Background Chorioamnionitis is a common underlying cause of preterm birth (PTB). It is hypothesised that polymorphisms in immunoregulatory genes influence the host response to infection and subsequent preterm birth. The relationship between histologic chorioamnionitis and 22 single nucleotide polymorphisms in 11 immunoregulatory genes was examined in a case-control study.
Background
Chorioamnionitis is a common underlying cause of preterm birth (PTB). It is hypothesised that polymorphisms in immunoregulatory genes influence the host response to infection and subsequent preterm birth. The relationship between histologic chorioamnionitis and 22 single nucleotide polymorphisms in 11 immunoregulatory genes was examined in a case-control study.Methods Placentas of 181 Caucasoid women with spontaneous PTB prior to 35 weeks were examined for histologic chorioamnionitis. Polymorphisms in genes  IL1A ,  IL1B ,  IL1RN ,  IL1R1 , tumour necrosis factor ( TNF ),  IL4 ,  IL6 ,  IL10 , transforming growth factor beta-1 ( TGFB1 ), Fas ( TNFRSF6 ), and mannose-binding lectin ( MBL2 ) were genotyped by polymerase chain reaction and sequence specific primers. Multivariable logistic regression including demographic and genetic variables and Kaplan-Meier survival analyses of genotype frequencies and pregnancy outcome were performed.
Methods
Placentas of 181 Caucasoid women with spontaneous PTB prior to 35 weeks were examined for histologic chorioamnionitis. Polymorphisms in genes  IL1A ,  IL1B ,  IL1RN ,  IL1R1 , tumour necrosis factor ( TNF ),  IL4 ,  IL6 ,  IL10 , transforming growth factor beta-1 ( TGFB1 ), Fas ( TNFRSF6 ), and mannose-binding lectin ( MBL2 ) were genotyped by polymerase chain reaction and sequence specific primers. Multivariable logistic regression including demographic and genetic variables and Kaplan-Meier survival analyses of genotype frequencies and pregnancy outcome were performed.IL1AIL1BIL1RNIL1R1TNFIL4IL6IL10TGFB1TNFRSF6MBL2Results Sixty-nine (34%) women had histologic evidence of acute chorioamnionitis. Carriage of the  IL10 -1082A/-819T/592A ( ATA ) haplotype [Multivariable Odds ratio (MOR) 1.9, P = 0.05] and  MBL2  codon 54Asp allele (MOR 2.0, P = 0.04), were positively associated with chorioamnionitis, while the  TNFRSF6 -1377A/-670G ( AG ) haplotype (MOR 0.4, P = 0.03) and homozygosity for  TGFB1 -800G/509T ( GT ) haplotype (MOR 0.2, P = 0.04) were negatively associated.
Results
Sixty-nine (34%) women had histologic evidence of acute chorioamnionitis. Carriage of the  IL10 -1082A/-819T/592A ( ATA ) haplotype [Multivariable Odds ratio (MOR) 1.9, P = 0.05] and  MBL2  codon 54Asp allele (MOR 2.0, P = 0.04), were positively associated with chorioamnionitis, while the  TNFRSF6 -1377A/-670G ( AG ) haplotype (MOR 0.4, P = 0.03) and homozygosity for  TGFB1 -800G/509T ( GT ) haplotype (MOR 0.2, P = 0.04) were negatively associated.IL10ATAMBL2TNFRSF6AGTGFB1GTConclusion These findings demonstrate that polymorphisms in immunoregulatory genes  IL10 ,  MBL2 ,  TNFRSF6  and  TGFB1  may influence susceptibility to chorioamnionitis.
Conclusion
These findings demonstrate that polymorphisms in immunoregulatory genes  IL10 ,  MBL2 ,  TNFRSF6  and  TGFB1  may influence susceptibility to chorioamnionitis.IL10MBL2TNFRSF6TGFB1

Background Acute infection of the amniotic fluid and chorioamnion is a common cause of preterm delivery before 34 weeks gestation. Consistent with previous reports [ 1 ], an Australian study found that 31% of preterm births (PTB) before 34 weeks had histologic chorioamnionitis [ 2 ]. An inverse relationship between delivery gestation and histologic chorioamnionitis was shown with histologic chorioamnionitis identified in 66% of births before 25 weeks. Chorioamnionitis usually results from ascent of pathogenic organisms such as Group B streptococcus and  Escherichia coli  [ 3 ], from the lower genital tract. A T helper cell type1 (Th1) cytokine host response occurs with the production of pro-inflammatory cytokines such as (IL-1β and TNF) produced mainly by activated monocytes/macrophages in amnion, chorionic and decidual tissue. The increased levels of these cytokines trigger prostaglandin F2 and E2 biosynthesis by the decidua and amnion, inducing uterine contractions and labour [ 4 ]. In the setting of infection, IL-1 and TNF have a synergistic effect on the acute phase cytokine IL-6 with increased production in trophoblasts and chorion and to a lesser extent decidua [ 5 ]. The intensity and duration of the inflammatory response is modulated by T helper cell type 2 cytokines and anti-inflammatory mediators such as IL-4, IL-10, IL-1 receptor antagonist (IL-1ra) and transforming growth factor beta-1 (TGFβ-1). The innate host defense system is also an important factor in pregnancy and the body's response to infection. Mannose-binding lectin (MBL), a protein of the complement system, opsonises pathogens independently of antibody for phagocytosis and influences inflammatory pathways [ 6 ]. There are several common polymorphisms in the promoter and coding region of the  MBL2  gene that profoundly influence circulating levels of functional, multimeric MBL. These genetic variants and low MBL levels are associated with risk and severity of infection in a variety of clinical contexts [ 6 ]. Fas, a cell surface receptor of the TNF/nerve growth factor superfamily, mediates apoptosis after binding Fas ligand [ 7 ]. Activated leucocyte populations, including T cells, express high amounts of Fas and evidence suggests that trophoblast Fas-mediated apoptosis is increased in women with chorioamnionitis [ 8 ]. The immune system operates as a complex regulatory network of cytokines and other mediators with a degree of redundancy that changes as pregnancy progresses [ 9 ]. This makes it difficult to assess the importance of single cytokines in inflammation and response to infectious agents when examined in isolation. The genes encoding these mediators contain coding and non-coding polymorphisms that influence level or function of the encoded mediators [ 10 ]. These polymorphisms may influence susceptibility to chorioamnionitis and adverse pregnancy outcome. Single nucleotide polymorphisms (SNPs) for  IL1  +3953 and  TNF  -308 are associated with human chorioamnionitis in  in vitro  studies [ 11 ] and carriage of the  TNF  -308 A allele has been reported as a risk factor for clinical chorioamnionitis in pregnant women [ 12 ]. However these reports were restricted to small numbers of cytokine genes and did not include the analysis of polymorphisms in molecules important in apoptosis and host defence. Examination of a range of immunoregulators and SNPs concurrently is more likely to identify which genes and alleles are of primary importance in response to intra-uterine infection. We hypothesized that polymorphisms in immunoregulatory genes may influence susceptibility to chorioamnionitis and subsequent preterm labour. To examine this hypothesis, we investigated multiple polymorphisms in immunoregulatory genes in a population of Australian Caucasoid women experiencing spontaneous PTB before 35 weeks and histologic chorioamnionitis.
