ΓΡΑΜΜΑΤΕΙΑ ΣΥΜΠΟΣΙΟΥ / SYMPOSIUM SECRETARIAT

CMYK ΓΡΑΜΜΑΤΕΙΑ ΣΥΜΠΟΣΙΟΥ / SYMPOSIUM SECRETARIAT ERA Ltd - 17, Asklipiou Str., 10680 Athens - Greece Tel.: +30 210 3634944, Fax: +30 210 3631690 E-mail: info@era.gr, Web Site: www.era.gr 2

Π ε ρ ι ε χ ό µ ε ν α / C o n t e n t s Σελίδα / page Πρόσκληση / Invitation...4 Οµιλητές - Συντονιστές / Faculty...5 Επιστηµονικό Πρόγραµµα / Scientific Programme...6 Γενικές Πληροφορίες / General Information...7 Περιλήψεις Οµιλιών / Lectures Abstracts...9-15 Wells D...10-11 Sakkas D...12 Rienzi L...13 De los Santos M.J...14 Davies S...15 3

CMYK Π ρ ό σ κ λ η σ η / I n v i t a t i o n Αγαπητοί συνάδελφοι, Έχει γίνει πλέον θεσµός στο χώρο της Υποβοηθούµενης Αναπαραγωγής το επίσηµο συµπόσιο που διοργανώνει η Εµβρυογένεση, µε θεµατολογία τις πλέον επίκαιρες εξελίξεις. Έτσι λοιπόν και φέτος σας καλωσορίζουµε, το Σάββατο 17 Σεπτεµβρίου, στο 6 o Επιστηµονικό Συµπόσιο στο ξενοδοχείο Hilton, που αφορά και αυτό όλες τις τελευταίες εξελίξεις του χώρου µας. Τόσο η θεµατολογία του συµποσίου όσο και και το αναγνωρισµένο κύρος των προσκεκληµένων οµιλητών, ευελπιστούµε ότι θα αποτελέσουν πόλο έλξης προς όλους εσάς που δραστηριοποιείστε ή έχετε γενικότερο ενδιαφέρον για την Υποβοηθούµενη Αναπαραγωγή. Σας αναµένουµε λοιπόν µε ιδιαίτερη χαρά για να µοιραστούµε µαζί µέσα από την ενεργό συµµετοχή σας, τις καταιγιστικές προόδους του χώρου µας. Με εκτίµηση και συναδελφικούς χαιρετισµούς, ρ Μηνάς Μαστροµηνάς Dr Stephen Davies 4

O µ ι λ η τ έ ς - Σ υ ν τ ο ν ι σ τ έ ς / Fa c u l t y Haris Cazlaris, PhD Senior Clinical Embryologist, Athens, Greece Stephen Davies, PhD Senior Clinical Embryologist, Scientific Director, «Embryogenesis» Assisted Conception Unit, Athens, Greece Elena Kontogianni, PhD Senior Embryologist-Geneticist, Scientific Director, IVF & GENETICS Center, Athens, Greece Minas Mastrominas, MD Director, «Embryogenesis» Assisted Conception Unit, Athens, Greece Laura Rienzi, PhD G.EN.E.R.A. Centre for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy Denny Sakkas, PhD Boston IVF, Waltham MA, and Dpt. of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven CT, USA Maria José de los Santos, PhD Directora de Laboratorio FIV, Doctora en Biología Laboratorio Fiv., Valencia, Spain Dagan Wells, PhD, F.R.C.Path. Scientific Leadership Fellow, University of Oxford, Founder & Director of Reprogenetics, UK 5

