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Abstracts of the 25th Annual Meeting of ESHRE, Amsterdam, the Netherlands, 28 June - 1 July, 2009 for time and ease of use, ambiguity and comprehension. Between October 1st use of telomeric probes in combination with centromeric probes (reciprocal and November 30th 2008, the survey was mailed twice to 26 Canadian fertility translocations), or alpha-satellite/locus-specific enumerator probes (Robertso- clinics and to 8 individual Canadian MDs, satelliting with these clinics. A modified survey was distributed online, to 392 American SART-member Here we present the development of a polymerase chain reaction (PCR)- clinics. Results were tabulated and summarized using SPSS.
based PGD approach for detection of chromosomal imbalances on embryos Results: 28 Canadian and 125 American surveys were completed (78% and derived from both reciprocal and Robertsonian translocation carriers. The pro- 32% response rates). For Canada, the largest proportion of surveys (50%) cedure involves testing of single blastomeres by fluorescent multiplex PCR was from Ontario. Respondents reported offering a total of 6927 stimulated analysis of polymorphic short tandem repeat (STR) markers located along IVF cycles per year, equivalent to 77% of the total cycles provided in the chromosomes involved by translocation.
Canada for 2007 (n ¼ 9019). The most common out-of-country treatment Material & methods: STR markers were selected to be located at either side of sought by Canadians was IVF with anonymous donor-eggs: 363 of 452 patients each breakpoint (reciprocal translocations) or at any point of the chromosomes treated (80%). Canadian respondents provided satellite monitoring for 431 involved (Robertsonian translocation). STR markers were also included to women undergoing out-of-country IVF. For patients entering Canada in determine the copy number of chromosomes 13, 14, 15, 16, 18, 21, 22, X, Y order to receive fertility treatment (n ¼ 146), the largest demand was for IVF in patients of advanced maternal age. Informativity testing of STR markers (73% of patients treated). 52% of respondents recommended specific destina- was performed for both partners of each couple. Only fully informative tion countries to their patients, but not specific providers. Confidence in markers presenting alleles not shared by the partners were selected. In order safety, effectiveness and ethicality were considered very important by 71 – to avoid misdiagnosis due to possible allele drop-out (ADO) occurrences, at 80% of respondents. Respondents felt that patients were most concerned least three STR for each chromosome were included in the protocol.
with effectiveness (88%) and safety (80%). 88% of Canadian respondents Embryos were diagnosed as “normal-balanced” if PCR results indicated two always provide the information requested by the destination clinic. Canadian signals (peaks) for each chromosome tested. Embryos were diagnosed as clinics were most interested in receiving information about complications of “unbalanced” if the PCR results showed a deviation from the “normal- treatment, number of embryos transferred and frozen.
balanced” signal pattern, such as trisomies (three peaks), monosomies (one For the United States survey, the largest proportion of responses came from peak) and nullisomies (no PCR signals).
the Southern US (31%). Respondents reported offering 35,387 stimulated IVF Results: Twelve PGD cycles were carried out for 12 couples carrying six cycles per year, equivalent to 41% of the total 85,326 stimulated cycles different reciprocal translocations and two Robertsonian translocations. The reported to SART for 2006. Responding US clinics reported treating 927 mean maternal age was 36.4+4.6 years. A total of 204 oocytes were collected, out-of-country patients, 51% of them with standard IVF. 36% of incoming 158 (77.5%) were MII, 126 (79.7%) fertilized and 110 embryos were biopsied patients were from Latin America and 23% from Europe. The largest pro- on day 3. PCR was successful in 102/110 (92.7%) blastomeres, accounting a portion of the 220 patients leaving the US in order to receive IVF or donor positive amplification on a total of 1048/1128 (92.9%) loci. Overall, 102 egg IVF, traveled to India / Asia: 41% and 52% respectively. Respondents (92.7%) embryos were successfully diagnosed, 52 of which resulted normal/ reported that confidence in treatment effectiveness and safety, as well as infor- balanced, 44 were unbalanced and 6 resulted to be haploid. PGS was included mation from other patients, were very important factors in patients’ decisions to in the PGD protocol of five couples, involving testing of 45 embryos, 40 come to their clinics. The majority of respondents felt that recent laboratory (88.9%) of which were successful diagnosed and 24 (60.0%) showed aneuploi- results and track sheets from previous cycles should always be sent with out- dies. Embryos suitable for transfer where identified in 10 cycles. Following of-country patients. Good concurrence was seen between Canadian and Amer- transfer of 23 embryos (mean 1.9+1.1), 7 women had a clinical pregnancy ican clinics’ ratings of key data that should be provided along with returning confirmed with fetal sacs and heart beat (70.0% pregnancy rate per embryo transfer). A total of 13 embryos implanted (56.5% implantation rate per Conclusions: The number of Canadians traveling to the United States for ART embryo transferred), for 10 of which heart beat was also detected. Only 2 is equivalent to approximately 5% of the total cycles performed in Canada.
