Concentrations Of sHLA-G Research Articles

Soluble immune checkpoints CTLA-4, HLA-G, PD-1, and PD-L1 are associated with endometriosis-related infertility.

Soluble immune checkpoint molecules constitute the emerging novel mediators in immune regulation. Their role in the pathogenesis of endometriosis has not been fully addressed. In this study, we aimed to investigate the relationship between the clinical manifestation of endometriosis-associated infertility and the level of four soluble immune checkpoints: sCTLA4, sHLA-G, sPD-1, and sPD-L1. The soluble immune checkpoint concentrations in serum and peritoneal fluid from 88 patients who underwent laparoscopy were evaluated by the enzyme-linked immunosorbent assay (ELISA). Clinical and hematological parameters were documented and analyzed. Endometriosis cases were evident to have significantly higher levels of serum sPD-L1 and all four molecules in peritoneal fluid compared to non-endometriosis control. Contrary, no significant differences were found in the concentration of serum sCTLA-4, sHLA-G and, sPD-1 between endometriosis and control group. There were significant positive correlations between serum and peritoneal fluid concentrations of sCTLA-4, sPD-L1, and sHLA-G. Serum sPD-L1 could discriminate endometriosis-related infertility to other pathological control. At a cutoff of 14,61 pg/mL, serum sPD-L1 had a sensitivity of 77% and specificity of 83%. Moreover, sPD-L1 level showed positive correlations with pelvic adhesion score and myeloid cell count. The elevated level of sPD-L1 in serum and immune checkpoint molecules in the peritoneal fluid could represent the hallmark of immune regulation in endometriosis. Serum sPD-L1 could serve as a potential noninvasive endometriosis biomarker. Also, the immune compartment related to the local immune checkpoint molecules may be implicated in biological mechanisms underlying endometriosis-related infertility.

Evaluation of the Level of Salivary sHLA-G in Children Aged 3-5 Years with or without Dental Caries.

Methods and Materials This analytic cross-sectional study was carried out on 83 healthy children aged 3 to 5 years of both genders, who were divided into three groups based on decayed dental surfaces (ds): group 1, caries-free children (CF, n = 29); group 2, children with 1 ≤ ds ≤ 3, 1 ≤ ds ≤ 4, and 1 ≤ ds ≤ 5 for age 3, 4, and 5 years, respectively (ECC, n = 20); and group 3, children with ds ≥ 4, ds ≥ 5, and ds ≥ 6 for age 3, 4, and 5 years, respectively (S-ECC, n = 34). The unstimulated saliva samples were collected, and the salivary sHLA-G concentration was measured by the ELISA kit. The SPSS Statistics v17.0 software and Mann–Whitney, Kruskal–Wallis, chi-square, and Spearman's rank correlation tests were used for statistical analysis. The level of significance was considered at p < 0.05. Results The mean concentrations of salivary sHLA-G in CF, ECC, and S-ECC groups were 3.18 ± 2.28, 5.64 ± 5.51, and 6.21 ± 6.03 ng/l, respectively (p = 0.047), and the mean salivary sHLA-G level was comparatively higher in children with dental caries than that of the CF group (p = 0.02), but there is no significant difference between ECC and S-ECC groups (p > 0.05). Spearman's rank correlation test showed a weak positive correlation (p = 0.039, r = 0.22), between the level of salivary sHLA-G and dental caries. Conclusion The present study provides some preliminary evidences on relationship between sHLA-G and dental caries in children.

Soluble HLA-G and TGF-β in couples attending assisted reproduction – A possible role of TGF-β isoforms in semen?

Soluble isoforms of the non-classical Human Leukocyte Antigen (HLA)-G as well as Transforming Growth Factor (TGF)-β is expressed in seminal plasma possibly influencing the pregnancy potential. We wanted to examine the association of seminal plasma sHLA-G, TGF-β1, TGF-β2 and TGFβ3 with pregnancy success in a cohort of 127 couples and 4 single women attending fertility treatment with the use of assisted reproduction technologies (ART). Soluble HLA-G, TGF-β1, TGF-β2 and TGF-β3 in seminal plasma did not fluctuate significantly over time. We did not find any impact of seminal plasma sHLA-G, TGF-β1, TGF-β2 and TGF-β3 on time-to-pregnancy measured as number of treatment cycles. There was a significant association between concentrations of seminal plasma sHLA-G and HLA-G variations in the 3’untranslated region (3’UTR) of the HLA-G gene, supporting and extending previous findings. Furthermore, by comparing seminal plasma concentrations of sHLA-G, TGF-β1, TGF-β2 and TGF-β3 in male subjects with reduced semen quality, male subjects with normal semen quality, and sperm donors, we found that TGF-β2 was significantly lower, and TGF-β3 was significantly higher, in seminal plasma from sperm donors. These findings suggest that TGF-β isoforms may influence semen quality and fertility.

