The starting dose of gonadotrophins was unchanged compared to ourprevious practice with agonists. Cetrotide 3 mg or 0.25 mg was introducedin the late follicular phase with at least 3 follicles having a diameter �or �10 mm and/or an estradiol level �400 pg/ml. The efficacy criteria were themean number of oocytes retrieved and inseminated.

Results: Demographic characteristics ( age, basal FSH, LH and E2, BMI)were comparable in both groups. Only the mean ART attempt was signif-icantly higher in the Cetrotide 0.25 mg group ( 3.5 vs 2.8, p �0.02). In bothgroups, E2 level on day of Cetrotide introduction and on day hCG areequivalent. In period 1 (Cetrorelix 3 mg), the mean number of oocytesretrieved increased from 8.2 to 10.6 within three months reaching a plateau.Similarly, in period 2 (Cetrorelix 0.25 mg), the mean number of oocytesretrieved increased from 4.7 to 9.9 within only 2 months. The sameobservation was made for the number of mature oocytes in both groups.When analysing Cetrorelix introduction, it appeared that from the beginningof each period to the end, the follicles diameter grew gradually from at least3 follicles �or � 10 mm to 3 follicles �or � 13 mm.

Conclusions: Our experience shows clearly that a new product of classhas to be introduced in common practice with careful evaluation. Thegradual increase in follicles diameter at Cetrorelix introduction shows thegrowing clinician confidence in preventing LH surge. Its major consequenceis the increase of the mean number of oocytes retrieved and inseminated,first step of ART.

Supported by: None.

ART: PREIMPLANTATION GENETIC DIAGNOSIS,MICROMANIPULATION AND OTHER LAB

TECHNIQUES

P-193

DNA microarray analysis in Down syndrome. I. H. Jung, S. M. Lee,S. H. Lee, J. H. Park, K. Y. Cha, K. W. Lee. Lab of Genetics & InfertilityMedical Ctr, CHA Gen Hosp, Coll of Medicine, Pochon CHA Univ, Seoul,South Korea; CHA Gen Hosp, Coll of Medicine, Pochon CHA Univ, Seoul,South Korea; Dept of Obstetrics and Gynecology, Kangbuk Samsung Hosp,Sungkyunkwan Univ, Sch of Medicine, Seoul, South Korea.

Objective: Down Syndrome (DS) is complex genetic disorder with men-tal retardation, skelectal defects, defects of metabolism in the brain. Themolecular mechanism of DS is not well understood and prenatal geneticdiagnosis is important in cliniques. cDNA microarray technology is used tothe analysis of gene expression levels for thousands of genes simulta-neously. We tried to find the DS biomarker for prenatal genetic diagnosis bycomparing the gene expression profiles from DS and normal amniotic cellsusing cDNA microarray technique.

Design: About 1100 human genes were analyzed in this experiment.After hybridization, this chip were scanned and analyzed. Comparing theexpression profile of DS with normal cell, we obtained the substractive geneexpression profiles and further analysis using 5 different DS samples.

Materials/Methods: We extracted total RNA from DS amniotic andnormal cells using Qiagen RNA prep kit. Each total RNA was labeled withcy-3 and cy-5. The chip hybridized for 16 hours in hyb-chamber. Afterhybridization, non-labeled probe is removed in washing step. Finally thechip was scanned by ScanArray 4000XL. We used Imagene software forgene expression data analysis. This software showed various graphic imagedata. From each quantified signal, we could compared expression levels.

Results: Performing clustering analysis, we found that 22 genes from1.1K genes analysis were differentially expressed in DS. The expressionlevel of lycyl oxidase gene in DS cells is about five times higher than thatof normal cell. Twenty of genes were increased by two times in DS. Onlyone of genes was decreased by a half in DS. Among 22 genes, 17 geneswere located in chromosome 21. These genes were related with proteinexpression, cell division, metabolism, cell structure, organism defense, andhomeostasis. We are doing of further analysis using five different DSsamples.

Conclusions: Difference expression data in DS and wild-type cells will bebasis of gene expression profile analysis. Most of genes did not show thelarge difference, but some of differentially expressed genes may be impor-tant in DS. We will discuss the role of these genes as the biomarker in DownSyndrome.

Supported by: Korean Health 21 R&D Project, Ministry of Health andWelfare, Korea (01-PJ10-PG6–01GN13–0002).

