Offizielles Organ: AGRBM, BRZ, DVR, DGA, DGGEF, DGRM, D·I·R, EFA, OEGRM, SRBM/DGE

Krause & Pachernegg GmbH, Verlag für Medizin und Wirtschaft, A-3003 Gablitz

Journal für

Reproduktionsmedizin und Endokrinologie– Journal of Reproductive Medicine and Endocrinology –

Andrologie • Embryologie & Biologie • Endokrinologie • Ethik & Recht • Genetik Gynäkologie • Kontrazeption • Psychosomatik • Reproduktionsmedizin • Urologie

Indexed in EMBASE/Excerpta Medica/Scopus

www.kup.at/repromedizinOnline-Datenbank mit Autoren- und Stichwortsuche

Alpha Biennial Conference, 15.–17. September 2006

Luzern (Abstracts)

J. Reproduktionsmed. Endokrinol 2006; 3 (4), 264-279

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ALPHA BIENNIAL CONFERENCE15.–17. SEPTEMBER 2006 IN LUZERN(ABSTRACTS*)

O1THE EFFECT OF IGF 2 ON HUMAN CRYO-PRESERVED OVARIAN TISSUE IN LONG-TERMCULTURES

R. Fabbri1, G. Pasquinelli2, C. Cani1,M. Derenzini2, S. Venturoli11Human Reproductive Medicine Unit,Obstetrics and Gynaecology and 2ClinicalPathology Section, Experimental Patho-logy, S. Orsola Hospital, Bologna, Italy

Introduction: Oocyte maturation ofcryopreserved ovarian tissue followedby in-vitro fertilization would be animportant option to treat infertility ofyoung women undergoing prematureovarian failure due to anticancer treat-ment. The aim of this study was toevaluate the effect of insulin-growthfactor 2 (IGF 2) on follicular and stromalcell preservation of human cryopreserv-ed ovarian tissue cultured for 16 weeks.

Material and Methods: The ovariancortex of five patients was collectedand immediately cryopreserved, aspreviously described [Fabbri et al.,2003]. After thawing, a specimen ofovarian cortex from each patient wasfixed for histological and ultrastructuralanalyses (control t0). The other pieceswere cultured at 37 °C in an atmos-phere of 6 % CO2 for 16 weeks, inminimum essential medium supple-mented with insulin-transferrin-sele-nium (ITS), follicle stimulating hormone(FSH), human serum (HS), N-acetyl-L-cysteine (NAC), antibiotics and IGF 2(medium A) or without IGF 2 (mediumB). The medium was changed everysecond day. After 16 weeks, pieces ofovarian tissue were fixed for histologi-cal and ultrastructural analyses (t16).The developmental stage of follicleswas classified using light microscopy(LM); follicular and stromal cell integ-rity was evaluated using transmissionelectron microscopy (TEM).

Results: TEM showed that the percentageof healthy follicles was increased in me-dium A (100 %) compared to medium B(50 %) after a 16-week culture period.

Furthermore, more growing follicles(i.e., secondary and preantral) wereobserved in tissue cultured in mediumA (LM observation). Conversely, TEMdid not show any difference in stromalcell preservation between medium Aand B, even if a slightly better structuralstromal cell integrity was observed inmedium A after 16 weeks of culturewith respect to control t0.

Conclusions: Our results suggest that IGF2 may promote the functional integrity ofthe follicles and stimulate follicular growthin an in-vitro culture. The advantage ofpreserving the integrity of the oocyte-granulosa-stroma interaction would be animportant task in improving the develop-ment of early follicles in culture.

O2THE EFFECT OF FSH AND NAC ON HUMANCRYOPRESERVED OVARIAN TISSUE CULTUREDFOR 32 WEEKS

R. Fabbri1, G. Pasquinelli2, B. Barzanti1,M. Derenzini2, S. Venturoli1

1Human Reproductive Medicine Unit,Obstetrics and Gynaecology and 2Clini-cal Pathology Section, ExperimentalPathology, S. Orsola Hospital, Bologna,Italy

Introduction: The fertility of women withpremature ovarian failure due to anti-cancer treatment might be preserved bycryopreservation and in-vitro culture ofovarian tissue. The aim of this study wasto evaluate the effect of follicle stimu-lating hormone (FSH) and N-acetyl-L-cysteine (NAC) on human frozen-thawedovarian tissue cultured for 32 weeks.

Material and Methods: The ovariancortex of a patient suffering from breastcancer was collected and immediatelycryopreserved, as previously described[Fabbri et al., 2003]. After thawing,pieces of ovarian cortex were culturedat 37 °C in an atmosphere of 6 % CO2for 32 weeks, in minimum essentialmedium supplemented with insulin-transferrin-selenium (ITS), human serum(HS), antibiotics, FSH and NAC. Themedium was changed every secondday. The developmental stage of folli-cles was classified by using light micro-scopy (LM); follicular and stromal cellintegrity was evaluated using transmis-sion electron microscopy (TEM).

Results: LM examination of corticalpieces after 32 weeks of culture showeda healthy early preantral follicle; TEMshowed that the follicle was character-ized by a polarized multilayer of granu-losa cells surrounding the oocyte andseveral erect microvilli extended from theoolemma into the zona pellucida; zonulaadherens-like and gap junctions wereobserved between granulosa cells andoolemma; the oocyte showed a regularly-shaped, outlined euchromatic nucleus;mitochondria were mainly located closeto the nuclear side; clusters of corticalgranules were found in the ooplasm.

Conclusions: These data suggest thatthe synergy in action of NAC and FSHplays an important role in follicle growthof ovarian tissue cultures. This is the firstreport showing morphological evidenceof a well-preserved preantral folliclerecovered from human frozen-thawedovarian tissue cultured for 32 weeks.

O3OOCYTE NUMBER AT PICK-UP IS A RELIABLEPREDICTIVE FACTOR TOWARDS CLINICALPREGNANCY RATES IN ART

C. E. Plancha1, S. C. Correia1, M. L. Rato1,P. Marques-Vidal2, D. Sobral1, G. Pinto1,P. Sá e Melo1, M. J. Carvalho1

1CEMEARE Centro Médico de Assistênciaà Reprodução, Lisbon, Portugal, 2Nutri-tion and Metabolism Unit, Instituto deMedicina Molecular, Lisbon MedicalSchool, Portugal

Introduction: Maternal age is thought torepresent the most important factor indetermining clinical pregnancy rateswhen using fresh eggs. Other factors,such as infertility factor, laboratory cul-ture conditions and the number of trans-ferred embryos have also been shownto influence clinical pregnancy rates inART. The objective of this study was toevaluate if the number of oocytes re-trieved per aspiration constitutes a cleardeterminant of clinical pregnancy ratesindependent of maternal age.

Material and Methods: Between July2000 and December 2005, 337 patientsunderwent 414 consecutive IVF/ICSIcycles. Inclusion criterion was the ob-tainment of at least one oocyte at pick-up. Patients were between 19 and

* Peer-reviewed and compiled by Mag. MarcVan den Bergh, IVF Laboratory Director,Kantonsspital Baden A.G., CH-5404 Baden,Switzerland (24 credits by SGGG, 15 EUMScredits).

For personal use only. Not to be reproduced without permission of Krause & Pachernegg GmbH.

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46 years of age, with a mean of 33.4years. Cycles were divided in groupsaccording to number of oocytes col-lected at pick-up as follows: 1–5 (n =97), 6–11 (n = 148), 12–17 (n = 104),18–23 (n = 37) and 24 or more (n = 24)oocytes. IVF/ICSI outcomes includingfertilization rate, cleavage rate, frozenembryos and clinical pregnancy rateswere compared between the groups.A multivariable logistics analysis wasperformed using SPSS v13.0 software.

Results: Patients with less than 6 oocytesat pick-up obtained a clinical pregnancyrate that was arbitrarily attributed thevalue 1. Patients with 6–11, 12–17 and18–23 oocytes at pick-up reached sig-nificantly higher clinical pregnancyrates (2.4×, 3.1× and 2.4×, respectively;p < 0.05) than the first group. Patientswith 24 or more oocytes collected atpick-up did not show a significant in-crease in clinical pregnancy rate whencompared with the first group. Theseresults were found to be independentof women’s age.

Conclusions: This study identifies thenumber of oocytes at pick-up as a reli-able predictive factor towards clinicalpregnancy rates in ART since it does notseem to be significantly influenced bymaternal age. Furthermore, it identifies6 as the minimum number of oocytesto collect at pick-up when aiming forthe best clinical pregnancy outcome.Oocyte number at pick-up may reflect,in a particularly effective way, the bio-logical age of the ovary, becoming thusmore significant than maternal chrono-logical age. Future studies should beaimed at confirming if oocyte numbersat pick-up still hold as a reliable predic-tive factor towards more comprehensiveparameters of success in ART such as“take home healthy baby” rates.

O4EVALUATION OF EMBRYO QUALITY ONDAY 2 VERSUS DAY 3

F. Prados, N. Ortiz-Piñate, G. Pérez-Bermejo, C. Hernández, O. Collado,I. BrunaUnidad de Reproducción, Hospital deMadrid-Monteprincipe, Madrid, Spain

Introduction: Delaying the transfer fromday 2 to day 3 increases the morphol-

ogy data available for embryo selectionand allows to relocate the embryos in theuterus at a more natural stage. However,some embryos do not develop properlyfrom day 2 to day 3. The aim of thisstudy was to compare the morphologi-cal quality of the embryos evaluated onday 2 and later on day 3; and to decideif the differences reflected variations inthe implantation potential or else weredue to an incorrect evaluation of themorphological parameters.

Material and Methods: The 231 IVFcycles with fresh embryo transfer inclu-ded in this study were performed during2005 and 2006, all of them had morethan 3 embryos and the patients re-ceived less than 400 FSH units per oocyteretrieved. The embryo transfers weredone either on day 2 or day 3 depend-ing on the day of the week. The day-2transfer group included 102 cyclesperformed on patients with an averageage of 34.2 years and 6.7 embryos percycle, while 119 day-3 transfers weredone on patients with an average age of34.6 years and 6.8 embryos per cycle.

Results: A total of 608 embryos wereevaluated on day 2 and day 3 in cyclesof day-3 transfer. For 342 (56.3 %) ofthose embryos, the grade attributed onday 3 was worse than on day 2, in 104(17.1 %) embryos it was better, while in162 (26.6 %) cases it was the same.Moreover, when we select among the226 embryos showing good quality onday 2 (4 cells, < 26% fragments, nomultinucleated nor unevenly sizedblastomeres, no defects in cytoplasmor ZP), only 154 (68 %) reached thecorrect 7–8 cells on day 3 and 16 (7.1 %)became arrested in 4 cells. When weselected the 87 embryos of good qualityon day 3 (8 cells, < 26 % fragments,no multinucleated nor unevenly sizedblastomeres, no defects in cytoplasm orZP), 82 of them (94.3 %) had 4 cells onday 2.

The implantation rate was 35.9 % (74/206) for day-2 and 33.9 % (87/257) forday-3 transfers (p > 0.05). The clinicalpregnancy rate (fetal heart beat) was51 % for day-2 (52/102) vs. 52.1 %(62/119) for day-3 transfers (p > 0.05).There were no abortions after day-2 and6 (9.7 %) after day-3 transfers (p < 0.05).The ongoing pregnancy rate was 51 %(52/102) for day-2 and 47.1 % (56/119)for day-3 transfers (p > 0.05).

