans- demonstrated an association with
to a stage at which the focus is no fer and a requirement for vitrification ploidy that is strong enough to supplant
longer on the ability to achieve a and subsequent warming of a selected the clinical application of PGT-A (1).
pregnancy but on the time to singleton embryo. Time-lapse imaging (TLI) has Historically, standard static embryo
pregnancy and live birth. Improve- been explored as a noninvasive mecha- morphology gradings have been used to
ments in the embryology laboratory nism for identifying an embryo that is select embryos for transfer. In practices
have allowed for blastocyst culture most likely to result in successful im- performing a high volume of PGT-A, it
and other methods aimed at selecting plantation via continuous image acqui- is common to define a criterion for bi-
the single best embryo for transfer. sition and study of individual embryo opsy that excludes embryos with poor-
One of these methods is trophectoderm morphokinetics. Multiple algorithms quality TE or inner cell mass (ICM)
(TE) biopsy at the blastocyst stage for have been designed to use embryo mor- grading from undergoing TE biopsy for
preimplantation genetic testing for phokinetics to predict embryos with a PGT-A. In this setting, low-quality em-
aneuploidy (PGT-A). A downside of higher probability of live birth upon bryos are discarded (2). However, studies
PGT-A is the need for a ‘‘freeze all’’ transfer; however, none of these have have shown that low-quality embryos
can result in successful pregnancies (3).
Received April 13, 2022; revised June 13, 2022; accepted June 14, 2022.
We sought to describe the morphoki-
M.M.Q. has nothing to disclose. P.M. has nothing to disclose. S.R has nothing to disclose. R.K.S. has netics of low-quality embryos and any
nothing to disclose. M.P.R. has nothing to disclose. relationship to embryonic ploidy in an
Reprint requests: Molly M. Quinn, M.D., Department of Obstetrics and Gynecology, Keck School of
Medicine at the University of Southern California, 2020 Zonal Ave, IRD 534, Los Angeles, Califor- attempt to elucidate features that could
nia 90033 (E-mail: [email protected]). predict euploid status from a low-
Fertil Steril Rep® Vol. 3, No. 3, September 2022 2666-3341
quality embryo. Our hypothesis is that
© 2022 The Authors. Published by Elsevier Inc. on behalf of American Society for Reproductive Med- morphokinetic parameters predict blas-
icine. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ tocyst quality as measured by static
licenses/by-nc-nd/4.0/).
https://doi.org/10.1016/j.xfre.2022.06.004 morphology; however, we predict that

