Whole blood RNA sequencing reveals a differential transcriptomic profile associated with cervical insufficiency: a pilot study.

BACKGROUND: The uterine cervix is a mechanical and immunological barrier against ascending infection during pregnancy. Cervical insufficiency (CI), a painless cervical dilation that occurs in the mid-trimester, is an important cause of extremely preterm birth. We hypothesized that women with CI have a differential transcriptomic profile. Therefore, we compared the transcriptomic profile of peripheral blood in women with CI and that of controls. METHODS: RNA sequencing was used to generate the global gene expression profiles of 11 women with CI and 4 controls, and differential expression analysis was performed to identify genes showing significant expression changes between the CI (n = 11) and control (n = 4) groups as well as between the CI-preterm (n = 7) and CI-term (n = 4) groups. Gene set enrichment was assessed in terms of Gene Ontology processes, and a subset of differentially expressed genes in CI was validated in a different sample-set by qRT-PCR and ELISA. RESULTS: Thirty genes were differentially expressed between the CI and control groups. Differentially upregulated genes in the CI group included neutrophil-mediated immunity-associated (DEFA3 and ELANE) and bicarbonate transport-related genes. The serum concentration of alpha defensin 3 was significantly higher in women with CI than in controls (P = 0.014). Analysis of differential gene expression according to pregnancy outcomes revealed 338 differentially expressed genes between the CI-term and CI-preterm groups. Immune and defense response to organism-associated genes and influenza A and NOD-like receptor signaling pathways were upregulated in the CI-term group. CONCLUSIONS: Our results revealed significant differences in the whole blood transcriptomic profiles of women with CI compared to those of controls. Different immune responses in women with CI may affect pregnancy outcomes.

COL1A1, COL4A3, TIMP2 and TGFB1 polymorphisms in cervical insufficiency.

OBJECTIVES: To investigate the association between selected single nucleotide polymorphisms (SNPs) with cervical insufficiency and its relationship with obstetric history. METHODS: Twenty-eight women with cervical insufficiency (case group) and 29 non-pregnant women (control group) were included. The SNPs sequenced included rs2586490 in collagen type I alpha 1 chain (COL1A1), rs1882435 in collagen type IV alpha 3 chain (COL4A3), rs2277698 in metallopeptidase inhibitor 2 (TIMP2), and rs1800468 in transforming growth factor beta 1 (TGFB1). RESULTS: We found a higher frequency of the normal allele in the control group (65.5%) and the homozygous mutated genotype in the case group (64.3%) for rs2586490 in COL1A1 (p=0.023). An unplanned finding in the cervical insufficiency group was a higher gestational age of delivery (median≥38 weeks) in the mutated allele than in the wild-type genotype (median of 28.2 weeks) for rs2857396, which is also in the COL1A1 gene (p=0.011). CONCLUSIONS: The findings of the present study corroborate the hypothesis that cervical insufficiency has a genetic component and probably involves genes encoding proteins in the extracellular matrix, in addition to inflammatory processes.

The genomics of prematurity in an era of more precise clinical phenotyping: A review.

Spontaneous preterm birth is a major public health problem, with a clear genetic component. Genetic association studies have evolved substantially in recent years, moving away from the traditional candidate gene analyses to newer approaches utilizing sophisticated analysis platforms to examine sequencing data, and shifting towards functional studies including methylation analysis. It is becoming increasingly evident that careful clinical phenotyping is crucial to high quality genetic association studies regardless of the assay or platform being used. Nonetheless, genetic studies of prematurity are hampered by numerous challenges including small sample sizes, incomplete phenotying, population stratification, and multiple comparisons. As the costs of sequencing and functional analyses continue to decrease, unbiased genome-wide assays will be more widely available. Researchers have met improved success recently when critically applying clinical phenotyping knowledge to group women prior to analyzing genotyping results. Eventually, as the analytic approaches evolve, it is likely that this methodology (combining precisely clinically phenotyped subjects with genome-wide data) will provide key information regarding the pathophysiology of prematurity, and provide potential new avenues for exploring innovative therapeutic strategies.

Polymorphisms in Genes Coding for Cytokines, Mannose-Binding Lectin, Collagen Metabolism and Thrombophilia in Women with Cervical Insufficiency.