Conclusions: The overall morphologicalquality grade attributed to the embryoson day 3 was lower than on day 2. Signi-ficantly more embryos seemed to dete-riorate than to improve (p < 0,01). Goodquality day-2 embryos which couldhave been chosen for transfer evolvedto lower-grade embryos and even 7,1 %were arrested at the 4-cell stage. If wewere mistaken by the evaluation ofday-2 morphology, we would expect abetter choice of embryos for transferwhen the selection is done on day 3.However, the efficacy of day-2 transferwas similar to that of day-3 transfer.The ongoing pregnancy rate wasslightly higher (not significantly) forday-2 transfers.

These results suggest that in good-prog-nosis patients, the morphological dataobtained from day-2 embryos are suffi-cient for a correct evaluation of theirimplantation potential. Further cultureuntil day 3 does not improve the accu-racy of the embryo selection for transferwhile some of the embryos may dete-riorate during this extra in-vitro incuba-tion period. Day-2 transfer seems to bepreferable for these patients.

O5PRACTICE OF MORPHOLOGICAL EVALUATIONOF OOCYTES, PRONUCLEAR STAGES ANDEMBRYOS IN GERMAN ART LABORATORIES

I. Hoppe1, M. Greuner2, V. Baukloh3

1for the German Society of HumanReproductive Biology (AGRBM), Uni-versitätsfrauenklinik Jena, Germany,2for the AGRBM, GMP Thaele, Happel,Giebel, Saarbrücken, Germany, 3for theAGRBM, Fertility Center Hamburg,Germany

Introduction: Identification of theembryo(s) with optimal developmentalcapacity is an internationally recog-nized method to increase pregnancyrates. At the same time, it lowers multi-ple pregnancy rates by applying SET orDET. Although this possibility does notexist in Germany (German EmbryoProtection Law), standardized evalua-tion of oocytes, pronuclear stages andembryos from clinical and scientificviews and in the context of qualitymanagement is necessary. However,little is known about the application of

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different morphological parameters inindividual ART laboratories.

Material and Methods: By means ofquestionnaires, members of the AGRBM– the German Society of ClinicalEmbryologists – were asked to anony-mously specify their practice of evalu-ating oocytes, pronuclear stages andembryos. Different established scoringsystems as well as individual morpho-logical criteria were considered.

Results: Out of 141 members of theAGRBM 65 (46.1 %) participated in thesurvey. For the specific stages, scoringsystems were used in different frequen-cies: oocytes –81.5 %, pronuclear stages–96.9 %, day-2 embryos –75.3 %, andday-3 embryos –80.8 % of participants.

For oocytes, evaluation of polar body(81.5 %) and cytoplasm (64.6 %) wasmost frequently performed while peri-vitelline space (49.2 %) and zona pellu-cida (49.2 %) characterization seemedto be considered less important. Forpronuclear stages, position (73.8 %)and size (78.5 %) of pronuclei werepreferentially evaluated. In addition,number (67.7 %), size (60.0 %) anddistribution (60.0 %) of nucleoli andpresence of the halo phenomenon(60.0 %) were frequently determined.On the other hand, appearance of cyto-plasm (57.0 %), polar body (55.4 %)and perivitelline space (41.5 %) wereless often considered.

In the case of embryo evaluation ondays 2/3, degree of fragmentation (63.8 %)and number of blastomeres (63.0 %)were the preferred morphological para-meters. Less commonly size (54.6 %)and multinucleation (29.9 %) ofblastomeres were assessed.

Conclusions: Assessment of morpho-logical parameters and their applicationas scoring systems during IVF/ICSI ispossible for oocytes, pronuclear stagesand embryos. In Germany, the pronu-clear stage is the crucial phase for prog-nostic evaluation. Therefore, the mostintensive morphological evaluationactivity is reported for this level. Con-siderable differences exist in the fre-quency and priority of individual mor-phological parameters between GermanART laboratories. A standardized proce-dure of morphological evaluation is there-fore urgently required. The AGRBMsees its task in appropriate training of its

German reproductive biologist mem-bers and in the establishment of inter-laboratory tests for the characterizationof gametes and embryos to ensure com-parable standards.

O6REVIEW OF ESTIMATION OF FRAGMENTATIONOF HUMAN EMBRYOS USING AN INTERNET-BASED QUALITY ASSURANCE SCHEME

J. StangerQAPonline, Newcastle, Australia

Introduction: The estimation of fragmen-tation is a component of all embryograding assessments, yet there are noclear guidelines or training process toensure all embryologists are uniform inassessments. Variation in assessmentsmay result in differences between staffboth in reporting and decision aspects,e. g. freezing or discarding.

Method: QAPonline is an international,online, image-based quality assuranceprogramme that allows individual parti-cipation. One scheme from the 2005series was selected for review for theassessment of embryo fragmentation.Fifteen images were available over theyear and up to 63 professional embryo-logists from 25 laboratories participatedeach month. The images were eitheranimated GIF files with multiple sectionsthrough the embryo or short videos andwere between the 2 and 8 cells (earlyday 2 to late day 3).

Results: Participation ranged between54 and 62 and the mean %-fragmenta-tion ranged from 1 % to 44 %. The CVwas above 15 % for all samples andwhere the mean value was in the criticalrange for allocation to A or B gradeembryos, the CV was above 25 % inall cases. Estimates for mean values forimages of 1–10 % fragmentation hadreplies ranging from 0–20 and CV> 68 %. Of more importance, for imageswith mean value between 11 % and30 % the range drifted between 10 %and 80 % and CV of 28–48 %. The rangein these embryos was such that thesame embryo may have been variablyallocated to either GOOD or POORstatus depending on the embryologist.The same schemes in 2004 and 2006exhibit similar variability. Preliminary

morphometric analysis from selectedimages indicates that the embryo volumewithin the perivitelline space is close to60–70 % and that the actual volume offragmentation as a % of total cell volumeis closer to the lower estimates. Therefore,the criteria for fragmentation of % of cellvolume or % of zona volume will conse-quently significantly influence each par-ticipant’s estimation.

Conclusion: In this brief QA programme,the variability in the assessment of frag-mentation raises concern over the train-ing and certification of competency. Allparticipants were experienced embryo-logists with many years’ experience andthis reflects that the description ofembryos may have been assumed to belargely self-evident but this data suggestotherwise. Description of embryos alwaysincludes an estimate of fragmentation,and variations as described above maytranslate into potential disparities indeciding the fate of embryos both withinand between clinics. Preliminary morpho-metric assessment suggests that fragmen-tation is overestimated. This reviewreports significant and concerning vari-ation in the estimation of fragmentationand suggests that closer attention isrequired for its definition and assess-ment of fragmentation.

O7SHLA-G IN EARLY HUMAN EMBRYOCULTURE AFTER ART

Z. Cerkiene1, 2, A. Eidukaite1, A. Usoniene2

¹VU Institute of Immunology, Vilnius,Lithuania, ²Vaisingumo Klinika, Vilnius,Lithuania

Introduction: HLA-G is a non-classicalHLA class I molecule that can be ex-pressed in membrane-bound or solubleform and is well known for its tolero-genic properties. Increasing interest isnow being addressed to the solubleforms because they might have prog-nostic value for the implantation andpregnancy process. The aim of ourstudy was to evaluate the correlationbetween embryo cleavage, morphologyand sHLA-G levels, to estimate theimpact of sHLA-G concentration onpregnancy outcome.

Material and Methods: The study wasperformed in a group of infertile patients

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with various infertility indications. IVF orICSI procedure was used for insemina-tion. After 72 h, on the day of transfer,embryo morphology was evaluated,ET according to embryo morphologywas performed and supernatants ofembryos were collected and stored at–90 °C until screening. For the quantita-tive measurement of soluble forms ofHuman Leukocyte Antigen G in cellculture supernatants we used ELISA.The ELISA kit from EHBIO Praha meas-ures total sHLA-G and the componentsare ready-to-use.

Results: A total of 46 couples partici-pated in this study. In all, 258 embryoculture supernatants were tested forsHLA-G. The soluble form of HumanLeukocyte Antigen G was detected in49 (19 %) embryo culture samples(range 0,4–9,9 IU/ml). The productionof Human Leukocyte Antigen did notdepend on morphological criteria of theembryo. No significant differences werefound between sHLA-G concentrationsin patients from IVF and ICSI groups.Soluble HLA-G had an impact on em-bryo cleavage rate and pregnancy out-come. Soluble HLA-G levels did notdiffer significantly with respect to theage of women and infertility indication.

Conclusions: Day-3 embryos secretesHLA-G into the surrounding medium;the concentration of which can be de-tected using ELISA. The concentrationof Human Leukocyte Antigen G corre-lates with the number of embryo blasto-meres but not with morphological crite-ria. sHLA-G might be a marker of em-bryo implantation potential after IVF/ICSI procedures, a factor which helpsto achieve and maintain pregnancy.

O8EMBRYO QUALITY ASSESSMENT BYRESPIRATION RATE MEASUREMENTS ANDIMAGE ANALYSIS OF TIME-LAPSE IMAGESDURING EMBRYO DEVELOPMENT

N. B. Ramsing1, H. Callesen2

1Unisense FertiliTech, Aarhus, Denmark,2Department of Genetics and Biotech-nology, Danish Institute of AgriculturalSciences, Tjele, Denmark

Objectives: Evaluate novel approachesto quantitative embryo quality assess-ment using unattended real-time imageanalysis of time-lapse images and non-invasive online measurement of meta-bolic activity.

Material and Methods: Bovine immaturecumulus-oocyte complexes were aspi-rated from slaughterhouse-derived ovaries,matured for 24 h before fertilization for22 h. Cumulus cells were then removedand presumptive zygotes were trans-ferred and cultured in synthetic oviductfluid medium. A novel high-resolutionmicrosensor technology was used toautomatically monitor the respirationrates of individual embryos throughouta 6-day culture period. Time-lapse im-ages and respiration rates were recordedautomatically by an EmbryoScope (Uni-sense FertiliTech, Aarhus) twice per hourduring this 6-day incubation.

Results: 99 bovine embryos were evalu-ated by time-lapse microscopy (48 %blastocyst rate). The complex automatedimage analysis procedure generated aquantitative measure of blastomereactivity for each image in the time-lapseseries. Pronounced peaks in blastomereactivity were found to be associatedwith cell divisions and the exact onsetand duration of cell divisions could bequantified directly based on position,shape and size of the recorded peaks.(Correctly identified divisional eventsby the fully automated routine > 95 %;n = 350). The derived timing of the firstcell divisions was shown to be relatedto the subsequent fate of the particularembryo: more than 90 % of the embryosthat developed to expanding blastocystscould automatically be identified. Therespiration of 88 embryos was investigat-ed in a different experiment. The respi-ration rate was initially stable around5 fmol/s and remained largely constant

until the 8-cell stage at about 75 hoursafter fertilization. Then the respirationrate increased gradually as the embryocompacted and developed into a blas-tocyst. A relationship was found be-tween morphological events and respi-ration patterns.

Conclusions: The novel methods andinstrumentation give unprecedentedquantitative information about the firstcell divisions. Based on our preliminaryfindings with bovine embryos, it islikely that the techniques can improveembryo selection in human IVF treat-ments.

