Comprehensive chromosome screeningccs

Comprehensive chromosome
screening alters traditional
morphology-based embryo selection:
a prospective study of 100
consecutive cycles of planned fresh
euploid blastocyst transfer
Eric J. Forman, M.D.,a,b Kathleen M. Upham, B.S.,a Michael Cheng, B.S.,a Tian Zhao, B.S.,a
Kathleen H. Hong, M.D.,a,b Nathan R. Treff, Ph.D.,a,b and Richard T. Scott Jr., M.D.a,b
a Reproductive Medicine Associates of New Jersey, Department of Reproductive Endocrinology, Basking Ridge; and
b University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Department of Obstetrics,
Gynecology & Reproductive Sciences, New Brunswick, New Jersey
Objective: To determine how often trophectoderm biopsy and rapid, real-time quantitative polymerase chain reaction (PCR)-based
comprehensive chromosome screening (CCS) alters clinical management by resulting in the transfer of a different embryo than
would have been chosen by traditional day 5 morphology-based criteria.
Design: Prospective.
Setting: Academic center for reproductive medicine.
Patient(s): Infertile couples (n ¼ 100; mean age 35 4 years) with at least two blastocysts suitable for biopsy on day 5.
Intervention(s): Prior to trophectoderm biopsy for CCS the embryologist identified which embryo would have been selected for
traditional day 5 elective single ET.
Main Outcome Measure(s): The risk of aneuploidy in the embryos that would have been selected on day 5 was calculated and
compared with the aneuploidy rate of the cohort of all embryos that underwent CCS testing. The aneuploidy risk was compared between
age groups.
Result(s): After quantitative PCR-based CCS, 22% (95% confidence interval 15%–31%) of the embryos selected by day 5 morphology
were aneuploid, which was lower than the 32% aneuploidy rate of the cohort. Patients R35 years had a higher risk of an aneuploid
blastocyst being selected by morphology than those 35 years old (31% vs. 14%). Among patients who had selection altered by
CCS, 74% (14/19) delivered, including 77% (10/13) after elective single ET. Most patients (77%) had an additional euploid blastocyst
vitrified for future use.
Conclusion(s): The CCS results alter embryo selection due to the presence of aneuploidy in embryos with optimal day 5 morphology.
Excellent outcomes were obtained when CCS-based selection was different than morphology-based
selection. (Fertil Steril 2013;100:718–24. 2013 by American Society for Reproductive
Medicine.)
Key Words: Single embryo transfer, eSET, preimplantation genetic screening, comprehensive
chromosome screening, aneuploidy
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Received February 26, 2013; revised April 25, 2013; accepted April 26, 2013; published online May 30, 2013.
E.J.F. has nothing to disclose. K.M.U. has nothing to disclose. M.C. has nothing to disclose. T.Z. has nothing to disclose. K.H.H. has nothing to disclose. N.R.T.
reports payment for lectures from the American Society for Reproductive Medicine (ASRM), Japanese Society for Assisted Reproduction, Penn State
University, Washington State University, Mayo Clinic, Applied Biosystems Inc., Texas Assisted Reproductive Technologies Society, and American Asso-ciation
of Bioanalysts; payment for development of educational material from ASRM; and patents pending. R.T.S. is a member of the advisory boards
of EMD Serono and Ferring Pharmaceuticals, and has received payment for lectures and travel expenses from Merck.
Reprint requests: Eric J. Forman, M.D., Reproductive Medicine Associates of New Jersey, 140 Allen Road, Basking Ridge, New Jersey 07920 (E-mail: eforman@
rmanj.com).
Fertility and Sterility® Vol. 100, No. 3, September 2013 0015-0282/$36.00
Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.fertnstert.2013.04.043
718 VOL. 100 NO. 3 / SEPTEMBER 2013

Preimplantation genetic screening has been proposed
primarily as a method to improve embryo selection for
patients with a poor prognosis for IVF success as a
result of previous implantation failures or advanced age (1).
On the contrary, for good prognosis patients, increased
utilization of blastocyst stage, elective single ET (eSET),
without aneuploidy screening, has been promoted to reduce
the risk of multiple gestation and its resulting sequelae (2).
Although prior studies using fluorescence in situ hybridiza-tion
did not improve eSET selection (3, 4), more accurate
comprehensive chromosome screening (CCS) methods have
demonstrated benefit (5, 6, 7). The degree to which CCS
impacts clinical management by altering embryo selection
for transfer and subsequent cyopreservation, even among
high-quality blastocysts, has not previously been quantified.
