Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging.
ABSTRACT In the current study, our aim was to demonstrate that EmbryoScope incubation conditions is comparable to standard laboratory incubation circumstances by comparing embryo quality, development and ongoing pregnancy rates between the EmbryoScope (ES) and a standard incubator (SI). We analyzed 478 embryos from 60 couples undergoing oocyte donation were included in the study.
All embryos retrieved from a patient were randomly distributed in the ES or SI. We calculated blastocyst development rate, blastocyst viability and ongoing pregnancy rate for embryo transfers from ES, SI and mixed (one embryo from the ES and one from the SI). Statistical analysis was conducted by Chi square tests, considering p < 0.05 significant.
No significant differences were found between the ES and SI from all the parameters evaluated.
Thus we concluded that time-lapse monitoring in the EmbryoScope does not impair embryo quality while allowing for morphological, spatial and temporal analysis of embryo development.
- SourceAvailable from: Thomas Ebner[show abstract] [hide abstract]
ABSTRACT: The association between oocyte morphology and subsequent fertilization rate and embryo quality in intracytoplasmic sperm injection (ICSI) is subject to considerable controversy. This retrospective study was carried out to investigate a possible prognostic value of first polar body morphology with regard to fertilization rate and embryo quality. A total of 70 consecutive ICSI cases was included in this study. The results showed that classification based on first polar body morphology revealed a significant correlation with fertilization rate (P < 0.025) and embryo quality (P < 0.001). Cytoplasmic criteria showed no correlation in this respect. Present data indicate that ICSI of oocytes with intact well-shaped first polar bodies yields higher fertilization rates and higher quality embryos.Human Reproduction 03/2000; 15(2):427-30. · 4.67 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The purpose of this study was to clarify developmental changes of early human embryos by using time-lapse cinematography (TLC). For human ova, fertilization and cleavage, development of the blastocyst, and hatching, as well as consequent changes were repeatedly photographed at intervals of 5-6 days by using an inverse microscope under stabilized temperature and pH. Photographs were taken at 30 frames per second and the movies were studied. Cinematography has increased our understanding of the morphologic mechanisms of fertilization, development, and behavior of early human embryos, and has identified the increased risk of monozygotic twin pregnancy based on prolonged incubation in vitro to the blastocyst stage. Using TLC, we observed the fertilization of an ovum by a single spermatozoon, followed by early cleavages, formation of the morula, blastocyst hatching, changes in the embryonic plates, and the development of monozygotic twins from the incubated blastocysts.American journal of obstetrics and gynecology 10/2008; 199(6):660.e1-5. · 3.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Mammalian preimplantation embryonic development is achieved by tightly coordinated regulation of a great variety of temporal and spatial changes. Therefore, it would be valuable to analyze these events three-dimensionally and dynamically. We have previously developed a live-cell imaging method based on the expression of fluorescent proteins, using mRNA injection and time-lapse florescence microscopy. However, with conventional fluorescent microscopy, three-dimensional images could not be obtained due to the thickness of the embryos and the optical problem in which ;out-of focus blur' cannot be eliminated. Moreover, as the repeated exposure of intense excitation light to the cell yields phototoxicity, long-term observation was detrimental to embryonic development. Here, we improved our imaging system to enable six-dimensional live-cell imaging of mouse preimplantation embryos (x, y and z axes, time-lapse, multicolor and multisample). Importantly, by improving the imaging devices and optimizing the conditions for imaging, such as intensity of excitation and time intervals for image acquisition, the procedure itself was not detrimental to full-term development, although it is a prolonged imaging process. For example, live pups were obtained from embryos to which two different wavelengths of excitation (488 and 561 nm) were applied at 7.5-min intervals for about 70 h, and 51 images were acquired in the z axis at each time point; thus, a total of 56,814 fluorescent images were taken. All the pups were healthy, reproductively normal and not transgenic. Thus, this live-cell imaging technology is safe for full-term mouse development. This offers a novel approach for developmental and reproductive research in that it enables both retrospective and prospective analyses of development. It might also be applicable to assessment of embryo quality in fields such as human reproductive technology and production animal research.Journal of Reproduction and Development 04/2009; 55(3):343-50. · 1.76 Impact Factor
ASSISTED REPRODUCTION TECHNOLOGIES
Embryo quality, blastocyst and ongoing pregnancy rates
in oocyte donation patients whose embryos
were monitored by time-lapse imaging
María Cruz & Blanca Gadea & Nicolás Garrido &
Kamilla Søe Pedersen & Mar Martínez &
Inma Pérez-Cano & Manuel Muñoz & Marcos Meseguer
Received: 8 October 2010 /Accepted: 16 February 2011 /Published online: 11 March 2011
# Springer Science+Business Media, LLC 2011
Purpose In the current study, our aim was to demonstrate
that EmbryoScope incubation conditions is comparable to
standard laboratory incubation circumstances by comparing
embryo quality, development and ongoing pregnancy rates
between the EmbryoScope (ES) and a standard incubator
(SI). We analyzed 478 embryos from 60 couples undergo-
ing oocyte donation were included in the study.
