Screening of single nucleotide polymorphism in matrix metalloproteinase-2 (MMP2) and tetraspanin CD63 genes in Chlamydia trachomatis-infected tubal ectopic pregnancy patients.

OBJECTIVE: Tubal ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Studies have suggested that infection-induced inflammatory responses are major risk factors for EP. The aim of the present study was to find an association between MMP2 and CD63 gene variants and risk of EP during Chlamydia trachomatis infection in an Indian population. METHODS: Fallopian tube samples of 120 EP and 120 tubal ligation women were collected. C. trachomatis was detected by PCR. The genotyping of MMP2 (rs17859882 G/T, rs7201A/C) and CD63(rs2231464 C/T, rs376086542 A/G) gene variants was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). RESULTS: The frequency of GG or GT genotype of MMP2 G/T polymorphism (rs17859882) was 66.6% in infected EP and 36.7% in uninfected EP and 22% in tubal ligation controls (P < 0.0001), while the frequency of AC or CC genotype of MMP2 A/C polymorphism (rs7201) was 66.6% in infected EP and 20.6% in uninfected EP and 13.5% in tubal ligation controls (P < 0.0001). The frequency of CT or TT genotype of CD63 C/T polymorphism (rs2231464) was 74% in infected EP and 21.8% in uninfected EP and 11.8% tubal ligation controls (P < 0.0001), while the frequency of AG or GG genotype of CD63 A/G polymorphism (rs376086542) was 48.1% in infected EP and 41.3% in uninfected EP and 18.6% tubal ligation controls (P < 0.0001). CONCLUSIONS: The present study revealed a strong association between the presence of gene variants MMP2 (rs17859882 G/T, rs7201A/C) and CD63 (rs2231464 C/T, rs376086542 A/G) and risk of tubal EP during C. trachomatis infection.

MicroRNA mediated regulation of oxidative stress and cytokines in Chlamydia trachomatis-infected recurrent spontaneous abortion: A case-control study.

PROBLEM: Increased oxidative stress (OS) and inflammatory responses are major underlying factors behind Chlamydia trachomatis-associated recurrent spontaneous abortion (RSA). miRNAs are known to regulate inflammation and OS and their dysregulation has been associated with compromised pregnancies. Therefore, aim of this study was to investigate the expression/correlation of OS biomarkers, cytokines and miRNAs in C. trachomatis-associated RSA. METHOD OF STUDY: Urine and non-heparinized blood samples were collected from RSA patients with history of >3 consecutive abortions (cases) and non-pregnant women with history of >2 successful deliveries (controls) attending Department of Obstetrics and Gynaecology, Safdarjung hospital, New Delhi. C. trachomatis detection was done in urine by PCR. miRNA expression was studied by microarray analysis and validated by real time-PCR. Evaluation of cytokines and antioxidant genes expression were done by real-time PCR. Level of OS biomarkers 8-hydroxy guanosine (8-OHdG) and 8-isporostane (8-IP) were measured by ELISA. RESULTS: Fifty circulating miRNAs were differentially expressed in infected patients compared with controls. Of these, four were overexpressed and 46 downregulated. Thirteen differentially expressed circulating miRNAs were selected to validate microarray results. miRs-8069, -3663-3p showed maximum upregulation/downregulation in infected versus control group. Expression of cytokines (IL-8, TNF-α, IFN-γ), antioxidant genes SOD2 and OS biomarkers (8-OHdG,8-IP) were increased while SOD1 was decreased in infected patients. miR-8069 showed significant positive correlation with cytokines, SOD2, 8-OHdG and 8-IP. miR-3663-3p showed significant positive correlation with SOD1. CONCLUSIONS: Overall results indicate circulating miRNAs are involved in pathogenesis of C. trachomatis-associated RSA and are potential modulators of cytokine signalling and OS in infected RSA.