P-194

Conservative preimplantation genetic diagnosis (CPGD). Andreas G.Schmutzler, Wen Chen, Jorg Weimer, Liselotte Mettler, Walter Jonat,Norbert Arnold. Univ Women’s Hosp, Kiel, Germany.

Objective: In order to increase implantation and pregnancy rates and todecrease pregnancy losses after IVF or ICSI, preimplantation genetic diag-nosis (PGD) for aneuploidy screening (AS) by blastomere or polar bodybiopsy was introduced. It is desirable to screen for aneuploidies noninva-sively by morphologic markers.

Design: Cytoplasm and first polar body of metaphase II oocytes weremorphologically classified. Oocytes which failed to fertilize were fixed andhybridized by FISH. Aneuploidies were correlated to morphologic markers.The markers of oocytes which fertilized and resulted in transferred embryoswere correlated to pregnancy rates.

Materials/Methods: Oocytes were assessed at 200 to 400 x magnification.Cytoplasm was classified into three groups: 1) ideal cytoplasm: clear cyto-plasm with uniform texture and homogenous fine granularity; 2) mildgranular cytoplasm: rough granularity, darkening of the whole cytoplasm;3) severe granular cytoplasm: granulation concentrated as a dark mass in thecentral part of the oocyte with a clear peripheral ring (centrally locatedcytoplasmic granulation, CLCG). First polar bodies were classified into fourgroups: 1) ideal: ovoid with smooth surface; 2) overmature: ovoid withrough surface; 3) fragmented; 4) large or very small. Oocytes were fixed atday 1 to day 4 after follicular puncture and stored at -20°C. Chromosomes13, 16, 18, 21 and 22 were hybridized by FISH. Aneuploidy was scored bystrict criteria (Dailey 1996). At embryo transfer the morphologic markers ofthe oocytes leading to the transferred embryo were noted. Data wereanalysed by chi-square tests.

Results: From 81 patients 258 oocytes were fixed. 180 oocytes were inmetaphase II, 135 well spread. Until now, 93 of these were analysed byFISH, 84 successfully (90%). 35% were euploid (29/84), nondisjunction(NDJ) was found in 26%, unbalanced predivision (UBP) in 13%, balancedpredivision (BPD) in 48%. BPD increased with culture time in vitro from35% on day 1 to 67% on day 3 and 4 (p �0.05), which was not the case forNDJ and UBP. For polar body morphology 112 oocytes were recruited. NDJand UBP increased from group 1 to 4 from 15% to 17%, 30% and 61% (p�0.01 for 1 vs 3 and 1 vs 4), BPD increased from 24% to 56% (p �0.05).For oocyte cytoplasm morphology 119 oocytes were recruited, 71 with idealmorphology, 31 with mild granularity, 17 with CLCG. NDJ plus UBP was21%, 29% and 47%, respectively. The difference between ideal cytoplasmand CLCG was significant (p �0.05). Pregnancy rates in cycles with 2 or 3transferred embryos where at least two embryos had ideal oocyte cytoplasmwas 42% (48/114), or 25%, if this was not the case (18/71) (p �0.05). Alsoimplantation was 18% with mostly ideal cytoplasm, 11% if not (p �0.05).

Conclusions: Every third unfertilized oocyte is aneuploid for NDJ andUPD, whereas BPD is at least in part an artefact due to long in vitro culture.Aneuploidies are indicated by not ideal oocyte cytoplasm or polar bodymorphology. This phenomenom is also reflected in pregnancy and implan-tation rates. Invasive PGD for AS might be reduced by a detailed morpho-logic screening of the oocytes. Genotype seems to correlate with phenotypealso in oocytes.

Supported by: Research Fund, University Women’s Hospital, Christian-Albrechts-University, Kiel, Germany.

P-195

In vitro development of human triploid zygotes reconstructed by pro-nuclear transfer. John Zhang, Yi Ming Shu, Lewis C. Krey, Hui Liu,Guang Lun Zhuang, Jamie Grifo. New York Univ Sch of Medicine, NewYork, NY; Zhongshan Univ Sch of Medicine, Guangzhou, China.

Objective: Nuclear transfer is a technique by which the nuclear genomeis removed and exchanged between oocytes, allowing assessment of thecomparative nuclear and cytoplasmic contributions to embryo development.In last years we evaluated the cytoplasmic influences on meiotic divisionsof human GV oocytes. The aim of the present study is to assess the impactof pronuclear (PN) transfer on in vitro development of human zygotes.