Expression and role of regulatory plasma cells in gravidas with systemic lupus erythematosus

Objective To investigate the expression and role of regulatory plasma cells in gravidas with systemic lupus erythematosus (SLE). Methods Gravidas with SLE were enrolled in Henan Provincial People′s Hospital from April 2013 to April 2018. They were divided into three groups including pregnancy control group, SLE stable group and SLE deterioration group. The ratio of CD3-LAG-3+ CD138high regulatory plasma cells was detected by flow cytometry. The concentrations of soluble human leukocyte antigen-G (sHLA-G) and anti-nuclear antibody Ig were detected by ELISA. Lymphocytes in peripheral blood of SLE deterioration group were isolated, and then cultured in RPMI1640 medium containing 10% fetal bovine serum and stimulated with HLA-G. Results Flow cytometry showed that the proportion of regulatory plasma cells in SLE stable group was (2.483±0.131 8)% and that in SLE deteriorating group was (1.662±0.130 4)%. There was a significant difference between the two groups (t=4.431, P=0.001 3). The concentrations of sHLA-G in SLE stable group and SLE deteriorating group were (36.50±3.510) ng/ml and (16.50±2.405) ng/ml, and the difference between the two groups was statistically significant (t=4.701, P=0.000 8). Correlation analysis showed that the concentration of sHLA-G was positively correlated with the proportion of regulatory plasma cells (r=0.747 1, P=0.000 9). The results of in vitro experiment showed that the proportions of B cells and regulatory plasma cells were (7.573±0.653 9)% and (1.593±0.187 9)% in SLE deterioration group and (3.732±0.717 8)% and (2.503±0.292 1)% in HLA-G group with statistical differences between the two groups (t=3.957, P=0.002 7; t=2.620, P=0.025 6). Conclusions The proportion of regulatory plasma cells and the concentration of sHLA-G were significantly decreased in pregnant patients with SLE, which was closely related to disease severity. HLA-G played an important role in promoting the proliferation of regulatory plasma cells. Key words: Regulatory plasma cell; Systemic lupus erythematosus; Pregnancy; HLA-G

HLA-G is upregulated in advanced endometriosis

ObjectiveTo assess whether the HLA-G immunomodulatory protein is potentially involved in the pathophysiology of endometriosis or disease progression. Study designCross-sectional observational study of 227 women who underwent laparoscopy, being 146 for endometriosis excision and 81 for elective tubal ligation (control group). Soluble HLA-G (sHLA-G) levels in the serum and peritoneal fluid (PF), as well as the HLA-G protein expression in matched eutopic and ectopic endometrium of women with and without endometriosis were evaluated by ELISA and immunohistochemistry assays, respectively. Women with endometriosis were separated into groups according to the initial (I/II, n = 60) and advanced (III/IV, n = 86) stages of disease. sHLA-G measurement was performed only in women with matched serum and PF samples in both the control (CTRL; n = 77) and endometriosis (EDT; I–II, n = 60; III–IV, n = 83) groups. HLA-G protein expression was evaluated in 26 women with deep endometriosis (I–II, n = 12; III–IV, n = 14) and 22 controls. ResultsHigher concentrations of sHLA-G (P = 0.013) in the serum but not in the PF were observed in women with advanced endometriosis compared to the control group. In situ expression of HLA-G protein was also higher in ectopic (P = 0.018) but not in eutopic endometrium of women with advanced endometriosis compared to control group. ConclusionOur findings suggest that HLA-G upregulation in advanced stages may contribute to the state of immunosuppression in endometriosis as disease progresses.