VOL. 3 NO. 3 / SEPTEMBER 2022 231

ORIGINAL ARTICLE: ASSISTED REPRODUCTION

morphokinetic parameters will have a more limited impact on syngamy, time to 2–8 cells, time to morula, time to start of
embryonic ploidy. blastulation, time to blastocyst, and time to expanded blasto-
cyst. Cleavage anomalies were recorded.
Embryonic biopsy for preimplantation genetic testing
MATERIALS AND METHODS was performed at the blastocyst stage in all embryos reaching
Trial Design and Study Population full blastocyst. On the day of the biopsy, 5–10 TE cells were
This is a secondary analysis of a sibling oocyte study of 2 gently aspirated. Biopsied cells were washed and cryopre-
different culture media systems designed to study early em- served before being sent for testing. Biopsied TE cells were
bryonic development within a time-lapse incubator (4). In analyzed for all 24 chromosomes by the testing laboratory
the primary study, embryo quality, morphokinetic parame- (PacGenomics, Agoura Hills, CA) using a next-generation
ters, and aneuploidy rates from TE biopsy were similar be- sequencing–based assay.
tween sibling embryos cultured in distinct media systems
from the time of gamete isolation. For this study, we focused
Predictors
on the static morphology, morphokinetics, and ploidy of
high-quality embryos vs. those of poor-quality embryos The primary predictors or exposure variables were morphoki-
that were graded insufficient for clinical use. Individuals netic parameters assessing time to specific developmental
planning in vitro fertilization with the intent of blastocyst endpoints from TLI (as delineated earlier). The age of the
culture and PGT-A were offered enrollment in the study oocyte from which an embryo was derived was dichotomized
before their treatment cycle between February 2018 and to <35 years or R35 years for stratified analysis.
November 2019. During this time frame, 631 patients were
eligible to participate and 200 patients consented to partic- Outcomes
ipation. The inclusion and exclusion criteria have been
described previously (4). One hundred seventy-six individ- The primary outcome was static blastocyst embryo morphology.
uals completed the study. High-quality embryos meeting the criteria for clinical use were
The study was approved by the institutional review board defined as blastocysts with expansion grade 3–6 according to
of the University of California San Francisco (IRB #17-22331) Gardner criteria and at least a B grading for ICM and TE (5).
and registered on clinicaltrials.gov (NCT 03503877). Written Expanded blastocysts with C grading for either t TE or ICM
informed consent was obtained from all study subjects before were defined as low-quality embryos and were deemed unsuit-
participation. able for clinical use. A secondary outcome was embryonic
ploidy determined by TE biopsy with PGT-A. A subanalysis
included the type of aneuploidy (simple, segmental, or complex).
Ovarian Hyperstimulation and Laboratory
Procedures Statistical Analysis
Ovarian stimulation was performed as described previously Outcomes were assessed for normality of distribution. Mean,
(4). Oocyte retrieval was performed according to clinic stan- standard deviation, medians, and interquartile ranges are re-
dard 36 hours after ovulatory trigger. A semen sample was ported. Chi-square was used as appropriate. Morphokinetic
obtained by masturbation within 1 hour of oocyte retrieval. evaluations were compared using Wilcoxon rank sum testing
The method of fertilization—via conventional insemination with Bonferroni correction to adjust for multiple compari-
vs. intracytoplasmic sperm injection—was determined by the sons. All analyses were performed in the statistical software
patient’s primary physician. package R version 3.6.3 (R Core Team, 2020).
At 16–18 hours, fertilization was evaluated by the exis-
tence of 2 pronuclei. All embryos were cultured in the Em-
bryoScopeþ time-lapse incubator (Vitrolife A/S, Viby J, RESULTS
Denmark). Once placed in the EmbryoScopeþ time-lapse Baseline characteristics and stimulation parameters of the
incubator, embryos were cultured at 37  C with 6.5% CO2 enrolled patients were reported previously (4). One hundred sev-
and 5.0% O2 for up to 6 days without media exchange. enty patients contributed embryos to the study. The median
The EmbryoScopeþ incubation chamber contains a built- number of oocytes collected was 13.5 (10.0–19.75). The median
in microscope and camera, allowing for continuous monitoring number of normally fertilized oocytes (2PN) per patient was
of embryonic development. An image acquisition software was 10.5 (7–15) and that of blastocysts formed was 7 (4–11). Blastu-
used to obtain high-contrast images every 10 minutes from lation rate (blastocysts/2PN) per patient was 71% (55%–85%).
several focal planes to create time-lapse videos. Conventional There were 935 high-quality blastocysts and 371 low-
embryonic assessment was made through observations at pre- quality blastocysts that did not meet the criteria for clinical
specified time points. Cleavage-stage embryos were assessed use. The distribution of static morphology grading at the
for cell number, symmetry, percentage fragmentation, evidence cleavage stage and the final blastocyst grading is depicted
of multinucleation, and progression of compaction. Blastocysts in Figure 1. Full automatic annotations were possible for
were evaluated to assess for blastocele volume and expansion, 864 high-quality and 328 low-quality blastocysts. Time-
ICM development, and TE organization. lapse imaging revealed a shorter time to 5 cells in embryos
Additional morphokinetic parameters were assessed with that subsequently became low-quality blastocysts: 48.4
time-lapse videos, including time to pronuclear fading or (42.4–48.7) hours vs. 50.2 (46.3–50.1) hours; P ¼ .02

232 VOL. 3 NO. 3 / SEPTEMBER 2022

 Fertil Steril Rep®

FIGURE 1

(A) Cleavage-stage embryo grading among high- and low-quality embryos. (B) Blastocyst stage final embryo grading among high- and low-quality
embryos (Gardner criteria). ICM ¼ inner cell mass. TE ¼ trophectoderm.
Quinn. Low-quality blastocyst morphokinetics. Fertil Steril Rep 2022.

