The Incidence of Mosaicism for Individual Chromosome in Human Blastocysts Is Correlated With Chromosome Length

Mosaicism, known as partial aneuploidies, mostly originates from mitotic errors during the post-zygotic stage; it consists of different cell lineages within a human embryo. The incidence of mosaicism has not been shown to correlate with maternal age, and its correlation with individual chromosome characteristics has not been well investigated. In this study, the results of preimplantation genetic testing for aneuploidy (PGT-A) derived from 4,036 blastocysts (930 IVF couples) were collected from 2015 to 2017. Via next-generation sequencing for comprehensive chromosome screening, embryo ploidy was identified as aneuploid, mosaic, and euploid. Total mosaicism was classified into two categories: “mosaic euploid/aneuploidy” (with mosaic aneuploidy between 20 and 80%) and “mosaic and aneuploidy” (a uniformly abnormal embryo superimposed with mosaic aneuploidies). Frequency of mosaicism was analyzed according to the function of chromosomal lengths, which divides involved chromosomes into three groups: group A (156–249 Mb), group B (102–145 Mb), and group C (51–90 Mb). The results show that the aneuploidy was more frequent in group C than in group A and group B (A: 23.7%, B: 35.1, 41.2%, p < 0.0001), while the mosaicism was more frequent in group A and group B than in group C [(Mosaic euploid/aneuploid) A: 14.6%, B: 12.4%, C: 9.9%, p < 0.0001; (mosaic and aneuploid) A: 21.3%, B: 22.9%, C: 18.9%, p < 0.0001; (Total mosaicism) A: 35.9%, B: 35.3%, C: 28.8%, p < 0.0001]. The significantly higher frequency of aneuploidy was on the shorter chromosome (< 90 Mb), and that of mosaicism was on the longer chromosomes (> 100 Mb). The length association did not reach significance in the patients with advanced age (≥ 36 years), and of the chromosome-specific mosaicism rate, the highest prevalence was on chromosome 14 (5.8%), 1 (5.7%), and 9 (5.6%). Although the length association was observed via group comparison, there may be affecting mechanisms other than chromosomes length. Eventually, twenty patients with mosaic embryo cryotransfers resulted in six live births. No significant correlation was observed between the transfer outcomes and chromosome length; however, the analysis was limited by small sample size.

Frequency of Sperm Aneuploidy in Oligoasthenoteratozoospermic (OAT) Patients by Comprehensive Chromosome Screening: A Proof of Concept

Background: Embryonic aneuploidy usually results in implantation failure and miscarriage. Considering significantly high frequency of sperm aneuploidy reported in oligoasthenoteratozoospermia (OAT) using fluorescence in situ hybridization (FISH) in limited number of chromosomes and lack of comprehensive chromosome screening (CCS) in OAT, the aim of this study was applying CCS in OAT sperm and comparison of the results with FISH findings. Methods: Five OAT patients with normal blood karyotypes and history of implantation failure were included. The successfully amplified samples, each containing two sperm, were analyzed by array comparative genomic hybridization (aCGH). FISH was utilized mainly depending on the aneuploidies found by aCGH to assess their frequencies in total sperm population. Results: In aCGH for 30 sperm, aneuploidy was found in 66% of samples. Following the study of 4300 sperm by FISH, an average of 55.46% aneuploidy was observed. No pregnancy was resulted with normal partners. Conclusion: Using aCGH, some abnormalities were observed that are not typically considered in sperm FISH studies. Despite small sample size of the comprehensive study, like other similar studies, the frequency of aneuploidies was considerable and similar to FISH. Aneuploidies revealed by aCGH at single sperm resolution were different from sperm population detected by FISH. Considering high frequency of aneuploidy in OATs sperm, preimplantation genetic testing for aneuploidy (PGT-A) can be used for in transfer of chromosomally normal embryos.

Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome in Patients with Impaired Reproductive Function

For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of fluorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.

Preliminary assessment of aneuploidy rates between the polar, mid and mural trophectoderm

SummaryThe objective of this study is to compare aneuploidy rates between three distinct areas of the human trophectoderm: mural, polar and a region in between these two locations termed the ‘mid’ trophectoderm. This is a cohort study on in vitro fertilization (IVF) patients undergoing comprehensive chromosome screening at the blastocyst stage at a private IVF clinic. All embryos underwent assisted hatching on day 3 with blastocyst biopsy and comprehensive chromosome screening. Biopsied blastocysts were divided into three groups depending on which area (polar, mid, or mural) of the trophectoderm was protruding from the zona pellucida and biopsied. Aneuploidy rates were significantly higher with cells from the polar region of the trophectoderm (56.2%) compared with cells removed from the mural region of the trophectoderm (30.0%; P = 0.0243). A comparison of all three areas combined also showed a decreasing trend, but this did not reach clinical significance, polar (56.2%), mid (47.4%) and mural trophectoderm (30.0%; P = 0.1859). The non-concordance demonstrated between polar and mural trophectoderm can be attributed to biological occurrences including chromosomal mosaicism or procedural differences between embryologists.

Preimplantation genetic testing for balanced complex chromosomal rearrangement carriers by comprehensive chromosome screening

Background: Balanced complex rearrangements (BCCRs) are balanced chromosomal structural aberrations that involve two or more chromosomes and at least three breakpoints. It is very rare in the population. Whether the couple of BCCRs benefit from preimplantation genetic testing (PGT) need to be further explored. Here, we reported the outcome of PGT in BCCRs carriers. Results: A total of 141 oocytes were retrieved from 7 couples within 10 PGT cycles, including 116 mature oocytes (MII), and 94 (81.03%) oocytes normally fertilized after intracytoplasmic sperm injection (ICSI). Then, 47 embryos were biopsied, including 8 embryos at the cleavage stage and 39 (41.49%) blastocysts. After comprehensive chromosome analysis, the balanced or normal embryo rate was 11.36% (5), the abnormal embryo rate was 88.63% (39), and 3 failed to amplify. Among them, the balanced or normal embryo rate was 33.33% (3) and the abnormal embryo rate was 66.67% (6) in the three-way rearrangements. The balanced or normal embryo rate was 5.6% (1) and the abnormal embryo rate was 94.4% (17) in double two-way translocations. The balanced or normal embryo rate was 5.9% (1) in exceptional CCRs, and the abnormal embryo rate was 94.1% (16). There were no significant differences among the three groups (P=0.11). In the 10 PGT cycles, there were 7 cycles in which no embryo could be transplanted and 3 cycles in which balanced or normal embryos underwent frozen-thawed embryo transplantation. One of the 3 cycles was clinically pregnant, and the prenatal diagnosis of amniocytes using G-band and SNP array at 16 weeks of gestation was 46, XN, and a boy was born alive and healthy. Conclusions: BCCR carriers have a high rate of obtaining abnormal embryos, but they can also have healthy offspring. For BCCR carriers with fertility needs, PGT is recommended to have related offspring, or they can choose sperm donor or ovum donation-assisted reproduction.

Blastocoel Fluid Biopsy

The identification of viable embryos for transfer is one of the main challenges in reproductive medicine. As chromosomal abnormalities are the major cause of implantation failure, preimplantation genetic testing of aneuploidy plays an important role in embryo selection. To make this approach more efficient, the possibility to retrieve informative DNA through a moderately invasive technique compared to the traditional forms of biopsy is appealing. Blastocoelic fluid is a valuable source of DNA. Its presence, as detected by whole genomic amplification, and the following analysis by comprehensive chromosome screening could add important information on the blastocyst ploidy condition and developmental potential.