PRESENTED RESEARCH USING VITROLIFE PRODUCTS
selection of
abstracts
asrm 2016
American Society for
Reproductive Medicine
Salt Lake City, October 2016
Vitrolife
Abstracts ASRM 2016
1
Oral presentations
Topic
Identifier
Time
Page
The difference in size between single pronuclei after ICSI and after
ICSI
C. Igashira et al.
O-42
12.30 PM
4
The morphokinetic characteristics of embryos derived from PCOS patients
N. Aono et al.
O-84
12:30 PM
4
0-208
12.00 PM
5
Monday October 17
Time-lapse
EmbryoScope time-lapse system
Wednesday October 19
Time-lapse
EmbryoScope time-lapse system
Development potential of oocytes with smooth endoplasmic reticulum
cluster and their behaviors observed by time-lapse recording system
C. Mizoguchi et al.
2
Abstracts ASRM 2016
Vitrolife
Poster abstracts
Poster abstracts will appear in numerical order following the below
scheme
Topic
Page
Time-lapse
EmbryoScope time-lapse system
6
Primo Vision time-lapse monitoring system
22
Embryo development
Vitrolife media and oil
24
Sequential media
26
Undisturbed embryo culture/single step medium
27
Vitrolife
Abstracts ASRM 2016
3
Oral Abstracts
Time-lapse
- EmbryoScope time-lapse system
O-42 Monday, October 17, 2016
12:30 PM
THE DIFFERENCE IN SIZE
BETWEEN SINGLE PRONUCLEI
AFTER ICSI AND AFTER IVF.
C. Igashira, J. Otsuki, K. Furuhashi, Y. Katada, T.
Sumimoto, K. Kishi, M. Matsuura, M. Mukai, C. Sumi,
Y. Tsuji, Y. Matsumoto, S. Kokeguchi, M. Shiotani.
Hanabusa Women’s Clinic, Kobe Hyogo, Japan.
OBJECTIVE: Some embryos derived from 1PN zygotes
after conventional IVF procedures (cIVF-1PN) have been
known to be diploid and have the potential to develop
into healthy babies. In contrast, most embryos derived
from 1PN zygotes after ICSI procedures (ICSI-1PN) have
been reported to contain abnormal chromosome configurations. In this study, we analyze the clinical outcomes
of cIVF-1PN and ICSI-1PN and investigate why ICSI1PN zytotes failed to produce pregnancy. DESIGN: A
retrospective study involving 7497 frozen-thawed single
blastocyst transfers. All patients received HRT and the
study spanned from January 2011 to December 2014.
Time-lapse observations were made on 79 1PN zygotes
from January 2013 to February 2015. MATERIALS AND
METHODS: Clinical pregnancy rates, miscarriage rates,
live birth rates and malformation rates resulting from
blastocysts derived from cIVF-1PN and ICSI-1PN were
compared with blastocysts derived from cIVF-2PN and
ICSI-2PN. Blastocysts derived from 1PN zygotes were
transferred when there were no embryos derived from
2PN zygotes. Among 79 1PN zygotes, which were observed by a time-lapse system, the size of the single pronucleus from 40 ICSI-1PN zygotes were compared with
those from 39 cIVF-1PN zygotes immediately before the
breakdown of their pronuclear membranes. RESULTS: A
total of 7497 frozen-thawed single blastocyst transfers
resulted in 3058 (40.8%) clinical pregnancies. Clinical
pregnancy rates in cIVF-1PN, cIVF-2PN, ICSI-1PN,
ICSI-2PN were 36.1% (26/72), 42.1% (2244/5332),
0% (0/20), 37.6% (814/2165) respectively. Among the
26 pregnancies derived from cIVF-1PN, 21 babies were
born, 4 women miscarried and 1 case lost contact. A
minor malformation (pneumothorax) was reported in 1
baby among the 21 postpartum babies. Among the 2244
4
Abstracts ASRM 2016
pregnancies derived from cIVF-2PN, 1681 babies were
born, 528 women miscarried and 35 cases lost contact.
Malformations were reported in 30 babies among the
1681 postpartum babies. There were no statistical differences in clinical pregnancy rates, miscarriage rates,
live birth rates and malformation rates between cIVF-1PN
and cIVF-2PN. In contrast, the pregnancy rate from ICSI1PN zygotes was zero, which was significantly lower than
that in ICSI-2PN. The average size of ICSI-1PN was
666.4µm2 (±105.2) and was significantly less than that
of cIVF-1PN 720.8mm2 (±114.4) (p=0.028). Student’s
t-test or the chi-square test was used for statistical analyses where appropriate. CONCLUSIONS: Zygotes derived
from ICSI-1PN produced blastocysts but no pregnancies,
while healthy babies were born from cIVF-1PN zygotes.
There was no statistical difference in pregnancy rates between blastocysts derived from cIVF-1PN and cIVF-2PN.
The smaller size of single pronuclei after ICSI compared
with single pronuclei formed after IVF may be related
to the disruption of microfilaments within the ooplasm
caused by the penetrating micropipette.
O-84 Monday, October 17, 2016
12:30 PM
THE MORPHOKINETIC
CHARACTERISTICS OF EMBRYOS
DERIVED FROM PCOS PATIENTS.
N. Aono,a,bR. Obata,a S. Maekawa,a N. Oka,a T.
Takeuchi,a H. Igarashi,b K. Kyono.b,a aKyono ART Clinic
Takanawa, Minatoku, Tokyo, Japan; bKyono ART Clinic,
Sendai, Miyagi, Japan.
OBJECTIVE: Polycystic ovary syndrome (PCOS) patients
are extremely sensitive to stimulation with exogenous
gonadotropins and are at an increased risk of developing
ovarian hyperstimulation syndrome (OHSS) during ART
treatment. Such patients are therefore likely to greatly
benefit from using the in vitro maturation (IVM) technique.
However, IVM oocytes have been found to exhibit lower
viability and developmental potential than in vivo matured
oocytes. The objective of this study was to compare
morphokinetics of early stage embryos from IVM oocytes
to that of mature oocytes obtained after controlled ovarian stimulation (COS) in PCOS patients, while mature
Vitrolife
oocytes from stimulated non-PCOS ovaries served as a
control. DESIGN: Retrospective study. MATERIALS AND
METHODS: From Jan. 2014 to Dec. 2015, zygotes
were divided into three groups according to oocyte
maturity and patient characteristics. A total of 66 zygotes
derived from 15 IVMcycles in PCOS patients (Group A),
33 zygotes from 8 COS cycles in PCOS (Group B), and
43 zygotes from 10 COS cycles in non-PCOS patients
(Group C) were involved. Following ICSI insemination,
morphokinetic parameters of each zygote were recorded and evaluated by a time-lapse monitoring system
(EmbryoScopeTM). RESULTS: In Group B, embryos developing to the 3-cell and 4-cell stage were significantly
slower than those in Group C (39.2 ± 6.6 hours vs. 35.1
± 67.2 hours, 43.2 ± 7.5 hours vs. 39.5 ± 6.7 hours)
(P <0.05). The time for the initiation of the compaction
and the time to the morula stage were remarkably shorter
inGroupA (75.5±13.6 hours, 82.9±8.5 hours) and Group
B(74.6 ± 17.6 hours, 83.4 ± 18.9 hours) than in Group C
(84.2 ± 7.5 hours, 96.8 ± 8.0 hours) (P <0.05). The time
for the initiation of blastulation and the time to full blastocyst stagewere significantly shorter i GroupA (92.8±7.4
hours, 104.3 ± 9.5 hours) than in Group C (102.6 ± 5.7
hours, 114.4 ± 7.0 hours) (P <0.05). CONCLUSIONS:
Embryos derived from PCOS patients, regardless of the
maturity at oocyte retrieval, showed faster development
from the initiation of compaction onwards in comparison
to non-PCOS patients. These results suggest that the
state of PCOS may cause changes in morphokinetic
behavior of early embryos.
O-208 Wednesday, October 19, 2016
12:00 PM
DEVELOPMENTAL POTENTIAL
OF OOCYTES WITH SMOOTH
ENDOPLASMIC RETICULUM
CLUSTERS AND THEIR BEHAVIORS
OBSERVED BY TIME-LAPSE
RECORDING SYSTEM.
from August 2010 to September 2015. During ICSI, three
experienced embryologists noticed oocytes containing
vacuole-like structures, which were defined as sERC by
their disappearance at the zygote stage. These embryos
were incubated and imaged by either high-resolution timelapse cinematography or EmbryoScope. Developmental
velocity, rate of clinical use in embryo transfer or cryopreservation, and achieving pregnancy were compared
between oocytes with [sERC(+)] and without sERC
[sERC(-)]. RESULTS: Of the 6,765 mature oocytes, 46
(0.7%) showed sERC(+). The developmental velocity of
embryos derived from sERC(+) oocytes was
significantly higher than that of sERC(-) oocytes, including
the time of 2nd polar body (PB) extrusion, pronuclei (PN)
formation, and syngamy from the ICSI procedure (in h:
2.5±0.6 vs. 2.9±1.0, 7.6±2.4 vs. 9.9±3.9, and 20.7±2.2
vs. 23.7±4.5, respectively; P<0.05). The sERC all disappeared by 4.3±1.2 h after ICSI in the period from 2nd
PB extrusion to PN formation. The rate of clinical use of
sERC(+) oocytes was 80.4% (37/46), of which 6
underwent fresh embryo transfer and 31 were cryopreserved. In total, 15 embryos were transferred (6 fresh
embryos, 9 frozen/thawed embryos), the pregnancy
rate was 40% (6/15), and miscarriage rate was 33.3%
(2/6). Moreover, the live birth rate was 50.0% (3/6) and
one is still ongoing. Healthy babies were born from oocytes with sERC(+). CONCLUSIONS: This study demonstrated that oocytes with sERC could develop into good
quality embryos and achieve successful pregnancy resulting in healthy babies. Further follow-up of the children
are clearly needed, and mechanisms of the emergence of
sERC and its impact on embryonic development are still
under investigation. Detailed studies with molecular
biological methods are necessary.
C. Mizoguchi,a K. Iwata,a M. Tsuneto,b K. Yumoto,b Y.
Mio.a aReproductive Centre, Mio Fertility Clinic, Yonago,
Japan; bMio Fertility Clinic, Yonago, Japan.
OBJECTIVE: We previously reported that oocyte morphology was associated with embryo quality and viability,
suggesting that controlled assessment of oocyte morphology is useful to predict human embryo quality. During
intracytoplasmic sperm injection (ICSI), we occasionally
observe oocytes with smooth endoplasmic reticulum
clusters (sERC) (Otsuki et al, 2004), which are thought to
disappear after fertilization and be closely associated with
poor prognosis in achieving pregnancy. This study aimed
to investigate the developmental potential of oocytes
with sERC and their behaviors using time-lapse recording system. DESIGN: Research study MATERIALS AND
METHODS: We collected the data of 6,765 matured oocytes retrieved after controlled ovarian hyper-stimulation
Vitrolife
Abstracts ASRM 2016
5
Poster Abstracts
Time-lapse
- EmbryoScope time-lapse system
P-84 Tuesday, October 18, 2016
FROZEN EMBRYO TRANSFER (FET)
OUTCOMES FROM DAY 4 EUPLOID
BLASTOCYSTS.
M. E. Thompson,a M. P. Portmann,a B. Scott, M. Kelly,b
M. J. Tucker,c I. Sasson,a M. Avella.a a Shady Grove Fertility
of PA, Wayne, PA; bSociety Hill Reproductive Medicine,
Philadelphia, PA; cIVF, Shady Grove Fertility, Rockville, MD.
OBJECTIVE: To assess the impact of biopsied and vitrified day-4 blastocysts on frozen embryo transfer (FET)
outcomes. DESIGN: This ongoing retrospective study
includes a pool of total 142 patients subjected to in vitro
fertilization (IVF) treatment between September 2013 and
February 2016. We compared IVF outcomes from blastocysts that underwent trophectoderm biopsy for preimplantation genetic screening (PGS) and were vitrified on day
4, day 5 or day 6. MATERIALS AND METHODS: Oocytes
were retrieved in Continuous Single Culture Medium supplemented with 10% SSS (Irvine Scientific), and denuded
from cumulus/corona cells by hyaluronidase treatment 2
to 4 hours before ICSI. Fertilization was assessed by the
presence of two pronuclei at 16-20 hr post sperm injection and from day 1 to day 6 post-fertilization, time-lapse
imaging (Embryoscope; Vitrolife) was performed to monitor embryo development. RESULTS: Day-4 blastocysts
exhibited moderate expansion compared to day-5 and
day-6, but presented a clear blastocoel cavity and a welldeveloped ICM at 90-98 hours after fertilization. Of note,
we observed a higher rate of euploidy among the day-4
versus the day-5 blastocysts (55.3% vs 43.9%), although
so far the low sample size of only 47 biopsied day-4 blastocysts prevented us from confirming statistical for this trend
(p=0.17, Fisher’s exact). The rate of euploidy among day-6
blastocysts (28.2%) was significantly lower than the day-4
blastocysts (p=0.016), and nearly so compared to the day5 blastocysts (p=0.065), although we would interpret this
with caution given the very low number of day-6 biopsied
blastocysts in our sample. Consistent with these data (as of
the date of submission of this abstract) out of 14 transfers,
10 patients have a documented live birth from a day-4
blastocyst transfer.
6
Abstracts ASRM 2016
CONCLUSIONS: Our results show promise in performing
PGS and transferring day-4 blastocysts on implantation
rates and pregnancy. Moreover, we propose late day-4 embryo observations as a valid alternative to the conventional
day-3 embryo check. There are reports of transferring ‘‘day
4’’ morula, but only few studies show blastocyst formation,
cryopreservation and subsequent embryo transfer at day 4.
We are currently recording data from more clinical cases
to substantiate our promising results and provide a better
perspective on the potential development and implantation
of day-4 blastocysts.
P-87 Tuesday, October 18, 2016
A NOVEL HIERARCHICAL
CLASSIFICATION METHOD BASED
ON MORPHOLOGY DYNAMICS OF
VITRIFIED-WARMED BLASTOCYSTS
TO FORECAST IMPLANTATION
POTENTIAL.
A. Coello, A. Cobo, A. Galan, L. Alegre, M. Nohales, M.
Meseguer. IVI, Valencia, Spain.
OBJECTIVE: The analysis of warmed blastocysts by time
lapse-imaging is providing new markers for implantation,
establishing quantitative values linked with clinical outcome.
The objective of this study is to identify predictive morphological variables for implantation and develop a hierarchical
model subdividing warmed blastocysts into categories with
different implantation potential. DESIGN: Retrospective
study. MATERIALS AND METHODS: The study included
435 thawed blastocysts with known implantation data
Vitrolife
which were evaluated using time lapse imaging. Blastocysts
were routinely placed in Embryoscope (UnisenseFertiliTech, (Vitrolife) Denmark) from immediately after
warming until transfer (>3.5h). Embryos were vitrified
and warmed with Cryotop method (KitazatoBiopharma).
Variables studied included initial and minimum thickness
of zona pellucida (ZP) (µm), initial and maximum area of
blastocyst (µm2), area of inner cell mass (µm2), expansion
(whether the embryo expands or not after warming) and
presence of collapse or contraction after warming procedure. After defining optimal ranges according to consecutive quartiles, a logistic regression analysis was performed
combining the variables described before and blastocyst
morphological classification criteria defined by the Spanish
Association of Embryologists (ASEBIR) into A, B, C or D.
RESULTS: We observed that expansion of warmed blastocysts is strongly correlated with impantation (32.8% in
blastocysts that expand (132/403) vs 9.4% in blastocysts
that do not expand (3/32); P=0.006). Throughout logistic
regression analysis the model identified the maximum area
of the blastocyst OR=0.41 (95%CI 0.22 - 0.77) followed
by the initial area OR=0.62 (95%CI 0.35 - 1.08) as the
most predictive variables characterizing implanting embryos
(blastocyst morphology was not considered relevant in
our model). We made a hierarchical model representing
a classification tree, which subdivided embryos into four
categories from A to D with decreasing expected implantation potential. CONCLUSIONS: We propose a hierarchical
model to classify warmed blastocysts according to their
probability of implantation. This model is not considering
relevant standard blastocyst morphology classification
when compared with morphology dynamics. To our knowledge, this is the first attempt of making a model for implantation using time lapse imaging in warmed blastocysts.