Background
Acute infection of the amniotic fluid and chorioamnion is a common cause of preterm delivery before 34 weeks gestation. Consistent with previous reports [ 1 ], an Australian study found that 31% of preterm births (PTB) before 34 weeks had histologic chorioamnionitis [ 2 ]. An inverse relationship between delivery gestation and histologic chorioamnionitis was shown with histologic chorioamnionitis identified in 66% of births before 25 weeks. Chorioamnionitis usually results from ascent of pathogenic organisms such as Group B streptococcus and  Escherichia coli  [ 3 ], from the lower genital tract. A T helper cell type1 (Th1) cytokine host response occurs with the production of pro-inflammatory cytokines such as (IL-1β and TNF) produced mainly by activated monocytes/macrophages in amnion, chorionic and decidual tissue. The increased levels of these cytokines trigger prostaglandin F2 and E2 biosynthesis by the decidua and amnion, inducing uterine contractions and labour [ 4 ]. In the setting of infection, IL-1 and TNF have a synergistic effect on the acute phase cytokine IL-6 with increased production in trophoblasts and chorion and to a lesser extent decidua [ 5 ]. The intensity and duration of the inflammatory response is modulated by T helper cell type 2 cytokines and anti-inflammatory mediators such as IL-4, IL-10, IL-1 receptor antagonist (IL-1ra) and transforming growth factor beta-1 (TGFβ-1).Escherichia coliThe innate host defense system is also an important factor in pregnancy and the body's response to infection. Mannose-binding lectin (MBL), a protein of the complement system, opsonises pathogens independently of antibody for phagocytosis and influences inflammatory pathways [ 6 ]. There are several common polymorphisms in the promoter and coding region of the  MBL2  gene that profoundly influence circulating levels of functional, multimeric MBL. These genetic variants and low MBL levels are associated with risk and severity of infection in a variety of clinical contexts [ 6 ].MBL2Fas, a cell surface receptor of the TNF/nerve growth factor superfamily, mediates apoptosis after binding Fas ligand [ 7 ]. Activated leucocyte populations, including T cells, express high amounts of Fas and evidence suggests that trophoblast Fas-mediated apoptosis is increased in women with chorioamnionitis [ 8 ].The immune system operates as a complex regulatory network of cytokines and other mediators with a degree of redundancy that changes as pregnancy progresses [ 9 ]. This makes it difficult to assess the importance of single cytokines in inflammation and response to infectious agents when examined in isolation. The genes encoding these mediators contain coding and non-coding polymorphisms that influence level or function of the encoded mediators [ 10 ]. These polymorphisms may influence susceptibility to chorioamnionitis and adverse pregnancy outcome. Single nucleotide polymorphisms (SNPs) for  IL1  +3953 and  TNF  -308 are associated with human chorioamnionitis in  in vitro  studies [ 11 ] and carriage of the  TNF  -308 A allele has been reported as a risk factor for clinical chorioamnionitis in pregnant women [ 12 ]. However these reports were restricted to small numbers of cytokine genes and did not include the analysis of polymorphisms in molecules important in apoptosis and host defence. Examination of a range of immunoregulators and SNPs concurrently is more likely to identify which genes and alleles are of primary importance in response to intra-uterine infection.IL1TNFin vitroTNFWe hypothesized that polymorphisms in immunoregulatory genes may influence susceptibility to chorioamnionitis and subsequent preterm labour. To examine this hypothesis, we investigated multiple polymorphisms in immunoregulatory genes in a population of Australian Caucasoid women experiencing spontaneous PTB before 35 weeks and histologic chorioamnionitis.Methods This case-control study was approved by the Women's and Children's Hospital Ethics and Research Committee. A total of 368 unrelated women of child-bearing age who had obstetric management at the Women's and Children's Hospital (WCH) and history of PTB before 35 weeks gestation were identified from the hospital data base. All women were approached in the first instant by letter of introduction and invited to participate in the study. For women living in the Adelaide metropolitan area, the study nurse made a home visit to those who volunteered for eligibility assessment. Geographical distance excluded 50, 66 did not respond, 40 were not eligible and 31 declined. Of the women enrolled some had more than one PTB. The PTB fitting the study criteria and closest to the enrolment date became the index birth. Written consent was obtained and a 9 ml peripheral blood sample was collected for isolation of leucocytes and DNA extraction. 181 women with a history of spontaneous preterm labour and subsequent PTB between 20 and 35 weeks in the index pregnancy had histological examination of the placenta performed. Histopathology staff members were unaware of the genetic results. Clinical data recorded included gestational age at delivery (calculated by ultrasonography at approximately 18 weeks). Obstetric factors known to place a woman at risk of preterm labour or preterm prelabour rupture of membranes (PPROM) (defined as prelabour rupture of membranes 3 or more hours before preterm delivery) were also recorded. Placental examination and swabbing was performed by staff histopathologists [ 13 ] and included examination of the placental tissue in blocks, extra-placental membranes in a "roll" (amnion, chorion and decidua) and the umbilical cord in cross section. Chorioamnionitis was defined as a dense polymorphonuclear leukocyte infiltration of the chorionamniotic component of the placenta and membranes (amnion, chorion, amniochorial membranes and/or chorionic plate (top part of the fetal side of the placenta). Inflammatory reaction confined to either the decidua or subchorial intervillous space of the placental disc, without infiltration of the chorion or amnion was recorded as negative chorioamnionitis [ 3 ]. Funisitis was defined as inflammation in one or more of the umbilical cord vessels (vasculitis) with or without inflammation in the Wharton's jelly (the supporting soft tissue around the vessel in the cord). The definition of acute chorioamnionitis included funisitis and/or amnionitis. In both groups a number of women had PPROM, occasional bleeding during pregnancy, recorded clinical chorioamnionitis or a history of prior term births. Tocolytic therapy and intravenous antibiotics were used according to obstetric protocols. All participants were Caucasoid, unrelated and at least 18 years of age at enrolment. Women with diagnosed autoimmune disease, pre-eclampsia, in vitro fertilisation treatment, confirmed uterine malformations, no spontaneous labour, ultrasound-confirmed fetal abnormality, insulin dependent diabetes, or multiple pregnancy were excluded. Demographic characteristics, a comprehensive medical and obstetric history, age, any smoking, alcohol consumption, substance use and clinical chorioamnionitis (defined as any three of the following – white blood cell >1500 × 10^9/L, three consecutive C reactive protein readings ≥ 15mg/L, pyrexia ≥ 38°C, uterine pain or tenderness) were verified from case records. Clinical chorioamnionitis was not used as a subgroup for polymorphic analysis because this did not include women with sub-clinical infection. Since the risk of chorioamnionitis is not as great in PTB after 35 weeks, women with PTB between 35 and 37 weeks were not enrolled [ 2 ]. Genes were chosen if reported to be associated with inflammatory disease[ 10 ] and if existing functional data suggested a role in chorioamnionitis and preterm delivery[ 5 , 11 ]. SNPs were selected where the minor allele has a frequency >10%. Several genes were known to contain polymorphisms at multiple sites that are of functional importance (e.g. the 5'and coding variants of  MBL2 ) [ 14 ], thus multiple variants were genotyped and the haplotype recorded. SNPs in the IL1 gene cluster,  TNF ,  IL4 ,  IL6 ,  IL10 ,  TGFB1 ,  TNFRSF6 , and  MBL2  genes and their relationship with histologic chorioamnionitis were therefore examined in a cohort of women with spontaneous PTB. Peripheral blood samples were blinded and tested at the Australian Red Cross Blood Service, Adelaide (ARCBS). Genomic DNA was extracted from ethylenediaminetetraacetic acid anticoagulated venous blood using standard methods as previously described [ 15 ]. SNPs for  TNF  (+488,-238,-308) [ 16 ],  IL1A  (-889),  IL1B  (+3962, -511),  IL4  (-590),  IL10  (-1082, -819, -592),  TGFB1  (-800, -509) [ 17 ],  MBL2  [-550, -221, codon 52 (Arg → Cys), codon 54 (Gly → Asp), codon 57 (Gly → Glu)] [ 18 ],  IL6  (-174), IL-1 receptor antagonist  IL1RN  (+11100), IL-1 type 1 receptor  IL1R1  (+970) and  TNFRSF6  (-1377, -670) [ 19 ] were genotyped using the polymerase chain reaction and sequence specific primers (PCR-SSP). Multiple polymorphisms were examined in the  IL10 ,  TNFRSF6 ,  TNF ,  TGFB1  and  MBL2  genes by PCR haplotyping. The term "haplotype" denotes ordered combination of alleles on a single chromosome, and is used where multiple polymorphisms are genotyped by PCR-haplotyping for a single gene. For PCR haplotyping combinations of forward and reverse allele-specific-primers were used to directly amplify alleles on the same chromosome [ 20 ]. This technique also provides a degree of redundancy, in that multiple reactions genotype a polymorphism, thus confirming results. All PCR-SSP reactions have been extensively used in the same laboratory. Internal control primers for conserved regions of the  DRB1  (major histocompatibility complex, class 11 DR beta 1) and  APC  (adenomatous polyposis coli) genes were included in every PCR mix to verify successful amplification. All genes except the  IL1A  had a repeat genotyping rate of less than 1%. The  IL1A  had a repeat rate of 10%. The sample size was calculated by using the statistical software package EpiInfo 6 (Version 6.04d, Centre for Disease Control and Prevention, Atlanta, GA). We estimated the study had sufficient power (1-β = 80%, P = 0.05) to detect a minimum of 17% difference in genotype frequency between chorioamnionitis and no chorioamnionitis for frequencies of approximately 10% (ie. 10% versus 27%). For a genotype frequency of 3% the minimum detectable difference was 10% (3% versus 13%). Allele and haplotype frequencies for polymorphisms were determined by direct counting from each chromosome. Frequencies of genetic variants were recorded as allele (proportion of positive chromosomes), genotype (proportion possessing homozygous or heterozygous combinations of alleles), and 'carriage' of an allele or haplotype (either in homozygous or heterozygous state). Alleles were said to be in Hardy-Weinberg equilibrium if the observed genotype frequencies did not differ significantly (P > 0.05) from those expected when analysed by Chi square. Gene variant frequencies were compared using 2 × 2 contingency table analysis and the Chi square test for independence using EpiInfo 6. Mean birth weights were compared by  t -test. Multivariable analysis was performed by stepwise backwards multiple logistic regression using SYSTAT 9.0 software (Systat version 1999 SPSS Inc). The basic model included genetic variables with P values < 0.15 obtained in univariate analyses. In each multivariable model, only one genotype or haplotype of each gene was selected for analysis. Selection of genotype/haplotype for the model was based on strength of association, the previously reported importance of a gene variant and homozygosity of alleles. At each backward step in the regression analysis P values ≥ 0.07 were rejected. Kaplan-Meier survival analysis (SYSTAT 9.0) was used to identify cumulative probability in maintaining pregnancy by gestation stratified by chorioamnionitis and gene variables. Breslow-Gehan Chi square was used to weight each gestational period by the total number at risk at that time so that earlier gestations receive greater weight than later gestations.