O9CORRELATION OF SPERM DNA FRAGMEN-TATION AS ASSESSED BY THE HALO SPERMASSAY WITH IVF OUTCOME PARAMETERS

A. Finn, V. Gross, J. A. Hill, L. ScottFertility Centers of New England,Reading, USA

Introduction: The introduction of intra-cytoplasmic sperm injection (ICSI) prov-ed to be highly efficacious for severeforms of male infertility. However, amale factor not resolved by the applica-tion of ICSI may be contributing toinfertility in some patients. Subtle malefactor may be identified by low normalmorphology despite otherwise normalsemen characteristics; low recovery ofsperm after gradient separation, arrestedembryo development, and lack of preg-nancy. The concept of sperm DNAfragmentation was introduced with theSCSA by Evensen et al. [Science 1980;210: 1131–3]. Since then, numerousassays have been developed in order toaccurately and easily assess the degreeof DNA fragmentation in human sperm.The halo assay is commercially avail-able to analyze sperm DNA integrityin-house and may offer insights into thesperm contribution to IVF/ICSI failurein mild male-factor couples.

Methods: Halo Sperm Kits were ob-tained from Indas Laboratories (Madrid,Spain) and used to evaluate DNA frag-mentation in pre-washed sperm sam-ples from control and subject men.Assays were performed according tothe instructions. DNA damage wasmeasured by chromatin dispersion or

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halo. A large or medium halo (similarto or larger than the smallest core dia-meter) represented little or no fragmen-tation; a small or no halo (smaller than1/3 of the smallest diameter of the core)showed large amounts of DNA damage.Control samples were collected fromthe male partners of IVF couples whowere < 37 years old and presenting fortreatment of female tubule pathology.Test samples were collected from themales of couples experiencing unex-plained IVF failure (n = 22) in the pres-ence of low sperm recovery and/or poorembryo development in combinationwith borderline oligospermia and/orteratospermia (10–20 mil/ml and lessthan 10 % normal forms) in at least 2previously failed IVF cycles.

Results: Control samples (n = 6) had anaverage DNA fragmentation rate of24.8 %; mean sperm recovery rate of66 %; fertilization rate of 79 %; meanage of 33.2 and female age 31.3; 57 %good day-1 score; 54 % good day-2score (even cell size and lack of multi-nucleation); low day-3 developmentalarrest (5.7 %) and 50 % clinical preg-nancy rate. Twenty-two test sampleswere analysed and showed an averagerate of DNA fragmentation of 45.9 %(p < 0.01); mean sperm recovery of39 %; 72 % fertilization rate; 56 %good day-1 score, 44 % good day-2score, and 39 % developmental arrestrate on day 3 (p < 0.01). The mean maleage was 36.0 and the mean female age34.1. Patients with low recovery (< 20 %)had a mean fragmentation rate of 55 %and a 43 % developmental arrest rate.Patients with poor day-3 embryo devel-opment (> 20 % arrested) had an aver-age fragmentation rate of 44 % and amean recovery rate of 32 %. In allcases, patients were repeat IVF failuredespite the replacement of morpho-metrically normal embryos. Five testpatients’ partners had a positive preg-nancy test. Four resulted in early bio-chemical losses, one is ongoing. Theongoing pregnancy rate for all under38-year-old patients in this practice is42 %.

Conclusions: These data indicate thateven in the presence of normal or onlysub-optimal sperm samples, the degreeof DNA fragmentation in the spermcould be a cause of pregnancy failure.In cases of poor embryo developmentand low sperm recovery, investigation

of the sperm DNA fragmentation ratesmay be a useful clinical test for unex-plained IVF failure patients. More dataare needed to validate this test and todevelop methods of treatment oncediagnosis is established.

O10PRELIMINARY RESULTS OF A PROSPECTIVESTUDY ABOUT SEMEN QUALITY IN VARIOUSGEOGRAPHIC REGIONS OF SWITZERLAND

M. Crausaz1, D. Goy-Eggenberger1,E. Stettler2, Y. Chollet3, M. Wisard3,A. Senn1, M. Germond1

1F.A.B.E.R. Foundation, Lausanne,Switzerland, 2Swiss Army, MedicalService, Ittigen, Switzerland, 3CHUV,Urology Unit, Lausanne, Switzerland

Background: Over the last decades,exposure to estrogens or related com-pounds in the environment has beensuspected to induce detrimental effecton male fertility and to be responsiblefor a decline in sperm concentrationand quality. In the absence of referencevalues for Swiss men, we started a pro-spective trial to better understand theparameters affecting male fertility. Thisstudy will extend over a period of3 years among voluntary young men,originating from all regions of Switzer-land, attending recruitment for militaryservice (conscripts), with the aim toinclude 3000 subjects.

Methods: A careful anamnestic evalua-tion of each subject was performedusing a validated questionnaire (Danishmodel), referring among others to theorigin of the parents, geographic loca-tion of the mother during pregnancy,medical history (cryptorchidism or othercongenital malformations), exposure totoxic agents, lifestyle and fertility historyof the subject. A complete urologicalstatus including clinical examinationand testicular ultrasound was perform-ed. Sperm, urine and blood sampleswere obtained from each volunteer.A Computer Assisted Sperm Analyzer(CASA) (SCA, Microptic SL, Spain) wasused for sperm analysis. A video se-quence of each sperm sample was re-corded and stored for later possible re-examination. The sperm morphologicalanalysis was performed by a trainedtechnician (Kruger’s criteria). A 10-year

follow-up is scheduled regarding fertilityand urogenital health history of eachsubject.

Results: Between September 2005 andApril 2006, 253 volunteers (5 % of theconscripts) accepted to participate in thetrial. Most volunteers originate from theFrench-speaking part of Switzerland.Demographic parameters of the cohortare (mean ± SD): age = 19.5 ± 1 years,weight = 72.4 ± 12 kg, height = 179 ±7 cm and BMI = 22.6 ± 3. Two azoo-spermic (0.8 %) and 3 cryptozoospermic(1.2 %) samples were detected. Spermvalues (except two azoospermic cases;n = 251) were (mean ± SD/median/range): abstinence length: 4.1 ± 4.6/3.1/0.5–31.1 days, volume: 3.05 ± 1.37/3.00/0.29–6.88 ml, sperm concentration: 71 ±69/53/0.2–570 × 106/ml, total spermcount: 226 ± 333/151/.8–3862 × 106,motile forms: 58 ± 20/59/7–96 %, rap-idly progressive forms: 39 ± 15/39/2.6–77 % and morphologically normalforms: 7.8 ± 5.0/7.5/0–25 %. Regardingthe subjects participating in the clinicalexamination (n = 141), 2.9 % had a totaltesticular volume < 30 ml, 25.5 % had avolume between 30–40 ml and 71.6 %were normal (≥ 40 ml); the mean ± SDsperm concentration in these categorieswere 10.6 ± 13.9 × 106/ml, 59.9 ± 56.6 ×106/ml and 76.6 ± 82.8 × 106/ml. Spermvalues below WHO references for vol-ume, concentration and mobility werefound in 56 (22 %) volunteers. In 31 ofthem, the low sperm concentration wascorrelated to a clinical finding: 6 cryp-torchidisms (unilateral or bilateral),7 varicocoeles, 7 other uro-genitaldiseases and 11 cases with a testicularvolume ≤ 30 ml. The 25 remainingcases need further investigation todetermine causes of low sperm values.

Conclusions: The preliminary resultsof this national prospective study showthat 22 % of the first 253 subjects havesperm parameters for volume, concen-tration or mobility below the WHOreference values. Cryptorchidism, uro-genital diseases or decreased testicularvolumes were frequently associatedwith the low sperm values. These pre-liminary results confirm the importanceof widening this study to the other geo-graphic regions of Switzerland and ofpursuing the follow-up of the subjectsin terms of fertility and urogenital dis-eases.

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O11IDENTIFICATION OF GENES DIFFERENTIALLYEXPRESSED IN HUMAN GRANULOSA CELLSFROM COMPETENT OOCYTES

M. Hamel, I. Dufort, C. Robert,M. C. Leveille, A. Leader, M. A. SirardCentre de Recherche en Biologiede la Reproduction, Laval University,Quebec, Canada

Introduction: To increase pregnancyrates in human IVF, usually two or moreembryos are transferred in each patient.Unfortunately, multiple pregnancies canoccur with potentially harmful conse-quences for the mother and the babies.The main objective of the project is tofind a way to predict embryo quality inorder to reduce the number to be trans-ferred. We made the hypothesis that theembryo quality depends on the finalmaturation of the follicle leading to acompetent or incompetent oocyte.Therefore, we believe that there aremarkers in granulosa cells from thefollicles bearing good oocytes.

Material and Methods: Granulosa cellsfrom consented patients were recoveredby individual follicle puncture with aspecial double lumen needle. Popula-tions of follicles (n = 5) leading to preg-nancy were subtracted from the nega-tive ones (n = 5) using SSH (SuppressiveSubtractive Hybridization) with the PCR-Select cDNA Subtraction Kit. ESTs sub-tracted products from SSH were se-quenced and compared against theGenBank database. Purified PCR prod-ucts from granulosa cells library, cumu-lus cells library were spotted on glassslides using a VersArray Chip WriterProrobot. Microarray analyses were per-formed in order to identify true granu-losa cell specific genes. Forward PCRproducts were used as probes for thefirst hybridization. For the second hy-bridization, populations of follicles(n = 15) leading to pregnancy and fol-licles (n = 15) from the negative oneswere used. To improve the genomecoverage, improve the number of can-didate genes and confirm candidategenes already found in our libraries,hybridizations with the human HG-U133_Plus_2 Affymetrix GeneChip®

with the 2 pools of patients were done(Plateforme des Biopuces, CREMO,Centre de recherche du CHUL, CHUQ).

Results: In the granulosa and cumuluscells library, 89 % and 68 % of the se-quences were from genes with knownfunction respectively, leading to a totalof 465 and 645 unique sequences ineach library. Some genes found in thetwo libraries as Cox-2, StAR, Interleukinand 3bHSD known in the literature tobe potential markers of granulosa cellsfrom competent follicle procedure, werefound in our subtracted library. A totalof 305 (46 % from human cells libraries)and 229 (78 % from human cells libraries)sequences had a strong hybridization(ratio more than 2 between competentand non-competent follicle) in the firstand second hybridization. For the humanAffymetrix GeneChip®, a total of 325 and505 genes had a strong ratio (more than2) for the two hybridizations, respectively.With the four hybridizations of the granu-losa chip and the human AffymetrixGeneChip®, a comparison betweencommon genes has been done and 96candidate genes have been identified.The differential expression of thesegenes is now being validated with real-time PCR to identify markers with highsensitivity and low false positive rate.The selected markers will then be usedwithin patients to assess their predictivevalue for single embryo transfer.

Conclusions: The microarray techno-logy is a very useful tool to discovernew genes and to provide informationin the context of oocyte competence.This technology will help to define thetranscriptome of granulosa cells presentin a competent oocyte and improve theselection of healthy oocytes and theselection of a single embryo with highpregnancy rates. (Research supportedby CIHR/IRSC and FRSQ).

O12EFFECT OF THE OCCURRENCE OF MULTI-NUCLEATED BLASTOMERES IN HUMANIVF TREATMENT

P. Fancsovits, G. Zs. Tóthné, F. Z. Takács,Á. Murber, Z. Papp, J. UrbancsekFirst Department of Obstetrics andGynaecology, Semmelweis University,Faculty of Medicine, Budapest,Hungary

Introduction: Multinucleation occurs ina high number of embryos in human

IVF treatments. However, data arelimited on the effect of multinucleatedblastomeres on embryo developmentand outcome of IVF treatment. Ourretrospective study analyses the effectof multinucleation on embryo quality andimplantation in a human IVF programme.

