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1.  Association of progesterone receptor polymorphisms with recurrent implantation failure after in vitro fertilization and embryo transfer 
Introduction
Progesterone is the hormone of pregnancy and is required for its initiation. The actions of progesterone are mediated by the progesterone receptor. Polymorphic variants of human progesterone receptor genes have been implicated in implantation failure.
Materials and methods
We, therefore, investigated the prevalence of H770H(C/T genotype), V660L polymorphism and a 306 bp Alu insertion in exon 7 of the progesterone receptor among women with history of recurrent implantation failure to determine whether any of these polymorphisms may serve as a risk factor for implantation failure. DNA was extracted from the buccal swabs obtained from 66 women experiencing implantation failure and 75 fertile control women. PCR amplification of fragments was purified and the DNA sequenced to identify the polymorphism. The frequencies for the three variants were 27% for H770H, 21% for V660L and 0% for the 306 bp Alu insertion in exon 7 among women with implantation failure compared with control women of 25% for H770H and 24%for V660L and 0% for the 306 bp Alu insertion in exon 7.
Discussion
No significant differences in the overall allelic frequency of progesterone receptor variants was seen when women experiencing recurrent implantation failure were compared with control women.
Conclusion
We conclude that the H770H and V660L and PROGINS progesterone receptor polymorphisms are not markers that can identify women at risk for recurrent implantation after IVF/ET.
doi:10.1007/s10815-008-9210-9
PMCID: PMC2582074  PMID: 18392676
Progesterone; Receptor polymorphisms; Recurrent implantation failure; RIF
2.  Association of progesterone receptor polymorphism with idiopathic recurrent pregnancy loss in Taiwanese Han population 
Purpose
Recurrent pregnancy loss (RPL) could be caused by insufficient progesterone in the luteal phase of menstruation and early pregnancy. Progesterone plays a critical role in oocyte maturation, embryo implantation and placenta maintenance in early gestation. This study was set out to investigate the association between polymorphisms of the progesterone receptor (PGR) gene and idiopathic RPL.
Methods
One hundred twenty-one women with a history of idiopathic recurrent pregnancy loss (RPL) and 179 control subjects were enrolled into the study. Six tag SNPs and two functional SNPs [PROGINS (rs1042838), +331 C/T (rs10895068)] of the progesterone receptor gene were genotyped.
Results
We found that the allele and genotype frequencies of the functional SNP [PROGINS (rs1042838)] were both significantly higher in patients with idiopathic RPL than in the control subjects (both P values = 0.006). In addition, the C-C haplotype, which consists of rs590688C > G and rs11224592T > C, is associated with a decreased risk of RPL (p = 0.004).
Conclusion
PROGINS polymorphism confers susceptibility to idiopathic recurrent pregnancy loss in Taiwanese Han women.
doi:10.1007/s10815-010-9510-8
PMCID: PMC3082658  PMID: 21086036
Progesterone receptor; PROGINS; Recurrent pregnancy loss; Tag SNP; Polymorphism
3.  Associations between androgen receptor CAG & GGN repeat polymorphism & recurrent spontaneous abortions in Chinese women 
Background & objectives:
Recurrent spontaneous abortion (RSA) is a reproductive problem that occurs in women in reproductive age with a frequency of 1-3 per cent. Previous studies have reported high levels of serum androgens to be associated with RSAs. At the molecular level, the effect of androgens is mediated through the activation of the androgen receptor (AR). The CAG and GGN repeat polymorphisms of the AR gene are associated with the AR activity. We hypothesize that the AR CAG/GGN repeat polymorphism may be associated with levels of serum androgens. Thus, this study as undertaken to evaluate the relationship between CAG/GGN repeats in exon 1 of the AR gene in women with RSAs.
Methods:
This case-control study was performed in Ningxia, PR China, including 149 women with RSAs and 210 controls. The CAG and GGN repeats of the AR gene were genotyped using a PCR-based assay and were analyzed using Peak Scanner Software v1.0 to determine the CAG/GGN repeat length.
Results:
CAG repeats ranged from 15 to 29 in the RSA patients, compared to 14 to 35 in the control group. The median value of CAG repeats was 22 for the RSA group and 24 for control group. The total AR CAG alleles (≤22 repeats), shorter AR CAG alleles (≤22 repeats), and biallelic means (≤22.5 repeats) were significantly different in the RSA group in comparison to the control group (P<0.001, P<0.01). The median value of the GGN repeats was 23 for the cases and 22 for controls. The total number of AR GGN alleles (≤23 repeats) was significantly different in the RSA group compared to the control group (P<0.5). There was no difference between the RSA group and the control groups in regards to shorter alleles, longer alleles, and biallelic means.
Interpretation & conclusions:
Our observation suggests that the CAG and GGN repeat length is shorter in women with RSAs as compared with controls and that shorter CAG and GGN repeats may be pathogenic for RSAs in Chinese women. Further studies need to be done in different ethnic populations.
PMCID: PMC4140038  PMID: 25027083
Androgen; androgen receptor gene; CAG repeats; GGN repeats; recurrent spontaneous abortion
4.  Progesterone receptor variation and risk of ovarian cancer is limited to the invasive endometrioid subtype: results from the ovarian cancer association consortium pooled analysis 
British Journal of Cancer  2008;98(2):282-288.