couples accepted to undergo to prenatal diagnosis, performed by chorion Eighty percent of these women seek anonymous donor egg IVF. Less than villus sampling (CVS) or amniocentesis, which confirmed the PGD results.
1% of US patients leave the country for fertility care and for them, the most popular destination is India / Asia, for standard or donor egg IVF. In the Conclusions: The above results demonstrate the feasibility and reliability of USA, approximately 3% of the total ART volume is made up of women our PCR-based PGD protocol for detection of chromosomal imbalances. The coming into the country for care. US clinicians stress the need for recent lab present technique has the potential to overcome to several inherent limitation data and previous stimulation track sheets. All parties surveyed rated effective- of the FISH procedure, such as suboptimal fixation, overlapping signals, split ness and safety of care as paramount in patient choice of destination.
signals, lack of signals, cross-hybridization, polymorphisms, limited avail- This research was supported by Assisted Human Reproduction Canada ability of the probes, combination of colours, decreasing of the accuracywith re-probing. This approach has the advantage to be rapid, low expensive,amenable to automation, involving an easy procedure and data interpretation.
Unlike FISH, with the presented protocol is also possible to distinguish the par-ental origin of chromosomes, allowing detection of uniparental disomies and the achievement of a DNA fingerprint for each embryo, useful for identification Session 33: Preimplantation genetic diagnosis of embryos that have implanted. Finally, because cell fixation is not necessary,the PCR-based protocol represents an easier procedure for management of transport PGD. Considering the encouraging preliminary clinical outcomeobtained, this approach has the potential to represent a valuable alternative toFISH-based PGD.
PCR-based detection of chromosomal unbalances on embryos: a possible future (r)evolution of PGD for chromosomal IVM with non-elevated E2 levels and PGD for BRCA1 mutation in a breast cancer patient. First report on mutation-free twin’spregnancy F. Fiorentino1, G. Kokkali2, A. Biricik1, B. Ismiailoglu1, R. De Palma1, D.
Stavrou2, A. Nuccitelli1, L. Arizzi1, M. Baldi1, K. Pantos2 D. Meirow1, B. Feldman2, A. Hourvitz1, J. Levron1, M. Brengauz1, K. Dotan2, 1GENOMA, Molecular Genetics Laboratory, Rome, Italy, 2Genesis Athens Hospital, Centre for Human Reproduction, Athens, Greece 1Sheba Medical Center, IVF unit, Tel Hashomer Ramat Gan, Israel, 2ShebaMedical Center, Danek Gertner Institute of Human Genetics, Tel Hashomer Introduction: Preimplantation genetic diagnosis (PGD) has been offered to carriers of balanced translocations as an alternative to prenatal diagnosis. Flu-orescence in-situ hybridisation (FISH) is the method of choice for detecting Introduction: Young breast cancer patients frequently wish to have biologic chromosome rearrangements. The FISH strategy involves the simultaneous children but reproductive decisions are difficult especially when fertility Abstracts of the 25th Annual Meeting of ESHRE, Amsterdam, the Netherlands, 28 June – 1 July, 2009 treatments are indicated. When hereditary breast and ovarian cancer gene performed soon after pick-up, which means between 35 to 37 hours post hCG (BRCA) mutations are present there is 50% chance of transmission of the administration (Gianaroli et al., 2007). In this study we investigated the usefulness mutation to the offspring which causes anxiety and concern. Ovarian stimu- of polarized microscopy to evaluate oocyte maturation stage (by assessing lation using fertility drugs frequently results in high E2 levels that is potentially meiotic spindle configuration) prior to IPB biopsy.