CMYK Ε πιστηµονικό Πρόγραµµα/Scientific Programme 09:00-09:50 Eγγραφές Registrations 09:50-10:00 Έναρξη Συµποσίου Μηνάς Μαστροµηνάς Welcome Address Minas Mastrominas 10:00-10:30 Μέρος 1 Προεδρείο: Έλενα Κοντογιάννη - Χάρης Καζλαρής Part 1 Chairpersons: Elena Kontogianni - Haris Cazlaris 6 Comprehensive chromosome screening: clinical data from new technologies Dagan Wells 10:30-11:00 New developments in the IVF laboratory Denny Sakkas 11:00-11:30 ιάλειµµα Kαφέ / Coffee Break 11:30-12:00 Μέρος 2 Προεδρείο: Έλενα Κοντογιάννη - Χάρης Καζλαρής Part 2 Chairpersons: Elena Kontogianni - Haris Cazlaris Oocyte cryopreservation by vitrification approach Laura Rienzi 12:00-12:30 Embryo morphokinetics, new horizons for embryo assessment Maria José de los Santos 12:30-12:45 Initial experience with the Embryoscope in clinical IVF Stephen Davies 12:45-13:00 Σχολιασµός - Συµπεράσµατα Closing Remarks 13:00 Ελαφρύ Γεύµα / Light Lunch

Γ ε ν ι κ έ ς Π λ η ρ ο φ ο ρ ί ε ς ΧΡΟΝΟΣ-ΤΟΠΟΣ Το 6 o Επιστηµονικό Συµπόσιο πραγµατοποιείται το Σάββατο 17 Σεπτεµβρίου 2011, στο Ξενοδοχείο Hilton (αίθουσα Galaxy) στην Αθήνα. ΓΛΩΣΣΑ Η επίσηµη Γλώσσα του Επιστηµονικού Συµποσίου είναι τα Αγγλικά, λόγω των ξένων οµιλητών. ΣΥΜΜΕΤΟΧΗ ΣΤΟ ΣΥΜΠΟΣΙΟ H συµµετοχή είναι ωρεάν και περιλαµβάνει: Παρακολούθηση των Επιστηµονικών Συνεδριάσεων, Πρόγραµµα & Περιλήψεις οµιλιών, Πιστοποιητικό Παρακολούθησης, ιάλειµµα Καφέ και Ελαφρύ Γεύµα. ΙΑΜΟΝΗ ΣΤΟ ΞΕΝΟ ΟΧΕΙΟ HILTON Ηµερήσια τιµή δωµατίου µε πρωινό και φόρους ΞΕΝΟ ΟΧΕΙΟ Μονόκλινο / ίκλινο HILTON ATHENS (5*) 180 ΓΡΑΜΜΑΤΕΙΑ ΣΥΜΠΟΣΙΟΥ Για οποιαδήποτε πληροφορία, εγγραφές, κρατήσεις δωµατίων κ.λπ. παρακαλούµε επικοινωνήστε µε την Γραµµατεία του Συµποσίου: ERA ΕΠΕ - Ασκληπιού 17, 106 80 - Αθήνα, τηλ.: 210 3634 944, 36 32 950, Fax: 210 3631 690 E-mail: info@era.gr, Web Site: www.era.gr Η Γραµµατεία θα λειτουργεί στο Συνεδριακό χώρο κατά την διάρκεια του Συµποσίου. G e n e r a l I n f o r m a t i o n DATE-LOCATION The 6 th Scientific Symposium is held on Saturday September 17 th, 2011, at the Hilton Hotel (meeting room Galaxy), in Athens. LANGUAGE The Official language of the Symposium is English, due to the foreign invited speakers. REGISTRATION The Registration to the Symposium is free of charge and includes: Attendance to the Scientific Sessions, Programme & Abstracts of lectures, Certificate of Attendance, Coffee Break & Light Lunch. HOTEL ACCOMMODATION IN HILTON ATHENS HOTEL Daily hotel rates with breakfast and taxes HOTEL Single / Double HILTON ATHENS (5*) 180 SYMPOSIUM SECRETARIAT For any information, registration, hotel booking etc. kindly contact the Symposium Secretariat: ERA Ltd - 17, Asklipiou Str., 10680 Athens-Greece Tel.: +30 210 3634944, Fax: +30 210 3631690 E-mail: info@era.gr, Web Site: www.era.gr The Secretariat will operate at the area during the Symposium. 7