There is evidence that progesterone plays a role in the aetiology of invasive epithelial ovarian cancer. Therefore, genes involved in pathways that regulate progesterone may be candidates for susceptibility to this disease. Previous studies have suggested that genetic variants in the progesterone receptor gene (PGR) may be associated with ovarian cancer risk, although results have been inconsistent. We have established an international consortium to pool resources and data from many ovarian cancer case–control studies in an effort to identify variants that influence risk. In this study, three PGR single nucleotide polymorphisms (SNPs), for which previous data have suggested they affect ovarian cancer risk, were examined. These were +331 C/T (rs10895068), PROGINS (rs1042838), and a 3′ variant (rs608995). A total of 4788 ovarian cancer cases and 7614 controls from 12 case–control studies were included in this analysis. Unconditional logistic regression was used to model the association between each SNP and ovarian cancer risk and two-sided P-values are reported. Overall, risk of ovarian cancer was not associated with any of the three variants studied. However, in histopathological subtype analyses, we found a statistically significant association between risk of endometrioid ovarian cancer and the PROGINS allele (n=651, OR=1.17, 95% CI=1.01–1.36, P=0.036). We also observed borderline evidence of an association between risk of endometrioid ovarian cancer and the +331C/T variant (n=725 cases; OR=0.80, 95% CI 0.62–1.04, P=0.100). These data suggest that while these three variants in the PGR are not associated with ovarian cancer overall, the PROGINS variant may play a modest role in risk of endometrioid ovarian cancer.
doi:10.1038/sj.bjc.6604170
PMCID: PMC2361465  PMID: 18219286
ovarian cancer; progesterone receptor; SNPs; PROGINS; pooled analyses; endometrioid ovarian cancer
5.  The progesterone receptor Val660→Leu polymorphism and breast cancer risk 
Breast Cancer Research  2004;6(6):R636-R639.
Background
Recent evidence suggests a role for progesterone in breast cancer development and tumorigenesis. Progesterone exerts its effect on target cells by interacting with its receptor; thus, genetic variations, which might cause alterations in the biological function in the progesterone receptor (PGR), can potentially contribute to an individual's susceptibility to breast cancer. It has been reported that the PROGINS allele, which is in complete linkage disequilibrium with a missense substitution in exon 4 (G/T, valine→leucine, at codon 660), is associated with a decreased risk for breast cancer.
Methods
Using a nested case-control study design within the Nurses' Health Study cohort, we genotyped 1252 cases and 1660 matched controls with the use of the Taqman assay.
Results
We did not observe any association of breast cancer risk with carrying the G/T (Val660→Leu) polymorphism (odds ratio 1.10, 95% confidence interval 0.93–1.30). In addition, we did not observe an interaction between this allele and menopausal status and family history of breast cancer as reported previously.
Conclusion
Overall, our study does not support an association between the Val660→Leu PROGINS polymorphism and breast cancer risk.
doi:10.1186/bcr928
PMCID: PMC1064075  PMID: 15535845
breast cancer; linkage disequilibrium; polymorphism; progesterone receptor
6.  Role of 14-bp deletion/insertion polymorphism in exon 8 of the HLA-G gene in recurrent spontaneous abortion patients 
BACKGROUND:
Human leukocyte antigen (HLA)-G belongs to the nonclassical Class I major histocompatibility complex, and is predominantly and specifically found on the extravillous cytotrophoblast cells of the placenta. HLA-G has been postulated as an important immunotolerant molecule in maintaining successful pregnancy and maternal tolerance of the semiallogenic fetus. Recent reports indicate that the 14-bp deletion/insertion polymorphism in exon 8 of the 3’UTR region of the HLA-G gene influences the HLA-G mRNA stability and isoform splicing patterns, thus modulating the levels of HLA-G expression.
AIM:
The aim was to study the 14-bp deletion/insertion polymorphism in exon 8 of the 3’UTR region of the HLA-G gene.
MATERIALS AND METHODS:
A total of 50 women with unexplained three or more recurrent spontaneous abortions (RSAs) and 41 normal healthy control women who have had normal pregnancies and were genotyped for the 14-bp deletion/insertion polymorphism were genotyped for the 14-bp deletion/insertion polymorphism by polymerase chain reaction for exon 8-specific primers
RESULTS:
It was found that the 14-bp allele deletion frequency was lower in patients (67%) versus controls (73%), while 14-bp allele insertion was higher among patients (33%) versus controls (9%). Similarly, the homozygous deletion halotype was higher among the controls (80.48%); the heterozygous insertion deletion haplotype (34%) and homozygous insertion haplotype (16%) were higher in RSA patients. The HLA haplotype HLA A*02:11_B*40:06:01:01 was increased among RSA women compared to controls.
CONCLUSION:
Our results suggest that 14-bp deletion/insertion polymorphisms might have importance in the outcome of pregnancy and the 14-bp deletion polymorphism in exon 8 of the HLA-G gene may be important from an evolutionary perspective of successful pregnancy.
doi:10.4103/0974-1208.92289
PMCID: PMC3276949  PMID: 22346082
HLA-G 14-bp deletion/insertion gene; India; RSA
7.  Genetic variation in the progesterone receptor gene and risk of endometrial cancer: a haplotype-based approach 
Carcinogenesis  2010;31(8):1392-1399.
Background: It is well established that estrogen increases endometrial cancer risk, whereas progesterone opposes the estrogen effects. The PROGINS allele of the progesterone receptor (PGR) gene reduces the function of PGR and has been associated with increased risk of the endometrioid type ovarian cancer. We investigated whether genetic variation in PGR is also associated with endometrial cancer risk using a haplotype-based approach. Methods: We pooled data from two endometrial cancer case–control studies that were nested within two prospective cohorts, the Multiethnic Cohort Study and the California Teachers Study. Seventeen haplotype-tagging single nucleotide polymorphisms (SNPs) across four linkage disequilibrium (LD) blocks spanning the PGR locus were genotyped in 583 incident cases and 1936 control women. Odds ratios (ORs) and 95% confidence intervals (CIs) associated with each haplotype were estimated using conditional logistic regression, stratified by age and ethnicity. Results: Genetic variation in LD block 3 of the PGR locus was associated with endometrial cancer risk (Pglobal test = 0.002), with haplotypes 3C, 3D and 3F associated with 31–34% increased risk. Among whites (383 cases/840 controls), genetic variation in all four blocks was associated with increased endometrial cancer risk (Pglobal test = 0.010, 0.013, 0.005 and 0.020). Haplotypes containing the PROGINS allele and several haplotypes in blocks 1, 3 and 4 were associated with 34–77% increased risk among whites. SNP analyses for whites suggested that rs608995, partially linked to the PROGINS allele (r2 = 0.6), was associated with increased risk (OR = 1.30, 95% CI = 1.06–1.59). Conclusions: Our results suggest that genetic variation in the PGR region is associated with endometrial cancer risk.
