gender for an X-linked disease can greatly enhance the process by whichembryos are decided to be eligible for transfer.

P-060

Chromosomal Constitution of Testicular Cells in 47,XXY Patients An-alyzed by FISH. M. Bergere1, R. Wainer2, V. Nataf1, A. Escalona1, M.Very1, J. Selva1. Departments of1Reproductive Biology and Cytogenetics,2Gynecology and Obstetrics, CHI Poissy-St Germain, 78303 Poissy, Paris-Ouest University, France.

Objectives: Evaluation of meiosis in 47,XXY or 46,XY gonia inazoospermic non mosaic Klinefelter patients by two ways 1) investigationof the correlation between the sex chromosomes complements of testicularcells biopsied and the efficiency of spermatogenesis 2) evaluation of theaneuploidy rate in eventual post meiotic cells.

Design: FISH analysis was performed on testicular cells in Klinefelterpatients. The respective incidences of normal diploid and aneuploid47,XXY cell lines and the post meiotic aneuploidy rate were evaluated inspermatids and spermatozoa.

Materials and Methods: Both karyotype and FISH analysis on lympho-cytes of 4 azoospermic patients were homogeneously 47,XXY. Testisbiopsy was performed in the 4 patients and a small sample was used forcytopathological analysis. In the IVF laboratory, if any testicular sperma-tozoa were found, all the biopsied tissue was frozen for a subsequent ICSIattempt, excepted one smear which was prepared for FISH analysis. Hy-bridization was achieved with a three directly labelled probe set for X, Yand 18 chromosomes (Chromoprobe, Cytocellt). DAPI counterstainedslides were observed with fluorescent microscope.

Results: Normal diploid cells were frequent in cases with spermatozoa.All the pachytene figures were observed 46,XY. In Sertoli Cell Only Syn-drome (SCOS), all the cells were diagnosed 47,XXY. Following tablesummarizes the FISH results.

Case

Number ofhybridized cells

observedDiploid

cells Spermatids Spermatozoa

1 35 XXY 100% 0 02 18 XXY 20% X,18 54% X, 18 (2)

XY 80% Y,18 39%X,Y,18 7%

3 72 XXY 22% X,18 44% X,18 (1)XY 78% Y,18 56% Y,18 (1)

Y,Y,18 (1)Y,18,18 (1)

4 36 XXY 37% X,18 55% X,18 (3)XY 63% Y,18 45% Y,18 (2)

X,18,18 (1)

Conclusion: All patients with spermatozoa displayed 46,XY cells; espe-cially the pachytene figures were 46,XY. The SCOS patient had a nonmosaic 47,XXY constitution in all the testicular nuclei examined. Ourresults support the hypothesis that only 46,XY gonia can achieve success-fully meiosis in Kinefelter patients, with a low aneuploidy rate for sexchromosomes in haploid cells.

P-061

Robertsonian Translocation in an Oligozoospermic Male Carrier: SpermFISH Study, ART Strategy and Genetic Counselling.M. Bergere1, M.Albert1, L. Allard1, R. Lombroso2, Y. Ville2, J. Selva1. Departments of1Biology and Cytogenetics,2Gynecology and Obstetrics, CHI Poissy-StGermain, 78303 Poissy, Paris-Ouest University, France.

Objective: Evaluation of a 13;14 translocation in a male patient requiringICSI concerning 1) risk of chromosomally unbalanced sperm, 2) indicationfor chromosome prenatal diagnosis, 3) number of embryos to be transferredafter ICSI balancing the risks of multiple pregnancies and prenatal diagno-sis.

Design: FISH analysis on sperm in a male carrier of a 13; 14 Robertso-

nian translocation before ICSI; fœtal karyotype and uniparental disomy forchromosome 14 were checked.

