Humrep-oral 1.108
Abstracts of the 22nd Annual Meeting of the ESHRE, Prague, Czech Republic, 18–21 June 2006
differences were found within any of these subgroups in terms of pregnancy or
(day 2 frozen embryos highly represented). Our results suggest that the best
implantation rates.
strategy is to select the embryos for freezing on day 3 and to carry out the
Subgroups A and B showed no differences in pregnancy (58% vs. 58%)
thawing on the day scheduled for the frozen embryo transfer.
or implantation rates (29% vs. 27%) when compared regardless of applica-tion of the technique. Subgroup C showed lower pregnancy (12% vs. 58%,p<0.001) and implantation rates (8% vs. 27%, p¼0.011) independentlyof undergoing AHA and lysed cell removal when compared with subgroups
FREE COMMUNICATION
A and B.
Conclusion: In our study, pregnancy and implantation rates in thawed embryo
Session 29 – Genetics—PGD
transfers seem to correlate with the survival of the embryo and the presence oflysed cells do not seem to inferfere in the correct development of the embryos.
Tuesday 20 June 2006
In our study, AHA and lysed cell removal did not alter the pregnancy orimplantation rates of thawed embryos. To assess the real impact of thistechnique higher number of patients is needed in order to have a relievable
Development of an improved preimplantation
statistical power.
genetic diagnosis test for fragile X syndrome andclinical application
P. Burlet1, N. Frydman2, N. Gigarel1, R. Gesny1, B. Aral1, G. Tachdjian2,
The influence of a restrictive selection of the embryos to be
V. Kerbrat3, E. Feyereisen3, J.P. Bonnefont1, R. Frydman3, A. Munnich1,
frozen and the day of thawing on the results of our cryopreservation
1Hoˆpital Necker, Genetique, Paris, France; 2Hoˆpital Beclere, Biologie de la
N. Ortiz-Pin˜ate, G. Pe´rez-Bermejo, C. Herna´ndez, A. Rubio, O. Collado,
reproduction, Clamart, France; 3Hoˆpital Beclere, Gynecologie obstetrique,
I. Bruna, F. Prados
Hospital de Madrid-Monteprı´ncipe, Unidad de Reproduccio´n,
Introduction: Fragile X syndrome (FXS) is the most common monogenic
Boadilla del Monte, Spain
cause of mental retardation. It is caused by mutations in the Fragile X Mental
Introduction: A good Frozen Embryo Transfer (FET) programme is compul-
Retardation-1 gene (FMR1), more than 95% of which involve hyperexpansion
sory prior to an effective reduction in the number of fresh embryos replaced
and hypermethylation of a polymorphic CGG trinucleotide repeat in the
without compromising the pregnancy rate per oocyte retrieval. Our purpose
50 untranslated region of the gene. Both males and females can be affected,
was to determine the effect of a strictly morphological selection of the embryos
leading to a high recurrence risk for this disorder, such that ‘at-risk' couples
to be frozen on the success of our cryopreservation programme. We also
often ask for preimplantation genetic diagnosis (PGD). PGD for FXS remains
compared the FET results obtained in cycles in which the embryos were thawed
difficult. First, technical difficulties have been reported. Two alternative meth-
the day before the transfer with those of embryos thawed a few hours before the
ods are used for single blastomere analysis, i.e. detection of the non expanded
CGG repeat allele, or use of linked polymorphic markers. Direct detection of
Material and methods: The human embryos included in this study were
the normal allele is difficult due to the lack of PCR sensitivity despite specific
frozen and thawed for transfer from 2001 to 2005 in our centre. Embryos
protocols (the expansion is refractory to PCR due to the high GC content of the
were generated from 256 homologous IVF cycles. Two periods of time were
repeat and surrounding sequences) and leads to a risk of allele drop-out (ADO).
compared. Cycles performed before 2004 (Group I) corresponded to cryopre-
In addition, this approach is suitable for only 63% of couples, the heterozyg-
servations in which all supernumerary multicellular embryos were frozen.
osity of the repeat in the normal population. The main drawback of the indirect
During the second period (Group II) embryos were strictly selected on the
method is the risk of recombination and/or ADO which may lead to misdiag-
basis of their morphological appearance prior to cryopreservation. Embryos
nosis, and the non-informativity of the markers which required to establishing a
were thawed one day before the transfer (49 FET) or the same day (107 FET).
diagnostic test for each family. A second limitation to PGD for FXS is that
All embryos in this study were cryopreserved using a slow-freezing protocol
women carrying premutation are at increased risk for premature ovarian failure.
with 1,2-propanediol as cryoprotectant.
