Single-cell profiling reveals immune disturbances landscape and HLA-F-mediated immune tolerance at the maternal-fetal interface in preeclampsia.

Background: Preeclampsia is a pregnancy-specific disorder that always causes maternal and fetal serious adverse outcome. Disturbances in maternal immune tolerance to embryo at the maternal-fetal interface (MFI) may be associated with preeclampsia onset. Recent studies have revealed the reduced expression pattern of HLA-F at the MFI in preeclampsia, while the mechanism of it mediating maternal fetal immune tolerance has not been revealed. Methods: Single-cell RNA sequencing on placental decidua was performed to reveal the immune disturbances landscape at the MFI in preeclampsia. Human Jar cells and NK-92MI cells were employed to study the role of HLA-F in trophoblasts and lymphocyte. Results: A total of 101,250 cells were classified into 22 cell clusters. Disease-related IGFBP1+SPP1+ extracellular villus trophoblast (EVT) was identified in the preeclamptic placental decidua, accompanied by newly discovered immune cellular dysfunction such as reduced ribosomal functions of NK populations and abnormal expression of antigen-presenting molecules in most cell clusters. Certain genes that are characteristic of the intermediate stage of myeloid or EVT cell differentiation were found to have unexplored but important functions in the pathogenesis of preeclampsia; specifically, we detected enhanced cell cross-talk between IGFBP1+SPP1+ EVT2 or SPP1+M1 cells and their receptor cell populations at the MFI of PE patients compared to controls. With respect to HLA-F, mIF staining confirmed its reduced expression in PE samples compared to controls. Over-expression of HLA-F in Jar cells promoted cell proliferation, invasion, and migration while under-expression had the opposite effect. In NK-92MI cells, over-expression of HLA-F increased the secretion of immunoregulation cytokines such as CSF1 and CCL22, and promoted adaptive NKG2C+NK cell transformation. Conclusions: We revealed the immune disturbance landscape at the MFI in preeclampsia. Our findings regarding cellular heterogeneity and immune cellular dysfunction, as revealed by scRNA-seq, and the function of HLA-F in cells provide new perspectives for further investigation of their roles in the pathogenesis of preeclampsia, and then provide potential new therapeutic target.

Evaluation of the angiogenic factors sFlt-1, PlGF, and the sFlt-1/PlGF ratio in preeclampsia and associated features.

PROBLEM: Soluble Fms-like tyrosine kinase-1 (sFLT-1) and placental growth factor (PlGF) were previously reported to play a key role in the pathogenesis of preeclampsia (PE). We tested the link between altered PlGF and sFLT-1 levels, and their ratio (sFlt-1/PlGF) with PE and PE-associated featured in Tunisian PE cases and age- and BMI-matched normotensive women. METHOD OF STUDY: Peripheral blood specimens from 88 women with PE, and 60 control women were tested for PlGF and sFLT by commercially available ELISA. RESULTS: Significant increases in sFlt-1 levels and in the sFlt-1/PlGF ratio, more than changes in PlGF levels were noted in PE subjects when compared to control women. Elevation in sFlt-1 and sFlt-1/PlGF ratio was observed at different percentile values in PE cases. The receiver operating characteristic (ROC) area under the curve (AUC) for sFlt-1, PlGF, and sFlt-1/PlGF ratio were 0.869 ± 0.031, 0.463 ± 0.048, and 0.759 ± 0.039, respectively. A systematic shift in sFlt-1, but not in PlGF, distributions for higher values occurred in PE subjects. A progressive increase in the adjusted OR paralleled increased sFlt-1 and the sFlt-1/PlGF ratio percentile values; no similar trend was noted for the PlGF percentiles. Increased sFlt-1 levels and sFlt-1/PlGF ratio were significantly correlated with dysmenorrhea, hypertension, baby weight, and C-section. In contrast, no correlation was found between PlGF and the PE-associated features tested. CONCLUSIONS: Increased sFlt-1 levels and corresponding sFlt-1/PlGF ratio, but not circulating PlGF levels, constitute an independent risk factor for PE.

