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Posters * Embryology (Embryo Selection)

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Posters * Embryology (Embryo Selection)

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Introduction: For parthenogenetic blastocyst formation, oocytes can be artificially activated by different physical and chemical stimuli. The main disadvantage of most parthenogenetic activating agents is that they cause a single rise in cytosolic Ca2 + concentrations, different from the Ca2 + oscillations seen during fertilization. In order to improve activation and development after parthenogenesis, we tried to use sperm injection and subsequent removal to mimic more closely the natural Ca2 + increases by releasing the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining. Therefore, we examined first the toxicity of Hoechst staining and UV-irradiation on artificially activated mouse oocytes. Alternatively, we used MitoTracker staining for removal of the sperm. Materials and Methods: In vivo matured mouse oocytes were collected from B6D2F1 females 14h post-hCG. Oocytes were activated by 10mM strontiumchloride in Ca-free KSOM + 2μg/ml cytochalasine D for 4h. One hour after start of activation, the oocytes were stained with Hoechst and irradiated with UV-light. Non-stained activated oocytes were used as positive control. In the first experiment, activated oocytes were stained for 10min with 0.5 or 1μg/ml Hoechst 33258 or 0.5 or 1μg/ml Hoechst 33342. In the second experiment, activated oocytes were stained with 0.5μg/ml Hoechst 33258 for 10, 20 or 30min. In a last experiment, ICSI was performed with sperm that was stained for 10min with 10μM MitoTracker Green FM and sperm was removed after 1h. Oocytes were cultured in KSOM medium and transferred into Cook Blastocyst medium on day 3. Blastocysts were differentially stained and numbers of inner cell mass (ICM) and trophectoderm (TE) were counted. Results: All oocytes in the first two experiments were activated and cleavage rate ranged from 94% to 100% in the different groups. In the first experiment, blastocyst formation was significantly higher in the positive control group (94%) compared with all other groups. For Hoechst 33258, blastocyst development was significantly higher with 0.5μg/ml (65%) compared to 1μg/ml (21%). For Hoechst 33342, blastocyst rate was 15% with 0.5μg/ml while no blastocysts could be obtained when 1μg/ml was used. The ICM/TE ratio of the blastocysts was significantly lower in the 0.5μg/ml Hoechst 33342 group (0.10 ± 0.06) compared with the groups with Hoechst 33258 (0.20 ± 0.10 for 0.5μg/ml, 0.23 ± 0.15 for 1μg/ml), but all groups were comparable with the positive control group (0.19 ± 0.08). Since the staining of sperm after ICSI was too rapidly fading away for efficient removal after 10min staining, we increased the staining time in the second experiment. Blastocyst rate was significantly higher in the positive control group (96%) compared with all other groups. Development to the blastocyst stage was significantly impaired when oocytes were stained for 20min (31%) or 30min (17%) compared to 10min (59%). No difference was detected in ICM/TE ratio of the blastocysts. In the last experiment, MitoTracker staining was not visible anymore in all oocytes after ICSI. In case the sperm head could be removed, 95% of oocytes died after 1 day. Conclusion: Our results show that both types of Hoechst combined with UV-irradiation have toxic effects on the development of parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. The effects are more severe with Hoechst 33342 than with Hoechst 33258. A higher concentration of Hoechst or longer staining times also significantly reduced blastocyst formation. We can conclude that using Hoechst staining and UV-irradiation should be avoided when working with mouse oocytes. This toxic effect is also relevant for human nuclear transfer since enucleation is frequently performed with Hoechst staining and UV irradiation. Since MitoTracker was also not succesfull for removal of the sperm, alternative methods for sperm identification should be investigated.
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
(21%). For Hoechst 33342, blastocyst rate was 15% with 0.5μg/ml while no
blastocysts could be obtained when 1μg/ml was used. The ICM/TE ratio of
the blastocysts was significantly lower in the 0.5μg/ml Hoechst 33342 group
(0.10 ± 0.06) compared with the groups with Hoechst 33258 (0.20 ± 0.10 for
0.5μg/ml, 0.23 ± 0.15 for 1μg/ml), but all groups were comparable with the
positive control group (0.19 ± 0.08). Since the staining of sperm after ICSI was
too rapidly fading away for efficient removal after 10min staining, we increased
the staining time in the second experiment. Blastocyst rate was significantly
higher in the positive control group (96%) compared with all other groups.
Development to the blastocyst stage was significantly impaired when oocytes
were stained for 20min (31%) or 30min (17%) compared to 10min (59%). No
difference was detected in ICM/TE ratio of the blastocysts. In the last experi-
ment, MitoTracker staining was not visible anymore in all oocytes after ICSI. In
case the sperm head could be removed, 95% of oocytes died after 1 day.
Conclusion: Our results show that both types of Hoechst combined with UV-
irradiation have toxic effects on the development of parthenogenetically acti-
vated mouse oocytes. Although activation and cleavage rate were not affected,
blastocyst formation was significantly reduced. The effects are more severe with
Hoechst 33342 than with Hoechst 33258. A higher concentration of Hoechst
or longer staining times also significantly reduced blastocyst formation. We
can conclude that using Hoechst staining and UV-irradiation should be avoided
when working with mouse oocytes. This toxic effect is also relevant for human
nuclear transfer since enucleation is frequently performed with Hoechst staining
and UV irradiation. Since MitoTracker was also not succesfull for removal of the
sperm, alternative methods for sperm identification should be investigated.
P-141 Evaluation of the proteomic analysis of embryo culture supernant
as a prognostic factor in IVF cycles
A. Exposito Navarro1, A. Ametzazurra2, D. Nagore2, L. Crisol1, F. Aspichueta1,
R. Mendoza1, R. Matorras1
1Assisted Reproduction Unit, Department of Obstetrics and Gynecology,
Cruces (Baracaldo) Bizkaia, Spain
2Proteomika, Parque Tecnológico de Bizkaia, Derio (Bizkaia) 48160, Spain
Introduction: Proteomic analysis of the embryonic secretome (proteins pro-
duced by the embryon and secreted into the surrounding medium) followed
by the identification of specific proteins critical for implantantion, may led to
the development of a non invasive viability assay to assist in the selection of
embryos for transfer.
The aim of the present study was identify and quantification of human cy-
tokines of cultured media in which embryos are cultured.
The MILLIPLEXTM MAP High-Sensitivity Human kit (Millipore) has been
used for the detection of cytokines in embryo culture supernatant.
Material and Methods: Principle of xMAP technology: This kit has been de-
veloped to be used with Luminex xMAP technology, based on a system of
suspended microspheres and detection using a Luminex 200 flow cytometer
(Luminex Corp).
The microspheres used are internally labelled with two fluorescent dyes. In
the assay, the antigen present in the sample (cytokine in this case) binds to the
capture antibody. The antibody is labelled with biotin (detection antibody) in
order that the fluorescence emitted can be detected and quantified.
We have analysed a total of 35 samples (< 50μl each) of embryo culture
supernatants. For this purpose, two sets of assays were carried out, one in April
2009, when 15 samples of embryo culture supernatant were analysed, and the
other one in December 2009, when 20 samples were analysed, in order to adjust
the methodology and confirm the results. The samples of supernatants come
from cultures of 48-hour post fertilization embryos in ISM1 culture medium
(ISM1™ Culture Medium, Medicult, Denmark), and from 72-hour post fertil-
ization embryos in ISM2 culture medium (ISM2™ Culture Medium, Medicult,
Denmark).
The kit used allows simultaneous quantification of the following panel of
human cytokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70),
IL-13, IFNg, GM-CSF and TNFα. It includes a standard for every cytokine (cy-
tokine stock solution at known concentration, from which a standard curve is
obtained), and a mixture of all the cytokines at minimum and maximum detect-
able concentrations (QC-1 and QC-2 respectively) for quality control.
The concentration of each cytokine present in the samples (embryo culture
supernatant) is obtained by extrapolation from the fluorescence emitted using
the standard curve for each cytokine.
17.5% (7/40), Group II 40.6% (52/128) (p = 0.026). For births after 37 weeks,
Group I rate is 72.5% (29/40) and Group II 46.9% (60/128) (p = 0.01).
Forty tree percent of all newborns (122/285) suffer from low birth weight
: 16.22% (6/40) of Group I newborns suffer from moderate hypotrophy and
32.53% (81/255) for Group II (p = 0.06)whereas 10% (4/40) of Group I new-
borns suffer from severe hypotrophy as do 12.45% (31/255) of Group II new-
borns (p = 0.56).
Maternal complication rate is 20.0% (8/40) for Group I and 30.47%
(39/128) for Group II (p = 0.67). Vascular pathologies account for 12.50% of
total cases (21/168). None of the Group I women were hospitalized for being
at risk of premature birth, whereas 10.94% (14/128) of Group II women were
hospitalized. Premature rupture of membranes affected 7.50% (3/40) of Group
I women versus 2.34% (3/128) for Group II. For Group I, term at birth without
maternal complications averages 35.9 weeks ( ±3.9) versus 34.2 ( ±4.2) in case
of complication (p = 0.21). For Group II the respective figures are the follow-
ing: 36.8 weeks ( ±2.4) versus 34.7 ( ±3.02) (p < 0.001).
Conclusion: This study gives objective information on the outcome of a triplet
pregnancy depending on its reduction to one or two embryos. None of the results
are decisive to prefer one type of multifetal pregnancy reduction over the other.
The most relevant criteria – returning home with at least one living child – does
not vary significantly between the two groups. These figures however allow for
better information and preparation of couples on the potential risks of multiple
pregnancies. It seems apparent that the couple’s request and decision should be
followed regardless of any strictly medical consideration.
POSTERS
EMBRYOLOGY (EMBRYO SELECTION)
P-140 Toxic effects of Hoechst staining and UV-irradiation on
preimplantation development of parthenogenetically activated mouse
oocytes
K. Versieren1, B. Heindryckx1, C. Qian1, J. Gerris1, P. De Sutter1
1University Hospital Ghent, Department of Reproductive Medicine, Gent,
Belgium
Introduction: For parthenogenetic blastocyst formation, oocytes can be artifi-
cially activated by different physical and chemical stimuli. The main disadvan-
tage of most parthenogenetic activating agents is that they cause a single rise in
cytosolic Ca2 + concentrations, different from the Ca2 + oscillations seen during
fertilization. In order to improve activation and development after partheno-
genesis, we tried to use sperm injection and subsequent removal to mimic more
closely the natural Ca2 + increases by releasing the oocyte activating factor.
Visualization of the sperm could be accomplished by Hoechst staining. There-
fore, we examined first the toxicity of Hoechst staining and UV-irradiation on
artificially activated mouse oocytes. Alternatively, we used MitoTracker stain-
ing for removal of the sperm.
Materials and Methods: In vivo matured mouse oocytes were collected from
B6D2F1 females 14h post-hCG. Oocytes were activated by 10mM strontium-
chloride in Ca-free KSOM + 2μg/ml cytochalasine D for 4h. One hour after
start of activation, the oocytes were stained with Hoechst and irradiated with
UV-light. Non-stained activated oocytes were used as positive control. In the
first experiment, activated oocytes were stained for 10min with 0.5 or 1μg/ml
Hoechst 33258 or 0.5 or 1μg/ml Hoechst 33342. In the second experiment, ac-
tivated oocytes were stained with 0.5μg/ml Hoechst 33258 for 10, 20 or 30min.
In a last experiment, ICSI was performed with sperm that was stained for 10min
with 10μM MitoTracker Green FM and sperm was removed after 1h. Oocytes
were cultured in KSOM medium and transferred into Cook Blastocyst medium
on day 3. Blastocysts were differentially stained and numbers of inner cell mass
(ICM) and trophectoderm (TE) were counted.
Results: All oocytes in the first two experiments were activated and cleavage
rate ranged from 94% to 100% in the different groups. In the first experiment,
blastocyst formation was significantly higher in the positive control group
(94%) compared with all other groups. For Hoechst 33258, blastocyst devel-
opment was significantly higher with 0.5μg/ml (65%) compared to 1μg/ml
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
With further development, embryo response trends downward, from posi-
tive (attraction), to negative (repulsion).
However, at any stage of development, among identical appearing embryos
cultured in optimized medium (KSOM + AA) mean response is significantly
more negative (p < 0.05) than embryos cultured in suboptimal medium (M16).
Following OET assay and return to culture conditions in KSOM + AA, em-
bryos continue to develop at a normal rate, and hatch.
Because the projected OET fields can be manipulated, in real-time, by the
operator, embryos can be physically sorted at the time of assay.
Conclusions: Light-induced dielectrophoresis provides a technically simple,
non-invasive, quantitative, and reproducible means to assay individual em-
bryos, so that the most developmentally mature embryos within any cohort of
otherwise indistinguishable embryos can be selected for IVF transfer.
We demonstrate how individual embryos can be manipulated in space, to
facilitate assay and expedite retrieval from the sorting chamber. (A short video
demonstration is provided).
P-143 The effect of long-term cryopreservation on embryo survival and
implantation
O. Bern1, D. Strassburger1, D. Komarovsky1, E. Kasterstein1, A. Komsky1,
B. Maslansky1, A. Raziel1, S. Friedler1, Y. Gidoni1, R. Ron-El1
1Med New Life, IVF and Infertility, Bucharest, Romania
Introduction: Patients who conceived in their fresh IVF treatment cycles nor-
mally wish to have their frozen-thawed embryos transferred after more then
2 years interval. Since prolongation of the freezing period of embryos is con-
sidered to be harmful to embryo quality we wished to evaluate the impact of
long and very long freezing periods on the survival and implantation rates of
the embryos.
Materials and Methods: Supernumerary embryos with good morphology
(equal sized blastomeres and ≤20% fragments) were frozen on the transfer day
of the fresh cycle. Freezing and thawing were performed with the MediCult kit
(Denmark)(slow freezing protocol/propanediol). In order to transfer the thawed
embryos on day 3 after ovulation (LH peak), all day 2 embryos were thawed on
day -1 of the transfer with overnight incubation. Day 3 embryos were thawed
on the morning of the transfer with an incubation of 2-4 h prior to the trans-
fer. Only intact embryos or embryos with ≤50% viable blastomeres (survived
embryos) were transferred. In non ovulatory patients the transfer took place
following three days of progesterone intake administered. 348 embryos in 92
thawed cycles, performed from 2006 to 2009 were evaluated in this retrospec-
tive study. All the patients delivered in their previous IVF trial. The outcome
of thawed embryos after long freezing period (L) (interval of 3-6 years) was
compared with the outcome of very long freezing period (VL) (6-11 years) in
terms of embryo survival rate, implantation, pregnancy and multiple pregnan-
cies rates.
Results: There were 58 thawed cycles with L and 34 ones with VL freezing
period of cryopreserved embryos. Mean patients’ age at the time of freezing
was 28 ± 4.2 and 27.7 ± 3.4 years, respectively and 32 ± 4.4 and 35 ± 5.4
years at the time of thawing. The intact embryos were 59% (127/215) out of all
thawed embryos for the L group and 67% (89/133) for the VL group. The over-
all survival rates were 81% (175/215) and 83% (111/133), respectively. Mean
number of transferred embryos per cycle was 2.8 ± 0.7 for the L group and
2.7 ± 0.6 for the VL group. The number of clinical pregnancies was 26 (45%)
for the L group and 16 (47%) for the VL group embryos. Implantation rates
were 21% (34/165) and 26.5% (25/94). The multiple pregnancies rates were
6/26 (23%) (4 twins and 2 triplets) for the L group and 7/16 (44%) (5 twins and
2 triplets) for the VL group. None of these differences was significant between
the two freezing periods.
Conclusion: This study demonstrated that the overall survival rates, implanta-
tion and pregnancy rates remained unchanged also after a very long freezing
period. Moreover, patients who previously conceived are prone to experience a
high number of multiple pregnancies with the transfer of their thawed embryos.
These data should be given to the patients when counseling them.
P-144 Comparison of IVF and ICSI in patients with one or two oocytes
J. Tang1, C. Fang1, M.F. Zhang1, T. Li1, G.L. Zhuang1
1The First Affiliated Hospital of Sun Yat-Sen University, Reproductive Medical
Center, Guangzhou, China
Fluorescence data analysis was carried out using SigmaPlot 11.0 software,
using a four-parameter logistic equation.
Results: In the first set of assays carried out in April 2009, (15 samples), a
level of fluorescence two-fold higher than the negative control (blank) was set
as the criteria for the results for each cytokine in each sample to be considered
significant and therefore included in the evaluation. However, in the December
assays (20 samples) (carried out twice to check the results), no fluorescence
values were discarded: on the contrary, all the values of fluorescence obtained
were included in the analysis, and concentrations were calculated for all the
cytokines which had a fluorescence signal above the minimum detection level
of the technique and falling within the range of the standard curve.
In general the fluorescence levels obtained for the 13 cytokines analysed in
the 35 samples of supernatants was very low, in most cases below the minimum
detection level of the technique, and hence they were not evaluable.
Conclusions:
1. The concentration of cytokines studied in embryo culture supernatants
was virtually zero or at least below the level of detection of this technique, so
the statistical analysis of the results is not justified.
2. The determination of cytokines using immunoassays based on suspen-
sion arrays is not a valid technique for evaluation of embryo quality in IVF.
P-142 A novel, quantitative, non-invasive method and technology to
guide selection of the most developmentally mature embryos for IVF
transfer
M.M. Garcia1, J.K. Valley2, P.S. Swinton3, W.J. Boscardin4, T.F. Lue1,
P. Rinaudo5, M.C. Wu2
1University of California San Francisco, Urology, San Francisco CA, U.S.A.
2University of California Berkeley, Electrical Engineering and Computational
Science, Berkeley CA, U.S.A.
3Gladstone Institute University of California San Francisco, Transgenic
Mouse CORE Lab Facility, San Francisco CA, U.S.A.
4University of California San Francisco, Biostatistics and Epidemiology, San
Francisco CA, U.S.A.
5University of California San Francisco, Obstetrics and Gynecology (Repro-
ductive Endocrinology and Infertility), San Francisco CA, U.S.A.
Introduction: The common pathway for the successful treatment of both male
and female infertility is the production of a healthy, viable pre-implantation
embryo.
IVF is limited in 3 critical domains: success (live birth) rate, morbidity (to
mother and fetus), and, cost (to patient, and health-care system).
Currently, embryos are chosen on the basis of visible (i.e. qualitative) mor-
phologic maturity. Although selection of the one or two “best” embryos for
transfer is essential to maximize success potential, embryo selection remains,
since its introduction in 1978, an entirely subjective endeavor. A more sensi-
tive, specific, and non-invasive means to quantitatively measure an embryo’s
developmental maturity would reduce the variability introduced by the current
standard, and likely, improve clinical outcomes.
Materials and Methods: We present a method which exploits the scaling elec-
trical properties of pre-transfer embryos, to quantitatively discern embryo de-
velopmental maturity, using light-induced dielectrophoresis termed Optoelec-
tronic Tweezers (OET).
We assayed 250 healthy mouse embryos cultured from the zygote stage
onward in KSOM + AA, and, clustered by stage of development (1-cell, 2-cell,
morula, early-, and late-blastocyst). We quantified each embryo’s response (at-
traction or repulsion) to the optically-induced OET field we projected onto
the sorting chamber slide. Linear regression, with developmental stage as a
response predictor, was performed. To assess whether OET can discriminate
between embryos that are identical in appearance but differ in development
potential, we compared OET responses, measured in a blinded fashion, among
identical paired cohorts of embryos, cultured from the zygote-stage onward
in either KSOM + AA or M16 medium. Wilcoxon rank-sum tests and Lev-
ene’s robust test for equality of variance were performed to compare paired
cohorts.
Results: OET response is a continuous outcome measure significantly corre-
lated with stage of developmental maturity (p < 0.05).
For embryos cultured in either KSOM + AA or M16, early stage embryos
(1-cell through compact morula) are attracted to the projected OET field (posi-
tive response).
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-146 Relationship between cleavage stage morphology and
comprehensive chromosome constitution
M.G. Katz-Jaffe1, J. Stevens2, S. McCormick2, R. Smith2, W.B. Schoolcraft3
1Colorado Center for Reproductive Med., Research, Lone Tree CO, U.S.A.
2Fertility Laboratories of Colorado, Embryology, Lone Tree CO, U.S.A.
3Colorado Center for Reproductive Med., Medical, Lone Tree CO, U.S.A.
Introduction: Cleavage stage morphology assessment is the predominant em-
bryo selection technique in human ART. The role of chromosome aneuploidy
screening in embryo selection is somewhat controversial and may be dependant
on the time point of embryo biopsy as well as the technique employed. To date,
studies have only described a limited association between these two embryo
selection methods that has been based on the assessment of only a handful
of chromosomes using FISH technology. In this study, we directly analysed
the relationship between cleavage stage morphology and a comprehensive as-
sessment of all 23 pairs of chromosomes in a large series of developmentally
competent blastocysts.
Materials and Methods: A total of 1,496 embryos underwent routine cleavage
stage morphology assessment on day 3 of development including blastomere
number and incidence of fragmentation. Following a further 48 hours of in vitro
culture, developmentally competent blastocysts with a clearly defined inner cell
mass were identified for trophectoderm biopsy. Comprehensive chromosome
screening (CCS) of all 23 pairs of chromosomes was performed on biopsied tro-
phectoderm cells by either metaphase comparative genomic hybridization (Re-
progenetics) or single nucleotide polymorphism microarray analysis (RMA-NJ).
Results: CCS results revealed 45.8% of human blastocysts to be euploid (cor-
rect number of chromosomes) with a mean maternal age of 37.4 ± 3.4 years.
From the group of aneuploid blastocysts (54.2%) every chromosome was im-
plicated in cell division errors including both losses and gains. In relation to
blastomere number (range 4 - > 10) no association was observed with chromo-
some constitution. The majority of aneuploid (59%) and euploid (60.5%) blas-
tocysts recorded 8 blastomeres on day 3 of embryonic development. Likewise
both slow (4-6 cells) and fast (9- > 10 cells) developing cleavage stage embryos
showed no association with the incidence of chromosome aneuploidy. Relative
to the degree of fragmentation, only embryos with < 30% fragmentation on day
3 reached the blastocyst stage and were identified for trophectoderm biopsy
with a clearly defined inner cell mass. The majority of both aneuploid (60%)
and euploid (58.5%) blastocysts were associated with < 10% day 3 fragmenta-
tion. However, no association was observed between the degree of fragmenta-
tion (range 0-30%) in the cleavage stage embryo and chromosome constitution.
In addition, no relationship was observed between the types of chromosome
errors (single, double or chaotic) and either day 3 blastomere numbers or the
incidence of fragmentation. In contrast, gender analysis revealed a probable
association between males and faster developing (9- > 10 cells) cleavage stage
embryos. The CCS technique employed had no bearing on any of these results
when analyzed separately.
Discussion: With the development of CCS techniques allowing for the analysis
of all 23 pairs of chromosomes, this study demonstrated no relationship between
cleavage stage embryo morphology and chromosome constitution in a large series
of developmentally competent blastocysts. Neither blastomere number nor the in-
cidence of fragmentation on day 3 of embryonic development could predict the
chromosome constitution of the subsequent developmentally competent blasto-
cyst. Trophectoderm biopsy may represent a more suitable time point for chromo-
some screening following the activation of the embryonic genome and the analysis
of only developmentally competent blastocysts. However, the clinical significance
of blastocyst CCS will need to be validated in a future randomized control trial.
Chromosome aneuploidies are a common occurrence in human reproduction con-
tributing to both implantation failure and early pregnancy loss, confirming the im-
portance of ongoing evaluation for inclusion in embryo selection.
P-147 In-vitro maturation of human germinal vesicle stage oocytes: role
of epidermal growth factors in the culture medium
I. Ben-Ami1, A. Komsky1, D. Strassburger1, O. Bern1, D. Komarovsky1, E.
Kasterstein1, B. Maslansky1, A. Raziel1, S. Friedler1, Y. Gidoni1, R. Ron-El1
1IVF and Infertility unit, Assaf Harofeh Medical Center Tel Aviv University
Israel, Tel-Aviv, Israel
Introduction: In-vitro maturation (IVM) of oocytes is a promising technique
to reduce the costs and avert the side-effects of gonadotropin stimulation for
Introduction: A great number of patients are poor responders to ovary stimula-
tion undergoing in vitro fertilization-embryo transfer (IVF-ET) treatment. Poor
responders have high cancel rates, low fertilization and pregnancy rates. Since
some poor responders have only one or two oocytes retrieved, how to select an
optimal fertilization technique is important to improve their pregnancy rates.
The goal of this study was to compare the results of conventional IVF with ICSI
in patients who have only one or two oocytes retrieved.
Patients and Methods: A total of 168 poor responders with one or two re-
trieved oocytes were analyzed retrospectively. On the day of oocyte retrieval,
sperm was considered as normal or abnormal via the World Health Organiza-
tion criteria before preparation. Patients with sperm concentration 1 × 106/ml
and 50% progressive motile spermatozoa (type A and B) after preparation of
the entire sample were performed IVF (106 cycles), otherwise were performed
ICSI (62 cycles). ICSI group and IVF group were analyzed according to the
woman’s age (< 35 or 35 years) and sperm quality (normal or abnormal). The
two groups were compared in terms of fertilization rates, high quality embryo
rates and pregnancy rates.