Progesterone-mediated immunoregulation of cytokine signaling by miRNA-133a and 101-3p in Chlamydia trachomatis-associated recurrent spontaneous abortion.

miRNAs regulate the expression of various genes involved in cellular and metabolic pathways in pregnancy related complications including recurrent spontaneous abortion (RSA). Modulation of progesterone and associated pro-inflammatory cytokines by miRNAs in Chlamydia trachomatis-associated RSA is still under investigation. Present study aimed to evaluate the expression/correlation of serum-circulating miRNAs-133a, 101-3p, 320b, 146b-5p, 24, 559, progesterone and few cytokines in C. trachomatis-positive spontaneous aborters. Non-heparinized blood and urine was collected from 120 patients with history of RSA (Group I) and 120 patients with ≥ 2 successful deliveries (Group II) attending Department of Obstetrics and Gynecology, Safdarjung hospital, New Delhi, India. C. trachomatis detection was performed by PCR and chlamydial load by real time PCR. Progesterone concentration was estimated by ELISA. miRNAs and cytokine expression was studied by quantitative real-time PCR and correlated with progesterone expression. Twenty six patients were found to be positive for C. trachomatis. miRNAs- 133a, 101-3p showed maximum upregulation in infected versus control patients. miRNA expression showed positive correlation with chlamydial load. Progesterone concentration showed significant decrease while cytokines (IL-6, IFN-γ, TNF-α) were significantly upregulated in C. trachomatis-positive patients. Positive correlation was observed between expression of miRNAs-133a and 101-3p and cytokines while negative correlation was observed with progesterone in infected RSA patients. Correlation between progesterone and cytokines was found to be significantly negative in infected RSA patients. Although further validation is required, the study concludes that miR-133a and 101-3p are of clinical importance and have a role in immunoregulation of progesterone and cytokines in infection associated RSA.

Intronic variants of LGALS13 gene encoding placental protein (PP13) are linked with increased risk of infection-associated spontaneous preterm birth.

PROBLEM: Spontaneous preterm birth (sPTB) is a global health issue. Studies suggest infection and infection-based inflammatory responses are major risk factors for sPTB. Considering the important role of anti-inflammatory proteins in pregnancy, the study aimed to find the association between anti-inflammatory LGALS13 gene variants IVS2-22 A/G (rs2233706) and IVS3+72 T/A (rs2233708) and the risk of sPTB during Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum infection in Indian population. METHOD OF STUDY: Placental samples of 160 sPTB and 160 term women were collected. Pathogens were detected by PCR. The genotyping of LGALS13 gene variants IVS2-22 A/G (rs2233706) and IVS3+72 T/A (rs2233708) was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). RESULTS: The frequency of AG or GG genotype of LGALS13 IVS2-22A/G polymorphism (rs2233706) was 75.5% in infected sPTB cases and 14.4% in uninfected sPTB cases and 7.3% in term birth controls (p < .0001), while the frequency of TA or AA genotype of LGALS13 IVS3+72T/A polymorphism (rs2233708) was 83.6% in infected sPTB cases and 18% in uninfected sPTB cases and 12.7% in term birth controls (p < .0001). The genotypic frequencies for both the variants of LGALS13 were statistically significant (p < .0001) in the infected sPTB versus uninfected sPTB and term birth controls. CONCLUSIONS: Study reveals strong association between the presence of immunological gene variants LGALS13 IVS2-22 A/G (rs2233706) and LGALS13 IVS3+72 T/A (rs2233708) and risk of sPTB during C. trachomatis, M. hominis and U. urealyticum infection.

Immune regulation by activation markers at feto-maternal interface in infection-associated spontaneous preterm birth.