A reduced ovarian response to stimulation protocols often results in few
Results: Group I: 57.0% of the 1062 embryos (2PN) obtained in the 150 oocyte
embryos available to test and thus to transfer. Diagnosing each embryo is
retrievals were frozen, while in the 106 retrievals of Group II we froze 41.5% of
therefore a true challenge.
the 884 embryos produced. 356 embryos (Group I) were thawed in 115 cycles
Methods: Recently, Multiple Displacement Amplification (MDA) has been
and 210 embryos were transferred in 101 FET resulting in to 25 ongoing
reported to yield large amount of DNA from single-cells. In order to increase
pregnancies (24.8% per FET). In 73 cycles from the Group II, 211 embryos
reliability and sensitivity of the FXS diagnosis, we developed a combined
were thawed and 127 were transferred in 65 FET, 22 of which resulted in
analysis of polymorphic markers (i.e. DXS548, DXS1215, FRAXAC1,
ongoing pregnancies (33.8% per FET). The implantation rate of Group II was
DXS998) with the study of the non-expanded CGG allele, and the amelogenin
significantly higher (26.8%) than that of Group I (15.7%). The cumulative
sequence for gender determination, using MDA as a first step.
ongoing pregnancy rate per oocyte retrieval was 62.0% for Group I and 59.4%
Results: Single-cell amplification efficiency was first assessed on single lym-
for Group II. For the second part of this study, 49 FET were performed 1 day
phocytes for both normal CGG alleles and the four linked microsatellite
after thawing 143 embryos, of which 86.7% survived. The implantation rate
markers. Amplification rate ranged from 88 to 95% with an ADO rate
was 16.2% and the ongoing pregnancy rate per transfer was 24.5%; rising to
comprised between 5 and 34%. Using this test, 5 PGD cycles were carried
26.8% for the 41 transfers in which the embryos were frozen 2 days after
out for five couples, and 53 blastomeres (26 embryos) were analyzed after
the oocyte retrieval. These results were compared with those of the 107 cycles
preliminary MDA. Amplification rate was increased by this technique from 41
in which 334 embryos thawed the same day of the transfer. In this case, the
to 64%, so that embryos with no results were rarer (19% vs. 45% without
survival rate was 74.6%, the implantation rate was 22.8% and the ongoing
MDA). Reliability of the test was considerably improved by combining both
pregnancy rate per transfer was 32.7%. This pregnancy rate rose to 37.0% for
direct and indirect analysis of the cells. The use of several polymorphic markers
the 81 FET in which the embryos were frozen on day 3 after retrieval.
indeed provides independent diagnostic confirmation. Particularly, the risk
Conclusion: A restrictive strategy for the selection of embryos prior to its
of misdiagnosis due to ADO at one locus was avoided by extrapolating the
freezing (Group II) lead to a higher ongoing pregnancy rate per FET and
information obtained from other loci, and the use of the amelogenin sequence,
significantly higher implantation rate than that obtained with a permissive
a method of sexing the embryos, enabled distinction between ADO and
strategy (Group I). However, the cumulative ongoing pregnancy rate per oocyte
hemizygosity. Furthermore, in cases of fully expanded alleles too large to be
retrieval was slightly lower in Group II. The careful selection of embryos due to
amplified by PCR, this test gives an internal amplification control. It can also
be frozen reduces the number of transfers that a patient has to go though
detect contaminations by showing exogenous alleles in linkage analysis and
without compromising her likelihood of pregnancy per oocyte pick-up. The
give some information on the ploı¨dy of the embryos.