CMYK 8

Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν L e c t u r e s A b s t r a c t s Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s 9

CMYK Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s Comprehensive chromosome screening: clinical data from new technologies Dagan Wells, PhD, FRCPath Senior Fellow in Reproductive Genetics, University of Oxford Director, Reprogenetics UK dagan.wells@obs-gyn.ox.ac.uk It is now more than two decades since the first clinical cycles of preimplantation genetic diagnosis (PGD) were undertaken, allowing couples at high risk of transmitting a serious inherited disorder to achieve healthy pregnancies and virtually eliminating the issue of pregnancy termination. As well as the detection of inherited disorders caused by mutation in an individual gene, screening of abnormalities related to chromosome rearrangements has also been a common indication for PGD. Approximately 1/160 infertile patients carries a chromosome rearrangement (most commonly a translocation), but the frequency can be as high as 1/15 for patients that have a history of recurrent miscarriage. Couples having PGD because of a chromosome rearrangement seek to avoid the high risks of congenital abnormality and spontaneous abortion caused by unbalanced chromosomal constitutions. Preferential transfer of genetically normal embryos from carriers of chromosome rearrangements has been associated with improved outcomes, notably a greatly reduced miscarriage rate. Research has clearly demonstrated that losses and gains of chromosomes (aneuploidy) are extremely common in human oocytes and embryos. The problem affects all patients undergoing infertility treatment and is not associated with any familial predisposition, rather it arises spontaneously. The exact frequency of aneuploidy is closely related to female age. Less than 10% of oocytes from young, fertile egg donors are chromosomally abnormal, while approximately three quarters of oocytes for women in the early forties have the wrong number of chromosomes. Aneuploidy is rarely compatible with survival to term and most affected embryos either fail to implant or miscarry. The high prevalence of aneuploidy, coupled with its lethality, has led to the concept of screening embryos prior to transfer. Aneuploidy is invisible to conventional morphological analysis and consequently screening methods are essential if the most viable embryos are to be indentified and selected for transfer. This approach, sometimes referred to as preimplantation genetic screening (PGS), has become the most widely practiced form of genetic testing applied to human oocytes and embryos. In theory, PGS should lead to an improvement in implantation and birth rates (especially in cases of elective single embryo transfer) and reduced rates of miscarriage and Down syndrome. However, several randomised controlled trials failed to observe the expected improvements in outcome. It now seems likely that the disappointing results of these PGS studies were due to the limitations of the technology used at the time (fluorescence in situ hybridisation FISH), as well as embryological issues that had not been fully appreciated (biopsy damage and chromosomal mosaicism). Newer methods appear to have overcome most of these deficiencies and emerging clinical data is encouraging. Unlike the older FISH techniques, new methodologies used for PGS allow the entire set of chromosomes to be tested, providing an unparalleled overview of the chromosomal status of the oocyte or embryo. The most widely used of the new generation of methods is microarray comparative genomic hybridisation (also known as microarray-cgh, array-cgh or acgh). Improvements in IVF outcome have been reported for IVF cycles where oocytes have been tested using acgh applied to the first, and in some cases sec- 10