Material and Methods: A total of 4041embryos from 835 IVF cycles performedbetween December 2001 and July 2005were analysed retrospectively. Embryomorphology and multinucleation wereassessed 2 and 3 days after fertilization.Embryo transfer was performed on day2 or 3, depending on the number ofembryos available for transfer. Cycleswere classified into 4 groups accordingto the frequency of embryos withmultinucleation (group I: 0 %; group II:0– < 25 %; group III: 25– < 50 %; andgroup IV: > 50 %). Correlation wasanalysed between frequency of multi-nucleation and embryo morphology(embryo-specific data), implantation andpregnancy rates (cycle-specific data).Pearson chi-square-test and Student-t-test were used for statistical analysis.

Results: Embryos containing multinucle-ated blastomeres had a significantlylower cell number (3.5 ± 1.3 vs. 4.1 ± 1.3;p < 0,0001) and a significantly higheramount of fragmentation (21.2 ± 13.9 vs.16.4 ± 12.4; p < 0,0001) than embryoswithout multinucleation. There was nocorrelation between patients’ age andfrequency of multinucleated embryos.Pregnancy rate was the lowest in groupIV (25.5 %) which was significantlylower (p < 0.02) than in group I (44.9 %),group II (50 %) and group III (47.6 %).Implantation rate was also lower ingroup IV (10 %) than in group I (19.3 %),group II (20.6 %) and group III (19.6 %),however, the differences between thegroups were not significant.

Conclusions: Embryo quality is stronglyinfluenced by the occurrence of multi-nucleation. However, multinucleationseems to be independent of maternalage. Pregnancy rate was affected onlyif more than 50 % of the embryos con-tained multinucleated blastomeres,which can be because of the lowernumber of embryos with normal nuclea-tion in those cycles.

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O13SELF-CORRECTION OF CHROMOSOMALLYABNORMAL EMBRYOS AND ITS IMPLICATIONFOR PGS

T. Frumkin, Y. Yaron, M. Malcov,T. Schwartz, T. Mey-Raz, T. Carmon,T. Cohen, F. Azem, A. Amit, D. Ben-YosefRacine IVF Unit & Prenatal DiagnosisUnit, Genetic Institute, Lis MaternityHospital Tel-Aviv Sourasky MedicalCenter, Israel

Introduction: Preimplantation geneticscreening (PGS) has been proposed asa method for improving success rates inpatients with repeated IVF failures. Thisapproach is based on the hypothesisthat such failures are the result of aneu-ploid embryos. It has been suggestedthat FISH analysis of blastomeres removedfrom pre-implantation embryos repre-sents the chromosomal constitution ofthe entire embryo. Whether this alsorepresents the chromosomal constitu-tion of the implanted fetus is as yet notclear.

Aim: To ascertain PGS results of aneu-ploid day-3 embryos with FISH reana-lysis at day 5 of development and evalu-ate whether self-correction occurs, andto what extent, during further cleavage.

Material and Methods: Fifteen repeat-edly failed IVF patients with good-qual-ity embryos underwent PGS. At day 3 ofdevelopment, 1–2 blastomeres wereaspirated and FISH analysis was perfor-med, using probes for chromosomes13, 15, 16, 17, 18, 21, 22, X, Y in tworounds of hybridization. Chromoso-mally normal embryos were transferredto the uterus on day 4/5. Chromosomal-ly abnormal embryos were re-analysedon day 5, using the same probe panels.

Results: A total of 52 embryos diag-nosed as aneuploid by PGS on day 3were re-analysed on day 5. On day 3,since the average cell number of thebiopsied embryos was 7.4 ± 1.3, theanalysis could be performed on 2 biop-sied blastomeres in 31 embryos (60 %).Altogether, 46 (88.5 %) of the aneuploidembryos underwent compaction orblastulation by day 5. FISH reanalysison day 5 was based on an average of7.6 ± 4.8 blastomeres/embryo, demon-strating that 13 (28 %) were partly or

entirely normally disomic (5 embryoshad 10–25 % disomic cells, 2 had 70 %disomic cells and 6 were 100 % normal).

Conclusions: PGS re-analysis on day5 of embryos designated as “aneuploid”demonstrated that some of these em-bryos are, in fact, mosaic normally diso-mic and some even completely disomicfor the chromosomes tested. This maybe explained by 1) FISH errors that leadto a 7–15 % mis-diagnosis, 2) a highdegree of chromosomal mosaicism inday-3 embryos, which cannot alwaysbe detected by PGS of 1 or 2 blastomeres,and 3) self-correction, a mechanism ofnatural selection which operates infavor of normal cells, probably moresignificant during cleavage and propa-gation towards the blastocyst stage.These findings also suggest that PGSresults must be interpreted with caution.

O14FF-MAS PROTECTS AGED OOCYTESFROM PREDIVISION AND MODULATESGENE EXPRESSION

U. Eichenlaub-Ritter1, S. Cukurcam1, 2,E. Vogt1, C. Hegele-Hartung2,B. Lindenthal21Universität Bielefeld, Fak. Biol., Gen-technol./Biotechnol., Bielefeld, Germany,2Res. Lab., Schering AG, Berlin, Germany

Introduction: Culture of denuded mouseoocytes in α-MEM predisposes them toprecocious chromatid separation (pre-division) [1]. Follicular meiosis-activat-ing sterol (FF-MAS) significantly reducespredivision of chromatids in youngoocytes [1]. Since loss of chromosomecohesion [2], predivision [3], mitochon-drial dysfunction [4], and permissivecheckpoint control [5] are discussed inage-related aneuploidy influences ofFF-MAS on chromosomal constitution,mitochondria and gene expression wereanalysed in young and aged mouseoocytes, particularly with respect topresence of chromatids and age-relatedaneuploidy at metaphase II.

Material and Methods: Oocytes fromyoung (1–5 months) or aged (≥ 9 months)CBA/Ca mice were matured in α-MEMwith and without 10 µM FF-MAS andassessed for chromosomal constitutionby spreading and C-banding [1]. Mito-

chondrial distribution was analysed bystaining with Mitotracker TM at meiosis I,at 6.5 h of culture with and without FF-MAS. Relative concentration of mRNAcoding for the meiotic cohesion proteinSMC1β and the checkpoint proteinMAD2 was compared between meta-phase II oocytes matured without andwith FF-MAS by real time fluorescentPCR with β-actin as standard.

Results: Aged oocytes were especiallysusceptible to induction of predivisionby maturation in α-MEM, supportingthe concept of transient loss of cohe-sion due to ageing. FF-MAS reducedpredivision significantly in aged andyoung oocytes and restored clusteringof mitochondria at the spindle. How-ever, no reduction in age-related non-disjunction by FF-MAS was observed,while FF-MAS did not increase hyper-ploidy rates, either. FF-MAS increasedrelative SMC1β-mRNA-concentration inyoung and especially in aged oocytes,and it slightly increased MAD2 mRNAin the aged group.

Conclusions: Some of the beneficialeffects of FF-MAS and its analogues onoocyte quality to improve developmen-tal potential of mammalian embryos(e. g. [6, 7]) may rely on protection ofoocytes from precocious chromatidsegregation and second meiotic errorsleading to aneuploidy in the embryo.FF-MAS appears to improve oocytequality by supporting mitochondrialactivity (for instance, by enhancing localsupply of high energy substrates orcalcium signalling during maturation).The protection from predivision mayalso relate to enhanced expression ofmeiotic cohesins that mediate tightattachment of sister chromatids, thuscounteracting partially the effect ofageing, and, possibly, improving check-point control [8]. However, the postu-lated loss of cohesion between chromo-somes during the long meiotic arrest ofaged oocytes in primordial folliclesprior to resumption of maturation [2]may be irreversible by the activity ofFF-MAS during resumption of matura-tion, and therefore probably cannotprevent susceptibility to first meioticnon-disjunction. In practical terms, theobservations suggest that aged oocytesare especially susceptible to aberrationsdue to sub-optimal media during in-vitro maturation, and that FF-MAS maybe of clinical relevance to improve

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oocyte maturation and quality by pro-tection from predivision.

References:1. Cukurcam S, Hegele-Hartung C, Eichenlaub-Ritter U. Meiosis-activating sterol protectsoocytes from precocious chromosome segrega-tion. Hum Reprod 2003; 18: 1908–17.2. Hodges CA, Revenkova E, Jessberger R,Hassold TJ, Hunt PA. SMC1beta-deficient fe-male mice provide evidence that cohesins are amissing link in age-related nondisjunction. NatGenet 2005; 37: 1351–5.3. Pellestor F, Andreo B, Arnal F, Humeau C,Demaille J. Mechanisms of non-disjunction inhuman female meiosis: the co-existence of twomodes of malsegregation evidenced by thekaryotyping of 1397 in-vitro unfertilizedoocytes. Hum Reprod 2002; 17: 2134–45.4. Eichenlaub-Ritter U, Vogt E, Yin H, Gosden R.Spindles, mitochondria and redox potential inageing oocytes. Reprod Biomed Online 2004;8: 45–58.5. Steuerwald N, Cohen J, Herrera RJ, Sanda-linas M, Brenner CA. Association between spin-dle assembly checkpoint expression and mater-nal age in human oocytes. Mol Hum Reprod2001; 7: 49–55.6. Marin Bivens CL, Grondahl C, Murray A,Blume T, Su YQ, Eppig JJ. Meiosis-activatingsterol promotes the metaphase I to metaphase IItransition and preimplantation developmentalcompetence of mouse oocytes maturing in vitro.Biol Reprod 2004; 70: 1458–64.7. Marin Bivens CL, Lindenthal B, O’Brien MJ,Wigglesworth K, Blume T, Grondahl C, Eppig JJ.A synthetic analogue of meiosis-activating sterol(FF-MAS) is a potent agonist promoting meioticmaturation and preimplantation development ofmouse oocytes maturing in vitro. Hum Reprod2004; 19: 2340–4.8. Cukurcam et al., Hum. Reprod. sub-mitted.

O15GENETIC MOSAICISM IN DONATED CRYO-PRESERVED 8-CELL EMBRYOS OF GOODQUALITY

K. Peeters1, K. Moordtgat2, E. VanAssche2,U. Punjabi1

1Center for Reproductive Medicine and2Center for Medical Genetics, Universityof Antwerp, Belgium

Introduction: The occurrence of geneticmosaicism is well documented amonghuman embryos. However, only fewstudies analysed mosaicism on wholeembryos with normal development onday 3. Most studies are confined to

embryos unsuitable for transfer or cryo-preservation on day 3 or to blastocystsor to genetically abnormal embryos.The aim of this study was to determinethe degree of mosaicism in donatedgood quality embryos on day 3 by ana-lysing as many blastomeres as possibleper embryo.

Material and Methods: Embryos includ-ed in this study were cryopreserved in1.5 M DMSO and 0.1 M sucrose onday 3 after IVF or ICSI for later replace-ment in another IVF cycle, they had atleast 6 blastomeres and less than 20 %fragmentation. Embryos were eitherdonated for research before thawing orwere unsuitable for transfer or overnightculture after thawing. In total, 34 embryoswere thawed by stepwise dilution of thecryoprotectants and fixed as wholeembryos in 0.1 % Tween, 0.01N HCl.FISH was carried out for chromosomes13, 18, 21, X and Y.

Results: 185 blastomeres of 28 em-bryos (mean of 6.6 blastomeres perembryo) gave interpretable results forat least 4 chromosomes in at least 4blastomeres.

