Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.

Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA, Animal Models Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA, TransViragen, Inc., Research Triangle Park, NC 27709, USA, Key Lab of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, Department of translational medicine, Department of surgery, Department of medicine, the first hospital, Jilin University, Changchun 130061, China.

Humanized mouse models have become increasingly important and widely used in modeling human diseases in biomedical research. Immunodeficient mice such as NOD- Rag1 -/- IL2RgammaC-null (NRG) or NOD- SCID - IL2RgammaC-null (NSG) mice are critical for efficient engraftment of human cells or tissues. However, their genetic modification remains challenging due to a lack of embryonic stem cells and difficulty in the collection of timed embryos after superovulation. Here, we report the generation of gene knockout NRG mice by combining in vitro fertilization (IVF) and CRISPR/Cas9 technology. Sufficient numbers of fertilized embryos were produced through IVF and a high rate of Fah gene targeting was achieved with microinjection of Cas9 mRNA, gRNA and single strand oligonucleotide DNA (ssDNA) into the embryos. The technology paves the way to construct NRG or NSG mutant mice to facilitate new humanized mouse models. The technology can also be readily adapted to introduce mutations in other species such as swine and non-human primates.