doi:10.1093/carcin/bgq113
PMCID: PMC2915632  PMID: 20547493
8.  Idiopathic Recurrent Pregnancy Loss: Role of Paternal Factors; A Pilot Study 
Background
This case-control study was designed with the aim of evaluating the role of sperm, oxidative stress and DNA damage in idiopathic recurrent pregnancy loss (iRPL). This pilot study is the first study done on the Indian population which reports the association between DFI, TAC and ROS in couples experiencing iRSA.
Methods
Twenty infertile men with a history of iRPL and 20 fertile controls (having fathered a child a year earlier) were included in the study which was performed in Laboratory for Molecular Reproduction and Genetics, India, from March 2010 to July 2011. The female partners of the participants were normal on gynaecological examination and had normal endocrine and blood profiles. Conventional semen analysis was performed (concentration, motility, morphology; WHO criteria, 2010) within 1 hour of sample collection. Levels of reactive oxygen species (ROS) were assessed by luminol-dependant chemiluminescence. The total antioxidant capacity (TAC) was quantified by ELISA. The Sperm chromatin structure assay (SCSA) was performed by flow cytometry to determine DNA fragmentation Index (DFI). Statistical analysis was performed using SPSS version 15 and parameters were compared by Mann-Whitney test. Pearson correlation test was used to find the correlation between parameters and a p-value <0.05 was considered significant. Receiver operating characteristics (ROC) curve analysis was applied to find out the cut-off value of DNA fragmentation index.
Results
No significant differences in age, seminal volume, liquefaction time, pH and sperm concentration were observed between the male partner of iRPL cases and the controls, but sperm morphology and motility were significantly (p <0.05) lower in the male partner of cases with idiopathic recurrent spontaneous abortion (RSA). The mean ROS levels observed were 47427.00 relative light unit (RLU)/min/20 million sperm in the male partners as compared to 13644.57 RLU/ min/20 million sperm in the controls (normal <15000 RLU/min/20 million). The mean TAC levels in the controls (6.95 mM trolox) were significantly (p <0.05) higher as compared to the male partners of women with IRPL (2.98 mM trolox). The average mean DFI of male partners were found to be 23.37±9.9 and the mean DFI of controls was 13.89±5.40. The mean DFI was significantly (p <0.05) higher when compared to the controls. The range of DFI in male partners was 8.50–44.07. However, in the controls the range was 7.70–23.50.
Conclusion
Sperm DNA integrity is critical for normal embryonic development and birth of healthy offspring. Oxidative stress due to the imbalance between raised free radical levels and low total antioxidant capacity is one of the critical causes of DNA damage. Thus assay of oxidative stress and sperm genomic integrity is essential in couples with iRSA following natural and spontaneous conception.
PMCID: PMC3719307  PMID: 23926513
Oxidative stress; Reactive oxygen species; Recurrent spontaneous abortion; Sperm DNA damage; Sperm chromatin structure assay
9.  Progesterone Receptor Gene Polymorphisms and Risk of Endometriosis: Results from an International Collaborative Effort 
Fertility and sterility  2010;95(1):40-45.
Objective
To investigate the association between self-reported endometriosis and the putative functional promoter +331C/T single nucleotide polymorphism (SNP) and the PROGINS allele.
Design
Control subjects from ovarian cancer case-control studies participating in the international Ovarian Cancer Association Consortium. The majority of controls are drawn from population-based studies.
Setting
An international ovarian cancer consortium including studies from the Australia, Europe and the United States,
Patients
5,812 White female controls, of whom 348 had endometriosis, from eight ovarian cancer case-control studies.
Interventions
None.
Main Outcome Measures
Genotypes for the +331C/T SNP and PROGINS allele and a history of endometriosis.
Results
The occurrence of endometriosis was reduced in women carrying one or more copies of the +331 T allele (OR=0.65; 95% CI: 0.43–0.98, p=0.042), whereas there was no association between the PROGINS allele and endometriosis (OR=0.94, 95% CI 0.76, 1.16).
Conclusions
Additional studies of the +331C/T variant are warranted given the current finding and the equivocal results of previous studies. The +331 T allele has been shown to result in a reduced PR-A to PR-B ratio and if the observed association with endometriosis is confirmed it would suggest that this ratio is important for this disease.
doi:10.1016/j.fertnstert.2010.06.059
PMCID: PMC3176720  PMID: 20719308
Endometriosis; progesterone receptor; ovarian cancer; PROGINS
10.  Association of progesterone receptor gene polymorphism with male infertility and clinical outcome of ICSI 
Purpose
To investigate the association of Progesterone Receptor (PR) gene variations and male infertility
Methods
DNA extraction, PCR and sequencing of PR gene, PROGINS insertion by PCR. Association of the variations with seminal parameters and outcomes of ICSI.
