Chromosomal abnormalities of the embryo are the most common cause of first-trimester pregnancy loss. In this single-center study, we assessed the frequency and the spectrum of chromosomal abnormalities in miscarriages for each year of maternal age from 23 to 44. Cytogenetic data were obtained by conventional karyotyping of 7118 miscarriages in women with naturally conceived pregnancies. Chromosomal abnormalities were identified in 67.25% of miscarriages. The total incidence of chromosomal abnormalities increased with maternal aging; however, its average change for a one-year increase in maternal age differed between age spans, equaling 0.704% in the span from 23 to 37 years and 2.095% in the span from 38 to 44 years. At the age of 38 years, the incidence rate surged sharply by 14.79% up to 79.01% and then increased progressively up to 94% in 44-year-old women. The spectrum of chromosomal abnormalities in miscarriages was the same for each year of maternal age from 23 to 44 years. However, the proportions of particular chromosomal abnormalities differed between karyotypically abnormal miscarriages in younger and older women. The proportions of trisomy 16, polyploidy, monosomy X, mosaic aneuploidies, and structural rearrangements decreased with increasing maternal age. In contrast, the proportions of multiple aneuploidies and regular trisomies 13, 15, 18, 21, and 22 showed an upward trend with maternal aging. To summarize, despite the increase in the total incidence of chromosomal abnormalities in miscarriages with maternal aging, the rate of change differs for younger and older women, being three times lower in the former than in the latter. Moreover, the proportion of some abnormalities in karyotypically abnormal miscarriages shows a steady growth, whereas the proportion of others becomes increasingly low with maternal aging, most probably due to the age-dependent prevalence of different molecular and cellular defects.

We report on the case of prenatal detection of trisomy 2 in placental biopsy and further algorithm of genetic counseling and testing. A 29-year-old woman with first-trimester biochemical markers refused chorionic villus sampling and preferred targeted non-invasive prenatal testing (NIPT), which showed low risk for aneuploidies 13, 18, 21, and X. A series of ultrasound examinations revealed increased chorion thickness at 13/14 weeks of gestation and fetal growth retardation, a hyperechoic bowel, challenging visualization of the kidneys, dolichocephaly, ventriculomegaly, increase in placental thickness, and pronounced oligohydramnios at 16/17 weeks of gestation. The patient was referred to our center for an invasive prenatal diagnosis. The patient’s blood and placenta were sampled for whole-genome sequencing-based NIPT and array comparative genomic hybridization (aCGH), respectively. Both investigations revealed trisomy 2. Further prenatal genetic testing in order to confirm trisomy 2 in amniocytes and/or fetal blood was highly questionable because oligohydramnios and fetal growth retardation made amniocentesis and cordocentesis technically unfeasible. The patient opted to terminate the pregnancy. Pathological examination of the fetus revealed internal hydrocephalus, atrophy of brain structure, and craniofacial dysmorphism. Conventional cytogenetic analysis and fluorescence in situ hybridization revealed chromosome 2 mosaicism with a prevalence of trisomic clone in the placenta (83.2% vs. 16.8%) and a low frequency of trisomy 2, which did not exceed 0.6% in fetal tissues, advocating for low-level true fetal mosaicism. To conclude, in pregnancies at risk of fetal chromosomal abnormalities that refuse invasive prenatal diagnosis, whole-genome sequencing-based NIPT, but not targeted NIPT, should be considered. In prenatal cases of trisomy 2, true mosaicism should be distinguished from placental-confined mosaicism using cytogenetic analysis of amniotic fluid cells or fetal blood cells. However, if material sampling is impossible due to oligohydramnios and/or fetal growth retardation, further decisions should be based on a series of high-resolution fetal ultrasound examinations. Genetic counseling for the risk of uniparental disomy in a fetus is also required.

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments—the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed.