Preeclampsia is associated with reduced renin, aldosterone, and PlGF levels, and increased sFlt-1/PlGF ratio, and specific angiotensin-converting enzyme Ins-Del gene variants.

We investigated the association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism with preeclampsia (PE) in Tunisian women. ACE I/D genotyping was done by PCR in 342 pregnant women with PE and 289 healthy pregnant women. The association between ACE I/D and PE and associated features were also evaluated. Decreased active renin concentration, plasma aldosterone concentration, and placental growth factor (PlGF) were observed in PE cases, while soluble fms-like tyrosine kinase-1 (sFlt-1)/PlGF ratio was significantly higher in the PE group. Distribution of ACE I/D alleles and genotypes were comparable between women with PE and control women. A significant difference in the frequency of the I/I genotype was seen between PE cases and control women according to the recessive model, with a trend towards association in the codominant model. Carriers of the I/I genotype had significantly higher infant birth weights compared to the I/D and the D/D genotype carriers. A dose-dependent relationship was also seen in VEGF and PlGF plasma levels and specific ACE I/D genotypes, with the lowest VEGF levels seen in the I/I genotype carriers compared to the D/D genotype carriers. Similarly, the I/I genotype carriers had the lowest PlGF levels compared to I/D and D/D genotype carriers. Furthermore, when studying the linkage between PE features, we found a positive correlation between PAC and PIGF. Our study suggests a role for ACE I/D polymorphism in the pathogenesis of PE, possibly through modulating VEGF and PlGF levels and infant birth weight, and highlights the relationship between PAC and PlGF.

Human forkhead box protein 3 gene variants associated with altered susceptibility to idiopathic recurrent pregnancy loss: A retrospective case-control study.

BACKGROUND: The pathogenesis of recurrent pregnancy loss (RPL) is multifactorial and not completely elucidated. Dysregulated immunity was implicated with RPL, in which regulatory T cells (Tregs) are key. As Tregs development and function are regulated by forkhead box P3 (FOXP3) transcription factor, and as FOXP3 expression is genetically determined, a role for FOXP3 polymorphisms in RPL pathogenesis was suggested. AIM: To investigate the association of rs2294021, rs2232365, rs3761548, and rs141704699 FOXP3 variants with idiopathic RPL in Lebanese women. METHODS: This retrospective case-control study included 386 RPL cases and 398 age-matched control women. Logistic odds ratios (OR) were estimated with 95% confidence interval after adjustment; a significance value of P<.05 was set. RESULTS: Significantly lower rs22944021 and rs2232365 minor allele frequency (MAF) was found in patients with idiopathic RPL in comparison with the control group. Furthermore, statistically significantly lower frequency of heterozygous and homozygous rs2294021 and rs2232365 genotypes was seen in controls, while significantly lower rs3761548 heterozygous genotype frequencies were found in the patient group. Obesity, antihypertension treatment, smoking, positive RPL family history, abortion state, and infertility treatment correlated negatively with rs2294021, while rs2232365 negatively correlated with obesity, and rs3761548 negatively correlated with infertility treatment. Marked linkage disequilibrium (LD) was noted among FOXP3 SNPs, with TGCC and CGAC haplotypes being positive, while CAAC, CACC, and TGAC haplotypes being negatively associated with RPL risk. Except for CGAC, the association of these haplotypes with RPL persisted after adjustment. CONCLUSION: FOXP3 gene variants and haplotypes are associated with altered incidence of RPL, proposing the role of Treg in RPL pathogenesis.

Contribution of -1031T/C and -376G/A tumor necrosis factor alpha polymorphisms and haplotypes to preeclampsia risk in Tunisia (North Africa).