Methods All embryos retrieved from a patient were randomly
distributed in the ES or SI. We calculated blastocyst
development rate, blastocyst viability and ongoing pregnancy
rate for embryo transfers from ES, SI and mixed (one embryo
from the ES and one from the SI). Statistical analysis was
conductedbyChisquare tests,consideringp<0.05 significant.
Results No significant differences were found between the
ES and SI from all the parameters evaluated.
Conclusions Thus we concluded that time-lapse monitoring
in the EmbryoScope does not impair embryo quality while
allowing for morphological, spatial and temporal analysis
of embryo development.
Keywords Embryoscope.Time-lapse image acquisition.
Embryo quality.Dynamic embryo evaluation.Monitoring
Assisted reproduction treatments (ARTs) in humans have
been highly successful in helping infertile couples. Devel-
opment of ART technologies allows observation of mor-
phological events during human embryonic development
. Information obtained from microscopic observations
have contributed significantly to the great success of ART
programs in humans and enhanced in vitro fertilization
(IVF) success rates. It is critical to identify viable high
quality embryos with the highest implantation potential.
Consequently, numerous criteria from embryo selection
have been proposed [2, 3] and among these, morphological
features have been used extensively in selecting excellence
embryos. However, embryo choice for transfer should not
be based only on cell number and morphological assess-
ment on the day of transfer .
Capsule EmbryScope incubation conditions and time-lapse image
acquisition does not affect embryo quality and reproductive outcome.
We evaluated the effects of dynamic assessment of embryo quality, a
tool that would give more information from the individual embryo in
order to improve selection.
This work was presented in the 66th Annual Meeting of the American
Society for the Reproductive Medicine in Denver (Colorado, United
M. Cruz (*):B. Gadea:N. Garrido:M. Martínez:
I. Pérez-Cano:M. Muñoz:M. Meseguer
Instituto Valenciano de Infertilidad,
N. Garrido:M. Meseguer
Instituto Universitario IVI Valencia,
Universidad de Valencia (IUIVI),
K. S. Pedersen
Instituto Universitario IVI,
Plaza de la Policía Local 3,
Valencia 46015, Spain
J Assist Reprod Genet (2011) 28:569–573
IVF technology allows for observations of early actions
of human fertilization and embryogenesis. Nevertheless,
concern about effects of handling the embryos has limited
the frequency of microscopic observations outside the
controlled incubation environment, thus the knowledge of
growth rates and morphological changes have deduced
largely from morphological appearance of embryos at few
discrete time points. Such “freeze-frame” images of the
active processes of growth and development necessarily
limit the information available to the observer and short-
lived processes may be missed entirely. In addition, the
dynamic and gradual nature of change in cellular morphol-
ogy can occult events which only become visible when
images are condensed into a continuous and coherent
Time-lapse image acquisition can minimize disturbance
to the culture environment by integrating the incubation and
inspection of embryos into one system. On the contrary,
culturing with intermittent observations requires the remov-
al of embryos from the incubator to their examination .
Time-lapse cinematography has advanced the understand-
ing of fertilization processes and development of early
human embryos. The opportunity to follow the dynamic
developmental patterns of embryos using time-lapse mon-
itoring will give us useful information for embryo selection.
Time-lapse monitoring allows a phenomenon observed on a
specific time point to be linked directly to an embryo’s
developmental capacity and implantation achievement .