As eSET is currently practiced, one embryo is selected by
temporal and morphological criteria from multiple blasto-cysts
deemed suitable for transfer on day 5 of development
(8). With rapid CCS, however, blastocysts undergo trophecto-derm
biopsy on day 5 with real-time, quantitative polymerase
chain reaction (qPCR)-based CCS performed. The best quality
embryo from among the euploid blastocysts is then selected
for transfer the following day (Figure 1). If the embryo
selected for fresh transfer after CCS is always the same
Fertility and Sterility®
embryo that would have been selected on day 5 (i.e., if the
best quality embryo is always euploid), then CCS would not
have the opportunity to improve outcomes.
The current study seeks to determine how often CCS
would alter selection for eSET and thus provide an estimate
of the degree to which CCS may improve fresh eSET outcomes.
To evaluate this prospectively, an embryologist identified the
embryo that would have been selected for traditional day
5 eSET in 100 consecutive cases. Trophectoderm biopsy and
qPCR-based CCS were performed and it was determined
how often the previously selected embryo would have a
predicted aneuploid karyotype, yielding an estimate for how
often CCS alters clinical care in different age groups.
MATERIALS AND METHODS
Population
The study population consisted of 100 consecutive patients
planning to undergo rapid CCS with trophectoderm biopsy
on day 5 and fresh transfer on day 6. All cycles were
performed at Reproductive Medicine Associates of New Jersey
between September 2011 and March 2012. To be eligible,
patients had to have at least two blastocysts suitable for
trophectoderm biopsy by late in the afternoon of day 5.
During the study period CCS was offered to all patients
undergoing IVF. It was recommended for patients of
advanced reproductive age, those with recurrent pregnancy
loss, a prior aneuploid ongoing pregnancy, or a prior failed
IVF cycle. This was a noninterventional, observational study
of consecutive ETs. All patients participated through an
institutional review board-approved protocol. In addition,
some patients having transfers during this time period were
participating in an institutional review board-approved
randomized controlled trial investigating CCS to optimize
single ET (ClinicalTrials.gov identifier NCT01408433). Their
participation in that trial did not impact this observational
study.
Patients whose embryos arrested before the blastocyst
stage or could not be biopsied until day 6 were not included
in the analysis. Patients who had a contraindication to fresh
transfer (risk of ovarian hyperstimulation syndrome [OHSS],
premature P elevation, abnormal endometrial proliferation)
were excluded. Patients who were planning to cryopreserve
all of their blastocysts for a future frozen ET were also
excluded.
Patients were treated per routine with an IVF protocol
selected by their treating physician. All stimulations included
LH activity in the form of purified menotropins (Menopur;
Ferring Pharmaceuticals) or low-dose hCG. Final oocyte
maturation was induced when at least two follicles reached
a maximal diameter of R18 mm and oocyte retrieval was
performed 36 hours later. All mature oocytes underwent
intracytoplasmic sperm injection (ICSI), even in cases with
normal semen parameters, to prevent genetic contamination
during CCS. All cleaved embryos underwent laser-assisted
hatching of the zona pellucida (ZP) on day 3 to facilitate
trophectoderm biopsy on day 5. Per practice routine, all
embryos were placed in extended culture regardless of the
size or quality of the cohort. Embryos were graded on day 5
FIGURE 1
Real-time, quantitative polymerase chain reaction (PCR)-based
comprehensive chromosome screening results after day 5
trophectoderm biopsy of four blastocysts from a 31-year-old patient
with a history of recurrent pregnancy loss. Had a day 5 elective
single ET been performed, the top embryo would have been
selected based on morphological criteria. After comprehensive
chromosome screening results were obtained, that embryo was
found to be aneuploid (47,XY,þ21). On the morning of day 6, the
embryo with the karyotype prediction of 46,XY was transferred,
although its morphology grade was 4BB, compared with 6AA for
the 47,XY,þ21 embryo. The patient delivered a healthy boy at 39
weeks, 4 days gestation.
Forman. CCS alters traditional embryo selection. Fertil Steril 2013.
