6 July, 2011 - La Diagnosi Genetica Preimpianto (PGD)


6 July, 2011 - La Diagnosi Genetica Preimpianto (PGD)
Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 July – 6 July, 2011
O-249 Treatment of endometriosis with an anti-L1CAM mAb ñ a mouse
D. Finas1, P. Hunold2, F. Koester1, P. Altevogt3, D. Hornung1
University of Luebeck, Department of Obstetrics and Gynaecology, Luebeck,
University of Luebeck, Clinic for Radiology and Nucear Medicine, Luebeck,
German Cancer Research Center, Translational Immunology, Heidelberg,
Introduction: Endometriosis is a progressive disease, which is frequently ­associated with serious pelvic pain possibly caused by increased nerve
growth. The neural cell adhesion molecule L1CAM is known as a regulator
of cell ­migration, proliferation, invasion and adhesion, but also as a stimulator
of neurite outgrowth. We have previously demonstrated the distribution and
­expression of L1CAM as an important mediator of the invasion of endometriotic implants and nerve outgrowth in patients with endometriosis. Therefore
we tested the effect of the intraperitoneal application of anti-L1 monoclonal
antibody (mAb) to reduce endometriosis implants and nerve outgrowth in an
endometriosis mouse model.
Material and Methods: Endometriosis was induced with a well published
mouse model by peritoneal auto transplantation of pieces of murine uterus of 5
weeks old B6C3F1 mice. Four weeks later, the lesions were measured by MRI
in vivo. We treated with repeated intra peritoneal application of anti-L1CAM
mAb 555 in different concentrations (verum) respectively IgG isotype (control)
twice weekly. Two weeks later, the lesions were measured by MRI and the
mice were sacrificed. Thus, we compared the pre- and post-therapeutic volumes of the endometriotic lesions in each group. Lesions were extracted and
endometriosis was histologically confirmed. Furthermore, number of nerves
within the endometriotic lesions was counted double blind after nerve specific
anti neurofilament immunohistochemistry in both groups. The study protocol
was a­ pproved by the Ministry of Nature and the Environment of SchleswigHolstein, Dept. of Animal Welfare.
Results: Five mice in each group were evaluated. Pretherapeutic mean lesion
volume (LV) by MRI was 25,0 mm3 (100 ± 83,3% (MW ± SD)) in the verum
resp. 25,3 mm3 (100 ± 38,6%) in the control group. The mean post therapeutic
LV was 27,0 mm3 (108,0 ± 80,0%) resp. 35,6 mm3 (140,9 ± 87,5%). This
shows clearly a reduced growth of endometriotic lesions under anti-L1CAM
mAb treatment compared to controls. Moreover, the nerve count within the
lesions showed a lower nerve density after antibody treatment (29,2 ± 10,4 vs.
69,9 ± 39,2 nerves per lesion).
Conclusion: The growth potential of endometriotic lesions was limited through
the intraperitoneal application of anti-L1CAM mAb. In addition, the concentration of peripheral nerves within endometriotic lesions was lower after treatment
with anti-L1CAM mAb. The concept of local intraperitoneal therapy should
be further evaluated. Endometriosis associated pain might be reduced by antiL1CAM mAb local therapy through limited nerve outgrowth. In a previous
study, anti-L1CAM mAb treatment was able to inhibit the intra-peritoneal
tumor growth and dissemination of ovarian carcinoma cells successfully. Our
here presented study demonstrates that this antibody could possibly also be
helpful for the treatment of endometriosis.
This study was supported by the German Research Foundation (DFG,
Ho 1832/6-1) and by a grant from Bayer-Schering Pharma Berlin, Germany
(“Grants for Targets”).
Wednesday 6 July 2011
10:00 - 11:45
O-250 PGD for translocations using array comparative genome
P. Colls1, J. Fischer1, T. Escudero1, K. Ketterson1, G. Harton1, S. Munne1
Reprogenetics, PGD Lab, Livingston, U.S.A.
Introduction: PGD for translocations using FISH has been successful in reducing the risk of miscarriage in carrier couples to about 10%. However, for older
translocation carriers with elevated risk of producing both aneuploid and unbalanced embryos, microarrays may be more advantageous. One objective of this
study was to validate array comparative genomic hybridization (aCGH) to determine the minimum fragment size detectable by this technique without incurring
a high error rate. Our database of 1325 PGD cases analyzed by FISH shows that
both translocated segments were > 30Mb long in 43.3% of couples, between 6
and 30Mb in 35.5% of couples, and less than 6Mb in the remaining 21.2%. Thus
we focused on the group with the smallest fragment being 6-30 MBs.
The second purpose of the study was to determine how many abnormal embryos would have been missed by FISH using probes only for the translocation.
Material and Methods: 10 patients with reciprocal translocations with the
smallest translocation segment being between 6 and 30 MB were recruited for
the study. In addition 15 Robertsonian translocations were also included for the
second objective. A reciprocal translocation was determined to be analyzable
if abnormalities in both chromosome arms of the translocation were detected.