Methods
This case-control study was approved by the Women's and Children's Hospital Ethics and Research Committee. A total of 368 unrelated women of child-bearing age who had obstetric management at the Women's and Children's Hospital (WCH) and history of PTB before 35 weeks gestation were identified from the hospital data base. All women were approached in the first instant by letter of introduction and invited to participate in the study. For women living in the Adelaide metropolitan area, the study nurse made a home visit to those who volunteered for eligibility assessment. Geographical distance excluded 50, 66 did not respond, 40 were not eligible and 31 declined. Of the women enrolled some had more than one PTB. The PTB fitting the study criteria and closest to the enrolment date became the index birth. Written consent was obtained and a 9 ml peripheral blood sample was collected for isolation of leucocytes and DNA extraction. 181 women with a history of spontaneous preterm labour and subsequent PTB between 20 and 35 weeks in the index pregnancy had histological examination of the placenta performed. Histopathology staff members were unaware of the genetic results.Clinical data recorded included gestational age at delivery (calculated by ultrasonography at approximately 18 weeks). Obstetric factors known to place a woman at risk of preterm labour or preterm prelabour rupture of membranes (PPROM) (defined as prelabour rupture of membranes 3 or more hours before preterm delivery) were also recorded. Placental examination and swabbing was performed by staff histopathologists [ 13 ] and included examination of the placental tissue in blocks, extra-placental membranes in a "roll" (amnion, chorion and decidua) and the umbilical cord in cross section. Chorioamnionitis was defined as a dense polymorphonuclear leukocyte infiltration of the chorionamniotic component of the placenta and membranes (amnion, chorion, amniochorial membranes and/or chorionic plate (top part of the fetal side of the placenta). Inflammatory reaction confined to either the decidua or subchorial intervillous space of the placental disc, without infiltration of the chorion or amnion was recorded as negative chorioamnionitis [ 3 ]. Funisitis was defined as inflammation in one or more of the umbilical cord vessels (vasculitis) with or without inflammation in the Wharton's jelly (the supporting soft tissue around the vessel in the cord). The definition of acute chorioamnionitis included funisitis and/or amnionitis. In both groups a number of women had PPROM, occasional bleeding during pregnancy, recorded clinical chorioamnionitis or a history of prior term births. Tocolytic therapy and intravenous antibiotics were used according to obstetric protocols. All participants were Caucasoid, unrelated and at least 18 years of age at enrolment. Women with diagnosed autoimmune disease, pre-eclampsia, in vitro fertilisation treatment, confirmed uterine malformations, no spontaneous labour, ultrasound-confirmed fetal abnormality, insulin dependent diabetes, or multiple pregnancy were excluded.Demographic characteristics, a comprehensive medical and obstetric history, age, any smoking, alcohol consumption, substance use and clinical chorioamnionitis (defined as any three of the following – white blood cell >1500 × 10^9/L, three consecutive C reactive protein readings ≥ 15mg/L, pyrexia ≥ 38°C, uterine pain or tenderness) were verified from case records. Clinical chorioamnionitis was not used as a subgroup for polymorphic analysis because this did not include women with sub-clinical infection. Since the risk of chorioamnionitis is not as great in PTB after 35 weeks, women with PTB between 35 and 37 weeks were not enrolled [ 2 ].Genes were chosen if reported to be associated with inflammatory disease[ 10 ] and if existing functional data suggested a role in chorioamnionitis and preterm delivery[ 5 , 11 ]. SNPs were selected where the minor allele has a frequency >10%. Several genes were known to contain polymorphisms at multiple sites that are of functional importance (e.g. the 5'and coding variants of  MBL2 ) [ 14 ], thus multiple variants were genotyped and the haplotype recorded. SNPs in the IL1 gene cluster,  TNF ,  IL4 ,  IL6 ,  IL10 ,  TGFB1 ,  TNFRSF6 , and  MBL2  genes and their relationship with histologic chorioamnionitis were therefore examined in a cohort of women with spontaneous PTB.MBL2TNFIL4IL6IL10TGFB1TNFRSF6MBL2Peripheral blood samples were blinded and tested at the Australian Red Cross Blood Service, Adelaide (ARCBS). Genomic DNA was extracted from ethylenediaminetetraacetic acid anticoagulated venous blood using standard methods as previously described [ 15 ]. SNPs for  TNF  (+488,-238,-308) [ 16 ],  IL1A  (-889),  IL1B  (+3962, -511),  IL4  (-590),  IL10  (-1082, -819, -592),  TGFB1  (-800, -509) [ 17 ],  MBL2  [-550, -221, codon 52 (Arg → Cys), codon 54 (Gly → Asp), codon 57 (Gly → Glu)] [ 18 ],  IL6  (-174), IL-1 receptor antagonist  IL1RN  (+11100), IL-1 type 1 receptor  IL1R1  (+970) and  TNFRSF6  (-1377, -670) [ 19 ] were genotyped using the polymerase chain reaction and sequence specific primers (PCR-SSP). Multiple polymorphisms were examined in the  IL10 ,  TNFRSF6 ,  TNF ,  TGFB1  and  MBL2  genes by PCR haplotyping. The term "haplotype" denotes ordered combination of alleles on a single chromosome, and is used where multiple polymorphisms are genotyped by PCR-haplotyping for a single gene. For PCR haplotyping combinations of forward and reverse allele-specific-primers were used to directly amplify alleles on the same chromosome [ 20 ]. This technique also provides a degree of redundancy, in that multiple reactions genotype a polymorphism, thus confirming results. All PCR-SSP reactions have been extensively used in the same laboratory. Internal control primers for conserved regions of the  DRB1  (major histocompatibility complex, class 11 DR beta 1) and  APC  (adenomatous polyposis coli) genes were included in every PCR mix to verify successful amplification. All genes except the  IL1A  had a repeat genotyping rate of less than 1%. The  IL1A  had a repeat rate of 10%.TNFIL1AIL1BIL4IL10TGFB1MBL2IL6IL1RNIL1R1TNFRSF6IL10TNFRSF6TNFTGFB1MBL2DRB1APCIL1AIL1AThe sample size was calculated by using the statistical software package EpiInfo 6 (Version 6.04d, Centre for Disease Control and Prevention, Atlanta, GA). We estimated the study had sufficient power (1-β = 80%, P = 0.05) to detect a minimum of 17% difference in genotype frequency between chorioamnionitis and no chorioamnionitis for frequencies of approximately 10% (ie. 10% versus 27%). For a genotype frequency of 3% the minimum detectable difference was 10% (3% versus 13%). Allele and haplotype frequencies for polymorphisms were determined by direct counting from each chromosome. Frequencies of genetic variants were recorded as allele (proportion of positive chromosomes), genotype (proportion possessing homozygous or heterozygous combinations of alleles), and 'carriage' of an allele or haplotype (either in homozygous or heterozygous state). Alleles were said to be in Hardy-Weinberg equilibrium if the observed genotype frequencies did not differ significantly (P > 0.05) from those expected when analysed by Chi square. Gene variant frequencies were compared using 2 × 2 contingency table analysis and the Chi square test for independence using EpiInfo 6. Mean birth weights were compared by  t -test.tMultivariable analysis was performed by stepwise backwards multiple logistic regression using SYSTAT 9.0 software (Systat version 1999 SPSS Inc). The basic model included genetic variables with P values < 0.15 obtained in univariate analyses. In each multivariable model, only one genotype or haplotype of each gene was selected for analysis. Selection of genotype/haplotype for the model was based on strength of association, the previously reported importance of a gene variant and homozygosity of alleles. At each backward step in the regression analysis P values ≥ 0.07 were rejected. Kaplan-Meier survival analysis (SYSTAT 9.0) was used to identify cumulative probability in maintaining pregnancy by gestation stratified by chorioamnionitis and gene