Blastomere removal from cleavage-stage mouse embryos alters placental function, which is associated with placental oxidative stress and inflammation

Abstract

Blastomere biopsy is an essential technique in preimplantation genetic diagnosis (PGD), a screening test that can detect genetic abnormalities of embryos before their transfer into uterus. Our results showed that the weights of fetuses derived from biopsied embryos were lower than that of non-biopsied counterparts at E12.5, E15.5, and E18.5. The ratio of fetal/placental (F/P) weights in the biopsied group was significantly lower than that in the non-biopsied group at E18.5. At E18.5, the mRNAs for selected glucose transporters, system A amino acid transporters, system L amino acid transporters, and imprinted genes were downregulated in the placentae of biopsied group, and the GLUT1 and CAT3 protein levels were decreased too. More apoptotic cells were detected by TUNEL in the placentae of biopsied group. Placentae from biopsied embryos exhibited lower levels of SOD and GSH. Furthermore, the concentration of MDA increased in the placentae from biopsied group. The levels of IL1B, IL6, and TNFA also significantly increased in the placentae of biopsied group. This study suggested that placental function may be sensitive to blastomere biopsy procedures, and placental oxidative stress and inflammation associated with blastomere biopsy may be critical factors of abnormal placental function and further influence the fetal development.

Figure 1. Blastocyst quality by differential staining (n = 15 per group).

(a) The number of total blastocyst cells. (b) The number of TE cells. (c) The number of ICM cells. (d) The ratio between ICM cell number and the total cell number. Asterisks indicate the statistical significance (*P < 0.001), VS. non-biopsied group.

Figure 2. Placental morphology at E18.5.

(a,b) Cross-sections of the entire placenta at E18.5, from non-biopsied group (a) and biopsied group (b) stained with H&E. L, labyrinth zone; J, junctional zone. (c–f) Representative microscopic views of placentae apoptosis measured by TUNEL staining in the labyrinth and junctional zone. Labyrinth zone from biopsied placenta (c); Junctional zone from biopsied placenta (d); Labyrinth zone from non-biopsied placenta (e); Junctional zone from non- biopsied placenta (f). (Scale bars: 10 μm).

Figure 3. Labyrinth and junctional areas and placental apoptosis at E18.5 (n = 6 per group).

(a) The area of labyrinth zone. (b) The area of junctional zone. (c) The ratio between labyrinth zone area and whole placenta area. (d) The ratio between junctional zone area and whole placenta area. (e) The percentage of TUNEL positive cells in labyrinth zone. (f) The percentage of TUNEL positive cells in junctional zone. Asterisks indicate the statistical significance (*P < 0.001), VS. non-biopsied group.

Figure 4. The expression of selected transporter genes and imprinted genes in placentae at E18.5.

Real-time PCR for the relative amounts of Glut1, Glut3, Snat1, Snat2, Snat4, Cat3, Lat1, Lat2,Igf2, Igf2r, H19, Igf2P0, and11βHsd2 mRNA in total placental tissue at E18.5 in the non-biopsied and biopsied groups (n = 6 per group). Asterisks indicate the statistical significance (*P < 0.05, **P < 0.01)VS. non-biopsied group.

Figure 5. The expression of GLUT1 and CAT3 protein in placentae at E18.5.

(a) Representative Western blottings of GLUT1, CAT3, and Actin in total placental homogenates, obtained from the non-biopsied and biopsied groups (3 litters per group). (b) Densitometric measurements of Western blotting autoradiograms (n = 3 per group). Asterisks indicate the statistical significance (*P < 0.001)VS. non-biopsied group.