Results: Fertilization rates were significantly higher in ICSI (106 cycles) group
than in IVF (62 cycles) group (83.72% vs. 63.75%, respectively, P = 0.001);
24.53% cycles in IVF group were complete fertilization failure, significantly
higher than ICSI group (9.68%), P = 0.018. There was no significant differ-
ence between ICSI group and IVF group in high quality embryo rates (61.76%
vs.70.00 %) and pregnancy rates (16.67% vs. 24.00%). Among all the patients,
108 poor responders were older than 35 years. In patients 35 years and with
abnormal semen parameter, ICSI (42 cycles) group gave a significantly higher
fertilization rate (83.93% vs. 55.56%, P = 0.001) and a significantly lower com-
plete fertilization failure rates (14.29% vs. 34.78%, P = 0.027) but no significant
difference in high quality embryo rates (69.57% vs.88.24%) and pregnancy rates
(15.15% vs.25.00%). In patients 35 years with normal semen parameter, ICSI
group (5 cycles) and IVF group (15 cycles) produced no significant difference
in fertilization rates (66.67% vs.47.83%), high quality embryo rates (33.33%
vs.80.00%) and pregnancy rates (0.00% vs.30.00%). In patients < 35 years with
abnormal semen parameter, ICSI group (14 cycles) and IVF (28 cycles) group
produced similar fertilization rates (86.96% vs.81.08%), high quality embryo
rates (55.56% vs.57.69%) and pregnancy rates (25.00% vs.18.18%).
Conclusion: In cycles with only one or two oocytes retrieved, high quality em-
bryo rates and pregnancy rates cannot be increased by ICSI, even with higher
fertilization rate.
P-145 Effects of the artificial shrinkage and assisted hatching before
vitrification on the development of the vitrified mouse expanding
blastocysts
D.S. Suh1, J.K. Joo1, J.R. Choi1, S.C. Kim1, M.S. JO1, K.H. Kim1, K.S. Lee1
1Pusan Natational University, Obstetrics & Gynecology, Busan, Korea South
Objective: This study was conducted to investigate the effects of the artificial
shrinkage and assisted hatching (PZD; patial zona dissetion) before vitrification
on the development of vitrified mouse expanding blastocyst.
Methods: Mouse 2-cell embryos were collected and cultured in G1.1 and G2.2
to expanding blastocyst. For artificial shrinkage (AS) the micro injection pi-
pette was inserted into blastocoele cavity and blastocoele fluid was aspirated.
For assisted hatching (AH) PZD method was used. Control group was -AS/-
AH and treatment groups were -AS/ + AH, + AS/-AH and + AS/ + AH. After
AS and AH mouse blastocysts were equillibrated in G10 and G10E20 for 3
mins, respectively, and vitrified in G25E25 after loading on capped pulled-
straw. Vitrified mouse blastocysts were thawed and cultured for 24 hrs. The
survival and hatching rate was compared among one control and three treat-
ment groups.
Results: The survival rates were 99%, 92% in + AS/ + AH and + AS/-AH
groups and 54%, 58% in -AS/-AH and -AS/ + AH group, respectively. The sur-
vival rate was significantly higher in + AS group than in -AS group (p < 0.01).
Hatching rates were 34%, 96% in -AS/-AH and -AS/ + AH groups and 41%,
100% in + AS/-AH and + AS/ + AH, respectively. The hatching rates was higher
in + AH group than in -AH group (p < 0.01). After thawing recovery rates were
100%. Loading on capped pulled-straw, that is effective and useful method on
vitrification.
Conclusion: This study showed that artificial shrinkage of blastocoele cavity
and assisted hatching (PZD) significantly improved the development of the vit-
rified mouse expanding blastocysts.
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were refrozen or 2. once-frozen embryos were not transferred due to inadequate
endometrial lining and thus had to be refrozen. The outcome of these twice-fro-
zen embryo transfer treatment cycles were compared with once-frozen embryo
transfer treatment cycles where all the embryos were generated from the same
fresh IVF/ICSI treatment cycle. Both first and second freezing and subsequent
thawing of embryos was performed using the slow freeze/thaw technique. The
main outcome measure was live-birth per embryo transfer.
Results: There were 44 women who had 52 twice-frozen embryo transfer
treatment cycles. These women had previously had 47 once-frozen embryo
transfer treatment cycles with all embryos generated from the same IVF/ICSI
treatment cycle. The mean age was 32 ± 4.4, reason for infertility 32.5% male
factor, 37.5% female factor, 17.5% mixed, 5% unexplained and 7.5% other.
The mean number of embryos transferred was 1.1 in both the once-frozen and
twice- frozen embryo transfer treatment cycles. The clinical pregnancy rate per
embryo transfer was 14/52 (27%) for twice-frozen embryo transfer and 12/47
(25.5%) for once-frozen embryo transfer ( P = 0.875). The live-birth rate per
embryo transfer was 7/52 (13%) for twice-frozen embryo transfer and 6/47
(13%) for once-frozen embryo transfer (P = 0.918).
Conclusions: Twice- frozen embryos appear to have a similar clinical preg-
nancy and live-birth rate as once-frozen embryos generated from the same
fresh IVF/ICSI treatment cycle, although the numbers are limited. Consider-
ation may be given to refreezing supernumerary once-frozen-thawed embryos
for future use.
P-149 BMP-2 and BMP-4 expression in early stages of neural tube
formation in human embryos
A. Namm1, A. Arend1, M. Aunapuu1
1Anatomy Institute of Tartu University, Histology and Embryology, Tartu,
Estonia
Introduction: Development, regional specification and morphogenesis of the
human brain seem to be controlled by Bone Morphogenetic Proteins (BMP’s).
BMP’s are members of TGF superfamily and play important roles in multiple
biological events. BMP’s were originally identified by the ability to induce the
formation of bone and cartilage. BMP’s have also important roles during em-
bryonic development of the embryonic pattering. In human embryonic develop-
ment BMP-2 and BMP-4 are critical signaling molecules required for the early
differentiation of the embryo and the establishment of the dorsal-ventral axis.
Despite of numerous investigations in animals, especially in rats and chicken,
only limited results exist about the developmental role of these regulators in the
formation of the human central nervous system. In our studies we investigated
BMP-2 and BMP-4 expression in the human embryos and we determined spa-
tial and temporal expression of BMP’s during the early stages of neural tube
development.
Material and Methods: Sixteen human embryos of Carnegie stages (CS)
14-20 were obtained by medical abortions. The study was approved by the Eth-
ics Review Committee on Human Research of the University of Tartu. The
embryos were fixed in 4% formaldehyde and embedded in paraffin according
to standard methods and were serially cut in transversal direction. The sec-
tions were stained with H&E and thionine for histological investigations. For
immunohistochemistry sections mounted on poly-L-lysine coated slides were
deparaffinized and rehydrated. Peroxidase activity was removed by 0.6% H2O2
in methanol. Then sections were washed in tap water and in PBS (pH = 7.4),
treated with normal 1.5% goat serum and incubated with the first antibody:
BMP-2 diluted 1:250 or BMP-4 diluted 1:100. In the next day sections were in-
cubated with secondary antibody. Peroxidatic activity was detected with DAB.
Sections were counterstained with hematoxylin. The BMP-2, BMP-4 labeling
was expressed by a subjective scale ranging from 0 to 3 (0 - no staining, 1 -
weak staining, 2 - moderate staining, 3- strong staining). Immunohistochemis-
try negative controls were performed by omitting primary antibody. The results
were estimated by two independent observers.
Results: Considerable BMP-2 and BMP-4 reaction appeared in embryos of CS
14 to 18. The strength of the reaction stayed between 0.6-1.6. Expression of
BMP’s was at low level in embryos of CS 20. In the present study we demon-
strate that the expression of BMP’s varied along the dorsal-ventral axis of neural
tube. Different intensity of immunostaining between the dorsal and ventral part
of the neural tube in the embryos of CS 14-18, but difference was not revealed
in the embryos of CS 20. The moderate and high level of BMP-4 expression
was detected in non-neural ectoderm. The reaction appeared in embryos of CS
in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro
are lower than those of in-vivo matured oocytes as in IVF indicating that opti-
mization of IVM remains a challenge. Recently, it was demonstrated that LH
exerts, at least in part of its action, on ovulation through stimulation of the
production of the epidermal growth factor (EGF) family members amphiregulin
(Areg) and epiregulin (Ereg) in pre-ovulatory follicles. The Areg and Ereg, in
turn, serve as paracrine mediators of LH. We aimed to investigate the effect of
supplementation of the EGF-like growth factors Areg and Ereg to the oocyte
culture medium on the IVM rate of human germinal vesicle (GV) stage oocytes.
We further aimed to compare the intracytoplasmatic sperm injection (ICSI) out-
come of the in vitro matured oocytes following incubation in IVM media either
with or without the addition of Areg and Ereg, including the rate of fertilization,
cleavage and embryo quality.
Materials and Methods: All the oocytes obtained from the follicular aspirates
were partially denuded from the surrounding cumulus oophorus using 0.1%
hyaluronidase for 1–2 min in order to ease the identification of GV stage eggs.
Following this, the presence of the GV was searched, using an inverted mi-
croscope (Diaophot 300; Nikon, Japan) with a magnification of X100; X200
and X400. The oocytes identified in their GV stage were separated from the
mature oocytes and cultured in IVM media (Sage/Cooper Surgical, CT, USA)
either with or without supplementation of recombinant human Areg (75 ng/
ml) and Ereg (75 ng/ml) for 24 hours. The oocytes were observed under an
inverted microscope (Diaophot 300; Nikon, Japan) after 24h incubation. Oo-
cyte maturation was determined by GV breakdown (GVBD)/metaphase I (MI)
or metaphase II (MII) stages. Oocytes reaching MII were used for ICSI. The
matured oocytes were injected with frozen thawed donor sperm. Culture of the
injected oocytes took place in 25 ml medium droplets (G1.2.Vitrolife) under
oil. Oocytes were observed for fertilization 16 ± 18 h after micromanipulation.
Normal fertilization was defined as the presence of two extruded polar bodies
(PBs) and two pronuclei. Cleavage was assessed 24 and 48 h later and the em-
bryos were classified according to their morphological appearance. Following
the attainment of day 2 embryos, they were all disposed.
Results: 81 human GV oocytes obtained after gonadotropin stimulation were
randomly cultured in IVM media (Sage/Cooper Surgical, CT, USA) either with
or without supplemented recombinant human Areg (75 ng/ml) and Ereg (75 ng/
ml) for 24 hours. Seventy six percent of the GV stage oocytes cultured in the
IVM media supplemented with Areg and Ereg reached MII stage at 24h versus
39% of the GV stage oocytes cultured in the standard IVM media (p = 0.001).
Significantly more MII oocytes underwent normal fertilization following ICSI
in the Areg and Ereg-supplemented IVM media than those cultured in the stan-
dard IVM media (57.1% versus 28.2%, p < 0.01), yielding higher rates of day
2 (54.8% versus 28.2% p < 0.05) and day 3 (47.6% versus 23%, p < 0.05) em-
bryos. There was no significant difference in the mean blastomere number of
day 2 and day 3 embryos attained from in vitro matured oocytes achieved from
Areg and Ereg-supplemented medium and those which were cultured in the
standard IVM medium (3.5 ± 1.3 and 5.7 ± 2.7 versus 4.1 ± 1.3 and 5 ± 1.6
respectively).
Conclusion: Supplementation of the maturation medium with Areg and Ereg
significantly improves the maturation rate of human GV oocytes in vitro, yield-
ing higher fertilization rate and larger number of human embryos.
P-148 A retrospective comparison of live-birth rate of once-frozen
versus twice-frozen embryo transfer
J. Koch1, M. Costello1, S. Kilani1
1IVF Australia, Fertility, Sydney NSW, Australia
Introduction: The first ever pregnancy arising from a frozen embryo was re-
ported in 1983. During the first thaw more embryos may be thawed than re-
quired and in some cases these supernumerary embryos have been refrozen,
to then become twice-frozen embryos. There is limited data on the outcome
of transferring twice-frozen embryos to the uterus, with no previous con-
trolled studies reporting on live-birth rate. The aim of this study was to compare
the pregnancy and live-birth rates in once versus twice-frozen embryo transfer
treatment cycles in the same cohort of women.
Material and Methods: This is a retrospective study of all women having IVF/
ICSI treatment between January 2003 and May 2009 at IVF Australia, Sydney,
Australia who had twice-frozen embryo transfer treatment cycles. These women
had twice frozen-embryos for one of two reasons; 1.multiple embryos were
thawed in the first thaw cycle and grown to blastocyst and excess blastocysts
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with good prognosis, including criteria of age 37 and less, no prior IVF failure,
and the availability of one or more high quality blastocysts. Oocytes under-
went ICSI and cultured in Sage media for extended culture. On day 5, eSET
cases were identified by using a scoring system that takes in to account embryo
morphology based on the size of the inner cell mass (ICM) and number of
trophectoderm cells and also if there was one or more blastocysts suitable for
cryopreservation.
Results: The mean age of the patient population was 31.1 ± 3.3 years. On av-
erage, 18 oocytes per patient were retrieved. Out of a total of 5738 oocytes
retrieved, 79.5% were injected, the majority (98.1%) of the fertilized oocytes
cleaved on day 2, 74.3% of the fertilized oocytes went on to blastocysts, and
53.3% of them were suitable for cryopreservation. In 314 eSET’s, 224 posi-
tive pregnancies (71.3%) were achieved with 205 clinical pregnancies (65.3%),
an implantation of 67.2% (211/314) and 188 ongoing pregnancies, yielding a
59.9% ongoing pregnancy rate. Furthermore, 73 patients came back for a frozen
embryo transfer (FET), of which 75.3% achieved a positive pregnancy, and a
clinical and ongoing pregnancy rate of 67.1% (49/73). That leads to a cumula-
tive ongoing pregnancy rate (fresh and frozen transfers combined) of 75.5%
(237/314) per oocyte retrieval. The added value of cryopreservation is 15.6%.
With today 124 healthy babies were born (59 boys and 65 girls).
Conclusions: Extended culture generates high pregnancy and implantation rates
even when we are transferring single embryos. A large cohort of good quality
blastocysts with a high percentage of them suitable for cryopreservation usually
qualified our patients who underwent an eSET. After 314 eSET’s, we observed a
high clinical pregnancy and implantation rate as well as a significant decrease in
the number of multiple-order pregnancies (less than 3%). Furthermore, combin-
ing the ongoing pregnancy of fresh and frozen embryo transfers, a cumulative on-
going pregnancy of more than 75% was observed. In conclusion, this data shows
that a successful implementation of eSET’s for clinical application is achievable
by using basic embryonic knowledge rather than relying on expensive equipment
and complex technology which is still under scientific development.
P-152 ICSI improve embryo quality in non-male factor patients with
suboptimal fertilization but not in those with normal fertilization by IVF
treatment
M.J. Chen1, H.F. Guu1, Y.F. Chen1, Y.J. Yih1, J.Y.P. Ho1, T.Y. Lin1, E.S.C. Ho1
1Veterans General Hospital of Taichung, Dept. of Ob/gyn, Taichung City,
Taiwan R.O.C.
Introduction: The purpose of this study is to investigate whether the perfor-
mance of ICSI for non-male factor infertility patients could improve the quality
of embryos in addition to guaranteeing the fertilization.
Material and Methods: The ART database from Jan 2002 till Sep 2009 was
reviewed (total 1169 cycles). We first excluded all male factor patients that
ICSI deemed mandatory and all those cycles with total fertilization failure by
IVF treatment. Then we chose those repeated cycles with first cycle by IVF
and second cycle by ICSI treatment (sequential IVF-ICSI: 72 cycles with 36
sets of patients) and all cycles with half IVF and half ICSI treatment (concomi-
tant IVF-ICSI: 45 cycles) for data analysis. Embryo quality (EQ) comparisons
between IVF and ICSI treatment were by (1) average embryos score from all
resulted embryos (aES), (2) average score from top three embryos (tES), and
(3) rate of good quality embryos (rGE). Paired samples statistics were carried
out by SPSS-PC ver.11.5 with p < 0.05 as statistical significance.
Results: For those with sequential IVF-ICSI treatment cycles (N = 25 sets)
with comparable embryo transfer (ET) timing, the aES, tES and rGE in IVF as
compared to ICSI cycles were 11.8 vs. 14.8 (p = 0.07), 14.1 vs. 19.0 (p = 0.002)
and 0.29 vs. 0.43 (p = 0.17). Subgroup analysis revealed that for those IVF cy-
cles (N = 8 sets) with fertilization rate (FR) 60% or more, those EQ parameters
were not statistically different. (p = 0.09, 0.21 and 0.73). However, for those
IVF cycles (N = 17 sets) with suboptimal FR (less than 60%), the EQ param-
eters become statistically different (p = 0.045, 0.004 and 0.183). For those with
concomitant IVF-ICSI treatment cycles with ET(N = 43), the EQ parameter in
IVF as compared to ICSI cycles were not significantly different as whole group
(p = 0.75, 0.16 and 0.31) and in those patients (N = 27) with optimal IVF FR
(p = 0.53, 0.58 and 0.23). However, in those IVF cycles (N = 16) with subop-
timal FR, the aES, tES and rGE were 12.8 vs. 13.6 (p = 0.58), 13.0 vs. 16.8
(p = 0.04) and 0.35 vs. 0.38 (p = 0.85).
Conclusions: These analyses revealed that in those patients with non-male fac-
tor infertility enrolling into IVF treatment with suboptimal fertilization result,
14-18. In addition, we noticed BMP-4 expression in embryos of CS 14 in the
roof plate of the developing neural tube and also BMP-4 expression in neural
crest cells was seen.
Conclusions: This study demonstrated important roles of BMP’s in the devel-
oping neural tube of human embryos. Our investigations have also confirmed
the idea that BMP-2 and BMP-4 are important mediators in the dorsal part of
human embryos and may initiate multiple pathways controlling the specifica-
tion, proliferation and differentiation of neurons in the developing neural tube.
P-150 Role of leptin in improvement of oocyte quality by regulation of
ovarian angiogenesis
J.K. Joo1, K.S. Lee1, Y.M. Choi2, J.D. Cho3
1Pusan National University Hospital, OB/GY, Busan, Korea South
2Seoul National University Hospital, OB/GY, Seoul, Korea South
3Elle-medi hospital, OB/GY, Changwon, Korea South
Introduction : Ovarian angiogenesis plays an important role in folliculogen-
esis. An active blood supply via ovarian angiogenesis seems to be essential for
the induction of oocytes with good quality. Leptin is an angiogenic factor which
regulates VEGF expression. This study was aimed to investigate whether leptin
administration during superovulation influences ovarian response, oocyte qual-
ity and VEGF expression in the ovary using different aged mice model.
Material and Methods : C57BL inbred female mice of two age groups (18-21,
and 29-31 weeks) were superovulated by intraperitoneal co-injection with 5 IU
of pregnant mare’s serum gonadotropin (PMSG) supplemented with recom-
binant mouse leptin at various doses (0.01, 0.1, 1 mg), followed by injection
with 5 IU of human chorionic gonadotropin (hCG) approximately 48h later.
Then, the mice were immediately paired with an individual male. The control
group was superovulated with PMSG and hCG without leptin. After 18 hours,
one-cell embryos were flushed and cultured for 4 days. Proteins were extracted
from ovaries removed just after the retrieval of one-cell embryos and VEGF
expression was examined by western blot.
Results : Treatment of 0.1 μg and 1 μg leptin significantly increased the number
and embryo development rate of one-cell embryos retrieved compared to the
control group. This positive effect of leptin was more significant with advanc-
ing female age. Ovarian VEGF expression was also significantly increased in
0.1 and 1 μg leptin-treated groups compared to the control group in both age
groups (P < 0.05).
Conclusions : Our present study showed that leptin administration with go-
nadotropins during superovulation in aged mice increased the ovarian response,
developmental competence of oocytes and ovarian VEGF expression. This re-
search may have potential clinical implications in the treatment of age-related
decline of fertility.
P-151 Elective single embryo transfer (eSET): A potentially safer
and successful approach for patients undergoing assisted reproductive
technology
C. Sipe1, E.J. Pelts1, J.M. Matthews1, S.R. Sanchez1, R.L.B. Brohammer1,
Y. Wagner1, J. Liebermann1, M. Uhler1, A. Beltsos1
1Fertility Centers of Illinois, IVF Laboratory, Chicago IL, U.S.A.
Introduction: The current established method for embryo selection in clinical
application is based on morphological and physical characteristics identified
using light microscopy. However, more and more voices are rising from the
clinical community arguing that morphology is not only subjective, but also a
relatively poor indicator of developmental potential. In turn, a variety of mini-
mal invasive approaches to assess embryonic developmental potential (such as
metabolism) are being developed. At the moment, most of these approaches are
still under scientific development and the vast majorities of IVF clinics are un-
able to afford proteomic/metabolomic studies, or are financially unable to equip
a laboratory with expensive equipment necessary to perform such studies. In
addition, most of these so-called non-invasive assays require major changes in
existing gamete/embryo culture and handling methods. Therefore, in an eSET
study performed at the Fertility Centers of Illinois (Chicago), we went back to
the basics of embryology using morphology criteria for the selection of good-
quality embryos, combined with a careful selection of patients for eSET, and
without changing the existing culture system.
Material and Methods: Between 2008 and 2009, records of a total of 314
autologous eSET on day 5 were reviewed. ESET was recommended to patients
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the retrieval of fewer oocytes leads to a higher number of fetal heart beats. In
the present study, deviation on normal oocyte morphology correlated with low
oocyte efficiency. Therefore, high oocyte yield could be considered an indi-
cator of low oocyte biological efficiency and cytoplasmic dysmorphisms may
contribute to biological wastage suggesting that protocols of minimal or mild
stimulation should be considered.
P-154 Developmental regulation and decisions in the human
preimlantation embryo
H. Van de Velde1, G. Cauffman1, A. Verloes2, C. De Paepe3, J. Sterckx1, H. Van
Ranst1, P. Devroey1, H. Tournaye1, I. Liebaers3
1UZ Brussel, Centre for Reproductive Medicine, Brussels, Belgium
2UZ Brussel, Department of Hematology, Brussels, Belgium
3UZ Brussel, Centre for Medical Genetics, Brussels, Belgium
Introduction: The zygote is the ultimate totipotent cell. Totipotency is lost dur-
ing preimplantation development, however it is unknown when and in which
cells. According to the inside-outside hypothesis of Tarkowski et al. (1967), the
outer cells of a morula become trophectoderm (TE) cells, whereas the inner
cells become inner cell mass (ICM) cells. The first morphologically visible sign
of differentiation becomes apparent at the fifth day of preimplantation develop-
ment during blastulation when the two lineages can be distinguished. Blastom-
eres of 2-cell, 4-cell and 8-cell stage human embryos are thought but not proven
to be totipotent. In order to allow the embryo to overcome perturbations in its
organization such as cell loss due to fragmentation, cryodamage and biopsy for
preimplantation genetic diagnosis or disturbance of the cleavage planes, the
blastomeres should be flexible and capable to develop into ICM and TE. This
has been called regulative development. The developmental decision to become
ICM or TE is crucial. We aimed (1) to investigate regulative development and
(2) to determine when the decision to develop into ICM or TE becomes ir-
reversible.
Material and Methods: Embryos were created in vitro with the approval of the
Belgian Federal Committee on medical and scientific research on embryos in
vitro and the Local Ethical Committee using gametes from consenting donors.
In addition, embryos which were diagnosed to carry a genetic disease were
used after informed consent. Only top quality embryos were used. Embryos
were biopsied in Ca2 + /Mg2 + free medium using laser biopsy. Blastomeres were
aspirated using a pipette with an inner diameter of 60-80 mm and put into an
empty zona pellucida (ZP). Reconstituted embryos were cultured individually
in sequential medium in microdroplets under oil up to day 6 of preimplanta-
tion development. They were analysed by immunocytochemistry and confocal
microscopy for the expression of NANOG (ICM) and HLA-G (TE) (data not
discussed).
Results: In order to investigate regulative development in vitro, we disturbed
the organization of the embryo by changing the position of the blastomeres
at the 4-, 8- and compacting/compacted stage. Seven tetrahedron 4-cell stage
embryos (day 2), eight 8-cell stage embryos (day 3) and eight compacting/com-
pacted embryos (14-28 cells on day 4) were disaggregated and the blastomeres
were randomly transferred into an empty ZP. Six, eight and seven reconsti-
tuted embryos respectively developed into good quality blastocysts with TE
and ICM.
In order to determine at which stage the decision to become ICM or TE
is irreversibly taken, we investigated the potency of the outer cells to develop
into blastocysts with TE and ICM. Seven compacting/compacted embryos were
manipulated by removing a limited number of outer cells (5-7) in order not to
confuse with inner cells and these cells were transferred into an empty ZP. Six
out of these ZPs with outer cells only developed into blastocysts with TE but
none of them appeared to have an ICM. Six out of the seven original embryos
(few remaining outer + all inner cells) developed into good quality blastocysts.
In order to exclude the possibility that 5-7 outer cells is not enough mass for the
development of an inner cell population within the embryo, outer cells of two
distinct embryos were transferred into an empty ZP (12-14 cells). Four embryos
reconstituted with outer cells only developed into good quality blastocyst with
TE and ICM, as well as the eight original embryos.
Conclusions: Interference with the localisation at the 4-cell, 8-cell and com-
pacting/compacted stage does not interfere with blastocyst formation, indicat-
ing that human preimplantation development is highly regulative. The decision
by the outer cells to become TE is not irreversibly taken at the compacting/
compaction stage of human preimplantation development.
ICSI might not only improve the availability of better quality embryos for
transfer (concomitant and sequential data) but also improve the average embryo
quality in general (sequential data). These effects were not demonstrated in op-
timal fertilization IVF patients. Verification by further prospective randomized
studies should be done.
P-153 Oocyte yield as predictor of biological efficiency in patients
younger than 35 years
F.B. Lopes1, R.C.S. Figueira2, D.P.A.F. Braga3, R.C. Ferreira2, T. Aoki1, A.
