Chromosome studies in human sperm nuclei using fluorescence in-situ hybridization (FISH)

Abstract

The use of chromosome specific DNA probes labelled with fluorochromes and especially the combination of several probes has been used to indirectly study the chromosome constitution of decondensed sperm nuclei by fluorescence in-situ hybridization (FISH), and has allowed to include this test in the protocol of study of infertile males. Still, if the test is to be valid, several strict conditions must be met, and some specific characteristics have to be taken into account. This becomes evident when comparing earlier results with more recent ones. The basic technical factors to be taken into account are the methods of chromatin decondensation, the number of spermatozoa and of individuals to study, the use of internal controls, the scoring criteria, the specificity of the probes and the possible existence of polymorphisms that may interfere with the detection of fluorescent signals. In the last 7 or 8 years, a large number of papers has been published, describing the incidence of aneuploidies in controls, in individuals in whom a tendency to non-disjunction was suspected and in infertile males. Studies in controls have shown a considerable intra- and inter-individual variability in the frequency of aneuploidies, the tendency of some chromosomes to undergo non-disjunction (chromosome 21 and the sex chromosomes) and the importance of alpha-satellite polymorphisms when using centromere probes. In the control population, the frequency of aneuploidy per haploid set has been estimated at approximately 6%. The incidence of aneuploidies in sperm nuclei for some of the chromosomes more frequently involved in trisomies is considerably higher than the incidence of these trisomies established through epidemiological data using the global incidence of chromosome abnormalities during the peri-implantation stage. In infertile males and in males with sex-chromosome abnormalities (usually with very low numbers of spermatozoa) the results show an increased incidence of sex chromosome aneuploidies and diploid (multi-aneuploid?) sperm nuclei. The results could be related to the higher incidence of chromosome abnormalities (especially sex-chromosome aneuploidies) observed in children conceived by intracytoplasmic sperm injection (ICSI).

PNA on human sperm: a new approach for in situ aneuploidy estimation

“…Owing to its relative simplicity and the commercial availability of numerous probes, FISH has become the standard technique for sperm analysis, despite the lack of specificity of some alphoid probes. 2 The PRINS reaction requires a thermocycler and sequential reactions, but provides a more specific and faster labelling procedure for the repeat DNA sequences. 3,16 When applied to human sperm preparation, both techniques need to be associated with a sperm nucleus decondensing treatment in order to made the targeted DNA region accessible.…”

“…To date, numerous chromosomal analyses on human sperm have been performed using fluorescence in situ hybridization (FISH) (for a review, see Downie et al 1 and Egozcue et al 2 ). These reports demonstrated the efficiency of the in situ labelling procedure on male gametes, but also pointed out the limitations of FISH on this biological material, which are essentially linked to the size of the probes and the reliability of the associated sperm decondensation treatments.…”

“…These reports demonstrated the efficiency of the in situ labelling procedure on male gametes, but also pointed out the limitations of FISH on this biological material, which are essentially linked to the size of the probes and the reliability of the associated sperm decondensation treatments. 2,3 The primed in situ (PRINS) reaction has offered an alternative approach for the direct chromosome analysis of human spermatozoa. Several studies have demonstrated that PRINS labelling was just as efficient as FISH on human sperm and exhibited higher specificity.…”

Comparison of Aneuploidy Frequency to Sperm FISH and Sperm Apoptosis Results in Embryos That Lost the Vitality

“…[10][11][12] Bu ça lış ma da sperm hüc re le ri X, Y ve 18 no lu kro mo zom la ra ait FISH prob la rı ile sa yı sal ano ma li var/yok şek lin de in ce len miş tir. İn-ce le me de X, Y ve 18 no lu kro mo zom la rın se çil mesi nin iki ne de ni var dır: Bun lar dan il ki cin si yet kro mo zom ano ma li le ri nin sperm ler de da ha sık kar-şı mı za çık ma sı, ikin ci si prob se ti nin kul la nı la bi lirli ği nin ko lay ol ma sıy dı.…”

“…4,9 Sperm ler de ya pı lan ça lış ma lar, cin si yet kromo zom la rı ile 21 ve 22 no lu kro mo zom la ra ait anoma li le ri nin sık lı ğı nın yük sek ol du ğu nu gös ter miş-tir. [10][11][12][13][14] Ma yo tik bo zuk luk gös te ren ol gu lar in ce lendi ğin de ise sperm dip lo i di si nin en sık rast la nan kro mo zom bo zuk lu ğu ol du ğu gös te ril miş tir. 15 Sperm ler de ki kro mo zom ano ma li le ri ile kro mo zomal ano ma li li em bri yo lar ara sın da ki iliş ki yi sap tamak için ya pı lan ça lış ma lar; cin si yet kro mo zom ano ma li le ri, oto zo mal di zo mi ler ve na dir gö rü len dip lo i di le ri or ta ya çı kar mak üze re yo ğun laş mış, trip lo id ge be lik ürün le rin de, dip lo id sper min olduk ça önem li bir ro le sa hip ol du ğu gö rül müş ve bu du ru mun ana faz I ev re sin de si nap tik ano ma li ler ne de niy le olu şa bi le ce ği öne sü rül müş tür.…”