Predicting the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles that use the testicular spermatozoa of azoospermic patients presents a challenge. Thus, the development of additional approaches to assessing the competence of a testicular-sperm-derived embryo without causing damage to gametes or the embryo is necessary. One of the key parameters in determining such developmental competence is telomere length (TL). We aimed to analyze TLs in spermatogenic cells from the testicular biopsy samples of azoospermic patients and determine how this parameter influences embryo competence for pre- and post-implantation development. Using Q-FISH, we studied the TL of the chromosomes in spermatogonia and spermatocytes I from the TESE biopsy samples of 30 azoospermic patients. An increase in TL was detected during the differentiation from spermatogonia to spermatocytes I. The patients’ testicular spermatozoa were used in 37 ICSI cycles that resulted in 22 embryo transfers. Nine pregnancies resulted, of which, one was ectopic and eight ended in birth. The analysis of embryological outcomes revealed a dependence between embryo competence for development to the blastocyst stage and the TL in spermatogenic cells. The TLs in spermatogonia and spermatocytes I in the testicular biopsy samples were found to be higher in patients whose testicular sperm ICSI cycles resulted in a birth. Therefore, the length of telomeres in spermatogenic cells can be considered as a potential prognostic criterion in assessing the competence of testicular-sperm-derived embryos for pre- and post-implantation development. The results of this study provide the basis for the development of a laboratory test for the prediction of testicular sperm ICSI cycle outcomes.

Background. The epigenome of gametes is formed under the control of the developmental programme and the influence of environmental factors. How cytosine oxidation patterns are formed and altered in human spermatogenesis remains obscure so far. The aim of the study was to assess 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) patterns in human spermatogenic cells and spermatozoa. Materials and Methods. The study was performed on testicular biopsy samples of 10 azoospermic patients and ejaculate samples of 5 sperm donors and 8 patients from infertile couples. The microscope slides were prepared for further indirect immunofluorescence to detect 5fC and 5caC and FISH to determine spermatogenic cell ploidy. Results. 5fC and 5caC were undetectable in mitotic and meiotic chromosomes of spermatogenic cells, and was present exclusively in some spermatogonia and spermatid interphase nuclei as well as in some ejaculated spermatozoa. The frequency of spermatozoa with 5fC and 5caC varied in a wide range and was higher in patients than in sperm donors (p=0,007, p=0,028). The increase in frequency of spermatozoa with 5fC and 5caC was accompanied with the decrease in frequency of morphologically normal and progressively motile spermatozoa. Conclusions. 5fC and 5caC are differentially distributed in human spermatogenic cells and spermatozoa. The immunocytochemically detected increase of 5fC and 5caC in individual spermatozoa is most likely induced by oxidative stress caused by effects of internal and external factors rather than developmental programme. The evaluation of 5fC and 5caC in spermatozoa can be potentially used as an additional criterion of ejaculate quality.

BACKGROUND: Epigenetic genome reprogramming is an important determinant of human embryo development. However, its mechanisms remain poorly elucidated, especially in genetically unbalanced embryos. AIM: The aim of this study is the analysis of DNA methylation and hydroxymethylation levels in trophectoderm and inner cell mass of genetically balanced and unbalanced human blastocysts. MATERIALS AND METHODS: Twenty-two IVF-derived human blastocysts were enrolled in the study; of these blastocysts, 15 were genetically unbalanced and 7 — genetically balanced. Detection of 5-methylcytosine and 5-hydroxymethylcytosine was performed on trophectoderm and inner cell mass nuclei by indirect immunofluorescence. RESULTS: In genetically unbalanced blastocysts, the DNA methylation level was elevated in both compartments. The DNA hydroxymethylation level, in contrast, was elevated only in inner cell mass, whereas trophectoderm cells retained the same level as in genetically balanced embryos. These changes equalized the inner cell mass and trophectoderm DNA hydroxymethylation levels in genetically unbalanced blastocysts, while in genetically balanced ones the 5-hydroxymethylcytosine content in inner cell mass lagged behind that in trophectoderm. CONCLUSIONS: Genetic imbalance is associated with differential epigenetic changes in trophectoderm and inner cell mass cells of human blastocysts: DNA methylation level increases in both compartments while DNA hydroxymethylation level increases only in inner cell mass. The trophectoderm cells in genetically unbalanced blastocysts retain the same hydroxymethylation level as in genetically balanced ones, suggesting a possible explanation of the ability of karyotypically abnormal embryos to implant.