Single blastomere removal from murine embryos is associated with activation of matrix metalloproteinases and Janus kinase/signal transducers and activators of transcription pathways of placental inflammation.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

UDP-glucuronosyltransferase 1a enzymes are present and active in the mouse blastocyst.

The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of ß-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development.