Identification of a Single Nucleotide Polymorphism of Vitamin D Receptor (VDR) and Vitamin D Binding Protein (VDBP) Gene and Its Dysregulated Pathway Through VDR-VDBP Interaction Network Analysis in Vitamin D-Deficient Infertile Females.

INTRODUCTION: The prevalence of female infertility in Pakistan is currently estimated at 22%, and emerging research suggests that vitamin D (VD) deficiency (VDD) may play a significant role in influencing female fertility. The focus of this study was to investigate the single nucleotide polymorphism (SNP) patterns within the VD binding protein (VDBP). The study aimed to explore dysregulated pathways and gene enrichment through an interaction network analysis, specifically focusing on the interplay between the VD receptor (VDR) and VDBP in females experiencing unexplained infertility (UI) coupled with VDD. METHODS: A cross-sectional study was conducted on VD-deficient, fertile, and UI female subjects. VDBP and VDR were assessed by enzyme-linked immunoassay and genotyping performed. FunRich (version 3.1.3; http://funrich.org/index.html) was employed for analysis of the identified proteins: VDR and VDBP and with their mapped gene datasets, gene enrichment, and protein-protein interaction (PPI) network. RESULTS: The mean VD and VDR values of infertile females were significantly lower than those of fertile females. VDBP in infertile females (median (IQR)): 296.05 (232.58-420.23)) was lower than that of fertile females (469.9 (269.57-875.55), (p=0.01)). On sequence analysis, a mutation rs 4588 SNP (Thr 436 Lys) was found in exon 11 of the VDBP gene of UI females, but no mutation in exons 8 and 9 of the VDR gene, with some insignificant intronic variants, was observed. The proteins such as plasma membrane estrogen receptor signaling pathway (p < 0.001), VDR, SMAD3, NCOR1, CREBBP, NCOA1, STAT1, GRB2, PPP2CA, TP53, and NCOA2 were enriched after biological pathway grouping when VDR was made the focused gene and directly interacting with VDBP. CONCLUSION: The females with UI exhibited significantly low VD, VDBP, and VDR. The plasma membrane estrogen receptor signaling pathway was enriched in VDD infertile females.

Endometriosis, infertility and MicroRNA's: A review.

The review aims to explore circulating small non- coding regulatory Ribonucleic Acids (miRNA) as biomarkers of endometriosis; a reproductive age group disorder. miRNA are linked with genetic, epigenetic and angiogenic factors, hormones, cytokines, chemokines, oxidative stress (OS) markers, mediators of inflammation, hypoxia, angiogenesis and altered immune system contributing to the pathogenesis of endometriosis. Hormonal imbalance occurs by decreased levels of miRNAs -23a and miRNAs -23b and increase in miRNAs -:135a, 135b, 29c and 194-3p. Angiogenesis by vascular endothelial growth factor is attributed to increased miRNAs -126, miRNAs -210, miRNAs -21, miRNAs -199a-5p and miRNAs 20A. OS upregulates miRNAs -302a by increased levels of Tumor Necrosis factor (TNF)-α, TNF- ß and Interleukin -1ß. Upregulation of miRNAs -199a and miRNAs -16 promotes inflammation and cell proliferation in the endometriotic lesions. The gold standard to diagnose endometriosis is laparoscopy, yet miRNA can be validated as diagnostic tool for detection, progression and prevention of endometriosis in large, independent cohorts of women, with and without endometriosis.

Kisspeptin and attributes of infertile males and females: A cross-sectional study in a subset of Pakistani population.

Kisspeptin, a peptide hormone, plays a pivotal role in fertility and neuroendocrine regulation of hypothalamo-pituitary-gonadal axis. Increased kisspeptin and reproductive hormones are responsible for fertility in male and females. This study aimed to explore the role of kisspeptin on hypothalamo-pituitary-gonadal axis by comparing the levels of kisspeptin in fertile and infertile subjects and identifying single nucleotide polymorphisms (SNPs) of KISS1 gene in exon 2 and exon 3 of infertile male and female cohorts. A cross-sectional study was carried out on 80 males (44 infertile and 36 fertile) and 88 females (44 in each group). Significantly high levels of kisspeptin (KP), follicle-stimulating hormone (FSH), luteinizing hormone and testosterone were observed in fertile male and female subjects except low FSH levels in comparison with infertile female subjects. One polymorphism in exon 2 (E1225K [G/A 3673]) and three in exon 3 (P1945A [C/G 5833]; Insertion of T at 6075; G2026G [C/G 6078]) in infertile group were detected, with low KP and hormonal levels. Male subjects had abnormal sperm parameters and unsuccessful attempt of intracytoplasmic sperm injection in females. Expression of SNP in exon 2 and exon 3 of KISS1 could be responsible for alteration in release of reproductive hormones and gonadal functions, hence causing infertility.

Cytokines and endometrial receptivity after intracytoplasmic sperm injection - A cohort study at Islamabad.