BACKGROUND: Spontaneous preterm birth (sPTB) is a global health concern. Studies reveal infections are majorly responsible for sPTB and immune activation markers play a role in regulation of maternal immune responses against pathogens during sPTB. AIM: To study the mRNA expression and correlation of activation markers (CD66a, ICAM1, ITGB1, TIM3, CD25, CD95) and associated cytokines (IL-1ß and IL-17)/prostaglandin receptors (EP2 and IP) in the placenta of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum-infected sPTB women. METHODS: Placental samples were collected from 160 sPTB and 160 term birth women. PCR was used for the detection of C. trachomatis, M. hominis, U. urealyticum. The mRNA expression of activation markers, cytokines and prostaglandin receptors was evaluated by real-time qPCR. RESULTS: The fold-change expression of CD66a, ICAM1, TIM3, CD25 and CD95 was 2.89, 5.5, 4.95, 6.44 and 6.95-fold (p < 0.001), respectively; while for cytokines- IL-1ß and IL-17 was 5.41 and 4.71-fold (p < 0.001), respectively and for prostaglandin receptors- EP2 and IP was 5.5 and 5-fold (p < 0.001) upregulated, respectively in infected sPTB women. Significant positive correlation was obtained among ICAM-1 and IL-1ß/EP2/IL-17, TIM3 and IP/IL-17. Significant negative correlation was obtained between CD66a and EP2/IL-17, CD25 and IL-1ß/EP2, CD95 and IL-1ß/EP2 in infected sPTB women. CONCLUSIONS: CD66a, ICAM1 and TIM3 may play role in inflammation and have potential for the clinical beginning of preterm labour during infection while CD25 and CD95 are possibly involved in immunotolerance at feto-maternal interface during C. trachomatis, M. hominis and U. urealyticum infection.

Chlamydia trachomatis infection regulates the expression of tetraspanins, activin-A, and inhibin-A in tubal ectopic pregnancy.

Mechanism of Chlamydia trachomatis causing tubal ectopic pregnancy (EP) is not well understood. Tetraspanins (tspans), activin-A, and inhibin-A might play a role in the development of pathological conditions leading to EP. The study aimed to elucidate the expression of tspans, activin-A, and inhibin-A with a role of associated cytokines in C. trachomatis-associated EP and analyze interacting partners of DEGs, with an expression of a few important interacting genes. Fallopian tissue and serum were collected from 100 EP (Group I) and 100 controls (Group II) from SJH, New Delhi, India. Detection of C. trachomatis was done by polymerase chain reaction (PCR) and IgG antibodies were detected by enzyme-linked immunosorbent assay. Expression of tspans, activin-A, inhibin-A, and cytokines was analyzed by real time (RT)-PCR and their interacting genes were assessed by STRING. Expression of few disease-associated interacting genes was studied by RT-PCR. A total of 29% (Group I) were C. trachomatis positive. Tspans and activin-A were significantly upregulated, while inhibin-A was significantly downregulated in Group Ia. ITGA1, TLR-2, ITGB2, and Smad-3 were a few interacting genes. Expression of ITGA1, TLR-2, and Smad-3 was significantly upregulated in C. trachomatis-positive EP. Results suggested dysregulated tspans, activin-A, and inhibin-A might play a role in C. trachomatis-infected tubal EP.

Microarray profiling of serum micro-RNAs in women with Chlamydia trachomatis-associated recurrent spontaneous abortion: A case control study.

Chlamydia trachomatis infection is a major cause of sexually transmitted diseases and adverse pregnancy outcomes such as recurrent spontaneous abortion (RSA). Micro-RNAs (miRNAs) have been known to be upregulated/downregulated in various reproductive-associated diseases such as ectopic pregnancy, preterm birth and pre-eclampsia. However, there is paucity of literature on miRNA profile in C. trachomatis-infected RSA. The present study aimed to determine the profile of serum miRNAs followed by their validation in C. trachomatis-infected RSA and to find target genes involved in biological pathways. Non-heparinized blood and first void urine were collected from 30 non-pregnant women with RSA and 30 non-pregnant women with ≥2 successful deliveries (controls) attending Department of Obstetrics and Gynaecology, Safdarjung hospital, New Delhi, India. C. trachomatis detection was done in urine by PCR and chlamydial load was determined by quantitative real-time PCR (qRT-PCR). miRNA expression was studied by microarray analysis followed by in vitro validation by qRT-PCR. Analysis of target genes/pathways was characterized in silico. 06 RSA patients were infected with C. trachomatis, while chlamydial load was found to be 6000-12,000 copies/ml. 110 circulating miRNAs were expressed differentially in infected RSA patients compared with controls. Of these, 16 were overexpressed and 94 downregulated. 06 differentially expressed circulating miRNAs were selected to validate the microarray results. qRT-PCR data confirmed the reliability of the microarray results: miR-4443, -5100, -7975 showed statistically significant upregulation, while miR-6808-5p, -3148, -6727-5p were significantly downregulated in infected RSA patients versus controls. Chlamydial load was positively correlated with these upregulated miRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that target genes of miRNAs in RSA are involved in AMPK, Akt and mTOR signaling pathways. Overall results indicate that differentially expressed circulating miRNAs are involved in pathogenesis of C. trachomatis-associated RSA and have the potential to be used as biomarkers for predicting RSA.