variables. Breslow-Gehan Chi square was used to weight each gestational period by the total number at risk at that time so that earlier gestations receive greater weight than later gestations.Results Sixty-nine (38%) women had histologic evidence of acute chorioamnionitis, and 112 (62%) had no such evidence. There were no significant differences between those with and without histologic chorioamnionitis for maternal age, race, history of smoking, alcohol consumption, substance use, new partner, cervical cerclage, placenta praevia after 22 weeks with bleeding, history of two or more PTBs before 35 weeks, miscarriage between 12 and 20 weeks or PPROM (Table  1 ). Histologic chorioamnionitis was positively associated with clinical chorioamnionitis [Univariate Odds Ratio (OR) 6.1, 95% Confidence Intervals (CI) 2.4–17, uncorrected P ≤ 0.0001], PTB before 29 weeks (OR 7.6, CI 3.4–18, P ≤ 0.0001) and positive placental culture (OR 3.0, CI 0.9–11, P = 0.04). Table 1 Demographic and clinical characteristics of women with preterm birth before 35 weeks. Variable Histologic Chorio-amnionitis n = 69 No Histologic Chorio-amnionitis n = 112 Significant OR (95% CI), and Uncorrected P values Maternal age (years) 29 (16–41) 30 (17–44) Delivery gestation (weeks) 28 (20–34) 30 (23–34) * Mean birth weight 1399 (± 676) 1897 (± 504) < 0.0001 Maternal age ≥ 35 years 11 (16%) 18 (16%) ns Maternal age ≤ 17 years 2 (3%) 2 (2%) ns Any smoking 22 (31%) 29 (26%) ns Any alcohol 44 (64%) 73 (65%) ns Any substance use 7 (10%) 10 (9%) ns Partner change for 14 (20%) 20 (18%) ns pregnancy Cervical cerclage 3 (4%) 2 (2%) ns Previous mid-trimester 4 (6%) 12 (11%) ns miscarriage (12–20 weeks) 2 or more preterm births 11 (16%) 20 (18%) ns Placenta praevia after 22 weeks with bleeding 1 (1%) 2 (2%) ns Documented clinical chorioamnionitis 22 (32%) 8 (7%) 6.1 (2.4–17), < 0.001 PPROM ≥ 3 hours 39 (57%) 74 (66%) ns Gestation < 29 weeks 33 (48%) 12 (11%) 7.6 (3.4–18), < 0.001 Documented positive placental culture 10 (14%) 6 (5%) 3.0 (0.9–11), 0.04 Neonatal sepsis 10 (14%) 0 19 (3 – 823), < 0.001 Neonatal death 6 (9%) 3 (3%) ns Post partum endometritis 5 (7%) 3 (3%) ns Descriptive statistical results are mean (± SD) or number (%) as appropriate, and maternal age and gestation as median (range). Characteristics are from index birth. PPROM = Preterm premature rupture of membranes 3 hours or more before delivery < 35 weeks. Univariate analyses were tested using Chi square analysis of 2 × 2 table, and uncorrected P values, Odds Ratio (OR), and 95% Confidence Intervals (CI), P values ≤ 0.05 are presented. Mean birth weights were tested by two-sample  t -test, assuming unequal variances. * See Figure 1, ns, not significant. All alleles were in Hardy-Weinberg equilibrium. All gene variants were tested for associations between the two groups. In preterm women without histologic chorioamnionitis frequencies for the  IL10 -1082A/-819T/-592A ( ATA ) haplotype [ 17 ],  MBL2  codon 54Asp (the MBL 'B' allele) [ 21 ],  TNFRSF6 -1377A/-670G ( AG ) haplotype [ 22 ] and  TGFB1 -800G/-509T ( GT ) homozygosity [ 17 ] were similar to reported Caucasian controls. In a comparison of women with chorioamnionitis and those without chorioamnionitis a comprehensive listing of gene carriage, alleles, genotype and haplotype frequencies is provided ( Additional file 1 ). Univariate analysis revealed that carriage of the  IL10 -1082A/-819T/-592A ( ATA ) haplotype (present in 49% of women with chorioamnionitis vs 33% without chorioamnionitis, OR 2.0, P = 0.03) and the  IL10 -819T and -592A alleles (50% versus 33%, OR 2.0, P = 0.03) were positively associated with histologic chorioamnionitis, as was carriage of the  MBL2  54Asp (B allele) (39% versus 25%, OR 1.9, P = 0.04) (Table  2 ). Although the frequency of the common  TNFRSF6 -1377 G/G genotype was higher in women with histologic chorioamnionitis (0.87 versus 0.73, OR 2.4, P = 0.03), the variant  TNFRSF6 -1377A/-670G ( AG ) haplotype (OR 0.4, P = 0.03) and homozygosity for the  TGFB1  haplotype -800G/-509T ( GT ) (OR 0.2, P = 0.03) were negatively associated with chorioamnionitis (Table  2 ). No other significant associations with gene variant frequencies were seen in univariate analysis. Table 2 Genes, alleles, haplotypes and genotypes in women with preterm birth before 35 weeks and chorioamnionitis. Gene Histologic Chorio-amnionitis n = 69 No Histologic Chorio-amnionitis n = 112 Univariate analysis OR (95% CI) Uncorrected P value Multivariable analysis OR (95% CI), P value IL10 Allele -819 T 34 (0.50) 37 (0.33) 2.0 (1.0–3.8), 0.03   -592 A 34 (0.50) 37 (0.33) 2.0 (1.0–3.8), 0.03 Haplotype -1082A/-819T/-592A 34 (0.49) 37 (0.33) 2.0 (1.0–3.8), 0.03 1.8 (1.0–4.0), 0.05 Genotype -819 C/T 28 (0.41) 28 (0.25) 2.1 (1.0–4-1), 0.03 -592 A/C 28 (0.41) 28 (0.25) 2.1 (1.0–4-1), 0.03 MBL2 Codon 54Asp ('B') 27 (0.39) 28 (0.25) 1.9 (1.0–3.9), 0.04 2.0 (1.0–4.0), 0.04 TNFRSF6 Allele -1377A 9 (0.13) 30 (0.27) 0.4 (0.2–1.0), 0.03 Haplotype -1377A/-670G 9 (0.13) 30 (0.27) 0.4 (0.2–1.0), 0.03 0.3 (0.2–0.9), 0.03 Genotype -1377 G/G 60 (0.87) 82 (0.73) 2.4 (1.0–6.3), 0.03 TGFB1 Haplotype -800G/-509T 2 (0.03) 14 (0.13) 0.2 (0.02–1.0), 0 .03 0.2 (0.04–0.9), 0.04 Homozygosity Data are presented as number and frequencies of alleles, genotypes and haplotypes. Univariate analyses were tested using Chi square analysis of 2 × 2 table, and uncorrected P values, Odds Ratio (OR), and 95% Confidence Intervals (CI), P values ≤ 0.07 are presented. Multivariable analyses included genetic variables with P values ≤ 0.15 using backward step multiple logistic regression. Multivariable model included carriage of  IL10 ATA ,  MBL2  codon 54Asp,  TNFRS6 -1377A/-670G ( AG ) haplotype, and homozygous  TGFB1 -800G/-509T,  TNF  +488 G/A, and  IL1R1  +970 T/T genotypes. MBL2  codon 54Asp (allele 'B') is a single nucleotide polymorphism in exon 1 codon 54 (Gly/Asp). TNFRSF6  is the gene symbol for Fas; ns, not significant. For confirmation of independence of association, a multivariable model was used including univariate variables with P values ≤ 0.15. This model included the above genetic variables (Table  2 ) plus  TNF  +488 G/A genotype (25% versus 15%, OR 1.8, P = 0.11) and  IL1R1  +970 T/T (6% versus 14%, OR 0.4, P = 0.08) from  Additional file 1 . Multivariable analysis confirmed that carriage of the variant  IL-10 ATA  haplotype [Multivariable Odds Ratio (MOR) 1.9, P = 0.05)] and  MBL2  codon 54Asp (MOR 2.0, P = 0.04) were independently associated with histologic chorioamnionitis, while homozygosity for the variant  TGFB1 -800G/-509T ( GT ) haplotype (MOR 0.2, P = 0.04), and carriage of  TNFRSF6 -1377A/-670G ( AG ) haplotype (MOR 0.4, P = 0.03) remained negatively associated with histologic chorioamnionitis. The remainder of SNPs tested showed no significant differences. To examine the cumulative probability of maintaining a pregnancy and effect of chorioamnionitis on gestation at delivery in the overall cohort, Kaplan Meier survival analysis was performed. Figure  1  shows that chorioamnionitis was more common in PTB at earlier gestations (Breslow-Gehan, Chi square 36 with 1 degree of freedom, P ≤ 0.001). In addition, the cumulative probability of maintaining a pregnancy was stratified on the carriage of  IL10 ATA  haplotype (Figure  2 ). Women with this  IL10 ATA  haplotype were more likely to deliver at earlier gestations (Breslow-Gehan, Chi square 3.5 with 1 degree of freedom, P = 0.06) (Figure  2 ). There was no evidence of the effect of  MBL2 ,  TGFB1 ,  or TNFRSF6  SNPs on gestation of PTB. Figure 1 Relationship of chorioamnionitis with gestation at delivery.  Kaplan-Meier survival estimates in 181 women where all gestations proceeded to delivery < 35 weeks. Effect of chorioamnionitis (C) versus no chorioamnionitis (NC), P < 0.001. Significance was tested using Breslow-Gehan Chi square analysis with 1 degree of freedom. Figure 2 Relationship of  IL10 ATA  haplotype with gestation at delivery.  Kaplan-Meier estimates in 181 women where all gestations proceeded to delivery before 35 weeks. Effect of  IL10 ATA  haplotype versus No  ATA , P = 0.06. Significance was tested using Breslow-Gehan Chi square analysis with 1 degree of freedom.