success rate for embryos thawed the day of the FET (mainly day 3 frozen
Conclusions: Embryonic transfers were carried out in all PGD cycles. One
embryos) was higher than that of embryos incubated 1 day prior to transfer
biochemical and one clinical pregnancy resulted, and a healthy child was
Abstracts of the 22nd Annual Meeting of the ESHRE, Prague, Czech Republic, 18–21 June 2006
born. MDA is a powerful tool improving both sensitivity and reliability of FXS
Pregnancy and birth outcomes following preimplantation
PGD. This single diagnosis procedure could be suitable to most patients carry-
genetic diagnosis with blastocyst biopsy compared with
S. McArthur, J.T. Marshall, D. Leigh, M. Traversa, A. Gee,
R.P.S. Jansen, K. de Boer
Comparison of the results of preimplantation genetic
Sydney IVF, Preimplantation Genetic Diagnosis, Sydney, Australia
diagnosis for single gene disorders combined with or withoutHLA typing
Introduction: For preimplantation genetic diagnosis analysis, embryos werecultured to the blastocyst stage of development on day 5 or 6 and blastocyst
G. Karlikaya1, H. Karadayi1, S. Sertyel1, S. Saglam1, B. Umay1,
biopsy—removal of trophoblast cells—used as the standard method of embryo
S. Kahraman1, F. Fiorentino21
biopsy, for both polymerase chain reaction (PCR) (where testing protocols have
Istanbul Memorial Hospital, Reproductive Endocrinology ART & Genetics
been developed for over 80 different diseases) and for fluorescent in situ
unit, Sisli Istanbul, Turkey; 2Laboratorio Genoma, Genetics, Rome, Italy
hybridization (FISH) analysis (for chromosome enumeration and translocation
Introduction: Preimplantation genetic diagnosis (PGD) for single gene dis-
testing). Here we report on pregnancy and birth outcomes following more than
orders (SGD) has become an alternative method to prevent the birth of an
100 births after PGD using blastocyst biopsy.
affected child. Also, many couples are now demanding PGD testing for the
Materials and methods: Embryos were cultured using Sydney IVF stage-
curative therapy of their affected children suffering from haematopoietic dis-
specific medium (Cook IVF). Cells for analysis were obtained via trophecto-
orders by selecting HLA-compatible healthy embryos. However, several
derm biopsy using a Hamilton-Thorne Zilos Tk Laser or FERTILASE
patient specific factors like advanced maternal age and diminished ovarian
near-infra-red 1380 nm diode laser, attached to an Olympus IX-70 inverted
reserve as well as low chance of finding HLA compatible healthy embryos limit
microscope. From December 2000 through December 2005, 671 PGD cycles
the successful outcome. In this study, we evaluate how the rate of HLA
were undertaken utilizing blastocyst biopsy. These cycles were analyzed for
compatible embryos could affect the outcome.
clinical pregnancy data and compared with results achieved using day 3 embryo
Materials and methods: This study was conducted at Istanbul Memorial
biopsy in our PGD program. Birth data and medical follow up were compared
hospital IVF and Genetics department between 2002 and 2006. 35 couples
for IVF embryos transferred at the stage of blastocyst (428 pregnancies) and
with 42 cycles in SGD group and 61 couples with 99 cycles in HLA±SGD
a PGD blastocyst biopsy cohort (87 pregnancies).
group were evaluated. After using standard controlled ovarian hyperstimulation
Results: To date there has been 107 births following blastocyst biopsy, includ-
protocols, ICSI was performed in all cases in order to eliminate the risk of
ing 17 from embryos frozen and thawed after blastocyst biopsy and PGD
contamination. On third day of embryo development, one or two blastomeres
analysis compared with 92 live births following day 3 embryo biopsy. There
were biopsied to analyze the single gene defect and/or HLA compatibility.
are an additional 52 ongoing pregnancies following blastocyst biopsy. Com-
Two-round PCR was performed after which minisequencing method was
paring IVF embryos that reach the stage of blastocyst development, biopsy of
performed to analyze the mutation of interest and STR analysis was performed
trophectoderm for PGD had no apparent detrimental effect on gestational age
to determine the HLA haplotypes. Selected embryos were transferred on fourth
(mean gestational age of 273 days in both cohorts), birth weight (mean birth
or fifth day. Mann-Whitney U test, Student t-test and chi-square test were
weight biopsied 3.38 kg vs. mean birth weight not biopsied 3.33 kg) or infant
applied to compare the results, where appropriate.
development (Table I).
Results: In comparison of SGD and HLA±SGD group, no significant differ-
ence was found between two groups in terms of patient's age (32.5±4.8 vs.