In addition, abnormal, non-transferred embryos previously analyzed by aCGH
were fixed and analyzed via fluorescence in-situ hybridization (FISH) in all
their cells to determine if the original aCGH PGD analysis was correct. FISH
was performed with probes above and below the breakpoints of the chromosomes involved in the translocation, plus probes for any other chromosome
shown abnormal by aCGH.
Results: All ten reciprocal translocations showed in their cohort of embryos
unbalanced chromosome rearrangements involving both translocated chromosome segments, including a case with the smallest fragment being 6MB in
length. 23 embryos were reanalyzed by FISH and all were found to be consistent with the aCGH diagnosis.
The rate of abnormalities for reciprocal translocations (av. Maternal age
32.5 years) was 87%: 32% of all embryos being unbalanced for the translocation only, 38.7% unbalanced plus other chromosome abnormalities, and 16%
abnormal but not unbalanced for the translocation. In comparison, the rate of
abnormalities for Robertsonian translocations (av. Maternal age 33.4 years)
was only 57%: 18% of all embryos being unbalanced for the translocation only,
16% unbalanced plus other chromosome abnormalities, and 19% abnormal but
not unbalanced for the translocation.
Discussion: PGD via FISH is limited in that only a few additional chromosomes can be tested simultaneously with a reciprocal translocation. Here we
report the use of aCGH for the simultaneous analysis of translocations and aneuploidy of all chromosomes. The present results indicate that aCGH can accurately detect reciprocal translocation segregation types involving an exchanged
fragment 6MB or larger. For fragments smaller than 6MB, the technique has not
been yet validated, however if three out of four of the fragments are larger than
6Mb, aCGH allows the detection of all unbalanced outcomes. Interestingly,
only 16-19% of abnormal embryos would not have been detected by FISH.
Given that FISH is a much simpler and cheaper technique, for young patients,
and patients with two terminal breakpoints, FISH remains a valid technique.
O-251 Sequential comprehensive chromosome analysys on polar bodies,
blastomeres and trophoblast: inefficacy of polar bodies approach in
predicting female meiotic aneuploidies
A. Capalbo1, F. Fiorentino2, R. Maggiulli1, S. Romano1, A. Borsatti2, A.
Joseph2, L. Spizzichino2, S. Bono2, A. Biricik2, S. Colamaria1, M.F. Ubaldi1,
L.F. Rienzi1
Genera, Reproductive Medicine, Roma, Italy
Genoma, Molecular Genetics Laboratory, Roma, Italy
Introduction: Polar bodies approach with comprehensive chromosome
a­ nalysis was proposed for preimplantation genetic screening in advanced reproductive age patients. The aim of the present study is to test the reliability of
this approach, by sequential biopsy and comprehensive analysis of the chromosomal segregation at different stage of development.
Material and Methods: Oocytes were biopsied for I polar body(PB1) and II
polar body(PB2) screening on day 1. The viable deriving embryos were biopsied through the same hole on day 3. Finally 3-5 trophoblast cells were obtained
from the blastocyst on day 5 or day 6. The cells were first lysed and DNA
amplified by whole genome amplification (WGA) using the SurePlex kit. WGA
products were then processed by Array-CGH according to the Perkin-Elmer
Constitutional Chip 4.0 protocol.
Results: Five patients over 40 years old were enrolled for preimplantation
diagnosis (with no male factor infertility). 44 MII oocytes were obtained,
of which 36 fertilized. Out of the 33 embryos obtained on day3 26 reached
Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 July – 6 July, 2011
blastocyst stage on day 5 or day 6. These embryos were sequentially biopsied at the three different stage of development. Successful biopsy and DNA
amplification was obtained in 76% of both polar bodies (86 and 86% for
PB1 and PB2, respectively), 94% of blastomeres and 100% of blastocyst.
A full diagnosis was thus obtained in 15/26 blastocyst (57.7%). Diagnosis
success rate was significantly lower for PBs as compared to blastomeres and
trophoblast cells (p=0.007). All the analyzed oocytes had chromosomal abnormalities assessed on PB, which originated from meiosis I errors (10/15)
and from meiosis II errors (14/15) or both (15/15). Despite different incidences, all chromosomes were involved in meiotic derived aneuploidies.
When looking to single chromosomal segregation on day 3, we observed
that misdiagnosis due to false positive in determining female meiotic derived aneuploidies in PBs was obtained in 15 out of 50 cases (30%). Moreover, 11 out of 50 (22%) meiotic derived female aneuploidies confirmed in
day 3 were rescued at blastocyst stage. Finally, 16 de novo mitotic errors
and/or male derived aneuploidies were recorded on day3, 14 of which confirmed at blastocyst stage (87.5%). The sensibility and specificity in predicting blastocyst chromosomal complement were significantly lower for
polar bodies approach 60,3% (95%CI 50,7 to 69,8) and 91,5% (95%CI 89.1
to 93.9) compared to day3, 77.8% (95%CI 70,4% to 85,3%) and 96.8%
(95%CI 95,3 to 98,3), respectively. As a result, only in 4 out of 15 blastocysts the resultant chromosomal complement would have been correctly
predicted by polar bodies strategy.