risky in hormone sensitive breast cancer. However, IVM introduced for women Materials & Methods: All patients undergoing an ICSI-PGD cycle on IPB for with polycystic ovarian syndrome (PCOS) can avoid this risk. This is the first single-gene disorder in our centre between September and December 2008 report of a pregnancy after combined in-vitro maturation (IVM) and pre- were enrolled in this study. Oocyte retrievals were performed 35 hours post- implantation genetic diagnosis (PGD) for BRCA1 in breast cancer patient.
hCG administration and oocyte denudation one hour later. Meiotic spindles Materials and methods: A 26 years old female was diagnosed with breast were assessed immediately after denudation with Oosight system (CRi, cancer estrogen receptor positive. The patient and her mother were positive Woburn, USA) followed by IPB biopsy. ICSI was finally performed on selected for BRCA1 gene mutation. IVF cycle initiated prior to chemotherapy in oocytes (maximum of 3 per patients according to the Law), between 6 – 8 hours purpose of preserving future fertility was canceled due to ovarian hyperstimu- post-biopsy, when diagnosis were available.
lation syndrome. Following chemo/radiotherapy and adjuvant Tamoxifen Results: A total of 60 cumulus-oocyte complexes have been obtained, of which therapy the patient was 3 years disease free, married and had irregular men- 39 displayed a IPB and were thus suitable for biopsy. When observed at polarized struations. She requested to stop therapy and attempt to conceive which was light microscopy 15 (38.4%) were at Metaphase II (MII) stage with a clear meiotic spindle in the oocyte cytoplasm, 13 (33.3%) were at Telophase I (TI) Fertility workout indicated good ovarian reserve FSH-3.9, AMH-5.15, AFC stage with a meiotic spindle in between the IPB and oocyte cytoplasm and in .40, however, due to PCOS she was infertile. Stimulation protocol resulted in 11 oocytes (28.2%) no spindle was detectable. Polar body biopsy was successful ovarian hyperstimulation and E2 levels were above 10,100pmole/l, IVF yielded in all MII stage oocytes (15/15) and all oocytes without meiotic spindle detectable 8 embryos but the patient did not conceive. To avoid high E2 levels in hormone (11/11), while in the TI group 3/13 oocytes degenerated (23.1%) due to cyto- sensitive breast cancer patient who suffered from PCOS, IVM technique was plasmic continuity and rupture during biopsy (P ¼ 0.04). Successful DNA ampli- offered. As the patient was BRCA1 gene mutation carrier and since infertility fication was obtained in 12/15 MII oocytes, 4/10 TI oocytes and 4/11 oocytes treatments were indicated PGD for BRCA1 was offered. Institutional review without MS detectable (P ¼ 0.04). The fertilization rate with the selected MII oocytes was 80.0%. In one case two TI oocytes were however used for ICSI Single cell molecular diagnosis protocol used non-direct informative familial (due to the lack of availability of healthy MII) and both developed to 1PN oocytes.
marker analysis. Genomic DNA samples of the patient and her parents were Conclusions: When IPB biopsy has to be performed, the use of polarized light screened for informativity in 8 different polymorphic markers located in or microscopy is useful to asses oocyte nuclear maturity which influences the adjacent to the BRCA1 gene on 17q21. Two markers, D17S951 and outcome of the procedure in terms of oocyte survival and successful DNA D17S1185, located 700 Kb upstream and downstream, respectively, from the amplification. In particular, TI oocytes may be damaged during biopsy result- gene sequence were found to be highly informative. The patient inherited a ing in oocyte degeneration. It can also be supposed that, in case of survival, the 93 bp allele linked to the mutated BRCA1 allele from her affected mother.
chromosomal arrangement of TI oocytes may be disrupted during aspiration. It Results: On 2nd day of spontaneous cycle E2 was 122 pmole/l and multiple fol- is suggested that timing of IPB biopsy should be adapted according to oocyte licles (5 – 6 mm) were present. On the 8th day E2 was 210 pmole/l and maximal nuclear maturation stage assessed by polarized light microscopy.