Only 8 embryos (28.6 %) were uni-formly diploid (n = 53 blastomeres) and9 (32.1 %) were limited mosaics (≥ 75 %2N-cells; n = 53). In these 17 genetically“normal” embryos, 11 blastomeres outof 106 were found to be abnormal(10.4 % false positive results). 6 embryos(21.4 %) were extensive mosaics (< 75 %and > 40 % 2N-cells; n = 39) with amean of 56.4 % diploid cells. 5 embryos(17.9 %) were abnormal (< 40 % 2N-cells; n = 40). Of these only one wasuniformly abnormal. In the remaining4 abnormal mosaics, 5 out of 36 blasto-meres were normal diploid (13.9 % falsenegative results). Of these embryos, 3were chaotic embryos without implan-tation potential but 1 was a trisomy 13with 1 diploid cell out of 6.

Conclusion: Mosaicism can be frequentamong day-3 embryos of good quality.

O16OBJECTIVE CHARACTERISATION OF PRONUC-LEAR ZYGOTES USING AN IMAGE ANALYSISSOFTWARE

F. Urner1, 2, A. Beuchat3, A. Chanson1,M. Germond1, 2, A. Senn1 ,2

1Medically Assisted Procreation Centre,Lausanne, Switzerland, 2F.A.B.E.R.Foundation, Lausanne, Switzerland,3Swiss Federal Institute of Technology,Biomedical Imaging Group, Lausanne,Switzerland

Introduction: Identifying embryos withhigh implantation potential is a pre-requisite for decreasing the number ofembryos transferred without compro-mising pregnancy outcome. In Switzer-land, analysis of 2PN-zygotes consti-tutes, through legal constraints, theunique way to identify viable embryos.This situation offers a unique opportu-nity to study the value of early zygotescoring for prognostic evaluation ofembryo implantation potential. In orderto reduce both the time needed and thesubjectivity of the 2PN-zygote scoring,a computer programme allowing a fastand semi-automatic quantitative measure-ment of a high number of features fromdigital images of 2PN-zygotes was de-veloped. The purpose of this study wasto determine to what extent this toolmight be useful in a clinical setting.

Material and Methods: A plug-in for theimage processing programme ImageJ(http://rsb.info.nih.gov/ij/) was devel-oped to analyse 2PN-zygote digitalimages. Zygotes were photographed17–20 h after insemination underHoffman contrast (Eyeware camera,Octax, Germany). The programmedetects automatically the oolemma andpronuclei outlines, measures distancesand angles, and calculates surfaceswithin the zygote. All measurements aresaved for subsequent statistical analysis.The main studied features were: PNsizes, PN position, cytoplasmic Haloarea, distribution of nucleolar precursorbodies (NPB). Each PN and its respec-tive NPB were analysed separately, PN1being the PN with the highest numberof NPB and possibly the male pronu-cleus. A sensitivity (% of true positives)and specificity (% of true negatives)analysis for different cut-off values ofthe measured parameters was done first

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on 118 transferred zygotes with knownindividual implantation outcome (set A).To limit the number of false positives(zygotes not implanted despite a goodscore), threshold values associated withhigh specificity (80 %) were selectedfor each parameter and their respectivepositive predictive values calculatedon basis of a mean implantation rate of15 %. To determine the importance ofthe above parameters on implantationof zygotes randomly allocated for trans-fer, images of 188 zygotes transferredon day 2–3, in consecutive non-se-lected IVF cycles were prospectivelyrecorded and analysed (set B).

Results: Specificity/sensitivity analysisshowed that, for all studied parameters,high specificity of 80 % was associatedwith limited sensitivity ranging from10 % to 31 % and positive predictivevalues ranging from 14 % to 27 %. Inset B, the only parameter which appearsto influence implantation was the disper-sion of NPB in PN2. Although not signifi-cant (p = 0.062), IR was higher (14/65,21.5 % vs. 14/123, 11.4 %) when atleast one zygote transferred presentedNPB global dispersion < 11 µm in PN2.

Conclusions: Computer-assisted analy-sis of zygotes allows fast and precisemeasurements of a number of morpho-logical parameters on digital images.When evaluated individually, the pa-rameters considered in this study didnot display a high predictive value ofimplantation. In a future study, we in-tend to use another statistical approachto evaluate the effect of combiningseveral of the measured parameters forprediction of implantation.

O17THE OCCURRENCE OF HIGH PRONUCLEUSSCORES IS NOT AGE DEPENDENT AND ISNOT RELATED TO THE TOTAL NUMBER OFRETRIEVED OOCYTES

M. J. Van den Bergh, M. Fahy-Deshe,K. Fluegel, S. Ruflin, C. Urech, A. Siragusa,J. Stutz, A. Kratzer, N. Fashing,M. K. HohlFertility Laboratory, Kinderwunsch-zentrum, Kantonsspital Baden, Switzerland

Introduction: Pronucleus Z1-score asdescribed by Scott et al. is associated

with a higher pregnancy rate. Little isknown about factors that might influ-ence the frequency of occurrence ofthe best Z-scores. We explored retro-spectively if the patient’s age and thetotal number of recovered oocytesinfluence the appearance of the high-est Z-scores.

Material and Methods: For 215 con-secutive ICSI treatment cycles with freshejaculated sperm, a total number of1145 zygotes were scored and classi-fied as Z1, Z2, Z3 and Z4; Z1 being thebest score. The 215 cycles were dividedin 3 age groups: < 34 years, ≥ 34 and< 38 years and ≥ 38 years old and thedistribution of the different Z-scoreswas analysed. The same 215 cycleswere divided in 3 groups according tothe total number of retrieved oocytes:< 5, ≥ 5 and < 10 and ≥ 10 oocytes andthe distribution of the Z-scores wasanalysed.

Results: The distribution in the threeage groups was for Z1: 17.8 % (91/511),16.6 % (61/368) and 19.2 % (51/266);for Z2: 16.6 % (85/511), 18.2 % (67/368)and 16.2 % (43/266); for Z3: 58.9 %(301/511), 53 % (197/368) and 56.7 %(151/266); for Z4: 6.6 % (34/511), 1.6 %(43/368) and 7.8 % (21/266). No signi-ficant differences were observed. Theimplantation rate in the 3 age groupswas 27.4 % (43/157), 22.8 % (29/127)and 9.6 % (11/115). The frequency ofZ1-score for less than 5 recovered was9 % (29/150), for ≥ 5 and < 10 oocytesalso 19 % (88/476) and for more than10 recovered oocytes 16 % (86/522); forZ2 the distribution was 15 % (23/150),15 % (85/473) and 17 % (87/522); forZ3 56 % (84/150), 52 % (247/473) and61 (318/522); a Z4-score occurred in9 % (14/150), 11 % (53/473) and in6 % (31/522). The implantation rates forthese groups were 14.7 % (16/109),20.4 % (36/176) and 27.2 % (31/114).The overall clinical pregnancy rate was32 % (69/215) including 9 twins and1 triplet. The mean clinical pregnancyrate after ICSI in 2004 was 25.9 % asreported by FIVNAT-CH (www.sgrm.org),the Swiss National Register.

Conclusions: The frequency of the bestpronucleus score (Z1) appears to beindependent of the patient’s age and ofthe number of retrieved oocytes.

O18Z-SCORE IS NOT PREDICTIVE FOR THEPOST-THAW SURVIVAL BUT FOR THEPREGNANCY RATE

M. J. Van den Bergh, M. K. Hohl,M. Fahy-Deshe, K. Fluegel, S. Ruflin,C. Urech, J. Stutz, A. Siragusa, A. Kratzer,N. FaschingFertility Laboratory, Kinderwunsch-zentrum, Kantonsspital Baden,Switzerland

Introduction: The assessment of thepronuclear morphology according to ascale proposed by Scott et al. is a com-monly used method to evaluate thecompetence of the obtained zygotes.This scoring system is especially valu-able in countries like Switzerland,where a maximum of 3 zygotes can bekept in culture and embryo freezing isprohibited. We evaluated the impact ofthe pronuclear score on the post-thawsurvival.

Material and Methods: For 438 zygotesfrozen with the classical propanediol-sucrose method (Lasalle & Testart) weanalysed the post-thaw recovery, thecleavage rate, the cleavage state, theembryo quality and the pregnancy ratein relation to the pronucleus Z-scoreobserved 18 hours after ICSI just beforecryopreservation.

Results: Of 44 zygotes with Z1-score30 (68 %) were intact after thawing,of 47 with Z2-score 35 (74 %), of 306with Z3-score 220 (72 %) and of 41with a Z4-score 25 (61 %). There wasno statistical difference in post-thawsurvival. The cleavage rate was similarin all groups: 90 % (27/30) for Z1-score,77 % (27/35) for Z2-score, 79 % (174/220) for Z3-score and 84 % (21/25) forZ4. No statistical differences were observ-ed. The mean embryo score – definedby the number of blastomeres, theirregularity and the presence of cytoplasmfragments – was 2.1 ± 0.86 for Z1,2.5 ± 1.28 for Z2, 2.4 ± 1.0 for Z3 and2.9 ± 1.28 for Z4. The difference inembryo scores between Z4 and Z3 orZ2 was significant (p = 0.02; p = 0.01).When at least 1 embryo was transferredafter thawing from a zygote with Z1-score, 38 % (8/21) positive pregnancytests per transfer were obtained, with aclinical pregnancy rate per transfer of

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29 % (6/21). When only embryos issuedfrom thawed zygotes with an initialscore of Z3 were transferred, we ob-tained 17 % (18/106) positive preg-nancy tests with a clinical pregnancyrate of 14 % (15/106). When no Z1s butat least 1 Z2-zygote contributed to thetransfer we observed a clinical preg-nancy of 8 % (2/24). The observed dif-ferences in pregnancy rates were sig-nificant. The overall clinical pregnancyrate per transfer obtained with those438 thawed zygotes was 18 % (30/168)and corresponds to the mean value of18.3 % reported by the national registerFIVNAT in 2004.

Conclusion: Embryos obtained afterthawing of zygotes with the best pronu-cleus score result in significantly higherpregnancy rates. The post-thaw survivaland cleavage is not related to the pro-nuclear score.

O19PREGNANCY RATES OF BIOPSIED CATTLEEMBRYOS

E. Mullaart, T. Otter, J. S. MertonHG, R&D, Arnhem, The Netherlands

Introduction: DNA analysis of an em-bryo biopsy is becoming an importantmethod to diagnose embryos for thepresence of particular genetic diseasesin humans but also in cattle. However,it is important that the pregnancy rate ofthe embryo is not too much diminishedby the biopsy procedure. With humanembryos it is often difficult to obtaininformation about the effect of needlebiopsy on pregnancy rates followingembryo transfer. For cattle embryos,however, this information (after bladebiopsy) is available for high numbers ofembryos and these data will be pre-sented here.

Material and Methods: Cattle embryoswere obtained after superovulation (SO)or by in-vitro production (IVP). For SO,female donor animals were treated withFSH, inseminated with semen and sub-sequently embryos were retrieved fromthe uterus by flushing at day 7 of theestrus cycle. For IVP, immature oocyteswere recovered by Ovum Pick Up,matured and fertilized in-vitro and sub-sequently cultured for 6 days.

Both SO and IVP embryos (both blasto-cysts and morulae) were biopsied byblade biopsy. During this procedure theembryo was immobilized on the bottomof a petri-dish and a small part (about10 cells) was removed with a knife. TheSO embryos (both biopsied and non-biopsied) were frozen using ethyleneglycol as a cryoprotectant and stored inliquid nitrogen until transfer. The IVPembryos were transferred fresh.