Preeclampsia is a gestational disorder characterized by hypertension and proteinuria. Excessive release of pro-inflammatory cytokines, particularly tumour necrosis factor-alpha, has been demonstrated to contribute to endothelial activation and poor trophoblast invasion in placental development, resulting in preeclampsia's clinical symptoms. Genetic polymorphisms of tumour necrosis factor-alpha can regulate its production and may play an important role in the pathogenesis of this disease. This study aimed to evaluate the association of five tumour necrosis factor-alpha gene promoter single nucleotide polymorphisms, or their haplotype combinations, with preeclampsia prevalence. This case-control study was conducted on 300 women with preeclampsia and 300 age-matched women with normal pregnancy from Tunisian hospitals. Genotyping of tumour necrosis factor-alpha -1031 T/C, -376 G/A, -308 G/A, -238 G/A, and +489 G/A SNPs was performed on DNA extracted from blood samples using PCR-restriction fragment-length polymorphism analysis. Statistical analysis was performed using the chi-square test. P < 0.01 were considered statistically significant to take into consideration the multiple comparisons. A significantly higher frequency of the minor allele -1031C (p < 0.001) was observed in preeclampsia cases compared to controls. Notably, the -1031C and -376A (CA) haplotype, which correlates with a higher production of TNF-α protein, had a higher incidence in women with preeclampsia (p = 0.0005). Conversely, the TG haplotype had a low frequency in preeclampsia cases compared to controls (p = 0.002) which suggests that it is associated with a reduced incidence of preeclampsia. These results suggest that tumour necrosis factor-alpha polymorphisms, in particular the -1031C/A, and the haplotype CA, contribute to an increased risk of preeclampsia in Tunisian women.

MMP-2 and MMP-9 Polymorphisms and Preeclampsia Risk in Tunisian Arabs: A Case-Control Study.

The abnormal production of matrix metalloproteinases (MMPs), especially MMP-9 and MMP-2, plays a pivotal role in hypertensive disorders of pregnancy, and as such, can influence the development of preeclampsia. These alterations may result from functional genetic polymorphisms in the promoter region of MMP-9 and MMP-2 genes, which modify MMP-9 and MMP-2 expression. We investigated the association of MMP-9 polymorphism rs3918242 (-1562 C>T) and MMP-2 polymorphism rs2285053 (-735 C>T) with the risk of preeclampsia. This case-control study was conducted on 345 women with preeclampsia and 281 age-matched women with normal pregnancies from Tunisian hospitals. Genomic DNA was extracted from whole blood collected at delivery. Genotypes for -1562 C>T and -735 C>T polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). An increased frequency of heterozygous MMP-9 -1562 C/T genotype carriers was observed in women with preeclampsia compared to healthy controls (p = 0.03). In contrast, the MMP-2 -735 C>T polymorphism was not significantly different regarding frequency distribution of the allele and genotype between healthy pregnant women and women with preeclampsia. Our study suggests that the MMP-9 -1562 C/T variant, associated with high MMP-9 production, could be a genetic risk factor for preeclampsia in Tunisian women.

IKZF1 genetic variants rs4132601 and rs11978267 and acute lymphoblastic leukemia risk in Tunisian children: a case-control study.