follicle diameter was 8 mm. FSH 75 IU/d was added for 4 days, on the 11th dayE2 was 600 pmole/l, leading follicle was 16 mm and 20 antral follicles werevisualized. Following HCG administration 14 immature oocytes were col- Genome-wide karyomapping for pgd of cystic fibrosis lected. Two metaphase I (MI) stage and 12 germinal vesicle stage (GV) were combines accurate linkage based testing with 24 chromosome cultured in IVM medium supplemented with FSH and LH. Within 2 days 5 eggs matured in-vitro (MII) and ICSI was performed only on these oocytes.
G. Harton1, B. Mariani1, A.R. Thornhill2, D.K. Griffin3, N. Affara4, Two good quality embryos underwent biopsy on the 5th day post aspiration and blastomers were sent for single cell evaluation. The 93 bp maternal 1Genetics and IVF Institute, Fairfax, Virginia, U.S.A., 2London Bridge Fertility allele linked to the mutated BRCA1 gene was not inherited in one blastomere Gynaecology and Genetics Centre, One St Thomas Street, London, United suggesting an unaffected embryo which was transferred on the same day. The Kingdom, 3Department of Biosciences, University of Kent, Canterbury, United patient conceived carrying a monochorionic biamniotic twin pregnancy.
Kingdom, 4Department of Pathology, University of Cambridge, Cambridge, Second trimester amniocentesis showed two 46 XY normal embryos that were negative for the BRCA1 gene mutation. At present the patient is at her33w of pregnancy.
Introduction: Karyomapping uses genome wide analysis of single nucleotide Discussion: This breast cancer patient suffering from infertility due to PCO polymorphism (SNP) genotyping of parents and offspring to ascertain the hap- completed successful assisted reproduction cycle using IVM technique lotypes of each parental chromosome. When applied to one or more cells biop- without elevation of E2 levels. Eggs that matured in-vitro were suitable for sied from a preimplantation embryo Karyomapping thereby identifies the single cell PGD analysis for BRCA gene mutation. The pregnancy following parental and grandparental origin of each chromosome or chromosome the IVM-PGD procedures solved patient’s infertility with no added risk and segment presen. For preimplantation genetic diagnosis (PGD), Karyomapping avoided passing mutated BRCA gene to patient’s progeny in an ethically has the advantage that there is no requirement to develop a patient or disease specific test for any single gene defect within the regions of the genomecovered by the SNPs, in addition, chromosomal aneuploidies, includingmeiotic trisomies and uniform monosomies, are identified simultaneously.
Use of polarized light microscopy to assess meiotic spindle Here we demonstrate that Karyomapping not only provides highly accurate configuration prior to first polar body biopsy for preimplantation genetic linkage based diagnosis for cystic fibrosis (CF) but also enabled two autosomal aneuploidies to be identified in embryos at the blastocyst stage.
Materials and Methods: PGD for CF was performed in a couple where both L. Albricci1, S. Romano1, R. Maggiulli1, A. Biricik2, L. Arizzi2, F. Fiorentino2, parents carry the common DF508 deletion by mechanical zona dissection and cleavage stage biopsy of one or two cells in embryos with 6 or more cells.