VOL. 100 NO. 3 / SEPTEMBER 2013 719

ORIGINAL ARTICLE: ASSISTED REPRODUCTION
and an embryologist experienced in trophectoderm biopsy
determined whether they were suitable for day 5 trophecto-derm
biopsy. To be biopsied, blastocysts had to reach at least
an expansion stage of 3, have a distinct inner cell mass (ICM),
and have an adequately cellular trophectoderm (9). Embryos
that were not suitable for biopsy by the afternoon of day 5
were considered not to be developing synchronously with
endometrial receptivity as they have been shown to have
lower implantation rates in fresh ETs (10, 11). These
embryos are therefore reassessed on day 6 for possible
trophectoderm biopsy and vitrification.
Embryo Selection
The biopsy was performed with an infrared diode laser by
removing approximately five trophectederm cells that had
herniated through the previously created breech in the ZP.
At the time of embryo biopsy, in addition to performing a
standard morphological assessment, the embryologist identi-fied
which blastocyst would have been selected for a day 5,
fresh single blastocyst transfer. The identity of the specific
embryo selected was sent to the principal investigator. The
selected embryo was otherwise treated as per routine.
The biopsies were processed for qPCR-based CCS as
previously described (12). In brief, multiplex amplification
of 96 loci (four for each chromosome) was performed and a
method of relative quantitation was applied to predict the
copy number status of each chromosome. This methodology
was designed to specifically identify whole chromosome,
not segmental, aneuploidy and was validated in preclinical
(12) and clinical studies (13, 14). A karyotype prediction
was made for each embryo by a certified cytogeneticist. The
laboratory technicians who processed the biopsy and the
genetics team that analyzed the qPCR data were not aware
of which embryo had been selected.
Transfers all occurred on the morning of day 6, before 6
complete days (144 hours) had elapsed from the time of oocyte
retrieval, as is standard for all fresh ETs at this center. Patients
received a transfer of one or two euploid blastocysts with the
best morphology. The transfer decision was not impacted by
the previous day's selection. Transfer order was determined
by the patient and her physician.
Statistical Analysis
The aneuploidy rate of the embryos that would have been
selected for a traditional day 5 single blastocyst transfer
was calculated and compared with the aneuploidy rate of
the overall cohort of blastocysts. Age group-specific
comparisons were also made by comparing the aneuploidy
rate of the selected blastocyst in patients 35 years old
with those R35 years old. Comparisons were made using a
c2 distribution with a P value of .05 considered significant.
The number of patients needed to treat (NNT) to prevent
the transfer of an aneuploid blastocyst was calculated as
the inverse of the aneuploidy rate of the selected day 5
blastocyst. For example, if there was a 50% aneuploidy
risk, the NNT would be 2 (1/0.5) (i.e., two patients would
have to use qPCR-based CCS and eSET to prevent the transfer
of one aneuploid blastocyst).
The impact of CCS on eSET delivery rates was estimated
using previously published implantation rates of predicted
aneuploid blastocysts (13) and the overall implantation rate
of euploid blastocysts in the current cohort. Based on these
estimates, the NNT was calculated and represents the number
of patients who would have to use CCS-based selection to
achieve an additional singleton delivery from eSET.
RESULTS
A consecutive series of 100 patients was evaluated. The
demographics of the patients are presented in Table 1. Overall,
22% (22/100; 95% confidence interval 15%–31%) of the
blastocysts that would have been selected for by traditional
day 5 morphological criteria were aneuploid and, therefore,
were not transferred. This was lower than the overall 32%
(192/597) aneuploidy rate of the entire cohort of biopsied
blastocysts (P¼.04; Table 2). The different types of aneu-ploidy
detected in the best quality day 5 blastocyst are
detailed in Table 3. The traditionally selected blastocyst
among patients 35 years old had an aneuploidy rate of
14% (7/51; 95% confidence interval 6%–25%), which
was lower than the aneuploidy rate of 31% (15/49; 95%
confidence interval 19%–45%) in the traditionally selected
blastocyst from patients R35 years old (relative risk 0.4,
95% confidence interval 0.2–1.0; P¼.04).
Clinical Outcomes
A total of 96 fresh transfers were performed. Two patients did
not have a transfer as all of their blastocysts were aneuploid
based on CCS test results. Both of these patients had a poor
IVF prognosis: a 42-year-old with three aneuploid blastocysts
and a 41-year-old with an elevated day 3 FSH of 18.8 IU/L
and five aneuploid blastocysts. One patient did not have a
transfer as initial CCS results were nondiagnostic (noncon-current)
and the four blastocysts were rebiopsied on day
6 and vitrified. One patient had one euploid blastocyst and
elected to undergo another fresh cycle in an attempt to obtain
additional blastocysts for transfer.