This novel classification may be useful to consider single
or double embryo transfer or indicate assisted hatching in
low score categories in frozen cycles. Supported by: The
Spanish Ministry of Economy and Competitiveness
(PI14/00523) through the Instituto de Salud Carlos III
program.
Reserve School of Medicine, Case University, Cleveland,
OH.
OBJECTIVE: Time lapse imaging and morphokinetics provide invaluable information on embryonic growth potential.
In this study we combine morphokinetics with chromosome
data from preimplantation genetic screening (PGS) to
evaluate the effect of cleavage anomalies, multinucleation,
cell kinetics and delayed blastulation on genetic status of
embryos. DESIGN: Retrospective analysis of morphokinetic
and PGS data from patients undergoing IVF. MATERIALS
AND METHODS: Normally fertilized zygotes were
cultured in the Embryoscope time-lapse incubation chamber. Embryo growth kinetics were evaluated by viewing
time lapse video footage. Time to specific cell stages was
assessed as well as the presence of multinucleation (MU),
direct uneven cleavage (DUC) and reverse cleavage (RC).
Trophectodermbiopsy was performed on expanded blastocysts, either day 5 or 6 of culture. The relationship between
the various parameters and chromosomal status was assessed. Statistical differences between treatment groups
were analyzed using the Chi square and students t-test; p
values <0.05 were considered significant. RESULTS: A
total of 853 zygotes were cultured in the Embryscope from
51 patients. The mean patient age was 37.6 ± 4.3. The
overall incidence of multinucleation, reverse cleavage and
direct uneven cleavage was 31%, 15% and 7%, respectively. PGS results were obtained for 292 biopsied blastocysts and 40% (n=118) were diagnosed as euploid.
Amongst biopsied blastocysts, 109 were from multinucleated embryos. The euploidy rate was not significantly different between MU and non-MU blastocysts (37%, 40/109vs
43%, 78/183, respectively). With reverse cleavage only 27
% of blastocysts were euploid (7/26) as compared to 42%
of non RC blastocysts (111/265 ) but differences did not
reach significance. Cell cycle timings and time to synchrony
were also examined . Table 1 shows the euploidy rate for
blastocysts with kinetics in desired range or out of range
(shown bolded).
TABLE 1. Kinetics and Chromosomal Status
Distribution of warmed blastocysts according to the model
category.
P-105 Tuesday, October 18, 2016
IS THERE AN INCREASE IN
ANEUPLOIDY RATE WITH DELAYED
BLASTULATION,MULTINUCLEATION
OR CLEAVAGE ANOMALIES?
N. Desaia P. Rambhia.b a OB-GYN/Women’s Health
Institute, Cleveland Clinic, Beachwood, OH; bCaseWestern
Vitrolife
CONCLUSIONS: Delay in formation of an expanded
blastocyst was associated with a lower euploidy rate.
Multiucleation, reverse cleavage and direct uneven cleavage did not appear to increase the rate of aneuploidy. A
larger data set is still needed to understand if these factors
have any bearing on the blastocyst’s chromosomal status.
Abstracts ASRM 2016
7
P-121 Tuesday, October 18, 2016
TIME-LAPSE IMAGING OF
MULTINUCLEATED EMBRYOS
AND THE ASSOCIATION WITH
ANEUPLOIDY DETERMINED BY
PREIMPLANTATION GENETIC
SCREENING.
L. R. Goodman, J. M. Goldberg, T. Falcone, N. Desai.
Women’s Health Institute, Cleveland Clinic, Cleveland, OH.
OBJECTIVE: To determine if embryo multinucleation visualized by continuous time-lapse imaging is associated with an
increased risk of aneuploidy. DESIGN: Prospective cohort
study MATERIALS AND METHODS: A series of patients
under the age of 38 years undergoing in-vitro fertilization
for unexplained infertility were recruited to participate when
at least 20% of their embryos exhibited evidence
of multinucleation (MU) on day two of continuous time
lapse culture in the EmbryoScopeTM. All expanded blastocysts underwent trophectoderm (TE) biopsy on day 5 or
6 for genetic screening. Un-biopsied early blastocysts, as
well as embryos arrested at cleavage/morula stage, were
also sent for analysis. Continuous variables were compared
with the student’s t-test, categorical with chi-squared and
logistic regression was performed to account for confounding variables. RESULTS: A total of 133 embryos from nine
couples were evaluated for genetic analysis. The average
age of patients was 31.9 +/- 3.2 years, who had 15.3 +/7.3 embryos with an average of 56.3 +/- 18.9% exhibiting
MU. Of the 133 embryos, 72 (54.1%) developed to the
expanded blastocyst stage and were able to undergo TE
biopsy. There was no difference in embryo development
to the expanded blastocyst stage between MU embryos
and those without evidence of MU (56.9 vs. 43.1%; p
0.37). When all embryos were evaluated, 57 (42.9%) were
euploid, 49 (36.8%) exhibited aneuploidy, 22 (16.5%) were
resulted as nonconcurrent and the remaining 5 (3.8%) had
no amplification. There was no difference aneuploidy rate
between MU and non-MU embryos (44.4 vs. 47.1%;
p=0.83). When accounting for confounding factors, MU
embryos were equally as likely to be genetically normal (OR
1.08 95% CI 0.48 - 2.46; p=0.85) and to make it to the
blastocyst stage (OR 0.69 95% CI 0.31 - 1.48; p=0.34)
as those without evidence of MU. Of the TE biopsied embryos with diagnosis (n=53), the rate of aneuploidy
was also found to be similar between MU and non-MU embryos (45.5 vs. 35.0%; p=0.57). When evaluating kinetic
parameters available though continuous time-lapse imaging, none of the timing of developmental milestones
differed by MU. Stage of arrest was also similar between
the two groups. CONCLUSIONS: In this study, there was
no increase in risk of aneuploidy in multinucleated embryos.
Additional data is needed to corroborate these findings.
Supported by: The Foundation for Embryonic Competence.
P-153 Tuesday, October 18, 2016
8
Abstracts ASRM 2016
CHROMOSOMAL CONSTITUTION
AND MORPHOKINETICS OF
ARRESTED AND NON-VIABLE
EMBRYOS.
K. Sorby,a,bE. Dimitriadis,a T. Osianlis.c a Hudson Institute
of Medical Research, Clayton, Australia; bMonash
University, Clayton, Australia; cRitchie Centre, Monash
University, Melbourne, Australia.
OBJECTIVE: To determine the chromosomal constitution
of embryos failing to develop to a clinically viable blastocyst following extended culture in vitro and whether the
morphokinetic behaviour of such embryos relates to their
chromosomal content. DESIGN: Prospective analysis of
morphokinetic and chromosomal data for non-viable embryos following independent determination of their unsuitability
for clinical use. MATERIALS AND METHODS: Embryos
were cultured in an Embryoscope time lapse incubator.
After six days in culture embryos not suitable for transfer or
freezing, due to cleavage arrest, multinucleation, failure to
cavitate, cellular degeneration or failure to form a blastocyst
of sufficient quality for clinical use, were subjected to whole
genome amplification and RESULTS: Interpretable results
were obtained from 136 embryos. Embryos deemed unsuitable for clinical use displayed an aneuploidy rate of 95.6%,
with only 6 embryos demonstrating an apparently euploid
chromosomal complement. Of these 6 embryos, 3 were excluded from use due to absence of an inner cell mass and
3 due to inadequate trophectoderm formation and/or cellular degeneration. Of note, every embryo returning a euploid
result underwent cavitation, leaving a 100% aneuploidy rate
for embryos that failed to reach the blastocyst stage. When
comparing embryos that did or did not attempt to cavitate
(excluding triploid embryos), embryos failing to reach this
stage contained on average twice the number of chromosomal errors (4.52 vs 2.25, p<0.0001). Unlike previously
reported data utilising clinically viable blastocysts, these
embryos exhibited a majority of monosomies over
trisomies (65.8% vs 34.2%), likely reflecting the poorer
developmental potential of monosomic embryos. Of interest, although triploid embryos are seen at the blastocyst
stage, of the 7 triploid embryos identified in this cohort, 6
arrested at either one cell (3 embryos) or following their first
cleavage division. The remaining triploid embryo reached
cavitation but was of extremely poor quality. This suggests
the rate of triploidy in early embryos may be significantly
higher than that seen in clinical blastocyst samples. In
addition to their standard aneuploidies, 15 embryos
contained a segmental aneuploidy, however, in only one
embryo was a segmental aneuploidy its only chromosomal
error. Similarly, 29 embryos contained additional mosaic
aneuploidies, below 50% increase or reduction but
distinctly identifiable as a gain or loss. CONCLUSIONS:
Embryos failing to reach the blastocyst stage in our culture
system displayed universal aneuploidy, a significant finding
when counseling our patients. The specific chromosomal
errors in non-viable embryos do appear to impact their morphokinetic parameters, however, further work is required to
Vitrolife
elucidate these potential relationships.
P-167 Tuesday, October 18, 2016
TIME-LAPSE EMBRYO
MORPHOKINETICS FOLLOWING
GNRH AGONIST OR HCG
TRIGGERING.
G. Oron, O. Sapir, R. Garor, Y. Shufaro, H. Pinkas, B.
Fisch, A. Ben-Haroush. IVF and infertility Unit, Beilinson
Hospital, Rabin Medical Center, Petach Tikva, Israel.
OBJECTIVE: GnRH agonist triggering is used instead of
hCG in antagonist cycles, in order to diminish ovarian hyperstimulation in high risk patients. GnRH agonist triggering acts by eliciting an endogenous surge of LH and FSH,
this effect on early embryonic development is still unknown.
Our aim was to compare embryo morphokinetic parameters
following GnRH agonist with hCG ovulation triggering, using the EmbryoScope time lapse monitoring system (TMS).
DESIGN: A retrospective cohort study. MATERIALS AND
METHODS: All TMS data of fresh ICSI cycles with antagonist protocol between 4/2013 - 1/2016 was analyzed.
Embryo morphokinetic parameters1 and pregnancy rates
were compared between the two groups. Timing of PBextrusion, PN-fading, and cell cleavage from division to 2
cells (T2) up to the 5-cell stage (T5), second cell cycle duration (CC2=T3-T2) and synchrony in division from 2-cell
to 4-cell blastomere embryos (S2=T3-T2) were compared.
Optimal CC2 was defined as R5 hours and optimal S2 was
defined as < 1 hour. A separate analysis for the embryo
culture medium: CSC (Irvine, USA), Global (Lifeglobal,
USA) and Sage (Cooper-Surgical, USA) was performed.
RESULTS: 1954 embryos derived from cycles triggered
with hCG (Group 1) and 606 embryos derived from cycles
triggered with GnRHa (Group 2) were analyzed (274 and
60 patients, respectively). Oocyte maturation rates and
fertilization rates were similar in both groups [84%and
72% in Group 1; 83% and 76% in Group 2]. Polar body
(PB) extrusion occurred earlier in Group 1 than in Group
2 (3.8h±2.2 vs 4.2h±3.6, p=0.015) and remained significant also in embryos cultured in CSC medium [Group 1
(n=1358) 3.9±23 vs Group 2 (n=379) 4.±4.1, p< 0.001].
Embryos cultured in Global medium had a shorter PN fading in Group 1 (n=352) than Group 2 (n=204) (24.8±4.1
vs 26.0±3.6; p<0.0001). At the 2-cell stage, the percentage of embryos with no multinucleation and the proportion
of symmetric-blastomere embryos was significantly higher
in Group 1than in Group 2 [49.9% and 36.2% (p<0.001)
and 87.1% and 82.9%; (p=0.033); respectively]. There
percentage of embryos that reached an optimal CC2 duration in Groups 1 and 2 was similar [79.1% (1543/1951)
and 76.1% (461/606), p¼0.1]. The percentage of embryos
with an optimal S2 was higher in Group 1 than in Group
2 [50.5% (985/1950) vs 43.2% (262/6060), p¼0.002].
Pregnancy rates in cycles with fresh embryo transfers were
similar in Groups 1 and 2 (40.2% and 35.6%; p¼0.5).
CONCLUSIONS: Morphokinetic parameters of embryos
derived following hCG or GnRHa ovulation triggering were
Vitrolife
overall similar. It is yet to be determined whether the longer
duration of several of the developmental kinetic parameters
in embryos following GnRh agonist triggering is of any clinical significance.
P-180 Tuesday, October 18, 2016
BLASTOCYST COLLAPSE.
VALIDATION OF PREVIOUS DATA.
R. Herrer Saura,a J. Marcos,b P. Valero,a V. Ramirez,aJ.
Serna,a E. I. Gil Arribas,c M. Meseguer.d aIVF, IVI Zaragoza,
Zaragoza, Spain; bIVF, IVI Murcia, Murcia, Spain; cIVI
Zaragoza, Zaragoza, Spain; dClinical Embryology, Valencia,
Spain.
OBJECTIVE: The appearance of time lapse incubators has
opened the possibility of studying dynamic embryo behaviors. Blastocyst collapse has been studied in different mammal models. Strong contractions seem to be deleterious for
embryo hatching. There is a high consumption of energy in
the process of contraction and re-expansion, which
could affect the extrusion of the blastocyst from the Zona
Pellucida (ZP) and, consequently, the possibility of implantation. DESIGN: Retrospective study to validate the
results obtained from IVI Murcia and IVI Valencia Marcos, J
et al, (Hum Reprod 2015) analyzing a second data set of
embryos, in a different laboratory and with different culture
conditions. Marcos et al. found that blastocyst collapse
affects implantation, but not hatching rate. Time of collapse and duration of contraction were similar in nonimplanted collapsed blastocyst (KID- CB) when compared
to implanted collapsed ones (KID+ CB). MATERIALS
AND METHODS: 340 cycles from the oocyte donation
program of IVI Zaragoza; 499 blastocysts (both fresh and
cryopreserved)were transferred, from whose 375 had
known implantation data (KID).Embryos where incubated
in Embryoscope (Vitrolife) at 37oC, 5% CO2, 5% 02 and
single step media (GLOBAL, Quermed). Only strong contractions where considered for the analysis (more than 50%
of Trofoectoderm (TF)detachment from ZP).Hatching rate,
implantation rate, duration and timeof contractions where
compared between contracted (CB) and non-contracted
(NCB) blastocysts. RESULTS: From the 499 blastocysts
transferred, 25.1% presented at least one contraction.
According with the study of Marcos et al, there were no
differences in hatching rate between CB (46.8%, [CI95%
38.0-55.6]) and NCB (43.5%, [CI95% 38.4-48.5]), but
implantation rate was statistically lower in CB (33.7%,
[CI95% 24.2-43.2] compared with NCB (47%, [CI95%
41.5-53.2], P=0.02). Taking into account only KID CB,
our data agree again with Marcos respecting to duration of
contraction, which is similar between KID- CB and KID+
CB (2.08 h (116.49-123.28h) vs 2.07 h (1.65-2.56h)),
butnot in the time of collapse (119.89 (116.49-123.28H)
KID- CB vs 111.2 (107.23-115.21) KID+ CB, p=0.0002).
CONCLUSIONS: Blastocyst collapse is a negative feature
of embryo dynamics, as implantation is affected. Our data
confirm the relevance of this parameter as it has been
validated in a different setting, with different patients and
Abstracts ASRM 2016
9
culture conditions. In consequence, this dynamic characteristic should be taken into account in the embryo selection
for transfer.
P-215 Tuesday, October 18, 2016
DYNAMIC ANALYSIS OF HUMAN
PARTHENOGENETIC ZYGOTES
INDUCED BY ARTIFICIAL OOCYTE
ACTIVATION.
A. Tanaka, K. Yumoto, K. Iwata, C. Mizoguchi, M.
Tsuneto, Y. Mio. Reproductive Centre, Mio Fertility Clinic,
Yonago, Japan.