Overexpression of immunomodulatory mediators in oral precancerous lesions

Human leukocyte antigen (HLA) G and E, programmed cell death 1 ligand 1 (PD-L1), IL-10 and TGF-β are proteins involved in failure of the antitumor immune response. We investigated the expression of these immunomodulatory mediators in oral precancerous lesions (oral leukoplakia-OL; n=80) and whether these molecules were related to the risk of malignant transformation. Samples of normal mucosa (n=20) and oral squamous cells carcinoma (OSCC, n=20) were included as controls. Tissue and saliva samples were analyzed by immunohistochemistry and ELISA respectively. Fifteen OL samples showed severe dysplasia (18.7%) and 40 samples (50%) presented combined high Ki-67/p53. Irrespective of the degree of epithelial dysplasia and the proliferation/apoptosis index of OL, the expression of HLA-G, -E, PD-L1, IL-10, TGF-β2 and -β3 was higher to control (P<0.05) and similar to OSCC (P>0.05). The number of granzyme B+ cells in OL was similar to control (P=0.28) and lower compared to OSCC (P<0.01). Salivary concentrations of sHLA-G, IL-10 and TGF-β did not allow for a distinction between OL and healthy individuals. Overexpression of immunosuppressive mediators in the OL reflects the immune evasion potential of this lesion, which is apparently independent of at cytological and proliferation/apoptosis status.

Serum and peritoneal fluid concentrations of soluble human leukocyte antigen, tumor necrosis factor alpha and interleukin 10 in patients with selected ovarian pathologies

BackgroundAlthough immune system plays a key role in the pathogenesis of both endometriosis and ovarian cancer, its function is different. Therefore, we hypothesized, that selected immune parameters can serve as diagnostic markers of these two conditions. The aim of this study was to compare serum and peritoneal fluid concentrations of sHLA-G, IL-10 and TNF-alpha in women with selected ovarian pathologies: benign serous cysts, endometrioma and malignant tumors. Clinical significance of using them for diagnostic purposes in women with serous ovarian cysts, endometriosis, and ovarian cancer, which in the future may improve the early diagnosis of ovarian diseases.Case PresentationThe study included women treated surgically for benign serous ovarian cysts, ovarian endometrioma and serous ovarian adenocarcinomas. Peripheral blood and peritoneal fluid samples were obtained intraoperatively. Patients with benign serous cysts, endometrioma and ovarian malignancies did not differ significantly in terms of their serum and peritoneal fluid concentrations of sHLA-G. Ovarian cancer patients presented with significantly higher median serum concentrations of IL-10 and TNF-alpha than other study subjects. Median concentrations of IL-10 and TNF-alpha in peritoneal fluid turned out to be the highest in ovarian cancer patients, followed by women with endometrioma and subjects with benign serous cysts. All these intergroup differences were statistically significant. Irrespective of the group, median concentrations of sHLA-G, IL-10 and TNF-alpha in peritoneal fluid were higher than serum levels of these markers.ConclusionsElevated serum and peritoneal fluid concentrations of IL-10 and TNF-alpha distinguish ovarian malignancies and endometriomas from benign serous ovarian cysts. In contrast to endometriosis, ovarian malignancies are characterized by elevated peritoneal fluid concentrations of IL-10 and TNF-alpha, elevated serum concentrations of IL-10 and low serum levels of TNF-alpha. Serum and peritoneal fluid concentrations of sHLA-G have no diagnostic value in differentiating between ovarian malignancies and endometriomas.