Design: Oocytes showing 3 PN and 2 polar bodies 12–16 h post IVF wereconsidered triploid and used after informed patient consent. Initially, 1, 2 orall 3 PN were removed from triploid zygotes and the resultant diploid,haploid or anuclear zygotes were cultured to assess their developmental

S180 Abstracts Vol. 78, No. 3, Suppl. 1, September 2002

potential. In the second study, exchange of 2 PN between triploid zygoteswas performed and in vitro development of the reconstructed triploidzygotes was compared with non-manipulated controls.

Materials/Methods: In the first study, 16, 17 and 13 triploid zygotes had1, 2 or all 3 PN removed from each of zygotes, respectively. Those zygoteswere sequentially cultured in G1/G2 medium for 5 days and their develop-ment was evaluated every 12 hours. Non-manipulated triploid zygotesserved as controls. In Study 2 two PN were removed from each of 28 pairsof triploid zygotes. Each removed 2PN karyoplast was inserted into theperivitelline space of one of paired triploid zygotes from which 2 of 3 PNalso had been removed; karyoplast - zygote complex fusion was induced byan electric pulse (1.8 kv/cm, 50 microseconds). Embryonic development atDay 5 was assessed between groups by chi-square analysis.

Results: In Study 1 upon removal of one or two PN, 10 of 16 (68%)diploid and 12 of 17 (70%) haploid zygotes developed beyond the 6-cellstage with 4 and 2 progressing further to 8–10 cells, which were similar tonon-manipulated ones (63%). On the other hand, lower proportion (30%,n�13) of anuclear zygotes (all 3 pronuclei removed) developed to 2–4 cellswith rest of them showing no cytokinesis. In the second study, 21 of 33(63%) reconstructed triploid zygotes developed beyond the 6-cell stage and2 of the cleaving embryos reached to the blastocyst stage; this did not differfrom controls (67%, n�62).

Conclusions: In vitro development of the reconstructed or control zygoteswas not influenced by their ploidy (1 to 3 PN). Neither nuclear manipulationnor electrofusion at the PN stage had any affect on subsequent embryodevelopment. Significantly, embryonic development consistently progresseswhen one or more PN is present suggesting that while both the ooplasm andzygotic genome may play independent roles in post-zygote development,they do interplay to coordinate cytokinesis at least to the 6-cell stage.Further studies will be carried out to evaluate the effect of synchronous andasynchronous cytoplasm on zygotic development.

Supported by: None.

P-196

Advancing maternal age compromises control of cell cycle checkpointfor aneuploidies in human embryos cultured in vitro. H. Asakura, K. P.Katayama, E. F. Stehlik, J. C. Stehlik, K. Winchester-Peden. IVF andCytogenetic Lab, Advanced Institute of Fertility, Milwaukee, WI.

Objective: Pregnancy and live birth rates following IVF-ET declinerapidly with advancing maternal age partly due to an increase of errors inmeiosis during female gametegenesis. In order to study genetic control ofearly embryo development, chromosomal abnormalities in human embryosat cleavage stage and their relevance to embryo development in extendedculture were investigated. Relationship between maternal age and frequencyof aneuploidies was also studied.

Design: Prospective experimental studyMaterials/Methods: 47 women underwent IVF-ET with preimplantation

genetic diagnosis (PGD) for screening of chromosomal abnormality. IRBapproved consents were signed. On day 3 of fertilization, blastomere biopsyand multi-probe fluorescence in situ hybridization (FISH) analysis forchromosome X, Y, 16 and 22 were performed. Biopsied embryos werecultured in sequential blastocyst media and day 5 morphologic grades wererecorded.

Results: Average maternal age and the number of embryos biopsied were34.1 � 4.5 y.o. (mean � S.D.) and 5.6 � 2.2 per patient, respectively. Total261 day 3 embryos were biopsied (grade 1:127, 2:104, 3–4:30). On day 5,70 blastocysts developed from the biopsied embryos (blastocysts formationrate: 26.8%). According to FISH criteria, embryos were judged as: 108(41.4% ) normal, 93 (35.6%) abnormal, and 60 (23.0%) not informative.Trisomy 22 was the most common aneuploidy. There was a significantlypositive correlation between the rate of FISH normality and day 5, but notday 3, embryo morphology. Complex aneuploidies involving multiple chro-mosomes were significantly more common among lower day 5 morphologicgrades. The rates of all abnormalities among informative embryos were52.1% (38/73) in Group A (age 19–32 y.o.), 49.4% (39/79) in Group B (age33–37 y.o.), and 55.1% (27/49) in Group C (age 38–43 y.o.). There was astrong positive correlation (p�0.09) between the rate of simple aneuploidiesof all abnormalities and maternal age: Group A: 39.4% (15/38), Group B:43.6% (17/39), Group C: 66.7% (18/27). There was an increasing trendamong informative embryos of frequency of autosomal monosomy ortrisomy as the maternal age increased: 4.1% (3/73), 6.3% (5/79), 12.2%