OBJECTIVE: The origin of the pronucleus (PN) in a single
PN zygote (1PN), and whether its genome is normal still
remains controversial. We recently established a novel
method of discriminating between maternallyand paternally-derived PN using immunofluorescence staining and
demonstrated the possibility that both the male and female
genome could be packed in 1PN in some cases. However,
currently analyzing karyotypes is an invasive technique,
limiting its clinical application. Therefore, we tried to
distinguish between normally-fertilized zygotes and 1PN
zygotes by their morphology or developmental behavior. In
this study, we used a microscope with timelapse system
to analyze the developmental time course and morphology of human 1PN zygotes, especially parthenogenetic
zygotes induced by artificial oocyte activation. DESIGN:
Research study. MATERIALS AND METHODS: This study
used 32 MII oocytes donated between October 2014
and August 2015 by patients who gave informed consent
for this study. Fresh or freeze-thawed MII oocytes were
activated electronically and the oocytes were observed
by EmbryoScope . We compared the developmental time
course and morphology between parthenogenetic zygotes
and normal 2PN zygotes fertilized by assisted reproductive
technology. RESULTS: There was no difference in the diameter of the PN in parthenogenetic zygotes compared to
the female PN in normal fertilized zygotes (28.9 2.2 vs 26.4
2.0 µm, respectively). There were significant differences
between normal 2PN and parthenogenetic zygotes for the
time from intracytoplasmic sperm injection or electronic
activation to the 2nd polar body (PB) extrusion (3.0±1.7 vs
2.3±0.5 h, respectively), from 2nd PB extrusion to syngamy
(20.8±4.1 vs 18.7±2.9 h, respectively), from syngamy to
1st cleavage (3.0±2.3 vs 3.9±1.1 h, respectively), and
from 1st cleavage to 2nd cleavage (9.8±4.8 vs 13.6±5.2
h, respectively). In addition, some parthenogenetic zygotes
(6 of 21) developed to blastocysts. CONCLUSIONS: The
time required from electronic activation to 2nd PB extrusion
and from 2nd PB extrusion to syngamy in parthenogenetic
zygotes was significantly shorter than in normal zygotes.
This may be because oocyte activation or decondensation
of the sperm nucleus is not required in parthenogenetic
zygotes. In addition, the time required from syngamy to 1st
cleavage, and from 1st cleavage to 2nd cleavage in parthenogenetic zygotes was significantly longer than in normal
embryos. Thus, differences in the time course of embryonic
10
Abstracts ASRM 2016
development in activated zygotes could be used to identify
the characteristics of parthenogenetic zygotes, even though
further studies are needed. Furthermore, although some
parthenogenetic zygotes develop to blastocysts, the clinical
use of zygotes with 1PN should be questioned. P-216 Tu
P-315 Tuesday, October 18, 2016
INCREASED ARREST PRIOR
TO COMPACTION IN EMBRYOS
DERIVED FROM TESTICULAR
SPERM.
P. K. Gill N. Desai. OBGYN/ Women’s Health Institute,
Cleveland Clinic, Beachwood, OH.
OBJECTIVE: Sperm play an essential role in embryonic genome activation and embryonic progression to blastocyst.
The purpose of this study was compare morphokinetics of
embryonic development in IVF-ICSI cycles in which sperm
were retrieved from the epididymis by percutaneous aspiration (PESA) or else through surgical extraction from the
testis (TESE). DESIGN: A retrospective analysis of the effect of epididymal and testicular sperm on early embryonic
development. MATERIALS AND METHODS: Kinetic data
and cycle outcomes were retrospectively analyzed for 76
azoospermic patients undergoing an IVF/ ICSI cycle with
percutaneous epididymal aspiration (PESA) or a testicular
sperm extraction (TESE). Normally fertilized zygotes
(n=389) were cultured in the EmbryoScope time lapse
incubator at 37±C with 6% O2 and 6% CO2. Time lapse
videos were viewed and annotated for cell division and
cleavage anomalies. The incidence of multinucleation, reverse cleavage and direct uneven cleavage was monitored.
Clinical pregnancy rate (CPR), implantation rate (IR) and
blastulation rate were also calculated. The chi square and
Student t-test were used as appropriate to compare PESA
and TESE cycle outcome data and embryo morphokinetics.
P values <0.05 were considered significant. RESULTS:
Clinical pregnancy and IR with TESE derived embryos
(51% and 31%, respectively) was similar to that with PESA
(57% and 37%, respectively). Embryos in the TESE group
were however significantly slower in reaching early kinetic
endpoints (t2,t4,t8). Table 1 contrasts growth kinetics
between the two treatment groups. The mean day 3 cell
count was lower with TESE (7.69 ± 2.09) vs PESA (8.65
± 2.73; p=0.03). embryos. Furthermore, an increased percentage of embryos in the TESE group failed to compact
(TESE 31%,vs PESA 16%; P<0.0001) and time to compaction was greater. Despite early differences, tM, tSB and
tEBL timings for embryos from both treatments were similar. We did however find that a significantly lower percentage of TESE embryos formed expanded blastocysts (TESE
34% vs PESA 52%; p<0.001). CONCLUSIONS: Delayed
developmental times were observed in embryos derived
from testicular sperm up until the stage of compaction, one
of the early indicators of embryonic genomic activation.
Compaction is a critical transition point for continued
embryo development. It is interesting that embryos from the
TESE group arrest at a higher rate prior to this stage
Vitrolife
but once over the hurdle , have similar kinetics as PESA
derived embryos. Prospective studies examining the severity of testicular dysfunction and sperm quality in TESE
patients and its relationship to embryo kinetics may help to
further elucidate these results.
Cycle Outcome and Kinetic Data
P-517 Wednesday, October 19, 2016
THE IMPACT OF EMBRYO
MORPHOKINETICS ON ICSI
OUTCOME: UNEXPALINED
INFERTILITY VS. MALE FACTOR
INFERTILITY.
N. Adel,a M. Elmahdy,b A. A. Aboali,a S. A. Hebisha,b A.
Elmasdy,a M. Galal.a aMadina Fertility Center, Alexandria,
Egypt; bObs. Gyn., Alexandria University - Faculty of
Medicine, Alexandria, Egypt.
OBJECTIVE: To evaluate morphokinetics of embryos using
Embryoscope (unisense Fertili-tech(Vitrolife), Denmark)
on ICSI outcome in patients with male factor infertility vs.
patients with unexplained infertility. DESIGN: Retrospective
cohort study. MATERIALS AND METHODS: 445 oocytes
were retrived from 48 patients with unexplained infertility
and 247 oocytes were retrived from 24 patients with male
factor infertility, fertilization of oocytes was performed by
conventional ICSI between April 2014 and january 2016
at Madina Fertility Center, Alexandria, Egypt. Embryos were
cultured and loaded into Embryoscope immediately after
injection and annotated for pattern time of cleavage
(tPNa: time of pronuclei appearance, t2: time of 2-cell,
t3: time of 3-cell, t4: time of 4 cell, t5: time of 5-cell,
CC2:second cell cycle t3-t2, S2: The time period of the
synchrony of the first cell cycle (t4-t3)), fertilization rate,
blastulation rate, pregnancy rate and implantation rate
were compared between the two studied groups.Statisical
analysis was conducted using Whitney test for comparing
Vitrolife
between the two groups, P values _< 0.05 considered significant. RESULTS: No significant difference was observed
between groups regarding fertilization rate and blastulation
rate. The pregnancy and implantation rates in patients with
unexplained infertility were significantly higher than those
with male factor infertility 52.08% (25/48) vs. 37.5% (9/24)
and 26.02% (38/146) vs. 16.66% (13/78), respectively;
(p<0.05). The mean time-points for patients with unexplained infertility were significantly higher at t2, t3, and t4
(31.13, 40.10, and 42.87, respectively) compared to t2,
t3, t4 of patients with male factor infertility (29.72, 38.07,
41.37, respectively, p<0.05) as shown in the following table: CONCLUSIONS: Morphokinetics of embryos derived
from patients with unexplained infertility showed a delay in
all time-points and resulted in higher pregnancy and implantation rates, while rapid motion of development showed
by morphokinetics of embryos derived from patients with
male factor infertility may negatively affect the pregnancy
and implantation rates.
P-519 Wednesday, October 19, 2016
DOES SPERM ORIGINHAVE AN
IMPACTON MORPHOKINETICS OF
HUMAN ZYGOTES?
T. Takeuchi,a N. Aono,a N. Oka,a R. Obata,a
N. Okuyama,a M. Machida,a K. Nagao,b H. Igarashi,c K.
Kyono.c a Kyono ART Clinic Takanawa, Minatoku, Tokyo,
Japan; bUrology, Toho University, Ota-ku, Tokyo, Japan;
c
Kyono ART Clinic, Sendai, Miyagi, Japan.
OBJECTIVE: ICSI with testicular sperm extraction (TESE)
is the choice of treatment for azoospermic patients irrespective of testicular pathology, i.e., obstructive (OA) or
non-obstructive azoospermia (NOA). However, lower fertilization and compromised embryonic development are often
observed particularly with NOA patients compared to those
with ejaculated spermatozoa (EJ). The objective of this
study was to assess the effect of azoospermic etiology on
morphokinetics of embryos fertilized with testicular sperm.
To do so, zygotes in OA and NOA patients were compared
with those in EJ patients by time-lapse monitoring (TM).
DESIGN: Comparative morphological assessment of ICSI
zygotes. MATERIALS AND METHODS: A total of 792 mature oocytes from 145 patients were ICSI inseminated with
OA (n=272), NOA (n=267) or EJ (n=433) sperm. After
ICSI, oocytes were individually cultured in a TM system
(Embryoscope). Time points of each morphokinetic event
were recorded up to the blastocyst stage, and the 3
groups were compared. Morphokinetic behavior was also
Abstracts ASRM 2016
11
compared between transferred embryos according to their
implantation potential. RESULTS: Average maternal age
was 33.5±4 years for NOA, which is the youngest out of
the 3 groups, 36.4±3 for OA, and 37.1±4 for EJ. While
fertilization rate in both NOA (54.3%) and OA (60.7%)
were lower (P <0.05) than EJ (69.7%), blastocyst formation
and implantation rates were all comparable. Time points for
PN disappearance, 2-cell stage, and 4-cell stage, as well
as the blastocyst, were longest in NOA (P < 0.01), while
the interval between the 4- and 5-cell stage was shortest
in NOA (P < 0.01). Those in OA embryos were all similar
to EJ, but PN appearance and time to morula were shorter.
Although the maternal age of implanted embryos
was younger than non-implanted counterparts, no morphokinetic difference was noted between the two. Among
implanted zygotes across the three groups, intervals between the 2- and 3-cell, and the 4- and 5-cell in NOA
were shorter than in EJ, but not different from that in OA.
CONCLUSIONS: NOA zygotes showed slower and less
synchronized cell cleavage than OA, which was similar to
EJ, indicating spermatogenic function has an impact on
early embryonic development. However, NOA embryos
that were suitable for transfer or capable of implantation
developed similarly to EJ zygotes.
P-534 Wednesday, October 19, 2016
EMBRYOQUALITY IS IMPROVED
BY PICSI SPERM SELECTION. A
RANDOMIZED CONTROLLED TRIAL.
L. Alegre,a N. Garrido,b J. Romero,c A. E. Palma,d J.
Remohi Gimenez,e M. Meseguer.f aClinical Embryology,
IVI Valencia, Valencia, Spain; bAndrology Laboratory,
Instituto Valenciano IVI Valencia, Valencia, Spain; cIVI
Valencia, valencia, Spain; dIVF Laboratory, IVI Panama,
Panama, Panama; eIVI Valencia, Valencia, Spain; fClinical
Embryology, Valencia, Spain.
OBJECTIVE: PICSI has been demonstrated to positively
select sperm with less aneuploidies, DNA fragmentation
and adequate maturation features. However, their impact
on the development and quality of embryos is unknown.
To evaluate whether microinjection with sperm selected
by PICSI (hyaluronic acid receptor binding ability) show
any improvement on embryo quality based on morphokinetic analysis in comparison with classic sperm selection.
DESIGN: Secondary analysis from an ongoing prospective,
randomized and triple-blinded trial, including a total of 144
infertile couples undergoing oocyte donation. MATERIALS
AND METHODS: Embryo quality parameters and in vitro
fertilization outcomes were compared between PICSI (72
patients) and control group, conventional ICSI (72 patients). A total of 535 embryos were analyzed in PICSI and
613 embryos in control group with Embryoscope or Eeva
for the main morphokinetic parameters. Also, the proportion
of embryos classified according to aneuploidy risk, blastocyst prediction and implantational potential (based on published algorithms) were compared. RESULTS: Statistically
significant differences between PICSI and ICSI groups
12
Abstracts ASRM 2016
were found when morphokinetic parameters were analyzed,
cc1: 22.4h (95%CI=22.0-22.8) vs. 21.8h (95%CI=21.422.1), t2: 29.0h (95% CI=28.3-29.7) vs. 30.24h
(95%CI=29.4-31.1), t3: 38.5h (95%CI=37.8-39.2) vs.
39.7h (95%CI=38.9-40.6), t4: 41.5h (95%CI=40.8-42.2)
vs. 43.0h (95%CI=42.1-43.8), t8: 65.2h (95%CI=63.866.6) vs. 67.5h (95% CI=66.1-68.8), t9+: 75.0h
(95%CI=73.4-76.5) vs. 78.6h (95%CI=77.1- 80.1), tEB:
111.6h (95%CI=110.3-112.8) vs. 113.9h (95%CI=112.3115.5) PICSI and control group respectively. Regarding the
embryo quality, expressed as the categories of aneuploidy
risk and implantation potential among groups, aneuploidy
likelihood (low, A type embryo) was 22% (95% CI=18.325.6) vs. 17% (95%CI=14.1-20.4), and D type embryo
(high aneuploidy likelihood) was 29% (95%CI=24.7-32.7)
vs. 35% (95%CI=30.6-38.6) for PICSI and ICSI respectively. Implantation potential classification between PICSI
and control group was 17% (95%CI=14.2-20.7) vs. 16%
(95%CI=13.4-19.3) (High likelihood, A type embryo) and
39% (95% CI=35.0-43.4) vs. 42% (95%CI=38.0-45.9)
(Low likelihood, E type embryo). No statistical differences
were found in the proportion of high quality embryos 18%
in PICSI group vs. 13% in control group based on Eeva
algorithm. CONCLUSIONS: PICSI leads to a significant
improvement of embryo quality, as reflected in the higher
implantation potential and lower risk of aneuploidies. The
better embryo quality obtained after its application the
higher clinical outcome would be expected with large scale
randomized control trials. Supported by: PI14/00523.
Spanish Ministry of Economy and Competitiveness.
Instituto de Salud Carlos III program
P-535 Wednesday, October 19, 2016
MORPHOKINETIC PARAMETERS
IN SPERM SELECTION BY
ANNEXIN-V SORTING PRIOR TO
ICSI IN OVUM DONATION PROGRAM
RESULTS FROM A PROSPECTIVE
RANDOMIZED TRIAL.
L. Alegre,a L. Romany,b N. Garrido,c A. Tejera,d J. Remohi
Gimenez,a M. Meseguer.e aIVI Valencia, Valencia, Spain;
b
Embryologist, Valencia, Spain; cAndrology Laboratory,
Instituto Valenciano IVI Valencia, Valencia, Spain; dIn Vitro
Fertilization Laboratory, IVI Company, Valencia, Spain;
e
Clinical Embryology, Valencia, Spain.
OBJECTIVE: Magnetic activated cell sorting (MACS)
procedure is able to separate reactive/positive sperm and
inject no apoptotic spermatozoa in the oocyte. Our objective was to determine if MACS-Anexin V selection improved
embryo morphokinetic parameters, development or implantation outcome, over the conventional swim-up technique.
DESIGN: Secondary analysis from a prospective, randomized and doubleblinded study. A total of 80 sperm
samples from patients undergoing ovum donation were
included. MATERIALS AND METHODS: We generated
two groups: MACS (Swim-up + MACS) and Swim-up
Vitrolife
group, before ICSI treatment. The MACS group included
37 couples and Swim-up 43. A total of 258 and 243
oocytes were analyzed respectively. With the use of Timelapse technology we did a complete embryo follow-up until
transfer with further morphokinetic analysis. RESULTS:
Similar results were obtained in implantation rates in both
groups 39.19% (CI95% 24.42_53.96) vs. 41.86%
(CI95% 28.46_55.26). Direct division from 1 to 3 cells
embryos incidence was 22.90% (CI95%17.74_27.99) vs.
