The peptide nucleic acids (PNAs) as probes for chromosomal analysis of human oocytes and preimplantation embryos

Abstract

Demonstrate the efficiency and the reliability of PNAs for the in situ chromosomal analysis of human oocytes and blastomeres Peptide Nucleic Acids (PNAs) are synthetic DNA mimics based on an uncharged polyamide backbone. Because PNA probes present multiple advantages in terms of affinity and specificity, we have tested this new type of probe on isolated human oocytes, polar bodies and blastomeres, in order to assess the possibility of using it for preimplantation diagnosis of aneuploidy. Using centromeric PNA probes specific for chromosomes 1, 4, 9, 16, X and Y, we performed multicolour labelling PNA reaction, sequential PNA reaction and combined PNA and fluorescence in situ hybridization (FISH) in 34 unfertilized oocytes and 27 blastomeres. Each PNA probe consists of a mixture of several short synthetic sequences (18 to 22 base units) specific for the centromeric sequence of the targetted chromosomes. The PNA probes were directly fluorochrome-labelled and supplied ready to use in hybridization buffer. The hybridization timing of PNA probes was 40 -60 minutes. In 27 oocytes, simple multicolour PNA assays were done. A sequential PNA procedure was experimented in 5 oocytes. Two other oocytes were utilized to test the combined PNA and FISH protocol. In 17 cases, corresponding polar bodies were obtained through the fixation procedure. Twenty-six oocytes (76.5%) displayed a normal pattern of signal according to the probes used. Eight oocytes (23.5%) displayed numerical abnormalities for the targetted chromosomes. Twenty-three blastomeres from 10 embryos were processed for PNA experiments. In 3 cases, the FISH technique was used as control in one blastomere. Only three embryos appeared to be diploid for the chromosomes tested. Four others displayed aneuploidy, and 3 embryos displayed mosaic pattern. Both rates and types of abnormalities scored in oocytes and blastomeres are in good agreement with the data of previous FISH studies The present study describes the first use of PNAs on isolated human oocytes and blastomeres for in-situ chromosomal identification. PNA probes allow a reliable chromosomal analysis and consequently, can provide an interesting adjunct to FISH for diagnostic analysis. The PNA technology possesses undeniable advantages for in-situ recognition of complementary DNA sequences. Because of the simplicity and the rapidity of the PNA procedure, these preliminary results point out the potential application of PNA in PGD program.