OBJECTIVE: To evaluate the role of tumour necrosis factor alpha and interleukin-6 in conception after assisted reproductive treatment. METHODS: The longitudinal cohort study was conducted from August 2014 to May 2015 and comprised patients undergoing intracytoplasmic sperm injection at the Islamabad Clinic Serving Infertile Couples, Islamabad, Pakistan. During the procedure, endometrial thickness was measured on the day of ovulation induction and serum tumour necrosis factor alpha along with interleukin-6 were measured on the embryo transfer day. Subjects were divided into groups, with non-pregnant females in Group A and those with confirmed foetal cardiac activity in Group B and non-pregnant females were divided into two groups. The difference between the groups and the association of cytokines with endometrial thickness were measured. . RESULTS: Of the 131 subjects, 79(60%) were in Group A and 52(40%) in Group-B. The mean level of interleukin-6 was 49.65±3.04 in Group B and 104.14±76.03 in Group A, with significant inverse correlation with endometrial thickness (p<0.001). The level of tumour necrosis factor alpha were significantly higher in Group-B compared to Group-A (p<0.001). CONCLUSIONS: The pro-inflammatory cytokine, tumour necrosis factor alpha, takes part in preparation of endometrial lining for implantation of embryo in assisted reproductive treatment procedures.

Serum tumour necrosis factor alpha in osteopenic and osteoporotic postmenopausal females: A cross-sectional study in Pakistan.

OBJECTIVE: To compare biochemical parameters serum tumour necrosis factor alpha, calcium, magnesium, bone-specific alkaline phosphatase and vitamin D in postmenopausal women. METHODS: This cross-sectional study was carried out from June 2015 to July 2016 at Jinnah Medical and Dental College, Karachi, and comprised postmenopausal women. Bone mineral density done by dual energy X-ray absorptiometryscan categorised subjects by World Health Organisation classification into normal (T score > -1) osteopenic (T score between -1 and -2.5) and osteoporotic (T score < -2.5). Biochemical parameters like tumour necrosis alpha, calcium, magnesium, bone-specific alkaline phosphatase and vitamin D were measured by solid phase enzyme amplified sensitivity immunoassay method. SPSS 16 was used to analyse the data. RESULTS: Of the 146 women, 34(23%) were normal, 93(67%) were osteopenic and 19(13%) were osteoporotic. There was significant difference in mean body mass index, serum tumour necrosis factor alpha and calcium in all the three groups (p<0.01). Significant mean difference was observed in serum calcium levels between normal and osteopenic, and between normal and osteoporotic group (p<0.05 each) without any significant mean difference between osteopenic and osteoporotic groups (p>0.05). A significant difference was observed for mean tumour necrosis factor alpha values between normal and osteoporotic groups (p<0.05). Tumour necrosis factor alpha showed negative correlation with bone mineral density in osteopenic and osteoporotic groups (p>0.05). CONCLUSIONS: Increased bone turnover in postmenopausal osteopenic women can be predicted by increased serum cytokine.

BCG vaccination is associated with decreased severity of tuberculosis in Pakistan.

Vaccination with Bacille Calmette-Guérin (BCG) is given at birth to protect against tuberculosis (TB) in Pakistan. The country ranks 6th amongst high-burden countries worldwide and has an incidence of 231/100,000 pyopulation. This was a cross-sectional multi-center hospital-based study. TB patients (n=218) with pulmonary (PTB, n=120) or extrapulmonary (ETB, 98) were recruited, and the presence of a BCG vaccination scar was documented. Cases were further classified into minimal, moderate and advanced PTB or less severe (L-ETB) or severe disseminated (D-ETB) disease. The association of age, gender and severity of TB infections with BCG vaccination of the individual TB cases was investigated. No difference was found of the BCG vaccination status of PTB and ETB cases, or in relation to age or gender. Patients under 29years of age comprised the largest group. There were more females with ETB than PTB. The largest group within ETB comprised those with tuberculous lymphadenitis (LNTB, 39%). A significantly greater number of LNTB cases had received BCG vaccinations than had those with pleural (unilateral) TB (p=0.004), and tuberculous meningitis (p=0.027) groups. Also, there were more immunized patients with pulmonary as compared with pleural disease (p=0.001). LNTB represents localized granulomatous disease and the observation of higher vaccination rates in this group suggests that BCG has protected against more severe forms of TB in this high-burden region.

CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

BACKGROUND: Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNgamma, TNFalpha and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. METHODOLOGY: We investigated live M. tuberculosis- and M. bovis BCG-induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNgamma, IL10 and CCL3, and mRNA expression of CCL2, TNFalpha, CCL3 and CXCL8 were determined. RESULTS: M. tuberculosis- and BCG-induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis-induced IFNgamma was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNgamma was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFalpha mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. CONCLUSIONS: The increased CCL2 and TNFalpha in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression.

BACKGROUND: Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG. RESULTS: M. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1-4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA. CONCLUSION: This study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.