24.30% (CI95% 18.89_29.67) comparing MACS vs
Swim-up groups respectively. Significant differences
were found in morphokinetic parameters, particularly at
cleavage stage embryos, results in MACS and Swim-up
groups respectively were compared; t2: 27.05h (CI95%
26,54_27,56) vs. 28.55h (CI95% 27.98_29.12), t3:
37.11h (CI95% 36.26_37.95) vs. 38.62h (CI95%
37.69_39.55), t4: 39.71h (CI95% 38.85_40.57) vs.
42.04h (CI95% 40.97_43.11), t6: 54.50h (CI95%
53.18_55.82) vs. 51.69h (CI95% 50.45_52.92),
t7_57.04h (CI95% 55.73_58.34) vs. 54.08h (CI95%
52.93_55.24). CONCLUSIONS: MACS technology application in unselected males undergoing ICSI on fertile
donors affects cleavage stage embryos, results in faster
cleaving embryos that may affect implantation potential.
Further studied should be performed to understand the
insights of this selection and whether this effect could be
observed in the outcome by large scale studies.
Morphokinetic parameters and statistical significance between groups (hours).
Supported by: PI14/00523. Spanish Ministry of Economy
and Competitiveness. Instituto de Salud Carlos III program.
P-539 Wednesday, October 19, 2016
OUTCOMES BETWEEN TWO
DIFFERENT METHODS: CLASSIC
ASSISTED HATCHING (AH) AND
ZONA THINNING
METHODS: Patients undergoing autologous IVF cycles
(n=270) with fresh embryo transfer on day 5 and embryo
culture in a time lapse system were included. Assisted
hatching was performed using a laser (Navilase, OCTAX
Microscience GmbH, Germany) on day 3 of development.
Patients were divided in 3 groups according to the type of
hatching technique applied: Group (n=22, 116 embryos):
classic assisted hatching technique involving 2 laser pulses
that opened a hole in the zona pellucida(ZP). The hole
was 1.5 times wider than the thickness of the ZP of each
embryo. Group ZT (n=26, 169 embryos): 25% of the ZP
perimeter was thinned to half of its thickness. Thinning was
performed using 15 consecutive pulses Control group
(n=222, 1451 embryos): no laser intervention. Embryos
were cultured in the Embryoscope (Vitrolife, Sweden) and
images were taken every 10 minutes. Kinetic markers in
hours post injection (hpi) included: time to start blastulation (tSB), blastocyst cavitation, blastocyst expansion,
initiation of hatching (tBiH) and the interval between the
start of blastulation and initiation of hatching (tBiH-tSB).
RESULTS: No differences were found in patients baseline
characteristics or in the embryo quality on Day 2, Day 3
and Day 5 of development. Blastocyst formation rate was
statistically higher in AH group (73.3%) when compared
to ZT (56.5%, p=0.0033) or control(60.5%; p=0.0064).
Similarly, blastocyst hatching rate was significantly higher
in AH vs. ZT and control groups (38.8%, 7.2% and 6.2%
respectively; p<0.0001). With respect to kinetic markers,
we found statistical differences in the time to hatching,
especially in tBiH-tSB [GC1] (10.2±4.7 for AH, 18.2±4.3
for ZT and 20.2±4.5 for control; p<0.0001). When analyzing clinical outcomes, implantation rate in AH group
(45.7%) showed to be significantly (p=0.0344) higher than
ZT group (33.3%). No differences were found when both
groups were compared to control (42.2%). Clinical pregnancy rate was significantly higher in AH group vs. control
(77.3% vs. 50.2%; p=0.0152) but no difference was found
between AH and ZT groups (77.3% vs. 65.2%). Ongoing
pregnancy rate was also higher in the study groups: 63.6%
in AH group vs. 38.1% in control group; p¼ 0.0078 and
65.2% in ZT group vs. 38.1% in control group;
p=0.0201. CONCLUSIONS: These preliminary results
suggest that: 1) Assisted hatching using a systematic laser
methodology is safe and does not impair embryo development. 2) Assisted hatching in general, increases the rate of
blastocyst hatching and AH appears to be more efficient
than ZT. 3) AH may help improving implantation rates, however, results must be confirmed in a larger study.
(ZT). L. Herrero,a N. Basile,b J. Garcia Velasco,b N. Costa
Borges,c G. Calderon.c aIVF laboratory, IVI Madrid, madrid,
Spain; bIVI Madrid, Madrid, Spain; cEmbryotools, Barcelona,
Spain.
P-543 Wednesday, October 19, 2016
EMBRYO MORPHOKINETICS AND
OVARIAN
RESERVE.
OBJECTIVE: To compare the effect of two laser assisted
hatching techniques on the blastocyst formation and hatching rates, and clinical outcomes. DESIGN: Retrospective
observational unicenter study. MATERIALS AND
OBJECTIVE: To evaluate the impact of ovarian reserve on
early embryo morphokinetic parameters in a time lapse
Vitrolife
S. Akarsu, F. Gode, B. F. Tamer, A. Z. Isik. In Vitro
Fertilization Centre, Izmir University, Izmir, Turkey.
Abstracts ASRM 2016
13
monitoring system. DESIGN: Retrospective study comparing time lapse monitoring analysis of embryos from three
groups of patients according to the ovarian reserve.
MATERIALS AND METHODS: A total of 200 infertile
couples including patients with diminished ovarian reserve
(Group1; n:41), normal ovarian reserve (Group2; n:62) and
polycystic ovary syndrome(Group3; n:97) were included in
the study.
Morphokinetic parameters of the study groups
The exclusion criteria were male factor, endometriosis and
recurrent implantation failure. All oocytes were fertilized
by intracytoplasmic sperm injection and embryos were
incubated in embryoscope taking images every 20 minutes.
The time from insemination to the following events were
analyzed: pronuclear fading (tpnf), and cleavage to 2,3,4,5
cells. The intervals between two consecutive cleavages
(the duration of second cell cycle (cc2: t3-t2); second
synchrony (s2:t4-t3)) and optimal ranges for morphokinetic
parameters of t5, s2 and cc2 in each group were also evaluated. The results were analyzed using one way anova to
compare timings and chisquare test to compare proportion.
A p-value of less than 0.05 was considered to be statistically significant. RESULTS: The morphokinetic parameters
including time to tpnf, t2, t3, t4, t5, cc2 ,s2, cc3, t5-t2 were
not statistically different between groups (p>0.05) (Table).
Data was analyzed according to different age groups including patients under and over 35 years. The morphokinetic parameters were not statistically different in patients. with
different age groups (p>0.05). The percentage of optimal
embryos according to t5 , s2 and cc2 were not statistically
different between groups(p>0.05) CONCLUSIONS: The
ovarian reserve status does not seem to affect the embryo
morphokinetic paramaters.
P-552 Wednesday, October 19, 2016
FOLLICLE SIZE AND
SYNCHRONICITY OF FOLLICULAR
DEVELOPMENT INFLUENCE
MORPHOKINETIC VARIABLES IN
HUMAN EMBRYOS.
S. Kahraman,a C. Pirkevi Cetinkaya,a M. Cetinkaya,a M.
Montag.b aAssisted Reproductive Technologies and
Reproductive Genetics Center, Istanbul Memorial Hospital,
Istanbul, Turkey; aIlabcomm GmbH, Sankt Augustin,
Germany.
14
Abstracts ASRM 2016
OBJECTIVE: To determine the influence of follicular size
(large and small) in synchronous and asynchronous follicle
development during stimulation on morphokinetic parameters of embryo development. DESIGN: This prospective
cohort study was conducted in a private IVF clinic between
July 2014 and September 2015. Strict inclusion criteria
(< 2 previous treatment cycles, age<_39 years,_>8
Cumulus Oocyte Complexes (COCs)) and exclusion
criteria (PGD or PGS indication, > 24 COCs retrieved
during pickup) were applied. Morphokinetic analyses were
performed only for correctly fertilized oocytes achieving the
blastocyst stage (n=1217) derived from 187 infertile patients. MATERIALS AND METHODS: Synchronous cycles
were defined as follicles of all sizes being present, whereas
asynchronous cycles were those clearly separated into a
small and a large cohort. Small follicles were defined
as <17mm on OPU day. Culture was performed in
EmbryoScope at 6% CO2 and 5% O2 using a single-step
culture medium with change on day 3. Morphokinetic
variables for all cleavage events up to the expanded blastocyst stage were annotated. Embryo selection was done
according to morphology. RESULTS: Embryos developing from small follicles were found to achieve all cleavage
times from 5-cell stage up to expanded blastocyst stage
earlier than those developing from large follicles. At the
end of the culture, when median tB values were compared,
blastocysts from small follicles developed 1.8h faster than
those from large follicles (p=0.0036). Next we looked at
the timings of embryos with known implantation data (KID).
For implanting blastocysts (KID positive) a statistically significant difference was observed for tB of small and large
follicles developed in asynchronous cycles (p=0.0012).
In asynchronous cycles, blastocysts from small follicles
were 2.67 h faster than those from large follicles. Also,
implanted embryos deriving from small follicles developed
in asynchronous cycles were faster than those developed in
synchronous cycles (tB=103.33h vs tB=104.63h, respectively). Although KID positives deriving from large follicles
developed from synchronous cycles had the same tB as
embryos deriving from small follicles developed in asynchronous cycles (tB=103.47h vs tB=103.33h, respectively), implanted blastocysts coming from large follicles and
developed in asynchronous cycles were clearly the slowest
with tB=107.83h. CONCLUSIONS: Constructing optimized morphokinetic algorithms for a specific centre needs
to take into account further variables such as follicular size
and synchronicity. This is of uttermost importance if the final
goal is to use models for identifying embryos for an elective
single embryo transfer. Supported by: Supported by the
Merck Serono Grant for Fertility Innovation (GFI 2014).
P-559 Wednesday, October 19, 2016
CAN BLASTOCYST EXPANSION
MORPHOKINETICS BE USEFUL IN
SELECTING A SINGLE EMBRYO?
A RETROSPECTIVE STUDY OF
Vitrolife
DOUBLE BLASTOCYST TRANSFERS
IN DONOR EGG BLASTOCYST
RECIPIENTS.
T. T. Huang,a T. Kosasa,a H. J. Ahn,b B. Kessel.a aOB/
GYN, University of Hawaii School of Medicine, Honolulu,
HI; bOffice of Biostatistics and Quantitative Health Science,
University of Hawaii School of Medicine, Honolulu, HI.
OBJECTIVE: The purpose of the study was to describe
and to compare the morphokinetics of blastocyst expansion in double embryo transfers resulting in either sustained
singleton or twin pregnancies. DESIGN: A retrospective
descriptive study designed to control for confounding
factors of endometrial receptivity and/or embryo transfer
difficulties. MATERIALS AND METHODS: This study compared 64 patients (32 pairs of blastocysts) having a 100%
implantation rate (IR) with 52 patients (26 pairs) having a
50% IR defined as having either two or one heartbeat
at 7 weeks gestation. After ICSI, all embryos were cultured
continuously in an Embryoscope until D5 transfer. Hourly
cross sectional area (CSA) measurements were retrospectively measured over 12 hours beginning from the time of
blastocysts formation (Tb). Embryo pairs were further stratified into ‘‘fast’’ or ‘‘slow’’ subgroups as follows: ‘‘100% IR
fast’’, ‘‘100% IR slow’’, ‘‘50% IR fast’’, and ‘‘50% IR slow’’.
Their expansion rates were compared at 4, 8, and 12 hours
from Tb (Kenny et al., 2006). RESULTS: Slope comparisons between the four groups over the 3 time intervals
resulted in several patterns having potential clinical implications: 1) The rank order of slope values (from highest to
lowest) was consistent at all 3 time intervals: ‘‘100% IR
fast’’, ‘‘50% IR fast’’, ‘‘100% IR slow’’, and ‘‘50% IR slow’’.
2) Comparisons of the stratified ‘‘fast’’ and ‘‘slow’’ embryos
within either the ‘‘100% IR’’ or the ‘‘50% IR’’ groups always
showed significant expansion slope differences beginning at 6 hours (P< 0.01) 3) Comparisons between the
‘‘fast’’ and ‘‘slow’’ embryos from either the ‘‘100% IR’’ or
the ‘‘50% IR’’ group showed a significant expansion slope
difference between only the ‘‘100% IR fast’’ and ‘‘50% IR
slow’’ (P< 0.002). 4) The expansion slope curve for the
‘‘50% IR slow’’ group was always outside of the range
defined by the ‘‘100% fast’’ and ‘‘100% slow’’ groups; in
contrast, that curve for the ‘‘50% IR fast’’ group was alwaysinside of that range. These results were used to define a
range of average expansion rates for implanting donor egg
blastocysts from the 100% IR group (e.g. 822-1036 u2 /
hr at 8 hours). The ‘‘fast’’ embryo expansion rates within the
50% IR group was always within this range (899 u2 /hr)
while the ‘‘slow’’ embryo rates in this 50% IR group were
outside of that range (657 u2 /hr). CONCLUSIONS: These
results first describe blastocyst expansion morphokinetics over the first 12 hours for egg donor blastocysts and
compared rates in implantations that form one versus two
sustained ongoing heartbeats. In pregnancies with a 50%
implantation rate, trailing embryos expanded at a rate outside that of those having positive implantation, while their
leading embryos expanded within the range having known
positive implantation. The results are consistent with an
Vitrolife
optimal expansion rate range and time to choose a single
embryo for transfer.
P-562 Wednesday, October 19, 2016
KINETICS OF THE EARLY IN
VITRO DEVELOPMENT OF HUMAN
HAPLOID ANDROGENOTES.
L. Escrich,a N. Grau,a Y. Galiana,a M. F. Insua,a J. Remohi
Gimenez,b M. Escriba.a,c aIVF Laboratory, University
Institute IVI Valencia, Valencia, Spain; bIVI Valencia,
Valencia, Spain; cIVI- Fundation, Valencia, Spain.
OBJECTIVE: To describe the early development of human
haploid androgenotes and to compare it to that of correctly
fertilized (control) embryos. DESIGN: Kinetic description of
the early in vitro development of 16 haploid androgenotes
compared with that of 20 control embryos. MATERIALS
AND METHODS: Androgenotes were produced by in vitro
fertilization of enucleated MII oocytes. Enucleation was
performed using PolScope technology. Once the spindle
had been identified, it was removed by aspiration using an
ICSI pipette. Ooplasts were then microinjected according
to the ICSI procedure. Data from control embryos were retrospectively collected from infertile couples enrolled in our
ovum donation program. In all cases, oocytes were cryopreserved by vitrification. Androgenotes and control embryos
were cultured in a time-lapse incubator for 3 days. The
following kinetic variables were evaluated: timing of cleavage from the 2- to 8-cell stage, and duration of the second
(cc2) and third cell cycles (cc3). RESULTS: Timings of
cleavage to the 2-,3-, 4-, 5-, 6-, 7-, and 8-cell stage were
statistically comparable in androgenotes and control embryos. In relation to indirect variables, cc2 was comparable
in both groups (averaged cc2: 11.9±1.2h; 95CI: 11.512.3h), while cc3 lasted longer in control embryos than in
androgenotes (15.5±4.5 hrs vs. 17.7±3.6hrs; p<0.05).
CONCLUSIONS: The kinetics of haploid androgenotes
are comparable to those of correctly fertilized biparental
embryos during the second cell cycle,
but not the third cell cycle. Supported by: IU-IVI Valencia.
P-578 Wednesday, October 19, 2016
SPINDLE POSITION AND SECOND
POLAR BODY ORIENTATION
ENABLES THE PREDICTION OF
EMBRYONIC DEVELOPMENTAL
POTENTIAL AFTER ICSI.
S. Kim, J. Eum, W. Y. Choi, S. Paek, S. Kwon, J. Kim, R.
Kim, Y. Hur, T. K. Yoon, W. Lee, D. Lee. Fertility Center,
CHA Gangnam Medical Center, College of Medicine, CHA
University, Seoul, Korea, Republic of.