Mesenchymal stem cells upregulate Treg cells via sHLA-G in SLE patients

BackgroundSoluble human leukocyte antigen-G (sHLA-G) is a non-classical HLA class I molecule, exhibiting strong immunosuppressive properties by inducing the differentiation of T regulatory cells (Treg). Mesenchymal stem cells (MSCs) transplantation alleviates disease progression in systemic lupus erythematosus (SLE) patients. However, the underlying mechanisms are largely unknown. ObjectivesTo explore whether sHLA-G is involved in upregulating effects of MSCs on Treg, which contributes to therapeutic effects of MSCs transplantation in SLE. MethodsThe serum sHLA-G levels of SLE patients and healthy controls were detected by ELISA. The percentages of peripheral blood CD4+ILT2+, CD8+ILT2+, CD19+ILT2+ cells and Treg cells were examined by flow cytometry. Ten patients with active SLE, refractory to conventional therapies, were infused with umbilical cord derived MSCs (UC-MSCs) and serum sHLA-G was measured 24h and 1month after infusion. The mice were divided into three groups: C57BL/6 mice, B6.MRL-Faslpr mice infused with phosphate buffer saline (PBS), and B6.MRL-Faslpr mice infused with bone marrow MSCs (BM-MSCs). Then, the concentrations of serum Qa-2 were detected. Peripheral blood mononuclear cells (PBMCs) were isolated from SLE patients and co-cultured with UC-MSCs for 3days at different ratios (50:1, 10:1, and 2:1) with or without HLA-G antibody, and the frequencies of CD4+CD25+Foxp3+ T cells were then determined by flow cytometry. ResultsThe concentrations of serum sHLA-G were comparable between SLE patients and healthy controls. However, there was a negative correlation between sHLA-G levels and SLE disease activity index (SLEDAI) scores in active SLE patients (SLEDAI>4). We found that serum sHLA-G levels were negatively correlated with blood urea nitrogen, serum creatinine and 24-hour urine protein in SLE patients. The sHLA-G levels were significantly lower in SLE patients with renal involvement than those without renal involvement. The expression of ILT2 on CD4+ T cells from SLE patients decreased significantly compared to that of healthy controls. A positive correlation between the frequencies of Treg and CD4+ILT2+ T cells was found in SLE patients. The levels of sHLA-G increased 24h post UC-MSCs transplantation. The concentrations of Qa-2 in BM-MSCs transplanted mice were significantly higher than those of control group. In vitro studies showed that MSCs increased the frequency of Treg cells in SLE patients in a dose-dependent manner, which was partly abrogated by the anti-HLA-G antibody. ConclusionsOur results suggested that MSCs may alleviate SLE through upregulating Treg cells, which was partly dependent on sHLA-G.

Soluble HLA-G concentrations in obese women during pregnancy and in cord blood

ContextLittle is known about soluble HLA-G (sHLA-G) concentrations in obese pregnant women with uncomplicated pregnancies. ObjectiveTo investigate the role of sHLA-G in obese pregnancies. DesignCase-control study, from 2013 to 2015. SettingA tertiary care centre. Patients168 healthy normal weight women and 59 overweight/obese women; to avoid the effect of preeclampsia on sHLA-G concentrations, cases were further divided in two groups: 42 with normotensive pregnancy and 17 who developed preeclampsia. Interventionsall the women enrolled received standard antenatal care and plasma sample collections were performed. Main outcome measuressHLA-G concentrations during pregnancy, before delivery and in cord blood. ResultsMaternal sHLA-G concentrations in overweight/obese with normotensive pregnancies increased by 14.7% (IQR=−26.4 to +89.6) in the 2nd trimester and by 19.6% (IQR=–33 to +104) before delivery and were significantly higher than in controls (p=0.024). Median cord blood sHLA-G concentrations were 53.5ng/ml (IQR=36–62.7) in the overweight/obese women with uncomplicated pregnancies (p<0.001 compared to controls) and 19.7ng/ml (IQR=7.5–36.3) in controls. Maternal concentrations of sHLA-G in the two trimesters and before delivery were significantly lower among subjects who developed preeclampsia than in controls (p<0.001) or in obese subjects with normotensive pregnancies (p<0.001). ConclusionssHLA-G concentratons are higher in normotensive overweight/obese women and their babies while lower in preeclamptic overweight/obese women and their cords. Obesity influences maternal and fetal sHLA-G concentrations during pregnancy, to optimize the reproductive success, while preeclampsia impairs the mother-offspring antinflammatory response.

Association of HLA-G*01:01:02:01/G*01:04:01 polymorphism with gastric adenocarcinoma

Human leukocyte antigen-G (HLA-G) plays an important role in tumor cell escape from immune surveillance and HLA-G polymorphisms might service as a potential risk factor for clinical outcomes in GAC (gastric adenocarcinoma). We investigated the association between HLA-G polymorphisms as well as soluble HLA-G level and accordance of GAC. This case-control study included 100 GAC patients and 102 unrelated Iranian individual’s samples as control. The clinical stages ranged from I to IV. PCR-RFLP method was carried out in order to specify the genotypes of the HLA-G gene. Concentrations of sHLA-G in serum were determined with the sHLA-G-specific enzyme linked immunosorbent assay (ELISA) kit. The G*01:04:01 and G*01:01:02:01 alleles were the predominant alleles in GAC patients and healthy controls. The G*01:01:03:01 and G*01:01:08 allele distributions are significantly higher among controls comparing to cases and seem to have protective effect (P value=0.026 and 0.007 respectively). There is a substantial differences in G*01:01:02:01/G*01:04:01 genotype frequencies between cases and controls (OR=2.8, P value<0.001). The G*01:01:03:01/G*01:04:01 and G*01:01:02:01/G*01:01:08 genotypes frequency are higher among controls in comparison to patients (P value=0.028 and 0.007 respectively). The polymorphisms in HLA-G could affect GAC induction and its outcome. Also, increased sHLA-G levels in serum might be a useful biomarker for diagnosis.