The embryos were transferred non-surgi-cally into recipient animals, whichwere at day 6, 7 or 8 of their estruscycle and pregnancies were scored5 months after transfer by rectal palpa-tion. The (raw) data were analysed bychi-square-test.

Results: The pregnancy rate for thenon-biopsied SO embryos was 60.4 %(n = 956) compared to 46.1 % (n = 421)for biopsied embryos (p < 0.001), indi-cating a 14.3 % lower pregnancy ratewith biopsied embryos. Despite a nu-meric difference, the pregnancy rate ofIVP embryos was not significantly af-fected (52.2 % [n = 697] and 44.4 %[n = 41] for non-biopsied and biopsiedembryos, respectively).

Conclusion: The use of the blade biopsytechnique on cattle embryos has anegative effect on the pregnancy rate.This effect is larger when embryos arefrozen after biopsy as is done for the SOembryos. Perhaps the combination oftaking a biopsy and subsequent freezingof the embryos is too harmful, resultingin a decrease in pregnancy rates. It isalso conceivable that a needle biopsy,as is routinely done with human em-bryos, is less harmful and results insmaller effects on pregnancy rates.However, needle biopsies can not bedone easily in a cost-effective manneron large numbers of embryos, while thisis possible with blade biopsy.

Despite the effect on pregnancy rate,blade biopsy followed by different DNAtests (for disease and sex) is a routineprocedure now in our company. Mo-mentary work is in progress to minimizethe effect of the biopsy method and toperform pre-amplification on the em-bryo biopsy in order to be able to domore (100–1000) different DNA tests.

O20REPLACEMENT OF CRYOVIALS BY HIGH-SECURITY STRAWS IN A HUMAN ZYGOTECRYOPRESERVATION PROGRAMME AFFECTSTHE SURVIVAL RATE BUT NOT THE CLINICALPREGNANCY RATE

M. Van den Bergh, M. Fahy-Deshe,K. Fluegel, S. Ruflin, C. Urech,A. Siragusa, J. Stutz, A. Kratzer, N. Fashing,M. K. HohlFertility Laboratory, Kinderwunsch-zentrum, Kantonsspital Baden,Switzerland

Introduction: Cryovials with or withoutring, used for freezing of gametes andembryos, are not leak-proof. Up to 45 %of the vials leak after 3 hours of submer-sion in liquid nitrogen, generating a riskof microbial contamination and explo-sion. Cryovials were replaced by heat-sealed high-security straws and ananalysis of the outcome in terms ofsurvival and pregnancy rate was made.

Material and Methods: Three differentprotocols for zygote-freezing were used.Group 1: cryovials in combination withfreezing with 1.5 Propanediol (PROH) inPhosphate Buffered Saline (PBS), group 2:high security straws in combination with1.5 PROH in PBS and group 3: high secu-rity straws in combination with 1.5 MPROH in Hepes Buffered Human TubalFluid Medium (MHTFM) supplementedwith 10 % Serum Substitute Supplement(SSS) and 0.1 M Sucrose. Freezing wasperformed according to the classicalLasalle-Testart protocol, with manualseeding at –7 °C and warming up in a37 °C water bath. Dilution of the cryopro-tectant was performed in 3 steps for thePROH-PBS groups and in 2 steps for thePROH-MHTFM group.

Results: In group 1: 765 zygotes werethawed, 553 (72 %) were intact and455 (59.5) did cleave by the next day,corresponding to a cleavage rate of82.2 % of the intact zygotes. A total of73 clinical pregnancies were obtainedafter 254 transfers corresponding to apregnancy rate (PR) per transfer of28.7 % or 24.8 % per thawing cycle,taking in consideration that 41 patientsdid not have a transfer. The implantationrate (IR) per replaced embryo was cal-culated from the observed foetal heartactivities as 19.7 % (90/455) or 11.8 %

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(90/765) per thawed zygote. Eleventwins and 3 triplets were observed. Ingroup 2: 297 zygotes were thawed, 137(46.1 %) were intact and 130 (95.9 %)cleaved. The clinical pregnancy rate pertransfer was 29.7 % (22/74) or 23.9 %(22/92) per thawing procedure. TheI.R. per replaced embryo was 20.8 %(27/130) or per thawed zygote 9.1 %(27/297). Four twin pregnancies but notriplets were observed. In group 3:159 zygotes were thawed, 117 (73.5 %)were intact and 105 (66 %) cleaved,corresponding to a 89.7 % cleavagerate. A 28 % (16/57) clinical pregnancyrate per transfer and a 26.6 % clinicalpregnancy rate were obtained. The I.R.per replaced embryo was 16 % (16/105)and 10.2 % per thawed zygote. Nomultiple pregnancies were observed.

Conclusion: Replacement of cryovialsby high-security straws for human zy-gote cryopreservation resulted in asignificant (p < 0,01) drop in the sur-vival rate. This drop was compensatedby changing to a Hepes-buffered me-dium containing 0.1 M sucrose and10 % SSS and a long-term effect on thecumulative pregnancy rate has beenreduced to a minimum.

Poster Presentation

P1PGD IN COUPLES WITH 47,XXY AND47,XYY KARYOTYPES AND/OR ABNORMALSPERMATOZOA

V. Peinado, L. Rodrigo, E. Mateu,A. Mercader, T. Pehlivan, I. Perez,M. Gil-Salom, J. Remohi, A. Pellicer,C. RubioInstituto Valenciano de Infertilidad,IVI Valencia, IVI Alicante, Spain

Introduction: Fluorescence in-situhybridization (FISH) studies on spermfrom patients with 47,XXY and 47,XYYkaryotypes show a high incidence ofsex chromosomal abnormalities and/ordiploidy, similar to those observed ininfertile patients with severe male fac-tor and normal karyotypes. The objec-tive of this study was to assess if theincreased incidence of sperm chromo-somal abnormalities observed in these

the control group (22.9% vs. 8.3%;p < 0.05) and the incidence of mosai-cism was also higher (25 % vs. 10.3 %).In group 2, there was an increasedincidence of abnormal embryos com-pared to controls (59.7 % vs. 33.5 %;p < 0.05). And again, there was a sig-nificant increase in embryos with sexchromosome disomy (15.8 %; p < 0.05)and mosaicism (36.6 %; p < 0.05). Nodifferences in PGD outcome were ob-served among the three groups, in termsof clinical pregnancies (37.5 %, 55.0 %and 43.8 %, respectively), implantation(41.7 %, 45.1 % and 35.3 %, respec-tively), and miscarriage rates (0 %,9.1 % and 14.3 %, respectively).

Conclusions: Couples with an abnormal47,XXY or 47,XYY karyotype and withan additional abnormal FISH result onsperm showed the highest risk of abnor-mal embryos in a PGD programme.Intermediate risk was observed in patientswith normal karyotype and increasedincidence of sex disomies and diploi-dies in sperm. The high incidence ofsex chromosome disomies and diploi-dies in sperm was positively correlatedwith an increased percentage of em-bryos with sex chromosomal abnor-malities and mosaicism.

P2PGD OUTCOME AND EMBRYO DEVELOPMENTIN PATIENTS WITH STRUCTURAL CHROMO-SOME ABNORMALITIES

E. Mateu, L. Rodrigo, A. Mercader,P. Buendía, T. Viloria, J. Remohí,A. Pellicer, C. RubioInstituto Valenciano de Infertilidad,Valencia, Spain

Introduction: The objective of this workis to evaluate the efficacy and clinicaloutcome of preimplantation geneticdiagnosis (PGD) using fluorescence in-situ hybridization (FISH) in coupleswith structural chromosome abnorma-lities.

Material and Methods: A total of 38 PGDcycles was performed in 27 coupleswith Robertsonian translocations, and46 PGD cycles in 41 couples with otherchromosomal rearrangements, includ-ing reciprocal translocations, inversionsand deletions (January 1997 to Decem-

two groups of patients could be trans-lated into a higher rate of embryonicchromosomal abnormalities on day-3embryos.

Material and Methods: Preimplantationgenetic diagnosis (PGD) for aneuploidyscreening was performed and threegroups of patients were included.Group 1 consisted of 8 PGD cycles in5 patients with 47,XXY or 47,XYYkaryotypes. Sperm FISH analysis forchromosomes 13, 18, 21, X and Y wasalso performed in 3 of these patients.Group 2 was formed of 48 PGD cyclesin 36 couples with normal karyotypesand classified as severe male infertilitybecause previous FISH analysis onsperm showed significant increases insex chromosome disomy and/or dip-loidy compared to normozoospermicfertile donors. A control group of 35PGD cycles in fertile young patientswho underwent PGD for sex-linkeddiseases was also included. Femaleage in all PGD groups was ≤ 37.Embryo biopsy was performed on day 3and chromosomes 13, 15, 16, 18, 21,22, X and Y were analysed by FISH.All probes were available from VysisInc., Downers Grove, Il., USA. Mosai-cism was estimated as discordant resultswhen two blastomeres from the sameembryo were analysed. Statistical analy-sis was done using chi-square-test,student-t-test and Welch-t-test whenappropriate.

Results: The incidence of chromoso-mal abnormalities in sperm samplesfrom 2 patients with 47,XXY and47,XYY karyotypes was significantly(p < 0.0001) increased compared tofertile donors, mostly due to higher sexchromosome disomy (1.5 % vs. 0.4 %)and diploidy (0.9 % vs. 0.2 %). Theseresults were similar to our previousobservations in severe male-factor pa-tients with normal karyotype and abnor-mal FISH results (0.88 % sex chromo-some disomy and 0.95 % diploidsperm).

In group 1, 52.1 % of embryos werechromosomally abnormal and even ahigher percentage of abnormal embryos(85.7 %) was observed in the subgroupof patients with an increased incidenceof sex chromosome disomy in sperm(p < 0.05 compared to control group).Sex chromosome aneuploidy was sig-nificantly increased compared to

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ber 2005). Embryo biopsy was per-formed on day 3 and one or two cellswere removed from each embryo. Inter-phase nuclei were analysed by FISHwith different combinations of centro-meric, locus specific and subtelomericprobes for each of the cases evaluated(Vysis Inc., Downers Grove, Il., USA).The embryos were cultured during theanalysis and normal/balanced embryoswere transferred on day 5.

Results: A total of 226 embryos wereanalysed from carriers of Robertsoniantranslocations, and 239 embryos fromcouples with other chromosomal rear-rangements. A high percentage of un-balanced embryos were observed inboth groups but it was significantlyhigher in patients with non-Robert-sonian abnormalities than in patientswith Robertsonian translocations (83.7 %vs. 68.6 % respectively; p = 0.0002).

Regarding the effect of the chromoso-mal imbalance in embryo development,blastocyst rates were significantly reduc-ed in unbalanced embryos from carriersof Robertsonian translocations com-pared to normal/balanced embryos(21.3 % vs. 49.1 %; p = 0.0006). Embryoswith monosomies showed the lowestblastocyst rate compared to normal/balanced embryos (17.5 % vs. 49.1 %;p < 0.0006), whereas embryos withtrisomies did not show differences withnormal/balanced embryos (29.4 % and49.1 %, respectively). Surprisingly, em-bryo development for other non-Robert-sonian structural abnormalities did notfollow this pattern, with similar blasto-cyst rates for both, normal/balancedand unbalanced embryos (61.3 % and50.3 %, respectively).