Conclusion: Polar bodies is an inefficient strategy due to difficulties related
to biopsy, handling and genomic DNA quality. Even when full diagnosis is
obtained, female derived meiotic aneuploidies cannot be accurately predicted
especially due to false positive results. As suggested by day 3 and day 5/6
subsequent analysis chromatids predivision, anaphase lag and previously acquired germinal mitotic derived aneuploidies accurately explain the observed
inconsistencies. Moreover, male and mitotic derived aneuploidies and correction mechanisms in act later in preimplantation development led to a very
low correlation between polar bodies screening and blastocyst chromosomal
O-252 Re-testing of no results embryos in PGD cycles by second biopsy
at blastocyst stage
P. Rubino1, L. Arizzi1, M.G. Minasi1, R. Pena1, F. Scarselli1, V. Casciani1,
A. Colasante1, S. Ferrero1, K. Litwicka1, M.T. Varricchio1, F. Fiorentino2,
A. Biricik2, F. Cucinelli3, Z.P. Nagy4, E. Greco1
European Hospital, Reproductive Medicine, Roma, Italy
GENOMA—Molecular Genetics Laboratories, Centre for Preimplantation
Genetic Diagnosis, Roma, Italy
San Camillo Forlanini, Obstetrics and Gynaecology, Roma, Italy
Reproductive Biology Associates, Centre for Reproductive Medicine, Atlanta
Georgia, U.S.A.
Introduction: Preimplantation genetic diagnosis (PGD) procedure allows the
testing of embryos before transfer for couples who are at risk of transmitting
a severe genetic disorder to their children. Cleavage stage biopsy, is the most
common approach for obtaining embryonic genetic material for PGD analysis. When diagnosing monogenic disorders in single cells using PCR based
protocols, there are some risks of PCR failure (“no-results”) or allele dropout
(“ADO” - incomplete results), potentially reducing the number of unaffected
embryos available for transfer.
In this preliminary study, we evaluated the possibility to reanalyze the “noresults” and the “ADO” embryos by blastocyst biopsy and subsequent vitrification for possible future embryo transfer.
Materials and Method: Between September 2009 and December 2010, 54
biopsy procedures on 368 fresh embryos (6,7 ± 3,1per patient) were performed in our Centre. Fifty cycles were completed with an embryo transfer.
All embryos that had six or more cells on day-3 were subjected to biopsy
following zona pellucida ablation using a 1.48 μm infrared laser. One blastomere from each embryo was removed and placed immediately in RNAseDNAse-free PCR tubes containing Lysis buffer and transferred to the Genetic Laboratory (Genoma Group-Rome). The diagnosis was performed by
multiplex single-cell PCR. Embryo transfer was performed on day-4 after
oocyte retrieval, as soon as test results were ready. The best quality embryos
considered normal for the tested mutation were replaced. All viable surplus
embryos were cryopreserved by vitrification. “No-results” (n = 13) and
“ADO” (n = 0) embryos that developed to the expanded blastocyst stage on
day-5, 6 or 7 were biopsied before vitrification. Due to the analysis of different linked markers for every mutation, no “ADO” embryos were obtained.
If no herniation of the trophectoderm cells from the hole created in the zona
on day-3 was observed, a gentle suction with the biopsy pipette was applied.
Four to six trophectoderm cells were dissected from each blastocysts using
about four laser pulses of 3ms duration as far as possible from the inner cell
mass. The biopsied cells were treated the same way as biopsied blastomeres.
The biopsied blastocyst were immediatly vitrified using the cryotop method
with Kitazato vitrification Media. Pregnancies were confirmed by ultrasound
detection of fetal heartbeat.
Results: The mean age of women was 34.2 ± 4.58, the average number of
embryos transferred was 2 ± 1. Twenty-five patients had a positive β-HCG test
(50%) and 22 pregnancies were confirmed by ultrasound diagnosis (44%) with
an implantation rate of 27,7%. The “no-results” embryo rate was 16,7%. Thirteen “no-results” embryos from 9 cycles that reached the expanded blastocyst
stage were re-biopsied before vitrification; 6 blastocysts were biopsied on day5, another 6 on day-6 and 1 on day-7. In 12 cases test results were obtained
(92%) and one case we had “no-results” again. Four of 9 patients have decided
to warm their embryos: 6 biopsied blastocyst were thawed, all of them survived
fully intact and 4 clinical pregnancies were obtained.
Conclusions: The ability of blastocysts to implant after trophectoderm biopsy
has been reported in previous studies. However, our preliminary data indicate
that biopsy of blastocysts developed from day-3 biopsied embryos does not
impair implantation potential even after cryopreservation using the vitrification
Patients undergoing assisted conception and PGD procedures to prevent a
genetically inheritable disease are in a particularly delicate condition. Reanalyzing the “no-results” and “ADO” embryos from a failed genetic testing, patients can have better chances to have unaffected embryos for a second attempt,
especially when no surplus unaffected embryos are available for freezing.