(6/49) for chromosome 16; 8.2% (6/73), 11.4% (9/79), 14.3% (7/49) forchromosome 22, in Group A, B and C. FISH normality among day 5blastocysts decreased, although not significant, as the maternal age ad-vanced.

Conclusions: Blastocyst culture in vitro served as a method to selectgenetically competent embryos that would have higher implantation effi-ciency after transfer. The majority of complex chromosomal abnormalities,probably representing postzygotic mosaicism, were blocked before blastu-ration. Our study, however, showed presence of simple aneuploidy is notincompatible with blastocyst development. This is consistent with highincidence of numeric chromosomal aberrations historically identified inearly pregnancy losses. There was a suggestion from our data that themechanism of genetic safeguard by cell cycle checkpoint might be morecompromised at advanced maternal ages, especially for simple aneuploidies.Blastocyst culture with PGD for aneuploidy screening may further enhanceefficiency of IVF-ET for a certain patient population.

Supported by: Not applicable.

P-197

A randomized controlled study to evaluate the effect of laser assistedhatching on human embryo development. Benjamin C. Wong, CatherineA. Boyd, Susan E. Lanzendorf. Jones Institute for Reproductive Medicine,Eastern Virginia Medical Sch, Norfolk, VA.

Objective: To evaluate the effect of laser hatching on human embryodamage and subsequent development using the Zona Infrared Laser OpticalSystem (ZILOS, Hamilton Thorne Research, Beverly, MA).

Design: This is a randomized control study.Materials/Methods: Following approval by the Institutional Review

Board at Eastern Virginia Medical School, frozen human embryos donatedfollowing in vitro fertilization were thawed using standard techniques.Embryos were cultured in HTF Medium (Irvine Scientific, Santa Ana, CA)supplemented with 10% Serum Substitute, Irvine) on Vero cell (ATCC,Rockville, MD) cocultures. Cleavage and morphology were evaluated everytwenty-four hours. Thirty-seven embryos were randomized into one of threegroups at the time of thaw: control, partial hatching or complete hatching.The laser hatching procedure was performed on an Olympus IX-70 InvertedMicroscope (Olympus America, Inc., Melville, NY) equipped with theZILOS. On Day 2 or 3 of development (Day 0 � day of insemination) allembryos were evaluated and zona thickness and embryo diameter recordedusing the ZILOS computer scale. Embryos randomized to complete hatch-ing underwent the production of a full-thickness defect in the zona usingthree pulses of laser treatment (2 msec at 135–140 mW power). Embryosrandomized to partial hatching underwent the creation of a defect in theouter half of the zona using the same laser settings. No laser treatment wasadministered to the control group. The dimensions of the defects weremeasured after the hatching procedure. The embryos were then cultured andmonitored for development to the blastocysts stage and completion of thehatching process. Statistical analysis was performed using the Kruskal-Wallis test for the baseline parameters and the Fisher’s exact test for theoutcome measurements.

Results: No significant difference was noted between the three studygroups for age of donor at oocyte retrieval, cell stage and embryo grade atthe time of freeze or thaw, zona thickness or embryo diameter. There wasno evidence of blastomere damage to any of the embryos undergoing thehatching procedure. There was no significant difference in blastocyst de-velopment (control: 41.7%, partial: 53.9%, complete: 66.7%) or in comple-tion of hatching from the zona (16.7%, 16.7%, 30.77%).

Conclusions: Laser hatching of human embryos using the ZILOS doesnot have an adverse effect on subsequent development. This preliminarydata shows a trend towards increased hatching in vitro following full laserhatching treatment and studies are ongoing to determine if the size of thezona opening has an effect on laser hatching outcome.

Supported by: Hamilton Thorne Biosciences Research, Beverly, MA.

P-198

The effect of different embryo biopsy procedures for PGD on blastocystformation and pregnancy. Basak Balaban, Aycan Isiklar, Bulent Urman.VKV American Hosp of Istanbul, Istanbul, Turkey.

FERTILITY & STERILITY� S181