The delivery rate was 65% per patient and 68% per
transfer. The implantation rate (number of fetal heart beats
divided by total number of embryos transferred) was 63%
(79/126). Among patients who had single euploid blastocyst
transfer, the delivery rate was 64% (42/66) and all were
singletons. Among those who had two euploid blastocysts
transferred, the delivery rate was 77% (23/30) including 10
twins and 1 triplet due to monozygotic twinning of an
TABLE 1
Characteristics of study population.
Mean ± SD (range)
Age (y) 35 4 (22–44)
Maximal day 3 FSH (IU/L) 7.0 2.3 (1.5–18.8)
Antim€ullerian hormone (ng/mL) 3.3 2.1 (0.3–8.6)
No. of oocytes retrieved 18 9 (5–56)
No. of blastocysts biopsied 6 4 (2–21)
No. of euploid blastocysts 4 3 (0–17)

embryo. Another triplet pregnancy miscarried at 12 weeks
gestation. Overall, the clinical miscarriage rate was 11%
(8/73).
Impact of Changing Selection with CCS
The 22 patients whose traditionally selected day 5 blastocyst
was aneuploid had a mean age of 37 years. Nineteen of these
patients had a fresh transfer of a different, CCS-selected,
euploid blastocyst with a delivery rate of 74% (14/19). The
delivery rate after single euploid blastocyst transfer in this
group was 77% (10/13), indicating that excellent outcomes
can be maintained with eSET even when the best quality
embryo by traditional morphological criteria is selected
against due to aneuploidy. There were three blastocysts
with the ability to result in viable gestations (two with
predicted trisomy 21 and one with predicted 47,XXY)
that would have been selected by traditional day 5
morphology-based criteria.
When performing eSET in this population, the number
NNT to prevent transfer of a predicted aneuploid blastocyst
was 5. For patients 35 years old, the NNT was 7; for those
R35 years old, the NNT was 3.
A previous nonselection study demonstrated that
embryos predicted to be aneuploid had a delivery rate of 4%
(13). In the current study population, euploid blastocysts
had an implantation rate of 63%. Thus, with traditional
morphology-based day 5 single blastocyst transfer, the
estimated expected delivery rate would be 50% (1 delivery
among 22 predicted aneuploid and 49 deliveries among
78 predicted euploid). After CCS, the expected delivery rate
would be 62% (62 deliveries among 98 predicted euploid
and 2 with no transfer). Thus, a relatively low aneuploidy
rate of 22% among selected blastocysts may translate into a
TABLE 2
Aneuploidy rate of blastocyst selected for elective single ET compared with overall cohort.
Aneuploidy rate of blastocyst
selected for day 5 eSET
Aneuploidy rate of cohort of
blastocysts
Relative risk (95% confidence
interval) P value
Overall 22% (22/100) 32% (192/597) 0.7 (0.5–1.0) .04
35 years-old 14% (7/51) 25% (83/336) 0.6 (0.3–1.1) .08
R35 years-old 31% (15/49) 42% (109/261) 0.7 (0.5–1.1) .14
Note: eSET ¼ elective single ET.
TABLE 3
Morphologic grading and comprehensive chromosome screening results for the embryos selected on day 5 that were aneuploid and the outcomes
of euploid embryo transfers.