Results
Four known SNPs in the PR gene were identified in the study of which three (rs3740753, rs1042838, rs104283) were co-inherited and in complete linkage disequilibrium with the PROGINS Alu insertion. There were no differences in their frequencies between fertile and infertile males. The rs2020880 was found at a very low frequency only in the controls but not in the infertile subjects. The sperm counts, fertilization rate, embryo quality or pregnancy rates were not different in individuals with or without PROGINS allele.
Conclusion
PR gene alterations are not associated with male infertility or ICSI outcome.
Electronic supplementary material
The online version of this article (doi:10.1007/s10815-013-0074-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s10815-013-0074-2
PMCID: PMC3800537  PMID: 23934021
Progesterone receptor; Polymorphism; PROGINS; Male infertility; ICSI; Azoospermia; Sperm counts
11.  The progins progesterone receptor gene polymorphism is not related to endometriosis‐associated infertility or to idiopathic infertility 
Clinics  2010;65(11):1073-1076.
OBJECTIVE:
This study aimed to determine the frequency of the PROGINS polymorphism in women with endometriosis‐associated infertility, in infertile women without endometriosis and in controls.
INTRODUCTION:
The human progesterone receptor gene has two isoforms that modulate the biological action of progesterone: isoform A, which is capable of inhibiting the activation of the estrogen receptors, and isoform B, which has the capacity to activate the estrogen receptors. Several polymorphisms have been described for this gene, among which one stands out: a polymorphism named PROGINS, which has been speculated to be related to the genesis of endometriosis by several studies with conflicting results.
METHODS:
This was a prospective study that included 148 patients with endometriosis‐associated infertility, 50 idiopathic infertile patients and 179 fertile women as controls. The PROGINS polymorphism was studied by PCR.
RESULTS:
Genotypes P1P1, P1P2 and P2P2 (P2 representing the PROGINS polymorphism) of the progesterone receptor gene presented frequencies of 93.9%, 5.4% and 0.7%, respectively, in the women with endometriosis‐associated infertility (p = 0.2101, OR = 0.51, 95% CI = 0.24‐1.09); 94.4%, 4.2% and 1.4%, respectively, in the patients with minimal/mild endometriosis (p = 0.2725, OR = 0.53, 95% CI = 0.20‐1.43); 93.5%, 6.5% and 0%, respectively, among the patients with moderate/severe endometriosis (p = 0.3679, OR = 0.49, 95% CI = 0.18‐1.31); 86.0%, 14.0% and 0%, respectively, in idiopathic infertile women (p = 0.8146, OR = 1.10, 95% CI = 0.46‐2.63); and 88.3%, 10.6% and 1.1%, respectively, in the control group.
CONCLUSION:
The data suggest that PROGINS is not related either to endometriosis‐associated infertility or to idiopathic infertility in the population studied.
doi:10.1590/S1807-59322010001100002
PMCID: PMC2999697  PMID: 21243274
endometriosis; polymorphism; PROGINS; progesterone receptor gene; infertility
12.  Role of Androgen Receptor CAG Repeat Polymorphism and X-Inactivation in the Manifestation of Recurrent Spontaneous Abortions in Indian Women 
PLoS ONE  2011;6(3):e17718.
The aim of the present study was to investigate the role of CAG repeat polymorphism and X-chromosome Inactivation (XCI) pattern in Recurrent Spontaneous Abortions among Indian women which has not been hitherto explored. 117 RSA cases and 224 Controls were included in the study. Cases were recruited from two different hospitals - Lakshmi Fertility Clinic, Nellore and Fernandez Maternity Hospital, Hyderabad. Controls were roughly matched for age, ethnicity and socioeconomic status. The CAG repeats of the Androgen Receptor gene were genotyped using a PCR-based assay and were analysed using the GeneMapper software to determine the CAG repeat length. XCI analysis was also carried out to assess the inactivation percentages. RSA cases had a significantly greater frequency of allele sizes in the polymorphic range above 19 repeats (p = 0.006), which is the median value of the controls, and in the biallelic mean range above 21 repeats (p = 0.002). We found no evidence of abnormal incidence of skewed X-inactivation. We conclude that longer CAG repeat lengths are associated with increased odds for RSA with statistical power estimated to be ∼90%.
doi:10.1371/journal.pone.0017718
PMCID: PMC3056719  PMID: 21423805
13.  Tumor Necrosis Factor-α Gene Polymorphisms in Korean Patients With Recurrent Spontaneous Abortion 
Reproductive Sciences  2013;20(4):408-413.
The objective of this study was to investigate the contribution of the tumor necrosis factor-α (TNF-α) gene polymorphisms to recurrent spontaneous abortion (RSA). The study participants consisted of 357 Korean women with RSA and 236 fertile women controls. Four TNF-α gene variants of all participants were analyzed by polymerase chain reaction–restriction fragment length polymorphism assay. The TNF-α -1031T>C and TNF-α -238G>A variants increased the risk of RSA TNF-α -1031TC+CC; adjusted odds ratio [AOR], 2.292; 95% confidence interval [CI], 1.547-3.395; P < .001; TNF-α -238GA+AA; AOR, 2.327; 95% CI, 1.038-5.217; P = .040), and these data were not different in a stratified analysis according to the number of consecutive spontaneous abortions. Also, the mutant genotypes of TNF-α -1031 and TNF-α -238 showed synergistic effects on increased RSA risk (-1031TC+CC/-238GA+AA; AOR, 4.054; 95% CI, 1.520-10.812; P = .005). In haplotype analysis, there were similar trends of data for combination analysis. In conclusion, the TNF-α -1031T>C and TNF-α -238G>A variants are possible genetic risk factors for RSA.
doi:10.1177/1933719112459237
PMCID: PMC4077515  PMID: 23202728
tumor necrosis factor-α; recurrent spontaneous abortion; polymorphism; risk factor
14.  An immunohistochemical study of CD83- and CD1a-positive dendritic cells in the decidua of women with recurrent spontaneous abortion 
Background
There are more and more women with recurrent spontaneous abortion (RSA). The mechanism of RSA is still unclear. Immunological factors have been postulated to play a role in the etiology of RSA. Dendritic cells (DCs) are the most potent antigen-presenting cells in the immune system, and the decidual DCs may take part in the occurrence of RSA. The difference in maturity status of decidual DCs among women with RSA and women with normal pregnancies is worthy of studying for its application to prevention and therapy.
