Tkach I. Peculiarities of chromosomal status in early reproductive loss in humans

The thesis is devoted to the study of karyotype peculiarities and telomeres areas of chromosomes at early reproductive losses (ERL) in humans. The initial concept of work was based on the assumption that the deficiency of telomeric DNA can be involved in the complex mechanism of pregnancy loss in the early stages of intrauterine development of a human, especially at the presence of an abnormal embryo karyotype. The frequency and spectrum of karyotype anomalies of the material of early reproductive losses of the western region of Ukraine (760 samples) have been investigated for the first time through the integrated application of the standard cytogenetic and iFISH methods for analysis the preparations, obtained by the "direct" method from uncultured chorionic villi. Karyotype abnormalities were found in 39.3% of cases. Genomic and numerical chromosomal abnormalities were observed with the following frequency in descending order: autosomal aneuploidy - 48.5%, polyploidy - 29.1%: prevailed triploidy- 26.1%, gonosomal aneuploidy - 22.4%: prevailed monosomy X - 21.1%. The results of our research showed that the dominant autosomal numerical anomalies were trisomy 16 - 21.1%, aneuploidy 15 - 9.4%, trisomy 22 - 6.4% and trisomy 21 - 6.0% in decreasing order. There was a tendency to a slight increase in the karyotype abnormalities in the material of pregnancies lost at 6-10 w.o.g. - 41.5% compared to at <6 w.o.g. (35.5%) and 11-14 w.o.g. (35.9%) . The part of karyotype abnormalities didn't differ in the material of sporadic (39.9%) and recurrent pregnancy losses (RPL) (38.2%). The contribution of genomic abnormalities in the material of lost pregnancies was higher in groups I (RPL in anamnesis) and II (single lost pregnancy) — 32.6% and 31.7%, respectively, and less in group III (sporadic pregnancy loss (SPL)) — 21.4%. The reverse trend was observed relative to the proportion of monosomy X, which was higher in group III - 25.0% compared with the indicator in group I (18.7%) and II (20.7%). Therefore, the frequency of genomic and chromosomal abnormalities in the material of ERL in women age <35 and ≥35years was analyzed. The overall frequency of identified karyotype abnormalities is not significantly different in the material of EPL of women of different ages (P>0.05), however, the contribution of karyotype abnormalities in ERL depends on the age of a woman, namely, with age significantly increases the proportion of aneuploidy due to autosomal trisomies (P <0.0001) and decreases the contribution of polyploidy (P <0.005) and monosomy X (P <0.005). Analysis of the frequency of the main abnormalities of the karyotype of a single lost pregnancy showed that the proportion of aneuploidy increases significantly with age (P <0.025) due to autosomal trisomies (P <0.0001) and decreases the proportion of polyploidies (P <0.025). A similar trend has been established in the analysis of SPL – the proportion of aneuploidies increases (P <0.05) due to autosomal trisomies (P <0.005) and decreases the proportion of polyploidies (P <0.05), and RPL (P <0.05 for aneuploidies; P <0.005 for autosomal trisomies; P <0.05 for polyploidies). Consequently, regardless the reproductive history, the frequency of autosomal trisomies increases and the genomic anomalies decrease in the material of ERL from women after 35 years. The lengths of telomere areas of chromosomes in a fetus of 5–12 weeks of intrauterine development have been investigated for the first time as a provocative factor of reproductive loss in humans. The relative telomere length in DNA isolated from samples of chorionic villi from lost pregnancies and induced abortions of 5-12 weeks gestation have been studied. Determination of relative length of telomeres (RTL) was performed using PCR with RT-PCR. Our results showed with a high degree of probability (P=0.000001) that ERL is characterized by short telomeres compared with induced abortions(IA). Information about telomere length in combination with the particularities of the karyotype in the post-implantation terms of intrauterine development is absent. Therefore, it seemed expedient to analyze the obtained indicators of the RTL depending on the presence/absence of chromosomal abnormalities in the examined material. Selected samples of the chorionic villi reproductive loss were characterized by the following karyotype: euploidy – 32 cases; triploidy – 13 cases; trisomy 16 – 13 cases; trisomy 21 – 10 cases; monosomy X – 12 cases, and IA – only with euploid karyotype. Established significant difference of RTL in material of ERL - (aneuploid: 0.29±0.03 and euploid: 0.64±0.12, P=0.0015 respectively). The results showed no significant variation in RTL among the various aneuploid groups. Detected significant difference in RTL between euploid ERL and IA (P=0.006). The obtained results allow us to assume that the process of telomere shortening is involved into the complex mechanism that leads to the early death of embryo.