Results
Sixty-nine (38%) women had histologic evidence of acute chorioamnionitis, and 112 (62%) had no such evidence. There were no significant differences between those with and without histologic chorioamnionitis for maternal age, race, history of smoking, alcohol consumption, substance use, new partner, cervical cerclage, placenta praevia after 22 weeks with bleeding, history of two or more PTBs before 35 weeks, miscarriage between 12 and 20 weeks or PPROM (Table  1 ). Histologic chorioamnionitis was positively associated with clinical chorioamnionitis [Univariate Odds Ratio (OR) 6.1, 95% Confidence Intervals (CI) 2.4–17, uncorrected P ≤ 0.0001], PTB before 29 weeks (OR 7.6, CI 3.4–18, P ≤ 0.0001) and positive placental culture (OR 3.0, CI 0.9–11, P = 0.04).Demographic and clinical characteristics of women with preterm birth before 35 weeks.VariableHistologic Chorio-amnionitis n = 69No Histologic Chorio-amnionitis n = 112Significant OR (95% CI), and Uncorrected P valuesDescriptive statistical results are mean (± SD) or number (%) as appropriate, and maternal age and gestation as median (range). Characteristics are from index birth. PPROM = Preterm premature rupture of membranes 3 hours or more before delivery < 35 weeks.Univariate analyses were tested using Chi square analysis of 2 × 2 table, and uncorrected P values, Odds Ratio (OR), and 95% Confidence Intervals (CI), P values ≤ 0.05 are presented. Mean birth weights were tested by two-sample  t -test, assuming unequal variances.t* See Figure 1,ns, not significant.All alleles were in Hardy-Weinberg equilibrium. All gene variants were tested for associations between the two groups. In preterm women without histologic chorioamnionitis frequencies for the  IL10 -1082A/-819T/-592A ( ATA ) haplotype [ 17 ],  MBL2  codon 54Asp (the MBL 'B' allele) [ 21 ],  TNFRSF6 -1377A/-670G ( AG ) haplotype [ 22 ] and  TGFB1 -800G/-509T ( GT ) homozygosity [ 17 ] were similar to reported Caucasian controls.IL10ATAMBL2TNFRSF6AGTGFB1GTIn a comparison of women with chorioamnionitis and those without chorioamnionitis a comprehensive listing of gene carriage, alleles, genotype and haplotype frequencies is provided ( Additional file 1 ). Univariate analysis revealed that carriage of the  IL10 -1082A/-819T/-592A ( ATA ) haplotype (present in 49% of women with chorioamnionitis vs 33% without chorioamnionitis, OR 2.0, P = 0.03) and the  IL10 -819T and -592A alleles (50% versus 33%, OR 2.0, P = 0.03) were positively associated with histologic chorioamnionitis, as was carriage of the  MBL2  54Asp (B allele) (39% versus 25%, OR 1.9, P = 0.04) (Table  2 ). Although the frequency of the common  TNFRSF6 -1377 G/G genotype was higher in women with histologic chorioamnionitis (0.87 versus 0.73, OR 2.4, P = 0.03), the variant  TNFRSF6 -1377A/-670G ( AG ) haplotype (OR 0.4, P = 0.03) and homozygosity for the  TGFB1  haplotype -800G/-509T ( GT ) (OR 0.2, P = 0.03) were negatively associated with chorioamnionitis (Table  2 ). No other significant associations with gene variant frequencies were seen in univariate analysis.IL10ATAIL10MBL2TNFRSF6TNFRSF6AGTGFB1GTGenes, alleles, haplotypes and genotypes in women with preterm birth before 35 weeks and chorioamnionitis.GeneHistologic Chorio-amnionitis n = 69No Histologic Chorio-amnionitis n = 112Univariate analysis OR (95% CI) Uncorrected P valueMultivariable analysis OR (95% CI), P valueIL10IL10AlleleHaplotypeGenotypeMBL2MBL2TNFRSF6TNFRSF6AlleleHaplotypeGenotypeTGFB1TGFB1HaplotypeData are presented as number and frequencies of alleles, genotypes and haplotypes.Univariate analyses were tested using Chi square analysis of 2 × 2 table, and uncorrected P values, Odds Ratio (OR), and 95% Confidence Intervals (CI), P values ≤ 0.07 are presented.Multivariable analyses included genetic variables with P values ≤ 0.15 using backward step multiple logistic regression. Multivariable model included carriage of  IL10 ATA ,  MBL2  codon 54Asp,  TNFRS6 -1377A/-670G ( AG ) haplotype, and homozygous  TGFB1 -800G/-509T,  TNF  +488 G/A, and  IL1R1  +970 T/T genotypes.IL10 ATAMBL2TNFRS6AGTGFB1TNFIL1R1MBL2  codon 54Asp (allele 'B') is a single nucleotide polymorphism in exon 1 codon 54 (Gly/Asp).MBL2TNFRSF6  is the gene symbol for Fas; ns, not significant.TNFRSF6For confirmation of independence of association, a multivariable model was used including univariate variables with P values ≤ 0.15. This model included the above genetic variables (Table  2 ) plus  TNF  +488 G/A genotype (25% versus 15%, OR 1.8, P = 0.11) and  IL1R1  +970 T/T (6% versus 14%, OR 0.4, P = 0.08) from  Additional file 1 . Multivariable analysis confirmed that carriage of the variant  IL-10 ATA  haplotype [Multivariable Odds Ratio (MOR) 1.9, P = 0.05)] and  MBL2  codon 54Asp (MOR 2.0, P = 0.04) were independently associated with histologic chorioamnionitis, while homozygosity for the variant  TGFB1 -800G/-509T ( GT ) haplotype (MOR 0.2, P = 0.04), and carriage of  TNFRSF6 -1377A/-670G ( AG ) haplotype (MOR 0.4, P = 0.03) remained negatively associated with histologic chorioamnionitis. The remainder of SNPs tested showed no significant differences.TNFIL1R1IL-10 ATAMBL2TGFB1GTTNFRSF6AGTo examine the cumulative probability of maintaining a pregnancy and effect of chorioamnionitis on gestation at delivery in the overall cohort, Kaplan Meier survival analysis was performed. Figure  1  shows that chorioamnionitis was more common in PTB at earlier gestations (Breslow-Gehan, Chi square 36 with 1 degree of freedom, P ≤ 0.001). In addition, the cumulative probability of maintaining a pregnancy was stratified on the carriage of  IL10 ATA  haplotype (Figure  2 ). Women with this  IL10 ATA  haplotype were more likely to deliver at earlier gestations (Breslow-Gehan, Chi square 3.5 with 1 degree of freedom, P = 0.06) (Figure  2 ). There was no evidence of the effect of  MBL2 ,  TGFB1 ,  or TNFRSF6  SNPs on gestation of PTB.IL10 ATAIL10 ATAMBL2TGFB1or TNFRSF6Discussion These results extend prior reports investigating gene polymorphisms and the pathogenesis of PTB and chorioamnionitis [ 11 ]. In this study we found significant positive associations between the carriage of the  IL10 ATA  haplotype,  MBL2  codon 54Asp and histologic chorioamnionitis, and negative associations with  TNFRSF6  and  TGFB1 . This is the first study to investigate  TNFRSF6  and  TGFB1  in combination with such a wide range of cytokine SNPs in histologic acute chorioamnionitis. Also, this is one of the few studies that have used multivariable and survival analyses for investigating adverse pregnancy outcomes such as chorioamnionitis and PTB. The differences in genotype distribution for  IL10  and  MBL2  may be of biologic importance in the pathogenesis of histologic chorioamnionitis. The function of IL-10 in pregnancy is of particular interest because of its role in the homeostatic control of an inflammatory immune response [ 23 ]. Although there have been conflicting reports the majority suggest that the  IL10 ATA  promoter haplotype and alleles comprising the haplotype are associated with low protein levels in Caucasians [ 24 ]. Low levels of IL-10 have been associated with PTB in rat models [ 25 ]. In a pregnant rhesus monkey model, combined intra-amniotic and maternal systemic IL-10 administration inhibited IL-1β-induced preterm uterine contraction [ 26 ]. Survival analysis showed that the  IL10 ATA  haplotype may influence the timing of PTB. IL-10 antagonises the synthesis and actions of pro-inflammatory cytokines such as IL-1 [ 27 ]. In chorioamnionitis, diminished IL-10 levels may provide an ineffective counter regulatory response to elevated maternal prostaglandin stimulated by the pro-inflammatory cytokines IL-1, IL-6 and TNF. Elevated levels of these pro-inflammatory cytokines in utero may serve as a maternal [ 28 ] and fetal [ 29 ] signal for the onset of premature parturition. The higher proportion of early PTB in women with the  IL10 ATA  haplotype may also be a reflection of failure of tocolysis in the presence of infection [ 26 ]. Our finding that the carriage of the  IL10 ATA  haplotype is more common in women with histologic chorioamnionitis suggests that this haplotype may be important in the pathogenesis of histologic chorioamnionitis and subsequent PTB. MBL is an important innate defense molecule active against a broad range of bacterial, viral, fungal and protozoan pathogens [ 30 ]. The  MBL2  variant alleles in exon 1 correlate with decreased circulating MBL. The single nucleotide substitution of an adenine for a guanine in codon 54 results in replacement of aspartic acid for a glycine in the MBL [ 31 ]. Functional MBL is multimeric, consisting of tetramers, pentamers and hexamers of triplets of the basic MBL peptide. This higher order structure is necessary for high avidity interactions between MBL carbohydrate recognition domains and repeated oligosaccharide moieties on pathogens. The MBL codon 54Asp 'B' allele disrupts assembly of  MBL2  peptide trimers resulting in lower stability and serum levels [ 32 ]. Low serum levels of MBL and  MBL2  genetic variants are associated with infection in a range of contexts including recurrent infection in children and adults [ 6 , 14 ], recurrent vulvovaginal candidiasis [ 33 ], recurrent miscarriage [ 34 ] and autoimmunity in common variable immunodeficiency disease [ 18 ]. The association between  MBL2  codon 54Asp and histologic chorioamnionitis suggests that this allele may result in low MBL levels, impaired opsonisation and clearance of pathogens, thus facilitating the development of chorioamnionitis. Fas protein is expressed on the surface of many cell types, such as lymphocytes, epithelial, fibroblasts and certain endothelial cells, and cytokines [ 7 ] and hormones [ 35 ] found in the placental microenvironment may modulate the immune response by regulating the expression of Fas and Fas ligand. In this study two polymorphisms in the Fas ( TNFRSF6 ) gene promoter were studied at positions -1377 and -670, and the -1377A/-670G ( AG ) haplotype was