32.5±5.3), number of mature oocytes (11.02±6.7 vs. 12.1±7.1) and number of
biopsied embryos (8.5±4.4 vs. 9.2±5.6) (p<0.05). While 69.7% of the embryos
were found to be suitable for transfer in SGD group after mutation analysis,
Clin. Preg per cycle
only 11.2% of the embryos were suitable for transfer in HLA±SGD group
Clin. Preg per transfer
since HLA compatibility of the embryos was also considered (p<0.001). In
Implantation rate
SGD group, only 2 out of 42 (4.8%) cycles were cancelled due to the lack of
Average no. embryos transferred
healthy embryos, while in HLA±SGD group, 37 out of 99 (37.4%) cycles were
cancelled due to the lack of healthy and HLA compatible embryos (p<0.001).
Although the pregnancy rates in transfer cycles of the two groups were similar
Conclusion: PGD with blastocyst biopsy provides patients with improved
(47.5% vs. 35.5%, p>0.05), the pregnancy rates were higher in SGD group
opportunities for preimplantation genetic diagnosis and pregnancies compared
according to the cycles initiated (45.2% vs. 22.2%, p<0.001). In SGD group,
with day 3 embryo biopsy, without detrimental effect on resulting live births
6 cases ended in early spontaneous miscarriage, 5 singleton pregnancies
and in most cases with normal subsequent follow-up. The benefits include
are ongoing, 10 healthy babies were born (5 singleton, 2 twins, 1 triplet).
(1) utilizing blastocyst culture and elective single embryo transfer (eSET) to
In HLA±SGD group, 8 cases ended in early spontaneous miscarriage and
reduce the risk of multiple pregnancies, (2) a higher implantation rate per
1 ectopic pregnancy was diagnosed, 9 pregnancies are ongoing (8 twins,
embryo transferred, (3) a higher clinical pregnancy rate, (4) a lower miscarriage
1 singleton), 5 healthy babies were born (3 singleton, 1 twin). In two cases,
rate and (5) a higher take home baby rate at normal birth weight.
hematopoietic stem cells (HSC) obtained from umbilical cord blood weresuccessfully transplanted.
Conclusion: Advances in IVF and molecular genetics not only improve the
Multiple displacement amplification as a method of whole
outcome in infertility cases but also provide to have a healthy child for the
genome amplification for preimplantation genetic diagnosis
couples who are carriers of a single gene disorder and give an opportunity to
S. Glentis1, S. SenGupta1, A. Thornhill2, I. Craft2, N. Carter3, L. Rickman3,
have an HLA compatible sibling for the curative therapy of their affected child
as well. However, low probability of finding both HLA compatible and healthy
1University College London, Obstetrics and Gynaecology, London, UK;
embryos for transfer limits the successful clinical outcome. Before starting each
2London Fertility Centre, Human Genetics, London, UK; 3The Welcome
cycle for SGD or HLA±SGD, this information should be discussed in detail
Trust Sanger Institute, Molecular Cytogenetics, Cambridge, UK
with the couples. More importantly, these couples, before planning theirfamily, should be informed by physicians about these options.
Introduction: Whole genome amplification (WGA) is a procedure aimed atachieving robust amplification of the entire genome from as little as 10 ng ofgenomic DNA. WGA has been used in many different areas of biology andmedicine, including genetic tests in samples with a limited amount of DNApresent, such as preimplantation genetic diagnosis (PGD). Different WGAmethods have been developed that can largely be separated into two groups:Polymerase chain reaction (PCR)-based methods, such as degenerate oligonu-cleotide primed PCR (DOP-PCR) and non-PCR-based methods like multiple
Abstracts of the 22nd Annual Meeting of the ESHRE, Prague, Czech Republic, 18–21 June 2006
displacement amplification (MDA). MDA is a relatively new non-PCR tech-
1309 being present in 4 families. A family history was described in ten cases
nique that utilizes phi29 DNA polymerase to amplify the entire genome in a
and two mutations occurred de novo in the affected partner. We initially
few hours. The purpose of this study was to evaluate MDA using three different
developed PGD tests to detect the mutation alone, but we rapidly set up
post-amplification methods (fluorescent PCR, comparative genomic hybridiza-
multiplex PCR combining mutation detection and indirect diagnosis using
tion (CGH) and microarrays) when the starting DNA template came from a
microsatellites D5S2027, D5S1965, D5S346, D5S421. We set up duplex and
single cell in order to achieve accurate results for PGD.