OBJECTIVE: To study the association between cervical insufficiency and single nucleotide polymorphisms in seven genes coding for pro- and anti-inflammatory cytokine-related factors, mannose-binding lectin 2 (MBL2), collagen1α1 (COL1A1), factor II and factor V Leiden genes. METHODS: In a case-control study, potential maternal biomarkers for cervical insufficiency were investigated in 30 women with a history of second-trimester miscarriage or preterm birth due to cervical insufficiency and in 70 control women. RESULTS: Homozygous carriers of the interleukin 6 (IL6) -174 genotype GG had an odds ratio (OR) of 3.1 [95% confidence interval (95% CI) 1.3-7.4, p = 0.01] and MBL2 genotypes coding for low or intermediate levels of plasma MBL had an OR of 3.3 (95% CI 1.2-9.0, p = 0.01) for cervical insufficiency compared with controls. Serum MBL levels were lower in women with cervical insufficiency than in controls (median 408 and 1,985 ng/ml, respectively, p < 0.01). CONCLUSIONS: Single nucleotide polymorphisms in the IL6 gene and the MBL2 gene and low MBL levels related to the latter polymorphism may increase the risk of preterm birth due to cervical insufficiency.

The association of beta-2 adrenoceptor genotype with short-cervix mediated preterm birth: a case-control study.

OBJECTIVE: To determine whether ß2 -adrenoceptor (ß2 AR) genotype is associated with shortening of the cervix or with preterm birth (PTB) risk among women with a short cervix in the second trimester. DESIGN: A case-control ancillary study to a multicentre randomised controlled trial. SETTING: Fourteen participating centres of the Maternal-Fetal Medicine Units Network of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. POPULATION: Four hundred thirty-nine women, including 315 with short cervix and 124 with normal cervical length. METHODS: Nulliparous women with cervical length <30 mm upon a 16-22-week transvaginal sonogram and controls frequency-matched for race/ethnicity with cervical lengths ≥40 mm were studied. ß2 AR genotype was determined at positions encoding for amino acid residues 16 and 27. MAIN OUTCOME MEASURES: Genotype distributions were compared between case and control groups. Within the short cervix group, pregnancy outcomes were compared by genotype, with a primary outcome of PTB <37 weeks. RESULTS: Genotype data were available at position 16 for 433 women and at position 27 for 437. Using a recessive model testing for association between short cervix and genotype, and adjusted for ethnicity, there was no statistical difference between cases and controls for Arg16 homozygosity (OR 0.7, 95% CI 0.4-1.3) or Gln27 homozygosity (OR 0.9, 95% CI 0.3-2.7). Among cases, Arg16 homozygosity was not associated with protection from PTB or spontaneous PTB. Gln27 homozygosity was not associated with PTB risk, although sample size was limited. CONCLUSIONS: ß2 AR genotype does not seem to be associated with short cervical length or with PTB following the second-trimester identification of a short cervix. Influences on PTB associated with ß2 AR genotype do not appear to involve a short cervix pathway.

Free fetal DNA levels in patients at risk of preterm labour.

OBJECTIVES: Our main goals were to evaluate the capability of ffDNA to increase the accuracy in prediction of preterm labour by cervical length and to explore potential mechanisms of disease associated with this pathology. METHODS: Fifty-six women, with male fetus, with cervical length assessment at between 22 and 24 weeks were included in the study and divided in 1) Short cervix (<15 mm) delivered at term (T = 20); 2) Short cervix delivered before 37 weeks (PT = 14); and 3) Patients who delivered at term with normal cervical length (N = 22). Maternal plasma samples were collected between 22 and 24 weeks of gestational age. PCR using primers against DYS14 gene were used to quantified ffDNA in plasma samples. Statistical analysis was done using ANOVA test and spearman´s correlation. RESULTS: The median gestational age at delivery for short cervix groups was 26 + 1 for PT and 39 + 3 for T. The control group delivered at a median gestational age of 39 + 6 weeks. ffDNA was detectable in all cases. There was no significant difference between the 3 groups. Similarly, no significant correlation was observed between ffDNA and gestational age at delivery (r = -0.23; p = 0.07). CONCLUSIONS: ffDNA does not increase the accuracy of short cervix at between 22 and 24 weeks for the prediction of preterm labour.