In essence, micro-cinematography offers unique possi-
bilities for non invasive studies of early embryogenesis in
humans. The EmbryoScope provides such an integrated
monitoring system consisting of a safe, controlled culture
environment including automated time-lapse high resolu-
tion image acquisition which allows for retrospectively
detection and testing for critical events during embryo
The objective of this study was to show that embryo
quality, blastocyst rate and reproductive success rates are
comparable for embryos cultured in a standard incubator or
in the EmbryoScope.
In a prospective cohort study, we analyzed 478 embryos
from 60 couples undergoing IVF treatment; in order to
minimize variations in oocyte quality only oocyte donation
treatments were included. Ovum donation is a useful tool
with which for studying external variables affecting assisted
reproduction treatments, since it reduces the variability of
oocyte quality associated with female infertility and also
reduces endometrial receptivity variability associated with
controlled ovarian stimulation protocols .
Donors were between 18 and 35 years old without
current or past exposure to radiation or hazardous chemical
substances, drug use, no family history or hereditary
chromosomal diseases, a normal karyotype, and tested
negative for fragile X syndrome and sexually transmitted
diseases as stated by Spanish law. All donors had normal
menstrual cycles of 26–34 days duration, normal weight
range (BMI 18–28 Kg/m2), no endocrine treatment (includ-
ing gonadotrophins and oral contraception) in the 3 months
preceding the study, normal uterus and ovaries at transvaginal
ultrasound (no signs of polycystic ovary syndrome), and
antral follicle count (AFC) >20 on the first day of gonadotro-
phin administration, after down regulation with GnRH
Age of the patients ranged from 32 to 45 years old. The
exclusion criteria were pathologies like endometriosis,
hydrosalpynx, obesity (BMI>30), uterine pathology (myo-
mas, adenomyiosis, endocrinopaties, trombophylia, chronic
pathologies, acquired or congenital uterine abnormalities),
recurrent pregnancy loss, or maternal age over 45 years old
Calculations of the sample size needed to confirm the
absence of differences between groups (non-inferiority)
were performed using the main endpoint of the study of the
blastocyst rates achievement, given that origin of the
embryos afterwards transferred did not depend on the
incubator. This calculus was based on the comparison of
two binomial proportions (blastocyst rate), assuming non-
inferiority hypothesis between ES and SI with respect to the
blastocyst rate, based on a pre-defined non-inferiority limit
of 0.66 for the odds ratio (OR) of Embryoscope versus
Standard Incubator (corresponding to limits of −9 to −10%
in the difference in proportion scale with an overall
Blastocyst rate of 50–60%). The non-inferiority criteria
were based on our clinical data and those differences which
were considered clinically relevant .
To guarantee that the culture conditions in both of
incubators are comparable, every day we check temperature
and the CO2concentration with external sensors in order to
confirm that they were in the values we fixed (6% CO2;
37.0°C); pH is not measured daily but we adjust it between
7.2 and 7.4 when incubators are restarted.
In all donors, ovarian stimulation was achieved by
pituitary desensitization using a GnRH analogue followed
by stimulation with gonadotrophins . The daily dose of
FSH was adjusted according to the donor’s ovarian
response based on serum estradiol levels and the number
and size of ovarian follicles as considered by transvaginal
ultrasonography. Oocyte recovery was scheduled 36 h after
hCG injection and conventional IVF or ICSI was performed
after oocyte aspiration.
For all the recipients who were still cycling, down-
regulation was carried out using a GnRH analogue. The day
570J Assist Reprod Genet (2011) 28:569–573
that the donor announced the onset of her period, the
recipient was informed to start with oral estradiol valerate
and lately, intravaginally micronized progesterone for
luteal-phase supplementation was used.
Successful fertilization was verified by confirmation of
two pronuclei 17–19 h after insemination/microinjection.
Embryos were randomly selected and evenly distributed in
the Embryoscope (n=238) and the standard incubator (n=
240); in the EmbryoScope images were acquired every
20 min in 7 focal planes. Embryos in the standard incubator
as well as embryos in the Embryoscope were standing apart
once a day to assess embryo quality; day 2 and day 3
embryos were categorized in four grades from A (high
quality) to D (low quality) depending on the number of
blastomeres, fragmentation, multinucleation and symmetry;
blastocysts were evaluated according to inner mass cell
(ICM), trophoctoderm (TE) and blastocoel expansion
Grade A embryos have 7–8 cell, <10% fragmentation,
even size and no multinucleation; grade B embryos have 7–
8 cell with 11–25 fragmentation, 9 evenly sized cell or
more and no multinucleation; grade C embryos have <6
cell, 26–35% fragmentation, uneven cell allowed but no
multinucleation; and grade D, if the embryo has 6 cells or
less and observation of multinucleation, >35% fragmenta-
tion or type IV fragmentation.
