The alpha chain of the human histocompatibility antigen HLA-G was identified as an array of five 37- to 39-kilodalton isoforms by the use of two-dimensional gel electrophoresis. Both cell-associated and secreted HLA-G antigens are prominent in first trimester villous cytotrophoblasts and are greatly reduced in third trimester cytotrophoblasts. Allelic variation was not detected, an indication that HLA-G is not obviously polymorphic in cytotrophoblasts. Among the following choriocarcinoma cell lines studied, HLA-G is expressed in JEG but not in Jar or BeWo. Expression of endogenous HLA-G genes has not been found in normal lymphoid cells. Thus, HLA-G is subject to both cell type-specific and developmental regulation and is expressed in early gestation human cytotrophoblasts.

Abstract. The specialized interaction between embryonic and maternal tissues is unique to mammalian development. This interaction begins with invasion of the uterus by the first differentiated embryonic cells, the trophoblasts, and culminates in formation of the placenta. The transient tumor-like behavior of cytotrophoblasts, which peaks early in pregnancy, is developmentally regulated. Likewise, in culture only early-gestation human cytotrophoblasts invade a basement membrane-like substrate. These invasive cells synthesize both metalloproteinases and urokinase-type plasminogen activator. Metalloproteinase inhibitors and a function-perturbing antibody specific for the 92-kD type IV collagen-degrading metalloproteinase completely inhibited cytotrophoblast invasion, whereas inhibitors of the plasminogen activator system had only a partial (20--40%) inhibitory effect. We conclude that the 92-kD type IV coilagenase is critical for cytotrophoblast invasion.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

ObjectiveTo assess whether embryonic DNA isolated from blastocyst culture conditioned medium (BCCM) combined with blastocoel fluid (BF) could be used for blastocyst stage non-invasive preimplantation genetic testing for chromosomal aneuploidy (non-invasive preimplantation genetic screening, NIPGS).Patients47 embryos from 35 patients undergoing IVF.InterventionsDNA analysis of combined BCCM plus BF in comparison with trophectoderm (TE) biopsy and/or whole blastocyst (WB)using next generation sequencing (NGS).ResultsEmbryonic DNA was successfully amplified in 47/47 NIPGS samples (28 frozen-thawed and 19 fresh culture samples) ranging from 6.3 to 44.0 ng/μl. For frozen-thawed embryos, the concordance rate for whole chromosome copy number per sample was equivalent between NIPGS vs. TE biopsy, NIPGS vs. WB and TE vs. WB samples taken from the same embryo was 87.5%; 96.4% and 91.7% respectively (P>0.05), and the rate of concordance per single chromosome was 99.3%, 99.7% and 99.7%, respectively (P>0.05). In fresh cases (Day 4 to Day 5/6 culture), the concordance rate for whole chromosome copy number per sample between NIPGS vs. TE samples taken from the same embryo was 100%, and the rate of concordance per single chromosome was 98.2% (P>0.05).ConclusionsA combination of BCCM and BF contains sufficient embryonic DNA for whole genome amplification and accurate aneuploidy screening. Our findings suggest that aneuploidy screening using BCCM combined with BF could potentially serve as a novel NIPGS approach for use in human IVF.