negatively associated with histologic chorioamnionitis. The -1377G/A is situated at the transcription factor SP-1 binding site [ 22 ], and the -670A/G substitution is located in the enhancer region and abolishes the binding site of the nuclear transcription element GAS (Gamma Interferon Activation Sequence). The functional significance of these polymorphisms has not been fully elucidated but they may modulate  TNFRSF6  transcription, Fas expression and thus apoptotic and inflammatory responses. Recent evidence suggests that cells are more sensitive to Fas-mediated apoptosis when levels of Fas expression increase [ 36 ]. Apoptotic cell death can modulate host containment of pathogens [ 19 ] and thus genetically determined variation in Fas-mediated cell death may influence histologic chorioamnionitis. Reported studies on gene polymorphisms and adverse pregnancy outcome have mostly examined SNPs in the genes encoding the pro-inflammatory cytokines TNF and IL-1. The  TNF  promoter -308A allele was associated with clinical chorioamnionitis in a small number of women [ 12 ]. The  in vitro  functional data of the effects of the  TNF  SNPs are conflicting and may be context dependent and influenced by strong linkage disequilibrium with other intra-and-extra-genic polymorphisms (e.g. with lymphotoxin alpha and classical HLA genes [ 37 ], not examined in this study). In contrast, in this study the  TNF -308A could not be identified as a susceptibility or severity factor for chorioamnionitis. SNPs of the IL1 cluster have been examined from different ethnic groups, in particular  IL1B  and  IL1RN  in women with a history of recurrent pregnancy loss [ 38 ], PTB [ 39 ], and in  in vitro  studies[ 11 ]. We did not find associations between genes of the IL1 cluster or  IL1R1  and histologic chorioamnionitis and this may partly be explained by differences in linkage disequilibrium patterns across different ethnic populations that can lead to different association results [ 40 ]. Our data support the role of  IL10 ,  MBL2  and  TNFRSF6  variants in determining the risk of histologic chorioamnionitis. However there are limitations to the study, such as small sample size. The range and number of gene polymorphisms examined and analysed by univariate and multiple logistic regression in this study is notable, but some correction for multiple analyses is required [ 41 ]. The appropriate method still remains a contentious issue. The Bonferroni method was not conducted, as this is a conservative method and leads to overcorrection [ 42 ]. Also our study is a hypothesis-generating exploratory study. SNP frequencies in women with and without chorioamnionitis are available to be included in meta-analysis to confirm or reject these findings ( Additional file 1 ).
Discussion
These results extend prior reports investigating gene polymorphisms and the pathogenesis of PTB and chorioamnionitis [ 11 ]. In this study we found significant positive associations between the carriage of the  IL10 ATA  haplotype,  MBL2  codon 54Asp and histologic chorioamnionitis, and negative associations with  TNFRSF6  and  TGFB1 . This is the first study to investigate  TNFRSF6  and  TGFB1  in combination with such a wide range of cytokine SNPs in histologic acute chorioamnionitis. Also, this is one of the few studies that have used multivariable and survival analyses for investigating adverse pregnancy outcomes such as chorioamnionitis and PTB.IL10 ATAMBL2TNFRSF6TGFB1TNFRSF6TGFB1The differences in genotype distribution for  IL10  and  MBL2  may be of biologic importance in the pathogenesis of histologic chorioamnionitis. The function of IL-10 in pregnancy is of particular interest because of its role in the homeostatic control of an inflammatory immune response [ 23 ]. Although there have been conflicting reports the majority suggest that the  IL10 ATA  promoter haplotype and alleles comprising the haplotype are associated with low protein levels in Caucasians [ 24 ]. Low levels of IL-10 have been associated with PTB in rat models [ 25 ]. In a pregnant rhesus monkey model, combined intra-amniotic and maternal systemic IL-10 administration inhibited IL-1β-induced preterm uterine contraction [ 26 ]. Survival analysis showed that the  IL10 ATA  haplotype may influence the timing of PTB. IL-10 antagonises the synthesis and actions of pro-inflammatory cytokines such as IL-1 [ 27 ]. In chorioamnionitis, diminished IL-10 levels may provide an ineffective counter regulatory response to elevated maternal prostaglandin stimulated by the pro-inflammatory cytokines IL-1, IL-6 and TNF. Elevated levels of these pro-inflammatory cytokines in utero may serve as a maternal [ 28 ] and fetal [ 29 ] signal for the onset of premature parturition. The higher proportion of early PTB in women with the  IL10 ATA  haplotype may also be a reflection of failure of tocolysis in the presence of infection [ 26 ]. Our finding that the carriage of the  IL10 ATA  haplotype is more common in women with histologic chorioamnionitis suggests that this haplotype may be important in the pathogenesis of histologic chorioamnionitis and subsequent PTB.IL10MBL2IL10 ATAIL10 ATAIL10 ATAIL10 ATAMBL is an important innate defense molecule active against a broad range of bacterial, viral, fungal and protozoan pathogens [ 30 ]. The  MBL2  variant alleles in exon 1 correlate with decreased circulating MBL. The single nucleotide substitution of an adenine for a guanine in codon 54 results in replacement of aspartic acid for a glycine in the MBL [ 31 ]. Functional MBL is multimeric, consisting of tetramers, pentamers and hexamers of triplets of the basic MBL peptide. This higher order structure is necessary for high avidity interactions between MBL carbohydrate recognition domains and repeated oligosaccharide moieties on pathogens. The MBL codon 54Asp 'B' allele disrupts assembly of  MBL2  peptide trimers resulting in lower stability and serum levels [ 32 ]. Low serum levels of MBL and  MBL2  genetic variants are associated with infection in a range of contexts including recurrent infection in children and adults [ 6 , 14 ], recurrent vulvovaginal candidiasis [ 33 ], recurrent miscarriage [ 34 ] and autoimmunity in common variable immunodeficiency disease [ 18 ]. The association between  MBL2  codon 54Asp and histologic chorioamnionitis suggests that this allele may result in low MBL levels, impaired opsonisation and clearance of pathogens, thus facilitating the development of chorioamnionitis.MBL2MBL2MBL2MBL2Fas protein is expressed on the surface of many cell types, such as lymphocytes, epithelial, fibroblasts and certain endothelial cells, and cytokines [ 7 ] and hormones [ 35 ] found in the placental microenvironment may modulate the immune response by regulating the expression of Fas and Fas ligand. In this study two polymorphisms in the Fas ( TNFRSF6 ) gene promoter were studied at positions -1377 and -670, and the -1377A/-670G ( AG ) haplotype was negatively associated with histologic chorioamnionitis. The -1377G/A is situated at the transcription factor SP-1 binding site [ 22 ], and the -670A/G substitution is located in the enhancer region and abolishes the binding site of the nuclear transcription element GAS (Gamma Interferon Activation Sequence). The functional significance of these polymorphisms has not been fully elucidated but they may modulate  TNFRSF6  transcription, Fas expression and thus apoptotic and inflammatory responses. Recent evidence suggests that cells are more sensitive to Fas-mediated apoptosis when levels of Fas expression increase [ 36 ]. Apoptotic cell death can modulate host containment of pathogens [ 19 ] and thus genetically determined variation in Fas-mediated cell death may influence histologic chorioamnionitis.TNFRSF6AGTNFRSF6Reported studies on gene polymorphisms and adverse pregnancy outcome have mostly examined SNPs in the genes encoding the pro-inflammatory cytokines TNF and IL-1. The  TNF  promoter -308A allele was associated with clinical chorioamnionitis in a small number of women [ 12 ]. The  in vitro  functional data of the effects of the  TNF  SNPs are conflicting and may be context dependent and influenced by strong linkage disequilibrium with other intra-and-extra-genic polymorphisms (e.g. with lymphotoxin alpha and classical HLA genes [ 37 ], not examined in this study). In contrast, in this study the  TNF -308A could not be identified as a susceptibility or severity factor for chorioamnionitis.TNFin vitroTNFTNFSNPs of the IL1 cluster have been examined from different ethnic groups, in particular  IL1B  and  IL1RN  in women with a history of recurrent pregnancy loss [ 38 ], PTB [ 39 ], and in  in vitro  studies[ 11 ]. We did not find associations between genes of the IL1 cluster or  IL1R1  and histologic chorioamnionitis and this may partly be explained by differences in linkage disequilibrium patterns across different ethnic populations that can lead to different association results [ 40 ].IL1BIL1RNin vitroIL1R1Our data support the role of  IL10 ,  MBL2  and  TNFRSF6  variants in determining the risk of histologic chorioamnionitis. However there are limitations to the study, such as small sample size. The range and number of gene polymorphisms examined and analysed by univariate and multiple logistic regression in this study is notable, but some correction for multiple analyses is required [ 41 ]. The appropriate method still remains a contentious issue. The Bonferroni method was not conducted, as this is a conservative method and leads to overcorrection [ 42 ]. Also our study is a hypothesis-generating exploratory study. SNP frequencies in women with and without chorioamnionitis are available to be included in meta-analysis to confirm or reject these findings ( Additional file 1 ).IL10MBL2TNFRSF6Conclusion In conclusion, the frequency differences in this study highlight the significance and biological relevance of genetic factors, IL-10, MBL and Fas regulation among women with acute chorioamnionitis and adverse pregnancy outcome.