triplex indirect diagnoses to propose a PGD whatever mutation is involved in
Materials and methods: Fresh buccal cells were used as a source of single cell
family cases. Protocols were optimized by modifying standard conditions,
DNA. The single cell lysis was done using standard Proteinase K or alkaline
particularly after a judicious primer choice, in order to add internal controls
lysis protocols, according to the WGA technique used. Control genomic DNA
in single cell PCR. Mutation detection strategies were based on: (i) sizing PCR
was obtained by conventional extraction methods from peripheral blood sam-
fragment for deletions; (ii) restriction length polymorphism, when the mutation
ples taken from the same subjects as the buccal cells. DNA from single cells
introduces a new restriction site; (iii) a new double allele specific PCR
was amplified either by MDA (Repli-g kit, Qiagen, UK) or by DOP (DOP
approach allowing the simultaneous detection of the wild type and mutated
primer, Oswel, UK) using previously validated protocols. The WGA products
allele. PCR conditions were optimized on a minimum of 60 single lympho-
from MDA were subjected to fluorescent PCR analysis to check the accuracy of
blasts from control cell lines or from single lymphocytes from affected patients.
WGA by MDA at 6 microsatellites. MDA products coming from single fibro-
Results: Seven different protocols were set up—two simplex, four duplex
blasts (which had previously been amplified) were also used to evaluate the
combining direct plus indirect diagnosis and one triplex indirect diagnosis.
PCR technique. Genomic DNA samples were used as positive controls. The
Once PCR conditions were optimized, 60 to 114 single cells were tested with an
MDA products were also used for CGH and microarray analysis to check
amplification rate ranging from 95 to 100%. A complete genotype was obtained
whether known aneuploidies could be detected. The results of MDA products
in 94% of cells with a PCR signal (81 to 99%) and a conclusive result in 99%
in CGH and microarrays were also compared with DOP products.
(95 to 100%). ADO occurred in 5% of heterozygous cells (1 to 14%). Ten
Results: (1) Microsatellite analysis: PCR analysis showed that MDA products
cycles were started, 9 reached the stage of biopsy and 7 had an embryo transfer
from single buccal cells and clumps (groups of 5–10 cells) gave accurate allele
procedure. A total of 75 blastomeres from 39 embryos were analyzed and 65
sizing in only 17.6% of loci tested, compared with the accuracy of genomic
(87%) blastomeres from 36 embryos gave a result. A total of 11 untransferred
DNA (98%) and MDA products from genomic DNA and single fibroblasts
embryos were reanalyzed and all confirmed PGD results. A positive HCG was
(100%). Allele dropout (ADO) was present at a rate of 15.7% in MDA products
obtained for 4 cycles: two biochemical and two ongoing pregnancies. A boy
from buccal cells, 7% in single buccal cells or clumps that were directly
was born but no prenatal or postnatal testing was performed to confirm PGD
subjected to PCR. ADO was absent in genomic DNA and MDA products
results. A single clinical pregnancy is still ongoing.
derived from single fibroblasts. (2) CGH: Analysis was possible from all the
Conclusion: We are now able to propose PGD to most couples at risk of
MDA products derived from genomic DNA (6/6) however the results were less
transmitting FAP to their offspring, whether the mutation is inherited or
clear than when compared with DOP-PCR as a method for WGA. When single
occurred de novo. Our current strategy is to use to multiplex PCR combining
buccal cells were used, MDA did not provide any results after CGH (0/4). (3)
either mutation detection and indirect diagnosis using microsatellites or indir-
Microarrays: When 200 ng of genomic DNA was applied to microarrays it
ect diagnosis for inherited cases with a private mutation. With our growing
presented good results. However, the same amount of MDA and DOP products
experience in workup, development of new protocols is less and less time
gave unreliable results with high background noise (0/5).
consuming which enable us to work on specific PGD tests for unavailable de
Conclusions: According to a growing body of literature, MDA seems more
novo mutation.