ond, polar bodies. Improvements in implantation and pregnancy rate have also been noted following application of acgh at the cleavage stage, this positive outcome despite the biological eccentricities of the cleavage stage (e.g. mosaicism). However, the stage at which acgh appears to confer the greatest benefit is when it is applied at the blastocyst stage, with several studies now indicating that implantation rates are increased 1.5-2 fold compared with controls. Results can be obtained using acgh within 24 hours of biopsy and fresh transfers are therefore possible, even after blastocyst biopsy. As an alternative to acgh, comprehensive chromosomal screening can also be carried out by assessing thousands of polymorphisms scattered along the length of each chromosome. This is achieved using a single nucleotide polymorphism (SNP) microarray. The transmission of the polymorphisms from the parents to the embryos allows the inheritance of each chromosome to be tracked, revealing instances when the wrong number of chromosomes are received by the embryo. Potentially, this sort of approach has the advantage that the gamete responsible for an abnormality (sperm or oocyte) can be determined and that cases of uniparental disomy (UPD), where both copies of a particular chromosome are inherited from the same parent, can be detected (acgh cannot detect UPD). Additionally, the use of SNP microarrays can allow chromosome screening to be combined with the diagnosis of single gene disorders: the inheritance of a defective gene revealed by the detection of associated SNP alleles (i.e. genetic linkage). The combination of these two types of analysis has, in the past, been extremely difficult to achieve. While SNP microarrays are associated with some advantages over other methods, concerns have been raised about the accuracy and clinical utility of this approach. The data obtained is not as straightforward as it may at first appear and needs to be cautiously evaluated. For example, embryos found to have UPD are generally excluded from transfer, however, not all forms of UPD are clinically significant. Additionally, it is not always possible to distinguish mitotic errors from those of meiotic origin. This has undoubtedly resulted in errors in assigning the gamete responsible for an aneuploidy with potential consequences for subsequent clinical treatment. Finally, it remains unclear whether all forms of meiotic error can be reliably detected using SNP microarrays. Thus, SNP microarrays hold promise, but further validation is needed before widespread clinical application. Another new approach for PGS is involves utilisation of real-time PCR, a DNA amplification method that allows quantification of individual loci on each chromosome, from which the copy number of the whole chromosome can be inferred. This method is attractive due to the high speed of analysis. Results are available after 4-5 hours, compared with ~12 hours for acgh and over 24 hours for SNP microarray. The rapidity with which results are obtained allows for maximum flexibility in the embryology laboratory in terms of the timing of embryo transfer. Currently, little data has been published concerning the accuracy or efficacy of this approach, but initial results are encouraging, especially when combined with analysis of blastocysts followed by fresh transfer. In summary, a variety of methods for the comprehensive chromosome screening of human oocytes and embryos are now available. Initial data using these techniques are suggestive of significantly improved outcomes following aneuploidy screening. Randomised trials to verify the efficacy of these methods are now underway. Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s 11

CMYK Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s New developments in the IVF laboratory Denny Sakkas, PhD Boston IVF, Waltham MA, USA and Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven CT, USA Although the clinical IVF Laboratory has undergone numerous changes in the past 30 years the two main stalwarts of the IVF lab, Culture Environment and Embryo Assessment are still to experience a radical transformation. Embryo culture media has improved exceptionally, however the vehicle for culturing embryos is only now appearing to change with the advent of microfluidic chambers, smaller incubators and incubators allowing real time imaging of the individual embryo. In contrast to the changes that have occurred in embryo culture media it can be argued that embryo assessment has undergone very little change because morphology and cleavage rate remain the mainstay of embryo assessment. As the number of ART cycles increases worldwide, improvements in the ability to quickly identify the best single embryo for transfer remains a critical goal for reproductive medicine. A number of technologies including the non-invasive measurement of glucose, lactate, pyruvate and amino acids have been present for over 20 years but were not adapted to clinical practice. The assessment of the embryo and correlation to viability using, genomic and proteomic profiling, and more recently, analytical examination of the embryonic metabolome has shown greater promise. In the next decade the IVF laboratory will be a very different location featuring automated embryo culture and imaging systems and a range of technologies that will allow the possibility of both invasive and non-invasive single embryo profiling using genomic, proteomic and metabolomic technologies to more accurately assess which embryo will lead to a normal live birth. 12