Maternal
age, y
Biopsied
blastocyts
Euploid
blastocysts
Day 5
morphology
of selected
embryo
Day 6
morphology
of selected
embryo
CCS predicted
karyotype
of selected
embryo
Morphology of
transferred
euploid
embryo 1
Morphology of
transferred
euploid
embryo 2
Transfer
outcome
28.8a 12 7 5BB 6BB 47,XX,+7 6BB 5BB Not pregnant
31.6b 4 1 5BB 6AA 47,XY,+21 4BB – Term singleton
33.2 12 8 5BB 5BB 47,XY,+22 6BA 6BA Preterm twins
33.3 6 4 4BB 5BB 47,XY,+16 6AA 6BA Biochemical loss
34.0 4 3 4BB 5BB 47,XY,+16 6AA – Term singleton
34.4 4 1 4AA 4CB 45,XY,16 6CB – Term singleton
34.5 5 2 4BB 6AA 45,XY,2 6AA 6BA Term twins
36.4 2 1 4AA 6BB 45,XY,13 6BB – Term singleton
37.1 3 1 4BB 6BA 47,XY,+7 6CB – Term singleton
37.3 5 4 4BB 6BB 45,XY,21 6AA 5BA Term singleton
37.4 4 1 4BB 5BB 45,XY,22 6BB – Term singleton
37.7 11 7 5AB 6BA 45,XY,13 6AA – Term singleton
37.8 7 3 4BB 6AA 46,XX,5+21 4BB 6BC Preterm singleton
37.9 8 4 4BB 5BB 45,XX,22 6BB – Term singleton
38.8 4 1 3BB 4BC 47,XXY 6CA – Not pregnant
39.3 6 2 4BB 6AC 47,XY,+21 6BC – Biochemical loss
39.5 5 1 4BB 5BA 44,XX,13,+16,20,21 5BB – Not pregnant
40.8 8 3 4AA 5BA 45,XY,22 6AA – Term singleton
40.9 5 0 6AA 6AA 47,XY,+15 – – No transfer
41.1 4 3 4BB 6BA 49,XY,+15,+19,+21 – – No transfer
42.7 3 0 3BB 6CB 45,XY,22 – – No transfer
44.2 3 1 4BB 5BB 48,XX,+15,+16 6AA – Term singleton
Note: CCS ¼ comprehensive chromosome screening.
a Anonymous oocyte donation.
b qPCR results demonstrated for this patient in Figure 1.

clinically significant improvement in single blastocyst
transfer delivery rates of 12% per cycle. Based on this
estimate, the NNT to achieve an additional singleton delivery
from eSET with CCS was 8.
Despite only cryopreserving euploid blastocysts for future
frozen ETs, 77 patients had at least one supernumerary
euploid blastocyst vitrified. Of those who had eSET, 51/66
(77%) had a euploid embryo cryopreserved.
DISCUSSION
This prospective study has demonstrated that in candidates
for fresh day 5 eSET, performing CCS results in a change in
embryo selection, when compared with traditional
morphology-based selection, a substantial proportion of the
time. The data from this study also suggest that this change
may result in clinically meaningful improvement in the
eSET success rate. It is not surprising that patients of
advanced reproductive age, who have a higher underlying
prevalence of embryonic aneuploidy, were more likely to
have selection altered by the CCS result.
Despite the benefits of singleton versus multiple preg-nancy,
efforts to promote eSET have been inadequate as
10% of ETs after IVF use this approach (15). The reluctance
to accept eSET likely relates to its lower per transfer delivery
rates when conventional methods of embryo selection are
used (16). Preimplantation human embryos have a substantial
risk of aneuploidy, regardless of maternal age, and if an
aneuploid embryo is selected for eSET, the cycle is destined
to fail. Although promising results have been demonstrated
in early studies using CCS to avoid transferring aneuploid em-bryos
(17), for CCS to improve the outcome after eSET, it must
result in a change from traditional methods of selection. That
is, the morphologically best embryo must sometimes be
selected against due to aneuploidy. This prospective study
demonstrates that blastocyst stage CCS changes embryo
selection even among high-quality expanded blastocysts.
Although eSET has primarily been promoted among young,
good prognosis patients, all infertility patients could benefit
from the reduced risk of multiple gestation, as long as overall
outcomes are not compromised. A retrospective study and a
small prospective study in a good prognosis population
demonstrated that CCS may improve the outcome of single
ET (5, 6). A randomized noninferiority trial demonstrated
that eSET after CCS results in similar ongoing pregnancy
rates as transferring two untested embryos in patients up to
age 42 years with age-appropriate ovarian reserve (7). There
are limited data on outcomes of eSET in an older population,
but a recent study of 264 eSETs without CCS in women aged
40–44 years found a clinical miscarriage risk of 37.5% and
live birth rate of 13.6% (18). Improvements in embryo
selection are likely to be required to make eSET a viable option
in this age group. The potential benefits of the improved
selection offered by CCS are not restricted to patients of
advanced age. Even in patients 35 years old, based on this
study's findings, one in seven eSETs with a high-quality blas-tocyst
would be expected to have a negligible chance for de-livery.
Transferring an untested blastocyst may increase the
risk for clinical miscarriage or an anomalous pregnancy (5).
The concept of eSET, especially in an older population,
has been recently questioned given the relatively low risk of
multiple gestation and the potential impact on overall success
after IVF (19). This argument minimizes the increased
obstetric risk in older patients and the worse obstetric
outcome from twin pregnancies compared with two singleton
pregnancies (20). The current study demonstrates that most
couples who want to have two children will be able to have
a second euploid blastocyst transfer after a successful cycle.