advantageous compared with other techniques of WGA when using 10 ng ofDNA as a starting template. However, when the starting template is from asingle cell results are not as unequivocally positive. Based on the results in the
Elective single embryo transfer compared with elective
present study, we speculate that the cell type from which the single cell DNA
double embryo transfer in preimplantation genetic diagnosis
template is derived may play a crucial role in the accuracy, reliability and
P. Donoso1, W. Verpoest1, E.G. Papanikolaou1, I. Liebaers2,
reproducibility of the results. Specifically, buccal cells may be suboptimal in
A. Van Steirteghem1, P. Devroey1
comparison to fibroblasts for the purposes of WGA using MDA. Further
1University Hospital Dutch-speaking Brussels Free University,
optimization and laboratory-specific validation may be required before this
Centre for Reproductive Medicine, Brussels, Belgium; 2University Hospital
technique can be applied routinely in clinical PGD.
Dutch-speaking Brussels Free University, Centre for Medical Genetics,Brussels, Belgium
Strategies and outcomes of preimplantation genetic
Introduction: Multiple pregnancies represent the main complication of ART.
diagnosis of familial adenomatous polyposis
Since the 1st of July 2003 the new Belgian legislation imposes on women lessthan 36 years a restriction to single embryo transfer (SET) in their first treat-
S. Viville1, N. Gardes2, C. Moutou21
ment cycle. Although this strategy has shown to be effective without affecting
Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire,
the pregnancy rates, concerns have been raised on its possible adverse effect on
CNRS/ INSERM/ ULP, Illkirch, CU de Strasbourg., France;2
the outcome of preimplantation genetic diagnosis (PGD) for inherited diseases
SIHCUS-CMCO CHU de Strasbourg, Service de Biologie de la
(monogenic disorders and translocations), since a lower pregnancy rate has
Reproduction, Schiltigheim, France
been observed compared with conventional IVF cycles. The aim of this study is
Preimplantation genetic diagnosis (PGD) was initially
to asses the impact of this legislation on the outcome of PGD cycles for
proposed to couples at risk of having a child affected by a severe genetic
monogenic disorders and translocations.
disorder (such as cystic fibrosis, myotonic dystrophy, spinal muscular
Materials and methods: We conducted a retrospective analysis of ICSI
atrophy). Fifteen years after the first PGD birth, new classes of indications
cycles with PGD for monogenic disorders and translocations, performed
emerged. These concern late onset diseases like Huntington's disease and, more
at our institution between January 2002 and December 2004, in women less
recently, inherited cancer. Because of the adulthood onset of hereditary
than 36 years of age in their first treatment. Two groups of patients were
cancer, prenatal diagnosis (PND) raises numerous issues on the acceptability
compared: The elective double embryo transfer group (e-DET group), per-
to terminate an affected pregnancy. This is the reason why PND for these
formed between January 2002 and June 2003 (before the new legislation) vs.
disorders is generally considered as unacceptable by couples as well as geneti-
the elective single embryo transfer group (e-SET group), performed since the
cists and legal or ethical authorities. PGD for hereditary cancer, even if subject
implementation of the new law (July 2003 to December 2004). The main
to controversy, seems to be a more acceptable option. We present our
outcome measures to be compared among the two groups where delivery rate
experience of PGD for familial adenomatous polyposis (FAP) between 2000
and multiple pregnancy rates. A separate analysis was also conducted for both
monogenic disorders and translocations. Statistical analysis was performed
Materials and methods: Twelve couples were referred between 2000 and
using the Fischer's exact test.
2005. Average female age at intake was 29.4 years (vs. 31.4 for all referrals).
Results: Fifty cycles were included in the e-DET group and 55 cycles in the
Nine different mutations were involved, the recurrent 5 bp deletion at codon
e-SET group. The mean±SD for age (30.2±2.7 vs. 29.7±3.1), number of
Abstracts of the 22nd Annual Meeting of the ESHRE, Prague, Czech Republic, 18–21 June 2006
cumulus-oocyte complexes (16.84±10.1 vs. 16.15±9.1) and number of zygotes
endometriotic lesions were removed. MSCTe findings were compared with
(13±7.6 vs. 12.5±7.2) were comparable between both groups. The two main
surgical and histological results.
indications in the monogenic disorder group were myotonic dystrophy (12%
Results: The endometriotic nature of all the bowel lesions removed at surgery
e-DET group vs. 12.8% e-SET group) and cystic fibrosis (14% e-DET group vs.