BACKGROUND: Associations between IKZF1 gene variants and Acute Lymphoblastic Leukemia (ALL) was recently reported. We examined whether the common IKZF1 polymorphisms rs4132601 T/G and rs111978267 A/G are associated with ALL among a Tunisian pediatric cohort. METHODS: This case-control study involved 170 patients with ALL and 150 control subjects. SNP genotyping was performed by TaqMan® SNP Genotyping Assay. RESULTS: The minor allele G of IKZF1 gene polymorphism rs4132601 T/G was significantly higher in ALL cases than in control subjects (P = 0.029), with 1.54-fold increased risk of ALL. The association of rs4132601 with ALL was seen under co-dominant (P = 0.009), recessive (P = 0.006), and additive (P = 0.027) genetic models, of which the co-dominant (P = 0.027) and recessive (P = 0.027) association remained significant after adjusting for covariates, and False Discovery Rate correction. In contrast, no association was noted for rs111978267 variant. Two-locus (rs4132601-rs11978267) IKZF1 haplotype analysis demonstrated association of GA (P = 0.053), with increased ALL risk [OR (95% CI) = 1.58 (1.00-2.51)], which remained significant after controlling for key covariates [aP = 0.046; aOR (95% CI) = 1.61 (1.01-2.57)]. CONCLUSION: We demonstrated the association of IKZF1 polymorphism rs4132601 T/G with increased risk of ALL among Tunisian pediatric cohort, with altered phenotypic changes among ALL patients.

Impact of obesity on the association of active renin and plasma aldosterone concentrations, and aldosterone-to-renin ratio with preeclampsia.

OBJECTIVE: To evaluate the association of active renin concentration (ARC), plasma aldosterone concentration (PAC) and aldosterone-to-renin ratio (ARR) with the risk of preeclampsia (PE), in particular according to the status of obesity. DESIGN: This retrospective case-control study involved 90 women with PE (mean gestation 35.6 ±â€¯3.6 weeks) and 90 age-matched control women with uncomplicated pregnancies (mean gestation 38.5 ±â€¯2.5 weeks). ARC and PAC were measured by radioimmunoassay; ARR calculated as PAC to ARC ratio. PE cases were stratified into 5 percentiles groups, and analyzed in multivariate logistic regression. RESULTS: Women with PE had significantly lower median ARC and PAC than control women, which were confirmed by percentiles analysis. Spearman correlation demonstrated negative correlation of ARC with body mass index, systolic/diastolic blood pressure. PAC correlated negatively with systolic/diastolic blood pressure, but positively with baby weight, ARC and ARR. On the other hand, ARR positively correlated with BMI and PAC, but negatively with ARC. Lower PAC was associated with PE, irrespective of body weight, while ARC levels were significantly lower in non-obese PE cases vs. control women. ARR was not significantly different between PE cases and control women, when stratified according to obesity. CONCLUSION: Low ARC and PAC in third trimester are more strongly associated with preeclampsia respectively in non-obese and obese women.

Contribution of microRNA-149, microRNA-146a, and microRNA-196a2 SNPs in colorectal cancer risk and clinicopathological features in Tunisia.

BACKGROUND AND AIM: Colorectal cancer (CRC) is a worldwide leading cause of mortality. Genetic studies have associated single nucleotide polymorphisms in genes encoding microRNAs with CRC risk but results are mostly inconclusive across variable ethnicities. In this study, we investigated the association of hsa-mir-149 rs2292832 C/T, hsa-mir-146a rs2910164 G/C and hsa-mir-196a2 rs11614913 C/T and explored their roles in clinicopathological features of CRC progression in an Eastern Tunisian cohort. SUBJECTS AND METHODS: Three hundred thirteen subjects were enrolled in our retrospective study including 152 CRC cases and 161 controls. Genotyping was assayed by RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) method. SPSS v.18.0, R and SNP Stats online software performed statistical analysis. RESULTS: Significantly higher hsa-mir-149C/T rs2292832 minor allele frequency was associated with increased risk of CRC [p = .03; OR = 1.54 (1.08-2.19)]. In addition, significant crude associations of hsa-mir-149C/T rs2292832 polymorphism were detected under codominant, dominant and additive models of inheritance. After adjusting for covariates and performing FDR correction, these associations did not remain. No associations were detected for hsa-mir-146a G/C rs2910164 and hsa-mir-196a2 C/T rs11614913. When performing stratified analysis of clinicopathological features according to genotypes, a significant association (p = .004) was found between hsa-mir-146a G/C rs2910164 and tumour differentiation grade. Regression analysis according to CRC progression features had demonstrated a trend toward significance in overdominant model of inheritance for hsa-mir-149C/T rs2292832 with a protective effect [p = .05; OR = 0.51 (0.26-1.02)]. CONCLUSION: Hsa-mir-149C/T rs2292832 and hsa-mir-146a G/C rs2910164 may influence CRC risk in an ethnicity-dependent manner by interfering with CRC progression parameters in Tunisian cohort.