Clinica Valle Giulia, GENERA Center for Reproductive Medicine, Roma, Italy Genetic analysis of the single cells by PCR and gel electrophoresis to identified Genoma, Molecular Genetics Laboratory, Roma, Italy four homozygous unaffected, nine carriers and 3 homozygous affected Introduction: Preimplantation genetic diagnosis (PGD) for chromosomal altera- embryos. Two hatching blastocysts were transferred on day 5 resulting in a tion and single-gene disorders (of maternal origin) using the first polar body (IPB) healthy liveborn girl. Five other embryos cryopreserved at the time of is the only approach possible in Italy where by Law inseminated/fertilized transfer were subsequently donated for research with the patients’ informed oocytes and embryos cannot be manipulated. Only after have obtained the consent. These embryos were thawed and three embryos at the blastocyst genetic diagnosis of each single oocyte (which takes between 4 to 8 hours), inse- stage biopsied and 3 – 10 cells removed from the trophectoderm. Lysis and mination can be performed. Thus, to avoid oocyte ageing the biopsy is normally whole genome amplification (WGA) was then carried out essentially according Abstracts of the 25th Annual Meeting of ESHRE, Amsterdam, the Netherlands, 28 June - 1 July, 2009 to the manufacturers instructions (Repli-g, GE Healthcare) and the parental 1Universitat Auto`noma de Barcelona, Biologia Cel.lular i Gene`tica Me`dica, genomic DNA and WGA products processed for genome-wide genotyping of Bellaterra, Spain, 2Universidade de Sa˜o Paulo, Departamento de Genetica, approximately 350K SNPs (Human CNV370 Quad v3; Illumina). Finally, Kar- Ribeira˜o Preto, Brazil, 3Fundacio´ Puig-Vert, Unitat de Reproduccio´, yomapping of the SNP genotype data was accomplished using dedicated Barcelona, Spain, 4Hospital Quiro´n, Unitad de Reproduccio´n, Alarco´n, Spain Background: In couples affected by a monogenic disease, Double-Factor Pre- Results: Karyomapping confirmed the CF status of all five embryos (2 homozy- implantation Genetic Diagnosis (DF-PGD) allows doubly selection of embryos, gous unaffected, 2 carriers and 1 homozygous affected) with multiple flanking i.e. free of the monogenic disease and being potentially euploid. The aim of DF- and intragenic SNP markers across the region of 7q which includes the CFTR PGD is to avoid the transfer of embryos diagnosed as being genetically normal locus. Furthermore, unlike the original analysis which could not distinguish for the couple’s causative mutation but without any information of chromoso- the parental origin of the deletion, the parental allele present in each of the car- mal alterations, which may lead to the transfer of aneuploid embryos, indeed riers was identified. In one embryo, a recombination event is located immediately with little chance of viability. Hence, the main objective of DF-PGD is to distal to the CFTR raising the possibility that conventional STR markers flanking increase the implantation rate in couples affected by monogenic diseases.
the gene may only have detected the recombination event and failed to give a The intention of this manuscript is to evaluate the feasibility and possible posi- definitive diagnosis. Karyomap analysis also revealed two autosomal aneuploi- tive effect on implantation of the DF-PGD after two years of clinical dies in two blastocyst stage embryos: one maternal monosomy 6 and one maternal trisomy 9, which was identified as a meiosis II error.
Methods: Eight couples affected by the recessive disease Cystic Fibrosis (CF), Conclusions: This preliminary data demonstrates that Karyomapping follow- one by Angelman’s Syndrome (AS) and another by von Hippel-Lindau disease ing WGA and SNP analysis from small numbers of embryo or trophectoderm (VHL), both dominant illnesses, participated in our study. Six of the patients cells biopsied from embryos at the blastocyst stage can provide highly accurate had no indication of PGS due to their maternal age being less than 35 (mean linkage based analysis for CF combined with detection of chromosomal aneu- 32.3 years old), but the other four patients suffered AMA (mean 39.25 years ploidy. With improved embryo survival following vitrification, therefore, it old). After oocyte fecundation by ICSI, the first polar bodies (1PB) were should now be possible to vitrify embryos at the blastocyst stage after biopsy biopsed and were then analysed using Comparative Genomic Hybridization and analyse the embryos for virtually any genetic disease and screen for aneu- (1PB-CGH) in order to screen for aneuploidies in the whole-chromosome ploidy of all 24 chromosomes simultaneously. This approach should make PGD complement. On day 3, a blastomere was biopsed from the evolutive by Karyomapping less expensive than conventional monogenic disease PGD embryos that was immediately amplified using Multiple Displacement because fewer embryos will be biopsied, more embryos will be euploid follow- Amplification (MDA), following by couple-specific, direct and indirect, ing growth to the blastocyst stage, and there is no need to custom develop tests mutation detection PCR protocol. On day 4, both monogenic detection and for each disease or couple interested in PGD.
1PB-CGH results were obtained allowing for a double selection of embryos free of the causative mutation and having originated from a potentiallyeuploid oocyte.