Methods
The EnVision two-step immunohistochemical staining technique was used to detect the expression of CD83 and CD1a in the decidua of women with RSA (30 cases) and normal pregnancies (30 cases). The maturity status, distribution and quantity of DCs in the two groups were observed. Observation of the staining and cell counting were done using microscope within 30 randomly selected high-power fields (HPF, 40 × 10). All data analyses were conducted with SPSS 17.0 and the statistical significance was set at P <0.05.
Results
The decidua from the two groups contained DCs that stained with the anti-CD83 and anti-CD1a antibody. Most of the decidual CD83+DCs from two groups were located in the stroma. There were more CD83+DCs clustered with other DCs in the stroma from women with RSA than normal pregnancies. Most of the CD1a+DCs in the decidua from the two groups are located close to maternal glandular epithelium. No difference in the location of CD1a+DCs was found in the decidua between two groups. The number of decidual CD83+DCs was statistically significantly higher in RSA women than in normal early pregnant women (14.20 ± 13.34/30 HPF versus 4.77 ± 2.64/30 HPF; t = 3.800, P = 0.001). The number of CD1a+DCs in the decidua was statistically significantly lower in RSA women compared with normal early pregnant women (3.97 ± 3.75/30 HPF versus 7.60 ± 6.08/30 HPF; t = 2.786, P = 0.008).
Conclusions
These findings suggest that the increase in the number of mature DCs and the decrease in the quantity of immature DCs in the decidua may be related to RSA. The maturation of decidual DCs may play an important role in the pathogenesis of RSA.
doi:10.1186/s40001-014-0076-2
PMCID: PMC4301856  PMID: 25563385
recurrent spontaneous abortion; decidua; dendritic cells; CD83; CD1a; immunohistochemistry
15.  HLA allele associations in idiopathic recurrent spontaneous abortion patients from India 
BACKGROUND:
Rejection of semiallogenic foetus in recurrent spontaneous abortion (RSA) has been postulated to be a consequence of genetic and immunological phenomena.
AIM:
To evaluate the role of human leukocyte antigen (HLA) alleles in RSA in Indian couples.
SETTINGS AND DESIGN:
A case-control study.
MATERIALS AND METHODS:
Eighty-one randomly selected couples with unexplained three or more RSAs and a control group of 97 couples with live birth belonging to the same ethnic background, referred to the Gynaecology Department, KEM Hospital were included in the case-control study. Serological HLA A and B typing was done followed by molecular subtypes, defined using PCR-SSOP technique for HLA A, B, and C in 40 couples and DRB1* and DQB1* in 28 couples which were then compared with appropriate case 46 and 88 controls.
RESULTS:
Serologically A3 (15.43% vs. 4.43%; odds ratio (OR) = 4.34; P = 0.0002) and B17 (25.3% vs. 11.34%; OR = 3.49; P = 0.0001) were increased. Haplotype A1-B17 was significantly increased. Molecular subtyping revealed that A*030102 (11.25% vs. 4.34%; OR = 3.00; P = 0.07), B*5701 (11.25% vs. 1.08%; OR = 13.10; P = 0.003), Cw*120201 (25% vs. 4.34%; OR = 10.50; P = 2.05E-05), HLA DRB1*030101 (17.85% vs. 3.40%; OR = 7.6; P = 0.0001), DRB1*150101 (32.14% vs. 13.63%; OR = 4.8; P = 0.0003), and DQB1*060101 (35.71% vs. 29.34%; OR = 2.3; P = 0.004) were significantly increased in patients. A differential association was noticed when compared with reported world RSA patients.
CONCLUSION:
The HLA alleles A*030101, B*5701, Cw*120201, DRB1*030101, and DRB1*150101 as well as their associated ancestral haplotype may play a significant role in development of RSA in India.
PMCID: PMC2700679  PMID: 19562059
Cw*120201; DQB1*050301 association; HLA B*5701; India; RSA
16.  Association between male infertility and either the +331G/A or the progins polymorphism of the progesterone receptor gene in a Chinese population 
Background:
Progesterone has been suggested to contribute to the regulation of spermatogenesis and to facilitate the production of viable sperm. Investigations have showed that polymorphism of progesterone receptor (PGR) is associated with some diseases.
Objective:
To analyze the potential relationship between male infertility and the +331G/A and progins polymorphisms of PGR gene.
Materials and Methods:
The cross-sectional study was carried out at the Department of Male Reproduction, Reproductive Medical Center, the Second Hospital of Jilin University. The restriction fragment length polymorphism (RFLP) technique was used to detect gene point mutations. Of the 145 semen samples analyzed, 35 were asthenozoospermic, 50 were oligoasthenozoospermic, 21 were azoospermic, 11 were teratozoospermic and 28 were from fertile male subjects.
Results:
Statistical analyses revealed that the genotypes of the +331G/A polymorphisms were in Hardy-Weinberg equilibrium in both the fertile (2=0, p=0.534) and oligospermic groups (2=0.021, p=0.537). Similarly, the genotypes of the progins polymorphisms were also in Hardy–Weinberg equilibrium in both the fertile (2=0, p=1) and oligospermic groups (2=0.005, p=1).