High plasma levels of HLA-G are associated with low birth weight and with an increased risk of malaria in infancy.

BackgroundThe immunosuppressive properties of HLA-G protein can create a tolerogenic environment that may allow Plasmodium falciparum to avoid host immune responses. There are known associations between high levels of circulating soluble HLA-G (sHLA-G) and either parasite or viral infections and it has been suggested that the induction of sHLA-G expression could be a mechanism via which infectious agents subvert host immune defence. The study presented here is the first to investigate the possible association between sHLA-G and malaria or malaria related risk factors in Benin.MethodsA parasitological and clinical follow-up of 165 mothers and their newborns from delivery through to one year of age was conducted in the Tori Bossito area of southern Benin. Plasma levels of sHLA-G were determined by ELISA in maternal peripheral and cord blood and again in infants' peripheral blood at 3, 6, 9 and 12 months of age. The associations between the levels of sHLA-G and malaria risk factors were investigated through multivariate mixed models.ResultsStrong correlations were observed between the maternal and cord plasma concentrations of sHLA-G. In multivariate analyses, high cord plasma levels of sHLA-G were independently associated with (i) low birth weight and (ii) an increased risk of P. falciparum infection in infancy.ConclusionThese results show for the first time the possible involvement of sHLA-G in generating immune tolerance during pregnancy-associated malaria. Soluble HLA-G may represent a useful marker of susceptibility to malaria in infants and be associated with the higher susceptibility to infection observed for LBW children.

The concentrations of soluble HLA-G protein are elevated during mid-gestation and decreased in pre-eclampsia

The aim of our study was to investigate the dynamics of the alterations of soluble human leukocyte antigen-G (sHLA-G) concentrations in sera of healthy non-pregnant women, as well as healthy pregnant women and patients with pre-eclampsia. Thirty five patients with pre-eclampsia, 52 healthy pregnant women, and 24 healthy non-pregnant women were included in the study. Sera concentrations of sHLA-G protein were determined using the immunoenzymatic ELISA method. Statistical analysis was performed using ANOVA and Mann-Whitney U tests. The concentrations of sHLA-G protein in sera of pregnant women in the first, as well as the second and third, trimesters of normal pregnancy were significantly higher in comparison with healthy nonpregnant women. The sera concentrations of sHLA-G in pregnant women in the second trimester of pregnancy were significantly higher compared to the first and third trimesters. The concentrations of sHLA-G in sera of patients with pre-eclampsia were significantly lower than in pregnant women in the third trimester of physiological pregnancy. The results of our study suggest that normal physiological pregnancy is associated with elevated sera concentrations of sHLA-G molecule. The increased concentrations of sHLA-G molecule in mid-gestation could suggest a role for the protein in the second phase of a physiological invasion of extravillous cytotrophoblast to spiral arteries. Furthermore, the results could suggest a role for the decreased sera concentrations of sHLA-G in the pathogenesis of pre-eclampsia.

HLA-G polymorphisms and soluble HLA-G protein levels in women with recurrent pregnancy loss from Basrah province in Iraq

HLA-G is a nonclassical, class I major histocompatibility complex (MHC) gene that exhibits immunomodulatory properties and likely plays a role in the maintenance of successful pregnancy. In this study, we investigated the role of HLA-G polymorphisms on risk for recurrent pregnancy loss (RPL) and on circulating levels of soluble (s)HLA-G in Iraqi women. DNA and plasma were obtained from blood samples collected at 9-12 weeks gestation from 50 women with RPL and 50 healthy pregnant women in Basrah province, Iraq. As measured by ELISA, median sHLA-G levels were significantly lower in the RPL cases compared to healthy controls (21.4 vs. 38.8 U/ml, respectively; P=0.025), and decreased with increasing maternal age (P=0.0051). However, HLA-G allele and haplotype frequencies did not differ significantly between cases and controls (P values ≥0.12 for all tests). In contrast, homozygosity for the C allele (CC) at a tri-allelic promoter polymorphism, -725C/G/T, was associated with lower concentrations of sHLA-G compared to the CG or CT genotypes (median levels 21.1 vs. 40.1 vs. 42.6 U/ml, respectively; P=0.0089). These results demonstrate that HLA-G genotype influences circulating sHLA-G levels during pregnancy but is not significantly associated with risk of RPL.

