The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with β2-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8α expression on PBMC without changing CD3δ expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

STUDY QUESTIONIs endometriosis associated with abnormally located endometrial basalis-like (SSEA1+/SOX9+) cells in the secretory phase functionalis and could they contribute to ectopic endometriotic lesion formation?SUMMARY ANSWERWomen with endometriosis had an abnormally higher number of basalis-like SSEA1+/SOX9+ epithelial cells present in the stratum functionalis and, since these cells formed 3D structures in vitro with phenotypic similarities to ectopic endometriotic lesions, they may generate ectopic lesions following retrograde menstruation.WHAT IS KNOWN ALREADYEndometrial basalis cells with progenitor potential are postulated to play a role in the pathogenesis of endometriosis and SSEA1 and nuclear SOX9 (nSOX9) mark basalis epithelial cells that also have some adenogenic properties in vitro. Induction of ectopic endometriotic lesions in a baboon model of endometriosis produces characteristic changes in the eutopic endometrium. Retrograde menstruation of endometrial basalis cells is proposed to play a role in the pathogenesis of endometriosis.STUDY DESIGN, SIZE, DURATIONThis prospective study included endometrial samples from 102 women with and without endometriosis undergoing gynaecological surgery and from six baboons before and after induction of endometriosis, with in vitro assays examining the differentiation potential of human basalis-like cells.PARTICIPANTS/MATERIALS, SETTING, METHODSThe study was conducted at a University Research Institute. SSEA1 and SOX9 expression levels were examined in human endometrial samples from women aged 18–55 years (by immunohistochemistry (IHC) and qPCR) and from baboons (IHC). The differential gene expression and differentiation potential was assessed in freshly isolated SSEA1+ endometrial epithelial cells from women with and without endometriosis (n = 8/group) in vitro. In silico analysis of selected published microarray datasets identified differential regulation of genes of interest for the mid-secretory phase endometrium of women with endometriosis relative to that of healthy women without endometriosis.MAIN RESULTS AND THE ROLE OF CHANCEWomen with endometriosis demonstrated higher number of basalis-like cells (SSEA1+, nSOX9+) in the functionalis layer of the eutopic endometrium compared with the healthy women without endometriosis in the secretory phase of the cycle (P < 0.05). Induction of endometriosis resulted in a similar increase in basalis-like epithelial cells in the eutopic baboon endometrium. The isolated SSEA1+ epithelial cells from the eutopic endometrium of women with endometriosis had higher expression of OCT4, NANOG, FUT4 mRNA (P = 0.05, P = 0.007, P = 0.018, respectively) and they differentiated into ectopic endometriotic gland-like structures in 3D culture, but not into mesodermal lineages (adipose or bone cells).LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTIONSmall sample size. Bioinformatics analysis and results depends on the quality of published microarray datasets and the stringency of patient selection criteria employed. Differentia...

Pig blastocysts isolated between Days 10 and 16 of pregnancy release the protease, plasminogen activator (PA), into the medium in a time-dependent manner when cultured in vitro. Production is biphasic. The initial phase (Days 10-12) coincides with the early elongation stages, while release during the second phase (Days 14-16) occurs during a time at which the DNA content of the blastocysts is increasing markedly. Uterine flushings from these pregnant animals contain the zymogen substrate for PA, plasminogen, presumably as a serum transudate. Plasminogen is present in highest amounts at Day 12. The blastocyst, therefore, has the potential ability to generate the broadly specific protease, plasmin, within the uterine lumen. However, during this same period, the endometrium secretes an inhibitor of plasmin into the uterine lumen. In pregnant animals the amount of plasmin inhibitory activity rose 7-fold between Day 10.5, when the blastocysts were spherical, and Day 12, when they had become filamentous. At Day 12 each uterine horn contained about 3 to 4 mg of plasmin inhibitor. A similar release of inhibitor can be initiated in nonpregnant gilts given a single, intramuscular injection of estradiol valerate on Day 11 of the estrous cycle. It is suggested that the initiation of estrogen production by the elongating blastocyst triggers the release of plasmin inhibitor by the maternal endometrium and that the inhibitor serves to prevent a proteolytic cascade of reactions initiated by blastocyst PA, which might otherwise damage the uterine epithelium.

Our data suggest that the presence of ectopic lesions in baboon causes changes in EuE miR expression as early as 3 months postinduction of the disease, and some of these changes may persist throughout the course of the disease. We propose that the marked down-regulation of miR-451 in both baboons and women with endometriosis increases the expression of multiple target genes. Increased expression of one of the target genes, YWHAZ, increases proliferation, likely contributing to the pathophysiology of the disease.