Significant differences were observed inthe percentage of cycles with at leastone normal/balanced embryo for trans-fer (81.6 % in Robertsonian transloca-tions vs. 47.8 % in non-Robertsonianstructural abnormalities; p = 0.003).Despite this, similar pregnancy (35.5 %and 54.5 %) and implantation (29.4 %and 35.3 %) rates were achieved inRobertsonian and non-Robertsoniancarriers. Likewise, the percentage ofmiscarriages was similar in both groups,and the cytogenetic study after hystero-embrioscopy in one miscarriage reveal-ed a trisomic foetus for chromosome 7(chromosome not involved in the rear-rangement). All the amniocentesis per-

formed showed normal or balancedkaryotypes as well as the karyotype intwo liveborns.

Conclusions: We can conclude thatthere is a high incidence of unbalancedembryos in carriers of both Robertsonianand non-Robertsonian rearrangements.

Additionally, unbalanced embryos canreach the blastocyst stage in both groupsof rearrangements. Although the blasto-cyst rates in normal/balanced and un-balanced embryos in non-Robertsonianrearrangements were similar, a signifi-cantly lower number of unbalancedembryos in Robertsonian translocationreached the blastocyst state, probablyas a reflection of the comparativelyhigher chromosome imbalance in theseembryos. Therefore, PGD is an effectiveand reliable approach in these couplesto decrease the risk of miscarriage andunbalanced offspring.

P3A UK EXPERIENCE OF OOCYTE CRYOPRESER-VATION – 5 YEARS ON

G. Aldis, S. Barlow, G. LockwoodMidland Fertility Services, Aldridge, UK

Introduction: Oocyte cryopreservationis an attractive proposition for manypatients, especially women whose ferti-lity is at risk. Although the post-thawsurvival rates in the past have beendisappointing, recent developments,such as the increase in sucrose concen-tration and the use of ICSI, have showna promising increase in oocyte survivaland pregnancy rates, approaching thoseof embryo freezing.

Material and Methods: A slow-freezeprotocol was adopted for the cryopre-

servation of Metaphase II oocytes using1.5 mol/l PROH and 0.3 mol/l sucrosefreezing solution. A total of 732 oocyteswere stored for 66 patients for purposessuch as fertility preservation (47 %) andreligious objections to embryo cryopre-servation (15.2 %). 11 patients under-went thaw cycles using the rapid-thawtechnique and the surviving oocyteswere inseminated using ICSI.

Results: Here we present the results ofour first thaw cycles. 88 oocytes werethawed and 59 survived (67 %). 47oocytes were injected, of which 30 ferti-lised normally (63.8 %). 28 embryoscleaved, giving a cleavage rate of 93.3 %and an average of 1.64 embryos weretransferred in 14 embryo transfers for11 patients. 3 clinical pregnancies re-sulted plus an additional biochemicalpregnancy. To date, we report the birthof three girls (1 twin) and one boy. Thisgives a live birth rate of 21.4 % perembryo transfer.

Conclusions: Although still small num-bers, our experience shows the survivalrates and live birth rates for oocytecryopreservation are encouraging towomen whose fertility may becomecompromised.

P4IMPORTANCE OF AN AIR PURIFICATIONSYSTEM IN THE IVF LABORATORY

M. Janssen, M. Nijs, A. Cox,E. Vanheusden, W. OmbeletGenk Institute for Fertility Technology,ZOL, Genk, Belgium

Introduction: The concentration of in-door pollutants can reach levels up to8 to 10 times the outdoor environment.These pollutants consist of chemical air

Table 1: Janssen et al.

Without WithAir purification filter Air purification filterPeriod 1 Period 2

Oocyte collections withembryo transfer 282 310Mean number of embryosper transfer 1.3 1.3Pregnancy rate per transfer 73/282 25.9 % 90/310 29 % p = 0.39Ongoing pregnancy rate 38/282 13.5 % 69/310 22.2 % p = 0.005

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contaminants (CACs), volatile organiccompounds (VOCs) and heavy metals.The most common pollutants detectedin an IVF laboratory are benzene, tolu-ene, freon, isoproponal, acetone andcarbon. This retrospective study evalu-ates the outcome of IVF/ICSI treatmentsduring 1 year and this before and afterthe installation of air purification filterson gas bottles and in all incubators inthe IVF laboratory.

Material and Methods: The outcome of282 subsequent IVF/ICSI cycles (period 1)is compared with 310 subsequent IVF/ICSI cycles from the same year. Thelatter 310 cycles were performed in thelaboratory where the air purificationsystem (Coda filter) had been installed(period 2).

Results: Table 1.

Conclusions: The implantation rate ofIVF/ICSI embryos significantly improvedafter the installation of an air purificationsystem in incubators in the IVF labora-tory. The presence of VOCs and CACshas a clear influence on the embryonidation and development of the earlypregnancy and has to be removed fromthe culture environment.

P5SPERM PARAMETERS AND DNAFRAGMENTATION

M. Nijs1, A. Cox1, M. Janssen1,E. Bosmans1, M. Laframboise1, A. AlonsoAbas2, C. De Jonge3, W. Ombelet1

1Genk Institute for Fertility Technology,ZOL, Genk, Belgium, 2Center for Statis-tics, University Hasselt, Diepenbeek,Belgium, 3Reproductive MedicineCenter (RMC), University of Minnesota,Minneapolis, USA

Introduction: Sperm DNA fragmentation(DF) can contribute to the abnormaldevelopment of embryos and to theirimplantation failure. The Sperm Chro-matin Structure Assay (SCSATM) can beused to determine the degree of DNAfragmentation (DFI) in human spermato-zoa.

The aim of this study is to investigate if acorrelation exists between sperm con-centration, motility and morphology of

a sperm sample and the DNA fragmen-tation as measured by SCSA.

Material and Methods: 212 patientsentering the IVF/ICSI programme wereasked to produce a semen sample (after2 days of abstinence) within 5–7 days ofthe actual IVF/ICSI attempt. Sperm sam-ples were evaluated for count, motilityand morphology (WHO criteria andKruger strict criteria). They were frozenwithin 30 minutes and shipped to RMC(USA) for SCSATM analysis. Statisticalanalysis included non-parametric linearregression.

Results: Table 2.

Conclusions: The data presented heredemonstrate a direct inverse correlationbetween DFI values and sperm concen-tration, motility and morphology.

P6INTEGRATION OF MULTIMORPHOLOGICALEMBRYO PARAMETERS FOR THE SELECTIONOF TOP-QUALITY EMBRYOS WITH THE BESTIMPLANTATION POTENTIAL

L. Samuelov, T. Schwartz, T. Cohen,A. Carmon, N. Mei-Raz, F. Azem, A. Amit,D. Ben-YosefRacine IVF Unit, Lis Maternity Hospital,Tel-Aviv Sourasky Medical Center,Tel-Aviv, Israel

Introduction: Implantation rate (IR) perembryo in IVF is low (10–15 %) whilethe multiple pregnancy rate with itswell-known complications, is high(20–30 %). Improved selection criteriafor top-quality transferred embryos withthe best implantation potential areneeded. Various morphological struc-tures in the embryo have been shownto correlate with implantation potential,and new parameters significantly im-proved selection criteria of top qualityembryos.

Aim: To evaluate several morphologicalparameters from fertilization throughday 3 of development in order to designan integrative embryo scoring systemfor improving selection criteria for topquality embryos with the best implanta-tion potential.

Methods: All patients ≤ 40years oldwith ≤ 3 previously failed IVF cycles,undergoing IVF/ICSI between October2004–October 2005 were included.A subgroup of all cycles with 100 %implantation rate (64 embryos) was alsoanalysed. Embryos were assessed at4 time points during development andtheir pronuclear morphology, timing offirst mitotic cleavage, cleavage rate andpattern, degree of fragmentation, theappearance of multi-nucleated blasto-meres and compaction were recorded.All parameters, individually and inte-gratively, were then correlated to clini-cal pregnancy rate (PR) and implanta-tion.

Results: There were 183 study patientsand 248 cycles. Significantly higherpregnancy rates were obtained withembryos displaying < 10 % fragmenta-tion and good cleavage patterns ondays 2 and 3 (p < 0.05). Embryos with> 7 blastomeres on day 3 were morelikely to implant than less advancedones (p < 0.028). Compacted embryosimplanted 3-fold more than non-com-pacted embryos (OR. 3.116). Analysisof the subgroup of all cycles with a100 % implantation rate revealed abetter prognosis for patients with maleand/or mechanical factor and for pa-tients who had already experiencedpregnancy. Among the implantedembryos, 71 % had early first cleavage,> 80 % had normal cleavage rate(4 and 8 cells on days 2 and 3, respec-tively) and 95 % had < 10 % fragmenta-tion. Good cleavage patterns (85 % ofimplanted embryos) and a single visiblenucleus (88 % of implanted embryos)were also good factors for predictingimplantation.

Conclusions: Comprehensive assess-ment/documentation of embryo mor-phology at various stages of develop-ment increases our ability to choosetop-quality embryos with the best im-plantation potential. Integrated analysisof these data will enable a more reli-able embryo scoring system for betterIR.

Table 2: Nijs et al.

Confidenceinterval 2.5 % 97.5 %Concentration 0.00003841 0.0369586Motility A+B 0.00012841 0.0006710Morphology 0.00069184 0.0040031

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P7REDUCED PERFORMANCE OF EMBRYOSFROM FROZEN OOCYTES

L. De Santis1, G. Coticchio2, I. Cino1,A. Bonu2, E. Rabellotti1, E. Papaleo1,F. M. Fusi1, A. Borini21IVF Unit, H S. Raffaele, UniversityVita-Salute, Milan, Italy, 2TecnobiosProcreazione, Bologna, Italy

Introduction: In the last few years, nu-merous pregnancies from cryopreservedoocytes have been achieved. However,detailed information on the possibleimpact of freezing-thawing on oocyteviability is still limited and in somecases controversial. In this study, wecompared the fertilization potential andearly development of sibling fresh andfrozen-thawed oocytes stored with twoalternative slow-cooling protocols.

Material and Methods: Oocytes in-cluded in this study were used for thetreatment of infertile couples. Controlledovarian hyperstimulation was inducedwith standard long protocol. After re-trieval, oocytes were cultured 1–2 hoursand assessed with respect to their mei-otic status after removal of surroundingcumulus cells. Only oocytes showingan extruded polar body I were eitherused fresh or frozen after culture for atotal period of time of about 4 hoursfollowing retrieval. Cryopreservationwas conducted with a slow-coolingprotocol based on a freezing solutioncontaining 1.5 mol/l PrOH as intracellu-lar cryoprotectant (CPA) and either 0.1(A) or 0.3 (B) mol/l sucrose as extracellu-lar CPA. Sperm microinjection was per-formed in fresh oocytes about 5 hoursfollowing retrieval, and in frozen oocy-tes about 1 hour after completion of thethawing procedure. Frequencies of ferti-lization, cleavage, as well as the pro-portion of good quality day-2 embryosgenerated from fresh and frozen-thawedoocytes were compared statistically withthe chi-square-test.

Results: Concerning cryopreservationprotocol A, 375 oocytes were thawed,with a survival rate of 21.9 %. Followingsperm microinjection, fertilization was69.8 % and 44.2 % in fresh and frozen-thawed oocytes, respectively (p < 0.05 %).In the fresh group, 126 (94.8 %) embryoswere obtained, while in the frozen co-

hort 29 (85.7 %) fertilized oocytes un-derwent cleavage. The proportion ofgood-quality embryos in the two classeswas 36 % and 23.8 % (p < 0.05), re-spectively. Eighty-four percent of em-bryos from fresh oocytes and 76 % ofembryos in the cryopreserved groupprogressed beyond the 2-cell stage(p < 0.05). With regard to cryopreser-vation protocol B (494 oocytes thawed),the survival rate was 59.1 %. The fre-quencies of fertilization and cleavagein fresh and cryopreserved oocyteswere 73.1 % and 76.7 % (p > 0.05),and 93.7 % and 89.5 % (p > 0.05),respectively. In the fresh and frozengroups, respectively 28.6 % and 27.0 %embryos displayed good morphology,while percentages of embryos withmore than two blastomeres were 91.8 %and 77.9 % (p < 0.05), respectively.