was confirmed at histology. Laparoscopy confirmed the absence of bowel
3.6% e-SET group). Translocations represented 20% (n¼10) of the e-DET
endometriosis 22 out of 23 women with entirely normal colon at MSCTe. In
group and 30.9% (n¼17) of the e-SET group. There was no statistically
one patient with complete obliteration of the pouch of Douglas caused by a
significant difference in the delivery rates between the e-DET and the e-SET
recto-vaginal nodule, MSCTe did not identify rectal involvement infiltrating
(32% vs. 25.5% p¼0.52). Multiple pregnancies were eliminated when e-SET
the muscular layer (1.4 cm). Among subjects with a diagnosis of bowel endo-
was performed (0% vs. 31.3% p¼0.45). When the cycles for translocations
metriosis at MSCTe (n¼75), additional four nodules were identified at surgery;
were excluded—only monogenic diseases analyzed—the delivery rates became
they were all located on the rectum and reached the serosa in two cases and the
almost identical (30% e-DET vs. 28.9% e-SET, p¼1.0). On the other hand,
muscolaris in other two cases. Therefore MSCTe identified 94.8% (110/116) of
when cycles for translocations were exclusively analysed a higher delivery rate
the bowel endometriotic nodules observed at surgery. MSCTe identified all
was achieved with e-DET than e-SET (40% vs. 17.6% p¼0.36), though it did
nodules located on sigmoid colon, cecum and ileum; 47 out of 52 (90.4%) rectal
not reach statistical significance.
nodules were diagnosed. MSCTe correctly determined the degree of infiltration
Conclusions: The implementation of a SET policy on young women
of the bowel wall in all the identified serosal bowel nodules. In 6 nodules
undergoing PGD for monogenic disorders enables a significant reduction of
reaching the submucosa, the depth of infiltration was underestimated by
multiple pregnancies without affecting the delivery rate. However, when PGD
MSCTe. A statistically significant positive correlation was observed between
is performed to select out translocations, the efficiency of single embryo
the diameter of the endometriotic nodules estimated at MSCTe and that meas-
transfer might be debatable and needs further evaluation in a higher series.
ured by the pathologist (Pearson's Correlation Coefficient, r¼0.974; p<0.001).
MSCTe had a sensibility of 98.7%, a specificity of 100%, a positive predictivevalue of 100%, and a negative predictive value of 95.7% in identifying womenwith bowel endometriosis.
Conclusions: MSCTe is effective in determining the presence, size and depth
FREE COMMUNICATION
of bowel endometriotic lesions.
Session 30 – Endometrium and endometriosis
Peritoneal macrophage depletion by liposomal
Tuesday 20 June 2006
bisphosphonate in a rat model of endometriosis affects both cytokinesand macrophage infiltration
M. Schachter1, H.D. Danenberg2, S. Friedler1, A. Raziel1, S. Mendelovic3,
Multislice spiral computerized tomography after colon
R. Ron-El1, D. Strassburger1, O. Bern1, G. Golomb4
distension with water enteroclysis in the diagnosis of bowel
1IVF and Infertility Unit, Assaf Harofeh Medical Center Tel Aviv University,
Zerifin, Israel; 2Hebrew University Hadassah Hospital, Cardiovascular
S. Ferrero1, E. Biscaldi2, E. Fulcheri3, N. Ragni1, V. Remorgida1,
Research Unit, Jerusalem, Israel; 3Dept. Pathology and Cytology, Assaf
Harofeh Medical Center Tel Aviv University, Zerifin, Israel; 4Hebrew
1San Martino Hospital and University of Genoa, Department of Obstetrics
University Jerusalem, Faculty of Pharmacology, Jerusalem, Israel
and Gynaecology, Genoa, Italy; 2San Martino Hospital and University of
Background: Activation of macrophages and peritoneal inflammation is cent-
Genoa, Department of Radiology, Genoa, Italy; 3San Martino Hospital and
ral to the initiation, implantation and perpetuation of endometriosis. Depletion
University of Genoa, Di.C.M.I. Unit of Anatomy and Histopathology, Genoa,
of macrophages and monocytes can be achieved by liposome mediated intra-
cellular delivery of biphosphonates (LBP) such as alendronate, which inacti-
Introduction: Although several radiological techniques have been proposed
vate phagocytotic cells. We previously examined the effect of LBP on
for the diagnosis of bowel endometriosis, data are inconclusive and no golden
development of endometriosis in a rat model and found a significant reduction
standard is currently available. This prospective study investigates the efficacy
of implant volume and adhesion formation after treatment with LBP. Our
of multislice spiral computerized tomography after colon distension with a
objective in this study was to examine the effect of LBP on peritoneal cytokines
water enteroclysis (MSCTe) in the diagnosis of bowel endometriosis.
and on endometriosis implant histology.