Determination of chlamydial load in recurrent miscarriage in relation to some female sex hormones: a case-control study.

INTRODUCTION: Chlamydia trachomatis is a frequent cause of adverse pregnancy outcomes including recurrent spontaneous abortion (RSA). However, regulation of infectious load by host immune response is unknown. Female sex hormones are known to affect C. trachomatis infection. The aim of this study was to determine correlation of chlamydial infectious load and gestational age with concentration of progesterone/estrogen in RSA. METHODOLOGY: Urine and non-heparinized blood were collected from patients with history of > 3 spontaneous abortions (n = 150, cases) and those with history of > 2 successful deliveries (n = 150, controls) from Department of Obstetrics and Gynecology, Safdarjung hospital, New Delhi, India. C. trachomatis positivity was determined by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) and chlamydial load by real-time PCR. Estrogen and progesterone concentrations were estimated by ELISA and correlated with chlamydial load. RESULTS: 22/150 case patients were positive for C. trachomatis. 2,000-10,000 copies/mL of chlamydial load were detected in infected RSA patients. Progesterone concentration showed significant decrease while estrogen concentration was significantly increased in C. trachomatis-positive RSA patients versus controls. Chlamydial load and estrogen concentration were positively correlated while progesterone concentration was negatively correlated with chlamydial load. Gestational age was positively correlated with concentration of estrogen and negatively correlated with concentration of progesterone in infected-RSA women. CONCLUSIONS: Overall findings suggest that interplay between chlamydial copy load, hormonal changes such as increased expression of estrogen and decreased expression of progesterone, and advanced gestational age may be contributing as deciding factors for ensuing RSA during C. trachomatis-infection.

Immunomodulation of cytokine signalling at feto-maternal interface by microRNA-223 and -150-5p in infection-associated spontaneous preterm birth.

Spontaneous preterm birth (sPTB) is a global health concern and it is the most prevalent cause of infant mortality and morbidity with occurrence rate of 5 - 18% worldwide. Studies suggest infection and infection-driven activation of inflammatory responses are the potential risk factors for sPTB. MicroRNAs (miRNAs) are thought to control the expression of several immune genes, making them crucial components of the intricate immune regulatory network and the dysregulation of miRNAs in placenta has been associated to several pregnancy-related complications. However, studies on possible role of miRNAs in immunomodulation of cytokine signalling in infection-associated sPTB are scarce. Present study aimed to investigate expression/ correlation of a few circulating miRNAs (miR-223, -150-5p, -185-5p, -191-5p), miRNA target genes and associated cytokines in sPTB women found infected with Chlamydia trachomatis/ Mycoplasma hominis/ Ureaplasma urealyticum. Non-heparinized blood and placental sample were collected from 140 sPTB and 140 term women visiting Safdarjung hospital, New Delhi (India) for conducting PCR and RT-PCR for pathogen detection and miRNA/ target gene/ cytokine expression, respectively. Common target genes of differentially expressed miRNAs were obtained from databases. The correlation between select target genes/ cytokines and serum miRNAs was determined by Spearman's rank correlation. 43 sPTB were infected with either pathogen and a significant upregulation of serum miRNAs was observed. However, miR-223 and 150-5p showed maximum fold-change (4.78 and 5.58, respectively) in PTB versus control group. IL-6ST, TGF-ß R3 and MMP-14 were important target genes among 454 common targets, whereas, IL-6 and TGF-ß were associated cytokines. miR-223 and 150-5p showed significant negative correlation with IL-6ST/ IL-6/ MMP-14 and positive correlation with TGF-ß R3/ TGF-ß. A significant positive correlation was found between IL-6ST and IL-6, TGF-ß R3 and TGF-ß. However, miR-185-5p and 191-5p were not significantly correlated. Although post-transcriptional validation is required, yet on the basis of mRNA findings, the study concludes that miR-223 and 150-5p are apparently of clinical importance in regulation of inflammatory processes during infection-associated sPTB.