Blastocysts were classified as viable or non viable:
viable if they were transferred and/or frozen and non viable
if they were arrested or they were poor quality embryos. We
also calculated ongoing pregnancy rate (OPR) for exclusive
transfers from we embryos cultured in the ES, the SI and
mixtures where one embryo from the ES and one embryo
from the SI were transferred (M).
Transfers were performed on day 3 and day 5, but
embryo selection criteria was based solely on the valuation
at the same time points (44 and 68 h after fertilization
procedure) on a morphology basis, both for the ES and SI
groups, hence the additional information provided by time-
lapse monitoring system was not used for embryo
The results were compared depending on embryo culture
environment by using a Chi squared test and significance
level of p<0.05; data are presented as proportions and 95%
confidence interval (95%CI).
The average number of oocytes that were received per
donor was 13.4 (CI95% 12.4–14.4). A total of 478 embryos
were analyzed from 60 couples with a median of 8 embryos
per couple (range 4–15; average 8.84). Contrast of embryo
quality grading at day 3, revealed similar distribution from
ES and SI groups respectively: A (n=79) 33.1% (95%CI
26.8–39.4) vs (n=85) 35.6% (95%CI 29.9–41.3); B (n=41)
17.1% (95%CI 12–22.2) vs (n=38) 15.7% (95%CI 11.3–
20.1); C (n=48) 20.4% (95%CI 15–25.8) vs 20.6% (n=50)
(95%CI 15.8–25.4) and D (n=70) 29.4% (95%CI 23.3–
35.5) vs (n=67) 28.1% (95%CI 22.7–33.5), p=0.941.
Blastocyst rate, the percentage of transferred and frozen
embryos and non viable discarded embryos were not
significantly different between the embryos from the standard
incubator and embryos cultivated in the embryo monitoring
system (Table 1). Blastocyts OR (Embryoscope vs. Standard
Incubator) were 1.158 (CI95% 0.739–1.689), confident
intervals were above the limits of the non-inferiority
hypothesis and in consequence sample size can be consid-
ered adequate. The proportion of viable embryos, resulting
from grouping those transferred and frozen were (n=68)
28.5 (95%CI 22.8–34.2) for embryos developed in the time-
lapse incubation system and (n=84) 35% (95%CI 29–41) for
those developed in the standard incubator, p=0.438.
When we compare how many embryos are transferred in
day 3 or day 5 in each incubator we did not find significant
differences between the Embryoscope and the Standard
Incubator (Table 2). From those embryos transferred in
day 3 (22/31) 71% (CI95% 55–87) from the Embryoscope
and (18/24) 75% (CI95% 57.7–92.3) from the classical
incubator were A or B embryos, as of those blastocyst
transferred in day 5 (17/19) 89.4% (CI95% 75.6–103.2)
from the time-lapse video system and (30/34) 88.2%
(CI95% 77.4–99) from the standard incubator were excel-
No significant variation was found between the ongoing
pregnancy rate for embryos incubated in the Embryoscope
(6/14) 42.8% (95%CI 16.9–68.7) and in the standard
incubator (8/19) 42.1% (95%CI 19.9–64.3) and mixed
transfers (8/24) 33.3% (95%CI 14.5–52.1), p=0.399.