(Table 1). Embryos that would become low-quality blastocysts however, TLI was unable to distinguish euploid vs. aneuploid
reached all subsequent morphokinetic milestones at a slower low-quality embryos.
pace. The time to blastocyst formation was 114.0 (106.4– Multiple prior publications have explored the use of mor-
113.9) hours vs 106.9 (101.3–107.4) hours, P< .0001 phokinetic timings gleamed from TLI to predict ploidy status
(Table 1). A similar pattern was observed when restricting among blastocysts. A recent review on the topic concluded
the morphokinetic analysis to euploid embryos graded low that although morphokinetic parameters from TLI may relate
vs. high quality (Supplemental Table 1, available online). to ploidy status, the predictive value was inadequate to replace
Among low-quality blastocysts, 280 were aneuploid, 83 PGT-A for aneuploidy screening (6). This was especially true
were euploid, and 8 had an indeterminate result. Morphokinetic when kinetic risk models developed at different centers were
parameters did not differ between euploid and aneuploid low- adopted before internal validation (7). Minasi et al. (8) explored
quality blastocysts (Table 2). High-quality embryos were more the relationship among standard morphology, morphokinetic
likely to be euploid (41.5% vs. 22.4%, P< .001). This was true development, and embryonic ploidy as determined by TE bi-
in a subgroup analysis of embryos derived from women aged opsy with PGT-A via array comparative genomic hybridiza-
% 35 years and >35 years (Table 3). Complex aneuploidy was tion in 928 blastocysts. Euploid embryos demonstrated a
more frequently identified in poor-quality embryos, particu- shorter time to start blastulation, expansion, and hatching
larly in women aged >35 years (Supplemental Table 2). than that demonstrated by aneuploid embryos. Notably, stan-
dard morphology was poorly predictive of ploidy with C grad-
ings for ICM among 17.1% of euploid blastocysts and C
DISCUSSION gradings for TE among 26.6% of euploid blastocysts (8). Simi-
In this secondary analysis of a large sibling oocytes study us- larly, Capalbo et al (3) found a 25.5% euploidy rate among
ing TLI and TE biopsy for PGT-A to evaluate the develop- poor-quality blastocysts, a rate of 30.1% when ICM was ‘‘C,’’
mental competence of blastocysts, we demonstrated that and a rate of 23.4% with a TE ‘‘C’’ score. Of note, this study
nearly a quarter of low-quality blastocysts were euploid; included only 153 total poor-quality blastocysts from 2

VOL. 3 NO. 3 / SEPTEMBER 2022 233

ORIGINAL ARTICLE: ASSISTED REPRODUCTION

TABLE 1

Morphokinetic parameters for high-quality and low-quality embryos in time-lapse imaging.

High quality Low quality
P value (Wilcoxon P value (Bonferroni
Parameter n Mean ± SD Median (IQR) n Mean ± SD Median (IQR) rank sum) corrected)
tPNfa 864 24.0 ± 3.6 23.6 (21.8–24.0) 328 24.5 ± 3.9 24.3 (22.0–24.5) .028 .396
t2b 864 26.5 ± 3.7 26.1 (24.3–26.5) 328 27.1 ± 4.1 26.9 (24.6–27.1) .011 .158
t3c 864 37.5 ± 4.7 37.5 (34.9–37.5) 328 37.2 ± 5.7 37.4 (34.2–37.2) .477 1
t4d 864 38.5 ± 4.6 38.2 (35.8–38.5) 328 39.3 ± 5.5 39.0 (35.6–39.3) .048 .668
t5e 864 50.1 ± 7.0 50.2 (46.3–50.1) 328 48.7 ± 8.5 48.4 (42.4–48.7) .001 .020
t6f 864 52.2 ± 6.5 51.9 (48.5–52.2) 328 52.8 ± 8.6 52.4 (47.4–52.8) .563 1
t7g 864 54.2 ± 7.4 53.3 (49.6–54.2) 328 56.0 ± 9.0 54.7 (49.9–56.0) .003 .0370
t8h 864 57.7 ± 8.9 56.1 (51.6–57.7) 328 60.3 ± 10.1 59.0 (53.0–60.3) < .001 < .001
tMi 864 88.3 ± 9.5 88.3 (82.1–88.3) 328 92.3 ± 10.0 91.5 (85.9–92.3) < .001 < .001
tSBj 864 99.0 ± 8.4 98.4 (93.4–99.0) 328 103.3 ± 9.1 102.7 (97.2–103.3) < .001 < .001
tBk 847 107.4 ± 9.0 106.9 (101.3–107.4) 312 113.9 ± 10.5 114.0 (106.4–113.9) < .001 < .001
cc2l 864 11.0 ± 2.3 11.3 (10.6–11.0) 328 10.1 ± 4.3 11.2 (10.0–10.1) .097 1
cc3m 864 11.6 ± 4.9 12.2 (10.9–11.6) 328 9.4 ± 6.6 11.3 (1.3–9.4) < .001 < .001
dbn 847 8.5 ± 3.6 7.8 (6.1–8.5) 312 11.0 ± 5.6 9.8 (7.0–11.0) < .001 < .001
Note: All times are presented in hours; tSB notation required for inclusion. IQR ¼ interquartile range.
a
From insemination to pronuclear fading.
b
Two-cell formation.
c
Three-cell formation.
d
Four-cell formation.
e
Five-cell formation.
f
Six-cell formation.
g
Seven-cell formation.
h
Eight-cell formation.
i
Morula formation.
j
Appearance of blastocele/start of blastulation.
k
Formation of blastocyst.
l
Duration of second cell cycle.
m
Duration of third cell cycle.
n
Duration of blastulation (tb–tSB).
Quinn. Low-quality blastocyst morphokinetics. Fertil Steril Rep 2022.