Conclusion:
Our results indicated that polymorphisms of the +331G/A and progins of the PGR gene are unrelated to male infertility, at least in a Chinese population.
PMCID: PMC4306983  PMID: 25653674
Progesterone receptor; +331G/A polymorphism; Progins polymorphism; Male infertility
17.  Autoantibody profile and other immunological parameters in recurrent spontaneous abortion patients 
Background:
An autoimmune cause and related immunological alterations resulting in recurrent spontaneous abortion (RSA) have been suggested in patients with unknown etiology.
Materials and Methods:
This study evaluated the autoantibody profile and other immunological parameters among RSA patients and normal pregnant women from Mumbai western India. Fifty RSA patients with unknown cause and greater than three consecutive abortions along with 50 normal pregnant women were studied for various auto antibodies such as ANA, anti-dsDNA, ANCA, AECA, 2 micro globulin, anti-HLA antibodies and ACLA using immunofluorescence microlymphocytotoxicity and ELISA. Immunological parameters such as HLA class I monoclonal antibody expression, CD3 (T cell), CD19 (B cell), and CD56 (NK cell) were estimated by flow cytometry.
Results:
The results revealed 34% positivity of all auto antibodies tested among patients. ANA(12%), ANCA (20%), AECA (24%), ACLA (8%), anti-dsDNA(0%), β2 microglobulin (14%), and anti-HLA antibodies(10%) among RSA patients were identified. An increased expression of HLA class I specific monoclonal antibody (10%) with HLA A3 (16%) specificity were found to correlate with shared HLA alleles among the RSA couples. Among normal pregnant (control) group ANA (2%), ANCA (2%), AECA (3%), ACLA (4%) and increased expression of CD56 with reduced HLA class I monoclonal were observed.
Conclusion:
Our findings suggest a possible role of various autoantibodies along with the related immunological parameters underlying RSA.
doi:10.4103/0300-1652.86126
PMCID: PMC3213746  PMID: 22082909
Autoantibody; Human leukocyte antigen; India; Recurrent spontanous abortion
18.  RsaI but not DraI polymorphism in CYP2E1 gene increases the risk of gastrointestinal cancer in Malaysians: a case–control study 
BMJ Open  2014;4(1):e004109.
Objectives
Our study aimed to investigate the association of CYP2E1 C-1019T RsaI and T7678A DraI polymorphisms and factors such as age, gender and ethnicity to the risk of gastrointestinal cancer (GIC) in Malaysians.
Design
Case–control study.
Setting
Malaysia.
Participants
520 consented healthy blood donors with no previous GIC record and 175 patients with GIC.
Measurements
C-1019T RsaI and T7678A DraI genotyping of CYP2E1 gene; direct sequencing.
Results
This study reveals that the variant c2 allele and carrier with at least one c2 allele of C-1019T single nucleotide polymorphism (SNP) significantly increased the risk of GIC but no significant association was found between T7678A SNP and combined analysis of C-1019T and T7678A SNPs to risk of GIC. The Malaysian Chinese had greater risk of GIC compared with the Malays, Indians and KadazanDusun. An increased risk of GIC was observed in individuals aged >40 years and women had a 2.22-fold and 1.58-fold increased risk of stomach and colorectal cancers, respectively, when compared with men.
Limitations
The future research should be conducted with a larger sample population and including the gene–gene and gene–environmental interactions.
Conclusions
Our study suggests that the rare c2 allele and carrier with at least one c2 allele of CYP2E1 RsaI polymorphism significantly elevated the risk of GIC and may be used as a genetic biomarker for early screening of GIC in Malaysians. The risk age-group has been shifted to a younger age at 40s and women showed a significant greater risk of stomach and colorectal cancers than men.
doi:10.1136/bmjopen-2013-004109
PMCID: PMC3902529  PMID: 24394801
Gastroenterology; Molecular Biology
19.  Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women 
Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.
doi:10.1590/S0100-879X2012007500081
PMCID: PMC3854186  PMID: 22584640
Genetic polymorphisms; Estrogen receptor gene; Progesterone receptor gene; Breast cancer susceptibility
20.  Polymorphisms, Mutations, and Amplification of the EGFR Gene in Non-Small Cell Lung Cancers 
PLoS Medicine  2007;4(4):e125.
Background
The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.
Methods and Findings
We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.
Conclusions
The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.
Masaharu Nomura and colleagues examine the distribution ofEGFR polymorphisms in different populations and find differences that might explain different responses to tyrosine kinase inhibitors in lung cancer patients.
Editors' Summary
Background.
Most cases of lung cancer—the leading cause of cancer deaths worldwide—are “non-small cell lung cancer” (NSCLC), which has a very low cure rate. Recently, however, “targeted” therapies have brought new hope to patients with NSCLC. Like all cancers, NSCLC occurs when cells begin to divide uncontrollably because of changes (mutations) in their genetic material. Chemotherapy drugs treat cancer by killing these rapidly dividing cells, but, because some normal tissues are sensitive to these agents, it is hard to kill the cancer completely without causing serious side effects. Targeted therapies specifically attack the changes in cancer cells that allow them to divide uncontrollably, so it might be possible to kill the cancer cells selectively without damaging normal tissues. Epidermal growth factor receptor (EGRF) was one of the first molecules for which a targeted therapy was developed. In normal cells, messenger proteins bind to EGFR and activate its “tyrosine kinase,” an enzyme that sticks phosphate groups on tyrosine (an amino acid) in other proteins. These proteins then tell the cell to divide. Alterations to this signaling system drive the uncontrolled growth of some cancers, including NSCLC.
Why Was This Study Done?
