| Background Non-invasive methods of assessing embryo quality are critical forpregnancy success following IVF, and currently there is no perfect markers in clinicalpractice for choosing embryo for transfer. The presence of soluble human leukocyteantigen-G (sHLA-G) in embryo culture supernatants (ECs) has been suggested as away to predict embryo quality and pregnancy success. There are still controversy dueto the different detection assays and the different culture conditions in laboratories.Objective To investigate whether sHLA-G can be a noninvasive marker of embryoimplantation potential through observation of the relationship between the presence ofsHLA-G in ECs and the IVF outcomes.Methods We focused on the standardization of IVF procedure and sHLA-G levelswere evaluated by ELISA or Electrochemiluminescence immunoassay. A single centerand a multi-center retrospective studies were conducted. Transfer cycles in which atleast one embryo selected for replacement expressed sHLA-G were grouped as positive. If none of the transferred embryos secreted sHLA-G, these cycles werecategorized as negative. We investigated the association between the presence ofsHLA-G with the pregnancy and implantation achieved by IVF-ET. And a prospectiveclinical controlled study was conducted for frozen embryo transfer cycles.Results1. sHLA-G presence was detected in26.5%of the113ECs from53ICSI cycles insingle center study. In the sHLA-G positive cycles,68.4%of patients (27/52) achieveda clinical pregnancy. In sHLA-G negative cycles, the pregnancy rate was only39.4%(12/40, P＜0.05). The IR in sHLA-G positive cycles was also significantly higher(51.3%) than that in sHLA-G negative cycles (24.3%, P＜0.05).2. Furthermore, a total2041ECs from924cycles were collected from9Chinese ARTcenters and analyzed for sHLA-G by ELISA method,25.5%of which were positive. In 87cycles where all embryos transferred were sHLA-G positive, PR and IR was49.4%and30.8%respectively, in306cycles with part of embryos transferred positive thecorresponding ratio were55.2%and32.9%respectively, and there was no statisticaldifference between the two groups. Pregnancy was obtained in53.9%(212/393)ofcycles when at least one transferred embryo contained sHLA-G, compared with37.3%(198/531)in cycles with none sHLA-G positive embryos transferred (P＜0.05), andthe IR in sHLA-G positive group was higher than that in negative group (32.5%vs23.0%, P＜0.05).Stratified analyzed by fertilization method, in IVF group, PR and IR were higher insHLA-G positive cycles than that in negative cycle(sPR:55.0%vs36.1%,IR:33.7%vs22.6%,P＜0.05); similar results could be observed in ICSI group(PR:51.3%vs37.2%,IR:29.4%vs22.6%,P＜0.05). Compared with sHLA-G negative cycles, higherPR and IR were obtained in sHLA-G positive cycles for women both≤35and＞35years old(≤35PR:54.6%vs40.0%,IR:34.4%vs25.6%,＞35PR:51.2%vs27.4%,IR:26.0%vs14.9%,P＜0.05). Increased PR and IR were observed in sHLA-Gpositive cycles compared with sHLA-G negative cycles with both Vitrolife andQuinn s culture media (Vitrolife:56.4%and33.9%vs41.3%and25.2%,Quinn s:54.1%and32.9%vs35.0%and21.4%, P＜0.05).In cycles with all embryos transferred successfully implanted, the percentage ofsHLA-G positive embryos was35.6%, higher than that in cycles with all embryostransferred failed to implant (19.2%, P＜0.05).Neither the miscarriage rate nor the ectopic pregnancy rate showed statisticaldifferences between two groups in all the nine centers.No correlation was found between embryo morphology score and sHLA-G expressionby Kendall rank correlation analysis.(tau_b=-0.057,P＜0.05).Multivariate logistic regression analysis showed that HLA-G expression, female ageand number of MII oocytes are independent parameters for the prediction of pregnancy.Among them, the sHLA-G status of embryos transferred were the most powerfulpredictors for the pregnancy outcome after ART (OR=2.170, P＜0.05). 3. The data from prospective study for FET showed PR and IR in the experimentalgroup with sHLA-G positive frozen embryos transferred were higher than those incontrol group with sHLA-G negative frozen embryos transferred (85.7%and55.6%vs52.0%and27.3%), the difference of IR was statistically significant (P＜0.05).4. We modified the ELISA kit for Electrochemiluminescence immunoassay assay,which could increase the assay sensibility by6.28fold. In a single center retrospectivestudies which utilized this assay, The PR and IR in sHLA-G positive cycles werehigher than those in negative cycles.5. We completed the package of lentivirus vector encoding HLA-G5as a tool forfurther study.Conclusions The results suggested sHLA-G in ECs could be utilized as a potentialmarker of embryo competency in ART programs.
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