Conclusion
In conclusion, the frequency differences in this study highlight the significance and biological relevance of genetic factors, IL-10, MBL and Fas regulation among women with acute chorioamnionitis and adverse pregnancy outcome.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions MA  contributed to study design, recruited study subjects, abstracted medical records, entered data, performed preliminary statistical analyses and drafted the manuscript. PH  provided design regarding immunoregulatory genes, interpretation of results and preparation of manuscript. CM  conceived methodology and supervision of PCR testing, interpretation of test results and preparation of manuscript. SH  supervised preparation of DNA and performed all PCR tests. JR  provided clinical authorisation and support for the study. Provided input regarding clinical database design, and provided critical analysis for obstetric correctness. HM  conceived and coordinated the study, conceived medical record review, reviewed statistical analyses and preparation of manuscript. All authors read and approved the final manuscript.
Authors' contributions
MA  contributed to study design, recruited study subjects, abstracted medical records, entered data, performed preliminary statistical analyses and drafted the manuscript.MAPH  provided design regarding immunoregulatory genes, interpretation of results and preparation of manuscript.PHCM  conceived methodology and supervision of PCR testing, interpretation of test results and preparation of manuscript.CMSH  supervised preparation of DNA and performed all PCR tests.SHJR  provided clinical authorisation and support for the study. Provided input regarding clinical database design, and provided critical analysis for obstetric correctness.JRHM  conceived and coordinated the study, conceived medical record review, reviewed statistical analyses and preparation of manuscript.HMAll authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:Supplementary Material Additional File 1 Gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation and histologic chorioamnionitis. Frequency distribution of immunoregulatory gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation stratified by histologic chorioamnionitis. Click here for file
Supplementary Material
Additional File 1
Gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation and histologic chorioamnionitis. Frequency distribution of immunoregulatory gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation stratified by histologic chorioamnionitis.Click here for file


This issue of CytoJournal contains an article on screening for anal intraepithelial neoplasia in high-risk male patients. This accompanying Editorial focuses on current understanding of this relatively new disease entity, with insights as to the potential role of screening cytopathology in the epidemiology, pathophysiology and clinical management of this HIV and HPV related anal lesion, which predominates in male patients living long-term with AIDS. Mention is made of techniques of obtaining samples, methods of preparation, and morphologic classification. Issues of anoscopic confirmation, as well as topical and surgical management are emphasized. The similarity of initial experiences in anal screening to problems encountered early in cervical cancer screening programs several decades ago, are highlighted.

Note For corresponding research article please see Arian et al, 2005 [ 15 ]
Note
For corresponding research article please see Arian et al, 2005 [ 15 ]The subject of anal screening cytology has entered the epidemiologic and cytopathologic literature as a topic of interest over the last decade, and is highlighted in this issue of CytoJournal in an article by Arain and colleagues. Until recently, anal cancer was not considered to be a neoplasm of major public health concern [ 1 ]. It occurred infrequently, usually in older people, affecting women more often than men; further, being a neoplasm of low incidence, it remained under the radar in terms of screening potential. In some respects, anal cancer mirrored cervical cancer in unscreened women, presenting late in the course of the disease with a variety of pelvic symptoms, and having a protracted, ultimately fatal, course. Patients invariably became social outcasts, suffering from intolerable fecal complications, which presented major nursing challenges. Two aspects of this scenario have changed. First was the fairly recent introduction of effective modern treatment regimes for invasive anal squamous cancers utilizing chemo-radiation, leading to improvement in morbidity and long term survival [ 2 , 3 ]. The second significant alteration was seen in epidemiology, and this is the area which has come to involve screening cytopathology. During the 1990s, in several European and North American cancer centers, an initially unaccountable increase in anal cancers was seen in younger people. It soon became apparent that HIV-positive homosexual males were affected in numbers greatly in excess of those expected. This was first noted in those urban areas in which large concentrations of homosexual men had been treated since the outbreak of the HIV epidemic. The term "males having sex with males" (MSM) was coined to cover these high-risk individuals. Preliminary information gleaned from screening programs in high HIV-positive incidence areas amongst MSM, showed detection rates of intraepithelial and early invasive neoplasia higher than any incidence ever recorded for cervical cancer screening. Further, HIV-negative homosexual MSMs, and HIV positive non-homosexual men (eg drug users) also exhibited an increased incidence of pre-malignant and invasive anal carcinomas. More recently, the syndrome of early onset anal cancer has been extended to include HIV-positive female patients, as well as females who are not HIV-positive, but who have genital HPV. All these groups show a higher incidence of abnormal anal cytopathology, though none quite as high as that found in HIV positive MSM. The unifying factor in most instances is ano-receptive intercourse, or extension of HPV infection from the genital tract to the anal mucosa, most obvious in the face of deficient immunity, or high viral load [ 4 - 6 ]. Epidemiologic studies have indicated that a prolonged preclinical phase precedes the onset of anal cancer in these high-risk groups. Just as in the cervix, there is a transitional zone (although not an abrupt squamocolumnar junction) in the anus. Rectal mucosa, with goblet cells, ends about one inch proximal to the external sphincter, giving way to a transitional epithelium, which in turn blends into a stratified squamous epithelium at the level of the anus. (External to the anus, any lesions which arise are considered to be primary skin lesions rather than anal lesions.) Almost all anal cancers develop in the transitional zone, where atypical, dysplastic and in-situ lesions are identifiable histologically. The cytologic counterparts are those of atypical cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL). Once this natural history had been demonstrated and confirmed, it seemed natural that exfoliative cytology could be investigated as an "anal Pap smear" [ 7 , 8 ]. As with the cervix, the technique of obtaining the anal sample is critical to the success of screening. Standard colonic preparation is not required, but the rectum should be emptied prior to obtaining the anal sample. Brushes, brooms and Dacron swabs have all been used, the type of spatula probably being less important than the skill of the operator. The instrument is inserted to a depth of one and a half inches beyond the external sphincter, and subsequently withdrawn in a firm downward spiral movement incorporating 10–12 rotations, to ensure the device has made contact with the full surface area of the transformation zone. Some have used direct smears onto glass slides with immediate wet fixation; most centers, however, employ immediate insertion of the scraping device into liquid fixative for thin layer preparation. This appears both to improve adequacy and preservation, and also to eliminate any fecal contamination; residual material in the vial can be used for HPV studies if required, or for the creation of a bank of teaching slides. There has been a relative dearth of literature on the cytomorphology of anal samples [ 9 - 11 ]. Classification according to Bethesda guidelines has been advised, implying similarity of the exfoliative cytology of atypical squamous cells of undetermined significance (ASC-US), anal intraepithelial lesions (A-SIL) and invasive squamous-cell neoplasms to those encountered in the cervix. This early in the development of the science of anal screening, and evident also in the current article in CytoJournal, there are indications that cytologic evaluation may not fully anticipate the severity of some lesions, when compared with biopsies taken simultaneously. Several groups have advised that all abnormal anal Paps should be followed by anoscopic evaluation, with biopsy confirmation when necessary. Anoscopy essentially mirrors colposcopy, enabling the viewer to see vascular abnormalities at magnification, and direct biopsies to the most abnormal-appearing areas. Despite these basic similarities, it is strongly stated in the literature that expertise in colposcopy does not equate to immediate competence in anoscopy; a significant learning curve exists for those wishing to acquire excellence in anoscopic technique, with associated accurate biopsy sampling. It is important that the anoscopist not be sidelined by visible condylomata, which may merely be sentinels of deeper flat lesions of higher grade. At the present time, lack of available expertise in this interventional follow-up of abnormal anal Paps may be the single limiting factor in any new screening program. It thus behooves those interpreting anal samples to be as proficient as they can be in pre-interventional assessment of the anal transformation zone. Experience over the last decade suggests that anal cancer may be as highly appropriate a target for screening as is the cervix, in selected populations. The neoplasm is frequently encountered in well-defined high-risk groups. It has a detectable pre-malignant phase, and is amenable to easy cytologic sampling. Cytodiagnosis is reasonably sensitive and highly specific, and histologic confirmation is relatively easily obtained by well-trained personnel. If there is a current area of deficiency in such programs, it may well be in the treatment of intraepithelial lesions, which, as yet, has not been adequately assessed in large numbers of patients. It is known that highly active anti-retroviral therapy (HAART) does not appear to alter the pathophysiology of anal lesions once initiated. A variety of topical agents such as podophyllotoxin and imiquimod have been tried, as has intralesional interferon; superficial ablative therapies including liquid nitrogen, electrocautery, laser and LEEP have been attempted with varying success rates. Circumferential surgical resection almost inevitably results in unacceptable loss of sphincter control and soiling, but anoscopy-directed limited excision may prove less morbid. The fact that so many options are available implies perhaps that no single modality is yet considered sufficiently effective, with minimal complication [ 12 , 13 ]. This, too, is reminiscent of the early years of management of cervical pre-neoplasia, when a host of methods was pursued in attempted elimination of focal lesions of the squamocolumnar junction. As in the cervix, human ingenuity will undoubtedly prevail, and one or two forms of extirpation will emerge as both efficacious and uncomplicated. An interesting consideration is whether or not anal cancer, and thereby anal cytopathology screening programs, would be of value in the developing world, particularly in Africa, India and China, where the bulk of the global incidence of HIV resides. This will depend on two very different factors. First is the nature of transmission. Unlike the situation in the developed world, AIDS in these regions is not essentially a disease of homosexual males; thus, without anal intercourse predominating, an upward trend in the incidence of anal cancer would seem unlikely. Anal screening programs would be unnecessary or cost- in effective in these communities. The second feature dictating the institution of screening programs in developing countries relates to antiretroviral therapy. Anal intraepithelial neoplasia and cancer are not encountered early in the progression of HIV/AIDS. Rather, they are late complications of patients living long-term with AIDS, usually implying patients living long-term on HAART [ 14 ]. Unless affordable very low cost antiretroviral drugs could be manufactured and distributed widely, it seems unlikely that patients in developing countries would survive into the time zone in which delayed neoplasms such as anal cancer become a public health priority.