Contribution of angiotensinogen M235T and T174M gene variants and haplotypes to preeclampsia and its severity in (North African) Tunisians.

BACKGROUND: Preeclampsia (PE) is a pregnancy-associated hypertensive disorder and a leading cause of maternal and neonatal morbidity and mortality. While its pathogenesis remains ill defined, several candidate genes for PE have been identified, but results remain inconclusive. We investigated the association of the angiotensinogen ( AGT) gene variants M235T and T174M with PE, and we analyzed the contribution of both variants to the severity of PE. METHODS: This case-control study enrolled 550 Tunisian pregnant women: 272 with PE, of whom 147 presented with mild, and 125 with severe PE, along with 278 unrelated age- and ethnically matched control women. AGT genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Significantly higher M235T minor allele frequency (MAF) was associated with increased risk of PE ( p < 0.001). Decreased frequency of heterozygous T174M genotype carriers were found in control women ( p = 0.015), suggesting a protective effect of this genotype (odds ratio (95% confidence interval) = 0.51 (0.29-0.89)). Two-locus haplotype analysis demonstrated MM and TT haplotypes to be negatively and positively associated with PE, respectively. MAF of M253T, but not T174M, was higher in the severe PE group, and carrying M235T or T174M minor allele was associated with increased body mass index ( p < 0.001) among unselected PE women. CONCLUSIONS: AGT M235T and T174M variants contribute to an increased risk of developing PE, and for M235T to PE severity.

Vascular endothelial growth factor single nucleotide polymorphisms and haplotypes in pre-eclampsia: A case-control study.

An association between vascular endothelial growth factor (VEGFA) gene variants and altered VEGF secretion and preeclampsia (PE) were described, often with inconclusive findings. An ethnic contribution to the association of VEGFA polymorphisms with PE and its associated features was also suggested. To investigate whether common VEGFA single nucleotide polymorphisms (SNP) are linked with PE and associated features in Tunisian women. A case-control study involving 300 women with PE, and 300 age-matched control women. Genotyping of VEGFA rs833052, rs699947, rs833061, rs1570360, rs2010963, rs25648, rs833068, rs833070, rs3025020, and rs3025039SNPs was done by real-time PCR. Minor allele frequency (MAF) of rs833052, rs699947, rs833061, rs1570360, rs2010963, rs25648, rs833068, rs833070, rs3025020, and rs3025039 VEGFA SNP, were not significantly different between PE cases and control women. In addition, there was lack of association of the genotypes of VEGFA SNPs with PE, irrespective of the genetic model used. Seven-locus (rs699947, rs833061, rs1570360, rs2010963, rs25648, rs833068 and rs833070) haplotype analysis demonstrated positive association of ATGCCAA, ACAGCAG and CCAGCGG, and negative association of CCAGCAA and ATGCCGG haplotypes with PE, all of which except for ACAGCAG remained associated with PE after correcting for multiple comparisons. Increased and reduced PE severity was associated with ATGCCAA, and with ATGCCGG and CCAGCAA haplotypes, respectively. Furthermore, carriage of CCGGTAG haplotype was associated with reduced risk of PE. Our study suggests that VEGFA haplotypes, more so than individual SNPs, play a role in PE pathogenesis in Tunisian women. These findings need confirmation in other ethnic populations.

FGF1 and FGF2 mutations in preeclampsia and related features.