Iaconelli Jr.1, E. Borges Jr.1
1Fertility - Assisted Fertilization Center, Clinical Department, São Paulo,
Brazil
2Fertility - Assisted Fertilization Center, IVF Laboratory, São Paulo, Brazil
3Fertility - Assisted Fertilization Center, Scientific Department, São Paulo,
Brazil
Introduction: Despite remarkable progress in both clinical and embryologi-
cal aspects of Assisted Reproductive Technologies, the number of viable em-
bryos and fetal heartbeat per retrieved oocyte during intracytoplasmic sperm
injection (ICSI) cycles are still disappointingly low. Aggressive protocols of
controlled ovarian stimulation aiming to maximize the number of retrieved
oocytes may lead to the recruitment of follicles that might have become atretic
in vivo. This, in turn, could compromise oocyte quality resulting in different
morphologic abnormalities. As a result, the assumption that more oocytes and
thus more embryos would improve treatment success is still controversial.
In order to enhance the knowledge on the consequences of ovarian response
on ICSI outcomes, this study was designed to evaluate whether oocyte yield
could be an indicator of oocyte biological competence. Moreover, as an at-
tempt to identify factors that may contribute to oocyte biological wastage,
we investigate whether oocyte dysmorphisms could affect its developmental
competence.
Material and Methods: Since maternal age, number of previous treatments
and male infertility factor could influence the evaluated parameters, this obser-
vational study excludes male factor cases and includes female patients younger
than 35 years old who underwent their first ICSI cycle. Patients were subdivided
according to the number of retrieved oocytes: 1 to 5 oocytes (Group-1, N = 56);
6 to 10 oocytes (Group-2, N = 85); 11 to 15 oocytes (Group-3, N = 79); 16 to 20
oocytes (Group-4, N = 39) and > 20 oocytes (Group-5, N = 76). Groups were
compared regarding clinical and laboratorial outcomes. Before sperm injection,
cytoplasmic (cytoplasmic granularity, smooth endoplasmic reticulum clusters
and vacuoles) and extracytoplasmic (perivitelline space size, perivitelline space
granularity, fragmented first polar body) oocyte abnormalities were recorded.
Regression analysis were performed to investigate the influence of oocyte dys-
morphisms on the percentage of viable embryos, high quality embryos and fetal
heart beat per retrieved oocyte.
Results: Similar normal fertilization rate (73.2% Group-1; 70.4% Group-2;
71.4% Group-3; 66.3% Group-4 and 69.0% Group-5, P = 0.3481), num-
ber of embryos transferred (2.0 ± 1.3 Group-1; 2.6 ± 1.2 Group-2; 2.6 ± 1.4
Group-3; 2.4 ± 1.4 Group-4 and 2.5 ± 1.2 Group-5, P = 0.4821) and pregnan-
cy rate (35.7% Group-1; 41.5% Group-2; 40.0% Group-3; 35.5% Group-4
and 43.5% Group-5, P = 0.1011) were found among the groups. However,
the percentage of oocytes that produced viable embryos (60.2% Group-1;
53.2% Group-2; 49.5% Group-3; 40.7% Group-4 and 40.0% Group-5,
P < 0.0001) and high quality embryos (34.7% Group-1; 27.2% Group-2;
23.0% Group-3; 22.0% Group-4 and 19.7% Group-5, P = 0.0016) decreased
significantly according to oocyte yield. The number of fetal heartbeat per re-
trieved oocyte (8.5% Group-1; 7.5% Group-2; 3.8% Group-3; 2.8% Group-4
and 1.8% Group-5, P < 0.001) was also statistically significantly higher in
patients with fewer oocytes collected. Evaluation of the oocyte morphology
was undertaken in 3408 metaphase II oocytes. We observed a significant
negative effect of the occurrence of oocyte intracytoplasmic dysmorphisms
and the number of fetal heartbeat per oocyte rate (OD: 1.48, CI: 1.03-1.36,
P = 0.018).
Conclusions: Our data shows that fertilization procedure, the first event of oo-
cyte loss during in vitro fertilization, is not affected by the number of retrieved
oocytes. Embryo development, described as responsible for the major oocyte
loss, is significantly compromised according to oocyte yield. Our study shows
that the higher the number of retrieved oocytes, the lower the chances for these
oocytes to become a high quality embryo. On the other side, we observed that
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embryo for each patient. Embryos were vitrified using Blastocyst Vitrifica-
tion Kit and warmed using Blastocyst Warming Kit (Cook, Sydney IVF) or
slow-frozen using G-FreezeKit Blast™ and thawed with G-ThawKit Blast™
(Vitrolife, Sweden).
Results: A total of 347 vitrified embryos were warmed, of which 317 survived
the process intact giving a survival rate of 91.4%. Only 274 slow-frozen em-
bryos survived the thaw from a cohort of 359 embryos giving a survival rate of
76.3% in this group. A pregnancy rate per embryo warmed of 27.1% (94/347)
was observed in the vitrification group versus a pregnancy rate per embryo
thawed of 19.8% (71/359) for the slow frozen group. Similarly a difference
was seen in clinical pregnancy rate/embryo thawed: 23.1% (80/347) for the
vitrification group and 15.6% (15.6%) for the slow frozen group. Finally, the
implantation rate per embryo thawed was found to be 30.5% (106/347) for
vitrified-warmed embryos but only 18.9% (68/359) for embryos which were
slow-frozen. The mean age for the vitrification group was 35.2 and 36.4 for the
slow-frozen group.
After adjusting for the difference in age between the two groups the preg-
nancy rate (p = 0.01), clinical pregnancy rate (p = 0.003) and implantation rate
(p = 0.001) per embryo thawed was found to be highly significantly improved
for the vitrified embryo group.
Conclusion: In the current climate of advocating elective single embryo trans-
fer, it is imperative to offer a cryopreservation programme with good survival
and pregnancy rates. CRGH has employed a very successful embryo vitrifica-
tion programme since 2007. This study has shown highly significant improve-
ment in survival, pregnancy, clinical pregnancy and implantation rates with
vitrified-warmed embryos.
P-157 Predictive value of embryos with top quality score does not
correlate with female age
T. Milachich1, L. Petkova1, D. Barov1, A. Shterev1
1SAGBAL Dr. Shterev Hospital, IVF Unit, Sofia, Bulgaria
Introduction: It is well known that numerous factors have influence on IVF/
ICSI outcome. Female age and embryo quality are among the factors with
highest priority. However, the correlation between these factors has not been
completely clarified. Thus the aim of this study is to search for any correla-
tions between the female age and the top quality scores of embryos trans-
ferred.
Material and Methods: We conducted a prospective randomize study of 1090
embryos for transfer, obtained from 452 couples after IVF/ICSI program. Aver-
age female age was 33.4 ± 4.95 (range 21-47 age). The main number of trans-
ferred embryos on day three was 2.3 ± 0.6. We have distributed embryos into
three groups (A, B and C) according to their quality on day 3 (62-64 hours post
insemination). This scoring consisted of the following parameters: cell number
and size, symmetry (distribution of blastomeres in the embryo), fragmenta-
tion and mononucleation. Thus the top quality embryo in group A has 7-8 cells
identical size, with less than 10% fragmentation, plus symmetry and mononu-
cleation in 1-3 cells (table 1):
Table.1
Quality of Cell - number Fragmentation Symmetry Mononucleated
embryos on day 3 and size (%) blastomeres
A 7-8 equal <10 Yes Yes
B 7-8 equal <10 Yes No
C <7>8, 7-8 non equal <10>10 Yes, No No
In accordance with the cohort of embryos per transfer in each woman, we
separated the patients in 3 groups: Group 1 (grade A); Group 2 (A + B, A + C)
and Group 3 (C, B + C).
In addition each patients group was divided into two subgroups – below and
above 35 years of age (table 2).
Results: Pregnancy rate in group 1 reached the average level of 52.6% per
embryo transfer. This study had shown that in the two subgroups 1.1. and
1.2. pregnancy rate (PR) per ET was similar (52.1% and 54.2%) (P > 0.05).
In contrast, the subgroups 2.1. and 2.2. revealed significant differences in PR
results and the same outcome was obtained in groups 3.1. and 3.2. (P 0.05).
The embryos belonging to group C in our morphological study had resulted in
barely 11.5% pregnancy rate and only 6.3% of older than 35 years women in
this subgroup got pregnant (table 2). Unlike the embryos of average and poor
P-155 The fate of the mosaic embryo: chromosomal constitution and
development of day 4, 5 and 8 human embryos
M.A. Santos1, G. Teklenburg1, N.S. Macklon2, D. Van Opstal3, G.H.
Schuring-Blom4, P.J. Krijtenburg4, J. de Vreeden-Elbertse1, B.C. Fauser1,
E.B. Baart5
1University Medical Centre Utrecht, Reproductive Medicine and Gynaecology,
Utrecht, The Netherlands
2University of Southampton, Obstetrics and Gynaecology, Southampton,
United Kingdom
3Erasmus Medical Centre, Clinical Genetics, Rotterdam, The Netherlands
4University Medical Centre Utrecht, Medical Genetics, Utrecht, The
Netherlands
5Erasmus Medical Centre, Obstetrics and Gynaecology, Rotterdam, The
Netherlands
Background: Post-zygotic chromosome segregation errors are very common
in human cleavage stage embryos after in vitro fertilisation, resulting in chro-
mosomally mosaic embryos. Little is known about the significance of mosa-
icism for the developmental potential. We assessed embryo development and
chromosomal constitution of embryos from compaction to the peri-implanta-
tion stage at day 8 after fertilisation.
Methods: From 112 donated cryopreserved day 4 human embryos, 21 were im-
mediately fixed and all cells analysed by fluorescent in situ hybridisation (FISH).
The remaining 91 embryos were thawed, with 54 embryos suitable for biopsy
of one or two cells. Biopsied cells were fixed and analysed by FISH for chro-
mosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. Biopsied embryos were kept in
standard culture conditions for 24h. On day 5, arrested embryos (n = 24) were
fixed completely, and developing blastocysts (n = 24) submitted to co-culture for
a further 72 hrs on a monolayer of decidualised endometrial stromal cells, fol-
lowed by fixation. Cell numbers were counted and all nuclei analysed by FISH.
Data derived from a previous FISH analysis done by our group on cryopreserved
good quality day 5 blastocysts (n = 36) were included in the present study.
Results: FISH analysis was successfully performed on 18 day-4 embryos.
According to our definition, 80% of embryos were mosaic and 11% showed
a chaotic chromosomal constitution. FISH analysis of two blastomeres from
morula-stage embryos showed that 54% of the embryos were mosaic, 40% nor-
mal, and 6% abnormal. Analysis of day 5 (n = 24), and day 8 embryos (n = 12)
showed a decrease in incidence of mosaic embryos over time, from 70% to
42%, respectively. A significant positive correlation was observed between the
total cell number and the percentage of normal cells in developing day 5 and
day 8 embryos but not in day 4 or arrested day 5 embryos.
Conclusions: These data suggest that a proportion of mosaic embryos undergo
developmental arrest between compaction and cavitation. If the embryo con-
tinues to develop, reduced proliferation or cell death of aneuploid cells may
be responsible for the increased proportion of chromosomally normal cells
throughout development of human embryos.
P-156 Vitrified versus slow-frozen blastocysts: a clinical audit for 706
embryos
S. Cawood1, A. Doshi1, S. Gotts1, P. Serhal1
1University College London Hospital, The Centre for Reproductive and
Genetic Health, London, United Kingdom
Introduction: Vitrification is widely becoming the preferred alternative to
slow-freezing for human embryos. Benefits include a reduced working time
and a potential of higher survival and implantation rates due to the reduced in-
cidence of ice crystal formation. The objective of this study is to retrospectively
compare the results of 359 slow frozen-thawed blastocysts with 347 vitrified-
warmed blastocysts.
Materials and Methods: Statistical data analysis was performed on all pa-
tients who came through for frozen thaw treatment between January 2006
and November 2009. CRGH employed a vitrification programme in late
2007 so those patients who had their embryos frozen after this date under-
went vitrification-warming in their frozen thawed cycle. A chi-squared test
(with continuity correction) was used to compare the two proportions and
two-sample t-tests to compare patient ages. Logistic regression was then
performed to compare pregnancy outcome, clinical pregnancy outcome and
implantation rate in the two groups. Finally, multiple regression was used
when assessing the effect of the group, after adjusting for age, on rates per
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-159 Development of integrated microfluidic chips for single embryo
physiology studies
S. Le Gac1, F. van Rossem1, T. Esteves2, M. Bioani2, A. van den Berg1
1MESA + Institute for Nanotechnology, BIOS the lab on a chip group, En-
schede, The Netherlands
2Max Planck Institute for Molecular Biomedicine, Munster, Germany
Introduction: Lab-on-a-chip devices provide integrated platforms for single
cell investigation and performing a series of operations without intervention
of the experimenter. These characteristics make them attractive for Assisted
Reproduction Technologies (ART) as microfluidics offers the possibility to
develop integrated technologies towards improved culture conditions and lim-
ited embryo manipulation. In this context, we propose to develop an integrated
microfluidic platform for ART, towards the development of a novel “tool” for
improvement in fundamental embryo research in In Vitro Fertilization (IVF)
centers. Specifically, such a platform is proposed for applications such as op-
timization of culture conditions, as well as non-invasive characterization and
monitoring of embryo development, towards eventual selection.
Materials and Methods: Microfabricated structures were produced using
PDMS (PolyDimethylSiloxane) a cell-friendly material conventionally em-
ployed for cellular investigations, and soft-lithography techniques. Two types
of structures were fabricated here; arrays of 800-μm wells and microfluidic sys-
tems containing a 2-mm diameter culture chamber. The latter were bonded to a
thin glass substrate. Naturally fertilized mouse embryos were introduced in both
types of structures at the one-cell stage and cultured there until they reached the
blastocyst stage. After this pre-implantation in vitro culture period, the resulting
embryos were implanted in pseudo-pregnant mice, for full-term development.
Results: mouse embryos were successfully cultured from the one-cell to the
blastocyst stage (four days) in both types of microstructures. Embryos cultured
in the PDMS micro-wells showed pre-implantation rates (79%) comparable to
embryos cultured in conventional Nunc four-well dishes (71%). Analysis of
ATP levels revealed similar values for all embryos, independently of the chosen
culture conditions. Importantly, we demonstrated that after implantation of the
in vitro cultured blastocysts into pseudo-pregnant mice, embryos cultured on
PDMS micro-wells achieve full-term development (i.e. viable pups) as suc-
cessfully as those cultured under standard conditions. Single embryos or pools
of embryos were successfully manipulated in the microfluidic systems and in-
troduced in the culture microchamber using a mild shear stress-free flow. The
development of a pool of embryos was monitored using microscopy.
Conclusions: We have so far demonstrated that PDMS microfabricated wells
and microfluidic systems can safely be applied for in vitro culture of mammali-
an embryos, thereby holding great potential for ART. The next step is to employ
our microfluidic chips in a study aiming to assess the impact of different culture
media compositions on mouse embryo full-term development. Following this,
we propose to extend the basic microchips to integrated platforms including
specific sensors to non-invasively study embryo physiology.
P-160 Oocyte morphometry parameters are correlated with oocyte
birefringence and women’s age
C. Valeri1, S. Pappalardo1, M. De Felici2, C. Manna3
1Center Biofertility, Embryology, Rome, Italy
2University of Rome Tor Vergata, Public Health and Cell Biology, Rome, Italy
3IVF Genesis Center, Reproductive Medicine, Rome, Italy
Introduction: Assesment of the zona pellucida and metaphase II spindle bire-
fringence by polarization light microscopy has been proposed to improve the
objective selection of good quality oocytes in IVF procedures. The aim of this
study was to evaluate several morphometric parameters of metaphase II (MII)
oocytes, including vitellus size (i.e. cytoplasm diameter), zona pellucida (ZP)
thickness and width of the perivitelline space, in relation with oocyte zona and
spindle birefringence and age of the oocyte-donating woman.
Materials and Methods: In the present study we analyzed the morphometric
characteristics of 314 MII oocytes obtained from stimulated ovaries of 72 pa-
tients in our Assisted Reproductive Technology (ART) program and women’s
age ranged from 24 to 46 years were considered. Patients were stimulated
with exogenous FSH and hCG was administred 36 h before follicular aspira-
tion. Live imaging of MII oocytes was performed non-invasively on a Nikon
Diaphot-300 inverted microscope (for analysis of morphometric parameters)
and by enhanced polarizing microscopy (OCTAX ICSI Guard™, OCTAX
quality (groups B and C), those of very good quality for transfer (group A) were
obtained in only 20.7% (226/1090) of all couples.
Table 2.
Groups Female age PR / ET (%)
1 All 52.6
1.1. (<35 age) 52.1
1.2. (>35 age) 54.2
2 All 39.4
2.1. (<35 age) 42.9
2.2. (>35 age) 28.6
3 All 11.5
3.1. (<35 age) 14.3
3.2. (>35 age) 6.3
average 31.0
Conclusions: Despite the fact that only 20.7% of all the patients have only
top quality embryos (grade A) per transfer, the chance for pregnancy in group
1 for both subgroups was similar in IVF/ICSI cycles. So, the results from our
study show that there is no correlation between female age and PR as far as the
embryo quality grade A is concerned. In cases when top quality embryos are
transferred the female age is not a crucial factor.
P-158 Mining the oocyte: unravelling the mechanisms of somatic
reprogramming and chromatin remodelling by manipulation of external
and intrinsic cues
T.C. Esteves1, S.T. Balbach1, M.J. Araúzo-Bravo1, M.J. Pfeiffer1, M. Boiani1
1Max-Planck Institute for Molecular Biomedicine, Cell and Developmental
Biology, Muenster, Germany
Introduction: The mammalian oocyte hosts a unique set of biological pro-
cesses, including the ability to haploidize its DNA, to remodel sperm chroma-
tin, and to support pre-implantation development. It has also the extraordinary
ability to reprogram (i.e. to confer pluri- and totipotency to) nuclei of somatic
cells after somatic cell nuclear transfer (SCNT). While paternal genome remod-
elling and somatic nuclear reprogramming may differ in some of the underlying
mechanisms, they also share common features (such as general remodeling pro-
cesses, leading to pronuclei formation). We aim to help unravelling such basic
requirements by evaluating the impact of induced changes in culture environ-
ment (i.e. media composition), or oocyte quality itself (i.e. ageing), on mouse
embryo pre-implantation development and physiology.
Materials and Methods: Oocytes from young B6C3 mice were used for in-
tracytoplasmic sperm injection (ICSI), or enucleated and used as recipients
for cumulus cells nuclei via SCNT. Different media of defined composition
were used for pre-implantation embryo culture, during which energy and redox
metabolism was assessed. SCNT and ICSI embryos were also produced using
oocytes from donors in reproductive decline (climacteric). Transcriptome and
reprogramming potential were assessed for SCNT embryos derived from young
and climacteric oocytes.
Results: When derived from the same pool of oocytes and cultured under the
same conditions, ICSI and SCNT embryos show differences in ATP, reactive
oxygen species (ROS), redox potential and amino acid uptake during the first
two cell divisions, pointing to differences at the time of chromatin remodelling
and nuclear reprogramming. Changing culture media composition revealed that,
while ICSI embryos seem more permissive to composition changes (shown as
similar pre-implantation rates for all media tested), blastocysts show a wider
variation in ATP and ROS levels for different media compared to SCNT coun-
terparts. Furthermore, despite changes in quality induced by ageing (as shown
by transcriptome differences), oocytes from young and climacteric donors sup-
port blastocyst development after ICSI and SCNT.
Conclusions: ICSI and SCNT embryos show differential response to manipula-
tion of culture environment, strongly suggesting differences in energy require-
ments to support chromatin remodeling and somatic nuclear reprogramming.
Results also show that ageing-induced changes in oocyte quality do not alter the
oocyte’s ability to remodel the paternal chromatin or reprogram the somatic ge-
nome, suggesting that genes of special relevance to the initial steps of these two
functions are among those showing stable expression during ageing. Further
pursuit of the molecular basis of the above-mentioned processes will include
an in-depth search for the oocytic factors, through extensive transcriptome and
proteome analysis.
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
There was no significant difference in patient age, and average number of oo-
cytes and ET between two groups.
The low O2 group showed a significantly high fertilization rate (p < 0.01) in
conventional IVF cycles compared to the high O2 group’s fertilization rate of
IVF, but not in ICSI cycles. Also the low O2 group exhibited the significantly
higher (p < 0.001, p < 0.03) clinical pregnancy and implantation rates than the
high O2 group.
Conclusions: Our results showed significant differences in fertilization (IVF,
not ICSI), clinical pregnancy and implantation rates between two groups.
It seems that a low oxygen is more effective for the outcome of in vitro fer-
tilization.
P-162 Lessons drawn by a 7 years experience with combined IVF-ICSI
attempts for couples with unexplained infertility
C. Wittemer1, C. Celebi1, S. Viville2
1C.M.C.O. - SIHCUS, ART Centre, Schiltigheim Cedex, France
Introduction: For couples with unexplained infertility having endured unsuc-
cessful intrauterine insemination treatments, a first ART attempt using classical
IVF is proposed. In order to avoid a complete fertilization failure, we systemati-
cally proposed since 2003 a combined IVF/ICSI technique; mature oocytes are
randomly split in two: one half is fertilized by IVF and the other one by ICSI.
This study presents the results of our 7 years’ experience and evaluates the
benefit of this strategy for the concerned couples.
Material and Methods: From January 2003 to December 2009, 499 couples
were concerned by the combined IVF/ICSI technique. They all presented a well
explored unexplained infertility [CA1] and failed to conceive after several IIU.
After oocyte retrieval, the cumulus spreading technique was used to identify the
mature oocytes which were randomly allocated to the IVF group or the ICSI
group. The combined IVF/ICSI technique was proposed only when at least 4
mature oocytes were retrieved. After fertilization, oocytes were individually
cultured and observed every day until embryo transfer or freezing at day 3.
Transferred embryos were chosen according to their quality and not to the tech-
nique.
Results: The women’s mean age was 33.2 ± 4.0 years and on average 10.1 ±
4.3 mature oocytes were retrieved. An average of 1.44 embryos was replaced
during 469 embryo transfers leading to a clinical pregnancy rate of 36.5%. Fur-
thermore, 185 frozen embryo transfers have been performed given a cumulative
pregnancy rate per couple of 44.6%, 636 embryos are still frozen[CA2].
According to the outcome of fertilization, the couples were allocated to 4
subgroups: 323 couples (64.7% of the studied population) with fertilization both
in IVF and ICSI (subgroup 1), 159 couples (39.8%) with fertilization failure in
IVF but fertilization in ICSI (subgroup 2), 10 couples (2.0%) with fertilization
in IVF but fertilization failure in ICSI (subgroup 3) and 7 couples (1.4%) with
fertilization failure both in IVF and ICSI (subgroup 4).
Total fertilization failure after IVF was observed in 166 cycles (33.3%) and
in 17 cycles (3.4%) after ICSI.
A special interest was focused on subgroups 1 and 2 which represent
[CA3] 96.6% of the studied population. No difference was seen concerning
women’s age, ovarian stimulation’s characteristics, number of mature oocytes
randomized for IVF (5.8 versus 5.3) and for ICSI (4.5 versus 4.4), fertilization
rate after ICSI (75.9 versus 73.2%) and pregnancy rate per transfer (38.9 versus
32.2%).
Even if the individual semen parameters were normal (WHO criteria), the
total number of motile and normal spermatozoa was significantly decreased in
subgroup 2 compared to subgroup 1 (32.6 versus 15.6 million, p < 0.05).
In subgroup 1 (fertilization in IVF and ICSI), the fertilization rate was sig-
nificantly lower in the IVF group compared to the ICSI group (59.3 versus
75.9%, p < 0[CA4].05).
Conclusion: This study shows that couples with unexplained infertility have
a high risk of total fertilization failure after classical IVF. The combined IVF/
ICSI technique highly decreased this risk from 33.3 to 3.4%. Furthermore a
better fertilization rate was obtained in the ICSI fertilized oocytes compared to
the IVF fertilized ones.
Patients with fertilization failure in IVF had a significantly lower semen
quality although individual semen parameters were in the range of the normal-
ity. This observation underlines the difficulty to define strict threshold semen
values which could predict fertilization outcome and suggests that interactions
between sperm and oocytes are implicated.
Microscience GmbH, Germany). Oocytes were classified into groups according
to zona pellucida birefringence (low or high zona birefringence) and meiotic
spindle visualization (presence or absence of the spindle). All morphometric
parameters were always calculated to the bigger axis of the oocyte, in corre-
spondence with the bigger perivitelline space.
Results: We found that highly birefringent zona were significantly thicker than
low birefringent ones (17.7 ± 0.3 μm vs 16.7 ± 0.3 μm, p < 0.01). Moreover,
oocytes with a highly birefringent zona and oocytes with a visible spindle dis-
played a thinner perivitelline space, as compared with low-birefringent and no-
spindle oocytes (p < 0.001 and p < 0.05, respectively). Finally, we found that
women age was inversely related to both cytoplasm diameter (r = 0.18, p <
0.001) and zona thickness (r = 0.23, p < 0.0001) of recovered oocytes.
Conclusions: Our results evidence a novel association of zona pellucida and
spindle birefringence with morphometric parameters of human oocytes (a
thicker zona is associated with a greater birefringence). Furthermore, we re-
port an intriguing decrease of oocyte size and ZP thickness in function of the
women’s age.
