◗ 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
Humrep-oral 1.108Abstracts 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,
British Society of Animal Science Does Big mean bad – The Science behind large scale production The Roslin Institute, Easter Bush Edinburgh 23 – 24 May 2013 Antimicrobial Resistance – Managing the risk Peter Harlech Jones - President BVA Is History Repeating Itself? Are we entering a post-antibiotic era?