OBJECTIVE: Previous studies have shown that the angle of
spindle is associated with fertilization and embryo development. Also, second polar body (2nd pb) orientation was
related to the early cleavage which affects the embryo development. However, the correlation between spindle and
Abstracts ASRM 2016
15
2nd polar body orientation was unknown. In present study,
we investigated the correlation between the angle of
spindle and the angle between the PN axis and the 2nd
pb, and the effect of such correlation on embryo quality.
DESIGN: This study was performed from February 2016 to
April 2016 in the Fertility center of CHA Gangnam Medical
Center. We analyzed 150 matured oocytes from 27 patients. The metiotic spindles were assessed by Polscope
before intracytoplasmic sperm injection (ICSI). Injected
oocytes were cultured individually in continuous single
culture medium (CSC medium, Irvine scientific, CA). After
16-18 hours, we measured the angle between the PN axis
and the 2nd PB. Then, the early cleavage was checked on
day 2 and embryo development was checked on day 3.
MATERIALS AND METHODS: After checking the angle
of spindle in mature oocytes, the oocytes were divided
into five groups according to the angle of spindle deviation from the PB position (0-5º, 6-15º, 16-45º, >46º and
non-visible). And then, the angle between the PN axis
and 2nd pb position was measured in pronuclear zygotes.
RESULTS: The angle between the PN axis and the 2nd pb
was increased with increasing angle of spindle (0-5º, 15.4º
; 6-15º, 17.9º; 16-45º, 22.8º;>46º, 77.1º). Also, the rate of
early cleavage and good quality embryos has declined with
increasing angle of spindle and angle between the PN
axis and the 2nd pb. However, non-visible oocytes showed
a lower incidence of the angle between the PN axis and
the 2nd pb (49.3º versus 77.1º) and higher incidence of
the rate of early cleavage (32.0% versus 14.3%) and good
quality embryos (32.0% versus 14.26%) as compared with
>46_ oocytes. To note that there was a tendency to be related to the rate of good quality embryos with embryo score
of Embryoscope. CONCLUSIONS: A significant relationship was examined between the angle of spindle and the
angle between the PN axis and the 2nd pb in the oocytes.
In addition, these correlated angles were associated with
embryo quality. Thus, this observation may give a new
indicator which can be used to predict the developmental
fate of embryos. Supported by: Grant of the Korea Health
Technology R&D Project through the Korea Health Industry
Development Institute (KHIDI), funded by the Ministry of
Health & Welfare, Republic of Korea (HI12C0055).
P-642 Wednesday, October 19, 2016
TROPHECTODERM (TE) CELL
TRANSPLANTATION: A FEASIBLE
TECHNIQUE TO IMPROVE
BLASTOCYST
QUALITY.
O. Perez,a H. Adriaanse,a G. R. Navarrete,a B. Tilley,a A.
Patel,a R. Gada,b K. L. Lee,b L. Lawrence,b M. R. Thomas,b
S. J. Chantilis.b aDallas Fertility Center, Dallas, TX; bDallasFort Worth Fertility Associates, Dallas, TX.
OBJECTIVE: The in vitro proliferative capacity of human TE
cells has been previously reported to be successful1. The
objective of this research is the successful transplantation
16
Abstracts ASRM 2016
and rapid proliferation of TE cells in blastocysts with low
numbers of TE cells. DESIGN: Research ongoing study.
MATERIALS AND METHODS: Patients consented the
use of non-viableday-6 embryos for the same cohort with
varying TE quality. Embryos designated as non-viable were
graded to be poor quality embryo not suitable for transfer or
freezing by non-study embryologists. A total of 11 TE transplantations were performed. Two different blastocysts were
selected for each transplantation: one with a high number
of TE cells (Donor) and one with a low number of TE cells
(Receptor). ATE biopsy was performed to remove approximately 10 TE cells. Subsequently, Receptor blastocysts
received these Donor TE cells. This procedure consisted
on making a 30 mm holeon the zona pellucida. TE cells
were placed gently inside Receptor blastocyts.After TE cell
transplantation, embryos were cultured individually with
blastocyst culture media (G2 Plus. Vitrolife) in a time-lapse
incubator (EmbryoScope) for approximately 22 hours. Each
receptor transplanted blastocyst was monitored by viewing time-lapse videos to verify the characteristics of the
cell attachment and proliferation. RESULTS: TE cells were
successfully introduced inside Receptor blastocysts. Donor
TE cells were not rejected by any recipient embryos. Cell
attachment was verified within 2 hours of incubation. After
cell attachment, transplanted cells proliferated to varying
degrees in the blastocysts. Approximately 50% of the transplanted blastocysts completely hatched with strong cell
adhesion. One of the transplanted embryos showed two
defined TE cell masses with no attachment between TE
areas. 10 out of 11 (91%) Recipient blastocysts showed
attachment and cell proliferation merging two different
sources of TE cells into a single well defined trophectoderm. CONCLUSIONS: TE cell number was increased in
Receptor trophectoderm when two sources of TE cells
were conjoined after TE transplantation. Proliferation of
TE cells improved the grade of Receptor blastocysts. We
hypothesized that TE proliferation on blastocysts could
possibly improve blastocyst cell trophectoderm morphology in which the development of TE is enhanced for robust
implantation. In this ongoing study, more transplantations
and genetic analyses of this merging layers of TE cells need
to be conducted to elucidate the effects on the Receptor
blastocysts.
P-647 Wednesday, October 19, 2016
A NEW APPROACH TO EVALUATE
THE INFLUENCE OF ADVANCING
MATERNAL AGE UPON MEIOTIC
SPINDLE MORPHOLOGY AND
DEVELOPMENTAL COMPETENCE IN
HUMAN OOCYTES.
R. Matsunaga,a S. Watanabe,a M. Miura,a Y. Kobayashi,a
N. Yamanaka,a M. Kamihata,a A. Kuwahata,a M. Ochi,a
T. Horiuchi.a aOchi Yume Clinic Nagoya, Nagoya, Japan;
a
b Department of Life Science, Prefectural University of
Hiroshima, Shobara, Japan.
Vitrolife
OBJECTIVE: The developmental competence of oocytes/
embryos decreases with advancing maternal age, and
meiotic errors gradually increase from when a woman
reaches her late 30s. The evaluation of meiotic spndle
morphology using a Polscope is an effective method for
investigating the effect of maternal age. However, very little
is known about the relationship between meiotic spindle
morphology in oocytes and their developmental competence as maternal age advances. DESIGN: Retrospective
study. MATERIALS AND METHODS: We analyzed 1496
mature oocytes from 461 patients undergoing letrozole
stimulation cycles between September 2013 and January
2016. Oocyte spindle morphology was analyzed prior to
intracytoplasmic sperm injection (ICSI) using a Polscope.
We divided oocytes into two groups according to patient
age: under 39 years (young, n=408) and over 39 years
(old, n=945). We compared spindle abnormality and type
(non visible, translucent, rotated, irregular shape) between
young and old groups. We also analyzed whether abnormal spindles affect fertilization rate, embryo developmental
competence, and embryo morphokinetics (time to pronuclear fading and the two cell stage from ICSI [tPNf and t2,
respectively] and abnormal cleavage rate of the first cleavage) in each group. Embryo morphokinetics were analyzed
using a time-lapse incubator (EmbryoScope). RESULTS:
There was no significant difference in the total rate of abnormal spindles between the young and old groups (8.8%
vs. 8.4%). However, the number of oocytes whose spindles
were not vertical to the plane of the oocyte’s equator was
increased in older oocytes (young vs. old: 2.5% vs. 5.0%).
In the older group, abnormal fertilization rates (maltipronuclear and monopronuclear) of oocytes with a nonvisible
spindle (38.5%) were significantly higher than oocytes with
a normal spindle (5.9%). Embryo morphokinetics were not
significantly different when compared between oocytes
with normal or abnormal spindles. Oocytes with abnormal
spindles were more prone to developmental arrest before
the 8-cell stage than oocyte with a normal spindle. In the
older group, blastocyst formation rates of oocytes with
abnormal spindle (22.6%) were significantly lower than
oocytes with normal spindles (55.6%). CONCLUSIONS:
Abnormal spindle location and morphology were more
prevalent in oocytes of advancing maternal age. We suggest that the increased incidence of abnormal spindle
is associated with a reduction in embryo developmental
competence.
P-648 Wednesday, October 19, 2016
TIME-LAPSE OBSERVATION CAN
HELP IMPROVE WORKFLOW
AND ENSURE THE CORRECT
OBSERVATION OF FERTILIZED
EMBRYOS.
R. Suzuki, H. Watanabe, H. Hasegawa, K. Tsukamoto,
M. Kobayashi, T. Kyoya, S. Saito, J. Kobayashi. Kanagawa
Ladies Clinic, Kanagawa-ken, Japan.
Vitrolife
OBJECTIVE: In recent years, there have been some reports
suggesting that embryos are evaluated as no pronuclei and
two polar bodies (0PN2PB) using conventional microscopic observation but the embryos observed by time-lapse
shows that these 2PN break down before the time when
we normally observe (2PN-BD). However there are very
few articles discussing the process and impact from such
PN breakdown.Here, we report whether we can reduce the
risk for incorrect observation of embryos following insemination by using time-lapse technology. DESIGN: From July
2014 to September 2014, 448 cycles with 2338 oocytes
were cultured for 6h following conventional IVF (cIVF) or
ICSI and observed at 20h after insemination in Embryo
Scope (Vitrolife)then we cultured the embryos into moist incubator until Day5.We used Veeck’s criteria and Gardner’s
criteria for embryo evaluation. Treatments used TESE-ICSI
and rescue-ICSI and calcium ionophore were excluded.
MATERIALS AND METHODS: We calculated the ratio for
which embryos were evaluated as 0PN2PB by conventional
microscopic observation but showed 2PN by time-lapse
observation. We also studied the cleavage rate, Day3 good
quality embryo rate, Day5 blastocyst rate, and Day5 good
quality blastocyst rate of these embryos. Furthermore, we
analyzed the time from PN appearance to its breakdown.
All statistical analyses were performed using chi squared
analysis. RESULTS: Among 281 embryos which were
evaluated as 0PN2PB using conventional microscopic
observation, 141 embryos were evaluatedas 2PN by timelapse observation. The cIVF group has 2PN-BD embryos
of 33.6% (38/113) and the ICSI group has the embryos
of 61.3% (103/168). The 2PN-BD embryos show same
development compared 2PN embryos in cIVF group.
However, in the ICSI group, Day3 good quality rate, Day5
blastocyst rate and Day5 good quality blastocyst rate were
significantly higher in the 2PN-BD group (P<0.01). For the
1825 embryos which were evaluated as 2PN by time-lapse
observation, the time from insemination to 2PN appearance was as follows; 8.6h±0.1h (5.5h-19.4h) for the cIVF
group, and 6.9h±0.1h (3.6h-22.3h) for the ICSI group. The
embryos which developed into 2PN laterthan 15 hours following insemination did not grow into good quality
blastocysts. Finally, the time of 2PN-BD was 15.9h
(ICSI) and 17.2h (cIVF) following insemination at earliest.
CONCLUSIONS: It is very difficult to get the information
of correct fertilization without using time-lapse system,
because there are some embryos whose pronuclei break
down early. Time-lapse technology can also help us improve workflow in the lab since actual observation does not
need to be done at a specific time.
P-653 Wednesday, October 19,
2016 BLASTOCYST METABOLISM,
AS DETERMINED BY A NOVEL
QUANTITATIVE APPROACH, IS NOT
IMPACTED BY CHROMOSOME
COMPLEMENT OR GENDER BUT IS
ALTERED WITH MATERNAL AGE.
Abstracts ASRM 2016
17
T. Schlenker,a A. Greene,a S. Lyons,b J. M. Stevens,a
J. Herrick,a J. Prenni,b J. Kirkwood,b C. Broeckling,b W.
B. Schoolcraft,a R. L. Krisher.a aColorado Center for
Reproductive Medicine, Lone Tree, CO; b Colorado State
University, Fort Collins, CO.
OBJECTIVE: To determine if the metabolic footprint of
single human day 5 blastocysts is associated with chromosome complement, gender, or maternal age. DESIGN:
Retrospective analysis. MATERIALS AND METHODS:
Individual embryos were cultured to the blastocyst stage in
25 µL of CCRM (in house prepared sequential) medium in
the EmbryoScope . Only medium collected from good quality blastocysts (grade 3BB and better) on D5 was included
in the analysis, following biopsy for comprehensive chromosome screening and vitrification. An internal standard
containing 20 isotopically labeled substrates was added
to all medium samples. A zwitterionic polymeric hydrophilic
interaction liquid chromatography (ZIC-pHILIC) column
was used to separate polar metabolites after MTBE extraction, followed by electrospray ionization (ESI) prior to triple
quadropole mass spectrometer (TQ-S) coupled to LC-MS/
MS. A standard curve was used for absolute quantitation of
each compound (Skyline software). Metabolic activity was
defined as the difference between medium with and without
an embryo. RESULTS: Embryos were derived from 16 patients (age 39.20.6 years, range 35-43). Medium samples
were analyzed from 20 euploid (13 female (F), 7 male (M))
and 20 aneuploid (6 F, 9 M) good quality D5 blastocysts.
None of the 29 metabolites quantified (including 21 amino
acids, glucose, lactate, pyruvate, citrate, alanyl-glutamine,
myo-inositol, ornithine and citrulline) differed significantly
in uptake or production between euploid and aneuploid
blastocysts, nor between male and female embryos, over
the 48 hour time period between D3 and D5. In contrast,
multiple differences in amino acid metabolism were related
to maternal age. A significant relationship exists between
maternal age and blastocyst metabolism of alanyl-glutamine, aspartic acid, methionine, phenylalanine and threonine. These amino acids all exhibited similar patterns of
metabolic change with age, in which embryos from women
35-37 produced the amino acid, embryos from women 3840 took up the amino acid, and embryos from women 4143 were intermediate in amino acid uptake. For example,
for aspartic acid (p<0.009), blastocysts in the 35-37 age
group produced 122.0±87.0, in the 38-40 age group they
took up 155.4±34.2 and in the 41-43 age group they took
up 15.0±75.4 pmol/embryo/48 hours. CONCLUSIONS:
Metabolism of these 29 analytes does not differentiate
embryos based on chromosome complement or gender.
However, maternal age has a significant impact on embryo
metabolism of amino acids. Further research is necessary to determine the impact of these changes on embryo
implantation potential.
P-655 Wednesday, October 19, 2016
BLASTOMERE EXTRUSION AND
ABNORMAL CLEAVAGE BEHAVIOR
18
Abstracts ASRM 2016
IN HUMAN EMBRYOS UNDER
TIME-LAPSE MONITORING:
POSSIBLE WAY OF EMBRYO
“SELFCORRECTION”?
N. Zaninovic, Q. Zhan, C. Norberg, Z. Ye, R. Clarke, Z.
Rosenwaks. Center for Reproductive Medicine, Weill
Cornell Medicine, New York, NY.
OBJECTIVE: To investigate the correlation between blastomere extrusions and abnormal cleavage behavior (direct
uneven cleavage (DUC) and karyokinesis without cytokinesis (NUK)) in human preimplantation embryos. DESIGN:
A retrospective analysis of time-lapse and PGS data.
MATERIALS AND METHODS: In this study, 5300 embryos from 624 preimplantation genetic screening (PGS)
cycles underwent blastocystbiopsy between January and
December 2015. Blastomere extrusions (blastomeres that
did not participate in embryo proper during blastulation),
DUCS, and NUKs were identified by time lapse monitoring
(EmbryoScope, Vitrolife, Sweden). Biopsied blastocysts
(BL) were testedby aCGH or SNP array and results were
classified as euploid (EUP),aneuploidy (ANU) or complex
abnormal (CxA). Data was analyzed usingthe c2 test.