P-161 Effect of low-oxygen compared with high-oxygen atmosphere on
the outcomes of in vitro fertilization, a prospective randomized study
H. Ryu1, C.Y. Park1, S.H. Min1, S.K. Choi1, C. Park2, S.H. Lee2, K.R. Kim2,
H. Jeong3, H.J. Chi4
1Mizmedi Hospital, I Dream (IVF) Infertility Research Center, Seoul, Korea
South
2Mizmedi Hospital, I Dream (IVF) Clinic Center, Seoul, Korea South
3Mizmedi Hospital Gangseo, I Dream (IVF) Clinic Center, Seoul, Korea South
4Mizmedi Hospital Gangseo, I Dream (IVF) Infertility Research Center, Seoul,
Korea South
Introduction: The fact that human embryos can grow at atmospheric oxygen
concentration (20%) has led to some confusion regarding the optimal concen-
tration for in vitro culture. Since the oxygen tension in the oviducts and uterus
of most mammalian species is only 2%–8%, excess oxygen from a 20% O2 gas
phase could create unfavorable conditions in the substrate to produce an excess
of free oxygen radicals. Although human embryos can successfully develop in
atmospheric oxygen levels, it is suggested that low oxygen concentrations mim-
icking physiological conditions improve embryo viability and morphology.
The aims of this study were to compare two different O2 concentrations dur-
ing the embryo cultured from ovum pick up to embryo transfer, and to examine
the effect of low O2 tension on the outcomes of in vitro fertilization.
Materials and Methods: We randomized 1065 cycles to culture under 6% CO2
in air (high O2), or 6% CO2, 5% O2, and 89% N2 (low O2) under 37.0 ºC for 4
years (20062009). Due to the cultivation in low oxygen tension or high oxy-
gen tension begins from the dish preparation stage, the randomization occurred
at the beginning of the ovarian hyperstimulation.
All oocytes and embryos of total 1065 cycles cultured during processing
in two different O2 concentrations. Sequential embryo cultures were generally
performed with GV series media (Vitrolife). Most embryos were transferred
mainly on day 3 after fertilization. The age of patients, number of oocytes re-
trieved and embryo transferred between the two groups were analyzed with the
T-test statistical analysis; the fertilization rate (IVF/ICSI) of oocytes, pregnancy
and implantation rates of two groups, in different O2 concentration-culture con-
ditions, were calculated with the χ2-test statistical analysis.
Results:
Table 1. Summary of patient characteristics and results of IVF outcomes in low O2 group or high
O2 group.
low O2tension high O2 tension P value
Number of cycles (n) 327 738
Mean of age (years) 35.1 ± 4.3 35.0 ± 4.4 NS
Mean no. retrieved oocyte (ea) 12.1 ± 8.8 12.7 ± 9.0 NS
Mean no. embryo transferred (ea) 3.3 ± 1.1 3.2 ± 1.1 NS
Fertilization rate (%)
IVF 82.5 76.2 0.01
ICSI 70.7 69.0 NS
Pregnancy Rate (%) 43.4 32.9 0.001
Implantation Rate (%) 16.1 13.3 0.03
* NS : no significant difference was noted.
* t-test and χ2-test were used for statistical analysis, and P values of < .05were considered significant.
Based on the two different O2 concentration conditions, we randomly divid-
ed into two groups, low O2 group (327 cycles) and high O2 group (738 cycles).
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
the other two registers. The rates of multiple births recorded on the three reg-
isters varied, but the difference was not statistically significant (24.1% on FIV-
CAT.NET, 27.9% on SEF-CAT and 28.1% on SEF-wCAT).
Conclusions: The data obtained from the voluntary ART register for own-egg
cycles are valid, but the data for donor-egg cycles are not. More studies are
necessary to determine the reasons for these differences.
P-164 NMR-based profiling of ovarian follicular fluid and plasma
C. McRae1, E. Baskind2, V. Sharma2, J. Fisher1
1University of Leeds, School of Chemistry, Leeds, United Kingdom
2St James’s University Hospital, Assisted Conception Unit, Leeds, United
Kingdom
Introduction: The success rates of infertility treatments such as in vitro fertili-
sation (IVF) and inter-cytoplasmic sperm injection (ICSI) are unsatisfactory,
and there is currently no reliable method of assessing the oocyte quality and
its potential.
It has been shown that follicular fluid (FF), the fluid surrounding an oocyte
as it develops within its follicle in the ovary, contains substances that are es-
sential in follicle growth and oocyte fertilisation. Therefore it may be possible
to identify such chemical markers within this fluid.
The aim of this study was to use high resolution nuclear magnetic resonance
(NMR) spectroscopy to profile the FF and blood plasma of women undergoing
natural cycle IVF or ICSI, followed by interrogation of the spectral data using
multivariate statistical analysis in order to detect and quantify biomarkers of
infertility. Natural cycles with unifollicular development enabled certainty of
the oocyte and ultimate embryonic origin. The effect of stimulating gonadotro-
phins could also be removed. To date, the majority of FF studies have utilised
animal models, furthermore specifically utilising the technique of NMR, to our
knowledge none have yet been reported on humans. For these reasons this is a
novel investigation.
Materials and Methods: FF was aspirated from 10 women undergoing natu-
ral cycle IVF or ICSI at The Assisted Conception Unit at St. James’s Hospi-
tal, Leeds, UK and centrifuged (2000 rpm, 5 min) immediately. Venous blood
samples were also collected and plasma obtained by centrifugation (200 rpm,
10 min).
For NMR the FF was defrosted and 200 μl mixed with 400 μl of a 0.17%
(w/v) solution of the sodium salt of 3-(trimethylsily)propionic-2,2,3,3-d4 acid
(TSP) in deuterium oxide (D2O). Plasma NMR samples were prepared in the
same way but with a centrifugation step (4000 rpm (900 g), 2 min) follow-
ing defrosting. 1H-NMR spectra were acquired on a Varian Unity Inova 500
spectrometer operating at 499.97 MHz proton frequency. The CPMG pulse
sequence [RD - 90° - (τ - 180° - τ)n - FID] was used with relaxation decay
(RD) = 2 s, τ = 1.5 ms and n = 150. The NMR spectral data were reduced,
normalised, pareto-scaled and mean centred before Principle components
analysis (PCA).
Results: This study was limited in number of patients and diversity of treat-
ment outcomes, due to the restrictions associated with the nature of the
sampling required. However, among this small cohort trends are arising
which need to be related to relevant biochemical processes. A distinction
can be made between the spectrum of a patient who experienced a bio-
chemical pregnancy and one who suffered a miscarriage, and all the other
patients: all but one of whom failed to get pregnant. It is pleasing to see
potential biomarker(s) emerging with NMR signals at 2.85, 2.89, 2.93, 3.13,
3.17 ppm. Metabolites typically found in these regions include glycerol,
albumin lysyl, creatine, citrulline and tyrosine, but to unambiguously deter-
mine this further experimentation including 2D NMR and spiking experi-
ments are required.
In addition, FF dynamics were investigated by comparing the NMR spectra
of FF taken at two times prior to ovulation. A difference in composition of FF
was observed between these times, particularly at 1.33 and 4.13 ppm, which
correspond to lactate.
Conclusions: Although the study is still early in its development, some inter-
esting and potentially valuable trends have so far been observed, and this work
has served as an efficient preliminary investigation, revealing that it may be
worthwhile to investigate FF composition among women undergoing infertility
treatment more extensively. To our knowledge this is the first study to combine
NMR spectroscopy and metabolomics techniques to identify chemical markers
of oocyte quality within human FF.
In this particular case of what is called “unexplained infertility”, a link
contribution of both oocytes and spermatozoa defect may be involved in the
fertilization process.
In this situation, ICSI should be performed on all retrieved oocytes in order
to optimize the results and the chance of pregnancy for these couples.
P-163 Validation of a voluntary assisted reproductive technology register
by comparison with an official one
F. Luceño Maestre1, J.A. Castilla Alcalá2, J.L. Gómez-Palomares3, Y. Cabello4,
J. Hernández5, J. Marqueta6, J. Herrero7, E. Vidal8, S. Fernández-Shaw9,
B. Coroleu10
1Centro Reproducción Humana, Reproduction Unit, Granada, Spain
2Hospital Virgen de las Nieves, Reproduction Unit, Granada, Spain
3FIVMadrid, Reproduction Unit, Madrid, Spain
4FIV Recoletos, Reproduction Unit, Madrid, Spain
5Hospital San Pedro, Obstetrics and Gynecology Service, Logroño, Spain
6Instituto Balear de Infertilidad, Reproduction Unit, Palma de Mallorca,
Spain
7Centro de Reproducción Asistida Sagrada Familia, Reproduction Unit,
Barcelona, Spain
8Hospital Clínico y Provincial, Reproduction Unit, Barcelona, Spain
9Centro García del Real, Reproduction Unit, Madrid, Spain
10Instituto Universitario Dexeus, Reproduction Unit, Barcelona, Spain
Introduction: Monitoring Assisted Reproductive Technology (ART) is essen-
tial for assessing the performance and impact of fertility treatment on birth
rates. There are two kinds of ART register in Europe: voluntary and official. The
validity of register data is very important with respect to the quality of register-
based observational studies. Our aim was to compare the agreement between
the voluntary and official ART registers.
Material and Methods: The data compared in this study were obtained for
the years 2005 and 2006: on the one hand, data were obtained from FIVCAT.
NET (an official assisted reproduction registry in the Health Department of the
Catalonia Regional Government, to which all authorized centres (both public
and private) performing assisted reproduction in the region are obliged to re-
port). On the other, data were obtained from the register of the Spanish Fertility
Society (SEF), provided on a voluntary basis. The data from the SEF register
were divided into two groups: 1) those from clinics in Catalonia (SEF-CAT); 2)
those from the rest of Spain, without Catalonia (SEF-wCAT). The techniques
compares were own and donated-egg cycles.
Results: The voluntary SEF register recorded 61.7% of the cycles reported to
the official FIVCAT.NET register. The distribution of clinics according to the
number of cycles provided did not vary significantly between FIVCAT.NET and
SEF-wCAT. In the case of own-egg cycles, SEF-CAT accounted for 77.2% of
the total number of cycles reported to FIVCAT.NET, while in cycles performed
with donated eggs, SEF-CAT represented 34.4% of the cycles reported to FIV-
CAT.NET. The variables analyzed in own-egg cycles (insemination technique
used, age of the women treated, numbers of embryos transferred, pregnancy
rates, multiple pregnancies and deliveries) were similar in the three groups stud-
ied. However, there were significant differences in donor egg cycles between the
voluntary and official registers. The utilization of IVF, ICSI and IVF + ICSI in
Catalonia varied between FIVCAT.NET and SEF-CAT. The differences are also
significant with respect to FIV-CAT cycles and those in the rest of Spain (30.6%
vs 19%; p < 0.001 in IVF cycles; 55.8% vs 73.2%; p < 0,001 in ICSI cycles;
and 13.6% vs 7.7%; p < 0.001 in IVF + ICSI cycles). The age distribution of the
patients registered on FIVCAT.NET was similar to that recorded by SEF-CAT,
and also coincided with the distribution corresponding to the rest of Spain.
The percentage of single-embryo transfers with donated eggs, as reported to
FIVCAT.NET, was different from that on the SEF-CAT register, and also from
that for the rest of Spain, although the difference was not statistically significant
(8.6% vs 3.4% vs 6.2%). The pregnancy rates per transfer were different on
each register. Thus, FIVCAT.NET differed from SEF-CAT as follows: 44.1%
vs 55.3%; p < 0.001; the corresponding figures for FIVCAT.NET with respect
to the rest of Spain were 44.1% and 49.9%; p < 0.001, respectively. Neverthe-
less, the difference between SEF-CAT and the rest of Spain was not statistically
significant. The rate of multiple pregnancies was also different among the three
registers analysed. Thus, on FIVCAT.NET and SEF-CAT, the respective values
were 20.6% and 33.3%; p < 0.001. For the rest of Spain, the rate of multiple
pregnancy was 29.7%, and therefore this was also significantly different from
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
the pronuclear stage in regard to spindle presence, second polar body extrusion
and pronuclear pattern formation.
Material and Methods: In this study we included 225 oocytes from 25 ICSI
patients. Immediately after ICSI oocytes were investigated under a polarized
light microscope system (PolarAide™, Octax, Bruckberg, Germany) for spin-
dle presence and zona pellucida properties. Up to 9 oocytes were then incu-
bated in a well-of-the-well (WOW) culture dish (Cryo-Innovation Ltd., Buda-
pest, Hungary) which was positioned on the stage of a compact digital inverted
microscope (Primo Vision, Cryo-Innovation Ltd.) which was placed inside an
incubator. The microscope was set to take a single picture of all 9 oocytes every
10 minutes over the total incubation time of 18 hours. Images were transferred
to a computer outside the incubator via a firewire cable and stored on hard
disk. Strictly at 18 h after ICSI oocytes were retrieved from the WOW dish and
placed in separate droplets in a standard culture dish under oil and examined
on an inverted microscope using a 40x Hofmann contrast objective. Pronuclei
were scored according to Scott et al. (2000) and up to 2 pronuclear oocytes were
cultured for transfer whereas the others were cryopreserved at the 2PN stage.
All images from one incubation experiment were used o generate a time-lapse
video. Based on this, the time interval between ICSI and the appearance of the
second polar body as well as the formation of pronuclei was recorded for every
individual oocyte. These time values were correlated with spindle presence,
pronuclear pattern and embryo development.
Results: Oocytes with the presence of a spindle as revealed by polarization
microscopy showed a faster extrusion of the second polar body (181 ± 55 min
versus 201 ± 52 min; P < 0.05) and a trend towards an earlier formation of
pronuclei compared to oocytes without a spindle. Pronuclear patterns Z1
and Z2 showed an earlier formation of pronuclei compared to patterns Z3/4
(477 ± 85 min versus 524 ± 102 min; P < 0.05). Oocytes which developed
into 4 cell stage embryos on day 2 were characterized by an earlier formation
of the pronuclei compared to embryos with less than 4 blastomeres on day 2
(463 ± 96 min versus 556 ± 65 min; P < 0.05).
Conclusions: The results of this study show that already in the first cell cycle
human oocytes after ICSI differ in regard to the time course of second polar
body extrusion and formation of pronuclei. This timing seems to have a direct
influence on the pronuclear pattern and on the first cleavage events. Our results
further support the idea, that pronuclear pattern formation is a time-dependent
process. Monitoring of the first cell cycle may offer another tool to predict
embryo development potential.
P-167 Comparison of with or without early cleavage assessment for
elective single embryo transfer on day 3
S.G. Lee1, Y.C. Lee2, S.M. Kang1, Y.J. Kang2, Y.K. Shin2, J.H. Jung2, J.H. Lim2
1Maria Fertility Clinic, Infertility Dept, Daegu, Korea South
2Maria Fertility Clinic, Infertility Dept, Seoul, Korea South
Introduction: Multiple pregnancies are associated with higher risks of ma-
ternal and fetal morbidity and mortality. To minimize higher order multiple
pregnancies after IVF/ICSI, single embryo transfer method has been regarded
as may be the best solution. Elective single embryo transfer (eSET) needs to
select embryo with the best implantation potential from a cohort. It has been
demonstrated that early cleavage embryos occurring by 2527h after insemina-
tion or ICSI have a higher implantation rate than non-early cleavage embryos,
and top quality embryos on day 3 have a higher implantation rate than non-top
quality embryos.
The aim of this study is to compare the efficacy of two embryo selection
methods that based on either early cleavage status or the combined cell mor-
phology with cell number on day 3 in IVF/ICSI.
Materials and Methods: A total of 345 women less than 40 years with their
autologous eSET in fresh IVF or ICSI using GnRH agonist long protocol
(n = 334) or GnRH antagonist protocol (n = 11) from January 2008 to Septem-
ber 2009 were included in this study. The patients were undergoing a first or
second trial of IVF or ICSI and had at least two embryos which were available
from which one was selected for transfer. Early cleavage status was assessed
at 2527 h after IVF/ICSI and eSET was performed on day 3. Patients were
divided into two groups. The group I (n = 189) has transferred with top quality
embryos derived from early cleavage embryos assessed on day 1. The group II
(n = 156) has transferred with top quality embryos which were not performed
assessment of early cleavage on day 1. The characteristics of top quality embry-
os were seven or more cells, less than 20% anucleated fragments and absence
P-165 Effect of early and late oocyte denudation on ICSI outcome
P. Boldi Cotti1, C. Colasante1, L. Perego1, L. De Lauretis1
1ICCS, Centro Fertilità, Milano, Italy
Introduction: It is well known that intracellular communication between cu-
mulus cells and oocyte is essential both for normal follicular differentiation
and oocyte developmental competence. Cumulus cells could secrete paracrine
growth factors or express adhesion molecules on their membranes which might
play an important role in nuclear and cytoplasm maturation.
Good results in Intra-Cytoplasmic Sperm Injection (ICSI) procedures de-
pend on both oocyte nuclear and cytoplasm maturity.
Nuclear maturity is easily identified by evaluating the extrusion of the first
polar body. In contrast, cytoplasm maturity, which is very important for oocyte
activation and fertilization and for embryo development, cannot be assessed
microscopically.
During the ICSI procedure, oocytes are denuded in order to be able to per-
form the injection and to then evaluate the nuclear maturity of the oocytes, thus
any potential positive effect of oocyte cumulus cells cannot exert influence.
The purpose of this retrospective study was to compare the effect of two dif-
ferent timings of cumulus cell removal and oocyte injection on ICSI outcome.
Matherials and Methods: One-hundred and fifty-nine women under the age
of 42 were entered into this study. Two hundred and three ICSI cycles were
divided in two groups according to the different timing of cumulus cell removal
and injection. In group 1 (no. cycles = 74) the oocytes were injected within 1 h
after denudation; in group 2 (no. cycles = 129) the oocytes were injected 1-3 h
after denudation.
There were no significant differences (p > 0.5) in the 2 groups in terms of
mean age, number of previous failures, number of oocytes collected, number of
oocytes injected and number of embryos transferred.
Oocytes were denuded singly, by a combination of enzymatic and mechani-
cal procedures.
Group 1 oocytes were injected immediately after cumulus cell removal,
whereas Group 2 oocytes were cultured in an incubator in cleavage medium
and were injected 1-3 h after denudation.
Clinical pregnancy was confirmed at six weeks by ultrasound examination.
Statistical analysis was performed by a chi-square test and results that yield-
ed p < 0.05 were considered statistically significant.
Results: Fertilization rate did not show important variations between groups
(77.6% vs. 78.3%; p = 0.8) Considering the quality of embryos, the results showed
that the top quality embryo rate derived from first group was significantly higher
compared to that derived from the second group (37.3 % vs. 27.9 %; p < 0.05).
Moreover, group 1 showed significantly higher pregnancy and implantation
rates than Group 2 (40.5% vs. 27.9% and 24.6% vs. 15.5%; p < 0,05).
Conclusions: The present study shows that a better pregnancy and implantation
rate may be achieved when oocytes are injected within 1 hour from cumulus
cell removal and the difference between the 2 groups is statistically significant.
There was also a significant increase in better quality embryos in Group 1 com-
pared to Group 2.
This could be explained by the fact that cumulus cells play a very important
role not only during early maturation steps but also after meiotic resumption,
and are involved in the acquisition of developmental competence.
These preliminary findings suggest that oocytes should be injected as soon
as possible after cumulus removal in order to improve the outcome of ICSI, but
further investigations are required to confirm these results.
P-166 Time-lapse based evaluation of human oocytes from ICSI up to
the pronuclear stage
M. Montag1, M. Köster1, A. Nikolov1, H. van der Ven1
1University of Bonn, Gynaecological Endocrin. & Reprod. Medicine,
Bonn-Venusberg, Germany
Introduction: The search for prognostic factors predicting embryo develop-
ment as well as the outcome of in-vitro fertilization (IVF) treatment attracts
much interest. One prognostic factor is the proper timing of early embryo de-
velopment up to the blastocyst stage. Continuous monitoring of embryo de-
velopment has become a realistic option due to new instrumentation and was
applied to follow the course of early cleavage embryos. However, the initial
phase of embryo development from the time of intracytoplasmic sperm injec-
tion (ICSI) up to the formation of the two pronuclei has been not investigated.
Here we show a time-lapse based evaluation of human oocytes after ICSI up to
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
during this time period. All parameters studied were statistically significant.
Collapsing the blastocoel prior to vitrification in the CryoTip is a highly effec-
tive method of human blastocyst cryopreservation.
P-169 A novel tilting embryo culture system (TECS) improves human
blastocyst quality clinically
T. Hara1, K. Naruse2, K. Matsuura2, T. Kodama1, K. Sato1, Y. Tateaki1, J.
Tanaka3
1Hiroshima Prefectural Hospital, Division of Reproductive Medicine, Hiro-
shima, Japan
2Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okaya-
ma University, Department of Cardiovascular Physiology, Okayama, Japan
3Graduate School of Biomedical Sciences Hiroshima University, Department
of Epidemiology Infectious Disease Control and Prevention, Hiroshima,
Japan
Introduction: A culture system producing high quality blastocysts capable
of implantation is critically important for elective single blastocyst transfer
(eSBT). In the Fallopian tube, human embryos are under not just hormonal
but also mechanical stimuli that could accelerate their development, such as
shear stress, compression and frictional forces. To apply appropriate mechani-
cal stimuli to embryos, we used a Tilting Embryo Culture System (TECS, Re-
productive BioMedicine Online in press) in which placing a culture dish on an
automatically tilting plate makes the embryos move back and forth along the
bottom of the dish. In a previous study using mouse embryos, we found that
TECS was safe for embryos and significantly increased the blastocyst cell num-
bers under physiologically normal levels of mechanical stimuli. In the pres-
ent study, we investigated whether TECS could improve the development of
human fresh embryos to be transferred, compared with a control static culture
system (CTRL).
Materials and Methods: Three hundred thirty-three retrieved oocytes from
24 IVF or ICSI cycles of 24 women were cultured in sequential media for 5–6
days. Retrieved oocytes were divided evenly into TECS and CTRL groups. In
the TECS group, the, dishes were subjected to a maximum 20° tilt for 10 min in
each direction at 1° per second. All embryos were evaluated at days 3, 5 and 6
using the Veeck or Gardner quality criteria.
The Gardner grades were converted into a blastocyst quality score (BQS)
as reported (Fertil Steril 2010 Jan 14. [Epub ahead of print]). We scored the ex-
pansion rate depending on the developmental stage and graded each embryo for
quality. For embryos graded 3AB, the final BQS = R(expansion) × R(ICM) × R
(TE) was equal to 3 × 3 × 2, giving a BQS score of 18.
The best-quality blastocyst of any cycle was defined to be the morphologi-
cally most advanced one with a grade of 3BB or more by day 6 in either the
TECS or CTRL groups. The best-quality blastocyst was transferred when pa-
tients were eligible for our eSBT policy. All blastocysts graded 3BB or better
were vitrified if the patients showed a risk of OHSS. This study was approved
by the IRB of Hiroshima Prefectural Hospital.
Statistical analysis was performed using the Mann-Whitney nonparametric
U-test and a one-sided upper bound test to compare the effectiveness of the rate
of producing best-quality blastocysts between systems: P < 0.05 was consid-
ered significant.
Results: The rates of cleavage, blastocyst formation and development to bet-
ter than grade 3BB blastocysts in the TECS system were 83% (90/108), 48%
(43/90) and 33% (30/90), respectively. For the CTRL group they were 86%
(89/104), 38% (34/89) and 23% (20/89). The difference in BQS grades between
TECS (mean = 21, n = 39) and CTRL (mean = 16, n = 30) was not statistically
significant (P = 0.12), however it would probably be significant with a larger
sample number. According to our previous report of the correlation between
BQS and cell numbers in blastocysts, the cell numbers would be enhanced by
TECS. The rate of producing best-quality blastocysts by the TECS method
(13/17) was greater than in the CTRL group (4/17) (P < 0.05).
Among ten best-quality blastocysts in the TECS group that were transferred,
eight developed to an ongoing pregnancy or successful birth. Seven couples did
not have a best-quality blastocyst in either system.
Conclusions: TECS could produce better quality blastocysts than the CTRL
system. It produced high rates of implantation, pregnancy and birth. TECS is a
promising culture method that enhances implantation competence of embryos
by exposing them to mechanical stimuli similar to those found in the Fallopian
tube.
of multinucleated blastomeres on day 3. The surplus embryos were cultured to
day 5 or 6 until blastocyst formation, and then vitrified using EM-grid follow-
ing artificial shrinkage.
Results: The group I and II did not differ significantly in the female age
(32.1 ± 2.9 yrs vs. 32.0 ± 3.1 yrs, P = 0.915), duration of infertility (48.6 ± 28.2
month vs. 47.9 ± 26.2 month, P = 0.798), cause of infertility, the number of
previous IVF cycle (0.2 ± 0.4 vs. 0.2 ± 0.4, P = 0.323), endometrial thickness
at hCG triggering (10.7 ± 1.4 mm vs. 10.8 ± 1.7 mm, P = 0.850), the number of
retrieved oocytes (11.5 ± 6.1 vs. 11.8 ± 5.9, P = 0.692), the number of fertilized
oocytes (8.1 ± 4.7 vs. 7.9 ± 4.4, P = 0.737). In the group I, ICSI was carried out
in 75 cases (39.7%) and in the group II in 54 cases (34.6%).
The rates of multiple pregnancy (1.0% vs. 1.5%) and the abortion (6.4% vs.
7.1%) were not significantly different between group I and group II. However,
implantation rate (52.9% vs. 42.3%, P < 0.05), clinical pregnancy rate (52.4%
vs. 41.7%, P < 0.05), and on-going pregnancy rate (46.0% vs. 34.6%, P < 0.05)
in the group I were significantly higher than those of group II. The number
of cryopreserved embryos was not significantly different between group I and
group II (3.2 ± 2.9 vs. 3.0 ± 2.6, P = 0.595).
Conclusions: Our results showed that the embryos derived from early cleavage
embryos may be associated with higher on-going pregnancy rate than embryos
with optimal embryo morphology on day 3. We concluded that the combination
of early cleavage embryos with embryo morphology would be an efficient tool
than embryo morphology alone in the selection of embryos for eSET on day 3.