According to implantation rate, we did not observe any
significant difference due to the reduced number of pure
Table 1 Blastocyst rate, proportion of transferred, frozen and discarded embryos incubate in the EmbryoScope vs. the standard incubator
Blastocyst CI95%Frozen CI95% TransferredCI95%DiscardedCI95%
Standard incubator (n=240)
J Assist Reprod Genet (2011) 28:569–573571
transfers from each incubator; the values were as follows:
for exclusive transfers from Embryoscope were (8/26)
30.8% (CI95% 13.0–48.5) vs (14/34) 41.0% (CI95 24.7–
57.7) obtained when we transfer two embryos from the
Embryo selection based on discrete routine assessment of
embryo morphology is not always associated with a higher
implantation or pregnancy rate. Therefore, other criteria
should be considered in the recognition of embryos with a
good projection for implantation or pregnancy. Time-lapse
cinematography imaging has rendered more information of
human embryonic development and growth behaviour in
comparison with conventional practice or intermittent
observation. Basically, this has shed more light on the
elementary descriptions of embryo development  and
enables compression of the entire course of early embryo
development into a short movie; hence this technique can
potentially progress in morphologic and chronologic anal-
yses of dynamic embryo developmental patterns, making an
important contribution to ART .
The combination of optimal incubation conditions for
early developing embryos and a time-lapse embryo
monitoring system are certainly of special meaning in
terms of gaining complete information of the development
of embryos and assessing their quality for transfer and
freezing. In the present study, we illustrate that embryos
incubated in the included culture and monitoring system
are not affected by the continuous observation associated
with the EmbryoScope. Hence, this system offers new
openings to get detailed and precise information about
morphological characteristics and cleavage status of grow-
ing embryos in combination with a safe and controlled
It is important to highlight that embryos cultured in an
incubator with an integrated time-lapse system are period-
ically exposed to light when digital images are acquired and
it is known that light exposure is an unnatural stress to
embryos and might affect embryo enlargement . Even
though the risk of impaired development in these culture
surroundings, each embryo were exposed to LED lighting
less than 50 s per day; however, we did not find significant
differences between embryos cultured with time-lapse
cinematography and embryos with discontinuous observa-
tions in a conventional incubator.
It should be noticed that extra information, for example
timing of early embryo development from the EmbryoScope
were not used for selecting high quality embryos for transfer
and freezing. Hence, we can conclude that embryo value and
reproductive outcomes do not diverge between the series in a
classic incubator and the EmbryoScope. The blastocyst
development rate and fraction of viable embryos was not
notably different between the two groups; even though when
we focus in the origin of the embryos (Embryoscope or
standard incubator) and in the day of the embryo transfer
(day 3 vs day 5), we also did not observe relevant differences,
confirming the usefulness of this new system.
These data, along with the similar results in ongoing
pregnancy rate in the three different groups (ES, SI and M)
confirm that this time-lapse monitoring system is suitable
for both research and clinical use. Validating the embryo
culture in the EmbryoScope subsequently allows studies
aiming at detecting new valuable selection parameters thus
optimizing selection of high quality embryos.
This experimental design is closely related to recent
publications  that assess time-lapse technology effec-
tiveness; not counting the methodological differences
between the two studies (we obtained the data from fresh
and ongoing cycles while they worked with frozen and
supernumerary embryos), both of works can emphasize the
time-lapse system as a tool with potential useful in basic
research and further clinical practices.
As we have mentioned previously, we analyze embryos
from oocyte donation programs in order to avoid introduc-
ing bias concerning oocyte quality, so we are likely
working with good prognosis females gametes; even
though, uterine receptivity is more stable avoiding interfer-
ence of controlled ovarian stimulation in uterine receptivity
 However, results presented in this paper could not be
necessary as relevant in infertile couples as are in this
model (oocyte donation).
It maybe expected that in the future, the employ of the
data from time-lapse observation will promote the improve-
ment of non invasive methods for the assessment of embryo
viability (15). Non invasive imaging is helpful not only for
explaining the morphological proceedings in human em-
bryos but also for evaluating the physiological importance
of these events during the early stages of development. The
introduction of this new technology is an exciting and
Transferred day 3 CI95% Transferred day 5CI95%
Table 2 Proportion of embryos
transferred depending on the
572J Assist Reprod Genet (2011) 28:569–573
powerful tool for viewing cellular activity and embryogen-
esis in a coherent, uninterrupted manner, otherwise not
available by analysis in real time.
In conclusion, the Embryoscope provides an adequate
culture environment for time-lapse imagingthatdoesnotaffect
embryo quality, blastocyst development or viability. It thus is a
useful device for monitoring the timing of early cleavages.
Galindo, María Roldán, Niels Ramsing and Karen Marie Hilligsøe for
their clinical and technical support in the management of this paper
Authors want to thank Sara Fortuño, Noemí
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