Soluble human leukocyte antigen‐G expression in hepatitis B virus infection and hepatocellular carcinoma

We investigated soluble human leukocyte antigen-G (sHLA-G) expression according to the phases of hepatitis B virus (HBV) infections and hepatocellular carcinoma (HCC). A total of 267 sera from anti-HBs positive healthy individuals (n = 50), chronic HBV carriers (n = 45), as well as patients with active hepatitis B (n = 46), liver cirrhosis (LC, n = 46) and early stage HCC (n = 80) were collected and assayed for sHLA-G. Relationships between sHLA-G levels and clinicopathologic parameters including HCC stages, differentiation grades, and levels of aminotransferases, HBV DNA and alpha-fetoprotein (AFP) were assessed. Concentrations of sHLA-G were higher in the active hepatitis B and HCC groups (median sHLA-G 53.7 and 178.8 U/ml, respectively) in comparison to other groups (P < 0.05), and there were no significant differences among sHLA-G levels of the anti-HBs positive, chronic HBV carrier and LC groups. Serum sHLA-G concentrations were not shown to be associated with clinicopathologic indices including the levels of aminotransferases, AFP, anti-HBs titer, HBV DNA, as well as HCC stages, numbers of tumor nodules, pathologic grades and presence of vessel invasion. The receiver-operating characteristic area under the curve (AUC) value of sHLA-G for differentiating HCC from LC was 0.98, which was greater than that of AFP (0.78) (P < 0.0001), and sensitivity and specificity of sHLA-G were, respectively, 90.0% and 95.7% for HCC when applying a cutoff level of 97.3 U/ml. Serum sHLA-G levels could be used as a diagnostic marker for HCC. Although sHLA-G levels did not reflect the severity of HBV infections and HCC, they were related with phases of the disease.

Levels of soluble human leukocyte antigen-G are increased in asthmatic airways

To the Editors: Human leukocyte antigen-G (HLA-G) is a non-classical, class Ib, major histocompatibility complex antigen, encoded by a gene on chromosome 6p21 within the HLA complex 1. HLA-G is constitutively expressed during pregnancy where it has a critical role in maintaining immune tolerance toward the allogenic fetus and placenta 2, 3, but has also been associated with inflammatory diseases such as psoriasis, multiple sclerosis, and ulcerative colitis, and with solid-organ transplantation 3, 4. We recently reported associations between variation in HLA-G and risk for asthma in Chicago-area asthma families, in multigenerational Dutch asthma families and in a birth cohort at high risk for developing asthma 1, 5. A role for HLA-G in asthma pathogenesis was further suggested by the demonstration of expression of a soluble isoform of HLA-G, sHLA-G5, in airway epithelial cells 1 and of increased circulating plasma levels of sHLA-G in children with atopic asthma 6. Because airway inflammation in asthma involves a T-helper cell (Th) type 2-skewing of lymphocytes similar to pregnancy, HLA-G is an attractive candidate molecule for promoting the immune profile characteristic of asthma. Localisation of HLA-G to airway epithelium suggests that its dysregulation could contribute to airway inflammation in chronic asthma. To evaluate this further, we hypothesised that HLA-G abundance would be increased in asthmatic airways. To test this hypothesis, we measured concentrations of sHLA-G in bronchoalveolar lavage (BAL) fluid obtained from 12 non-asthmatic control subjects and 15 subjects with mild persistent asthma. The use of human subjects was approved by the University of Chicago Institutional Review Board (Chicago, IL, USA). Asthma was diagnosed using National Asthma Education and Prevention Program guidelines. …

How to predict implantation? No correlation between embryonic aneuploidy and soluble human leukocyte antigen G-concentrations