P-168 Collapsing of the blastocoel by laser prior to vitrification with
the CryoTip improves embryo survival and pregnancy rates after embryo
transfer
A. Dorfmann1, K. Carroll1, M. Sisson1, M. Geltinger1, S. Yap1, M. Iwaszko1
1Genetics & IVF Institute, Embryology, Fairfax VA, U.S.A.
Introduction: It has been suggested by some authors that collapsing the blas-
tocoel cavity of human blastocysts prior to cryopreservation by vitrification
may improve success rates. After observing that blastocysts which were less
expanded at the time of vitrification seemed to better survive the process, we
decided to test the effectiveness of purposely collapsing the blastocoel cavity
immediately prior to vitrification. The current study examines our results with
embryos donated to research as well as our initial series of clinical cases utiliz-
ing a laser to collapse the blastocoel cavity prior to vitrification.
Materials and Methods: IVF and embryo culture was carried out in Vitrolife
G series media. Vitrification was done using the closed system CryoTip (Irvine
Scientific) in standard vitrification media (equilibration solution = 7.5% eth-
ylene glycol/7.5% DMSO; vitrification solution = 15% ethylene glycol/15%
DMSO). Only high quality blastocysts showing good development of both inner
cell mass (ICM) and trophectoderm were vitrified on day 5 or 6 of develop-
ment. Immediately prior to vitrification, each blastocyst was exposed to a single
500μs pulse from a Zilos TK laser (Hamilton Thorn). The laser pulse was aimed
at the junction of the trophectoderm and the zona pellucida at the opposite pole
from the ICM. In the first phase of this study, embryos donated to research were
assigned to one of two groups: A – Collapse with laser or B – Control. Given
the positive results of this phase, we moved ahead and began collapsing the
blastocoel cavity on clinical cases. We compared the results of cases in which
the blastocoel was collapsed prior to vitrification with those from the previous
six months which had not been collapsed prior to vitrification. The warming
and transfer cycles were performed concurrently between June and December
2009. Results were compared using the Fisher’s exact test.
Results: In phase 1, 33 embryos were vitrified; 20 were collapsed (Group A)
and 13 were not collapsed (Group B). Survival was 20/20 in the collapsed
group vs. 7/13 in the non-collapsed group; p < 0.001. Twenty four hours post
warming, 12/20 from group A re-expanded vs. 3/13 from group B; p < 0.05.
Collapsed blastocysts appeared morphologically healthier after warming. In the
clinical phase of this study, a total of 55 collapsed blastocysts (Group A) and 71
non-collapsed (Group B) were warmed. The rate of survival was 52/55 (95%)
for Group A vs. 49/71 (69%) for Group B; p < 0.001. Transfers of previously
collapsed embryos yielded a clinical pregnancy rate of 65% (19/29) vs. 39%
(11/28) for non-collapsed embryos; p < 0.05.
Conclusions: Collapsing the blastocoel cavity increased both the overall sur-
vival rate and the quality of the blastocysts tested after vitrification using the
closed CryoTip system. After implementation in our clinical IVF program, the
clinical pregnancy rate for frozen embryo transfers increased from 39% to 65%
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
Introduction: It is generally accepted that human ovarian follicles that would
undergo atresia are rescued by hormonal stimulation during assisted reproduc-
tive technology cycles. Without stimulation, these follicles would complete
their atretic death program normally and be cleared from the ovary. As a con-
sequence of stimulation, some oocytes are held within these possibly compro-
mised follicles and are considered to be of reduced quality compared to oocytes
fated for ovulation within follicles not previously committed to atresia. In an
attempt to detect differences in follicle quality due to this differential fate, the
aim of this study was to assess autophagic and apoptotic cell death in cumulus-
granulosa complex by measuring lysosome activity in situ using a simple proto-
col based on a Lystotracker Red molecular probe and DNA staining.
Material and Methods: Sixty-seven cumulus cell samples were collected during
oocyte retrievals in 13 patients after written informed consent. All patients received
a long standard stimulation protocol and retrievals were performed 36 hours after
human chorionic gonadotrophin (hCG) injection. Cells were placed on glass slides
labeled with a unique sample identifier matched to individual oocytes and kept hy-
drated in droplet of Ca++/Mg++-free phosphate buffer solution (PBS). Cumulus cells
were washed in a droplet of Lysotracker Red (Molecular Probes) diluted 1:5000 in
PBS for 10 minutes at room temperature (RT) in the dark. Afterwards, the samples
were washed 3 times in PBS and subsequently fixed in 4% paraformaldehyde (pre-
pared fresh weekly in PBS, final pH = 7.2) for 30 minutes at RT. Finally, cells were
rinsed in PBS and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) in
antifade. Samples were stored at 4oC until visualization. Assessment of lysosome
activity consisted of epifluorescence microscopy visualization performed blindly
by two unique operators. A minimum of 200 cells was counted by each operator
and lysosome activity scored as a ratio of positive cells/counted cells. The number
of apoptotic cells was also counted and statistical analysis performed to correlate
the two cell types using the Chi-square test.
Results: The lysosome activity was measured and, according to the value, eggs
were divided into 3 groups: A (21 samples) 0-5.9%, B (42 samples) 6-20.9%
and C (4 samples) 21-45%. None of the samples displayed higher than 45%
activity. The number of apoptotic cells was evaluated and correlated to the rate
of lysosome activity within these groups. In group A just 38.0% (8/21) of the
samples showed marked apoptotic cumulus cells; in group B (35/42) the rate
increased significantly to 83.3% (p = 0.0001) and to 100% (4/4) in group C.
Conclusions: The preliminary data suggest a possible correlation between the
Lysotracker Red absolute fluorescence of the samples and the rate of marked
apoptotic cells with a pre-apoptotic nucleus. Translating this to the clinical field
might allow for better selection of the right cumulus-enclosed oocyte without
compromising egg viability. The rate of rescued follicles could also be related
to stimulation protocols leading to eventual modulation of the gonadotrophin
dosage accordingly.
P-172 Maintaining the human pre-zygote culture environment through
early cleavage after ICSI insemination may improve implantation and
pregnancy rates
V. Casciani1, P. Rubino1, M.G. Minasi1, A. Colasante1, F. Scarselli1, A.M.
Lobascio1, L. Arizzi1, E. Iammarrone1, K. Litwicka1, S. Ferrero1, A. Tocci2, C.
Piscitelli3, F. Cucinelli4, Z.P. Nagy5, E. Greco1
1European Hospital, Centre for Reproductive Medicine, Rome, Italy
2Nuova Villa Claudia, Centre for Reproductive Medicine, Rome, Italy
3Villa Margherita, Centre for Reproductive Medicine, Rome, Italy
4San Camillo Forlanini, Obstetrics and Gynaecology, Rome, Italy
5Reproductive Biology Associates, Centre for Reproductive Medicine, Atlanta,
Georgia
Introduction: In conventional IVF it is common routine to change culture me-
dium on day-1, after decumulation of the inseminated oocytes. During this pro-
cedure cumulus-corona cells are removed and the fertilized oocytes are placed
into a new drop of culture medium. This practice of moving fertilized oocytes in
new drops on day-1 is adopted also when ICSI insemination is performed. Most
laboratories place the inseminated oocytes on day-0 into fertilization or cleav-
age medium after ICSI, and move them into a fresh drop of cleavage medium
on day-1. However, with the use of ICSI this practice may not be necessary
and ICSI inseminated oocytes may be kept in the same cleavage medium from
day-0 to day-3. In the present study we aim to compare two different methods
of embryo culture, consisting in changing or not the culture media on day-1, in
order to determine the optimal condition for achieving better results in terms of
embryo development, pregnancy and implantation rate.
P-170 Comparison of slush nitrogen with standard liquid nitrogen
vitrification of sibling human oocytes
M.G. Minasi1, F. Scarselli1, P. Rubino1, V. Casciani1, A. Colasante1,
M. Lobascio1, E. Alviggi1, S. Ferrero1, K. Litwicka1, E. Iammarrone1,
F. Cucinelli2, P.G. Giannini3, A. Tocci4, Z.P. Nagy5, E. Greco1
1European Hospital, Reproductive Medicine, Rome, Italy
2San Camillo Forlanini, Obstetrics and Gynecology, Rome, Italy
3Villa Margherita, Reproductive Medicine, Rome, Italy
4Nuova Villa Claudia, Reproductive Medicine, Rome, Italy
5RBA Centre, Reproductive Medicine, Atlanta, U.S.A.
Introduction: In recent years several different vitrification protocols have been
proposed for human oocytes cryopreservation. Optimization of the technique is
key to achieve improved outcomes. The principle of vitrification is to prevent ice
crystal formation. High cryoprotectant concentrations and very fast cooling rates
are employed to achieve this goal. Liquid nitrogen has a temperature of -196°C
when used in routine applications. However, when applying negative pressure
(slush nitrogen) it is possible to obtain lower temperature such as -210°C. It has
been suggested that when using the slush nitrogen it may be possible to achieve
a faster cooling rate thus optimizing vitrificationn and obtaining improved out-
comes. The present study aims to evaluate the efficiency of vitrification in terms
of survival, maintenance of meiotic competence, fertilization and embryo quality
in sibling human oocytes cryopreserved with slush or standard liquid nitrogen.
Materials and Methods: from December 2008 to December 2009, 97 oocyte
freezing cycles were performed (mean age ± SE of 35.41 ± 0.34). Oocytes were
cryopreserved using cryotops as carrier employing solutions of 1.33M (7.5%)
and 2.66M (15%) ethylen glycol (EG), 1.06M (7.5%) and 2.12 (15%) dimethyl
sulfoxide (DMSO) and 0.5M sucrose. Oocytes were randomly divided in two
groups for each patient: half of the oocytes were plunged into standard liquid
nitrogen (LN2 group) and the other half into slush nitrogen (SN2 group). The
slush nitrogen was obtained with the use of a rapid-cooling LN2 chamber (Vit
Master). During the study period, 92 oocytes were warmed at room temperature
using serial dilution of sucrose 1M, 0.5M and 0.0M. All survived oocytes were
examined using the Oosight/SpindleView system immediately after the warm-
ing and again after two hours of incubation. Statistical analysis was performed
using Fisher’s exact test at the level of P < 0.05.
Results: 44 and 48 oocytes vitrified with LN2 and SN2, respectively, were
warmed. 37 oocytes survived (84.1%) in the LN2 group and 33 (68.8%) in the
SN2 group (P = 0.09). Eight oocytes (21.6%) in the LN2 group and 9 oocytes
(27.3%) in the SN2 group showed meiotic spindle (MS) when observed at the Pol-
scope immediately after warming (NS). After two hours of incubation, 28 of LN2
(75.7%) and 23 of SN2 (69.7%) oocytes showed reassembled MS (NS). 36 and
31 oocytes were injected in the LN2 and the SN2 group, respectively. 28 (77.8%)
oocytes fertilized normally in the LN2 group and in 18 (58.1%) oocytes in the SN2
group (NS). Excellent or good quality embryos were 25 (89.3%) in the LN2 group
and 16 (88.9%) in the SN2 group (NS). Statistically significantly higher number of
oocytes fertilized (and developed to excellent/good quality embryos) in the LN2
group when taking into account the number of oocytes warmed in each group.
Conclusions: The results of the present study do not support previous sugges-
tions that SN2 may contribute to an improved outcome for oocyte vitrification.
Unexpectedly, fertilization and embryo development was superior when “stan-
dard” LN2 was used for vitrification. We may only speculate that at the origin of
this phenomenon there could be a technical situation related specifically to the
SN2 usage rather than difference in temperature of the liquid nitrogen. Working
space is highly reduced when using SN2 (Vit Master) compared to the working
space when LN2 is used. Reduced working space on the other hand, may limit
the speed of manipulation, an essential element of efficient vitrification proce-
dure, consequently, resulting in suboptimal outcomes. It is warranted to perform
additional studies to further clarify the contribution of different physical and
technical parameters for the optimization of the oocyte vitrification procedure.
P-171 Lysosome and apoptotic activity in cumulus cells: do we rescue
too much?
A. Borini1, N. Tarozzi1, D. Fiorentin1, M.A. Bonu1, M. Nadalini1, J. Johnson2,
L. De Santis3, V. Bianchi1
1Tecnobios Procreazione, Centre for Reproductive Health, Bologna, Italy
2Yale University School of Medicine, Obstetrics Gynecology and Reproductive
Sciences, New Haven CT, U.S.A.
3University Vita Salute, Scienze Natalità H.S. Raffaele, Milano, Italy
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dose was gradually increased every 2 days. Daily injections of progesterone in
oil 50 mg was added by cycle day 15 and continued until the pregnancy test.
Endometrial preparation was synchronised to the day of transfer.
Primary outcomes were rates of implantation, clinical pregnancy, delivery
rates. Secondary outcomes were rates of miscarriage.
Result: The results of the assessed parameters are given in the Table.
Conclusion: The present study showed that transfer of blastocysts is preferable
over cleavage stage in frozen-thawed cycles. Furthermore, extended in-vitro
culture of human multicellular embryos to day5 yielded similar outcome to
blastocyst cryopreservation. Therefore, the feasible strategy for the embryology
laboratory appears to be cryopreservation of blastocysts.
Thaw and ET Thaw and ET Thaw Day3 ET
Day3 Day5 Day5
Total cycles thawed (N) 1761 282 168
Cancellation rate (%) 2,4 2,2 5,55
Total cycles with transfer (N) 1719 276 159
Mean age ± SD 32,8 ± 5,2 31,3 ± 4,6*** 32,4 ± 4,3
Survival rate (%) 85,99 83,42 87,71
Mean embryos transferred ± SD 3,0 ± 0,7*** 2,2 ± 0,6 2,2 ± 0,7
Clinical pregnancy per transfer/ 56,7***/ 55,3*** 71,4/ 69,9 76,7/ 72,6
thaw cycle (%)
Implantation rate (%) 28,3*** 51,1 51,3
Miscarriage rate (%) 20,1 15,2 13,1
Abortion (N) 11 4 2
Delivery rate per transfer/ 44,6***/ 43,5*** 59,1/ 57,8 65,4/ 61,9
thaw cycle (%)
Extrauterine pregnancies (N) 17 3 0
*excl. Extrauterine pregnancies
** P 0.0001
P-174 MicroRNAs regulated by DNA methyltransferase control mice
blastocyst development
Y.M. Lee1, H.W. Chen2, P. Wu1, C.R. Tzeng3
1National Yang-Ming University, Department and Institute of Pharmacology,
Taipei, Taiwan R.O.C.
2National Taiwan University, Department of Toxicology, Taipei, Taiwan R.O.C.
3Taipei Medical University, Department of Obs/Gyn, Taipei, Taiwan R.O.C.
Introduction: Epigenetic regulation is very important in early embryo devel-
opment for it can affect genomic imprinting, gene reprogramming and gene
expression. Previous study showed that 93% of genes expressed in early em-
bryo are found to exhibit a CpG island over their transcription start point. In
addition, various studies have indicated that epigenetic regulation influences
gene control mainly through DNA methylation, histone modification and mi-
croRNAs (miRNAs). Furthermore, the function and expression of microRNAs
may be regulated by the processes of DNA methylation and histone modifica-
tion although the involved miRNAs and their downstream genes still remain
unknown. In our study, characterization of microRNAs expression profiles in
embryos of different developmental stages and understanding the mechanisms
and dynamics underlying the reprogramming process would be demonstrated to
analyze the complex gene regulatory networks.
Materials and Methods: The 5 weeks old ICR mice were superovulated and
the ICR mice embryos were collected and cultured in human tubal fluid me-
dium. The miR profiling of ICR mice embryos from morula to blastocyst with
or without Dnmt inhibitors treatment. Embryos treated 24hrs with or without
Dnmt inhibitors were collected for total RNA extraction. Products were ampli-
fied from 10 ng of total RNA samples with the TaqMan MicroRNA Assay. The
miR targets were predicted with some modification from the comprehensive
databases of experimentally supported animal miR targets software including
pathway analysis for mouse, MiRNAmap and PicTar. Mir predicted targets
were confirmed by SYBR green real time PCR analysis.
Results: To examine whether DNA methylation could regulate microRNA
expression, 450 microRNA amplification was based on the mature sequence
of specific microRNAs and probes were also designed uniquely for individual
analysis of different microRNA expression. Comparing the control and the
DNMT inhibitor treated groups, above the total of 187 screened microRNAs,
48 microRNAs were downregulated at least 5 folds, in another hand, 17 mi-
croRNAs were upregulated at least 5 folds. In our study, several important
microRNAs have been reported to play critical roles in early embryo develop-
ment. mir-21, mir-143, mir290, mir20a, let-7e, let-7g has been reported impor-
tant in embryo implantation, mir-96, mir-296, mir-34b could play some roles in
Material and Methods: Data were collected from September 2008 to Novem-
ber 2009 on 272 couples requiring ICSI insemination. After ICSI (day-0), oo-
cytes were individually placed into 35μl drops of Cleavage Medium (SAGE)
covered with mineral oil. After 24h (day-1) oocyte fertilization was evaluated
and the 2PN zygotes were either kept in the same dish (Group “noCH”, N =
131; mean age = 38.5 ± 4.52 ± SD) or moved to a new identical dish containing
fresh Cleavage Medium (Group “CH”, N = 141; mean age = 38.4 ± 3.94 ± SD).
Embryos were then maintained in the same dish until embryo transfer which
occurred on day-2 or day-3. Statistical analysis were performed with χ2 test at
the level of P 0.05.
Results: In groups “noCH” and “CH”, 411 and 472 oocytes were inseminated
and 80.5% and 79.5% fertilized (NS), respectively. Proportions of type A + B
and C + D embryos were 91.5% versus 88.8% and 11.8% versus 13.6% (NS),
respectively. In groups “noCH” and “CH”, 288 and 325 embryos were trans-
ferred respectively, and 52 and 48 patients had positive β-HCG (39.7%, Group
“noCH”; 34.0% Group “CH”; NS); 46 and 36 patients had clinical pregnan-
cy (35.1%, Group “noCH”; 25.5% Group “CH”; NS, P = 0.085); 59 and 47
gestational sacs were detected with an implantation rate of 20.5% and 14.5%
(P 0.05).
Discussion: The results of the present study show that fertilization rate and
embryo development and quality are similar in both groups, however, implan-
tation rate is higher when no change over of embryo was performed on day-1
after ICSI insemination. When changing culture medium on day-1, all metabo-
lites are removed, including those that are secreted by the embryo, and fresh
nutrients are available for the fertilized egg. We hypothesize that, by changing
culture media on day-1, autocrine factors are removed resulting in a reduced
competence of the developing embryos to implant. However, when we ana-
lyzed our data separately for day-2 and day-3 embryo transfers, we observed
a statistically significantly higher positive b-HCG and clinical pregnancy rates
in addition to a much higher implantation rates in “noCH” compared to “CH”,
when embryos were transferred on day-2. This additional analyses may reflect
that those embryos transferred on day-2, after their media were changed on
day-1, had less time to benefit the effects from those autocrine factors which
themselves have secreted during the time in culture. Conversely, no difference
in outcomes were seen when only day-3 embryo transfers were analyzed, in-
dicating that embryos, after day-1 change over, had more time to benefit from
autocrine factors. These observations may shed light on the importance of fac-
tors secreted by the early human embryo and help to identify a more beneficial
culture strategy.
P-173 Effectiveness of frozen embryo transfers by comparing outcomes
from day 3, day 5 and post-thawed extended culture of day 3 until day 5
N. Mesut1, H.N. Ciray1, A. Mesut1, T. Aksoy1, M. Bahceci1
1Bahceci Clinic, IVF, Istanbul, Turkey
Introduction: The efficacy of cryopreservation programs is an important con-
sideration of every IVF clinic. Storage of materials in the laboratory requires
space and considerable amount of embryologists’ time as well as financial bur-
den to the couple. It is a matter of debate among IVF professionals on which
stage to preserve embryos; namely cleavage versus blastocyst. Both strategies
may possess advantages and disadvantages and currently there is not any solid
evidence to favor one towards another.
The aim of this retrospective clinical study was to evaluate the effective-
ness of frozen embryo transfers by comparing outcomes of day 3 with day 5
embryos and also with post-thaw extended culture of thawed day 3 embryo
until day 5 prior to transfer.
Material and Methods: Between 2004 to 2009 embryos obtained from pa-
tients were frozen and subsequently thawed by employing three transfer strate-
gies; transfer of thawed day 3 embryos, transfer of thawed day5 blastocysts,
and transfer of blastocysts arising from post-thaw extended culture of day3
embryos. Clinical and embryological parameters of these three groups were
retrospectively analyzed.
Supernumerary cryopreserved embryos were those which displayed 6
cells and 20% fragmentation and good quality blastocysts ( 3BB, Gardner
score), for day 3 and 5, respectively. All embryos were cryopreserved in cry-
ovials using slow-freezing and -thawing protocols specific to day3 and day5
embryos, using Quinn’s Advantage® media.
In frozen cycles, embryo transfers were performed in hormone replacement
cycles. Patients received injections of oestradiol (2 mg) on cycle day 1 and the
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
3Institute for the Care of Mother and Child, Embryology, Prague, Czech
Republic
4Institute for the Care of Mother and Child, Gynecology, Prague, Czech
Republic
5Prague Fertility Centre, Gynecology, Prague, Czech Republic
Introduction: Multiple different embryo selection strategies have been pro-
posed up to date. The most usual evaluation of morphological parameters is
based on daily microscopic observations. However, this classical approach can
only reveal limited and static information, omitting the dynamics and timing of
early mitotic divisions. Here we analyze the chronology of early mitotic events
by continuous human embryo monitoring, demonstrating a strong correlation of
cell cycle timing and cleavage synchrony with the outcomes of pregnancy tests.
Materials and Methods: A total of 210 human pronuclear embryos were
subjected to time-lapse monitoring (PrimoVision, Cryo-Innovation, 1
picture/10 min; with the institutional ethical approval) under standard culture
conditions. The exact timing of the three interphases (IP) occurring after the
two-cell stage (IP2 defined as the period between 2 and 3 cells stages, IP3
between 3 and 5, and IP4 between 5 and 9 cells embryo), and the synchrony of
daughter cell cleavages in these three cycles (i.e. timing of the transition from
3 to 4 cells, from 5 to 8 and from 9 to16 cells) were analyzed. After 5 days of
culture, the morphologically best blastocysts with distinct embryoblasts were
transferred, and the above data were correlated with the outcomes of preg-
nancy tests (fetal heart beat).
Results: In 28 embryos giving viable pregnancies (12 singletons and 8 twins),
the durations of IP2 and IP3 were similar, taking 12-14 hrs. The IP4 between 5
and 9 cells stage was taking 22-24 hrs. In these embryos, the sister blastomeres
cleaved in a very synchronous manner: from 3 to 4 cell stage in 10-20 min;
from 5 to 8 cells in 30-50 min, and from 9 to 16 cells within 40-70 min. The
timing of early events correlated strongly with the abilities of the embryos to
develop to fully expanded blastocysts. 78 out of 92 (85 %) analyzed embryos
that cleaved in the above described ranges reached the blastocyst stage within
5 days of culture. On the other hand, when the cleavage of some cells occurred
during the IP period (frequently occurring at the beginning of IP) it always
led to asynchronous and/or uneven cleavage of the resulting daughter cells.
They were frequently arrested within the next few cleavages and partly/fully
abnormal embryo development occurred. The sooner the untimely cleavage oc-
curred – with the respect to the order of IP’s - the more abnormal development
was detected. None of 18 embryos exhibiting abnormal premature cleavage
during the IP2 period (mostly in three cells stages) developed to blastocyst
stage. 8 (19 %) of 42 embryos detected in 5 cell stage before ending IP3 reached
blastocyst, and finally, 19 (32 %) of 58 embryos with some cleaved cells in the
IP4 interval formed blastocysts.
Discussion: Taking in account the biological importance of S-phase during in-
terphase, there is not a surprise that timing of the early postfertilization process-
es correlates with viability of the resulting embryos. As we show in this study,
any untimely premature cleavage was associated with the time-dependent im-
pairment of embryonic development. In these cases, the insufficient duration of
the IP period may not be apparent when counting blastomeres once a day, and
can only be revealed by a precise time-lapse monitoring. Taken together, per-
manent embryo monitoring opens a new opportunity for exact measurement of
the early phases of human embryo development and provides a novel powerful
strategy for objective early embryo pre-selection.
P-177 Development potential and clinical implication of embryos having
either one pronucleus, (1PN) or no pronuclei (0PN)
Y. Aoi1, H. Takahashi1, H. Saitou1, C. Takiue1, N. Kawakami1, M. Tone1, R.
Hirata1, S. Terada1, N. Yoshioka1, T. Habara1, N. Hayashi1
1Okayama Futari Clinic, Obstetrics and Gynecology, 285-1 Tsudaka
Okayama, Japan
Introduction: We set out to assess the development potential and clinical im-
plication of embryos with one pronucleus (1PN) or no pronuclei (0PN). As they
have poorer development potential and implantation rates than zygotes with
two pronuclei. Therefore, the purpose of the present study was to discover the
incidence of 1PN or 0PN zygotes in our standard IVF population. We examined
appearance rates, embryo development and pregnancy rates of 1PN or 0PN
zygotes.
Materials and Methods: We have carried out a retrospective cohort study with
the index cycles identified for this study chosen after review of all IVF cycles
cell differentiation, mir-134, mir-138, mir-290, mir-296, mir-322, mir-452 and
mir-128b is expressed during pre-implantation development and might regulate
embryo cell compaction and blastocyst differentiation. To verify the function of
our epigenetic regulated microRNAs, we used several microRNA target gene
prediction software such as PicTar and Miranda to validate the microRNA tar-
get genes. By these softwares, we obtain thousands of target genes for each
microRNA, we compare the predicted target genes with the microarray data of
preimplantated embyos that includes the genes regulated during morula to blas-
tocyst. Hundreds of genes were selected after comparing the array data with our
predicted target genes, 20 important genes involved in embryo development,
stem cell, cell growth and differentation, were selected for SYBR green QPCR
confirmation, such as mir-34b/c, mir-124a targeted Jag1, mir-23a/b, mir-29b,
mir-322 and mir-381 targeted YY1, mir-433-3p targeted Hif1a and mir-124a,
mir-375 targeted SP1.