Materials and Method: A 32 year old patient and his 27 year old wiferequired assisted reproduction. The spermogram showed oligozoospermia(2.1 millions per ml). The karyotypes, in both patients were 45,XY,t(13;14)and 46,XX respectively. FISH analysis on sperm used subtelomeric probes(Cytocell, amplitech°) labelled with biotin and digoxigenin for 13q and 14qand DAPI counterstained hybridized slides were observed with a fluorescentmicroscope equipped with FITC, Cy3 DAPI filters associated with a Per-ceptive Scientific International analyzing system. ICSI was performed on 15oocytes in metaphase; 13 ovocytes were fertilized, 2 embryos were trans-fered, 7 embryos were frozen. This resulted in a singleton pregnancy and achorion villi biopsy was performed for karyotype and molecular biology inorder to check the parental origin of chromosomes 14.

Results: 107 spermatozoa were analyzed and 86% (n593) could bescored. 43% (n540) showed normal FISH signals: single spot for both13qter and 14qter (thus either balanced or normal). 57% were abnormal:11.8% (n511) were disomic for chromosome 13, thus potentially leading toembryos with trisomy 13. The risk is higher than expected from publishedresults on prenatal diagnosis and chromosomal analysis of fertile sperm inthis situation. 13.9% (n513) were disomic for chromosome 14. The highproportion of abnormalities especially disomy 13 and the need for invasiveprenatal diagnosis made us opt for a transfer limited to two embryos;chorion villi analysis of the resulting monofetal pregnancy revealed anormal karyotype without uniparental disomy 14.

Conclusion: Evaluation of ICSI embryos for chromosomal risk by FISHanalysis of sperm in a male carrier of a translocation is useful to provideadequate counselling for prenatal diagnosis after ICSI and to choose thenumber of embryos to be transfered. Indeed published evaluations ofchromosomal risks for fertile patients with translocation may be inadequatefor infertile couples requiring ICSI. This work was supported by a clinicalresearch grant APHP CRC n°96053

References

Cytogenetic analysis of sperm from a male heterozygous for a 13;14Robertsonian translocation. Martin RH, Hum Genet, 1988 Dec, 80:4,357–61.

Paternal uniparental disomy for chromosome 14. American Journal ofMedical Genetics 1997, 70:74–79.

P-062

Pregnancies Following Preimplantation Genetic Diagnosis of Chromo-somal Translocations in Couples with Reduced Fertility. 1H. Bicker-staff, 1C. T. Yeong,1P. Scriven, C. Mackie Ogilvie,1F. O’Mahoney, P.Braude, et al.1Center for Preimplantation Genetic Diagnosis, Guy’s and StThomas’ Hospital, London, UK.

Objective: Chromosome rearrangements may be reciprocal translocations(involving the breakage of two non-homologous chromosomes with ex-change of segments), Robertsonian translocations (involving breakpoints atthe centromeres of two acrocentric chromosomes) or inversion of a segmentwithin a single chromosome. During meiosis, any of these rearrangements(depending on the mode of segregation) may lead to chromosome imbalanceand hence reduced fertility, pregnancy loss or viable abnormal offspring.Preimplantation genetic diagnosis using chromosome specific probes andFISH technology has the potential to identify normal/balanced karyotypesleading to the replacement of unaffected embryos.

Design: A prospective cohort study of all cases referred to the PGDCentre at Guy’s and St Thomas’ Hospital.

Materials and Methods: Patients underwent PGD cycles involving ovar-ian stimulation and egg collection as for standard IVF treatment. Embryobiopsy was performed on Day 3 with the removal of one or two blastomeresfor analysis as previously described (Scriven et al1998). Transfer of theunaffected embryos was performed on Day 4. Pregnancy was confirmed byultrasound at 4 weeks after embryo transfer.

Results: A total of nine cycles of PGD for six couples with chromosomerearrangements was performed. The mean age was 35.1 (range 31–39). Twopatients had Robertsonian translocations, four had reciprocal translocationsand 1 had a pericentric inversion. An average of 16.5 eggs (range 10–23)was retrieved with a mean fertilization rate of 53%. One cycle was a frozenthawed cycle. A total of 80 embryos were biopsied with 100% survival rate.

S114 Abstracts Vol. 74, No. 3, Suppl. 1, September 2000