Molecules that inhibit the tyrosine kinase activity of EGFR (for example, gefitinib) dramatically shrink some NSCLCs, particularly those in East Asian patients. Tumors shrunk by tyrosine kinase inhibitors (TKIs) often (but not always) have mutations in EGFR's tyrosine kinase. However, not all tumors with these mutations respond to TKIs, and other genetic changes—for example, amplification (multiple copies) of the EGFR gene—also affect tumor responses to TKIs. It would be useful to know which genetic changes predict these responses when planning treatments for NSCLC and to understand why the frequency of these changes varies between ethnic groups. In this study, the researchers have examined three polymorphisms—differences in DNA sequences that occur between individuals—in the EGFR gene in people with and without NSCLC. In addition, they have looked for associations between these polymorphisms, which are present in every cell of the body, and the EGFR gene mutations and allelic imbalances (genes occur in pairs but amplification or loss of one copy, or allele, often causes allelic imbalance in tumors) that occur in NSCLCs.
What Did the Researchers Do and Find?
The researchers measured how often three EGFR polymorphisms (the length of a repeat sequence called CA-SSR1, and two single nucleotide variations [SNPs])—all of which probably affect how much protein is made from the EGFR gene—occurred in normal tissue and NSCLC tissue from East Asians and individuals of European descent. They also looked for mutations in the EGFR tyrosine kinase and allelic imbalance in the tumors, and then determined which genetic variations and alterations tended to occur together in people with the same ethnicity. Among many associations, the researchers found that shorter alleles of CA-SSR1 and the minor forms of the two SNPs occurred less often in East Asians than in individuals of European descent. They also confirmed that EGFR kinase mutations were more common in NSCLCs in East Asians than in European-descent individuals. Furthermore, mutations occurred more often in tumors with allelic imbalance, and in tumors where there was allelic imbalance and an EGFR mutation, the mutant allele was amplified more often than the wild-type allele.
What Do These Findings Mean?
The researchers use these associations between gene variants and tumor-associated alterations to propose a model to explain the ethnic differences in mutational frequencies and responses to TKIs seen in NSCLC. They suggest that because of the polymorphisms in the EGFR gene commonly seen in East Asians, people from this ethnic group make less EGFR protein than people from other ethnic groups. This would explain why, if a threshold level of EGFR is needed to drive cells towards malignancy, East Asians have a high frequency of amplified EGFR tyrosine kinase mutations in their tumors—mutation followed by amplification would be needed to activate EGFR signaling. This model, though speculative, helps to explain some clinical findings, such as the frequency of EGFR mutations and of TKI sensitivity in NSCLCs in East Asians. Further studies of this type in different ethnic groups and in different tumors, as well as with other genes for which targeted therapies are available, should help oncologists provide personalized cancer therapies for their patients.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040125.
US National Cancer Institute information on lung cancer and on cancer treatment for patients and professionals
MedlinePlus encyclopedia entries on NSCLC
Cancer Research UK information for patients about all aspects of lung cancer, including treatment with TKIs
Wikipedia pages on lung cancer, EGFR, and gefitinib (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0040125
PMCID: PMC1876407  PMID: 17455987
21.  MYBPC3 gene variations in hypertrophic cardiomyopathy patients in India 
BACKGROUND
Hypertrophic cardiomyopathy (HCM) is a complex cardiac muscular disorder, inherited as an autosomal dominant disease with variable penetrance. Cardiac myosin-binding protein C (MyBPC) is the predominant myosin-binding protein isoform in the heart muscle. One hundred forty-seven mutations have been detected in MYBPC3, accounting for 15% of all HCM cases.
OBJECTIVE
To screen exons 16, 18, 19, 22, 24, 28, 30, 31 and 34 in the MYBPC3 gene in Indian HCM patients.
METHODS
Sixty control and 95 HCM samples were collected from cardiology units of the CARE Hospital (Nampally, Banjara Hills, Secunderabad, India) for genomic DNA isolation followed by polymerase chain reaction and single-stranded conformational polymorphism analysis.
RESULTS
Screening of the exons revealed two variations – one novel frame shift mutation in exon 19 at the nucleotide position 11577^11578 and one novel single nucleotide polymorphism (SNP) in codon 1093 of exon 31, coding for glycine with a C>T transition (GGC/GGT), in addition to the seven known SNPs mainly in the intronic region and one known missense mutation D770N in this population.
CONCLUSION
The novel frame shift mutation identified in exon 19, D570fs, with the insertion of an adenine residue in codon 570 coding for aspartate, results in a premature termination codon that produces a truncated protein lacking myosin- and titin-binding sites, explaining the role of the nonsense-mediated decay pathway. A novel SNP identified in codon 1093 of exon 31 was found to be a synonymous codon, which may have a regulatory effect at the translational level, attributing to affinity differences between codon-anticodon interactions. The screening of this gene may be relevant in the Indian context.
PMCID: PMC2644567  PMID: 18273486
Hypertrophic cardiomyopathy; MYBPC3; Nonsense-mediated decay pathway; Novel mutation
22.  The progesterone receptor PROGINS polymorphism is not related to oxidative stress factors in women with polycystic ovary syndrome 
Background
Women with PCOS have been reported to be at increased risk of a number of gynaecological neoplasias, including endometrial, breast, and ovarian cancer. Studies of the possible association of genetic variation in progesterone receptor polymorphism with risk of ovarian and breast cancer have concentrated on a variant known as PROGINS.
Methods
Ninety-five young women with PCOS and 99 healthy control women were included in our study. All subjects underwent venous blood drawing for complete hormonal assays, lipid profile, glucose, insulin and PROGINS polymorphism genetic study.
Results
In PROGINS polymorphism results; in both control and the patient groups T1/T1 has been detected in high levels. But for genotype (p = 0.178) and allele (p = 0.555) frequencies both of the groups give similar results. Statistically significant difference has been detected on serum FSH levels for T1/T1 genotype according to T2/T2 genotype.