O-253 Outcomes of 327 cycles on preimplantation human leukocyte
antigen typing with or without mutation analysis
C. Beyazyurek1, C.G. Ekmekci1, H.A. Tac1, N. Ajredin1, H. Yelke1, S.
Istanbul Memorial Hospital, Reproductive Genetics Centre, Istanbul, Turkey
Introduction: Preimplantation HLA typing technique allows the birth of children who are potential donors of stem cells for their affected siblings. This
technique can be used for acquired diseases such as leukemia or can be used
for single gene disorders (SGD) such as thalassemia in combination with preimplantation genetic diagnosis (PGD) technique. The purpose of this study is to
share the one of world’s largest experiences on HLA typing cycles and assess
the effect of clinical parameters on reproductive outcome of patients referred
for thalassemia.
Materials and Methods: 171 couples have undergone 327 HLA typing cycles. 262 cycles were performed for HLA typing in combination with mutation
analysis and 65 cycles were performed for the sole purpose of HLA typing.
For most of the cases, one blastomere was removed from day 3 embryos by
laser technique. Mutation analysis was done using either minisequencing or restriction fragment length polymorphism (RFLP) method combined with linked
short tandem repeats (STR) marker analysis, and HLA typing was performed
using polymorphic STR markers scattered through the HLA gene cluster.
Results: 17.5 % of the diagnosed embryos were found to be HLA matched.
Embryo transfer was performed in 212 cycles. 34.9% clinical pregnancy rate
(CPR) was achieved per transfer. 59 healthy and HLA compatible children were
born. 21 sick children have been cured through haemopoietic stem cell transplantation and 23 children are awaiting transplantation.
Conclusion: The data demonstrated that, once a mutation-free and HLAcompatible embryo was found, CPRs did not differ statistically significantly
by the presence of some cycle-related limitations such as diminished ovarian
reserve. Preimplantation HLA typing is an effective therapeutic tool for curation of an affected sibling even for the poor prognosis patients. The reported
21 sick children who have been cured by Haemopoietic Stem Cell Transplantation (HSCT) is the largest number available from one center. Despite the
lower probability of finding suitable embryos for embryo transfer, the data
presented in this report shows the feasibility and the practicability of PGDHLA application.
Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 July – 6 July, 2011
O-254 The combined experience of three European centres with
preimplantation genetic diagnosis for Huntington disease
M. De Rademaeker1, C. Moutou2, M. Van Rij3, J. Dreesen3, M. De Rycke4,
I. Liebaers4, S. Viville5, J. Geraedts3, C. De Die3
Universitair Ziekenhuis Brussel, Centre of Medical Genetics, Brussel,
Université de Strasbourg, Hôpitaux Universitaires de Strasbourg, Service de
Biologie de la Reproduction--SIHCUS-CMCO
Maastricht University Medical Centre, Clinical Genetics, Maastricht,
The Netherlands
Universitair Ziekenhuis Brussel, Centre for Medical Genetics, Brussel,
Université de Strasbourg, Hôpitaux Universitaires de Strasbourg, Service de
Biologie de la Reproduction--SIHCUS-CMCO
Introduction: Huntington disease (HD) is an autosomal dominant progressive
neurodegenerative disorder, seriously affecting quality of life of patients and
their family members. The disease causing mutation is an expanded CAG in the
HTT gene on chromosome 4 (4p16.3). Reproductive options for gene carriers
or at risk persons include prenatal testing and preimplantation genetic diagnosis
(PGD). The combined experience of three European centers is presented.
Materials and Methods: Data on all intakes and outcome of PGD treatment
cycles between 1995 and 2008 were collected. Three hundred thirty one couples had an intake for PGD for direct or exclusion testing PGD for HD. Single
cell testing methods were very similar in our 3 PGD centres. The single cell
test started as simplex PCR, but is now performed by multiplex fluorescent
PCR co-amplifying from 2 to 6 loci for direct or exclusion testing. PGD based
on direct testing is allowed in all three centers. Exclusion was, but is no longer
allowed in the Netherlands.
Results: 68% (225/331) of the couples requested direct testing and 32%
(106/331) exclusion PGD. 12% (40/331) of couples showed a history of fertility problems necessitating IVF/ICSI. 39% (129/331) of females had at least one
previous pregnancy and 21% (68/331) had experienced at least one pregnancy
termination after PND for HD. Significantly more females had experienced
a termination of pregnancy (TOP) (25%) (57/225) in the direct testing group
compared to the exclusion group (10%)(11/106) (p = 0.0027). For the exclusion
group more couples in Brussels (53%) (20/38) than in Strasbourg (22%)(13/59)
had at least one previous pregnancy (p = 0.007). In Brussels 21% (8/38) of
the couples had a history of pregnancy termination(s) after exclusion PND for
HD whereas in Strasbourg there was only one TOP after PND in the exclusion
group (1.7%)(1/59)(p = 0.0045).
Remarkably, in Strasbourg 8 couples had terminated a pregnancy without
PND. For the three centers the mean number of biopsied embryos per cycle to
oocyte retrieval (OR) was 5.85 (2277/389). Mean number of embryos transferred per cycle was 1.65 (511/310). 105 positive HCG tests in 84 females occurred. The clinical pregnancy rate was 21.6% per cycle to OR and 27.1 % per
transfer. 77 deliveries resulted in the birth of 90 children (65 singletons, 11
twins and 1 triplet). Prenatal diagnosis to confirm PGD results was performed
more frequently in Brussels ((41.3%) (19/46) of clinical pregnancies) than in
Maastricht (10%) (2/21) and never in Strasbourg.