BACKGROUND: Fibroblast growth factor (FGF) 1 and FGF2 were previously linked with preeclampsia (PE), possibly through altering decidual and placental FGFR2 expression. Since common variation in FGF1 and FGF2 might influence FGF1 and FGF2 activity, this study evaluated whether common FGF1 and FGF2 variants are linked with PE and associated features. METHODS: The association between FGF1 rs34011 and FGF2 rs2922979 SNPs and PE were tested in 300 women with PE, and 300 age-matched control women. RESULTS: The allelic distribution of FGF1 rs34011 (P < 0.001) but not FGF2 rs2922979, variants were significantly different between PE cases and control women. Marginal association of FGF2 rs2922979 was seen after controlling for key covariates. Setting homozygous major allele genotype (1/1) as reference, significantly higher frequencies of heterozygous rs345011, and reduced frequency of heterozygous rs2922979 genotype carriers were seen in PE cases; the distribution of the remaining genotypes were comparable between cases and controls. Carriage of rs2922979 minor allele correlated with fasting glucose (P = 0.02), while the presence of rs34011 minor allele was not correlated with PE-associated features. CONCLUSIONS: Our study suggests that the genetic variants of FGF1 rs34011, more so than FGF2 rs2922979, may play a role in PE pathogenesis in Tunisian women. These findings need confirmation in other ethnic populations.

Placental growth factor and vascular endothelial growth factor serum levels in Tunisian Arab women with suspected preeclampsia.

The angiogenic factors vascular endothelial growth factor (VEGF) and placental growth factor (PIGF) play a central role in the process of angiogenesis. We evaluated the association of free PIGF and free VEGF levels and the risk of preeclampsia (PE) among Tunisian Arab women, and established the range of VEGF and PIGF in normal healthy pregnancies, between 24 and 42weeks of gestation. This retrospective case-control study included 345 women with PE, and 289 women with uncomplicated pregnancies. PIGF and VEGF plasma levels were quantitated by commercially-available ELISA. Compared to control women, plasma PIGF concentrations were lower in women with PE at all gestation age intervals (P<0.0001), compared to VEGF levels which were significantly lower in women with PE but only during early gestation age intervals ([29-32[ and [32-35[). High odds for developing PE, and correspondingly higher associations, were associated with low PIGF values (less than the 5(th) percentile), at all gestation age intervals. The only exception was recorded for the [29-32 [interval, which was not statistically significant. PIGF testing, recorded at 29-37weeks of gestation, had a higher specificity (93-100%) than sensitivity, and the positive predictive values ranged from 90% to 100% for 24-37weeks of gestation. This indicates that it mainly detects non-PE healthy women as well, and thus may be useful as a screening test, though currently unreliable for diagnostic purposes. Reduced PIGF levels during different gestation age intervals, and reduced VEGF levels during early gestation age intervals are also associated with subsequent development of PE in our population; the gestational age interval adjusted-5(th) percentiles of PIGF provide reference ranges for this marker in normal pregnancy.

Association of common eNOS/NOS3 polymorphisms with preeclampsia in Tunisian Arabs.

We investigated the association of endothelial nitric oxide synthase (NOS3) polymorphisms -786T>C, 27-bp repeat 4b/4a, and Glu298Asp with preeclampsia (PE). This was a case-control study involving 345 unrelated Tunisian women with PE and 289 unrelated age- and ethnically matched control women. The -786C allele was significantly increased in PA patients when compared to healthy controls (P=0.015). In contrast, MAF of Glu298Asp (P=0.103) and 4b/4a (P=0.168) were not significantly different between the study groups. Higher frequencies of heterozygous Glu298/298Asp and homozygous -786T/-786T genotypes were seen in PE cases compared to healthy subjects. The combination of genotypes 221 (-786T>C, Glu298Asp, 4a/4a) was more in PE cases compared with control women (17.68% vs. 8.36%; P=0.029). Multivariate regression analysis confirmed this association. Genetic variation at the NOS3 locus represents a genetic risk factor for increased susceptibility to PE.