Galectins are critical regulators of cytokine signalling at feto-maternal interface in infection-associated spontaneous preterm birth.

INTRODUCTION: Spontaneous preterm birth (sPTB) is a global health issue. Studies suggest infections are chiefly associated with sPTB and galectins (gals) play a role in regulation of innate and adaptive maternal immune response against pathogens during sPTB. The aim of this study was to describe the gene expression of gal -1, -3, -8, -9, -13 in relation to gene expression of cyclooxygenase-2 (COX-2) and the cytokines IL-8, IL-10, TNF-α, IFN-ϒ in the setting of sPTB and confirmed infection with Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum. METHODS: Placental samples were collected from 120 term control and 120 sPTB pregnancies. PCR was used to detect specific pathogens. Gene expression of galectins, cytokines, and COX-2 was performed using real time qPCR. RESULTS: Fold-change expression of gal -1, -3, -8, -9, -13 was 5.13, 6.11, 1.14, 5.23 and 7.16 (p<0.001), respectively; while IL-10, IL-8, TNF-α, IFN-ϒ and COX-2 was 6.29, 6.55, 6.35, 6.36 and 2.73-fold upregulated (p<0.05), respectively in infected sPTB. Gal-1 was positively correlated with IL-10 (r=0.49, p=0.003) while gal-3 showed significant correlation with IL-8 (r=0.42, p=0.0113), TNF-α (r=0.65, p=< 0.001) and COX-2 (r=0.72, p=0.001). However, gal-8 was not significantly correlated with any cytokine. Gal-9, -13 were negatively correlated with IFN-ϒ (r=-0.45, p=0.006) and IL-8 (r=-0.39, p=0.018). DISCUSSION: Gal-1, -9, -13 are anti-inflammatory and might play role in immune-tolerance while gal-3 is pro-inflammatory and possibly responsible for immunogenic response, having potential to anticipate the clinical beginning of preterm labour during infection.

Association of functional superoxide gene polymorphism with chlamydia trachomatis-associated recurrent spontaneous abortion.

BACKGROUND: Oxidative stress generated by Chlamydia trachomatis infection is associated with reproductive complications such as recurrent spontaneous abortion. Aim of prospective study was to evaluate whether single nucleotide polymorphisms (SNPs) of SOD1 and SOD2 gene are associated with C. trachomatis-infected recurrent spontaneous abortion (RSA). METHODS: 150 patients with history of RSA and 150 patients with history of successful deliveries were recruited from Department of Obstetrics and Gynecology, Safdarjung hospital, New Delhi, India. Urine and non-heparinized blood samples were collected and C. trachomatis was detected by polymerase chain reaction (PCR). Using qualitative real time PCR, SNPs rs4998557 (SOD1) and rs4880 (SOD2) were screened in enrolled patients. Level of 8-hydroxyguanosine (8-OHdG), 8-isoprostane (8-IP), progesterone and estrogen was determined by enzyme-linked immunosorbent assays and correlated with SNPs. RESULTS: Significant differences were found in frequency of AA genotype of SOD1 gene among RSA patients versus controls, (82% and 54.66%, respectively; p = 0.02; OR 0.40; CI 95%). Frequency of AA genotype of SOD1 gene among RSA patients with C. trachomatis infection was 87.33%, while in uninfected RSA patients was 71.33% (p < 0.0001; OR 8; CI 95%). No significant relation was found between SOD2 (rs4880) genotype and RSA. Furthermore, significant increase in 8-OHdG, 8-IP and estrogen and significant decrease in progesterone was observed among patients carrying AA genotype. CONCLUSIONS: Findings suggest the clinical importance of AA genotype along with 8-OHdG, 8-IP and estrogen and progesterone in screening C. trachomatis-infected RSA women.

