Effects of Intrauterine Air Bubbles on Embryonic Development in Mice

Abstract

During murine embryo transfer, air bubbles frequently are loaded with embryos into the transfer catheter, but the role of air bubbles on embryonic development is unclear. This study shows that intrauterine air disrupted embryo spacing, induced deciduoma, and impaired postimplantation development. RNA sequencing showed that the gene expression profile of air-induced deciduoma differed significantly from that of embryo-induced decidua but is similar to tetraploid-induced deciduoma. A subset of 33 common genes was upregulated in the embryo-induced decidua compared with air- or tetraploid-induced deciduoma. These data suggest that the inner cell mass (ICM) plays a key role in regulating decidualization and that the trophectoderm is an intermediate that relays ICM-derived signals to other target cells. Our results may provide an innovative approach for detecting the developmental status of embryos in human reproductive medicine.

Figure 1.

Primers and amplicon length of evaluated genes.

Figure 2.

Intrauterine air–induced decidualization. (A) Air (2.0 µL) was infused into a single uterine horn of a pseudopregnant day 3.5 mouse. After 4 d, the right horn showed decidualization. (B) Relationship between the volume of intrauterine air infusion and decidualization. (C) A large intrauterine air bubble (asterisk) was observed immediately after air infusion into uterine horns. (D) Microbubbles (arrowhead) were detected at 2 h after air infusion. (E) Air was infused into a single uterine horn of a pseudopregnant day 3.5 mouse. After 1 d, the mouse was anesthetized and the tail veins injected with 0.1 mL of 1% trypan blue in saline. The unilateral uterine horn contained a diffuse blue area. (F) A natural day 4.5 pregnant uterine horn displayed distinct and isolated blue bands after the trypan blue injection.

Figure 3.

Intrauterine air disrupted postimplantation embryonic development. Air were infused into a single uterine horn (asterisk) of pregnant mice. (A) Day 1.5. (B) Day 2.5. (C) Day 3.5. (D) Day 4.5. The contralateral uterine horn without air infusion was used as a control. On day 7.5, fetal development was significantly superior in the (E through H) control horns than in the (I through L) air-infused horns.

Figure 4.

Decidualization on day 7.5 induced by different deciduogenic stimuli. (A) Natural day 3.5 blastocysts were transferred into a single uterine horn of a pseudopregnant day 2.5 mouse.(B) Natural day 3.5 blastocysts and 2.0 µL of air were transferred into a single uterine horn of a pseudopregnant day 2.5 mouse.(C) Tetraploid embryos were surgically transferred into the right oviduct of a pseudopregnant day 0.5 mouse, followed 72 h later by surgical infusion of 2.0µL of air into the left uterine horn.(D) Germinal vesicle oocytes were transferred into a single uterine horn of a pseudopregnant day 2.5 mouse.

Figure 5.

Heatmap of genes differentially expressed between day 7.5 embryo-induced decidua compared with air- or tetraploid-induced deciduoma. Yellow to black to pink indicates a gradient of high to low expression. A total of 33 genes were upregulated in the embryo-induced decidua compared with the air- and tetraploid-induced deciduoma. No genes showed significant differences in expression between air- and tetraploid-induced deciduoma.

Figure 6.

(A) Gene ontology enrichment chart of upregulated genes in biologic process category and (B) the protein interaction network of genes highly expressed in embryo-induced decidua.