RESULTS: The overall incidence of blastomere extrusions
in PGS embryos was 16.5% (877/5300), which were significantly lower in embryos of women greater than or equal
to age 42 (14.1%, 165/1170) and higher in age group
35-37 and 38-40 (18.2 % (163/895) - 18.5% (222/1200),
respectively). The extrusion incidences were significantly
higher in BLs exhibiting DUCS (66.8%, 152/232) and
NUKs (50.3%, 81/161)when compared to BLs without
abnormal behavior (8.9% (171/1913),all p<0.0001). The
same trends were confirmed among all oocyte age groups.
DUC Daughter blastomere extrusions occur in 85.2%
(132/155) of DUC BLs. Similarly, NUK daughter blastomeres were extruded in 53.1% (43/81) of all NUK BLs.
The total blastomere extrusion rate in all eupoloid blastosysts was significantly higher in both DUCS BLs(69.2%,
83/120) and NUKs BLs (41.6%, 32/77) compared to
BLs without abnormal behavior (10.2% (97/955), all
p<0.0001). In eupolidembryos, DUCS daughter blastomeres were extruded in 85.5% (71/83) of DUCS BLs and
NUKs daughter blastomeres were §extruded in 43.8%
(14/32) of NUKs BLs. CONCLUSIONS: The incidence of
blastomere extrusion was elevated in embryos exhibiting
abnormal cytokinesis and karyokinesis. Euploid blastocysts
derived from these embryos had a higher incidence of
extrusions, especially daughter blastomeres resulting from
abnormal cleavage behavior, suggesting a possible mechanism of embryo self-correction.
P-656 Wednesday, October 19, 2016
EARLY STAGE EMBRYOS THAT HAVE
BEEN ABNORMALLY CLEAVED STILL
PRODUCE GOOD IMPLANTATION
OUTCOMES AND SUCCESSFUL
Vitrolife
PREGNANCIES, BUT ONLY IF THEY
DEVELOP INTO BLASTOCYSTS.
Y. Kobayashi,a R. Matsunaga,a M. Miura,a N. Yamanaka,a
M. Kamihata,a S. Watanabe,a A. Kuwahata,a M. Ochi,a
T. Horiuchi.b aOchi Yume Clinic, Nagoya, Japan; bDepartment of Life Science, Prefectural University of Hiroshima,
Shobara, Japan.
OBJECTIVE: Although many studies have used dynamic
analysis of embryo cleaving, using a time-lapse embryo
monitoring system, to report low developmental potency in
abnormally cleaved early stage embryos, and that these abnormal embryos can occasionally develops into blastocysts,
there are few reports on implantation outcomes and pregnancy prognosis using such embryos.We measured the effect of cleaving on early stage embryos using a time-lapse
embryo monitoring system. We report implantation outcomes and pregnancy prognosis. DESIGN: Retrospective
study. MATERIALS AND METHODS: We collected eggs
from 378 women (treatment cycle: 488, average age: 40.1)
between January 2013 and June 2013 and offered blastocyst culture and dynamic analysis of 1217 embryos. We
cultured embryos and then monitored their development
using EmbryoScope (Vitrolife). We classified embryos into
those: dividing first into three cells or more (AC1 group),
dividing first into two cells, then five or more (AC2 group),
and dividing first into two cells, then four (NC group).
Successful blastocysts were frozen, and a single freeze
thaw embryo transfer was performed. Blastocyst development, pregnancy, and miscarriage rates were compared
between the three groups. RESULTS: Successful blastocyst development rates were 11.5% (22/192, AC1 group),
23.2% (33/142, AC2 group), and 53.2% (470/883, NC
group). There was a significant difference between the
groups (P<0.05). Pregnancy rates were 33.3% (4/12, AC1
group), 41.2% (7/17, AC2 group), and 48.5% (141/291,
NC group), and miscarriage rates were 50.0% (2/4, AC1
group), 28.6% (2/7, AC2 group), and 34.8% (49/141,
NC group). Pregnancy, and miscarriage rates showed no
significant difference between the groups. Birth defects
and deformities were not observed. CONCLUSIONS:
Abnormally cleaved embryos seem inappropriate for early
embryo transfer because they result in a low rate of blastocyst development. However, once they have developed into
blastocysts, they result in good implantation outcomes and
successful pregnancies. Therefore, we suggest that they
should be considered as potential embryo transfer candidates.
P-657 Wednesday, October 19,
2016 EVALUATION OF EARLY
CYTOKINETIC TIMEPOINTS BY
TIMELAPSE MICROSCOPY.
G. F. Celia, K. J. Fresa, T. F. Chi, D. J. Kotze, S. Bocca, S.
Oehninger. Ob/Gyn, The Jones Institute for Reproductive
Medicine, Norfolk, VA.
Vitrolife
OBJECTIVE: To use Time-Lapse Microscopy (TLM) to
identify early cytokinetic time-points indicative of implantation potential. DESIGN: Retrospective Data Analysis.
MATERIALS AND METHODS: 68 patients (average age
33.6 4.9 years), retrieved from Jan 2015-Mar 2016, underwent IVF with ICSI, TLM culture (Embryoscope, Goteborg,
Sweden), and embryo transfers resulting in either 100%
(Preg: 17) or 0% (NP: 52) implantation. These patients
were selected as the fate of all transferred embryos was
known. 129 embryos were transferred on day 3 (65) or
day 5 (64) at the discretion of the physician. TLM records
were reviewed to determine the timing of the first through
third cytokinesis. The timing of divisions relative to fertilization and each other was compared between implanting
and non-implanting embryos. RESULTS: Review of TLM
revealed an average time to first cytokinesis of 28.1 ±3.2
(NP) and 27.5±2.7 (Preg) hrs post ICSI. Comparisons
between the groups did not reveal a significant difference in timing (P=0.33). Time to second (37.9 ±3.2 NP
vs 27.5±2.7 Preg) and third cytokinesis (28.1±3.2 NP
vs 27.5±2.7 Preg), typically 2-cell to 3-cell and 3-cell
to 4-cell, similarly did not show any difference between
groups (P=0.56 and P=0.523, respectively). In 34 cases,
the embryos appeared to cleave directly from 2 to 4 cells,
although the shutter speed (20 minutes) limited interpretation of these events. These embryos were found to have
a significantly greater implantation potential (odds ratio
= 2.59, P = 0.043). A final analysis of the time between
1st and 3rd cytokinesis in each group was made. Raw
data showed no difference between groups (P = 0.347),
however further investigation revealed complete implantation failure in embryos requiring >15 hours to complete
these divisions. All significance was interpreted at P<0.05.
CONCLUSIONS: The purpose of this study was to identify
specific time points during earlier embryo cleavage that are
predictive of implantation potential. Although no raw time
points demonstrated such a relationship, the increased implantation potential of embryos with a rapid 2 cell to 4 cell
conversion, and the complete failure of all embryos which
took >15 hours to complete 1st through 3rd cleavages are
potential selection criteria with TLM. Furthermore, the latter
marker may prove useful in laboratories lacking TLM culture,
as minimal observation is required to identify and exclude
these embryos from transfer.
P-658 Wednesday, October 19, 2016
IS ICSI WITH CALCIUM IONOPHORE
AFFECTING HUMAN EMBRYO
DEVELOPMENT?
J. A. Aguilar,a M. Ojeda,a E. Taboas,a M. Perez,a E. Munoz.b
a
IVF Laboratory, IVI Vigo, Vigo, Spain; b Gyneacology, IVI
Vigo, Vigo, Spain.
OBJECTIVE: Artificial Oocyte Activation (AOA) with
Calcicum Ionophore (Ica), has shown to be a successful alternative choice for those patients with previous
total fertilization failure after ICSI. However, the information concerning its impact on the embryo development is
Abstracts ASRM 2016
19
scarce. The aim of this study is to analyze the fertilization,
pregnancy and implantation rates, as well as direct cleavage rate, blastomere multinucleation incidence, and embryo
morphokinetics parameters in patients who underwent ICSI
with AOA using an ICa. DESIGN: Retrospective cohort
study of 65 couples with severe male factor, under 1 mill/
ml spermatozoa. We compared 271 oocytes microinjected
with an Ica from 36 patients who had a previous fertilization rate under 30% in previous ICSI cycles (Ica group), to
232 oocytes from 29 couples with similar sperm characteristics (concentration under 1mll/ml) (control group)
between January 2011 and December 2015 in IVI Vigo
Clinic. MATERIALS AND METHODS: AOA was carried out by microinjecting the oocytes with spermatozoa
in a Ica buffered solution. Then, they were incubated for
twenty minutes in a culture media + Ica solution in a 37oC,
6%CO2 20%O2 atmosphere, and finally cultured in a time
lapse monitoring incubator at 37oC 6% CO2, and 20%O2.
X2, t-Student and Mann-Whitney tests were applied
when suitable for statistical treatment of data. RESULTS:
Fertilization rate was similar among groups, 54,2% (Ica) vs
58,1% (p=0.06). Pregnancy rate was 54.2% in Ica group
vs 58,1% in the control group (p=0.06). Implantation rate
did not show any statistical differences neither. There
were no differences between groups in abnormal fertilization rate (1,3 and 4 pronucleai), although the proportion
was higher in Ica group. The proportion of multinucleated
blastomeres at two and four cells stage between groups
was similar; p=0.8 and p=0.009 respectively, as well as
the direct cleavage rate (p=0,447). Time at which second
polar body was extruded (tPB2), was briefer at Ica group
(p=0.001). CONCLUSIONS: ICSI with AOA by ICa seems
not to affect neither fertilization, pregnancy and implantation
rates, nor the proportion of multinucleated blastomeres, or
direct cleavage. Concerning morphokinetics, only tPB2 is
affected by ICa, what could be related with the resumption
of meiosis and calcium levels within the oocyte.
2015, embryos were cultured in sequential media (SM)
using Quinn’s Advantage (QA) Cleavage Plus media up
to Day 3 and transferred to QA Blastocyst Plus Media up
to Day 6. From September 2015 to April 2016, embryos
were cultured using Sage One-Step Media (OSM) up to
Day 6 with no refreshing of media on Day 3. Embryos for
both groups were incubated in either Embryoscope (ES) or
Planer (PL) incubators. Fertilization rate, blastocyst development on Day 5 and Day 6, and total blastocyst development was compared for both culture media systems.
Blastocyst development was calculated as the percentage
of blastocysts formed from the number of fertilized oocytes
(2pns). These parameters were also compared between
the ES and PL incubation systems. Comparisons of blastocyst development rates were statistically analysed using a
Chi-squared test and adjusted using a Bonferroni correction. RESULTS: A total of 1152 oocytes (73 patients)
were cultured with sequential media and a total of 1035
oocytes (67 patients) were cultured with One Step media.
No differences in patient age or egg maturation rate were
noted in each group. There was a significant increase in the
total blastocyst development rate in OSM compared to SM
(62.2% vs 54.4%; P=0.035). No differences in blastocyst
development were noted between the incubators irrespective of which culture media was used. CONCLUSIONS:
The data indicate that One Step Media significantly
improves the overall blastocyst development rate and is
capable of maintaining culture conditions from fertilization
to blastocyst development in both ES and PL conditions,
when compared to sequential media. The use of One Step
media can greatly benefit the efficiency of the IVF lab, since
it reduces the time for making dishes and changing media
on Day 3, and also eliminating any disturbances to the
embryo during culture.
Blastocyst development in Sequential and One-Step media
using Embryoscope and Planer incubators
P-663 Wednesday, October 19, 2016
BLASTOCYST DEVELOPMENT
USING SEQUENTIAL MEDIA
VERSUS ONE-STEP MEDIA IN
EMBRYOSCOPE AND PLANER
INCUBATORS.
K. Kaskar,a D. P. Hamilton,b K. Miller,b P. W. Zarutskie,a
W. E. Gibbons.a aDepartment Ob/Gyn, Baylor College
of Medicine, Houston, TX; bFamily Fertility Center, Texas
Children’s Hospital, Houston, Texas.
OBJECTIVE: To compare the blastocyst development rate
of embryos cultured in sequential media versus a one-step
media. DESIGN: Retrospective analysis of embryos of patients undergoing in-vitro fertilization treatment over a twoyear period from April 2014 to April 2016. MATERIALS
AND METHODS: Only IVF patients with oocytes inseminated using intra-cytoplasmic sperm injection and whose
embryos were cultured to Day 5 and did not have a Day 3
transfer were included in the study. From April 2014 to July
20
Abstracts ASRM 2016
Vitrolife
P-664 Wednesday, October 19, 2016
IS THE ADDITIONAL INFORMATION
FROM TIME-LAPSE MONITORING
USEFUL IN EMBRYO ASSESSMENT.
J. X. Zhang,a M. Pavone,a A. K. Lawson,b J. Robins.c aObstetrics and Gynecology, Northwestern University Feinburg
School of Medicine, Chicago, IL; bNorthwestern University,
Chicago, IL; cNorthwestern University Feinberg School of
Medicin, Chicago, IL.
OBJECTIVE: Traditional fixed-time evaluation (FTE) of fertilization at 18 hours after fertilization and embryo morphology
on day 3 could miss information on fertilization status if the
pronuclei appear (PNa) after 18 hours or pronuclei fade
(PNf) before 18 hours after fertilization, as well as informationon embryo development before day 3. This study
examined the developmental potential of embryos with
PNa greater than 18 hours or PNf less than 18 hours after
fertilization, and of embryos with irregular division (ID) or
with multinucleated blastomeres (MN) during days 1 and 2
using time lapse monitoring (TLM). This information would
have been missed when using traditional FTE. DESIGN:
Retrospective cohort. MATERIALS AND METHODS: The
study included all embryos that were cultured in incubators
equipped with TLM capabilities (EmbryoScope, Vitrolife)
at an urban academic IVF center between August of 2015
and February of 2016. The developmental potential of embryos with PNa >18 hours or PNf <18 hours after fertilization as well as embryos with ID (1 cell directly divided into
3 or 4 cells) or MN (at least blastomere was multinucleated) before day 3. Developmental potential was defined
as development to 8-cell stage on day 3, blastocyst by
day 6, and implantation rate after transfer of one embryo
or two embryos with identical classification. Differences
were analyzed by a Chi-squared test (Fisher’s exact test).
RESULTS: A total of 1599 embryos from 291 cycles of
IVF-ET were analyzed. The mean age of the patients was
36.2 years old with a std of 4.1. The overall clinical pregnancy (confirmed by ultrasound observation) rate was 41%.
Very few embryos were observed to have their PNa after
18 hours of fertilization (n=5) and none of these embryos
developed to 8-cell stage by day 3 or blastocyst stage by
day 6. None of these embryos were selected for transfer. PNf before 18 hours after fertilization occurred in 25
embryos (1.6%). These embryos’ developmental potential
was reduced by at least 50% (p<0.01), compared with
embryos with PNf>18 hours (8-cell development: 32% vs
65%; blastocyst development: 20% vs 55%, and implantation rate: 14% vs 28%). Embryos with ID or MN during
days 1 and 2 had much poorer developmental potential
compared with embryos without these anomalies (8-cell
development: 13% vs 77%, p<0.001; blastocyst development: 4% vs 67%, p<0.001; and implantation rate: 5% vs
29%, p<0.05). CONCLUSIONS: Compared to traditional,
Vitrolife
fixed-time evaluation (FTE) of in-vitro-produced embryos,
time-lapse monitoring (TLM) can generate additional
information on embryo development that FTE may miss.
Compared to TLM, FTE may mis-read fertilization status on
a small percentage of embryos that have limited developmental potential. Information on abnormal development
during the first two days in culture can assist in the identification of those embryos that have very poor developmental
potential.
P-667 Wednesday, October 19, 2016
A NOVEL, HIGHLY SENSITIVE
TANDEMMASS SPECTROMETRY
METABOLOMICS APPROACH
PREDICTS OUTCOME OF POOR
QUALITY DAY 5 BLASTOCYSTS.
R. L. Krisher,a S. Lyons,b A. Greene,a J. M. Stevens,a J.
Herrick,a J. Kirkwood,b J. Prenni,b C. Broeckling,b W. B.
Schoolcraft.a aColorado Center for Reproductive Medicine,
Lone Tree, CO; bColorado State University, Fort Collins,
CO.