Materials and methods: Ninety-eight women with symptoms suggestive of
Material and methods: Twenty-four adult female Sabra strain rats were
colorectal endometriosis underwent MSCTe. To reduce bowel peristalsis and
subjected to an endometriosis model, by resection of one uterine horn, and
colonic spasm, 20 mg of joscine N-bromuro (Buscopan; Boehringer Ingelheim,
suture of 6 open uterine squares to the mesentery in each rat. Three additional
Florence, Italy) were administered intravenously immediately before the water
rats were subjected to a sham operation which included resection of the uterus
enema. Colonic distension was achieved by introducing 2000–2300 ml of water
and nylon suturing to the mesentery with no uterine squares. The 24 rats were
(37C). All patients received an intravenous injection of Iopamidol (Bracco,
then divided randomly to two treatment and one control group, and treated with
Milan, Italy) with an iodine concentration of 370 mg/ml. The rate of intra-
4 weekly intraperitoneal injections of alendronate in liposomes. Two treatment
venous injection of contrast material was set at 2.5 ml/s with an automatic
doses were employed, 1 mg/kg per injection (low dose) and 10 mg/kg per
power injector for all examinations. Bolus-tracking software designed to mon-
injection (high dose). Four weeks after the initial surgery, the rats were
itor organ contrast enhancement (SmartPrep; GE Medical Systems, Wisconsin,
sacrificed. Histopathological sections and avidin-biotin immunohistochemistry
USA) was used to maximize the quality of MSCTe image. All patients were
with staining for macrophages in implants were carried out with mouse anti-rat
scanned on a 16-row MSCT scanner (LightSpeed, GE Medical Systems,
macrophage antigen ED1. The density of macrophage infiltration was scored
Wisconsin, USA). The scan parameters were 16·0.625 mm collimation;
by counting stained macrophages per HPF, expressed as the average number of
rotation time 0.7 s; tube voltage 120 kV; effective mAs 370. The axial plane
stained macrophages counted per 800 background cells. Cell-free peritoneal
and multiplanar reconstructions were evaluated; images were independently
fluid was collected at the time of sacrifice, and subsequently analysed for
reviewed at a PACS (picture archiving and communications system) work-
concentrations of tumor necrosis factor alpha (TNFa) and monocyte chemo-
station by two observers; disagreement between observers was resolved by
tactic protein 1 (MCP1) using commercially available ELISA kits.
consensus in a joined session. Locations, number of nodule/s, size of the
Results: Immunohistochemistry demonstrated a significantly reduced pattern
nodule/s, and depth of bowel wall infiltration were determined. Within
of macrophage infiltration in the low-dose treatment as opposed to the control
20 days after the radiological examination, independently from the findings
group, (staining score 107±144 vs. 300±124, p<0.02) although no significant
of MSCTe, all women underwent laparoscopy. At surgery, after adequate ade-
differences were found between the high-dose and control group for infiltration
siolysis, terminal ileum, cecum, sigmoid colon and rectum were systematically
score (320±174 vs. 300±124). Peritoneal TNFa was significantly reduced in
examined to verify the presence of endometriotic lesions; all visible bowel
the high-dose group as opposed to low-dose and control groups, 0.52±1.03,8.27±12.04 and 7.97±8.6 pg/ml, for high dose, low dose and control groups,
Source: http://www.laboratoriogenoma.it/writable/relazioni/ESHRE%202006%20kar.pdf
◗ Autoanticorps etautoantigènes de la peau Autoanticorps et autoantigènes de la peau ◗ Les dermatoses bulleuses auto-immunespage 5 René-Louis HUMBEL, Président du GEAI ◗ Méthodes de détection Laboratoire de Biochimie et Immunopathologie - Centre Hospitalier de Luxembourg des autoanticorps associés aux dermatosesbulleuses auto-immunes
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