Aberrant gene expression of superoxide dismutases in Chlamydia trachomatis-infected recurrent spontaneous aborters.

Study aimed to characterize the expression of antioxidant genes SOD1 and SOD2 in Chlamydia trachomatis-induced recurrent spontaneous aborters and further determine their role by in silico analysis. First void urine was collected from 130 non-pregnant women with history of recurrent spontaneous abortion (RSA) (Group I) and 130 non-pregnant women (Group II; control) attending Obstetrics and Gynecology Department, SJH, New Delhi, India. C. trachomatis detection was performed by conventional PCR in urine. Gene expression of SOD1 and SOD2 was performed by quantitative real-time PCR. Further, its interacting partners were studied by in silico analysis. 22 patients were positive for C. trachomatis in Group I. Significant upregulation was observed for SOD2 gene in C. trachomatis-infected RSA patients while SOD1 was found to be downregulated. Increased concentration of oxidative stress biomarkers 8-hydroxyguanosine and 8-isoprostane was found in C. trachomatis-infected RSA patients. Protein-protein interaction (PPI) of SOD proteins and its interacting partners viz.; CCS, GPX1, GPX2, GPX3, GPX4, GPX5, GPX7, GPX8, CAT, PRDX1, TXN, SIRT3, FOXO3, and AKT1 were found to be involved in MAPK, p53 and foxo signaling pathways. Molecular pathways involved in association with SODs indicate reactive oxygen species (ROS) detoxification, apoptotic pathways and cell cycle regulation. Overall data revealed alleviated levels of SOD2 gene and decreased expression of SOD1 gene in response to C. trachomatis-infection leading to production of oxidative stress and RSA.

Differential expression of matrix metalloproteinases in the fallopian tube of women with Chlamydia trachomatis-associated tubal ectopic pregnancy.

Chlamydia trachomatis is an established risk factor for ectopic pregnancy (EP) in fallopian tube (FT). Matrix metalloproteinases (MMPs) have potential role in disease pathogenesis, however, dysregulation of extracellular matrix by MMPs/TIMPs (tissue inhibitors of MMPs) in infection-associated EP remains unknown. The aim was to study the expression of MMP-2, -9, -14/TIMP-1, -2, -3 in C. trachomatis-positive tubal EP patients. The study comprised of 100 tubal EP (Group I) and 100 tubal ligation patients (Group II; controls) enrolled from Department of Obstetrics and Gynaecology, VMMC and Safdarjung hospital, New Delhi (India) for collection of FT. Detection of C. trachomatis MOMP was done by PCR while quantitative expression of MMPs/TIMPs was studied by real-time PCR. Data was statistically evaluated by Graphpad prism. Overall, C. trachomatis was found in 18/100 tubal EP patients. After ruling out Neisseria gonnorhoeae and Mycoplasma genitalium, Group I was divided into Group Ia (C. trachomatis DNA-positive) and Group Ib (C. trachomatis DNA-negative; internal controls). Significant upregulation of MMP-2, -9, -14 and downregulated TIMP-1, -2, -3 were found in Group Ia versus controls (Groups Ib/II) (p < 0.05). Fold-change in MMP was significantly higher in Group Ia versus controls ('p' < 0.05). Maximum 5.5-fold upregulation was found in MMP-2. It is apparent by molecular analysis that differential expression of MMPs/TIMPs, particularly enhanced MMP-2 leads to tubal EP in C. trachomatis DNA-positive women.

Differential expression of urine-circulating micro-RNAs in Chlamydia trachomatis-induced recurrent spontaneous aborters.