To determine if soluble human leukocyte antigen-G (sHLA-G) concentrations in spent culture media may assist in identifying the normal embryo for implantation. Prospective blinded comparative study. Reproductive genetic and reproductive medicine centers. One hundred and sixteen embryos obtained from eight patients undergoing in vitro fertilization (IVF) with preimplantation genetic diagnosis (PGD). Culture media obtained 2 days after fertilization were analyzed for sHLA-G concentrations using an enzyme-linked immunosorbent assay (ELISA) assay. A sHLA-G concentration of >or=1.9 mIU/mL was considered a positive predictor for successful implantation. Polar bodies and blastomeres from day-3 embryos were tested by PGD for 5 to 11 chromosomes: 8, 9, 13, 15, 16, 17, 18, 21, 22, X, and Y. The results of the sHLA-G concentrations were compared with the results of the PGD analyses. We found an sHLA-G concentration >or=1.9 mIU/mL in 48% (56 out of 116) and normal PGD results in 52% (57 out of 116) of embryos. Of the embryos with normal PGD results, 46% (26 out of 57) had sHLA-G concentrations >or=1.9 mIU/mL. Among the embryos with sHLA-G >or=1.9 mIU/mL, 46% (26 out of 56) had normal PGD results, and 21% of embryos displayed both normal PGD results and sHLA-G >or=1.9 mIU/mL. No correlation between concentrations of sHLA-G in embryo culture media and PGD results of an embryo's aneuploidy were observed.

Correlation between preimplantation genetic diagnosis results and soluble HLA G concentrations in embryo culture media

The transfer of a single embryo is the goal of assisted reproductive technology programs to minimize the risk of multiple gestations. The difficulty has been to decide which embryo to transfer. Measurement of soluble HLA-G (sHLA-G) concentrations in culture media has been reported to predict implantability of embryos. The question arose whether HLA-G culture media concentrations were associated with chromosomal complement of the embryo. To answer this question, culture media from embryos undergoing preimplantation genetic diagnosis (PGD) were analyzed for sHLA-G concentration. The results of the sHLA-G concentrations were compared with the results of the PGD analysis. Prospective comparative study. Culture media from 100 embryos obtained 2 days after fertilization by intracytoplasmic sperm injection were analyzed for sHLA G concentrations using an ELISA kit (Ex Bio/BioVendor, CARI, Chicago, IL). A sHLA G concentration of ≥2 U/ml was considered a positive result. On the morning of day 3 of development, embryos were biopsied in calcium and magnesium free Global media. Fluorescent in situ hybridization with directly labeled probes (Abbott/Vysis; Des Plaines, IL) for chromosomes 13, 14,15,16,17, 18, 21, 22, X and Y was performed in two cycles. Soluble HLA G concentration ≥2 U/ml was found in 45 (45%) and <2 UI/ml in 55 (55%) of embryo culture media. PDG results were interpreted as normal chromosomal complement for those chromosomes studied in 59 (59%) and abnormal in 41 (41%) of embryos. Of 41 embryos reported to have a normal karyotype, 16 (40%) had sHLA G concentrations ≥2 U/ml and 25 (60%) had sHLA G concentrations <2 U/ml. Among the 45 embryos that had culture media concentrations of sHLA G ≥2 U/ml, 24 (54%) displayed normal PGD results and 21 (46%) had abnormal PGD results. sHLA G concentrations were <2 U/ml in culture media of 55 embryos among which 33 (60%) had normal and 22 (40%) had abnormal PGD results. No correlation between concentrations of sHLA-G in embryo culture media and PGD results were observed.

HLA-G and its role in implantation (review)

Human leukocyte antigen G (HLA-G) is thought to play a key role in implantation by modulating cytokine secretion to control trophopblastic cell invasion and to maintain a local immunotolerance. The literature is reviewed to provide a description of the genetic background, properties of the protein, and the function of HLA-G. Data are presented on potential clinical applications of HLA-G including the use of evaluation of HLA-G gene polymorphisms in the diagnosis of patients experiencing recurrent pregnancy loss and evaluation and testing of soluble HLA-G (sHLA-G) in embryo culture media for the selection of embryos for transfer after in vitro fertilization (IVF). The literature supports a central role of HLA-G for successful implantation. Of couples experiencing recurrent pregnancy loss, 32% demonstrated the -1725G HLA-G polymorphism. Our data showed that when embryos were selected for transfer after IVF based on culture media concentrations of sHLA-G > or = 2 U/ml and good morphologic grade, a 65% pregnancy rate compared with a 0% pregnancy rate in those with <2 U/ml sHLA-G. HLA-G is important for successful implantation in human beings. The HLA-G -725 promoter polymorphism is a risk factor for recurrent miscarriage. Measurement of sHLA-G in embryo culture media can help select embryos for transfer after IVF allowing fewer embryos to be transferred in an attempt to lower multiple gestation rates.