TABLE 2

Morphokinetic parameters for euploid and aneuploid low-quality embryos in time-lapse imaging.
Euploid Aneuploid
P value (Wilcoxon P value (Bonferroni
Parameter n Mean ± SD Median (IQR) n Mean ± SD Median (IQR) rank sum) corrected)
tPNfa 73 24.7 ± 3.5 24.7 (22.2–24.7) 247 24.4  4.1 24.2 (21.9–24.4) .571 1
t2b 73 27.3 ± 3.6 27.4 (24.9–27.3) 247 27.0  4.3 26.8 (24.4–27.0) .463 1
t3c 73 37.5 ± 5.1 37.8 (34.4–37.5) 247 37.0  5.8 37.0 (34.1–37.0) .453 1
t4d 73 39.5 ± 5.3 39.3 (36.8–39.5) 247 39.1  5.5 38.9 (35.5–39.1) .654 1
t5e 73 48.2  7.7 49.0 (41.9–48.2) 247 48.7  8.6 48.1 (42.7–48.7) .845 1
t6f 73 52.5  7.5 52.6 (47.5–52.5) 247 52.6  8.7 51.8 (47.4–52.6) .630 1
t7g 73 55.9  8.6 55.5 (51.2–55.9) 247 55.7  9.0 54.5 (49.7–55.7) .721 1
t8h 73 60.6  10.0 58.6 (53.3–60.6) 247 60.1  10.2 59.1 (52.4–60.1) .813 1
tMi 73 93.5  10.2 94.1 (87.8–93.5) 247 91.6  9.7 91.1 (85.8–91.6) .132 1
tSBj 73 103.6  9.5 102.6 (97.9–103.6) 247 102.9  8.8 102.6 (96.8–102.9) .656 1
tBk 71 113.7  9.8 113.5 (106.9–113.7) 233 113.7  10.6 113.9 (106.2–113.7) .959 1
cc2l 73 10.2  4.7 11.3 (10.0–10.2) 247 10.1  4.3 11.2 (9.9–10.1) .559 1
cc3m 73 8.7  6.2 11.1 (1.0–8.7) 247 9.5  6.6 11.4 (1.4–9.5) .349 1
dbn 71 10.6  5.4 9.6 (6.6–10.6) 233 11.1  5.7 10.0 (7.2–11.1) .380 1
Noe: All times are presented in hours, tSB notation required for inclusion. IQR ¼ interquartile range.
a
From insemination to pronuclear fading.
b
Two-cell formation.
c
Three-cell formation.
d
Four-cell formation.
e
Five-cell formation.
f
Six-cell formation.
g
Seven-cell formation.
h
Eight-cell formation.
i
Morula formation.
j
Appearance of blastocele/start of blastulation.
k
Formation of blastocyst.
l
Duration of second cell cycle.
m
Duration of third cell cycle.
n
Duration of blastulation (tb–tSB).
Quinn. Low-quality blastocyst morphokinetics. Fertil Steril Rep 2022.

234 VOL. 3 NO. 3 / SEPTEMBER 2022

 Fertil Steril Rep®

TABLE 3

Ploidy status of high-quality and low-quality embryos by age £35 years or >35 years.
Characteristics High quality Low quality
Group n % n % P value (chi-square)
All Euploid 388 41.5 83 22.4 < .001
Aneuploid (all) 530 56.7 280 75.5
Undetermined 17 1.8 8 2.2
Age (y) Euploid 146 48.8 42 32.6 .008
%35 years
Aneuploid 147 49.2 84 65.1
Undetermined 6 2.0 3 2.3
Age (y) Euploid 242 38.1 41 16.9 < .001
>35 years
Aneuploid 383 60.2 196 81.0
Undetermined 11 1.7 5 2.1
Quinn. Low-quality blastocyst morphokinetics. Fertil Steril Rep 2022.

