Outcomes of BRCA pre-implantation genetic testing according to the parental mutation origin: a cohort study.

BACKGROUND: The process of gamete formation and early embryonic development involves rapid DNA replication, chromosome segregation and cell division. These processes may be affected by mutations in the BRCA1/2 genes. The aim of this study was to evaluate BRCA mutation inheritance and its effect on early embryonic development according to the parental origin of the mutation. The study question was approached by analyzing in vitro fertilization cycles (IVF) that included pre-implantation testing (PGT-M) for a BRCA gene mutation. METHODS: This retrospective cohort study compared cycles of pre-implantation genetic testing for mutations (PGT-M) between male and female patients diagnosed with BRCA 1/2 mutations (cases), to a control group of two other mutations with dominant inheritance (myotonic dystrophy (MD) and polycystic kidney disease (PKD)). Results were compared according to mutation type and through a generalized linear model analysis. RESULTS: The cohort included 88 PGT-M cycles (47 BRCA and 41 non-BRCA) among 50 patients. Maternal and paternal ages at oocyte retrieval were comparable between groups. When tested per cycle, FSH dose, maximum estradiol level, oocytes retrieved, number of zygotes, and number of embryos available for biopsy and affected embryos, were not significantly different among mutation types. All together 444 embryos were biopsied: the rate of affected embryos was comparable between groups. Among BRCA patients, the proportion of affected embryos was similar between maternal and paternal mutation origin (p = 0.24). In a generalized linear model analysis, the relative oocyte yield in maternal BRCA patients was significantly lower (0.7, as related to the non BRCA group)(p < 0.001). Zygote formation and blastulation were not affected by the BRCA gene among paternal cases (P = 0.176 and P = 0.293 respectively), nor by paternal versus maternal BRCA carriage (P = 0.904 and P = 0.149, respectively). CONCLUSIONS: BRCA PGT-M cycles performed similarly compared to non-BRCA cycles. Inheritance rate and cycle parameters were not affected by the parental origin of the mutation.

Effect of the aflatoxins B1 and M1 on bovine oocyte developmental competence and embryo morphokinetics.

Aflatoxins are considered as reproductive toxins for mammalian species. Here, we studied the effect of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1 (AFM1) on the development and morphokinetics of bovine embryos. Cumulus oocyte complexes (COCs) were matured with AFB1 (0.032, 0.32, 3.2, 32 µM) or AFM1 (0.015, 0.15, 1.5, 15, 60 nM), then fertilized and the putative zygotes were cultured in an incubator equipped with a time-lapse system. Exposing COCs to 32 µM AFB1 or 60 nM AFM1 reduced the cleavage rate, whereas exposing them to 3.2 or 32 µM AFB1 further reduced the blastocyst formation. A delay was recorded for the first and second cleavages in a dose-dependent manner for both AFB1- and AFM1-treated oocytes. A delay was recorded in the third cleavage in the AFM1-treated group. To explore potential mechanisms, subgroups of COCs were examined for nuclear and cytoplasmic maturation (n = 225; DAPI and FITC-PNA, respectively), and mitochondrial function was examined in a stage-dependent manner. COCs were examined for their oxygen consumption rates (n = 875; Seahorse XFp analyzer) at the end of maturation, MII-stage oocytes were examined for their mitochondrial membrane potential (n = 407; JC1), and putative zygotes were examined using a fluorescent time-lapse system (n = 279; IncuCyte). Exposing COCs to AFB1 (3.2 or 32 µM) impaired oocyte nuclear and cytoplasmic maturation and increased mitochondrial membrane potential in the putative zygotes. These alterations were associated with changes in the expression of mt-ND2 (32 µM AFB1) and STAT3 (all AFM1 concentrations) genes in the blastocyst stage, suggesting a carryover effect from the oocyte to the developing embryos.

Seasonal variation in the morphokinetics of in-vitro-derived bovine embryos is associated with the blastocyst developmental competence and gene expression.

Summer heat stress is a major cause of reduced development of preimplantation embryos. Nevertheless, seasonal effects on embryo morphokinetics have been less studied. We used a non-invasive time-lapse system that allows continuous monitoring of embryos to study the seasonal impact on embryo morphokinetics. The experiments were performed during the cold and the hot seasons. Cumulus-oocyte complexes were aspirated from ovaries, in-vitro-matured, and fertilized. Putative zygotes were cultured in an incubator equipped with a time-lapse system. The cleavage and blastocyst formation rates were lower in the hot vs. the cold season (p < 0.01). The kinetics of the embryos differed between seasons, reflected by a delay in the second cleavage in the hot vs. the cold season (p < 0.03). The distribution of the embryos into different morphological grades (good, fair, and poor) throughout the first three cleavages differed between seasons, with a higher proportion of good-grade embryos in the hot season (p < 0.03). Cleaved embryos were categorized as either normal or abnormal, based on their first cleavage pattern. Normal cleavage was defined as when the first cleavage resulted in two equal blastomeres and further classified as either synchronous or asynchronous, according to their subsequent cleavages. Abnormal cleavage was defined as when the embryo directly cleaved into more than two blastomeres, it cleaved unequally into two unevenly sized blastomeres, or when the fusion of already divided blastomeres occurred. The proportion of abnormally cleaved embryos was higher in the hot season vs. the cold one (p < 0.01), reflected by a higher proportion of unequally cleaved embryos (p < 0.02). In the cold season, abnormally cleaved embryos had a lower potential to develop into blastocysts relative to their normally cleaved counterparts (p < 0.001). Blastocysts that developed in the cold and the hot seasons differed in the expression of genes that related to the cell cycle (STAT1; p < 0.01), stress (HSF1; p < 0.03), and embryo development (ZP3; p < 0.05). A higher expression level was recorded for the STAT1 and UHRF1 genes in blastocysts that developed from unequally vs. the synchronously cleaved embryos (p < 0.04). We provide the first evidence for a seasonal effect on embryo morphokinetics, which might explain the reduced embryo development during the hot season.