Conclusions: Couples opting for exclusion had significantly less previous
pregnancies ans less pregnancy terminations (p = 0.0027). The results obtained
are similar to usual PGD success rates. PGD seems to be a good option for
(potential) carriers of the HD gene to have unaffected children.
O-255 Segmental chromosome imbalance in human oocytes and
preimplantation embryos: incidence, origin and clinical relevance
D. Wells1, E. Fragouli2, P. Colls3, S. Alfarawati2, S. Munne3
University of Oxford, Nuffield Dept of Obstetrics and Gynaecology, Oxford,
United Kingdom
Reprogenetics UK, Inst. of Reproductive Sciences, Oxford, United Kingdom
Reprogenetics, Livingston, New Jersey, U.S.A.
Introduction: Recently, it has been reported that loss and gain of chromosome fragments (segmental imbalances) are common in human embryos. A
small study of 23 cleavage stage embryos reported that 70% were affected by
segmental imbalance (Vanneste et al 2009). This alarmingly high incidence
led to concern that this form of genetic instability could be a major contributor to the poor efficiency of in vitro fertilisation treatments. Segmental e­ rrors
occurring in embryos may also explain the origin of many constitutional
chromosomal abnormalities detected during pregnancy or at birth, a principal
cause of mental retardation and birth defects. Given the wide-ranging clinical implications of segmental chromosome anomalies, verification of their
frequency in a larger number of samples and in different clinics is urgently
required. We aimed to determine the true incidence and origin of segmental
chromosome anomalies, revealing whether they should be considered a cause
for clinical concern.
Materials and Methods: We conducted a study of 1,524 human preimplantation embryos (cleavage stage and blastocyst) and 368 oocytes, derived from
nine different IVF clinics. Micromanipulation allowed biopsy of polar bodies
from oocytes, blastomeres from cleavage stage embryos and trophectoderm
cells from blastocysts. Biopsied cells were subjected to whole genome amplification, followed by microarray comparative genomic hybridisation (arrayCGH) using a well validated microarray technique, providing accurate detection of losses and gains of chromosomal fragments >3Mb in size.
Results: A total of 1892 samples were tested (368 oocytes, 586 cleavage stage
embryos and 938 blastocysts), revealing that 3.3% of oocytes were affected by
segmental imbalance. This rose to 20% in cleavage stage embryos (calculation
based on assessing all cells of the embryo), before declining to 6% in blastocysts. Chromosome breakage did not occur at random, with frequent breakage
at centromeres. In many cases loss of one whole chromosome arm was accompanied by duplication of the other arm. This likely represents isochromosome formation. Patient and embryo-specific tendencies to produce segmental
abnormalities were noted, with some patients producing more affected embryos
than expected by chance (P<0.05) and embryos with segmental errors being
at increased risk of multiple chromosome breakages occurring simultaneously
Conclusion: This large study demonstrated that segmental abnormalities are
relatively common among human oocytes and embryos, although their incidence was lower than has recently been suggested. Based upon our results,
we calculate that the true incidence of cleavage stage embryos with segmental
imbalance is <20%. Discrepancies in the incidence of segmental chromosome
abnormalities recorded in different studies may be related to variation in patient
populations or to differences in ovarian stimulation or embryo culture systems.
This raises the possibility that segmental abnormalities might be reduced by
optimising stimulation and culture protocols. The tendency for some patients to
display multiple affected embryos also argues for procedure or patient-specific
effects. Some segmental errors were of meiotic origin, but most arose during
the first few mitoses following fertilisation. The incidence of segmental imbalance declined as embryo development progressed, indicating that abnormal
cells probably fail to divide or undergo apoptosis. Based on these results, it is
likely that few embryos containing segmental anomalies ultimately produce
children. Cell loss due to the presence of segmental imbalance may have a
negative impact on embryo viability (as does cell loss via other mechanisms),
but such anomalies are probably not associated with a high risk of abnormalities at birth, due to elimination of affected cells at early embryonic stages. This
is the first study to assess the timing, location and frequency of de novo chromosome breakage in human oocytes and embryos and indicates that segmental
imbalance is likely to be clinically relevant, although less so than some have
previously hypothesised.
O-256 Identification of differentially methylated cell cycle genes in
human embryos with chromosomal mosaicism
A. Kashevarova1, E. Tolmacheva1, N. Sukhanova2, I. Lebedev1
Institute of Medical Genetics Russian Academy, Cytogenetic Laboratory,
Tomsk, Russia C.I.S.
Institute of Medical Genetics Russian Academy, Genetic Clinic, Tomsk, Russia C.I.S.
Introduction: Preimplantation embryos as well as miscarriages are characterized with a high frequency of chromosomal mosaicism indicating an increased
level of postzygotic mitotic errors during early stages of embryogenesis. However the underlying causes remain illusive. Recently human pathology has
been shown to involve both genetic and epigenetic factors. Previously we have
shown a high frequency of RB1 and P14ARF epimutations in diploid-aneuploid
miscarriages (Tolmacheva et al., 2008; Kashevarova et al., 2009). The present
work was aimed to identify and describe a spectrum of differentially methylated
cell cycle genes responsible for chromosome segregation and mosaicism origin.
Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 July – 6 July, 2011
Material and Methods: Cytotrophoblast (CT) and extraembryonic mesoderm (EM) of 6 miscarriages (11.5 ± 2.1 weeks) with diploid-aneuploid
mosaicism and RB1 epimutations were analyzed using Infinium HumanMethylation27 BeadChip (Illumina, USA) covering 27,578 CpG-sites of 14,495
genes. Four out of 6 embryos had both disomic and trisomic cell lines. Two
abortions were trisomic for chromosome 16, one for chromosome 10, and the
other one for chromosome 21. One abortion was disomic for Y chromosome.
The 6th embryo was mosaic for monosomy 13. Four miscarriages (9.9 ± 2.7
weeks) with normal karyotypes and 2 induced abortions (8.8 ± 1.8 weeks)
were used as control. DNA methylation analysis was performed according to
the manufactures recommendations. Data were analyzed using GenomeStudio
Methylation Module software. DiffScore > |20| was used to determine significant differences in methylation level of each CpG-site between two samples.
Genes associated with biological process “cell cycle” were selected using the
Gene Ontology classification (GO:0007049, cell cycle, http://www.geneontology.org).
Results: Twelve of 247 cell cycle genes present on the BeadChip were differentially methylated in CT and 7 genes in EM of diploid-aneuploid miscarriages.
Two genes differentially methylated in CT participate in chromosome segregation regulation (LZTS1 and ESPL1), 4 genes regulate G1/S transition (CCND2,
CDKN2A, RASSF1, and DBC1), and 6 genes are involved in apoptosis (TP73,
PYCARD, BCL2, BRCA2, AHR, and VHL). Differentially methylated in EM
CHFR and RCC2 regulate chromosome segregation, RASSF1 – G1/S transition, and ALOX15B, TP73, VHL, MYBL2 – apoptosis. None of these genes but
LZTS1 and CDKN2A was differentially methylated in diploid miscarriages.
DLG7, LZTS1, CDKN2A, and DCC were differentially methylated in their CT
and EM respectively. Three out of 4 diploid miscarriages had one or both differentially methylated DLG7 and LZTS1 in CT. These genes are essential for
proper M-phase progression and chromosome segregation. The only gene hypermethylated in CT of the remaining embryo was CDKN2A regulating G1/S
transition. DCC methylated in EM regulates apoptosis and presumably trophoblast invasion.
Conclusions: Abnormal methylation of cell cycle genes was shown in miscarriages with normal karyotype and diploid-aneuploid mosaicism. However
the spectrum of genes, their combinations and processes they are involved are
differ between these two groups of embryos. We hypothesized that inactivation
of genes essential for chromosome segregation will lead to the origin of cells
with numerical chromosome aberrations but further the abnormal cells will be
arrested and eliminated as in the case of diploid miscarriages. If G1/S transition
and apoptosis genes are deregulated as well (like in abortions with chromosomal mosaicism) then, apparently, it will favor abnormal cells survival and
chromosomal mosaicism formation.
This study was supported by grants of Federal Program N P806 and P1161
and State Contract for National Educational Center N 02.740.11.0281.
1 Tolmacheva et al., Rus. J. Genet., 2008, vol. 44, no. 11, pp. 1266-1271.
2 Kashevarova et al., Rus. J. Genet., 2009, vol. 45, no. 6, pp. 749-755.
Wednesday 6 July 2011
12:00 - 13:15
Wednesday 6 July 2011
O-257 Formation of distinct cell types in the mouse embryo
M. Zernicka-Goetz1
University of Cambridge, Wellcome - CRC Institute, Cambridge,
United Kingdom
12:00 - 13:00
A crucial question in mammalian development is how cells of the early embryo
differentiate into distinct cell types. These cell fate decisions are taken when
cells undertake waves of asymmetric divisions, which generate one daughter
cell on the inside and one on the outside of the embryo. Inside cells, so called
inner cell mass (ICM), provide stem cell population for all cells of the future
body, while outside cells differentiate into the trophectoderm, the extra-embryonic tissue with signalling properties. But how the fate of these inside and outside cells is decided and what decides whether a cell divides symmetrically or
asymmetrically have remained unknown. To address both of these questions we
combined live-cell lineage studies with gain and loss-of function approaches to
assess the function of key cell fate-determining genes in this process. Our studies indicate that both cell polarisation and cell position regulate the expression
of cell fate-determining genes.
Moreover, we found that the natural heterogeneity among early embryo
cells biases their fate. Such bias would maximise chances of developmental
success while retaining developmental plasticity.