Conclusions: This study we demonstrated the miR profiles in early embryo
development and also identified the DNA methyaltion-regulated specific miRs,
we also confirmed that these miRs could target to growth and cell differentiated
related genes which are vital in preimplantation development. This study might
improve our knowledge of epigenetic-regulated miRs in early embryo growth
and cell differentiation during pre-implantation embryo development.
P-175 Morula stage embryos on day 5: what to expect?
I. Antonova1, T. Milachich1, L. Petkova1, M. Yunakova1, P. Chaveeva1,
A. Shterev1
1SAGBAL Dr. Shterev Hospital, Embryology, Sofia, Bulgaria
Introduction: Extending embryo culture to blastocyst stage provides better op-
portunities for selection of embryos with higher developmental potential and
decreases the number of transferred embryos. Thus the frequency of multiple
gestations is lower. Replacement of blastocysts enhances the reproductive out-
come of IVF.
On the other hand, culturing the embryos to day 5 for transfer is related
with number of risks: monozygotic twinning, no spare embryos for freezing,
not achieving the blastocyst stage etc.
The aim of this study was to evaluate the pregnancy outcome in patients
with morula stage transfer on day 5. Also we assessed the predictive values of
different parameters.
Material and Methods: The current study includes 63 IVF/ICSI cycles with
embryo transfer on day 5, separated into 2 groups:
Group A – 30 patients with transfer of morula stage embryos on day 5
Group B – 33 patients with transfer of blastocyst stage embryos on day 5
In both groups we have analyzed: average number of retrieved oocytes,
fertilization rate, embryo quality on day 3, embryo quality on day 4, blastocyst
formation on day 5 and pregnancy rate.
Results: There was no statistical difference between the two groups in: average
number of retrieved oocytes Group A (9.8) and Group B (12). Fertilization rates
(82% Group A versus 83% Group B) and embryo quality on day 3 (Group A -
34% good, 22% average and 44% bad; Group B - 33% good, 28% - average and
39% bad embryos) were also similar in both groups (P > 0.05).
In contrast of these data, there was a significant difference in embryo de-
velopment on day 4 in first group (16.5% compacted morula stage and 83.5%
cleavage stage) versus the second one (58.5% compacted morulas and 41.5%
cleavage stage).
Blastocyst formation on day 5 in second group was 50% (29% morula
and 21% cleavage stage embryos respectively). No blastocysts were observed
in group A.
Pregnancy rate in Group B was higher comparing to Group A (42.4% versus
20.0%) (P 0.05).
Conclusions: Embryo transfer of morula stage embryos on day 5 is related to
lower pregnancy rates. No difference in embryo quality was observed between
two groups on day 3. Slow developing embryos on day 4 could be a predictive
factor for high risk for absence of blastocyst stage on day 5.
P-176 Permanent embryo monitoring and exact timing of early
cleavages allow reliable prediction of human embryo viability
D. Hlinka1, M. Dudas2, J. Rutarova3, J. Rezacova4, S. Lazarovska5
1Prague Fertility Centre, embryology, Prague, Czech Republic
2P. J. Safarik University, Institute of Biology and Ecology, Kosice, Slovakia
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
the assessed group, patients who had an early-cleaved embryo transferred were
designed as EC (early-cleaved) group and patients who had no early-cleaved
embryos transferred were defined as NEC (not early-cleaved) group. Statistical
analysis comparing the three groups (not assessed, EC and NEC) was Kruskal-
Wallis test and differences in implantation rates were calculated by Chi-square
test.
Results: Forty-nine percent of patients in the assessed group had at least one
early-cleaved embryo. Patient’s age was similar among the NA (31.1 ± 3.6),
EC (30.7 ± 3.3) and the NEC groups (30.6 ± 2.9) (P > 0.05) as well as ovar-
ian reserve and mean number of cycles. There were no differences between
assessed and NA groups on mean number and quality of zygotes neither on
the quality of embryos transferred. Single embryo transfer took place in 270
patients: 131 patients in the NA group and in 139 in the assessed group. From
the NA group, 61 embryos chosen to be transferred were EC (44%) and 78
were NEC (56%). Implantation rates were not significantly different among
the three groups: 28%, 25% and 27% for NA, EC and NEC groups, respec-
tively (P > 0.05).
Conclusions: The current study is the first to demonstrate that when applying
the policy of single embryo transfer on day 2 in a GnRH-antagonist protocol,
assessment of early cleavage is not helpful in choosing an embryo with the best
implantation potential. Furthermore, it should be taken into account that when
choosing an embryo for transfer, the presence of early cleavage appears not to
be a powerful predictor for embryo implantation in those patients.
References:
1 Yang et al. Early-cleavage is a reliable predictor for embryo implantation in the GnRH
agonist protocols but not in the GnRH antagonist protocols. Reprod Biol Endocrinol
(2009) 7:20 doi:10.1186/1477-7827-7-20.
2 Yang et al. Early cleavage does not predict treatment outcome following the use of
GnRH antagonists in women older than 35. Fertil Steril (2007) 88:1573-8.
P-179 Retrospective evaluation of the outcomes of a sequentially
cultured human blastocyst transfer programme by slow controlled-rate
freezing
J. Silva1, M. Cunha1, P. Viana1, J.M. Teixeira da Silva1, C. Oliveira1, A.
Gonçalves1, N. Barros1, M. Sousa2, A. Barros1
1Centro de Genética da Reprodução, Prof. Alberto Barros, Porto, Portugal
2Institute of Biomedical Sciences Abel Salazar UMIB University of Porto, Dpt.
Microscopy Lab. Cell Biology, Porto, Portugal
Introduction: The main purpose of IVF is the transfer of a single embryo to re-
duce the risk of multiple pregnancies. The use of an efficient sequential culture
method has enabled to select those embryos with capability to develop to a pre-
implantation stage, hereby increasing the treatment efficiency, as blastocysts
have a higher implantation potential. To increase the rates of pregnancy and
delivery, extended culture is also dependent on a successful freezing blastocysts
programme. In our centre, we kept until present the slow freezing method due
to the excellent results obtained. Our experience with day 5 embryo transfer
programme was evaluated retrospectively during 2005-2008, with new-born
outcomes being here presented.
Materials and Methods: 122 blastocyst thawing cycles were performed, in the
majority of them for couples that failed to achieve a pregnancy with fresh trans-
fer. Blastocyst score according to Gardner and Lane (2000). 75% of the cases
were ICSI cycles. The mean female age was 34.6 years (24-50). Blastocysts were
cultured in sequential media ISM1/BlastAssist (Medicult) or G-1-Plus/G-2-Plus
(Vitrolife). Freeze-thaw procedures were according to Medicult BlastFreeze/
BlastThaw protocols (Ménézo and Veiga, 1997; Virant-Klun et al., 2003). Freez-
ing was at 37ºC using BlastFreeze. Embryos were aspirated into a PETG clear
rigid straw, 0.15ml/91mm (Cryo BioSystem), generally 1-2 blastocysts/straw.
Programmed slow freezing was performed in a Planer K10 apparatus (Middle
Sex, UK). Thawing was performed in the day of embryo transfer (ET), at room
temperature, under a bell-shaped glass with a 5%CO2 stream of flow, using
BlastThaw. Embryos were then cultured in a humidified incubator with 6%CO2,
5%O2 and 89%N2, at 37ºC, in Medicult IVF-medium and then in BlastAssist
until ET. Only blastocysts, contracted, partially or completely reexpanded, with
<50% degeneration were replaced (mean 1.7 blastocysts/transfer). Blastocysts
were placed in UTM medium for ET. For those cases in which embryo culture
was performed with Vitrolife media, embryos were placed in G-IVF-Plus, G-2-
Plus, and finally in EmbryoGlue. The mean time from thaw to ET was about 2h.
Ultrasonically-guided ET was performed in the large majority of the cases by
performed in our program from January 2006 through May 2009. 1572 cases
from 929 patients were collected 18450 oocytes were investigated. These 464
zygotes of 1PN arose from 305 patients and 348 cycles and these 2347 zygotes
of 0PN arose from 660 patients and 913 cycles. All embryos were maintained at
37°C in humidified atmosphere of 6.5% CO2, 5% O2, 88.5% N2. Within 4 hours
of retrieval, oocytes were inseminatied or fertilized ICSI. 18 hours after insemi-
nation or ICSI were cultured, we checked zygotes having 2PN or 1PN or 0PN
or 3PN. 1PN and 0PN were cultivated separated from 2PN, and cultured same
condition. Developed blastocysts of 1PN and 0PN were frozen, single or mixed
with 2PN zygote. We transferred the frozen-thawed 1PN or 0PN embryo as last
resort after obtaining informed consent from the couple.
Results: The 1PN and 0PN rates were 3.3%(225/6693), 12.7%(2347/6693)
from conventional IVF and 2.0% (239/11757), 10.0%(1182/11757) from
ICSI. From 1PN of conventional IVF and ICSI cleavage rates on day 3 were
74.2%(cIVF) and 72.0%(ICSI), good quality embryos were 28.0%(cIVF)
and 18.8%(ICSI). From 0PN of conventional IVF and ICSI cleavage rates on
day 3 were 35.8%(cIVF) and 32.8%(ICSI), and good quality embryos were
19.0%(cIVF) and 3.0%(ICSI). Frozen blastocysts of 1PN were 41 zygotes
(cVF) and 13 zygotes (ICSI),and transferred 18 zygotes (cIVF) and 4 zygotes
(ICSI). Frozen blastocysts of 0PN were 38 zygotes (cIVF) and 49 zygotes
(ICSI), and transferred 26 zygotes (cIVF) and 35 zygotes (ICSI). The pregnancy
rate, including 1PN zygotes, transferred were 41.2%(cIVF) and 0%(ICSI),and
only 1PN zygotes transferred were 33.3%(cIVF) and 0%(ICSI). All pregnancy
cycles of 1PN zygotes transferred from cIVF. There were no miscarriages, and
children were born from 7 cycles. Healthy children have been born after trans-
fer of 1PN embryos. The pregnancy rate including 0PN zygotes, transferred
were 33.3%(cIVF) and 27.3%(ICSI),and only 0PN zygotes transferred were
16.7%(cIVF) and 18.2%(ICSI). Children were born from 4, 6 cycles, and on
going 1, 2 cycle. There were 2, 2 miscarriages.(cIVF, ICSI) Healthy children
have been born after transfer of 0PN embryos too. The occurrence of congeni-
tal anomalies in embryo transfer of 1PN or 0PN zygotes cycles was similar to
those in embryo transfer of 2PN zygotes.
Conclusions: Embryo development of 1PN and 0PN zygotes had poorer devel-
opment potential and implantation rates than 2PN zygote, but pregnancy was
attained in cycles of 1PN and 0PN zygotes transferred. Pregnancy and birth
resulting from transfer of a blastocyst ovserved 1PN or 0PN, it may become the
index of the normal growth after that. Therefore, we have counseled patients
that, with no other suitable embryos available for transfer, there is a very low
likelihood of viable pregnancy after transfer of 1PN or 0PN zygotes. It would
be the least preferable form of transfer in clinical application.
P-178 Early-cleavage does not predict implantation in day 2 elective
single embryo transfers in GnRH-antagonist cycles
J. Montagut1, F. Bonald1, N. Guillen1, V. Guitard1, E. Balu-Genvrin1, E. Crae1,
D. Nogueira1
1IFREARES, Laboratoire de Biologie de la Reproduction, Toulouse, France
Introduction: The evaluation of early cleavage has been widely applied as an
additional parameter to predict the embryo with the best implantation potential.
Nevertheless, the importance of early cleavage assessment is still debatable.
Previous studies have suggested that contradictory results might be accounted
to the differences in patient’s characteristics and stimulation protocols 1,2. These
cited studies, however, included 2-3 embryos transferred per patient mixing
early-cleaved embryos with embryos not presenting early cleavage. The present
study was conducted to evaluate whether the assessment of early cleavage can
predict the implantation potential when a single embryo is transferred on day 2
in patients receiving a GnHR-antagonist stimulation protocol.
Materials and Methods: This prospective study included consecutive patients
having 37 years old with primary infertility, a first or a second IVF/ICSI cycle
from August 2008 to October 2009. All patients included received the same
protocol associating recombinant FSH (Gonal-F; Merck Serono or Puregon,
Shering-Plough) and GnRH-antagonist Cetrorelix (0.25 mg Cetrotide; Serono
Laboratories). A total of 286 patients presenting at least one zygote on day 1
post-insemination were randomly distributed as not assessed (NA) group and
assessed group for early cleavage evaluation at 25-26 hours post-insemination.
Within the assessed group only embryos with two even cells were considered
as “early-cleaved”. The choice for transfer was based on embryo quality on
day 2 and zygote morphological pattern on day 1. When there were embryos of
equal quality, preference for transfer was given to the early-cleaved embryo. In
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analysed samples using the Relative Expression Software Tool (Pfaffl et al.,
Nucleic Acids Res 2002: e36).
Results: When comparing cumulus cells of ETSPs between the two different
ovarian stimulation protocols, only minor differences in gene expression of
the selected candidates were found. Cumulus cell samples of FFOs from the
GnRH agonist stimulation protocol showed major differences from samples
of ETSPs from the same protocol. In contrast, cumulus cell samples of FFOs
from the GnRH antagonist stimulation protocol had a gene expression profile
more similar to that of ETSPs. Out of the 17 analyzed genes, only expression
of endothelin receptor type A (EDNRA) was shown to be significantly different
between cumulus samples from FFOs and ETSPs, irrespective of the ovarian
stimulation protocol.
Conclusions: These preliminary results indicate that oocytes with a high de-
velopmental potential, determined by their ability to result in pregnancy, origi-
nate from follicles with common gene expression characteristics, possibly the
most mature follicles. Moreover, they suggest an overall higher quality of the
cohort of oocytes retrieved after the GnRH antagonist stimulation protocol.
These data support the hypothesis that ovarian stimulation with GnRH antago-
nist co-treatment allows only the most mature follicles to develop up to oocyte
retrieval. Since EDNRA was shown to be differentially expressed in cumulus
cell samples from oocytes associated with pregnancy, expression levels of this
gene may be indicative of oocyte quality.
P-181 Culture embryos under low oxygen conditions improves clinical
results
P. Gámiz Izquierdo1, J.M. De los Santos1, A. Tejera1, A. Pellicer2, J.L.
Romero1, A. Galán1, C. Albert1, M.J. De los Santos1
1Institut Universitari - IVI Valencia, IVF Laboratory, Valencia, Spain
2Institut Universitari - IVI Valencia, OB/GYN, Valencia, Spain
Introduction: Mammalian embryos can be successfully cultured under a gas
phase containing either atmospheric (20%) or reduced (5%) O2 concentration.
Low oxygen tension during in vitro culture is intended to mimic the in vivo
conditions of both human oocytes and embryos and therefore be beneficial for
embryo development and implantation. A number of studies have been carried
out with contradictory results. Very recently a prospective randomized clinical
trial has been performed on blastocyts transfer in women undergoing first cycle
of blastocyts.
We wanted to evaluate whether low oxygen tension was also useful on day
3 embryos transfers in egg recipient women, where the oocyte variable is con-
trolled, in terms of fertilization rate, percentage of morphologically optimal
embryos, implantation rate and ongoing pregnancy rates.
Material and Methods: A prospective study was conducted in a total of 939
cycles, 458 (3192 embryos) were cultured in an atmosphere of 5% O2 concen-
tration and 481 cycles (3754 embryos) in ambient oxygen atmosphere using
Sanyo MCI 5M incubators.
Oocytes and embryos were cultured in 50μl drops of HTF under mineral oil.
Day 3 embryo transfers were performed using Wallace catheters. The param-
eters analyzed to asses the embryo quality were cell number, degree and pattern
of fragmentation, symmetry and size of the cells, multinucleation and the aspect
of cytoplasm. Statistical analysis was performed with Fisher’s exact test, and
statistical significance was defined by P-values of < 0.05.
Results: No significant differences were found between the two groups
on fertilization rates (70.9% versus 71.8%, respectively). However, the
percentage of good quality day 2 an day 3 embryos were significantly
improved when 5% oxygen was used, 60,0% versus 54.0% on day 2 and
64.1% versus 58.0% on day 3 respectively. Low oxygen tension showed
statistical increase on clinical pregnancy rate (55.9% versus 46.2%), ongo-
ing pregnancy rate (46.3% versus 38.0%) and implantation rate (38.2%
versus 31.7%).
Conclusions: Better quality day 2 and day 3 human embryos were achieved by
culturing the oocytes and embryos under 5% oxygen tension. We observed an
improvement in clinical results when human embryos were cultured under low
oxygen concentration compared to culture under atmospheric oxygen condi-
tions. We believe that culture under low percentage of oxygen is more similar
to natural conditions in the uterus (Fisher et al.,1993), and may decrease the
formation of reactive oxygen species (Goto et al.,1993 ). This could explain
that the implantation and pregnancy rate obtained are higher with this type of
culture conditions.
the same gynaecologist, using a Sure View Wallace Embryo Replacement Cath-
eter (Smiths Medical Int.). All ET were performed in a programmed cycle. To
prepare the endometrium, oral Estradiol 2mg tablets were given bid, from days
2-7 of the menstrual cycle, followed by 2mg tid, since day 8. An ultrasound was
performed by day 15 of the cycle. Embryo transfer was programmed to about
3 days after an ideal endometrial appearance. Two endovaginal progesterone
100mg tablets were added tid, beginning 2-3 days before ET.
Results: The outcome rates were: 76% of survival, 48% biochemical preg-
nancy, 43% clinical pregnancy, 42% ongoing pregnancy, 29% implantation and
37% births. The spontaneous abortion rate was 7% (7-13%). Topic pregnan-
cies represented 42%, and ectopic 0.8%. Single pregnancies represented 76%,
twins 22% and trigemelar 2% (one case in 2006). The male (51%) to female
(49%) ratio was 1.0. No morphological and chromosomal abnormalities were
detected.
Conclusions: A recent review on slow freezing of human blastocysts showed
excellent results: 76-95% survival, 31-69% pregnancy and 23-43% of implan-
tation rates (Youssry et al., 2008). The present results confirm and update previ-
ous studies based on blastocyst slow freezing and thawing, and suggest that this
technique is not yet to be replaced in those centres with high rates of blastocyst
survival, pregnancy, implantation and birth after thawing.
Due to development of sequential media, extended culture to the blastocyst
stage and an efficient freeze-thaw procedure for blastocysts became possible,
thus increasing the success of IVF, and decreasing the number of multiple preg-
nancies. This technique should thus be introduced as a routine procedure in all
IVF laboratories.
P-180 The effect of ovarian stimulation with GnRH agonist or
antagonist co-treatment on cumulus cell gene expression in relation to
oocyte quality
C. van de Werken1, H. Jahr2, J.S.E. Laven1, E.B. Baart1
1Erasmus MC, Obstetrics and Gynaecology Division of Reproductive Medi-
cine, Rotterdam, The Netherlands
2Erasmus MC, Orthopaedics, Rotterdam, The Netherlands
Introduction: In a previous study, oocytes retrieved after mild, GnRH an-
tagonist, ovarian stimulation were shown to develop into a higher proportion
of chromosomally normal embryos than oocytes retrieved after convention-
al, GnRH agonist, ovarian stimulation(Baart et al., Hum Reprod 2007:980).
Since oocyte maturation is linked to and guided by follicular development,
it is hypothesized that the two types of ovarian stimulation influence oo-
cyte development through its hormone-responsive follicular microenviron-
ment, the cumulus granulose cells. Differences in gene expression profiles
might be expected in cumulus cell samples from women undergoing ovarian
stimulation with GnRH agonist co-treatment, or with a GnRH antagonist.
Therefore, the aim of this pilot project was to compare gene expression of
a selected panel of genes in cumulus cell samples from both stimulation
protocols and link this to the observed developmental potential of the cor-
responding oocytes.
Material and Methods: Women < 38 years of age with a regular indica-
tion for IVF and a partner with normal semen parameters, were randomly
selected from our routine IVF program. Patients underwent either ovarian
stimulation (150 IU rec FSH/ day) with GnRH agonist long protocol co-
treatment, or with GnRH antagonist co-treatment, starting 150 IU rec FSH/
day on cycle day 2. After oocyte retrieval, a part of the cumulus cells was
removed from each oocyte and stored at -20°C in lysis buffer (RLT buffer,
Qiagen). The oocytes were inseminated and cultured separately in order to be
able to link cumulus cell samples to the observed developmental potential of
the corresponding oocyte. In total, 72 cumulus cell samples from 8 patients
undergoing the GnRH agonist protocol and 46 cumulus cell samples from 8
patients undergoing the GnRH antagonist protocol were collected. In each
group, 3 patients became pregnant after single embryo transfer. For each of
these patients, two cumulus cell samples were selected for analysis: one cor-
responding to an oocyte which failed to fertilize (FFO) and one resulting in
the embryo transfer with subsequent pregnancy (ETSP). In the cumulus cell
samples, expression levels of COX-2, HAS-1, MMP-2, -3, -9, -16, CSPG2,
TIMP-1, -2, COL3, CTSK, ADAM17, BMP-2, AURKB, IGFBP4, EDNRA and
FST were determined by quantitative reverse transcription polymerase chain
reaction (qRT-PCR). Gene expression was normalized to the expression of an
index of “housekeeping” genes and compared between different subgroups of
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the definition of a good quality embryo (GQE) has to be validated. In this
prospective study, the impact of CASS on the definition of a GQE has been
evaluated.
Materials and Methods: A total of 502 transferred embryos (single embryo
transfer cycles) from 459 patients, younger than 36 years participating in the
IVF/ICSI program for the first or second time, were evaluated in this prospec-
tive study. Embryos were evaluated by one embryologist using a CASS (Ferti-
Morph, Image House, Copenhagen, Denmark). This system allowed to record
sequential images of the same oocyte or embryo by automatically focusing
through the complete embryo at 5 μm intervals. Using these image sequences,
the number and size of blastomeres and the degree of fragmentation was evalu-
ated semi-automatically. Implantation was defined as a positive β-hCG value
(>25IU) 14 days after embryo transfer. Statistical analyses were performed
using Chi-square test (significance level 0.05).
Results: Overall, the implantation rate (IR) per embryo transferred was
32%. There was a significant impact of the number of blastomeres on IR
(p = 0.004). The IR of a 8 cell stage (39%) embryo was significantly higher
compared to an 7 cell stage embryo (24%) (p < 0.0001). No differences were
found in IR when an embryo had 8, 9 or 10 blastomeres on day 3. Moreover,
no differences were found when these embryos were subdivided in catego-
ries based on the number of blastomeres on day 2 (2, 3, 4 or 5 cell stage).
The degree of fragmentation measured by CASS had no influence on the IR.
Embryos with equally sized blastomeres had a higher IR (37%) compared
to embryos with unequally sized blastomeres (29%) (p = 0.07). Based on
these results, a GQE was defined as an embryo with 8,9 or 10 equally sized
blastomeres. The IR of these embryos was significantly higher compared to
the other embryos (43% versus 28%) (p < 0.0001). In contrast to the standard
definition of a GQE, the fragmentation was excluded from the definition by
CASS.
Conclusions: For the first time, a definition of a GQE on day 3 is given based
on computer assisted evaluation in a unique setting of single embryo transfer
cycles on day 3. These results are an important step in the validation of a CASS
for the selection of embryos in a non-invasive way.
References:
1 Arce et al. Hum Reprod 2006;21:2141-8.
2 Paternot et al. 2009 Reprod Biol Endocrinol, 7:105.
3 Garrisi et al. Fertil Steril 1993,59:366-74.
4 Hnida et al. Hum Reprod 2004;19:288-93.
P-184 Effect of platelet activating factor (PAF) on embryonic
development and implantation in human in-vitro fertilization (IVF)
H.K. Hwang1, H.M. Kim1, J.H. Lee1, Y.J. Jung1, A. Kang1, M.J. Kook1,
J.Y. Jung1, S.J. An1, H.C. Kwon1, S.J. Lee1
1Mirae and Heemang Ob/Gyn Clinic, Infertility Dept., Seoul, Korea South
Introduction: Platelet-activating factor (PAF) plays a significant role in fer-
tility. Preimplantation stage embryos produce PAF which is required for de-
velopment. PAF’s mechanism is receptor-mediated and its presence has been
reported in the developing mouse and human embryo. In previous our data,
exposure of preimplantation stage mouse embryos results in higher develop-
ment potency and implantation rates. The study aims to evaluate the effect of
PAF on developmental potency and implantation in human IVF.
Materials and Methods: In this prospective observational study, we analysed
142 IVF/ICSI cycles from July 2009 to December 2009. They underwent IVF
program according to GnRH-antagonist with recombinant FSH protocols. Pa-
tients were allocated into the different culture condition. We used the Quinn’s
Advantage Cleavage Medium as a plain medium (control group) and added
0.5 nM PAF (1-O-Hexadecyl-sn-glycero-3-phosphorylcholine) to define (study
group) the effect. Embryo transfer was performed on day 3 or 5. The luteal
phase was supplemented with 8% vaginal progesterone.