The fact that their cryopreserved embryos are all individually
vitrified euploid blastocysts makes it likely that future frozen
transfers will be single ETs as well.
Although similar cumulative outcomes may be achieved
by sequentially performing frozen single ETs of untested
embryos, the practicality of this approach is in question.
Most clinicians report deviating from American Society for
Reproductive Medicine transfer guidelines after a failed IVF
cycle (21). Patients experience increased depression and
anxiety after IVF failure (22), adding pressure to transfer
more embryos in the subsequent cycle. In reality, many
patients are likely to opt for transfer of two embryos after
failing with a fresh eSET.
To improve acceptance of eSET by patients and clinicians,
there is a critical need to optimize the outcome of the first
eSET. Improving embryo selection is an important step in
that direction. A variety of noninvasive methods have been
proposed to enhance selection for eSET including analysis
of the transcriptome of cumulus cells (23, 24, 25), the
embryonic secretome (26, 27), the metabolomic profile of
spent culture media (28, 29), nutrient uptake and utilization
(30), and time-lapse imaging parameters of early develop-ment
(31, 32). However, none of these modalities has been
shown to improve selection for eSET in a prospective
clinical trial. Should benefit be documented from one of
these approaches, it could be applied in parallel with an
invasive approach as described in the present study and
may further enhance selection among high-quality, euploid
blastocysts.
Although the study population included a wide range of
ages, all patients produced at least two high-quality blasto-cysts
by day 5 and were true candidates for fresh eSET.
Although patients whose embryos blastulated on day 6 did
not receive fresh ETs, there is evidence that outcomes may
be improved in these patients by vitrifying their blastocysts
for a future frozen ET (33). Furthermore, there is emerging
evidence that early blastocysts selected for day 5 eSET are
significantly less likely to implant than expanded blastocysts
(8). Because vitrified euploid blastocysts maintain high
implantation rates (5, 17), further studies are required to
determine whether vitrification and future frozen eSET is an
optimal strategy and how often CCS would alter selection in
these cases.
Although this study suggests that qPCR-based CCS may
improve embryo selection and assist in optimizing eSET,
this technology is currently available at a limited number of
centers. There are other CCS platforms, such as single
nucleotide polymorphism arrays, that have been extensively
validated in preclinical studies (34) and the predictive
value of an abnormal result has been determined using a

nonselection trial (13). The rapid nature of the qPCR technique
used in the present study (relative to array-based methods)
allows for transfers within the same cycle when embryos
develop synchronously and blastulate by day 5. However,
excellent outcomes may be achieved by vitrifying all tested
blastocysts and sending biopsy samples to a reference labora-tory
(17). Finally this approach may be cost effective when
compared with array-based CCS methodologies as the added
expense of whole genome amplification is not required.
It should be noted that this treatment approach does
introduce additional laboratory procedures for some patients.
For example, ICSI is recommended when genetic testing is
performed to eliminate the risk of contamination from sperm
or granulosa cells. In the current study, 68% of patients had a
medical indication for ICSI. Although there is concern about
increased risks of congenital malformations in babies
conceived after ICSI (35), these may relate to the underlying
male factor infertility rather than the procedure itself.
Increased risk has not been documented in couples with
normal semen parameters who use ICSI for preimplantation
genetic screening rather than due to severe male factor
infertility (36). Assisted hatching is also required for all
embryos being placed in extended culture as it facilitates
the trophectoderm biopsy procedure, which has been shown
to be safer than cleavage stage biopsy (37). Several prospec-tive
trials have demonstrated that assisted hatching is not
harmful in an unselected infertile population (38). Although
there is concern that hatching may increase the risk of
monozygotic twinning (39), centers with experience in
extended culture and micromanipulation have not found
this to be the case (40). The low risk of monozygotic twinning
in this study and other centers using assisted hatching
for CCS (5, 7) are reassuring, but continued collection of
safety data will be important if this approach is to be widely
implemented.
In summary, high-quality blastocysts selected by tradi-tional
day 5 morphology-based criteria have a substantial
risk of aneuploidy across age groups. When selection is
altered to prioritize euploidy versus morphology, patients
achieve excellent outcomes with high implantation rates
and low clinical miscarriage rates. Future research to identify
noninvasive markers of reproductive potential may further
enhance selection among euploid blastocysts.
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