Genetics of the cervix in relation to preterm birth.

Preterm birth is the most significant problem encountered in obstetrics in the developed world. Genetic factors are thought to play a role in a proportion of preterm births, and candidate genes have been studied in several areas relevant to parturition. Abnormal cervical function, a clinical spectrum, including cervical insufficiency (CI), is a contributing factor to the overall problem of preterm birth. There are many risk factors and etiologies for CI. However, it is becoming clear that, at least in part, a genetic predisposition to CI plays a role in the condition. Specifically, genes related to connective tissue metabolism and inflammation have been shown to be associated with CI.

Discussion: 'the interleukin-10 gene and cervical insufficiency' by Warren et al.

In the roundtable that follows, clinicians discuss a study published in this issue of the Journal in light of its methodology, relevance to practice, and implications for future research. Article discussed: Warren JE, Nelson LM, Stoddard GJ, et al. Polymorphisms in the promoter region of the interleukin-10 (IL-10) gene in women with cervical insufficiency. Am J Obstet Gynecol 2009;201:372.e1-5.

Polymorphisms in the promoter region of the interleukin-10 (IL-10) gene in women with cervical insufficiency.

OBJECTIVE: Our objective was to determine whether polymorphisms in the promoter region of the interleukin-10 gene are more common in women with cervical insufficiency compared with controls. STUDY DESIGN: We conducted a case-control study. Histories and blood were obtained from 121 cases and 157 controls. DNA was extracted and purified by using Puregene kits. Samples were analyzed for the interleukin-10 -1082 G/A polymorphism and interleukin-10.G microsatellite in the interleukin-10 gene promoter region. RESULTS: The -1082 G/A polymorphism in the promoter region of the interleukin-10 gene occurred with similar frequency in cases and controls. The interleukin-10.G microsatellite contained 10 alleles (G6-G16). The G13 allele was present more frequently in cases (24.1%) compared with controls (14.6%) (P = .05). CONCLUSION: Interleukin-10 is known to down-regulate inflammation. The G13 allele in the interleukin-10.G microsatellite occurred more frequently in women with cervical insufficiency compared with controls, suggesting that alterations in inflammatory processes may play a role in cervical insufficiency.

Connective tissue and related disorders and preterm birth: clues to genes contributing to prematurity.

To identify candidate genes contributing to preterm birth, we examined the existing literature on the association between known disorders of connective tissue synthesis and metabolism and related diseases and prematurity. Our hypothesis was that abnormal matrix metabolism contributes to prematurity by increasing risk of preterm premature rupture of membranes (PPROM) and cervical incompetence. Based on this review, we identified gene mutations inherited by the fetus that could predispose to preterm birth as a result of PPROM. The responsible genes include COL5A1, COL5A2, COL3A1, COL1A1, COL1A2, TNXB, PLOD1, ADAMTS2, CRTAP, LEPRE1 and ZMPSTE24. Marfan syndrome, caused by FBN1 mutations, and polymorphisms in the COL1A1 and TGFB1 genes have been associated with cervical incompetence. We speculate that an analysis of sequence variation at the loci noted above will reveal polymorphisms that may contribute to susceptibility to PPROM and cervical incompetence in the general population.

Collagen 1Alpha1 and transforming growth factor-beta polymorphisms in women with cervical insufficiency.

OBJECTIVE: To estimate whether polymorphisms in the collagen 1Alpha1 gene (COL1Alpha1) and the transforming growth factor-beta gene (TGF-beta;1) are more common in women with cervical insufficiency than in those without the condition. METHODS: Medical, obstetric, and family histories and blood were obtained from women with (n=121) and those without (n=165) cervical insufficiency. DNA was extracted and purified by using commercial DNA isolation kits. Samples were analyzed for variants in two genes, the COL1A1 intron 1SP1 and TGF-beta Arg-25-Pro polymorphism, by using an allele-specific polymerase chain reaction assay. RESULTS: Thirty-four of 125 (27.2%) women with cervical insufficiency had at least one first-degree female relative affected. The frequency of the homozygous TT genotype in the COL1A1 gene was increased in women with a history of cervical insufficiency compared with controls (10.8% compared with 3.1%, P=.04). The TGF-beta polymorphisms (ArgPro and ProPro) also were increased in cases (38.3% compared with 14.6%, P<.001). CONCLUSION: Over one fourth of women with cervical insufficiency have a family history of cervical insufficiency, and the COL1A1 intron 1SP1 and TGF-beta Arg-25-Pro polymorphisms are associated with the condition. These observations suggest that, in part, cervical insufficiency is mediated by genetic factors. LEVEL OF EVIDENCE: II.

