Soluble HLA-G (sHLA-G) in human embryo culture media: a useful marker for assessing embryo quality, and implantation potential for IVF

Abstract

Objective: Soluble HLA-G (sHLA-G) is known to be a critical modulator of local maternal-fetal immunological relationships during pregnancy. The detection of sHLA-G in embryo culture media suggests that sHLA-G may have a role in evaluating embryo quality and implantation potential after IVF. Therefore, we compared sHLA-G molecule expression in the media bathing individual embryos selected for transfer 72 hrs post fertilization by intracytoplasmic sperm injection (ICSI) with implantation and clinical pregnancy rates. We also compared sHLA-G molecular expression between morphologically “poor” and “good” quality embryos. Design: Retrospective cohort study Setting: Multi-site, private practice Patients: Infertile women 28–43 years old (n = 30) with own oocytes undergoing IVF. Interventions: Thirty women undergoing in vitro fertilization (IVF), all of whom had at least 2 embryos available for transfer, were included in the study. All oocytes were fertilized by ICSI and cultured individually in a 50 μl of P-1 (Irvine Scientific) media for 60–67 hr. The embryos were evaluated for embryo transfer (ET), whereupon they were transferred into 50 μl of Blastocyst (Irvine Scientific) media for ET with each embryo being further cultured for an additional 72 hr. Thereupon, 20–30 μl of P-1 medium was collected from the media in which the individual embryos were cultured. A specific sHLA-G ELISA monoclonal anti-HLA-G antibody and W6/32 anti-class I antibody was used in a sandwich ELISA assay to detect for the presence of sHLA-G in each individual sample. Culture media from trophoblast-derived JEG-3 cell line was utilized as a positive control in order to assess the specificity of the ELISA. Primary measures of outcome: The level of sHLA-G expression in each individual sample of P-1 medium relative was correlated with embryo quality as assessed on day 3 post fertilization using the Graduated Embryo Scoring (GES) method, and with subsequent pregnancy and implantation rate. Results: We tested for sHLA-G antigens expressed in the media surrounding each individual embryo. Embryos were classified in three groups based on such ranges. In Group I, the culture media of all embryos with a GES of 20–50 that were <7cells cleaved following 72 hrs in culture, poor sHLA-G expression (<0.120±0.017). No pregnancies occurred in this group. Group IIcomprised embryos that had attained 7–9 cells and had a GES of comprised embryos that had attained cells and had a of 70–100, but demonstrated weakly positive sHLA-G expression (mean 0.237±0.051) in the media. No pregnancies occurred in this group. Group III, embryos comprised those that reached to the 7 to 9 cell stage and each had a GES score of 70–100, but in addition showed strongly positive sHLA-G expression (mean 0.246±0.045). Six (20%) out of thirty patients had strongly positive sHLA-G expressing embryos on day 3. The clinical pregnancy and implantation rates following the transfer of these embryos were 84% (5/6) and 43% (9/21) per embryo, respectively. In addition, there was a positive correlation between the amount of sHLA-G in the culture media and the GES as well as the implantation rate per embryo. None of the atretic, arrested, or abnormally fertilized embryos revealed any sHLA-G in the media. Conclusions: The presence and concentration of sHLA-G in the culture medium 72 hours following fertilization by ICSI could provide a useful indicator measure of subsequent embryo implantation potential.