Conclusions: In addition to ensuring ahigher survival rate, the use of increasedsucrose concentration in the freezingsolution (protocol B) coincides with afertilization rate that appears unchangedcompared to the unfrozen control. Onthe contrary, fertilization ability of oocy-tes stored with protocol A appears com-promised to a certain extent. Cleavagerate results were moderately reducedwith both protocols, although differ-ences did not attain statistical signifi-cance. Furthermore, irrespective of theprotocol used, stored oocytes developinto embryos with decreased morpho-logical quality (protocol A) and abilityto progress beyond the second cell-cycle division (protocols A and B). Thismay explain why in the large majorityof the experiences conducted so farneither protocol has proven itself ableto generate implantation rates compara-ble to those of fresh oocytes.

P8QUALITY CONTROL OF IUI/IVF DISPOSABLESAND PRODUCTS: A TWO-YEAR SURVEY

M. Janssen1, M. Nijs1, A. Cox1,E. Vanheusden1, R. Martens2,D. Wissmann2, H. Ruis2, W. Ombelet1

1Genk Institute for Fertility Technology,ZOL, Genk, Belgium,2ZBC Geertgen,De Mortel, The Netherlands

Introduction: Consumables, media andother products can be a source of toxic-

ity to the culture system of oocytes,spermatozoa and embryos.

It is crucial to identify those consum-ables/products that contain toxic sub-stances and that will have a possibletoxic effect during oocyte collection,sperm preparation, artificial insemina-tion, embryo culture and transfer.

Material and Methods: All new consum-ables and new batches were submittedto a quality control (QC) programmebefore being accepted for use in the IVFculture during two years, from 2003 till2005. Seventy-four products includingplastics, consumables, tubings andsurgical gloves were submitted to thehuman sperm survival test (SpST). TheSpST monitors the sperm survival, i.e.motility, of a normal semen sample(WHO criteria) over 24 and 96 hoursand this after being exposed to a con-sumable or product, and compares thisoutcome to the same sample that hasnot been exposed (control). When theSpST index dropped below 0.85, theproducts/consumables were consideredto be toxic.

Results: Five types of products usedduring the oocyte collection procedurewere found to be toxic: an SpST indexof 0 was noted (zero sperm viability)24 hours post exposure. The productswere 8 brands of non-powdered surgi-cal gloves, 2 types of hysterometers andone type of tubing used for the oocytecollection needle. These products andmaterials all contained silicone, latex ornitrile components. The uncoated poly-styrene cover of a petridish, used up tothat point for the ICSI procedure, alsoshowed a toxic effect. The cover of onetype of semen receptacle, coated withsilicone, could also be identified astoxic by the SpST.

Discussion: The SpST clearly identified5 types of products with absolute toxic-ity to human spermatozoa. As a conse-quence, these products/consumableswere rejected for use in the IVF systemand an alternative had to be sourced.

Conclusion: The SpST is an inexpensiveand easy method to identify potentiallytoxic products and consumables usedfor all IVF procedures. The inclusion ofthe SpST in our continuous monitoringprogramme of IVF results (fertilizationrate, embryo development, mulinuclea-

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tion, implantation rate and ongoingpregnancy rate) has been proven to bea valid strategy.

P9ASSISTED HATCHING BY LASER FORFROZEN-THAWED HUMAN EMBRYOS

M. Nijs, D. Stevens, A. Cox, M. Janssen,W. OmbeletGenk Institute for Fertility Technology,ZOL, Genk, Belgium

Introduction: Cryopreservation of hu-man embryos could leave the embryoswith a hardened zona pellucida (ZP),resulting in a lower hatching and subse-quently lower implantation potential.Assisted hatching by laser prior to trans-fer could facilitate the hatching proce-dure of the frozen-thawed embryos.This prospective randomised studyevaluates the possible beneficial effectof lasering of the ZP as an assistedhatching.

Material and Methods: Day-3 humanembryos (grades A and B) were frozenand thawed using the slow Propane-diol-Sucrose protocol. The embryoswere placed in culture until day 4.Embryos were allocated to the control(zona intact) group or to the laser group(zona lasering) according to their thaw-ing number. Prior to transfer, an open-ing was created in the ZP with two300 mW laser pulses using the Hamil-ton Thorn Zilos-system.

Results: Table 3

Conclusions: Assisted hatching did notimprove the implantation rate of frozen-thawed day-3 embryos. A trend towards

lower implantation rates could be ob-served when transferring one or twoembryos with a lasered ZP. The nega-tive impact of the laser pulse on furtherembryo development has to be investi-gated in more detail since this methodis also used routinely for lasering the ZPprior to biopsy in a PGD procedure.

P10PROSPECTIVE EVALUATION OF ICSI GUARDSPINDLE VISUALIZING HARDWARE FOR USEDURING ICSI

J. Meriano, J. Alexis, O. Cabaca, P. Bingil,R. D’Onofrio, K. CadeskyLifeQuest Centre for ReproductiveMedicine, Toronto, Canada

Hypothesis: Visualization of oocytespindle used in combination with ICSImay improve ART outcome.

Objective: We wished to determinewhether most spindles were adjacent topolar body as first thought and whetherthe use of a spindle visualizing appara-tus would help in the patient cycle.

Introduction: ICSI guard is a birefrin-gence filter and software system allow-ing visualization of oocyte spindle.Theoretically, if the spindle is not adja-cent to the polar body, the embryologistmay make proper adjustments beforeICSI.

Design: Prospective sequential analysisof 44 ICSI cycles in which the ICSIguard was used to visualize (group 1)the oocyte spindle before ICSI, and 44ICSI cycles in which the ICSI guard wasnot used (group 2) (as a concurrentcontrol). All ICSI patients (except PESA

and TESA) ≤ 42yr enrolled were ob-served for spindle appearance beforeICSI.

Material and Methods: The study wasperformed in a private health care faci-lity. Glass bottom ICSI dishes wereused. Oocyte retrieval, sperm prepara-tion and ICSI were performed as previ-ously described.

Results: Average age was 36.4 yrs ingroup 1 and 35.98 yrs in group 2. Nodifference in age, maturation, and ferti-lization or blastocyst rates was seenbetween the two groups. 476 oocyteswere retrieved from 42 patients ingroup 1 of the study. Of these, 345 weremature (MII) and 257 (74.5 %) fertilizednormally with 2 pronuclei. ICSI guardwas used in group 1 before ICSI. Con-trol group (group 2) was observedsequentially.

20.5 % of the oocytes examined didnot show a spindle. A spindle was seenin 274 oocytes. We found that only31 % (107) of the oocytes in which wedid visualize a spindle had the spindleadjacent to the polar body (in the“12 o’clock” position relative to polarbody). 48 % of spindles seen were at1 and 2 “o’clock” position, 12 % wereon or below the equator of the oocyte,which is a potentially dangerous positionwhen the polar body is at 12 o’clock,possibly leading to chromosomal scatterand aneuploidy.

Discussion: The ICSI guard was usefulin visualizing the spindle in 80 % of alleggs observed. It was unexpected tofind that only 31 % of spindles were at12 o’clock position and 12 % were onor below oocyte equator. ICSI positionwas adjusted for those oocytes. Ex-trapolating over a year’s time wherewe would inject a projected 4500oocytes, the number potentially “res-cued” theoretically is approximately540 eggs. More data is being added tothis study.

Table 3: Nijs et al.

Control Laser groupZona Intact Assisted Hatching

80 Single Embryo transfer 45 35Pregnancy rate per transfer 13/45 28.9 % 4/35 11.4 %Implantation rate 10/45 22.2 % 4/35 11.4 %60 Double Embryo transfer 31 29Pregnancy rate per transfer 9/31 29 % 6/29 20.7 %Implantation rate 11/62 17.7 %* 5/58 8.6 %

*2 twins

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P11IVF-LABORATORY PRACTICES IN FINLAND

L. KuusipaloNorth Carelian Central Hospital,Joensuu, Finland

Introduction: Application of the EUtissue culture directive will start shortlyin Finland. The quality of laboratorieswill be monitored by government au-thorities. How to define quality? Howdo Finnish IVF laboratories actuallyperform their activities? Are they all thesame?

Method: Telephone interview was usedto gather information of IVF laboratoriesin Finland. Seventeen out of 20 labora-tories agreed to take part in this study.The responsible IVF biologist answered45 questions in a telephone interview.

Results: The technical equipment varieda little among laboratories. Sperm countand study of follicular fluid is oftendone without heated microscope stage.Average number of oocytes per ovumpick-up is 7–13. Over 20 oocytes perpatient are nowadays rare in most cli-nics. Pipetting using mouth piece suc-tion is still in use in some clinics, as itis very ergonomic. Cells are cultured inmicrodroplets under oil, open 1 ml dishor 4-well dish with or without oil. Oil isalways used to cover an ICSI dish, and

in one clinic to cover denuding solutionin a 4-well dish.

Embryos are mostly evaluated throughICSI microscope. In some clinics, theyare studied from a computer display inlarger size. In some labs, the images ofall embryos are stored, and in some theones that are transferred. The classifi-cation of embryos has two major appro-aches: one concentrating on the levelof fragmentation (valid day-2 embryosshould not have more than 20 percentfragmentation), and the other on thequality and number of blastomeres(correct number of blastomeres plusnormality and elasticity of them is vital,the amount of fragmentation is irrel-evant).

Embryos with malformed, granular, va-cuolised or unevenly sized blastomeresare not utilized in some labs. Some labsaccept lower quality in fresh transfers,and some freeze them. The policy forembryos with multinucleated blasto-meres differs: some disqualify embryos,some use them when the patients wantso, and some use them as the last option.

Thawing of frozen embryos is done bydipping the ampule or by strawing in a37, 30 or 20 °C water bath, or by thawingthe straw in air. Also, self-made freezingand thawing solutions are used withsuperior results (90 % of embryos in-tact). Finishing the thawing protocol is

done either by moving the embryo fromtable-warm media straight to 37 °Cmedia, or by allowing the last thawingmedia to gradually warm up. Not alllaboratories utilize outside quality con-trol for sperm analysis. Staff meetingsare commonly held once a week, insome clinics daily, once a month oronce a year. Half of the clinics utilizepsychological counselling to discussdemanding cases or communication inwork environment. The work load perperson was very different between clinics.

The quality in IVF-laboratory work can bedetermined by fertilization and cleavagerates, amount of first-class embryos, bio-chemical and clinical pregnancies, andthe “take home baby” rate. Some labora-tories were not aware what their ownrates are.

Conclusions: It will be difficult for govern-ment authorities to define quality in aFinnish IVF laboratory, as the labs are verydifferent from each other. Pregnancy is acombination of many factors, and thelaboratory is just one of them. The parti-cipants of the enquiry emphasized theimportance of practice and team-work.IVF is modern handicraft, with ICSI beingthe master class. In practice, the labora-tory can rely on its own work quality, ifthe fertility rate is higher than 65–70 %,cleavage rate of fertilised oocytes is 90 %and first-class embryos are regularly pro-duced.

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