Oocyte cryopreservation by vitrification approach L. Rienzi, F.M. Ubaldi G.EN.E.R.A Centre for Reproductive Medicine, Clinica Valle Giulia, Via G. De Notaris 2b, 00197 Rome, Italy Cryopreservation of embryos at different stage of development is an indispensable part of assisted reproductive techniques. Frozen embryo transfer contributes already 25% of all births achieved by assisted reproduction worldwide, and with systematical application, it is estimated that up to 40% of implantations can be derived from frozen embryos. In many clinics, birth rates after transfer of cryopreserved embryos are close or identical to those achieved with their fresh counterparts, increasing considerably the overall success rate of ART procedures measured by delivery per oocyte aspiration rates. However, legal issues and moral concerns may restrict the application of embryo cryopreservation. Additionally, due to the lack of a partner it cannot be applied in many cases of fertility preservation with medical or social indications, and may create controversial issues in case of divorce or separation of partners. The most feasible solution for these problems is oocyte cryopreservation. Unfortunately, in spite of the relative early successes, widespread application of oocyte cryopreservation was hampered for a long time by inconsistent efficiency of the available cryopreservation methods. Stepwise adjustments of traditional slow freezing protocols as well as optimization of minimum volume vitrification methods have resulted in breakthroughs in this field. Recent prospective randomized studies in oocyte donation programs have found no significant differences between fresh and vitrified oocytes regarding the in vitro and in vivo developmental potential. To test the laboratory efficacy of oocyte vitrification as a routine application for the standard infertile population we have performed a prospective non-inferiority trial (Rienzi et la., 2010). We have found that oocyte vitrification procedure followed by ICSI is not inferior to the fresh insemination procedure, with regard to fertilization and embryo developmental rates. We have then also estimated the cumulative reproductive outcome of a cohort of infertile couples undergoing ICSI and oocyte vitrification in restrictive legal conditions (limitation in the number of inseminated oocytes, embryo cryopreservation forbitten) (Ubaldi et al., 2010). In this prospective longitudinal cohort study, the cumulative ongoing pregnancy rates obtained by the insemination of fresh and vitrified oocytes from the same cohort were calculated as primary outcome measures. Moreover, the effect of basal and cycle characteristics on clinical outcomes were assessed. The overall ongoing pregnancy rates obtained in the fresh, and first and second warming cycles were 37.4, 25.0 and 27.3%, respectively. The overall cumulative ongoing clinical pregnancy rate observed per stimulation cycle was 53.3%. Maternal age was the only characteristic found to influence the reproductive outcome, with an inverse correlation between the age >40 and the ongoing pregnancy rates (P =0.04, by multivariate regression analysis). We can conclude that high cumulative ongoing pregnancy rates can be obtained with transfers of embryos derived from fresh and cryopreserved oocytes in a typical infertile population. Female age significantly affects outcomes in this system. We believe that due to the latest advancements in the vitrification approach, cryopreservation of oocytes offers now new perspectives in Assisted Reproductive Technology. Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s 13