Results: No statistical differences were observed between control and study
group in mean age (34.9 ± 3.6 vs 35.7 ± 3.3), the number of oocytes retrieved
(14.4 ± 11.8 vs 11.8 ± 9.8) and the number of embryos transferred per patient
(2.8 ± 1.0 vs 3.0 ± 1.0). The good embryo, clinical pregnancy and implantation
rates in study group (53.7%, 37.5% and 14.7%) were higher than control group
(45.4%, 34.5% and 9.3%), but it was not statistically significant. Miscarriage
rates were 14.7% in control and 16.7% in study group.
Conclusions: The results show that the proportion of good embryo, pregnancy
and implantation rates are higher in the PAF group. The present study suggests
P-182 Cumulus cell gene expression in ICSI patients predicts embryo
development and pregnancy
T. Adriaenssens1, S. Wathlet1, I. Segers1, G. Verheyen2, H. Van De Velde2, W.
Coucke3, P. Devroey2, J. Smitz1
1Vrije Universiteit Brussel (VUB), Follicle Biology Laboratory, Brussels,
Belgium
2UZ Brussel, CRG, Brussels, Belgium
3Scientific institute of Public Health, Clinical Biology, Brussels, Belgium
Background: Cumulus cell (CC) gene expression is proposed as a non de-
structive analysis method to predict oocyte competence in assisted reproductive
techniques (ART). There are however important between-patient differences in
CC gene expression for which correction is mandatory. This can be achieved
when expression results are combined with patient and cycle characteristics
using a multivariable regression analysis/model.
Material and Methods: CC from 25 ICSI patients stimulated with a GnRH-
antagonist and recombinant FSH protocol were collected individually and
oocytes were individually fertilized and cultured. All patients had a single
embryo transferred with an implantation rate of 11/25 and a live birth rate
of 9/25. The following patient and cycle characteristics were recorded: age,
BMI, number of days of gonadotrophin stimulated, ovarian response (amount
of oocytes retrieved /daily dose of gonadotrophins), and serum levels of FSH,
LH, E2 and Progesterone on day of hCG. CC were analyzed for the expres-
sion of SDC4, PTGS2, VCAN, ALCAM, GREM1, TRPM7, CALM2 and
ITPKA using quantitative PCR. The within-patient variation in expression
was related to oocyte maturity and the developmental potential of the oocyte.
Models predictive for embryos or blastocysts with a normal developmental
speed and 10% fragmentation were developed using multivariable regres-
sion analysis using the expression values and patient and cycle characteristics
as variables. Finally CC gene expression was studied in function of successful
transfer and pregnancy.
Results: This is the first proof of concept study in GnRH-antagonist and rFSH
stimulated patients. Mature oocytes have higher PTGS2 and TRPM7 and lower
VCAN expression in their surrounding CC. All genes, except VCAN, had a
positive correlation with good embryo morphology and were used in prediction
models for good embryo or blastocyst development (P < 0.01). Most models
combine expression values of two genes with two patient or cycle character-
istics. SDC4 expression is the most potent predictor for blastocyst quality on
day5. Higher expression of all genes (except CALM2) was also related to im-
plantation and pregnancy (P < 0.05).
Conclusion: Multivariable prediction models using the current genes could
be useful when fertilization and embryo or blastocyst culture are restricted for
legal reasons. This strategy also proved useful for pregnancy prediction but
requires confirmation in a larger patient population.
P-183 The impact of a computer assisted scoring system on the
definition of a good quality embryo on day 3
G. Paternot1, T.M. D’Hooghe1, S. Debrock1, C. Spiessens1
1U.Z. Leuven, Leuven University Fertility Center, Leuven, Belgium
Introduction: Standard evaluation of embryos by an embryologist is based
on direct microscopic evaluation. This results in the definition of a good
quality embryo (GQE) being a 7- or 8-cell stage with less than 25% of frag-
mentation and equal to slightly unequal blastomeres. This direct microscopic
evaluation shows a number of disadvantages. Firstly, a high inter-observer
variability has been reported.1-2 Secondly, the absence of a clear defined stan-
dard method leads to a loss of important information. Thirdly, the evaluation
time has to be kept as short as possible to prevent exposure to suboptimal
conditions since fluctuations in pH and temperature have deleterious effects.3
New technology using multilevel imaging by a computer assisted scoring
system (CASS) has the potential to overcome most of these disadvantages.
Firstly, it is possible to store multilevel images of the embryo, in a fast way,
limiting the exposure time to less optimal conditions. Secondly, these multi-
level images allow embryologists to assess embryo quality in the same way
as an exploration at the inverted microscope.3 Thus allowing an evaluation
in more detail without any time restrictions. Thirdly, embryo characteristics
can be analyzed semi-automatically resulting in a better and more precise
way of scoring which may reduce the inter-observer variability.2,4 Before a
CASS can be used to select the embryo with the highest implantation chance,
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P-186 Comparison of the effects of two in vitro culture media on embryo
fragmentation and pregnancy outcome
M. Binda1, R. Campo1, G. Van Kerkhoven1, V. Frederickx1, A. Serneels1,
P. Roziers1, I. Vranken1, A.S. Lopes1, A. Van Nuland1, S. Gordts1,
P. Puttemans1, M. Valkenburg1, S. Gordts1
1LIFE (Leuven Institute for Fertility and URG), Leuven, Belgium
Introduction: The aim of this study was to compare the effects of two in vitro
culture media, ISM1 (MediCult, Jyllinge, Denmark) and GM501 (Gynemed
GmbH & Co. KG, Lensahn, Germany), on embryo fragmentation and pregnan-
cy outcome. Primary outcome parameters were pregnancy and take-home-baby
rates. The secondary parameter was the correlation of embryo fragmentation
rate with pregnancy outcome.
Material and Methods: From June to September 2007, the embryos of 172 pa-
tients entering the Unit Reproduktieve Geneeskunde at Heilig Hart Ziekenhuis
(Leuven) IVF/intracytoplasmic sperm injection (ICSI) program were randomly
allocated to either the standard sequential culture medium ISM1 (control group)
or a new universal medium (GM501) (study group). Blastocyst culture, preim-
plantation genetic diagnosis (PGD) patients, testicular sperm extraction (TESE),
and egg donation procedures were excluded from the study. Conventional embryo
transfer was performed on day 2 or 3. For embryo scoring, the number of blasto-
meres, the cell similarity, and the fragmentation rate were taken into account. The
fragmentation rate was classified into three groups: no fragmentation, less than
30% fragmentation, and more than 30% fragmentation. On the day of the transfer,
the embryo morphology was scored and the number of embryos to be transferred
was determined in accordance with Belgian legislation. Data were analyzed using
the Fisher’s exact test. P < 0.05 was considered statistically significant.
Results: A total of 172 patients were randomized: 87 for ISM1 group and 85
for GM501 group. Five patients (four in the ISM1 group and one in the GM501
group) were excluded from the study because no transfer was performed; there-
fore, 83 and 84 transfers were performed for ISM1 and GM501 groups, re-
spectively. There were no significant differences (P > 0.05) with regard to age
(34.7 ± 5.0 vs. 34.5 ± 5.1), number of oocytes per cycle (7.5 ± 4.7 vs. 7.0 ± 3.9),
number of embryos per transfer (1.5 ± 0.8 vs.1.7 ± 0.8), and technique used for
fertilization (100% IVF/100%ICSI/ 50%IVF-50%ICSI: 24.1%/61.7%/13.3%
vs 21.4%, 61.9%, 16.6% ) between the control group and the study group.
We did not observe any differences between the ISM1 control group and
GM501 study group with regard to fertilization (68.4% vs. 72.8%), pregnancy
(32.5% vs. 27.4%), and implantation (17.8% vs 20%) rates, ongoing pregnancy
(22.9% vs. 21.4%), and take-home-baby (27.7% vs. 27.4%) rates. No differ-
ences in the number and symmetry of the blastomeres were found between the
embryos transferred in the two groups. However, fewer non-fragmented embry-
os (25.4% vs. 49.2%, p < 0.0001) and a higher percentage of minimally frag-
mented embryos, i.e., < 30% fragmentation rate, (70% vs. 47.6%, p = 0.0003)
were available for transfer when GM501 was used.
Conclusions: In conclusion, in vitro culture of embryos in Gynemed GM501
medium yielded pregnancy outcome results comparable to those of embryos
cultured in ISM1, in spite of the higher embryo fragmentation rate in GM501.
Because embryo culture in GM501 is a one-step procedure, and the medium has
a long shelf life of 6 months, this product could contribute to higher efficiency
in IVF laboratories.
P-187 How we could rescue undocumented embryos with a common
paternity test?
A. Rodríguez-Arnedo1, J. Ten1, J. Guerrero1, B. Lledo2, M.A. Carracedo1,
J.A. Ortiz2, J. Llacer3, R. Bernabeu3
1Instituto Bernabeu, Embryology Dept., Alicante, Spain
2Instituto Bernabeu Biotech, Molecular Biology Dept., Alicante, Spain
3Instituto Bernabeu, Reproductive Medicine Dept., Alicante, Spain
Introduction: It is now well established that there is an extensive dialogue
between the fertilizing sperm and the egg, leading to the activation of the egg
and to the sperm head decondensation. This is followed by the pronuclear for-
mation, syngamy, and the first cleavage. Pronuclei appear, routinely at 12-20 h
post-insemination. Occasionally, pronuclei can disappear before the assessment
of fertilization, considering these eggs as not fertilized. The dividing zygotes
are really “undocumented embryos”, and so, no viable neither for transfer nor
to freeze. If these embryos are discarded in a short embryonic cohort, we can
decrease the pregnancy options of these couples.
that PAF helps the embryonic development in in-vitro condition and this im-
proved outcome may increase the cumulative pregnancy rate.
P-185 Oocytes hormonal microsurroundings, fertilization potential and
embryos morphometric parameters in women with different androgenic
state at IVF cycles
O. Somova1, A. Feskov2, I. Feskova2, N. Chumakova2, O. Zozulina2,
Y.E. Zhilkova1
1Center of Human Reproduction “Clinic of Professor Feskov A.M.”,
Embryological Laboratory, Kharkiv, Ukraine
2Center of Human Reproduction “Clinic of Professor Feskov A.M.”, IVF
Department, Kharkiv, Ukraine
Introduction: The search of non-invasive prognostic markers to identify
and select the best oocytes and viable embryo(s) in IVF cycles is important.
Investigations of hormonal, cytokine profiles of oocyte microsurroundings
in follicular fluid (FF), metabolomic profiling of culture media, et cetera si-
multaneously with new morphological criteria for embryos are intensively
developed. However there are few researches on relation between peculiari-
ties of endocrine, paracrine and autocrine microenvironment of oocytes from
women with initially different hormonal status and morphological features of
the obtained embryos, their viability and IVF outcome. The aim of the work
was to study the hormonal profile of oocytes microsurroundings, their fertil-
ization potential and morphometric peculiarities of the transferred embryos
in patients with different androgenic status who achieved or not pregnancy
at IVF/ICSI cycles.
Material and Methods: 64 women aged of 32.3 ± 2.4 years were divided
in 2 groups, depending on their androgenic status: normoandrogenia (NA,
n = 46) and hyperandrogenia (HA, n = 18). All patients underwent a con-
trolled ovarian stimulation in GnRH analogue protocols. Levels of testos-
terone (T), estradiol (E2), sex hormone binding globulin (SHBG), leptin in
follicular fluid (FF) from the largest follicles, content of E2 in blood serum on
days of oocytes aspiration and embryo(s) transfer (Day 0 and Day 5 respec-
tively) were determined with ELISA kits. Index of free androgens (IFA) was
calculated. Number, morphological quality of oocytes and embryos assessed.
Morphometric analysis of the best embryos on Day 3, which became blas-
tocysts on Day 5 and were transferred (1-2 blastocysts/transfer), conducted
with the computer program OCTAX EyeWare (Germany). Statistic analysis
was performed using Mann-Whitney and Student tests. P-value < 0.05 was
considered as significant.
Results: The T level in FF of patients with HA was 2.4 times higher than in
patients with NA (P < 0.05). IFA also increased and was equal to 14.8 ± 2.8
versus 5.4 ± 0.9% in group NA (P < 0.05). There were no differences in E2
and SHBG levels in groups. The leptin content in FF was 1.6 times less in HA
group (P < 0.05). The total number of oocytes did not differ between groups,
but the amount of atretic oocytes was greater, the number of embryos and
their quality were considerably less in HA group. The diameter and total vol-
ume of the best embryos, zona pellucida (ZP) thickness did not differ between
the groups, but the absolute and relative volumes of blastomeres composed
embryos were higher in HA group (P < 0.05). The pregnancy rate in HA group
was 33 versus 50% in NA group. While comparing women with HA who
achieved pregnancy (n = 6) or not (n = 12), we revealed that serum E2 levels
on Day -1 and Day 5 were 2 times higher at patients who became pregnant
and were equal to its levels at women with NA. SHBG and leptin levels in
FF, the number of non-atretic oocytes, good embryos were also significantly
greater and the embryo ZP thickness was on 20% less at pregnant HA women
and didn’t differ from NA pregnant patients. T level and IFA were higher in
both pregnant and non-pregnant HA patients. In contrast to these patients,
hormonal microenvironment of oocytes from women with NA (pregnant or
no) varied in less degree. Moderate differences observed in SHBG and T lev-
els. The considerable changes were in oocyte/embryo quantity and quality,
however morphometric parameters of the transferred embryos didn’t differ
significantly.
Conclusions: There is a difference between hormonal microsurroundings of
oocytes from women with different androgenic status, their fertilization po-
tential and morphometric parameters of the best embryos. It is necessary to
develop the complex approaches to embryo selection in dependence on patient
endocrine status. Leptin, testosterone/SHBG levels, ZP thickness are crucial for
achieving pregnancy at women with HA.
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
For the groups with one previous FBT, the pregnancy rate with good-quality
embryo transfers was lower with razor AH than control {36.6% (34/93)/ 53.6%
(75/140): P < 0.05}.
For the groups with two previous FBT, the pregnancy rate was lower with
razor AH than control {36.1% (26/72)/ 54.0% (27/50): P < 0.05}.
However, the pregnancy rates of zona free and laser AH with one and two
FBT were not significantly different with control. Furthermore, the results of
the groups with no and three or more previous FBT were no significantly differ-
ent among three AH and control.
Next, the pregnancy rates were compared in four age groups (less than 25
years, 25-34, 35-39, and 40 or older).
< Transfer of poor-quality embryos >
The pregnancy rates of poor-quality embryo transfers with or without AH
among all age groups were not significantly different.
< Transfer of good-quality embryos >
In the groups of age 25-34, the pregnancy rate with good-quality embryo
transfers was lower with laser AH than control {29.4% (5/17)/ 60.6% (114/188):
P < 0.05}, but the pregnancy rates of razor AH and zona free were no signifi-
cantly different with that of control.
The pregnancy rates of good-quality embryo transfers among three age
groups (less than 25, 35-39, and 40 or older) with the three AH methods and the
control were not significantly different.
Conclusions: This study indicated that razor AH is not a useful technique com-
pared with zona free and laser AH in FBT, and utility of AH in all cryopre-
served embryos might not be effective regardless of the quality of blastocyst,
number of the previous FBT performed, and female age. Therefore, embryo
transfer without AH should be the first option for infertile couples to achieve
implantation.
P-189 Frozen-thawed human embryo outcomes under natural and
stimulated cycles: a retrospective study
A. Ferrieres1, M. Poulain1, V. Loup1, T. Anahory1, H. Dechaud2, S. Hamamah1
1Hôpital Arnaud de Villeneuve, Reproductive Biology Unit, Montpellier
Cédex 5, France
2Hôpital Arnaud de Villeneuve, Reproductive Clinical Unit, Montpellier
Cédex 5, France
Introduction: Beyond the alterations of human embryos after slow freezing-
thawing, the preparation of endometrium for frozen-thawed embryos replace-
ment is still debated. The objective of this study was to compare embryo out-
come after freezing-thawing procedure and implantation rates during natural
and exogenous hormone stimulation cycles in which frozen-thawed embryo
transfer (FET) was carried.
Material and Methods: A retrospective study was performed in our academic
center for assisted reproductive technology (ART). Two hundred and five FET
cycles were analysed regarding cycle regimen: group A (n = 101 cycles initi-
ated, 88 transfers, 22 at the blastocyst stage) had FET under natural cycle after
monitored spontaneous ovulation and group B (n = 104 cycles initiated, 88
transfers, 24 at the blastocyst stage) had FET in a gonadotrophin stimulated
cycle after monitored triggered ovulation by hCG and luteal phase support by
vaginal progesterone. Main outcome measures were the embryo outcome after
freezing-thawing, implantation and ongoing pregnancy rates.
Results: The two groups were comparable with respect to their demographic
characteristics and reproductive history, including age (32.7 years ± 0.3 in
group A vs 32.5 years ± 0.3 in group B, p = 0.4), fresh cycle outcome and causes
of infertility. There were no significant differences between the two groups with
regard to the number of thawed and transferred embryo per FET (3.1 ± 0.1 and
2.0 ± 0.05 respectively in group A vs 2.8 ± 0,1 and 2.1 ± 0.06 respectively
in group B, p = 0.09 and 0.2 respectively). The mean embryo survival index
before transfer was comparable in the two groups (67.4% in group A vs 68.9%
in group B, p = 0.3). The implantation and ongoing pregnancy rates were sig-
nificantly increased under stimulated FET cycles (8.9% and 14.8% respectively
in group A vs 14.1% and 27.3% respectively in group B, p = 0.04 and 0.02
respectively) and the ongoing pregnancy rate concerning the FET at the blas-
tocyt stage was significantly higher in the group B (9.1% in group A vs 45.8%
in group B, p = 0.002).
Conclusion: The replacement of frozen-thawed embryos under stimulated
cycle was associated with higher implantation and ongoing pregnancy rates
when compared with a natural cycle. Natural cycle is currently advised for FET.
The fluorescence in situ hybridation (FISH) technology has been applied to
evaluate the chromosomal pattern of these embryos. However, this technique
presents limitations, because we can not differ between paternal and maternal
contributions.
The aim of this study was to ascertain the real ploidy of these embryos with
an accuracy method, using DNA technology in order to increase the success
rate per cycle or to prognose a next treatment in case of no pregnancy.
Materials and Methods: Since March 2009, 23 undocumented embryos com-
ing from fifteen ICSI treatments were collected in our in vitro fertilization Unit.
These embryos appeared both in donated (9) and in own oocytes (6) cycles.
Pronuclei appearance were examined 16-20h after fertilization. Oocytes with
no pronuclei were cultured and observed routinely. The embryos resulting from
these zygotes were included in this study. DNA from parents were obtained
and isolated from bucal swab. Embryos were placed in alcaline lysis buffer
followed of two incubations at -20ºC for 30 minutes and 65ºC for 10 minutes
respectively. After neutralization, cells lysates were used directly for multiple
displacement amplification (MDA, GEhealthsciences).
In order to identify the paternal-maternal origin of the DNA from the un-
documented embryos, the AmpFlSTR Identifiler PCR Amplification Kit (Ap-
plied Biosystems) was used. The PCR reaction includes a multiplex containing
15 loci for main STR and amelogenin.
For a comparison of the genetic profiles of embryos, paternal and maternal
samples were carried out to identify the origin of the DNA.
Results: The 23 undocumented embryos obtained had DNA amplification and a
positive paternity test. Eighteen embryos (78.27%) were disomic embryos (2n)
with paternal and maternal contributions, and 4 for of them (17.39 %) were 2n
embryos with only maternal contribution. Only one embryo (4.34%) was hap-
loid (n) or uniparental disomic with maternal origin.
Conclusion: The majority of discarded embryos coming from catalogued “not
fertilized” oocytes are chromosomical normal, and consecuently suitables for
transfer or freeze. We are increasing the number of cases to confirm this results.
A great number of couples could benefit from these findings, optimizing their
chance for pregnancy. To our knowledge, this is the first report evaluating un-
documented embryos by means of a paternity test.
P-188 Efficacy of assisted hatching in frozen-thawed blastocyst transfer
K. Usui1, Y. Nakajo1, M. Ota1, H. Hattori1, T. Kyoya1, T. Takisawa1, K. Kyono1
1Kyono ART Clinic, Gynecology, Sendai, Japan
Introduction: For cryopreserved embryos, the freeze-thaw process might fur-
ther exacerbate hardening of the zona pellucida (ZP). The artificial rupture or
thinning of ZP before embryo transfer, assisted hatching (AH), has been pro-
posed to foster spontaneous hatching and improve embryo implantation rate.
Although AH has been performed in the patients with frozen-thawed blastocyst
transfer (FBT) at our clinic, it remains unclear whether AH is useful for implat-
ntation. Therefore, we evaluated the efficacy of AH in FBT.
Material and Methods: This retrospective study included 1335 FBT cycles,
including 1) 582 razor AH {a quarter of ZP was ruptured with the help of metal
micro blade (MICRO FEATHER BLADE; FEATHER®)}, 2) 106 zona free (ZP
was removed from the embryo with the help of metal micro blade and gentle
pipetting), 3) 86 laser AH {a quarter of ZP was ruptured with the help of laser
(ZILOS-tk; HAMILTON THORNE BIOSCIENCES)}, and 4) 560 no AH (con-
trol), between March 2007 and November 2009. Clinical pregnancy rates of
the three AH methods depending on the quality of blastocyst, the number of
previous FBT performed, and female age were compared with the pregnancy
rate of control.
Results: The sample was divided into four groups depending on the numbers of
the previous FBT (0, 1, 2, 3 or more), and the pregnancy rates were compared
among those groups.
< Transfer of poor-quality embryos with a grade of “BC”, “CB”, or “CC”
by Gardner’s criteria >
For the groups with no previous FBT, the pregnancy rate with poor-quality
embryo transfers was significantly lower with razor AH than control {18.8%
(6/32)/ 50.9% (29/57): P < 0.01}, but the pregnancy rates of zona free and laser
AH were not significantly different compared with that of control. Furthermore,
the pregnancy rates of razor AH, zona free, and laser AH with more than one
FBT had no significant differences compared with that of control.
<Transfer of good-quality embryos with a grade of “AA”, “AB”, “BA”, or
“BB” >
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
the relative increase towards the metabolically more active TE just prior to
implantation in both diet conditions may represent a general mechanism facili-
tating adaptation to intrauterine environments whilst tight control of the ICM
environment is maintained.
Support by the BBSRC, University of Southampton School of Medicine
and Wellbeing of Women is gratefully acknowledged.
P-191 Improved human embryo quality in days 3 and 5 with a low
oxygen closed culture system
N. Prados1, M. Ruiz1, J. García-Ortega1, P. Vime1, M.J. Hernáez1, M. Crespo1,
M. Fernández-Sánchez2, A. Pellicer3
1IVI Sevilla, Laboratorio Embriología Clínica, Sevilla, Spain
2IVI Sevilla, Human Reproduction, Sevilla, Spain
3Institut Universitari - IVI Valencia, Human Reproduction, Valencia, Spain
Introduction: Oocytes and human embryos do not come across ambient oxy-
gen levels (20%) in vivo. Low oxygen systems (from 5 to 10%) have been
proposed for in vitro embryo culture, especially for blastocysts culture, with
unclear results. We wanted to see the impact over embryo quality in our setting
of a novel closed embryo culture chamber with low oxygen and very stable
conditions.
Material and Methods: This prospective study was conducted at IVI Sevilla,
Spain, from October to December of 2009. We included 657 embryos from
83 different patients. First cycle ICSI patients with blastocyst transfer indica-
tion (regardless of any other characteristic) were divided in two groups using
our standard patient distribution. For the control group, we allocated three of
our standard incubators for this group at 6% CO2. No more than 4 patients
were assigned to a single incubator at the same time. Low oxygen culture was
performed in the Ac-tive®(IVF)-N system (Russkin, UK) which is a totally
closed workstation where embryos do not enter in contact with atmospheric
conditions from ICSI until the transfer. The conditions in this system were set
at 5%O2 6%CO2. In the study group, we included no more than 12 patients
in chamber at the same time, though theoretically the maximum number of
patients could be more than twice. All other embryo media and conditions
and the scoring (ASEBIR guidelines) were the same in both groups. Control
group included 338 embryos from 43 patients and the study group included
319 embryos from 40 patients. Blastocyst transfer was scheduled if four or
more good quality embryos were scored on day 3. If not, day 3 transfer was
performed.
Results: No differences were found among the cycle characteristics of both
groups (number of oocytes, number of zygotes or number of transferred em-
bryos). There were no differences in cell number on day 2 embryos but study
group embryos on day 2 were more symmetric (41,0% vs. 59,0%; OR = 1,23
[IC95: 1,065-1,429]) and less fragmented (6,1% vs. 12,1%; OR = 1,07 [IC95:
1,016-1,120]). The distribution of embryo quality (grades A to D) was signifi-
cantly different (p = 0,002) with a higher proportion of good quality embryos
(45,8% vs. 59,9% [A or B]; OR = 1,31 [IC95: 1,13-1,52]).
On day 3, study group embryos had more high numbered embryos
(p < 0,001) with no difference in fragmentation or compactation but with better
symmetry (33,5% vs. 45,6%; OR = 1,21 [IC95: 1,09-1,35]). Distribution of em-
bryo quality was again much better in the study group (p < ,001) with a higher
proportion of good quality embryos (35,9% vs. 54,7% [A or B]; OR = 1,52
[IC95: 1,28-1,81]).
There were more patients with four or more good quality embryos in the
study group (which where scheduled as day 5 transfer 30% vs 53%; OR = 1,74
[IC95: 1,01-2,98]). In the patients who underwent day 3 transfer there were
no differences in the distribution of embryo quality or the number of frozen
embryos.
In patients who underwent day 5 transfer, although both groups had a simi-
lar blastocyst rate, they were of better quality (A, B grade) in the study group
(4,2% vs. 25,4%; OR = 6,05 [IC95: 2,67-13,75]).