Association between the morphokinetics of in-vitro-derived bovine embryos and the transcriptomic profile of the derived blastocysts.

The time-lapse system is a non-invasive method that enables a continuous evaluation through embryo development. Here, we examined the association between the morphokinetics of the developing embryo and the transcriptomic profile of the formed blastocysts. Bovine oocytes were matured and fertilized in vitro; then, the putative zygotes were cultured in an incubator equipped with a time-lapse system. Based on the first-cleavage pattern, embryos were categorized as normal or abnormal (68.5±2.2 and 31.6±2.3%, respectively; P<0.001). A cleaved embryo was defined as normal when it first cleaved into two equal blastomeres; it was classified as synchronous or asynchronous according to its subsequent cleavages. An abnormal pattern was defined as direct, unequal, or reverse cleavage. Direct cleavage was classified as division from one cell directly into three or more blastomeres; unequal cleavage was classified as division that resulted in asymmetrically sized blastomeres; and reverse cleavage of the first division was classified as reduced number of blastomeres from two to one. Of the normally cleaving embryos, 60.2±3.1% underwent synchronous cleavage into 4, 8, and 16 blastomeres, and 39.7±3.1% cleaved asynchronously (P<0.001). The blastocyte formation rate was lower for the synchronously vs. the asynchronously cleaved embryos (P<0.03). The abnormally cleaved embryos showed low competence to develop to blastocysts, relative to the normally cleaved embryos (P<0.001). Microarray analysis revealed 895 and 643 differentially expressed genes in blastocysts that developed from synchronously and asynchronously cleaved embryos, respectively, relative to those that developed from directly cleaved embryos. The genes were related to the cell cycle, cell differentiation, metabolism, and apoptosis. About 180 differentially expressed genes were found between the synchronously vs. the asynchronously cleaved embryos, related to metabolism and the apoptosis mechanism. We provide the first evidence indicating that an embryo's morphokinetics is associated with the transcriptome profile of the derived blastocyst, which might be practically relevant for the embryo transfer program.

Melatonin slightly alleviates the effect of heat shock on bovine oocytes and resulting blastocysts.

Heat stress is associated with increased production of reactive oxygen species (ROS) and disruption of bovine oocyte function. Here, we examined whether the antioxidant melatonin can alleviate the deleterious effects of heat stress on oocyte developmental competence. Cumulus-oocyte complexes were matured for 22 h at 38.5 °C (control) or for 22 h at 41.5 °C (heat shock) with or without 1.0 × 10-7 M melatonin. At the end of maturation, a subgroup of oocytes was examined for nuclear and cytoplasmic maturation, ROS level and mitochondrial membrane potential. A second subgroup of oocytes underwent fertilization (18 h), and putative zygotes were cultured in an incubator equipped with a time-lapse system for ∼190 h. Cleavage rate and the proportion of blastocysts, as well as embryo kinetics were recorded. Expanded blastocysts were collected and their transcript abundance was evaluated. Heat shock increased ROS and reduced the proportion of oocytes that resumed meiosis and reached the metaphase-II stage. Exposing oocytes to heat shock with melatonin alleviated these effects to some extent, expressed by a marginal reduction in ROS level and increased proportion of metaphase-II stage oocytes. Neither the distribution of oocyte cortical granules nor polarization of the mitochondrial membrane differed between control and heat-shocked oocytes cultured with or without melatonin. Heat shock reduced the proportion of embryos that cleaved and developed to blastocysts, characterized by alterations in kinetics of the developed embryos expressed by a delay in the first cleavage, second cleavage and blastocyst formation for heat-shock vs. control groups. Melatonin did not restore the competence or kinetics of embryos developed from heat-shocked oocytes. However, expanded blastocysts developed from heat-shocked oocytes treated with melatonin expressed a higher transcript abundance of genes associated with mitochondrial function, relative to the control and heat-shock group. In summary, melatonin improved the oxidative status of heat-shocked oocytes to some extent and had a beneficial effect on maternal mitochondrial transcripts in the developed blastocysts.