Studies behind mechanisms of Chlamydia trachomatis-induced recurrent spontaneous abortion is still in its infancy. Possible strategy for preventing recurrent spontaneous abortion at molecular level is needed. Despite its multifactorial aetiology, Chlamydia trachomatis is important cause of RSA. However, mechanism leading to RSA in C. trachomatis-positive patients is not understood and novel strategies are needed. It is hypothesized that microRNAs play important role in RSA regulation during infection. Study aimed to elucidate expression/role of urine-circulating miRs-320b, 221-3p, 146b-5p,-16,-24,-559 in recurrent spontaneous aborters with C. trachomatis infection and to find their target genes by bioinformatic analysis. First-void urine was collected from 30 non-pregnant women with RSA (Group I) and 30 non-pregnant women with ≥2 successful deliveries (Group II; Controls) attending Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College, Safdarjung hospital, New Delhi (India). PCR was performed to detect C. trachomatis. Expression of miRNAs was studied by quantitative real-time PCR while target genes/functional annotations were predicted by GO/KEGG databases. Data was statistically evaluated. 05 RSA patients were C. trachomatis-positive. Group I was subdivided into Group Ia (C. trachomatis-positive RSA; n = 5) and Group Ib (C. trachomatis-negative RSA; internal controls). miR-320b, -221-3p, -146b-5p, -16, -24 were significantly upregulated (miR-16 showed maximum 4.3 fold-change) while miR-559 was downregulated (0.5 fold-change) in Group Ia versus controls ('p'<0.001). Bioinformatic analysis revealed that target genes of miRNAs in RSA are involved in apoptosis and AMPK signalling pathways. Results showed differential expression of miRNAs implyingmiR-16 and miR-559 as potential biomarkers of RSA in infected women. Furthermore, network of genes of differentially expressed miRNAs regulates RSA by targeting gene function in apoptosis, cell adhesion and angiogenesis.

Does tumour necrosis factor alpha-induced cyclooxygenase-2 expression lead to spontaneous abortion in Chlamydia trachomatis-infected women.

Cytokines might be involved in spontaneous abortion by triggering inflammatory mediators (Cyclooxygenases (Cox)) leading to spontaneous abortion in Chlamydia trachomatis (Ct)-infected women. Study aimed to quantitate the expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-8) and Cox-2 in endometrial curettage tissue (ECT) of Spontaneous Aborters (SA). SA (n = 135) and 120 age-matched controls were enrolled from SJH, New Delhi, India. PCR was performed for detection of Ct MOMP gene (537 bp) in ECT. mRNA expression of pro-inflammatory cytokines and Cox-2 was assessed by real-time qPCR. Data was statistically evaluated. 14.8% SA were diagnosed as Ct-positive. Elevated expression of TNF-α, IFN-γ, IL-8 and Cox-2 was observed in Ct-infected SA. Ct-positive recurrent aborters showed significantly higher cytokine expression. Significant positive correlation was found between expression of Cox-2 and TNF-α in infected SA. Data suggested an increased expression of Th-1 cytokines, particularly TNF-α that induced Cox-2 expression in ECT, leading to spontaneous abortion in Ct-infected pregnant women.

Cytokine-induced expression of nitric oxide synthases in Chlamydia trachomatis-infected spontaneous aborters.

Purpose: The aim of study was to evaluate expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in Chlamydia trachomatis (CT)-infected spontaneous aborters (SA). Materials and methods: Endometrial curettage tissue was collected from 140 SA (sporadic SA- 70; recurrent SA- 70) (Group I) and 140 age-matched controls (Group II) from Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi, India. Polymerase chain reaction was performed for diagnosis of CT. The expression of iNOS/ eNOS/ IFN-γ/ TNF-α was assessed by real-time polymerase chain reaction (PCR). Results: 15.7% SA were CT-positive (Group I); none in controls. Sporadic spontaneous aborters (SSA) (n = 8/70), recurrent spontaneous aborters (RSA) (n = 14/70) diagnosed as CT-positive (Group-I). Significant upregulation of iNOS/ eNOS was found in CT-positive SSA/RSA compared with CT-negative SSA/RSA and healthy controls. TNF-α and IFN-γ were expressed in CT-positive SSA/RSA compared with negative SSA/controls. iNOS showed a significant strong positive correlation with TNF-α and IFN-γ in CT-infected SA. eNOS showed a significant positive correlation with TNF-α and no correlation with IFN-γ in CT-infected SA. TNF-α was positively correlated with IFN-γ. Conclusions: Significantly high expression of iNOS/ eNOS and proinflammatory cytokines affected pregnancy in CT-infected RSA, thereby implying that there occurs cytokine-induced expression of nitric oxide synthase (NOS).

