Author(s): Xiaoqin Ye [1, 7]; Kotaro Hama [2, 7]; James J. A. Contos ; Brigitte Anliker ; Asuka Inoue ; Michael K. Skinner ; Hiroshi Suzuki [3, 6]; Tomokazu Amano ; Grace Kennedy ; Hiroyuki Arai ; Junken Aoki ; Jerold Chun (corresponding author) 
Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies . Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2-4). Targeted deletion of LPA3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase 2 (COX2) (ref. 5), was downregulated in LPA 3 -deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2 ), which are critical for implantation . Exogenous administration of PGE 2 or carbaprostacyclin (a stable analogue of PGI2 ) into LPA3 -deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.
Multiple factors can adversely affect successful pregnancy. Two of these factors are failed synchronization between embryonic and endometrial development during implantation and occurrence of multiple gestations (especially monochorionic gestation), which can result in fetal demise [1, 6, 7, 8, 9]. These factors are particularly important for the clinical success and efficacy of assisted reproductive technologies. One molecular factor that has been previously implicated in female reproduction is the small, bioactive phospholipid LPA . LPA has a range of influences that are mediated by at least four G-protein-coupled receptors, LPA1-4 (ref. 2). Deletion of LPA1 and LPA2 in mice revealed roles for these receptors in neural development, craniofacial formation, neuropathic pain and altered cellular signalling, but without obvious effects on female reproduction [11, 12, 13, 14]. These results suggested that LPA signalling in female reproduction might be mediated by other LPA receptors including LPA3 (formerly known as Edg7) (refs 3, 4), LPA4 (ref. 15), unidentified LPA receptor(s), or possibly non-receptor pathways. Towards identifying LPA-dependent mechanisms affecting reproduction, we targeted LPA3 for deletion. LPA3 is a receptor with distinct signalling properties and a preference for unsaturated LPA species [2, 3, 4].
Functional deletion of LPA3 was achieved by replacing a fragment covering the untranslated region and the start codon in exon 2 with a neomycin-resistance gene in reverse orientation in R1 embryonic stem cells (Supplementary Figs 1 and 2). The LPA 3 -deficient mice were born with normal mendelian frequency without sexual bias (Supplementary Table 1), and appeared grossly normal (data not shown). However, LPA3 -deficient females produced litter sizes of less than 50% compared with that...