Contribution of cryopreservation of blastocysts biopsied Results: One-hundred-fifteen 1PBs were obtained, achieving a satisfactory at the cleavage-stage to the success of a pre-implantation genetic diagnosis CGH in ninety-nine of them (86.2%). During the clinical case, only 1PB- CGHs from developing embryos were analysed (77), 45.45% of them being Y. Khalaf1, T. El-Toukhy1, A. Kamal1, E. Wharf1, J. Grace1, V. Bolton1, aneuploid (35/77). Twenty of the aneuploid 1PBs (57.2%) were obtained from patients with an age of less than 35 years old, whereas the other 15 1Guy’s and St Thomas’ Hospital, Women’s Health, London, United Kingdom 1PBs (42.8%) were from AMA patients.
Eighty-one blastomeres were biopsied and successfully amplified with Introduction: An elective single embryo transfer (SET) policy has not been MDA, 45 being non-affected by the respective disease (i.e. wild-type homozy- applied to pre-implantation genetic diagnosis (PGD) for inherited genetic dis- gotes or heterozygote carriers for CF and wild-type homozygotes for VHL and orders because of concerns regarding post-thaw survival of biopsied embryos.
AS). In 35 of the genetically healthy blastomeres (66.7%) the 1PBs-CGH was Our objective was to evaluate the survival and pregnancy potential of embryos also available, 15 of them (42.9%) were potentially euploid, therefore they biopsied for PGD at the cleavage-stage and cryopreserved at the blastocyst classified as DF-PGD transferable embryos. The other 20 (57.1%) originated stage and its contribution to the overall success of an elective SET policy in from an aneuploid oocyte. All of the other 10 non-affected embryos with no informative CGH were tagged as being PGD transferable.
Materials and Methods: From January 2006, all couples who had two or more Nine out of the fifteen DF-PGD transferable embryos and eight out of the ten transferable PGD blastocysts biopsied on day three of culture were offered PGD transferable embryos were selected morphologically for transfer to 10 single blastocyst transfer and cryopreservation of surplus blastocyst(s) using patients. Three out of the nine (33.3%) DF-PGD embryos and one out of the a slow freezing technique. We compared the outcome of 32 cryo-thawed eight (12.5%) PGD embryos did implant and developed, resulting in the PGD cycles with that of 191 cryo-thawed conventional IVF/ICSI cycles per- birth of four healthy babies. Even though the implantation rate of the DF- formed between January 2006 and July 2008. We also compared the PGD group is almost three times higher than the PGD group, these differences outcome of all fresh PGD cycles performed before and after January 2006.
Results: The blastocyst survival rate was similar between the PGD and IVF/ Conclusion: Despite the reduced number of cycles performed using DF-PGD, ICSI groups (87% vs 88%, P ¼ 0.94). There was no significant difference in it seems to be a good tool to increase the implantation rate in couples affected the implantation and clinical pregnancy rates between the two groups (35% vs 29%, p ¼ 0.45 and 34% vs 36%, P ¼ 0.77, respectively). During the same Acknowledgements This research study has been funded by the Ministerio de period, the multiple pregnancy rate in the fresh PGD programme dropped Sanidad y Consumo Fondo de Investigacio´n Sanitaria Instituto de Salud Carlos from 36% to 10% (OR ¼ 0.20, 95% CI 0.08 – 0.48, P , 0.001) with no III (FIS-ISCIII; PI 051395), the Grup de Suport a la Recerca de la Generalitat de Catalunya 2005SGR00495 and by the Ca`tedra de Recerca EUGIN. The first Conclusion: The survival and implantation potential of biopsied PGD embryos author has a predoctoral grant from the Generalitat de Catalunya cryopreserved at the blastocyst stage is comparable to that of non-biopsied IVF/ ICSI cryopreserved blastocysts. Elective SBT and cryopreservation of surplusblastocyst(s) for later use should extend to include PGD for inherited geneticdisorders.
Two-year experience of double-factor preimplantation genetic diagnosis: first preliminary results A. Obradors1, M. Rius1, G. Daina1, J.F. Cuzzi2, O. Martı´nez-Pasarell3,E. Ferna´ndez4, M. Oliver-Bonet1, J. Benet1, J. Navarro1


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