Re-Examination of PGT-A Detected Genetic Pathology in Compartments of Human Blastocysts: A Series of 23 Cases.

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.

Telomere Length in Human Spermatogenic Cells as a New Potential Predictor of Clinical Outcomes in ART Treatment with Intracytoplasmic Injection of Testicular Spermatozoa.

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.

Prenatal Detection of Trisomy 2: Considerations for Genetic Counseling and Testing.

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.