Rapid Multicolor PRINS For Numerical Chromosome Anomalies Detection In Human Spermatozoa

Objective: Numerical chromosome anomaly was one of the most important kinds of human chromosme diseases by inducing pregnancy loss, miscarriage, infant death, congenital malformations and nerve damage. Primed in situ labeling (PRINS), a novel method combining features of FISH and polymerase chain reaction, can be used as an alternative to FISH of numerical chromosome anomalies detection. Since PRINS has been wildly used in human genome research and medical chromosome screening, the design and verification of the chromosome-specific-primer were mainly based on repetitive sequences (alpha satellites) at cloned fragments from the centromere of specific chromosome. Since 2001, as the completion of human genome mapping, discretional parts of whole human genome sequence could be conveniently retrieved on the Internet, accurately compared and rapidly analyzed by new computer/net-tools. Given all these advancements, people should reassess the validity and reliability of traditional PRINS-primers by using more complete and updated genome information and technologies. To investigate and evaluate an updated and reasonable multicolor PRINS protocol for numerical chromosome anomaly analysis in human sperm, we use new genomic network tools for confirming traditional primers in multicolor PRINS, explore and evaluate a more reasonable PRINS protocol for numerical chromosome anomaly analysis in human sperm.Methods: The match information between traditional primers and whole Homo sapiens genome sequences was updated by using new genomic network tools. Almost whole traditional primer panel has been compared. The BLAST results proved the effectiveness of traditional primers in most cases.We also set up a more stable method of multicolor PRINS protocol in human cultured lymphocyte metaphase cells, and evaluate its veracity. The labeling characteristics of various target sequences and fiuorochromes were analyzed by dual-color preliminary experiments; Using the non-ddNTP-blocking triple-color PRINS method, primer 18, 21, X and Y were used to label chromosomes in metaphase cells from peripheral blood in order to setup stable methods of multicolor PRINS. A peripheral blood sample from a Klinerfelter syndrome patient (47, XXY) had also been studied to test the feasibility of multicolor PRINS for rapid diagnosis of numerical chromosome abnormity and prove the correspondence between the number of signals and chromosomes. The results of PRINS were compared with the chromosome G banding analysis.PRINS labeling may be used for direct estimation of disomy rate in human sperm. Before the PRINS reaction, the sperm slides were denatured in 3M NaOH solution for 4-5 min pretreatment allowing simultaneous decondensation and denaturation of sperm nuclei. The applying conditions of the primers and the combinations of target-dUTP-fluorochrome were optimized and proportional labelings for three chromosomes in human lymphocyte metaphase were observed within 3.5 hours. We estimated incidences of disomy and for 2 autosomes and 2 gonosomes in sperm samples from 2 normal fertile men.Results: Within 2.5 hours, three chromosomes had been simultaneously marked in same sperm nucleus. The best detection flux was up to triple color for suitable application of sperm multi-PRINS reaction, and the frequency of one-color-labeling with reached 99%. For each chromosome, a minimum of 5,000 (autosomes) or 10,000 (gonosomes) sperm nuclei were analyzed. The mean disomy rate of autosomes ranged from 0.125%(chromosome 18) to 0.152%(chromosome 21). For gonosomes, the mean rate ranged from 0.085%(chromosome X) to 0.102%(disome XY). Similar to the results of dual-color PRINS reported before, the disomy of 21 is a little higher than other chromosomes. Also the disomy of autosome is a little higher than gonosome, but there was no significant statistical difference among these values. In diploidy, as the frequencies ranged from 0.035% to 0.083%, heterogeneity was also not obvious. It even can be seen that the disomy rates of both two donors were higher than the diploidy rates (autosomes), which may imply that more disomy occurred than disomes in meiosis process.Conclusion: PRINS was seemed to be a rapid and reliable way to detect numerical chromosome anomalies with high flux in human lymphocyte metaphase cells and sperm nuclei. It was suggested that the primer sequence would always be compared with the whole genome information to infer its reliability before the synthesization, and there would also be a pre-testing to confirm the efficiency of the primer with unclear BLAST results. In chromosome detection with multicolor PRINS technology, strictly controlled reaction conditions are indispensable.