Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion.

Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

Elevated frequencies of micronuclei in pregnant women with type 1 diabetes mellitus and in their newborns.

Pregestational diabetes mellitus (type 1 and type 2) affects about 1% of the obstetric population. In diabetes, persistent hyperglycemia can be a source of DNA damage via overproduction of reactive oxygen species (ROS). Using the cytokinesis-block micronucleus (CBMN) test, we measured the frequencies of micronuclei (MN) per 1000 binucleated (BN) cells in pregnant women (mothers) with type 1 diabetes mellitus (T1DM) and in their newborns. Peripheral blood lymphocytes were collected from 17 pregnant women with T1DM and cord-blood lymphocytes from their 17 newborns. The control group included 40 pregnant women (mothers) without diabetes mellitus (DM) and their 40 newborns. In the group of pregnant women with T1DM, the mean number of MN per 1000 BN cells was 2.35 (±1.07), significantly (p<0.001) higher than in the control group of pregnant women (0.86±0.90). The frequency value in the group of newborns of T1DM mothers was 1.42 (±0.60), significantly (p<0.05) higher than in the corresponding control group (0.67±0.79). The value in the group of mothers with T1DM was significantly (p<0.05) higher than in their newborns. Comparing mothers without DM with their newborns, no significant frequency differences were observed. No significant correlations were observed between MN frequencies in mothers with T1DM and either the frequencies in their newborns, the duration of diabetes, or HbA1C levels. Our results indicate that T1DM is accompanied by increased frequencies of MN in pregnant women and their newborns.

Effects of the polysaccharide beta-glucan on clastogenicity and teratogenicity caused by acute exposure to cyclophosphamide in mice.

Alterations that could lead to the cancer development may also be related to an adverse development of offspring in experimental animals. Some functional foods, which contain the polysaccharide beta-glucan, have been described as being effective in the prevention of clastogenic damage. Based on that finding, the aim of the present study was to determine the efficacy of this sugar polymer in the mutagenic and teratogenic damage control. Two sets of females, pregnant and non-pregnant, were evaluated. The results indicated that beta-glucan was effective in preventing clastogenic damage in pregnant as well as non-pregnant females. In addition, pregnant females were more susceptible to mutagenic damage. However, teratogenic effects were not prevented effectively, although there was a trend toward a reduction in level of malformations. Despite beta-glucan did not prevent malformations, it increased fetal viability and reduced number of post-implantation losses and resorption, thereby enhancing reproductive performance in females.

Relevance of experimental models for investigation of genotoxicity induced by antiretroviral therapy during human pregnancy.

The current incidence of human immunodeficiency virus (HIV-1)/AIDS affects around 7000 pregnant women in the United States. When given during pregnancy, the nucleoside analog 3'-azido-3'-deoxythymidine (AZT) significantly reduces maternal-fetal transmission. It has been previously shown that AZT is incorporated into DNA, where it causes mutations in the HPRT and TK genes. It also changes cell cycle gene expression, and induces S-phase arrest, micronuclei, chromosomal aberrations, sister chromatid exchanges, telomeric attrition, and other genotoxic effects in cultured cells. A predicted consequence of these events is genomic instability that together, with clastogenicity may contribute to the carcinogenic potency of AZT. Various aspects of genotoxicity are explored in this contribution seeking to understand the multiple effects of this antiretroviral agent in animal models and humans. This mini-review describes some of the experimental models used to elucidate the genotoxicity induced by antiretroviral therapy during human pregnancy. The use of diverse methods to detect biomarkers of exposure, such as an AZT-specific radioimmunoassay, micronuclei bearing intact chromosomes, and telomeric DNA attrition highlight the role of in vitro models to elucidate exposure and risk. The relevance of the in vitro models is followed by the introduction of the role of the nucleoside analogs in transplacental carcinogenesis along with the description of a transplacental perfusion model and a transplacental carcinogenesis rodent model. In a more direct clinical application the use of AZT-DNA incorporation as a biomarker of exposure, in experiments conducted in vivo in Erythrocebus patas monkeys and in humans, addresses the possibility of elucidation of potential cancer risk in those infants exposed in utero. Two relevant aspects of this contribution are the potential application of some of the models described in this mini-review, as diagnostic tools in antiretroviral-exposed populations, and the use of these models to understand the nature of the genotoxicities and minimize the undesirable side effects of the antiretroviral therapy.

Comparison of 4', 6'-diamidino-2-phenylindole and Giemsa stainings in preimplantation mouse embryos micronucleus assay including a triple dose study.

Analysis of micronuclei (MN) in preimplantation embryos is a good method for the evaluation of cytogenetic damage induced by occupational and environmental mutagen during early pregnancy. To examine whether conventional Giemsa staining produced the same accuracy of micronuclei as the DNA-specific 4', 6'-diamidino-2-phenylindole (DAPI) staining in preimplantation embryo induced by maternal exposure to chlorpyrifos, we conducted assays on 469 mouse (3 groups) preimplantation embryos micronucleus. Slides were stained with DAPI. After DAPI staining, the slides were de-stained and restained with Giemsa. Giemsa staining showed similar frequencies in MN to DNA-specific DAPI staining in all three groups. Both staining techniques revealed significant increases in frequency of MN in the treated group in comparison to the control group. Both methods showed a statistically significant correlation between MN frequency and the dose of chlorpyrifos. Compared with DAPI staining, the sensitivity of Giemsa staining was 85.0%, 86.0% and 90.9% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. The specificity was 97.9%, 91.4% and 96.5% for control, 40 mg/kg, and 80 mg/kg chlorpyrifos treated group, respectively. Thus, we recommend that Giemsa staining technique be a standard staining method in detecting MN of preimplantation embryos induced by occupational or environmental hazards.

Dynamic blastomere behaviour reflects human embryo ploidy by the four-cell stage.

Previous studies have demonstrated that aneuploidy in human embryos is surprisingly frequent with 50-80% of cleavage-stage human embryos carrying an abnormal chromosome number. Here we combine non-invasive time-lapse imaging with karyotypic reconstruction of all blastomeres in four-cell human embryos to address the hypothesis that blastomere behaviour may reflect ploidy during the first two cleavage divisions. We demonstrate that precise cell cycle parameter timing is observed in all euploid embryos to the four-cell stage, whereas only 30% of aneuploid embryos exhibit parameter values within normal timing windows. Further, we observe that the generation of human embryonic aneuploidy is complex with contribution from chromosome-containing fragments/micronuclei that frequently emerge and may persist or become reabsorbed during interphase. These findings suggest that cell cycle and fragmentation parameters of individual blastomeres are diagnostic of ploidy, amenable to automated tracking algorithms, and likely of clinical relevance in reducing transfer of embryos prone to miscarriage.