A Study of Soluble HLA-G 1 Protecting Porcine Endothelial Cells Against Human Natural Killer Cell-Mediated Cytotoxicity

Human natural killer (NK) cells, which can mediate direct lysis of porcine endothelial cells, play an important role in xenograft rejection. HLA-G, which is a critical molecule in maintaining maternal immune tolerance of semi-allogenic fetus, is able to protect susceptible target cells from lysis induced by NK cells. In this study, we investigated whether soluble HLA-G 1 (sHLA-G 1) protected porcine xenogeneic cells against human NK cell-mediated lysis. Methods The human sHLA-G 1 genomic DNA (pcDNA3-sHLA-G 1) was transfected into a B lymphoblastoid cell line 721.221 (LCL721.221) by nucleofector. The sHLA-G 1 expression of the transfected LCL721.221 cells was identified by RT-PCR and Dot-ELISA. The sHLA-G 1 protein was purified by affinity chromatography on anti–HLA-ImAb W6/32 coupled to cyanogen-bromide–activated Sepharose 4B from culture supernates of transfectants. Various concentrations of sHLA-G 1 protein (0, 2, 4, 6, or 8 μg/mL) were added to a NK cell-mediated xenogenic cell lysis system with either NK92 cells or fresh human peripheral blood mononuclear cells (PBMCs) cocultured with the porcine endothelial cells line. A LDH release assay was used to evaluate NK cell-mediated cytotoxicity. Results sHLA-G 1 provided significant protection of porcine endothelial cells against human NK-mediated cytotoxicity in a dose-dependent manner. The rates of NK92 cell-mediated cytotoxicity were reduced to 83.4 ± 5.7% (2 μg/mL), 56.6 ± 9.3% (4 μg/mL), 39.3 ± 10.2% (6 μg/mL), and 31.2 ± 4.9% (8 μg/mL) versus 96.9 ± 3.0% in the control group ( P < .01). Similarly, adding 6 μg/mL sHLA-G1 reduced the mean rate of PBMC-mediated cytotoxicity ( n = 4) to 5.8 ± 1.6% from 23.9 ± 1.3% in the control group ( P < .01). Conclusions These results indicated that sHLA-G 1 protected xenogeneic porcine endothelial cells against attack by human NK cells, thus providing a new approach to overcome NK-mediated immunity to xenografts.

Intrathecal synthesis of soluble HLA-G and HLA-I molecules are reciprocally associated to clinical and MRI activity in patients with multiple sclerosis

The aim of this study was to provide further insight into the effective contribution of classical soluble HLA-A, B and C class Ia (sHLA-I) and non-classical soluble HLA-G class Ib (sHLA-G) molecules in immune dysregulation occurring in multiple sclerosis (MS). We evaluated by enzyme-linked immunosorbent assay (ELISA) technique intrathecal synthesis and cerebrospinal fluid (CSF) and serum levels of sHLA-I and sHLA-G in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) MS patients stratified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also tested, as neurological controls, 91 patients with other inflammatory neurological disorders (OIND) and 92 with non-inflammatory neurological disorders (NIND). Eighty-two healthy volunteers served as further controls for sHLA-I and sHLA-G determinations. An intrathecal production of sHLA-I and sHLA-G detected by specific indexes was significantly more frequent in MS patients than in controls (p<0.01). An intrathecal synthesis of sHLA-I was prevalent in clinically (p<0.02) and MRI active (p<0.001) MS, whereas a CSF-restricted release of sHLA-G predominated in clinically (p<0.01) and MRI stable (p<0.001) MS. sHLA-I levels were low in the serum of clinically active (p<0.001) and high in the CSF of MRI active (p<0.01) MS. Conversely, sHLA-G concentrations were decreased in the serum of clinically stable MS (p<0.01) and increased in the CSF of MRI inactive MS (p<0.001). The trends towards a negative correlation observed between CSF and serum concentrations and intrathecal synthesis of sHLA-I and sHLA-G in patients without evidence of clinical and MRI activity confirmed that intrathecal production and fluctuations in CSF and serum concentrations of sHLA-I and sHLA-G were reciprocal in MS. Our results suggest that, in MS, a balance between classical sHLA-I and non-classical sHLA-G products modulating both MRI and clinical disease activity in opposite directions may exist.