Proteomic Analysis of the Human Anterior Pituitary Gland.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.

Differential expression of circulating Th1/ Th2/ Th17 cytokines in serum of Chlamydia trachomatis-infected women undergoing incomplete spontaneous abortion.

The study aimed to elucidate role of Th1/Th2/Th17 cytokines in the immunopathogenesis of spontaneous abortion in Chlamydia trachomatis (Ct)-positive first-trimester aborters. Endometrial curettage tissue and serum were collected from 145 aborters (spontaneous abortion (SA) group, n = 85; recurrent miscarriage (RM) group, n = 60) and 120 controls attending Department of Obstetrics & Gynecology at Safdarjung hospital, New Delhi (India). Polymerase chain reaction was used to detect Ct plasmid/MOMP, while commercial cytometric bead array kit was utilized to estimate circulating serum cytokines. 13.7% aborters were Ct-positive, however, none was found to be infected among controls. IFN-γ, TNF-α, IL-2, IL-6 and IL-17A cytokines were significantly increased in SA group/RM group (Ct-infected) versus controls. IL-4 showed no difference between groups, while IL-10 was significantly elevated in controls versus Ct-infected subjects in SA group/RM group. Furthermore, IFN-γ, TNF-α, IL-6, IL-17A cytokines were significantly elevated in Ct-positive RM group versus Chlamydia-infected SA group. However, IL-2, IL-4 and IL-10 cytokines showed no significant difference between Ct-positive SA group versus infected RM group. Positive correlation was found between few cytokines (TNF-α and IFN-γ/IL-17A; IL-17A and IFN-γ/IL-6) in Ct-positive aborters. Our study clearly established the role of Th1/Th2/Th17 cytokines in the pathogenesis of spontaneous abortion in Ct-infected subjects and found that Chlamydia-positive recurrent aborters had a predominant Th1/Th17 bias.

Involvement of matrix metalloproteinases and their inhibitors in endometrial extracellular matrix turnover in Chlamydia trachomatis-infected recurrent spontaneous aborters.

Mechanism underlying Chlamydia trachomatis disease pathology is not completely understood. It was hypothesised that dysregulation of extracellular matrix by matrix metalloproteinases (MMPs) might be involved in sequelae of events leading to abortion in C. trachomatis-infected women. Hence, this study was undertaken to elucidate the expression of MMP-2/MMP-9/TIMP-1/TIMP-3 in infected recurrent spontaneous aborters (RSA) and simultaneously ascertain effect of few cytokines (TNF-α, TGF-ß1, TGF-ß2)/cyclooxygenase-2 (Cox-2) on MMPs. Endometrial curettage tissue was collected from 135 RSA (group I) and 120 age-matched controls (group II) at Department of Obstetrics and Gynecology, SJH, New Delhi (India). PCR was performed for diagnosis of C. trachomatis MOMP/plasmid gene. Expression of MMP-2/MMP-9/TIMP-1/TIMP-3 mRNA was assessed by quantitative real-time PCR. Data were statistically evaluated. 15.6% (n = 21) RSA (Group-I) were diagnosed as C. trachomatis positive. Significant upregulation of MMP-2/MMP-9 and decreased TIMP-1/TIMP-3 were found in C. trachomatis-positive RSA (group I) versus control women (group II). MMP-2 was found positively correlated with cytokines/Cox-2. Significant positive correlation was observed between MMP-2/MMP-9 and C. trachomatis copy load. Overall data suggested that dysregulated MMPs/TIMPs promoted excessive endometrial matrix degradation which, in turn, affected pregnancy leading to spontaneous abortion in infected women.