centers. This finding that poor-quality blastocysts have poten- live birth per retrieval cycle. Future investigations should
tial for euploid status is important when many clinics exclude evaluate the reproductive potential of low-quality euploid
embryos for biopsy on the basis of static morphology grading blastocysts in larger cohorts.
(2).
Although we demonstrate a shorter time to 5-cell forma- Acknowledgments: The authors acknowledge the work of
tion in embryos that subsequently became low-quality em- the embryology team at the University of California San Fran-
bryos, the significance of this finding is uncertain. cisco’s Center for Reproductive Health, without which this
Notably, 5-cell formation is before embryonic genome acti- study would not be possible. Furthermore, they thank PacGe-
vation. Prior research limited by the assessment of embry- nomics for providing preimplantation genetic testing for
onic ploidy at the cleavage stage by array comparative aneuploidy and CooperSurgical for supplying embryo culture
genomic hybridization reported some ability to distinguish media. Additionally, the authors appreciate the assistance of
between aneuploid and euploid embryos on the basis of mor- Jørgen Berntsen, M.Sc. and Mark Larman, Ph.D. with data
phokinetic development to the 4-cell stage (9). Specifically, analysis.
the investigators demonstrated a greater standard deviation
in time to early cell divisions among embryos with meiotic
errors (10) and an increased risk for falling outside optimal REFERENCES
ranges proposed for 5-cell formation to 2-cell formation 1. Mumusoglu S, Yarali I, Bozdag G, Ozdemir P, Polat M, Sokmensuer LK, et al.
and duration of third cell cycle (9). Although this differential Time-lapse morphokinetic assessment has low to moderate ability to predict
euploidy when patient- and ovarian stimulation-related factors are taken
was demonstrated in the time to 5-cell formation between
into account with the use of clustered data analysis. Fertil Steril 2017;107:
high and low-quality embryos within our study, it was not 413–21.e4.
seen when comparing the time to 5-cell formation between 2. Knudtson JF, Robinson RD, Sparks AE, Hill MJ, Chang TA, Van Voorhis BJ.
low-quality euploid and aneuploid blastocysts. Common practices among consistently high-performing in vitro fertilization
A significant limitation of our study is that low-quality programs in the United States: 10-year update. Fertil Steril 2022;117:42–50.
euploid embryos were not transferred. As a result, we are 3. Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, et al. Cor-
unable to describe the reproductive potential of these low- relation between standard blastocyst morphology, euploidy and implanta-
tion: an observational study in two centers involving 956 screened
quality blastocysts. In the study by Capalbo et al (3),
blastocysts. Hum Reprod 2014;29:1173–81.
poor-quality euploid embryos were eligible for transfer, 4. Quinn MM, Marsh P, Ribeiro S, Simbulan RK, Hickman C, Berntsen J, et al.
and 7 of 13 (53.8%) of these resulted in ongoing implanta- Aneuploidy rates and morphokinetic parameters of embryos cultured in
tion. This outcome was not different from euploid embryos distinct culture media: a sibling oocyte study. Hum Reprod 2022;37:226–34.
that were graded as average quality (3). These data raise 5. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst
the question of whether embryology laboratories should score affects implantation and pregnancy outcome: towards a single blasto-
cyst transfer. Fertil Steril 2000;73:1155–8.
consider poor-quality embryos suitable for clinical use either
6. Zaninovic N, Irani M, Meseguer M. Assessment of embryo morphology and
for transfer untested or when PGT-A is planned. This ques-
developmental dynamics by time-lapse microscopy: is there a relation to im-
tion reveals the tension between an approach that favors plantation and ploidy? Fertil Steril 2017;108:722–9.
high levels of embryo selection designed to minimize time 7. Kramer YG, Kofinas JD, Melzer K, Noyes N, McCaffrey C, Buldo-Licciardi J,
to pregnancy and an alternative that maximizes cumulative et al. Assessing morphokinetic parameters via time lapse microscopy

VOL. 3 NO. 3 / SEPTEMBER 2022 235

ORIGINAL ARTICLE: ASSISTED REPRODUCTION

(TLM) to predict euploidy: are aneuploidy risk classification models univer- 9. Basile N, del Carmen Nogales M, Bronet F, Florensa M, Riqueiros M, Rodrigo L,
sal? J Assist Reprod Genet 2014;31:1231–42. et al. Increasing the probability of selecting chromosomally normal embryos
8. Minasi MG, Colasante A, Riccio T, Ruberti A, Casciani V, Scarselli F, et al. by time-lapse morphokinetics analysis. Fertil Steril 2014;101:699–704.
Correlation between aneuploidy, standard morphology evaluation and 10. Chavez SL, Loewke KE, Han J, Moussavi F, Colls P, Munne S, et al. Dynamic
morphokinetic development in 1730 biopsied blastocysts: a consecutive blastomere behaviour reflects human embryo ploidy by the four-cell stage.
case series study. Hum Reprod 2016;31:2245–54. Nat Commun 2012;3:1–12. 1251.

236 VOL. 3 NO. 3 / SEPTEMBER 2022