Conclusion
No relation has been detected between the inflammatory and oxidative stress factors, and PROGINS polymorphism alleles. This may be because the PCOS patients are young and their BMI means are normal and their CIMT and oxidative stress markers are like healthy women.
doi:10.1186/1475-2840-6-29
PMCID: PMC2094702  PMID: 17919323
23.  Analysis of single nucleotide polymorphisms of CRYGA and CRYGB genes in control population of western Indian origin 
Indian Journal of Ophthalmology  2009;57(3):197-201.
Aim:
Polymorphisms in γ-crystallins (CRYG) can serve as markers for lens differentiation and eye disorders leading to cataract. Several investigators have reported the presence of sequence variations within crystallin genes, with or without apparent effects on the function of the proteins both in mice and humans. Delineation of these polymorphic sites may explain the differences observed in the susceptibility to cataract observed among various ethnic groups. An easier Restriction Fragment Length Polymorphism (RFLP)-based method has been used to detect the frequency of four single nucleotide polymorphisms (SNPs) in CRYGA/CRYGB genes in control subjects of western Indian origin.
Materials and Methods:
A total of 137 healthy volunteers from western India were studied. Examination was performed to exclude volunteers with any ocular defects. Polymerase chain reaction (PCR)-RFLP based method was developed for genotyping of G198A (Intron A), T196C (Exon 3) of CRYGA and T47C (Promoter), G449T (Exon 2) of CRYGB genes.
Results:
The exonic SNPs in CRYGA and CRYGB were found to have an allele frequency 0.03 and 1.00 for ancestral allele respectively, while frequency of non-coding SNP in CRYGA was 0.72. Allele frequency of T90C of CRYGB varied significantly (P = 0.02) among different age groups. An in-silico analysis reveals that this sequence variation in CRYGB promoter impacts the binding of two transcription factors, ACE2 (Member of CLB2 cluster) and Progesterone Receptor (PR) which may impact the expression of CRYGB gene.
Conclusions:
This study establishes baseline frequency data for four SNPs in CRYGA and CRYGB genes for future case control studies on the role of these SNPs in the genetic basis of cataract.
doi:10.4103/0301-4738.49393
PMCID: PMC2683433  PMID: 19384013
Cataract; γ-crystallins; Indian; polymerase chain reaction; restriction fragment length polymorphism; single nucleotide polymorphism
24.  Methylenetetrahydrofolate Reductase C677T and A1298C Mutations in Women with Recurrent Spontaneous Abortions in the Northwest of Iran 
ISRN Obstetrics and Gynecology  2012;2012:945486.
Introduction. Recurrent spontaneous abortion (RSA) is a significant obstetrical complication that may occur during pregnancy. Various studies in recent years have indicated that two common mutations (C677T and A1298C) of the methylenetetrahydrofolate reductase (MTHFR) gene are risk factor for RSA. This study was carried out to determine the influence of (C677T and A1298C) of the methylenetetrahydrofolate reductase (MTHFR) gene mutations with RSA. Materials and Methods. A total of 139 women were included in this study: 89 women with two or more consecutive miscarriages and 50 healthy controls. Total genomic DNA was isolated from blood leukocytes. To determine the frequency of the two common C677T and A1298C MTHFR gene mutations in the patients and controls, we used two methods, amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. Results. There is no significant difference in the prevalence of 677T/T genotype among women with RSA and healthy controls (P = 0.285). Also no statistically significant difference in the frequency of A1298C MTHFR gene mutation was detected between the two groups (P = 0.175 ). Conclusion. In conclusion, the results indicate that the Amplification Refractory Mutation System-PCR method was in complete concordance with the results obtained by standard PCR-restriction fragment length polymorphism method. The results also show no significant difference in MTHFR C677T/A1298C genotype distribution among the two groups; therefore, further studies on larger population and other genetic variants to better understand the pathobiology of RSA are needed.
doi:10.5402/2012/945486
PMCID: PMC3504415  PMID: 23209927
25.  Interleukin-6 and interleukin-10 gene polymorphisms and recurrent pregnancy loss in Romanian population 
Background: Approximately 10-14% of the clinically acknowledged pregnancies end with spontaneous abortion at Caucasian population. Possible immunologic causes of recurrent miscarriages have been extensively researched. The change in the cytokines balance synthesis in favor of those synthesized by Th2 cells with an increase of interleukin 6 (IL6) and interleukin10 (IL10) secretion is considered essential for maintaining a normal pregnancy.
Objective: The study objective was to establish an association between interleukin 6 and 10 genes polymorphisms and etiology of recurrent pregnancy loss.
Materials and Methods: The genetic polymorphism of interleukin 6 and 10 genes were studied by PCR-RFLP in the DNA of 69 women with recurrent pregnancy loss and 64 control women with at least one successful pregnancy and without known pregnancy loss. Statistical analysis was performed using Fisher test and differences were considered statistically significant with a p<0.05.
Results: Our results demonstrated a role for -819 C/T but not for -592 C/A IL10, -1082 A/G IL10 and -174G/C IL6 polymorphisms in idiopathic recurrent spontaneous abortion (RSA) in Romanian population. Frequency of genotype -592 CC/-819 CC was higher in the control group than in experimental group (p=0.005). In contrast, genotype -592 AC/-819 CT was more frequent in the experimental group (p=0.05). In this study we have not detected genotype -174 C/C in IL6 gene in patients with spontaneous abortions, nor in the control group.
Conclusion: This study demonstrated a possible association between IL-10 -819 C/T promoter polymorphism and idiopathic RSA among Romanian patients.
PMCID: PMC4248146  PMID: 25469134
Interleukin; Pregnancy loss; Polymorphisms

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