Comparison of 2, 5, and 20 % O2 on the development of post-thaw human embryos

Abstract

Purpose: The objective of this study is to investigate the effect of 2, 5, and 20 % O2 on post-thaw day 3 human embryo culture until blastocyst stage.

Methods: One hundred fifty-five day 3 human embryos were used. One hundred twenty out of 155 embryos were recovered after thawing. Surviving embryos were distributed into 2, 5, or 20 % O2 groups and cultured for 2.5 days. At the end of culture, blastocyst formation was assessed, and then, embryos were collected for RT-qPCR or immunofluorescence analysis.

Results: Using visible blastocoel to define blastocyst formation, 58.7 % (27/46) of surviving day 3 embryos formed blastocyst at 2 % O2, 63.6 % (28/44) at 5 % O2, and 66.7 % (20/30) at 20 % O2. The difference in blastocyst formation rates was not significant. Average blastocyst cell number was 119.44 ± 11.64 at 2 % O2, 142.55 ± 22.47 at 5 % O2, and 97.29 ± 14.87 at 20 % O2. Average apoptotic rate was 4.7 % ± 0.4 % for blastocyst formed at 2 % O2, 3.5 % ± 0.7 % at 5 % O2, and 5.8 % ± 1.1 % at 20 % O2. Apoptosis rate was significantly lower for blastocysts formed at 5 % O2 (p < 0.05). Compared with gene expression levels at 5 % O2, which were arbitrarily set as "1," 20 % O2 is associated with significantly higher expression of BAX (2.14 ± 0.47), G6PD (2.92 ± 1.06), MnSOD (2.87 ± 0.88), and HSP70.1 (8.68 ± 4.19). For all genes tested, no significant differences were found between 2 and 5 % O2.

Conclusion: The result suggests that development of cryopreserved human embryos from day 3 to blastocyst stage benefits from culture at 5 % O2.

Keywords: Apoptosis; Human embryo; Oxygen; PCR; Stress.

Figures

Fig. 1

Blastocyst cell numbers were not significantly different among the three O2 groups. Apoptosis rate was significantly lower in 5 % O2 compared with 2 and 20 % O2. a Embryos were cultured from thawed day 3 embryos for another 2.5 days to blastocyst stage. This figure shows average blastocyst cell number in each group. b Apoptosis was significantly lower at 5 % O2 (p < 0.05), but no difference was found between 2 and 20 % O2. Original data was log transformed, and the comparison among three groups was done by one-way ANOVA followed by Dunnett’s post hoc test. Distinct letters a and b on top of the histogram bars indicate a significant difference between the comparison groups (p < 0.05). Data is presented with average ±SEM

Fig. 2

Representative blastocysts cultured at 2 or 20 % O2 with nuclear and apoptosis staining. a, d Active caspase-3 staining of apoptosis for blastocysts cultured at 2 and 20 % O2, respectively. b, e Same embryos as in a and d, respectively, showing DAPI staining of nucleus. c, f Merged image of a and b and d and e. Micron bar represents 25 μm

Fig. 3

The mRNA expression level of six marker genes showed no significant difference between 2 and 5 % O2. The mRNA levels of BAX, MnSOD, G6PD, and HSP70.1 were significantly higher in 20 % O2 than in 5 % O2. Data were analyzed using ANOVA followed by Dunnett’s post hoc test. The same pair of letters a and a indicates no significant difference between the comparison groups. Distinct letters a and b indicate a significant difference (p < 0.05). Data is presented with average ±SEM