OBJECTIVE: To investigate whether the metabolic footprint
of single human blastocysts can differentiate between good
quality day 5 blastocysts and poor quality day 5 blastocysts
that are or are not capable of producing a good
quality blastocyst on day 6. DESIGN: Retrospective
analysis MATERIALS AND METHODS: Individual embryos
were cultured to the blastocyst stage in 25 µL of CCRM (in
house prepared sequential) medium in the EmbryoScope.
Medium samples (n=88) were collected on day 5 following 48 hours of culture from wells containing a good quality
(grade 3BB and better, vitrified/transferred) or poor quality
(assessed again on day 6) blastocyst. Control medium
from wells without an embryo in the same dish were also
collected. An internal standard containing 20 isotopically
labeled substrates was added to all samples. A zwitterionic
polymeric hydrophilic interaction liquid chromatography
(ZIC-pHILIC) column was used to separate polar metabolites after MTBE extraction, followed by electrospray
ionization (ESI) prior to triple quadropole mass spectrometer (TQ-S) coupled to LC-MS/MS. A standard curve was
used for absolute quantitation of each compound (Skyline
software). Metabolic activity was defined as the difference between the concentration of each metabolite in
medium with and without an embryo. Data was analyzed
using ANOVA (NCSS 10) and Fisher’s LSD MCT when
P<0.05. RESULTS: Patients (n=19; 39.5±0.6 years)
produced an average of 4.6±0.7 blastocysts (range 1-10).
Each blastocyst was categorized as good quality day 5
(G; n=41), poor quality day 5/good quality day 6 (PG;
n=20) or poor quality day 5/poor quality day 6 (PP; n=27).
Uptake or production of 29 metabolites was quantified,
including 21 amino acids, glucose, lactate, pyruvate,
citrate, alanyl-glutamine, myo-inositol, ornithine and citrulline. The metabolic footprints of G and PG blastocysts
were indistinguishable on day 5. However, PP blastocysts
Abstracts ASRM 2016
21
produced more glutamine (p=0.0001) and alanine
(p=0.0455) than either G or PG blastocysts (glutamine;
G=2413.0_143.6, PG=2702.5_239.5, PP=4194.5_505.3
pmol/embryo/48 hrs: al nine; G=2983.3_281.6,
PG=2870.5_351.6, PP=4138.0_502.9 pmol/embryo/48
hrs). CONCLUSIONS: Using this novel metabolomics
platform, we are able to accurately and sensitively quantify
the uptake and production of 29 metabolites by single human blastocysts. This analysis revealed significant metabolic differences in poor quality blastocysts on day 5 that
are related to embryo competence, but that could not be
determined by morphology alone.
P-680 Wednesday, October 19, 2016
EMBRYO KINETICS AND
IMPLANTATION RATE BY TIMELAPSE MICROSCOPY.
L. Yang, M. Peavey, K. Kaskar, K. Miller, C. T. Valdes, T. L.
Woodard, P. W. Zarutskie, W. E. Gibbons. Obstetrics
and Gynecology, Baylor College of Medicine, Houston, TX.
OBJECTIVE: Non-invasive time-lapse microscopy is
used to monitor embryo development in culture to assist in embryo grading and transfer selection. Prior studies demonstrate conflicting results as to whether embryo
morphokinetics is predictive of blastulation or implantation.
This study examines the relationship between embryo morphokinetics and implantation rate with patient demographics during the initial phase of time-lapse imaging. DESIGN:
Retrospective cohort analysis of embryos that underwent
timelapse imaging and subsequent transfer at Baylor
Family Fertility Center from 2014 to 2016. MATERIALS
AND METHODS: A total of 183 embryos that underwent
time-lapse imaging and subsequent transfer were analyzed
retrospectively. Only transfers with single or two-embryos
that all implanted (n=131) and those that failed implantation (n=52) were included. Embryos were cultured
in EmbryoScope time-lapse incubator (Vitrolife).
Morphokinetic parameters (pronucleus (PN) to blastocyst
formation) were correlated with clinical data including
oocyte age, maternal anti-Mullerian hormone (AMH), peak
estradiol, body mass index (BMI), and Society for Assisted
Reproductive Technology diagnosis. Data was analyzed
by multivariate analysis of covariance Fisher’s exact test,
and Chi-square tests. RESULTS: Embryos with implantation had a shorter time from fertilization to 9 cell formation
compared to embryos without implantation, which trended
towards significance (66.4 vs. 73.3h, p=0.081). When
data was analyzed from time of PN fading, we observed
the same results. When controlled for differences in patient
age, AMH, and peak estradiol levels, we found a trend in
delayed appearanceof the blastocele in embryos that failed
implantation (99 vs.94h, p=0.075). No statistical difference was observed in time to PN fading, 2, 3, 4, 5, 6, 7, or
8 cell and time from 2-3, 3-5, and 3-4, 5-8 cell. Embryos
that implanted were associated with decreased oocyte age
(35.57 vs 33.18yr, p=0.0003), higher maternal AMH (4.41
vs. 2.87ng/mL) and higher peak estradiol levels (3010 vs.
22
Abstracts ASRM 2016
2411pg/mL). A lower BMI trended towards, but did not
reach, significance (21.82 vs. 26.86kg/m2, p=0.0811). No
difference was found when the annotator of the morphokinetics or patient diagnosis was analyzed. CONCLUSIONS:
Implantation success of embryos undergoing IVF correlated with a trend in delayed time to 9 cell and blastocele
appearance from PN fading or fertilization in embryos that
failed to implant. Clinical factors favoring implantation
included maternal factors of decreased oocyte age and
increased ovarian reserve, consistent with previous reports.
Much larger numbers will be forthcoming and needed to
determine if morphokinetic parameters can aid embryo
selection within subsets of age and ovarian reserve.
Time-lapse
- Primo Vision
monitoring system
P-133 Tuesday, October 18, 2016
THE TIMING OF CYTOKINESIS
IN EMBRYOS WITH PATERNALLY
OR MATERNALLY DERIVED
CHROMOSOMAL ABNORMALITY
USING TIME-LAPSE SYSTEM.
M. J. Ryu, S. Bark, J. Kim, M. J. You, H. S. Kim, M. H.
Kim, H. J. Jeong, M. Chung. Seoul Rachel Fertility Center,
Seoul, Korea, Republic of.
OBJECTIVE: To investigate the differences of the timing of
cytokinesis with embryos of paternally derived and maternally derived chromosomal aberration in euploid blastocysts, aneuploid blastocysts and non-biopsy embryos.
DESIGN: A retrospective cohort study from July 2013 to
October 2015. This study were analysed embryos of 60
ICSI-PGS patients diagnosed with chromosomal aberration. 168 embryos were cultured in time-lapse system
(Primo Vision) and images were recorded. A total of 70
embryos underwent trophectoderm (TE) biopsy on 5 day
and 6 day were analysed using array comparative genomic
hybridization (aCGH). MATERIALS AND METHODS: We
were classified into 2 categories, paternally derived vs maternally derived; 1) developing time of euploid blastocysts
(n=14), aneuploid blastocysts (n=56) and non-biopsy
embryos (n=98), 2) phenotype of cytokinesis of euploid,
aneuploid blastocysts and non-biopsy embryos. The timing
of cytokinesis were evaluated; cell divisions (tS, tSC, t2-t5,
t8, tSB, tB) and interval (cc2, cc3, s1, s2, s3). RESULTS:
No significant differences were found time of division
and intervals between euploid and aneuploid blastocysts
with paternally derived or maternally derived chromosomal aberration. A significant differences were showed
between euploid blastocysts and non-biopsy embryos;
tSC (23.3±1.6 and 26.7±3.6, p=0.0276) in maternally
Vitrolife
derived chromosomal aberration. A significant differences
were showed between euploid blastocysts and non-biopsy
embryos; tM (92.1±4.0 and 99.5±8.8, p=0.0322) and tSB
(99.3±5.7 and 113.6±11.0, p=0.0021) in paternally derived chromosomal aberration. Also, there was a statistically
significant difference in euploid blastocysts and aneuploid
blastocysts respectively between paternally derived and
maternally derived chromosomal aberration; tS (21.7±1.4
and 25.1±2.6, p=0.0209; 22.6±2.2 and 24.8±3.1,
p=0.0056), tSC (23.3±1.6 and 27.0±2.6, p=0.0116;
24.9±2.9 and 26.9±3.1, p=0.0117), t2 (23.6±1.6 and
27.3±2.6, p=0.0102; 25.2±2.8 and 27.2±3.1, p=0.0164)
and t4 (35.8±2.2 and 39.3±2.9, p=0.0388; 36.6±4.7 and
39.4±3.6, p=0.0240). Although there was no significant
differences, they showed abnormal phenotype of cytokinesis in aneuploid blastocysts and non-biopsy embryos of
paternally derived and maternally derived chromosomal aberration respectively; direct cytokinesis (3.1% and 32.7%,
8.3% and 25.6%), reverse cytokinesis (6.3% and 22.0%,
8.3% and 4.7%) and chaotic cytokinesis (0.0% and 14.5%,
4.2% and 14.0%). CONCLUSIONS: These findings may
indicate that time-lapse system provides cytokinetic differences of embryos diagnosed with paternally or maternally
derived chromosomal aberration. Significant differences
were found cytokinetic parameters between maternally
derived and paternally derived chromosomal aberration
with euploid or aneuploid blastocysts. Also, there wasn’t
showed abnormal phenotype of cytokinesis euploid.
(vitrolife Sweden), and rate of clinical outcome. Moreover in
IVM cycles, these time points were also compared between
transferred embryos that implanted or did not implant.
RESULTS: IVM zygotes showed significantly lower good
quality embryo on day3, blastocyst formation, and good
blastocyst rates compared with IVF. There were no differences in time points of cell division (tPNa to t8), pregnancy
and abortion rates between the two groups. However, there
were significant differences in synchrony of the second cell
cycles (t4-t3:S2) (S2:4.5±5.0h vs. 1.42.3h, P=0.0002),
and the rate of direct cleavage of one to three, or there to
five cells was significantly higher in the IVM group (90/194
46.4% vs. 9/48 18.8%, P=0.0016). In the IVM group, there
were no differences in development time points between
implanted and non-implanted embryos. CONCLUSIONS:
IVM zygotes showed increased abnormal cleavage compared with IVF; however, there were no significant differences in pregnancy rate or abortion rate between IVM and
IVF. We rejected these abnormal cleaved embryos for transfer, indicating that in order to improve the pregnancy rate it
is important to exclude the abnormal cleaved embryo, such
as direct cleavage, from embryo transfer in IVM treatment.
P-393 Wednesday, October 19, 2016
DOES IN VITRO MATURATION
(IVM) HAVE AN IMPACT ON
MORPHOKINETIC PARAMETERS IN
COMPARISON WITH IVF?
H. Hattori,a Y. Nakajo,a N. Aono,b H. Igarashi,a K. Kyono.a
a
Kyono ART Clinic, Sendai, Miyagi, Japan; b Kyono ART
Cllinic Takanawa, Minatoku, Tokyo, Japan.
OBJECTIVE: Polycystic ovarian syndrome (PCOS) patients
are at higher risk of ovarian hyper stimulation syndrome
(OHSS) due to the high dose of gonadotropin in IVF. To
avoid the possibility of OHSS, IVM techniques have been
developed and employed in clinical settings. However,
IVM delivers lower pregnancy rates and birth rates than
IVF. The objective of this study was to assess whether IVM
treatment affects embryo development events together
with morphokinetic parameters compared with IVF, and to
identify the morphokinetic parameters specific to embryos
that were suitable for implantation in IVM cycles. DESIGN:
Retrospective study. MATERIALS AND METHODS: The
subjects were 41 couples who underwent 50 cycles of IVM
at our clinic, from May, 2013 to Nov., 2015. After the failure
of IVM treatment, 12 couples underwent standard IVF
treatment. We assessed 194 embryos derived from IVMICSI and 48 embryos derived from IVF-ICSI. Their treatment outcomes were analyzed with morphokinetic events
(cell division and interval of cell cleavage) by Primovision
Vitrolife
Abstracts ASRM 2016
23
Poster Abstracts
Embryo Development
- Vitrolife media and oli
P-555 Wednesday, October 19, 2016
FRAGMENT REMOVAL OF THE
FRAGMENTED HUMAN DAY2
EMBRYOS SIGNIFICANTLY
INCREASED SUBSEQUENT
DEVELOPMENT AND CLINICAL
OUTCOMES.
S. Kim,a Y. Kim,a S. Kwak,a J. Park,a C. Yoo,a H. Sun,b
J. Kim,b K. Lee,b H. Chi.a aBabydream Research Center,
Mamapapa&Baby Ob/Gy Clinic, Ulsan, Korea, Republic of;
b
Mamapapa&Baby OB&GY clinic, Ulsan, Korea, Republic
of.
OBJECTIVE: The purpose of this study was to assess the
beneficial effect of fragment removal on the subsequent
development and pregnancy outcome of the fragmented
human day 2 embryos. DESIGN: The study included 96
patients who underwent day 3 ET program from March
2015 to October 2015. The patients were divided into two
groups; Fragment removal (FR) group (n=48) and control
group (n=48). There were no differences between the
FR and the control groups in the number of blastomeres
(4.0±1.3 vs. 4.0±1.3) and the morphological grade of
embryos (3.1±0.6 vs. 3.1±0.5) before fragment removal on
day 2. MATERIALS AND METHODS: Fragment removal
was performed on the grade 3 embryos (10 %< degree
of fragmentation <25%) of the FR group.We performed
assisted hatching with laser and fragments removal on
day2. Before fragment removal, the fragmented embryos
were incubated in G-PGD medium (Vitrolife) under paraffin
oil (Ovoil, Vitrolife) for 30 minutes. Microsurgical fragment
removal was performed with handmade suction micro-pipet
with outer diameter of 30mm. RESULTS: There were no
differences in the characteristics of the patients between
the FR and the control groups; mean age of the patients
(36.2±4.3vs. 36.2±4.1), endometrial thickness (10.4±2.5
vs. 10.0±2.0), AMH levels(4.4±4.5 vs. 4.2±2.9), and mean
number of retrieved oocytes (8.7±5.7 vs. 9.2±4.8). No differences were also observed between the two groups infertilization rate (76.0±17 vs. 71.0±22%) and mean number
of ET (2.0±0.4 vs. 2.0±0.3). After fragment removal and
24 h culture in vitro, although the number of blastomeres
(7.0±1.6 vs. 6.8±1.6) was comparable in the two groups,
embryo morphological grade (2.0±0.8) of the FR group
was significantly improved compared to that (3.2±0.5
24
Abstracts ASRM 2016
p<0.01) of the control group. Clinical pregnancy (43.8%)
and implantation rates (28.1%) of the FR group were
significantly higher than the rates (23.0 and 14.7%, respectively, P<0.05) of the control group. CONCLUSIONS:
Early fragment removal on day 2 significantly improved the
subsequent development and pregnancy outcomes of the
fragmented embryos. These results suggest that early fragment removal effectively prevents secondary degeneration
of fragmented embryos by eliminating the release of toxic
substances as soon as possible.
P-659 Wednesday, October 19, 2016
AFFIXING LABELS ON CULTURE
DISHES DECREASE THE
DEVELOPMENT RATE OF MOUSE
EMBRYOS.
H. Kawano,a K. Nakata,a M. Kamoshita,b J. Ito,b N.
Kashiwazaki,a N. Yamashita.a aYamashita Syonan Yume
Clinic, Fujisawa, Japan; bLaboratory of Animal Reproduction
Department of Animal Science & Biotechnology School of
Veterinary, Azabu University, Sagamihara, Japan.
OBJECTIVE: Temperature, humidity, oxygen concentration, bacteria, and airborne volatile organic compounds
(VOCs) have been reported to influence the development
of mammalian embryos during in vitro culturing. Identifying
and managing culture embryos, we are able to use a pen or
labels for writing patient’s information on the plastic ware.