CMYK Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s Embryo morphokinetics, new horizons for embryo assessment Maria José de los Santos 1, Alberto Tejera 1, Javier Herrero 1, Karen Marie Hilligsoe 2, Marcos Meseguer 1 1 IVI Valencia, Spain, 2 Unisense Fertilitech Aarhus, Denmark For many years embryo morphology alone was used as a main and unique tool to select embryos before transfer in IVF laboratories worldwide. The experience given by the years showed to embryologists to recognize those parameters that were more associated to further embryo developmental and implantation potential such as, number of blastomeres, percentage of fragmentation, presence of mulitnucleation, etc..the introduction on the early nineties of PGS allowed not only to increase the cycle success rates in certain group of patients but also to demonstrate the association of certain embryo morphological parameters with the risk of carrying chromosomal abnormalities. Nowadays the omics- based technologies are on their way of offering additional support to the conventional morphological analysis of embryos in order to increase the accuracy of embryo selection before transfer. However, these techniques are still expensive, requiring validation and adaptation to the speed of the reproductive cycle. On the contrast, image analysis system can become a more reliable and real method. So far most of the morphological embryo evaluation, are based on static and limited measurements of embryo development events, so we may be loosing valuable data concerning embryo viability. In this sense time-lapse observation opens up the opportunity for optimizing current morphological grading establishing the morphokinetic of embryo development through the continuous registration of the exact time of the main events from fertilization to blastocyst stage allowing not only setting up average values and confidence intervals for each of the events based on a considerable statistical material but providing novel kinetic parameters which may aid in the selection of viable embryos. We have used an automatic embryo monitoring system to generate a multivariable model to classify embryos depending on its implantation probabilities. By the use of morphological data together with new variables like timing of 1 st (2 cells) (t2) 2 nd (3 cells) (t3) 3 rd (4 cells) (t4) and 4 th (5 cells) (t5) division a logistic regression model has been defined. ROC curve analysis to determine the predictive properties of these model with respect to probability of implantation presented 0.720 (CI95% 0.645-0.795). With the data we can create a hierarchical model with the corresponding decision tree procedure which defined embryos into 5 categories from A to E with extra plus (+) or negative tag (-) with the intention to be used for embryo selection in a routine basis providing therefore an approach for diagnosis of embryo competence in assisted reproduction 14

Initial experience with Embryoscope TM in clinical IVF Stephen Davies Clinical Embryologist, Embryogenesis The limitations of assessing embryo viability by morphology alone are one of the reasons why we have to transfer multiple embryos in order to increase the chances of achieving a pregnancy after IVF treatment. All other non-invasive methods to date have proven to be unreliable and impractical, so until new robust methods are developed, morphology is still the method of choice in clinical embryology. For this reason, several companies have recently developed incubators with inbuilt microscope and camera facilities to monitor and record embryo development. The convenience of having a purpose-built system which allows visualisation of embryo development whilst maintaining optimal culture conditions is an significant improvement in clinical embryology. The embryologist can now have full access to details which by any other monitoring method to date are not so readily available. Embryo grading can now be expanded to include morphology in conjunction with timeframe of characteristic cellular events and cell cleavage patterns which either follow or deviate from a predicted normal developmental cycle. Thus, providing a much more comprehensive system to assess embryo viability (Wong et al, 2010 Nature Biotechnology 28 (10) p1115-1121) We introduced the Embryoscope TM in our laboratories during April/May 2011. After an initial trial period with abnormally fertilised embryos, we started with clinical cases by incubating half of the fertilised oocytes after ICSI in Embryoscope TM & the other half as normally in conventional incubators. We soon realised that we were missing essential information from embryos cultured in conventional incubators, so we quickly converted to incubating all the embryos from specific cases of interest in the Embryoscope TM. The patients we selected were (i) Previous failures with good fertilisation & morphologically good embryos. (ii) Fertilisation after TESE. (iii) PGS - where we would also obtain whole genome data after arraycgh. (iv) Cases with potentially many embryos which we routinely split the oocytes into two different culture media. To date we have completed 22 cases in which all embryos were incubated in Embryoscope TM and embryos for transfer or vitrification determined by modified grading criteria. The outcomes to date are: (a) ongoing clinical pregnancies (n=16), (b) negative hcg (n=5), (c) 1 case with no embryo transfer because all embryos assessed as abnormal after PGS. We are gaining in confidence that the modified selection criteria we have at present is helping us to improve our ability to sub-select developing embryos into the categories of (a) best for transfer, (b) suitable for vitrification, and (c) most probably non-viable. Our aim is to reduce the number of embryos transferred during clinical IVF without reducing the pregnancy rates. Π ε ρ ι λ ή ψ ε ι ς Ο µ ι λ ι ώ ν / L e c t u r e s A b s t r a c t s 15

CMYK Με την ευγενική χορηγία των With kind sponsorship of