O-258 X chromosome inactivation a key event during early
C. Patrat1, I. Okamoto1, D. Thépot3, N. Peynot3, P. Fauque4, N. Daniel3, P.
Diabangouaya1, J. Renard3, V. Duranthon3, E. Heard1
Mammalian Developmental Epigenetics Group, CNRS UMR 3215/INSERM
U934, Institut Curie
Université Paris Diderot – Assistance Publique Hôpitaux de Paris – ­Service
de Biologie de la Reproduction, Hôpital Bichat-Claude Bernard, Paris,
INRA UMR 1198 Biologie du Développement et de la Reproduction, Jouy en
Josas Cedex France,
Laboratoire de Biologie de la reproduction CECOS, CHU de Dijon, Université de Bourgogne
Introduction: In mammals, X-chromosome dosage compensation is achieved
by inactivating one of the two X chromosomes in females. The developmental
regulation of X chromosome inactivation (XCI) has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA,
Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell
mass of the blastocyst and random XCI subsequently occurs in epiblast cells.
But multiple strategies for initiating XCI could exist for mammals. The aim of
the study was to analyse XCI initiation in rabbit and human pre implantation
Material and Methods: We examined the expression of XIST RNA, which is
the key regulatory molecule underlying X inactivation, the kinetics of epigenetic marks such as histone modifications induced by XIST RNA and the silencing of X-linked genes. Single cell techniques, involving RNA Fluorescence In
Situ Hybridisation (FISH) and immunofluorescence with antibodies specific for
different epigenetic marks have been used to assess the activity and epigenetic
status of the X chromosome during development.
Results: We show that in both rabbit and human pre-implantation embryos,
Xist is not subject to imprinting and X inactivation initiates much later than in
mouse embryos. Furthermore, Xist is up-regulated on both X chromosomes in a
high proportion of rabbit and human embryo cells, even in the ICM, in contrast
with the mouse where X inactivation is strictly monoallelic from the outset. In
rabbits, this triggers XCI on both X chromosomes, implying that the choice of
which X chromosome will finally become inactive occurs downstream of Xist
up-regulation. In humans, XCI is not triggered by the blastocyst stage, despite
the up-regulation of XIST.
Conclusion: Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for
dosage compensation during early embryogenesis, probably as the regulation
of processes such as XCI have to display substantial plasticity to accommodate
evolutionary changes.
Wednesday 6 July 2011
12:00 - 13:00
Abstracts of the 27th Annual Meeting of ESHRE, Stockholm, Sweden, 3 July – 6 July, 2011
Genes differentially expressed with fold change ≥ 2 were identified (summarized in the table below):
Hybridization (conditions compared) NOA vs. OA NOA vs. Normal OA vs. Normal No. of genes
Up-regulated in condition 1 Down-regulated in condition 1
541 1530 433 557
NOA: RNA from Non-obstructive Azoospermia; OA: RNA from Obstructive Azoospermia
Normal: Representative RNA from normal individuals, purchased from a company
A new scoring system was followed. The method was efficient in determining the percentage overlap and the potential markers, wherever needed the
consensus was derived from gene-lists across studies. In graphs 2 and 3 (figures
not shown), there was a high percentage overlap between the genes from the
experiment with that of the database block with a high reliability score, which
proves our hypothesis. Any block with a percentage value, greater than the expected random chance of occurrence (% overlap between the genes from the
experiment with the total genes in the database), is considered as reliable block,
from which the potential markers can be identified.
We developed a new method to derive a more reliable expression pattern
of genes, using the existing mass-scale data - from one tissue and condition at
a time. The approach involved manual extraction (biocuration) of maximum
number of available data sets, development of a database, and deriving a consensus expression pattern across ‘comparable’ multiple studies (addressing
same location and condition) for each gene. The new database and associated
software serve as a ‘gene expression prediction platform’. This platform performed better than any other system in providing straight forward expression
information for randomly selected genes. Experimental and in silico validations
confirmed the higher reliability of the database. The new approach has been
used to identify more dependable potential biomarkers for clinically relevant
Conclusions: A) The gene expression platform for mammalian testis provides
highly reliable information in various aspects. B) Experiments and in silico
analysis show that the gene expression patterns derived from the new platforms
are highly reliable; the genes with higher reliability, as per the database, were
repeated frequently in the experimental data set for similar conditions. C) The
analysis of the experimental results also indicated a threshold level for the reliability of the score. D) New sets of potential biomarkers identified are very
promising as they contain many novel genes. E) The information could be very
useful for basic research as well.
Acknowledgements: Department of Information Technology & Department of
Biotechnology, Government of India & Agilent Technologies, India.
P-429 Defective deacetylation of H4K12 in human oocytes is associated
with advanced maternal age and chromosome misalignment
J.H. van Doorninck1, C. Eleveld2, M. van der Hoeven1, E. Birnie3,
E.A.P. Steegers2, R.J. Galjaard4, J.S.E. Laven5, I.M. van den Berg1
Erasmus MC, Obstetrics & Gynaecology Subdivision Reproductive Medicine
and Dpt Clinical Genetics, Rotterdam, The Netherlands
Erasmus MC, Obstetrics & Gynaecology, Rotterdam, The Netherlands
Erasmus MC, Institute of Health Policy and Management, Rotterdam, The
Erasmus MC, Clinical Genetics, Rotterdam, The Netherlands
Erasmus MC, Obstetrics & Gynaecology Subdivision Reproductive Medicine,
Rotterdam, The Netherlands
Introduction: Chromosome segregation errors during human oocyte meiosis
are associated with low fertility in humans and the incidence of these errors
increases with advancing maternal age. Studies of mitosis and meiosis in yeast
and animals suggest that defective remodelling of chromatin plays a causative
role in aneuploidy. We analyzed the histone acetylation pattern during the final
stages of human oocyte maturation to investigate whether defective epigenetic
regulation of chromatin remodelling in human oocytes is related to maternal
age and leads to segregation errors.