The subject of anal screening cytology has entered the epidemiologic and cytopathologic literature as a topic of interest over the last decade, and is highlighted in this issue of CytoJournal in an article by Arain and colleagues. Until recently, anal cancer was not considered to be a neoplasm of major public health concern [ 1 ]. It occurred infrequently, usually in older people, affecting women more often than men; further, being a neoplasm of low incidence, it remained under the radar in terms of screening potential. In some respects, anal cancer mirrored cervical cancer in unscreened women, presenting late in the course of the disease with a variety of pelvic symptoms, and having a protracted, ultimately fatal, course. Patients invariably became social outcasts, suffering from intolerable fecal complications, which presented major nursing challenges. Two aspects of this scenario have changed. First was the fairly recent introduction of effective modern treatment regimes for invasive anal squamous cancers utilizing chemo-radiation, leading to improvement in morbidity and long term survival [ 2 , 3 ]. The second significant alteration was seen in epidemiology, and this is the area which has come to involve screening cytopathology.During the 1990s, in several European and North American cancer centers, an initially unaccountable increase in anal cancers was seen in younger people. It soon became apparent that HIV-positive homosexual males were affected in numbers greatly in excess of those expected. This was first noted in those urban areas in which large concentrations of homosexual men had been treated since the outbreak of the HIV epidemic. The term "males having sex with males" (MSM) was coined to cover these high-risk individuals. Preliminary information gleaned from screening programs in high HIV-positive incidence areas amongst MSM, showed detection rates of intraepithelial and early invasive neoplasia higher than any incidence ever recorded for cervical cancer screening. Further, HIV-negative homosexual MSMs, and HIV positive non-homosexual men (eg drug users) also exhibited an increased incidence of pre-malignant and invasive anal carcinomas. More recently, the syndrome of early onset anal cancer has been extended to include HIV-positive female patients, as well as females who are not HIV-positive, but who have genital HPV. All these groups show a higher incidence of abnormal anal cytopathology, though none quite as high as that found in HIV positive MSM. The unifying factor in most instances is ano-receptive intercourse, or extension of HPV infection from the genital tract to the anal mucosa, most obvious in the face of deficient immunity, or high viral load [ 4 - 6 ].Epidemiologic studies have indicated that a prolonged preclinical phase precedes the onset of anal cancer in these high-risk groups. Just as in the cervix, there is a transitional zone (although not an abrupt squamocolumnar junction) in the anus. Rectal mucosa, with goblet cells, ends about one inch proximal to the external sphincter, giving way to a transitional epithelium, which in turn blends into a stratified squamous epithelium at the level of the anus. (External to the anus, any lesions which arise are considered to be primary skin lesions rather than anal lesions.) Almost all anal cancers develop in the transitional zone, where atypical, dysplastic and in-situ lesions are identifiable histologically. The cytologic counterparts are those of atypical cells of uncertain significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL). Once this natural history had been demonstrated and confirmed, it seemed natural that exfoliative cytology could be investigated as an "anal Pap smear" [ 7 , 8 ].As with the cervix, the technique of obtaining the anal sample is critical to the success of screening. Standard colonic preparation is not required, but the rectum should be emptied prior to obtaining the anal sample. Brushes, brooms and Dacron swabs have all been used, the type of spatula probably being less important than the skill of the operator. The instrument is inserted to a depth of one and a half inches beyond the external sphincter, and subsequently withdrawn in a firm downward spiral movement incorporating 10–12 rotations, to ensure the device has made contact with the full surface area of the transformation zone. Some have used direct smears onto glass slides with immediate wet fixation; most centers, however, employ immediate insertion of the scraping device into liquid fixative for thin layer preparation. This appears both to improve adequacy and preservation, and also to eliminate any fecal contamination; residual material in the vial can be used for HPV studies if required, or for the creation of a bank of teaching slides.There has been a relative dearth of literature on the cytomorphology of anal samples [ 9 - 11 ]. Classification according to Bethesda guidelines has been advised, implying similarity of the exfoliative cytology of atypical squamous cells of undetermined significance (ASC-US), anal intraepithelial lesions (A-SIL) and invasive squamous-cell neoplasms to those encountered in the cervix. This early in the development of the science of anal screening, and evident also in the current article in CytoJournal, there are indications that cytologic evaluation may not fully anticipate the severity of some lesions, when compared with biopsies taken simultaneously. Several groups have advised that all abnormal anal Paps should be followed by anoscopic evaluation, with biopsy confirmation when necessary. Anoscopy essentially mirrors colposcopy, enabling the viewer to see vascular abnormalities at magnification, and direct biopsies to the most abnormal-appearing areas. Despite these basic similarities, it is strongly stated in the literature that expertise in colposcopy does not equate to immediate competence in anoscopy; a significant learning curve exists for those wishing to acquire excellence in anoscopic technique, with associated accurate biopsy sampling. It is important that the anoscopist not be sidelined by visible condylomata, which may merely be sentinels of deeper flat lesions of higher grade. At the present time, lack of available expertise in this interventional follow-up of abnormal anal Paps may be the single limiting factor in any new screening program. It thus behooves those interpreting anal samples to be as proficient as they can be in pre-interventional assessment of the anal transformation zone.Experience over the last decade suggests that anal cancer may be as highly appropriate a target for screening as is the cervix, in selected populations. The neoplasm is frequently encountered in well-defined high-risk groups. It has a detectable pre-malignant phase, and is amenable to easy cytologic sampling. Cytodiagnosis is reasonably sensitive and highly specific, and histologic confirmation is relatively easily obtained by well-trained personnel. If there is a current area of deficiency in such programs, it may well be in the treatment of intraepithelial lesions, which, as yet, has not been adequately assessed in large numbers of patients. It is known that highly active anti-retroviral therapy (HAART) does not appear to alter the pathophysiology of anal lesions once initiated. A variety of topical agents such as podophyllotoxin and imiquimod have been tried, as has intralesional interferon; superficial ablative therapies including liquid nitrogen, electrocautery, laser and LEEP have been attempted with varying success rates. Circumferential surgical resection almost inevitably results in unacceptable loss of sphincter control and soiling, but anoscopy-directed limited excision may prove less morbid. The fact that so many options are available implies perhaps that no single modality is yet considered sufficiently effective, with minimal complication [ 12 , 13 ]. This, too, is reminiscent of the early years of management of cervical pre-neoplasia, when a host of methods was pursued in attempted elimination of focal lesions of the squamocolumnar junction. As in the cervix, human ingenuity will undoubtedly prevail, and one or two forms of extirpation will emerge as both efficacious and uncomplicated.An interesting consideration is whether or not anal cancer, and thereby anal cytopathology screening programs, would be of value in the developing world, particularly in Africa, India and China, where the bulk of the global incidence of HIV resides. This will depend on two very different factors. First is the nature of transmission. Unlike the situation in the developed world, AIDS in these regions is not essentially a disease of homosexual males; thus, without anal intercourse predominating, an upward trend in the incidence of anal cancer would seem unlikely. Anal screening programs would be unnecessary or cost- in effective in these communities. The second feature dictating the institution of screening programs in developing countries relates to antiretroviral therapy. Anal intraepithelial neoplasia and cancer are not encountered early in the progression of HIV/AIDS. Rather, they are late complications of patients living long-term with AIDS, usually implying patients living long-term on HAART [ 14 ]. Unless affordable very low cost antiretroviral drugs could be manufactured and distributed widely, it seems unlikely that patients in developing countries would survive into the time zone in which delayed neoplasms such as anal cancer become a public health priority.in