Conclusions: The Ac-tive®(IVF)-N system provides a real continuous tempera-
ture and low oxygen culture. In low oxygen incubators, embryos must be scored
in microscope stages in ambient air. In this study, most morphologic parameters
are better in the study group, more blastocysts transfers are performed and the
general quality is improved. The differences appear even on day 2 embryos.
This kind of closed embryo culture systems seems to be very promising and
could be a standard in some years, if these results are confirmed with pregnancy
and outcome rates.
However the incomplete monitoring of natural cycle leads to a low success of
pregnancy. A slight stimulation protocol for FET cycles should be an alternative
particularly for FET at the blastocyst stage.
P-190 Maternal dietary protein intake influences conceptus
differentiation and biosynthesis levels before implantation in the mouse
model
J. Eckert1, G. Premkumar1, F. Lock1, S. Brooks2, S. Haque1, I.T. Cameron1, Y.
Cheong1, T.P. Fleming2
1University of Southampton, Developmental Origins of Health and Disease,
Southampton, United Kingdom
2University of Southampton, School of Biological Sciences, Southampton,
United Kingdom
Introduction: Changes in maternal diet during gestation can induce develop-
mental adaptations influencing conceptus growth and metabolism. Such fetal
adaptations can increase the risk of adult onset diseases. Human epidemiology
and animal experimental data support this ‘Developmental Origins of Health
and Disease (DOHaD)’ hypothesis yet our understanding of when and how this
is induced remains limited.
Nutrient conditions within the mother can alter fetal growth regulation me-
diated by sensing energy status and adapting biosynthesis levels (eg mTOR
or AMPK). Of potential relevance for ART, we have previously shown that
postnatal phenotypic changes can be induced exclusively during the preim-
plantation period, becoming detectable by the blastocyst stage. At this stage,
the first cell lineage divergence into inner cell mass (ICM, giving rise to the
embryo proper) and trophectoderm (TE, giving rise to placental lineages)
emerges, coinciding with major metabolic transitions. Blastocyst environment
can influence gene expression, cell numbers and lineage allocation, suggest-
ing mechanisms whereby energy status and metabolic signalling in response
to different environments affect anabolic activity and growth potential of the
conceptus. However, we know little about directionality of the responses to
nutrition levels. Here, we compare cell allocation, protein biosynthesis and
energy/stress status indicated by AMPKβ1 distribution in blastocysts derived
from mothers fed isocaloric diets with different protein content exclusively
during the preimplantation period.
Material and Methods: Naturally mated female MF1 mice (7 1/2-8 1/2
weeks of age) were randomly allocated to either a low protein diet (9% ca-
sein, LPD), high protein diet (30% casein, HPD) or control diet (18% ca-
sein, control) on day of plug (d 0.5). On day 3.5, mothers were sacrificed and
overall protein synthesis of blastocysts flushed from the uterus was measured
using the CLICKit-AHA and TAMRA detection technology (Invitrogen). Al-
ternatively, embryos were differentially labelled for cell lineage allocation
or fixed to determine localisation of AMPKβ1 by immunofluorescence and
confocal microscopy. Embryos from at least 10 mothers were examined for
each endpoint. Data were analysed using a 2-way ANOVA followed by Bon-
ferroni’s test.
Results: Both maternal diets altered cell allocation in blastocysts directing
cells towards the TE lineage just prior to implantation (d3.75) yet via differ-
ent routes. Whilst HPD-blastocysts reduced the ICM (p < 0.001) maintaining
TE and total cells, thus indirectly forcing cells towards TE (p < 0.001), LPD-
blastocysts increased the TE-lineage (p < 0.05) directly with more total cells but
unchanged ICM. Blastocysts from HPD mothers synthesised significantly more
protein compared to controls and LPD (p < 0.001; 50% upregulation). Maternal
protein restriction, in contrast, caused a mild initial reduction in blastocyst pro-
tein biosynthesis compared to controls (15%; p > 0.1). Distribution of AMPKβ1
as indicator of cellular stress, although very variable between embryos and cell
lineages, also responded to maternal diet. AMPKβ1 was increasingly found
in nuclei of the TE in response to either protein challenge compared to con-
trols. However, whilst HPD induced a diffuse distribution across nucleus and
cytoplasm in more than 50% of blastocysts, LPD blastocysts showed a more
localised pattern.
Conclusions: We show that maternal protein intake, either over- or mild un-
dernutrition, influences embryonic lineage differentiation and biosynthesis lev-
els suggesting that maternal-embryonic crosstalk regulates conceptus growth
potential before implantation. Mechanisms include AMPK signalling which
proved to be highly versatile in the early embryo. Increased AMPK distribution
within TE nuclei after both dietary challenges suggests general stress responses
in the embryonic lineage directly exposed to intrauterine environments. Thus,
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
Material and Methods: By using the PicTar algorithm, four potential miR-
135a targets were identified. They were Complexin 1 (CPLX1), G1/S- specific
Cyclin D2 (CCND2), E3 ubiquitin ligase Seven In Absentia Homolog 1 (SIAH1)
and Checkpoint Suppressor 1 (CHES1). Direct interactions between miR-135a
and the 3’-untranslated region (3’UTRs) of potential targets were validated via
miRNA luciferase reporter assay; and the luciferase activities were normalized
with beta-galactosidase activities. The expression levels of the potential targets
mRNA and protein were also examined using quantitative real-time PCR and
Western Blotting, respectively in HeLa cells with knockdown (miRCURY LNA
knockdown Probe) and force-expression (miR-135a precursor molecules) of
miR-135a.
Zygotes were collected from ICR mice. MiR-135a inhibitor and/or SIAH1
specific antibody was microinjected into the cytoplasm of the zygotes, control
zygotes were injected with scrambled inhibitor or water. The effects of the treat-
ment on embryo development and protein expression were determined.
Results: Among the four potential miR-135a targets studied, SIAH1 was
confirmed as a miR-135a target. There were two miR-135a potential binding
sites on the 3’UTR of SIAH1. Luciferase reporter assay for these binding sites
showed a significant increase of luciferase activities in miR-135a knockdown
HeLa cells (n = 3, p < 0.05). Besides, inhibition (n = 6) and forced-expression
(n = 3) of miR-135a increased and decreased respectively the SIAH1 protein
level significantly (p < 0.05). However, no alteration of SIAH1 mRNA level
was observed in miR-135a knockdown HeLa (n = 6, p > 0.05). This indicated
that the regulation of miR-135a on SIAH1 expression was at the translation
level.
To confirm the results obtained in cell line, the roles of miR-135a and
SIAH1 were studied in embryos. We found that the increase expression of miR-
135a in the zygote stage when compared with that of oocytes coincided with
the decrease of SIAH1. Microinjection of miR-135a inhibitor into developing
zygotes elevated the expression of SIAH1, and decreased the number of zy-
gotes developed to the 2-cell stage (p < 0.01). The growth arrest was partly nul-
lified by injection of SIAH1 specific antibody into miR-135a inhibitor treated
zygotes.
Conclusions: MiR-135a is important in the first cleavage division of mouse
embryo and partially exerts its function through inhibiting the expression of
SIAH1.
References:
1 Berezikov, E., Guryev, V., van de Belt, J., Wienholds, E., Plasterk, R. H., & Cuppen, E.
(2005). Phylogenetic shadowing and computational identification of human microRNA
genes. Cell, 120(1), 21-24.
2 Bernstein, E., Kim, S. Y., Carmell, M. A., Murchison, E. P., Alcorn, H., Li, M. Z., et al.
(2003). Dicer is essential for mouse development. Nat Genet, 35(3), 215-217.
3 Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F., & Hannon, G. J. (2004). Pro-
cessing of primary microRNAs by the Microprocessor complex. Nature, 432(7014),
231-235.
P-194 Rescue ICSI of oocytes that failed to appear the fertilization corn
and cytoplasmic flare 6 hours post-insemination in conventional IVF
T. Wada1
1Ochi Yume Clinic Nagoya, Laboratory, Nagoya, Japan
Introduction: We have performed rescue ICSI of oocytes that failed to ex-
trude the second polar body 6 hours post-insemination in conventioal IVF since
2004. In rescue ICSI, the criteria for choosing unfertilized oocytes are very
important. Recently, using time-lapse cinematography, the appearance of a fer-
tilization corn in surface of oocytes, and cytoplasmic flare in the ooplasma dur-
ing fertilization were observed about 2.2 hours after extrusion of second polar
body (Mio, JMOR 23, 27-35, 2006). Therefore, the objective of this study was
to investigate whether the fertilization corn and cytoplasmic flare were added as
new criteria for judging fertilized oocytes in addition to the extrusion of second
polar body for Rescue ICSI.
Material and Methods: From April 2004 to December 2008, we studied res-
cue ICSI of oocytes that failed to extrude the second polar body, and did not
find fertilization corn and cytoplasmic flare as the additional criteria, individu-
ally for 612 cycles and 1619 cycles induced by clomiphene-HMG-GnRHa ad-
ministration. Conventional IVF was perfomed 3 hours after ovum pick up, and
the final sperm concentration was adjusted to 1*10^5/ml. In Rescue ICSI, the
spindle was fixed at the 12 o’clock position if it could be visualized using ICSI
Guard (OCTAX) and the first polar body was fixed at the 12 o’clock position if
the spindle could not be visualized. The oocytes were examined for fertilization
P-192 Selection of high potential embryos through culture in poly-
(dimethylsiloxane) micro-wells and time-lapse imaging
S. Hashimoto1, N. Kato2, K. Saeki2, Y. Morimoto3
1IVF Namba Clinic, Division of Research, Osaka, Japan
2School of Biology-Oriented Science and Technology, Kinki University, Wa-
kayama, Japan
3IVF Namba Clinic, Medical Office, Osaka, Japan
Introduction: It has been shown that extending culture of embryos to the blas-
tocyst stage increases the pregnancy rate in assisted reproductive technology.
However, the extended culture increases the risk of spontaneous abortion. If
high potential embryos can be selected at Day 2/3 of culture, problems asso-
ciated with extended culture might be reduced. Recently, we obtained time-
lapse images of development of individual human embryos with a combination
of an individual culture in non-porous poly-(dimethylsiloxane) micro-wells
(PDMSMWs) and a microscope inside an incubator (Hashimoto et al., 2009).
In the present study, we used this system to assess the relationship between the
time course of the embryo development and the developmental competence to
morphologically normal blastocysts by taking time-lapse images of embryos
cultured in PDMSMWs.
Materials and Methods: A PDMSMW plate with cylindrical micro-wells (300
μm in diameter and 200 μm deep) was produced by a soft-lithographic tech-
nique and cured under low pressure. It was placed on the bottom of a well of
a 4-well dish. All patients gave informed consent. A total of 76 frozen-thawed
pronuclear embryos were cultured in PDMSMWs individually. An embryo was
placed in each micro-well covered with 1-mL culture medium and was cultured
for 120 h. Time-lapse images of embryos were captured 6 times per hour over
5 days. Time of the first cleavage was regarded as time 0. In addition, we mea-
sured times required for second and third cleavage division of embryos. The
quality of blastocysts was evaluated according to Gardner & Lane (1999). In
this study, blastocysts that were scored at least 3AA, without C, after 96 h of
culture were regarded as high-scoring. The data were statistically compared
using a Mann-Whitney U-test.
Results: Thirty-seven embryos (48.7%) developed to the blastocyst stage. Em-
bryos that developed to high-scoring blastocysts took about the same time to
develop to the 4-cell stage (13 h) and the 8-cell stage (32 h) as embryos that
developed to low-scoring blastocysts. However, in embryos that developed
to high-scoring blastocysts, the time required from 3- to 4-cell stage (second
cleavage division, 1.0 h) was shorter (P < 0.05) than the time in embryos that
developed to low-scoring blastocysts (3.2 h). Time required from 5- to 8-cell
stage (third cleavage division) was also short in embryos developed to high-
scoring blastocysts (P < 0.05; high-scoring blastocyst: 13.2 h vs. low-scoring
blastocyst: 22.0 h).
Conclusions: These results lead to propose that high potential embryos that
develop to high-scoring blastocysts can be selected at Day 2/3 by assessment of
the synchronization of early cleavage timing of each blastomere using a time-
lapse imaging system composed of an individual culture method in PDMSMWs
and a microscope inside the incubator.
P-193 MicroRNA-135a regulates early embryo development through
mediating expression of E3 ubiquitin ligase seven in absentia Homolog 1
(SIAH1)
C.O.N. Leung1, R.T.K. Pang1, W.M. Liu1, K.F. Lee1, W.S.B. Yeung1
1The University of Hong Kong, Obstetrics and Gynaecology, Hong Kong,
Hong Kong
Introduction: MicroRNAs (miRNAs) are single-stranded 19-25 nucleotide
non-coding RNA molecules capable of regulating gene expression at both tran-
scription and translation levels (Denli et al., 2004). There are more than 700
known human miRNAs. It is estimated that they regulate more than 30% of all
protein coding genes (Berezikov et al., 2005). MiRNAs are involved in many
physiological process and diseases. Knockout mice lacking miRNA process-
ing machinery Dicer died at embryonic day 7.5 (Bernstein et al., 2003). To
investigate whether miRNA played a role in early embryo development, we had
determined the miRNA profile of mouse preimplantation embryos. We found
that miR-135a was specifically expressed in the zygotes and decreased upon
development, consistent with the involvement of the miRNA in early embryo
development. This study aimed at evaluating the role of miR-135a in embryo
development through in silico and in vitro approaches.
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Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
P-196 Development of tripronucleated ICSI-derived embryos using real
time assessment
J.M. de los Santos1, L. Escrich1, N. Grau1, A. Pellicer2, J.L. Romero1,
M.J. Escribá1
1Instituto Valenciano de Infertilidad (IVI), IVF Laboratory, Valencia, Spain
2Medical school. Universidad de Valencia., Obstetrics & Gynaec department,
Valencia, Spain
Introduction: Up to now, the discontinuous morphological assessment of the
embryos, along the in vitro culture is commonly used to assist embryologists
in the embryo selection process. The Embryoscope (Unisense, Denmark) is
an incubator, equipped with an image system that captures 5 focal planes, at
9 minutes time-lapsed periods along the 6 days of the culture. Thus, movies of
development are obtained with a minimal variation of the culture conditions.
Assessment of the movie allows us to discover new landmarks on development
and to determine at what exact time relevant developmental events occurred.
In the present work, we presented our preliminary results on in vitro de-
velopment of tripronucleated (TPN) ICSI-derived embryos, vitrified and
warmed and subsequently cultured in either an Embryoscope or under standard
conditions.
Material and Methods: After ICSI, 43 TPN zygotes were identified and vitri-
fied using the cryotop methodology. After warming, TPN were cultured in IVF
(Vitrolife, Göteborg) medium for the 3 initial first days and then, transferred
into CCM (Vitrolife, Göteborg) medium for three additional days. Culture
conditions were 37ºC and 5%CO2 in air. Thirty-seven vitrified/warmed TPN
zygotes were cultured under standard culture conditions and six TPN zygotes
were cultured in the Embryoscope. The Embryoscope’s culture differs from
standard one in the volume of the medium used for culture (20 μl vs. 50 μl) and
in the continuity/discontinuity of the embryo observations (with concomitant
alteration of the culture conditions).
Around every 24 hours, we checked the embryo stage (number of blastom-
eres, morula/blastocyst stage) in order to determine the embryo developmental
ability, measured as the number of embryos that reached the 2- or 4-cell, 8-cell
and morula/blastocyst stages (Chi square tests). Along with the assessment of
the Embryoscope’s movie, we also determined at which time did the first and
the second cleavage occur (hours, hrs; ANOVA test) and how many cells did the
TPN ICSI-derived embryos at the first cleavage have. These results were com-
pared to those obtained from standard embryo assessment and culture, when
possible.
Results: TPN zygotes, cultured in Embryoscope or under standard conditions,
cleaved (66.7% vs. 89.2%, p = 0.39), reached the 8-cell (33.3% vs. 73.0%,
p = 0.86) and the blastocyst (33.3% vs. 10.8%; p = 0.39) stages, at comparable
rates. All day-3 TPN embryos progressed on development to the blastocyst
stage when they were cultured in the Embryoscope; however, when embryos
were cultured under standard conditions, only 14.8% of day-3 embryos pro-
gressed to blastocyst (p = 0.049).
Following the screening of the TPN embryo development movie, we observed
that all TPN progressed on the first division at 35.5 ± 10.8hrs, rendering 2-cell
embryos in all cases. Soon after, the second cleavage occurred simultaneously on
both blastomeres in three of four embryos (35.7 ± 4.96hrs), whereas in one, only
one blastomere cleaved, rendering a 3-cell embryo at around 34.2hrs of culture.
Conclusions: In conclusion, development to the blastocyst stage is not affected
by the culture/assessment system. However, a beneficial effect on the last de-
velopmental days was observed on TPN embryos cultured in the Embryoscope;
the minimum volume of the culture (20μl) or the fewer alterations on culture
might explain this effect. More data are needed to confirm such suggestions.
Finally, from a basic research point of view, the Embryoscope technology
confirms earlier suggestion, related to the first cleavage of the TPN ICSI-derived
zygotes by which the single centriole inheritance is indicated, which reinforces the
dyginic origin of the third pronucleus, opposite to TPN IVF-derived embryos.
Authors would like to thank Biosense Inc and IMPIVA (IMIDTF 2009/134)
for supporting these studies.
P-197 Artificial oocyte activation: a method to assess the oocyte
cytoplasmic competence
M. Escribá1, N. Grau1, L. Escrich1, J.M. de los Santos1, A. Pellicer2,
J.L. Romero1
1Instituto Valenciano de Infertilidad (IVI), IVF Laboratory, Valencia, Spain
2Medical school, Universidad de Valencia, Obstetrics & Gynaec department
10-13 hours after rescue ICSI, and two pronuclei (2PN) were judged as normal
fertilization. Cleavage of the oocytes and blastocyst development were assessed
on day 2 and day 5. All blastocysts were vitrified and stored for a single blasto-
cyst transfer in the next cycle.
Results: The rescue ICSI in the failure of 2PB extrusion (1PB group) or the
no appearance of fertilization corn and cytoplasmic flare (no flare group) per-
formed 18.6% (114/612) or 15.2% (246/1619) of total cycle, respectively. Nor-
mal fertilization rate of rescue ICSI in the no flare group (84.8%, 408/481) was
similar to that in the 1PB group (80.9%, 165/204), but 3PN rate in the no flare
group (3.3%, 14/481) was significantly (P < 0.05) lower than that in the 1PB
group (7.8%, 16/204). The percentage of good cleaved embryos at day 2 was
no significantly difference between the no flare group (17.9%, 73/408) and the
1 PB group (17.0%, 28/165), but the percentage of good blastocysts at day 5
in the no flare group (27.0%, 110/408) was significantly (P < 0.01) higher than
that in the 1PB group (16.4%, 27/165). Pregnancy rates of a single blastocyst
transfer were no significantly difference between both groups (20.5%, 17/83
and 21.4%, 6/28).
Conclusions: These results clearly confirmed that rescue ICSI using fertiliza-
tion prediction based on fertilization corn and cytoplasmic flare, in addition to
the extrusion of second polar body, was very useful for preventing the incidence
of polyspermy, and producing good blastocysts at day 5.
P-195 Time-lapse video evidence of murine embryonic developmental
regression in sub-optimal culture conditions using Embryoscope™
T. Elliott1, J. Kahn1, J. Lowderman1, G. Wright1, C. Chang1, D. Bernal1,
H. Kort1, Z. Nagy1
1Reproductive Biology Associates, IVF Lab Suite 600, Atlanta GA, U.S.A.
Introduction: As documented, sub-physiological culture environment in IVF
frequently lead to embryo developmental arrest and to degeneration, however,
continuous observation on morphological changes are not described. The ob-
jective of the present study was to perform a detailed observation on the effect
of suboptimal culture conditions on mouse embryo development using time-
lapse video recording with Embryoscope™.
Time-lapse video recording and analysis of embryonic development of fertil-
ized single cell mouse embryos to the blastocyst stage was performed using Em-
bryoscope™. Poor or abnormal development rate when noted in specific assays
was documented in video form. Due to the unhumidified incubation area and the
use of new culture slides for the Embryoscope™, in this particular study a control
slide was compared to a slide overlaid with culture media instead of oil.
Materials and Methods: Mouse embryo development (from the zygote to
the blastocyst stage) was performed by Embryoscope™ (Unisense FertiliTech
A/S, Denmark). The custom Embryoscope™ culture-slides were prepared per
usage instructions using Global® media (LifeGlobal®, http://www.lifeglobal.
com/). The control slide had 1.2 ml of Ovoil overlaid, the test slide had 1.2ml
Global® media overlay. Every 20 minutes a picture was taken by the Embryo-
scope™. Eleven mouse embryos were placed into each group. Development of
the mouse embryos in the test group was compared to the control.
Results: All 11 embryos reached the blastocyst stage by 70 hours in the control
slide.
In the test slide, 4 embryos were discounted from the study because of a
lack of ability to visual all stages of their development due to media condensa-
tion on the slide lid obscuring the light to the Embryoscope™. All remaining
8 embryos had died after 60 hours. The earliest embryonic demise occurred
at 49 hours. Of the 8 embryos observed, four reached the 2 cell stage, two
reached the 3 cell-stage and one developed to 4 cell-stage. Cleavage stages
greater than 2 cells were observed from 7 to 18 hours in duration. All three and
four-cell stage embryos then regressed to the two cell stage before completely
degenerating.
Conclusions: Suboptimal culture environment has led to degeneration of all
test embryos by the end of the observation period, while all control embryos
in the physiological culture environment reached the blastocyst stage. Interest-
ingly, some of test embryos has demonstrated a regression process (from 3-cell
stage to the 2-cell stage or from the 4-cell stage to the 2 cell stage) before
complete degeneration, indicating that this pattern may be quite typical during
a degeneration process in suboptimal conditions.
This is the first time, according to the author’s knowledge, that embryonic
developmental reversal, as a typical pattern prior degeneration, has been ob-
served and recorded as video.
by guest on January 25, 2016http://humrep.oxfordjournals.org/Downloaded from
i193
Abstracts of the 26th Annual Meeting of ESHRE, Rome, Italy, 27 June – 30 June, 2010
individualised dose of recombinant FSH (based on AMH and AFC in addition
to the standard markers), in patients undergoing controlled ovarian stimula-
tion in their first cycle of IVF/ICSI. The primary outcome was the proportion
of women who had an appropriate ovarian response (defined as having 5-14
oocytes), as well as the number of poor (< 5 oocytes) and exaggerated respond-
ers (>14 oocytes). The secondary outcome was the ongoing clinical pregnancy
rate.
Material and Methods: A total of 286 women were included in this ran-
domised controlled trial. They all were entering their first cycle of IVF/ICSI
using the long agonist protocol and had both ovaries present. Randomisation
was achieved using a computer-generated list of numbers that were printed and
placed into sequentially numbered opaque sealed envelopes (SNOSE). Patients
in the individualised group received an rFSH dosage that was calculated based
on the AMH level and AFC, in addition to standard markers by using tables that
were drawn up based on previous publications. Standard patients received an
rFSH dose based the protocol used at the ACU, where the dose was calculated
depending on women’s age, early follicular FSH, E2 and the presence of PCO.
Results: In the standard group 71 patients (60.7%) had an appropriate response
(defined as 5-14 oocytes) compared to 53 patients (50%) in the individualised
group (P = 0.14). Fourteen patients (12%) in the standard group and 19 patients
(17.9%) in the individualised group were poor responders (retrieved < 5 oo-
cytes) (P = 0.26). An exaggerated response (retrieved > 14 oocytes) occurred
in 32 patients (27.4%) in the standard group compared to 34 patients (32.1%)
in the individualised group (P = 0.47). The ongoing clinical pregnancy rate
per initiated cycle was 25.2% in the individualised group, and 39.3% in the
standard group (P = 0.02). The mean total dosage of rFSH used in the indi-
vidualised group was 2495 units compared to 2611 units in the standard group
(P = 0.43).
Conclusion:No significant difference was found in terms of ovarian response
for patients given either an individualised or standard dose of rFSH. A signifi-
cant difference was found in the clinical pregnancy outcomes, with women in
the individualised group having lower pregnancy rates compared to those in the
standard group. Total rFSH dosage was higher in the standard group, however,
the difference between the two groups was not statistically significant. This
study, therefore, found no additional benefit from measuring AMH and AFC in
addition to a standard regime that is already tailored to considering markers of
ovarian reserve.
P-199 Which stage is suitable for vitrification from the practical point of
view, two pronuclear oocyte (2PN) or blastocyst (BL)?
S. Miyaji1, S. Mizuno1, L. Horiuchi1, A. Haruki2, A. Fukuda2, Y. Morimoto3
1IVF Osaka Clinic, Reproductive technology, Osaka, Japan
2IVF Osaka Clinic, Doctor, Osaka, Japan
3IVF Namba Clinic, Doctor, Osaka, Japan
Introduction: Cryopreservation of gamete, zygote or embryo is indispensable
technology for present human ART. Recently vitrification method is applied on
human embryos at any stages such as two pronuclear oocyte (2PN), cleaved
embryo and blastocyst. However, it has not been determined yet which devel-
opmental stages of embryo are suitable for vitrification. In the present study, the
efficiency of vitrification was compared between 2PN and BL from the point
of not only post thaw survival and subsequent embryonic development in vitro,
but also practical point of view.
Materials and Methods: Eight hundred one 2PNs of 126 IVF/ICSI patients
from January to December, 2009 were used for the analysis. They were divided
into 2 groups (A and B). Group A consists of 372 2PNs (55 patients) that were
vitrified at 2PN and cultured to BL after thawing for frozen-thawed transfer.
Group B consists of 106 good quality BLs derived from 429 2PNs (71 patients)
which were developed to BL in vitro and vitrified for future frozen-thawed em-
bryo transfer. In the first study, development rates to