Material and: Human surplus oocytes of different meiotic maturation stages
(GV, MI and MII, n = 401) were collected from standard IVF/ICSI treatments
(MEC + , no objection). Oocytes were stained with antibodies against acetyl
groups on different lysines on the tail of histone 4 (H4K5, H4K8, H4K12,
H4K16) and against the spindle component α-tubulin followed by epifluorescent or confocal microscopy.
Results: GV oocytes had an intense staining of the chromatin for all four histone 4 lysine acetylations. Similar to mouse oocytes, human oocytes showed a
general pattern of deacetylation during maturation to the MI and MII stages.
However, in MI as well as MII oocytes, the percentage of residual acetylation varied between different modifications. We focussed on H4K12 because
acetylation of H4K12 has been demonstrated to be associated with age and
aneuploidy in mouse oocytes. Residual H4K12 acetylation was more frequently
found in oocytes obtained from older women, with a significant correlation between defective de-acetylation and maternal age (r = 0.185, p = 0.007). 88% of
the oocytes with residual acetylation had misaligned chromosomes while only
33% of the oocytes that showed correct deacetylated chromatin had misaligned
chromosomes (p < 0.001).
Conclusions: We conclude that defective deacetylation during human female
meiosis increases with maternal age and is correlated with misaligned chromosomes. As MII oocytes with misaligned chromosomes are predisposed to segregation errors, our data imply that defective regulation of histone deacetylation
could be an important factor in age-related aneuploidy.
P-430 Clinical application of array comparative genomic hybridization
in preimplantation genetic diagnosis for reciprocal and robertsonian
F. Fiorentino1, L. Spizzichino1, S. Bono1, A. Biricik1, G. Kokkali2, L. Rienzi3,
F.M. Ubaldi3, E. Iammarrone1, A. Gordon4, K. Pantos2
GENOMA - Molecular Genetics Laboratory, Preimplantation Genetic
­Diagnosis Section, Rome, Italy
Genesis Athens Hospital, Centre for Human Reproduction,
Athens, Greece
G.EN.E.R.A, Centre for Reproductive Medicine, Rome, Italy
Bluegnome Ltd, Bluegnome Ltd, Cambridge, United Kingdom
Introduction: Fluorescence in-situ hybridisation (FISH) is the most widely
used method for detecting unbalanced chromosome rearrangements on preimplantation embryos. FISH is known to have several well-documented technical limitations which may lead to incorrect interpretation of the results and
a potentially adverse outcome. The clinical application of a molecular-based
assay, known as array comparative genomic hybridization (Array-CGH), used
to simultaneously screen for unbalanced translocation derivatives as well as
aneuploidy of all 24 chromosomes is described in this study.
Material and Methods: This is a prospective longitudinal cohort study performed in order to evaluate the clinical efficiency of Array-CGH in a specific
category of patients. Cell biopsy was carried out on cleavage stage embryos
(day 3). Single cell were first lysed and DNA amplified by whole genome
amplification (WGA). WGA products were then processed in 24 hours by Array-CGH using high density arrays 24sure + , BlueGnome. Balanced/normal
euploid embryos were then selected for transfer on day 5 of the same cycle.
The chromosomally abnormal embryos that developed to blastocyst stage were
reanalyzed in order to confirm PGD results.
Results: Twenty-eight consecutive cycles of PGD were carried out for 23 couples
carrying 18 different balanced translocations. Overall, 187/200 (93.5%) embryos
were successfully diagnosed, 16% (30/187) were normal or balanced, 17.1%
(32/187) were unbalanced for the translocation and normal for aneuploidy, 39.6%
(74/187) had aneuploidy and were unbalanced, 27.3% (51/187) were normal or
balanced but showed aneuploidy of chromosomes not involved in the translocation. The re-analysis at blastyocyst stage were concordant for all embryos followed-up. Despite high levels of mosaicism, all day 3 aneuploid embryos were
again diagnosed as abnormal after re-analysis on trofectoderm cells, confirming at the end the previous results regardless of the actual abnormal genotype.
Embryos suitable for transfer were identified in 17 cycles (73.9%), with transfer
of 22 embryos (mean 1.3 ± 0.5). Twelve couples achieved a clinical pregnancy
(70.6% per ET), with a total of 14 embryos implanted (63.6% per transferred
embryo). Six patients delivered 7 healthy babies, while the other pregnancies
(1 twins and 5 singletons) are currently ongoing beyond 20 weeks of gestation.
Conclusions: The data obtained demonstrates that array-CGH is able to detects
chromosome imbalances on embryos. Unlike some alternative techniques, this

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