There has been concern that the VOCs originated from the
ink of felt-tip pens, which are too small to be removed with
the HEPA filter, might cause the decrease of the developmental rate of culture embryos. On the other hand, verification report is less about the labels. This study was conducted to investigate effects of affixing labels on culture dishes
on development of the embryos in the mouse. DESIGN:
Prospective experimental Animal study. MATERIALS AND
METHODS: We performed ovarian hyperstimulation on
8-week-old female BDF1 mice by administering eCG 7.5
IU or hCG 7.5 IU. After rearing these females in the same
cage with males of the BDF1, fertilized eggs were collected from oviducts of the plug-positive females. Plastic
dishes (Falcon) with a 35-mm aperture were prepared with
five 20-µL drops of M16, covered with mineral oil (Vitrolife),
placed in a 60-mm dish (Falcon), and then cultured at rest
in an incubator (HC-3100 ASTEC). Five to ten embryos
Vitrolife
were cultured per drop. Labels using acrylic adhesive
were affixed to the inside of the lid of the 60-mm dish, and
culturing was performed at 37_C, 5% CO2, 5% O2, 90%
N2, and saturated humidity. The number of affixed labels
totaled 0 (control group), 1 (group A), 7 (group B), or 13
(group C), and development of the cultured mouse embryos from the 1-cell stage was investigated for 96 hours.
Statistically analysis between experimental groups was
determined by one-way ANOVA followed by Tukey test for
multiple comparisons. Following the approval by the Animal
Care and Use Committee of Azabu University, we used
263 mouse embryos in this study, which was performed
at the Azabu University in theduration from September
2015 to December 2015. RESULTS: The cleavage rate
of the control group and groups A, B, and C were 97.5%,
97.1%, 12.0%, and 9.5%, respectively, with significant
(P<0.001) decreases in groups B and C compared with
the control; the developmental rate to the blastocyst stage
were 88.6%, 0%, 0%, and 0%. As increasing the number
of label, the incidences of cleavage of the cultured embryos were reduced, and there was no embryo developed
to the blastocyst stage in all of the label-affixed groups.
CONCLUSIONS: Affixing labels using acrylic adhesive on
culture dishes have detrimental effects on the development of mouse embryos at 1-cell stage. It is concluded
that careful consideration should be given to the adhesive’s
components when using label stickers for culture dish
management.
P-660 Wednesday, October 19, 2016
WASHING MINERAL OIL USED FOR
MICRODROP OVERLAY DOES NOT
IMPROVE STABILITY OF MEDIA
OSMOLALITY.
J. E. Swain,a A. E. Batcheller,b W. B. Schoolcraft,c N.
Bossert.b a CCRM IVF Network, Lone Tree, CO; b CCRM
Minneapolis, Edina, MN; c Medical Director, Colorado
Center for Reproductive Medicine, Lone Tree, CO.
OBJECTIVE: Evaporation of culture media and the resulting
increase in osmolality is a concern in some IVF laboratories,
especially considering the increased use of non-humidified
benchtop incubators. This concern may be increased when
using single-step embryo culture media and uninterrupted
culture over periods of up to 7 days. Some conjecture that
washing of oil ‘‘saturates’’ the oil and prevents absorption
of media components from microdrops, thereby possibly
protecting growth conditions. The impact of extended uninterrupted culture and washing of mineral oil used for overlay
on osmolality of microdrops was examined. DESIGN: Basic
research study. MATERIALS AND METHODS: Microdrops
of media (25ul) were prepared in a laminar flow hood at
room temperature by pipetting 12.5µl of media onto the
surface of a 35mm dish, covering with 3.5ml of unwashed
or washed mineral oil (Ovoil, Vitrolife), removing the media
and adding 25µl of fresh media. Washed oil was prepared
by adding culture media in a 1:5 ratio to the oil, inverting 30
times and allowing to settle. Dishes were made every 24hr
Vitrolife
over 7 consecutive days (168h) and placed into a nonhumidified benchtop incubator at 37_C (G185, K-systems).
At the end of 7 days, all dishes were removed and osmolality of microdrops measured using a vapor pressure osmometer (Wescor). Positive controls included media directly
out of the bottle (con) and microdrops under oil for 10min
(con drops). The resulting 16 treatments drops treatments
were measured 3 times each. Data were analyzed using
ANOVA and Tukey analysis and presented as the mean
±SEM, p<0.05. RESULTS: Mean osmolality of con media
and con drop were similar (263±1.0 and 265±2.1mOsm,
respectively). Osmolality of microdrops under washed
and unwashed oil increased over time and were both
significantly higher than con and con drops after 48h of
culture. No significant differences were apparent between
washed and unwashed oil within any time point examined.
CONCLUSIONS: Washing of mineral oil for use with
microdrop overlay had no protective impact on stabilizing
media osmolality compared to unwashed oil when utilized
for up to 7 days in a non-humidified incubator environment.
Both types of oil overlay resulted in significant osmolality increase over time compared to controls after 48h of culture.
This osmolality increase should be considered when trying
to optimize growth conditions within the IVF laboratory.
Microdrop Osmolality (mOsm) After Time in Culture
P-679 Wednesday, October 19, 2016
MEDIA OSMOLALITY CHANGES
OVER 7 DAYS FOLLOWING
CULTURE IN A NON-HUMIDIFIED
BENCHTOP INCUBATOR.
J. E. Swain,a W. B. Schoolcraft,b N. Bossert,c A. E.
Batcheller.c aCCRM IVF Lab Network, Lone Tree, CO;
b
Colorado Center for Reproductive Medicine, Lone Tree,
CO; cCCRM Minneapolis, Edina, MN.
Osmolality (mOsm) of media over time
OBJECTIVE: Environmental stressors encountered in the
IVF laboratory can negatively impact embryo development.
Introduction of benchtop incubators and use of single-step
Abstracts ASRM 2016
25
uninterrupted culture methods aim to reduce harmful
environmental deviations and improve system stability.
However, evaporation and the subsequent increase in
media osmolality could be induced from culture in the nonhumidified environment present in many benchtop incubators, which could be amplified due to extended culture
time with no media refreshment; especially in laboratories
who culture embryos for up to 7 days. This study examines
the impact of extended culture times in a non-humidified
benchtop incubator on media osmolality. DESIGN: Basic
research study. MATERIALS AND METHODS: Microdrops
of media (25ml) were prepared in a sterile airflow hood at
room temperature by pipetting 12.5ml of media onto the
surface of a 35mm dish, covering with 3.5ml of unwashed
mineral oil (Ovoil,Vitrolife), removing the media and adding
25ul of fresh media. Dishes were made every 24h over 7
consecutive days (168h) and placed into a 37_C nonhumidified benchtop incubator (G185, K-systems). Identical
dishes were prepared and cultured in a37_C humidified
box incubator (MCO-18AIC, Sanyo). At the end of 7 days,
all dishes were removed and osmolality measured using
a vapor pressure osmometer (Wescor). Positive controls
included media directly out of the bottle (con) and microdrops under oil for 10min (con drops). The resulting 16
treatments were measured 3 times. Data are presented as
the mean ±SEM and analyzed using ANOVA and Tukey
analysis, p<0.05. RESULTS: Mean osmolality of con and
con drops were 263±1.0 and 265±2.1 mOsm, respectively. These values did not significantly differ. Osmolality of
microdrops following 1-7 days of culture in a dry incubator
increased over time, differing significantly from con after
24h and differing from con drops and humidified incubator
drops after 72h. Omolality in a humidified incubator remained unchanged and similar to con and control drops at
all time points examined. CONCLUSIONS: Uninterrupted
culture for up to 7 days in a non-humidified incubator
resulted in an increase in media osmolality over time, while
osmolality of microdrops in a humidified incubator remained
unchanged. These findings may vary based on volume of
media and oil overlay used. Care must be taken when implementing use of dry-culture incubators or uninterrupted
culture within the IVF laboratory to avoid creation of damaging culture conditions.
Sequential media
P-670 Wednesday, October 19, 2016
SUCCESSFUL PREGNANCIES
AFTER VITRIFIED EMBRYO
TRANSFER OF HUMAN EMBRYOS
CULTURED IN RECOMBINANT
ALBUMIN.
M. Murakami, A. Egashira, K. Tanaka, H. Otsubo, S.
Mizumoto, T. Kuramoto. Kuramoto Women’s Clinic,
Fukuoka, Japan.
OBJECTIVE: Human serum albumin (HSA), a commonly
used protein source for ART, can contribute to biological
26
Abstracts ASRM 2016
variation and possibly disease transmission. Additionally,
mammalian embryos cultured in blood serum are known to
have impaired qualities. Recombinant human albumin (rHA)
might reduce these risks, but little is known about neonatal data after transferring embryos cultured in rHA. Our
previous randomized controlled trials (RCTs) revealed that
the replacement of HSA with lower amounts of rHA during
human embryo culture yielded good quality (GQ) embryos
(1, 2). Here, clinical data, including perinatal outcomes associated with vitrified ET of these embryos, were analyzed.
DESIGN: Follow-up study of previously conducted RCTs.
MATERIALS AND METHODS: Our RCTs included a total
of 136 patients who underwent IVF/ICSI treatment at our
clinic between July 2012 and March 2015, and each patient
had R4 2PN oocytes 18 h after insemination (1, 2). Embryo
culture (n=1,255) was performed in G1/G2 media containing either 0.5 mg/mL rHA (group A) or 5 mg/mL HSA
(group B), and GQ embryos were vitrified by day 6. Clinical
data were evaluated for patients with vitrified embryos in
groups A (n=67) and B (n=68). RESULTS: Patient age was
similar for groups A and B (35.6_0.5 vs. 35.7_0.4 years,
respectively). The total number of vitrified day 2/3 embryos
and day 5/6 blastocysts were 51 and 106 in group A and
48 and 175 in group B, respectively. By the end of March
2016, groups A and B underwent 80 (day 2/3 ET=15,
day 5/6 ET=65) and 94 (day 2/3 ET=15, day 5/6 ET=79)
ET cycles with vitrified/warmed embryos, respectively. All
embryos survived after warming. The number of embryos
transferred (1.13±0.04 vs. 1.16_0.04), implantation rates
(46/90 (51.1%) vs. 41/109 (37.6%)), clinical pregnancy
rates per ET (44/80 (55.0%) vs. 38/94 (40.4%)), and
ongoing/delivered pregnancy rates per ET (36/80 (45.0%)
vs. 30/94 (31.9%)) did not differ between groups A and B,
respectively. Twins occurred in 1/34 (group A) and 1/29
(group B) of live deliveries. Groups A and B had similar
perinatal outcomes, including gestational age (39.4±0.2 vs.
38.7±0.4 wk) and birth weight (3195±91 vs. 3059±94 g).
Birth defects occurred in 0/35 (group A) and 2/30 (group
B) neonates. CONCLUSIONS: The viability of human embryos cultured in rHA media was comparable to or slightly
better than that of embryos cultured in conventional HSA
media, resulting in live births after vitrified ET. Our findings
demonstrate the feasibility of using defined culture media to
yield GQ embryos, and subsequently, successful pregnancies. Further studies, including those focused on improving rHA media to increase blastocyst yield and with more
participants, are being performed to validate the efficacy.
Using rHA will help develop a standardized ART system to
eliminate potential risks associated with the use of serum
proteins.
P-681 Wednesday, October 19, 2016
CYTOPLASMIC PITTING
ASSOCIATED WITH SINGLE MEDIUM
CULTURE AND ITS IMPACT ON THE
ICSI RESULTS.
M. V. Paz, J. Cicar_e, F. Lo Menzo, L. Domenech, P.
Perfumo, V. B. Ventura. Servicio Medicina Reproductiva.
Vitrolife
Grupo Gamma, Rosario, Argentina.
OBJECTIVE: To determine if single medium embryo culture
is equivalent to a sequential one regarding embryo quality,
pregnancy rate, abortion and ongoing pregnancy. Analyze
the presence of cytoplasmic pitting, its relationship to the
medium used and its impact on ICSI results. DESIGN:
Observational prospective study. MATERIALS AND
METHODS: 194 cycles of patients younger than 40 yearsold with fresh embryo transfer were included. The sequential
media used were G1 and G2 (Vitrolife), and single medium
CSC (Irvine) without renewal on day 3. The presence of
cytoplasmic pitting was identified on day 3 of culture. In order to analyze its impact on the results, only the transfers of
two embryos with or without pitting were studied, including
44 patients. Comparisons between groups were performed
using Pearson’s chi-squared tests or Fisher’s exact tests.
Logistic regression models were adjusted for clinical pregnancy and ongoing pregnancy according to the explanatory
variables: culture medium, pitting, low ovarian response,
male factor, day of transfer and number of embryos transferred. RESULTS: Multivariate analysis for clinical pregnancy suggests a favorable trend toward sequential media
(p=0.047). A significant association between the presence
of pitting and single medium culture was found (CSC
34.39% vs G1/G2 2.23%; p<0.001). This morphological
feature was beneficial in terms of blastulation, pregnancy
and implantation rates; however, it is not significant for abortion or ongoing pregnancy. CONCLUSIONS: According to
the explanatory variables, it is suggested that the embryo
culture in G1/G2 sequential media increases the probability
of achieving a clinical pregnancy when compared to single
medium CSC without renewal on day 3. The results of this
observational study show a beneficial effect of the presence
of pitting on day 3 on the blastulation and pregnancy rates,
which should be further examined in order to be confirmed.
Results according to the presence or absence of cytoplasmic pitting
Undisturbed embryo culture/
single step medium
OBJECTIVE: Vitrification is an alternative method to slow
freezing,which reduces chilling sensitivity and crystallization
damages. However vitrification requires high concentrations
of cryoprotectants (CPs) which exposes cells to osmotic
and toxicity damages. We modified the minimal drop size
(MDS) technique by exposing the embryos only to equilibration solution (ES), after that partial drying and finally plunged
into liquid nitrogen (LN). DESIGN: To compare survival and
blastulation rates of day 2 embryos resulting from one and
three pronuclei (1PN and 3PN) undergoing conventional
vitrification or vitrification using only equilibration solution (ES). MATERIALS AND METHODS: Day 2 embryos
resulting from 1and 3PN oocytes were randomized into
two groups: Group A- embryos that were vitrified utilizing
the vitrification cooling kit according to the manufacturer
instructions (Origio-Sage); Group B- one or two embryos
were transferred with minimal volume of culture medium to
the top of the drop of ES with a free-fall for 10 minutes. The
drop containing the embryo/s with a volume of < 0.15µL
was transferred to a Kitazato CryoTop Vitrification Device
and hold for 30 seconds before plunging into LN. Warming
was performed with the vitrification warming kit (OrigioMedicult). The embryos were cultured to the blastocyst
stage in G-TL medium (Vitrolife Sweden). The outcome of
the warmed groups A and B embryos was compared in
terms of survival and blastulation rates. RESULTS: There
were 71 warmed embryos in group A and 62 in group B.
Mean patients’ age was 32.6±5.9 and 32.9±5.6 years
respectively, and the mean number of blastomeres was
4.6±1.8 and 4.6±1.6. Mean number of abnormal fertilizations (1PN and 3PN) per patient was 2±2.1 in both roups.
The rates of intact embryos were 56% (40/71) in group A
and 66% (41/62) in group B. The overall survival rate (intact
embryos and embryos withR50% viable blastomeres) was
91% (65/71) and 89% (55/62) respectively. The blastulation rate was 21% (14/65) and 20% 11/55) which is
comparable with the rate in our center (19%) for embryos
emerging from 1 and 3PN. All the above figures are with
no statistical significance. CONCLUSIONS: In the present
study we have demonstrated that using a minimal volume
of ES (MDS technique, Arav 1989) enables vitrification of
embryos if partially dried before cooling in liquid nitrogen.
The 30 sec dehydration of the drops permits the vitrification
of the drops probably due to increase in the viscosity when
the water in the sample dehydrates and the concentration in
the solution increases. Also, the fact that the volume of the
drop decreases along with the dehydration, contributes to
the success of this method. The simplicity of the technique
may reduce toxicity of the cryoprotectants and improve the
safety of the vitrification process.
P-88 Tuesday, October 18, 2016
SUCCESSFUL VITRIFICATION
OF HUMAN EMBRYOS USING
EQUILIBRATION SOLUTION.
O. Bern,a Y. Natan,b R. Ron-El,a A. Arav.b aIVF, Assaf
Harofeh, Beer Yaacov, Israel; bFertileSafe Ltd., Ness. Ziona,
Israel.
Vitrolife
Abstracts ASRM 2016
27
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Abstracts
ASRM 2016
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