The human embryo following biopsy on day 5 versus day 3: viability, ultrastructure and spindle/chromosome configurations.

RESEARCH QUESTION: Are there any differences in viability, spindle abnormalities and mitochondrial and other organelle structures amongst embryos biopsied on day 3 versus day 5 before and after vitrification? DESIGN: A total of 240 day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects (PGT-M) (n = 115) or for aneuploidies (PGT-A) (n = 125) were divided into two groups: (i) 120 blastocysts treated for viability, spindle/chromosome configuration (SCC) analysis and transmission electron microscopy (TEM) analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20); (ii) 120 embryos were re-biopsied at the blastocyst stage and treated for viability, SCC and TEM analysis (fresh n = 20, n = 20, n = 20 and following vitrification/warming n = 20, n = 20, n = 20). Also, 60 vitrified blastocysts biopsied only on day 5 that were rejected for transfer following PGT-M (n = 6) or PGT-A (n = 54) were treated following warming for viability (n = 20), SCC (n = 20) and TEM analysis (n = 20). RESULTS: No differences were observed in SCC and ultrastructure between embryos biopsied on day 5 and day 3 but following vitrification higher numbers of abnormal spindles, distension of mitochondria, multivesicular bodies, lipofuscin droplets, altered cell junctions and occasionally excessive accumulation of glycogen granules were evident. The fresh day 3 biopsied group also had a lower incidence of damaged (propidium iodide-stained) cells compared with the fresh day 3+5 (P = 0.02) and the vitrified day 5 (P = 0.001) biopsied groups. CONCLUSIONS: Biopsies on day 5 and day 3 do not adversely affect embryo viability, SCC or ultrastructure, although following vitrification minimal embryo quality-dependent increases in spindle abnormalities and cell damage are observed.

Viability assessment using fluorescent markers and ultrastructure of human biopsied embryos vitrified in open and closed systems.

RESEARCH QUESTION: Are there any differences in viability and ultrastructure amongst embryos biopsied on Day 5 versus Day 3 following vitrification in open and closed systems and compared to fresh embryos? DESIGN: One hundred human embryos (40 blastocysts biopsied on Day 5 and subsequently vitrified in open or closed systems and 60 Day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects and for aneuploidies were either treated fresh [n = 20] or vitrified [n = 40] in open or closed systems) and following warming and culture for 4 h were subjected to viability staining with carboxyfluorescein-diacetate succinimidylester/propidium iodide or processed for transmission electron microscopy. RESULTS: No statistically significant differences were observed in the viability of human biopsied embryos following vitrification in open and closed systems. Compared to fresh embryos, vitrified ones had a higher incidence of damage (propidium iodide-stained cells) irrespective of the vitrification method (P = 0.005). These damaged cells were more prominent in Day 5 biopsied blastocysts and mainly located at the position of cutting. Characteristic lipofuscin droplets (representative of apoptosis) and a higher number of vacuoles and distension of mitochondria were also more evident in vitrified embryos, although this was not statistically assessed. CONCLUSIONS: Vitrification in open and closed systems does not adversely affect the viability and ultrastructure of Day 5 and Day 3 biopsied embryos as revealed by the minimal yet statistically significant cell damage observed. This damage may be compensated by the embryos, which in their attempt to fully recover following vitrification, potentially enable 'rescue' processes to eliminate it.

Effects of moist cold stratification on germination, plant growth regulators, metabolites and embryo ultrastructure in seeds of Acer morrisonense (Sapindaceae).

Breaking of seed dormancy by moist cold stratification involves complex interactions in cells. To assess the effect of moist cold stratification on dormancy break in seeds of Acer morrisonense, we monitored percentages and rates of germination and changes in plant growth regulators, sugars, amino acids and embryo ultrastructure after various periods of cold stratification. Fresh seeds incubated at 25/15 °C for 24 weeks germinated to 61%, while those cold stratified at 5 °C for 12 weeks germinated to 87% in 1 week. Neither exogenous GA3 nor GA4 pretreatment significantly increased final seed germination percentage. Total ABA content of seeds cold stratified for 12 weeks was reduced about 3.3-fold, to a concentration similar to that in germinated seeds (radicle emergence). Endogenous GA3 and GA7 were detected in 8-week and 12-week cold stratified seeds but not in fresh seeds. Numerous protein and lipid bodies were present in the plumule, first true leaves and cotyledons of fresh seeds. Protein and lipid bodies decreased greatly during cold stratification, and concentrations of total soluble sugars and amino acids increased. The major non-polar sugars in fresh seeds were sucrose and fructose, but sucrose increased and fructose decreased significantly during cold stratification. The major free amino acids were proline and tryptophan in fresh seeds, and proline increased and tryptophan decreased during cold stratification. Thus, as dormancy break occurs during cold stratification seeds of A. morrisonense undergo changes in plant growth regulators, proteins, lipids, sugars, amino acids and cell ultrastructure.