[Marfan syndrome in pregnancy: presentation of four cases and discussion]. / Das Marfan-Syndrom in der Schwangerschaft: Vorstellung von vier Kasuistiken und Diskussion dieser Koinzidenz.

The coincidence of Marfan syndrome and pregnancy means a high risk for mother and child, as it is associated with cardiovascular and obstetric complications. We report our experience of four pregnancies with the Marfan syndrome. The course of pregnancy, the peripartum management and both the maternal and neonatal outcomes of four pregnant women with the Marfan syndrome, who were treated in our department between 1995 and 2005, were retrospectively analysed. The pregnancies of two women were complicated by premature rupture of membranes (36 (th) gestational week) and premature uterine contractions with cervical incompetence (30 (th) gestational week), respectively. One patient developed class 3 (NYHA) heart failure in the 3 (rd) trimenon. Two out of four women had mild cardiovascular disease and could deliver vaginally. In the other two cases a primary Caesarean section was performed at the 36 (th) week of gestation because of severe cardiovascular morbidity. No patient had a progressive aortic dilatation, dissection or rupture. The neonatal outcome was uneventful in all cases. Three newborns underwent a genetic evaluation for the Marfan syndrome, in two of them mutations in the fibrillin 1 gene were detected. Women with the Marfan syndrome should be counselled pre-conception and observed by an interdisciplinary team during pregnancy. If the aortic root diameter is < 40 mm, without progression in pregnancy, and in the absence of severe valve insufficiency, then pregnancy is in most cases well tolerated and vaginal delivery can be performed.

The effect of combined therapy with ritodrine, erythromycin and verapamil on the frequency of micronuclei in peripheral blood lymphocytes of pregnant women.

The main aim of this study was to investigate the genotoxic effect of combined pharmacotherapy applied in post-operative treatment of cervical cerclage in pregnant women over six days. This study included 19 phenotypically healthy pregnant women in mid-trimester with a diagnosis of cervical insufficiency, mean age 28+/-5.33. The frequency of micronuclei (MN) was estimated in peripheral blood lymphocytes of patients before surgical intervention and after the end of applied pharmacotherapy by application of cytokinesis block micronucleus (CBMN) test. Mean value of baseline MN frequency was 6.84+/-2.91 MN/1000 binucleated cells, and after the end of the applied therapy, 10.32+/-4.27 MN/1000 binucleated cells (P<0.001) were found. The data of cell proliferation index showed that the combined therapy did not induce significant difference in cell kinetics (P>0.05). Our results showed that combined pharmacotherapy treatment over six days significantly increased the frequency of MN in peripheral blood lymphocytes of pregnant women.

Relationship between tumor necrosis factor-alpha genotype and success of emergent cerclage.

The guanine to adenine substitution at the -308 position in the tumor necrosis factor-alpha (TNF-alpha) gene promoter region results in a 5-fold greater cytokine response to an inciting event. We investigated whether this polymorphism is associated with cervical incompetence and adverse pregnancy outcome after emergent cerclage. Women with a diagnosis of cervical incompetence requiring an emergent cerclage between 15 and 24 weeks were enrolled. Women without pregnancy complications were recruited as controls. DNA extraction from peripheral blood and polymerase chain reaction (PCR) amplification of a 144-base pair segment of the TNF-alpha gene were performed with subsequent sequencing. Twenty-three women underwent emergent cerclage and participated in the study, 13 (57%) of whom delivered after 28 weeks. Twenty-three women served as controls. There were no differences in the TNF-alpha polymorphism between women with cervical incompetence and controls or between women with cervical incompetence who delivered before versus after 28 weeks. The TNF-alpha polymorphism was not associated with cervical incompetence or with delivery prior to 28 weeks in women who received an emergent cerclage.