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ORIGINAL RESEARCH |
From the 1Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Salt Lake City, Utah; and 2Taueret Laboratories, Salt Lake City, Utah.
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ABSTRACT |
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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-ß 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-ß 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-ß 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
These observations raise the question of whether genes related to cervical connective tissue play a role in the pathophysiology of this condition. There are two genes that have been associated with abnormalities of connective tissue and extracellular matrix, the collagen 1alpha1 (COL1A1) gene and the transforming growth factor-ß (TGF-ß) gene. Grant et al4 first described a G to T polymorphism in the first intron of the COL1A1 gene at the binding site for the transcription factor Sp1. This polymorphism has been associated with low bone mineral density and increased incidence of osteoporotic fracture.4 Others also have found an association between bone density and polymorphisms in this region of the gene.5 Similarly, polymorphisms of the gene encoding for TGF-ß, leading to increases or decreases in TGF-ß, influence the interaction between cells and extracellular matrix. In turn, these polymorphisms have been associated with disease states characterized by perturbations of connective tissue. Thus, our objectives were to estimate whether 1) polymorphisms in genes relevant to connective tissue metabolism or 2) a family history of cervical insufficiency is more common in women with the condition compared to controls.
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MATERIALS AND METHODS |
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The study was approved by the Institutional Review Board of the University of Utah, and each woman gave written informed consent before study participation. Patient enrollment occurred from August 2004 through March 2006. Medical and obstetric histories were ascertained by patient interview and verified by review of the patients’ medical records. Family histories were obtained solely through the patient interview and noted to be positive if cerclage placement were performed in the family member. Blood was collected from participants in yellow top tubes (acid citrate dextrose) and was centrifuged for 15 minutes at 2,000 revolutions per minute. Buffy coats were then extracted and frozen at –80°C until use.
DNA extractions were performed with a commercial DNA isolation kit, Puregene, (Gentra Systems, Minneapolis, MN). Briefly, buffy coat from approximately 6–8 mL of whole blood was collected. Red blood cells were lysed and separated from the nuclei pellet by centrifugation. Proteins were precipitated by using a nucleic acid–specific protein precipitation solution (Gentra) and separated from the DNA by centrifugation. The DNA-containing supernatant was collected, and the DNA was precipitated with 100% isopropanol and collected by centrifugation. The precipitated DNA was washed with 70% ethanol and again collected by centrifugation. The DNA sample was resuspended in a solution containing 10 mmol/L Tris hydrochloride, pH 7.5, and 1 mmol/L ethylenediaminetetraacetic acid. Concentrations of the samples were determined with ultraviolet spectrometry by measuring an A260 absorbance. Purity was determined by an A260/A280 ratio. Samples were diluted to a standard concentration and stored at –20°C.
Samples (30 ng per sample) were amplified for the TGF-ß1 (Arg25Pro) and COL1A1(Sp1) polymorphisms using an allele-specific polymerase chain reaction (PCR) method. Briefly, each reaction contained 0.5 µmol/L of a primer whose 3'-most base was specific to one allele, 0.5 µmol/L of a primer whose 3'-most base was specific to the second allele, and 0.5 µmol/L of a 5'-6-FAMM fluorescently labeled third primer that was on the opposite strand to the two allele-specific primers. The fluorescently labeled third primer would generate a PCR product with either one or both of the allele-specific primers, depending on the genotype of the sample. In addition to the primers, the PCR reaction contained 1x PCR buffer II (Applied Biosystems [ABI], Foster City, CA), 1.5 mmol/L MgCl2, 200 µmol/L of each deoxyribonucleotide triphosphate (dNTP), and 0.6 units of Taq Gold Polymerase (ABI). The PCR reactions were cycled in an ABI 9700 thermocycler as follows: denaturation at 96°C for 11 minutes, five cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 20 seconds. This was followed by 30 cycles of 94°C for 10 seconds, 55°C for 10 seconds, and 72°C for 20 seconds. After PCR, 0.1 µL of the PCR reaction was added to 10 µL of deionized formamide containing an internal sizing standard. The polymorphisms were detected by using an ABI 3730XL fluorescent capillary electrophoresis instrument. The alleles were discriminated based on the size of the PCR fragments generated. The alleles differed by 4 base pairs due to the differing primer lengths used to generate the PCR products. Genotypes were determined by using an ABI allele calling software, Genemapper.
Observed genotype frequencies were determined and Hardy-Weinberg calculations performed. Expected genotype frequencies were compared with the actual genotype frequencies in our study population. The actual genotype frequencies detected in women with cervical insufficiency and those in controls also were compared. Odds ratios were calculated, and 
2 analysis and Fisher exact test were used to compare allele frequencies between cases and controls. In addition, cases with and those without a family history were compared with regard to the presence of the polymorphisms. Two-sided P values were used in all comparisons.
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RESULTS |
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Cases and controls were matched for race and were similar with respect to age and gravidity (t test). Our cases were 80.6% Caucasian and 19.3% Hispanic. The mean gestational age of losses attributed to cervical insufficiency was 20.2 weeks (range 12–25 weeks).
In women with a history of cervical insufficiency, 34 of 125 (27.2%) women had a family history, defined as a first-degree relative with the condition, compared with none in the control group. There was no family history of cervical insufficiency in controls.
Table 1 summarizes the genotype frequencies of the COL1A1 and TGF-ß polymorphisms in cases and controls. 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; odds ratio [OR] 3.8, 95% confidence interval [CI] 1.4–10.3). The TGF-ß polymorphisms (ArgPro and ProPro) also were increased in cases (38.3% compared with 14.6%, P<.001; OR 1.9, 95% CI 1.2–3.2).
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Of the 108 cases in which adequate family history and blood samples were available, 33 (30%) had a family history of cervical insufficiency. Table 2 summarizes the allele frequencies for both the COL1A1 and TGF-ß genes in women with cervical insufficiency who did and those who did not have a family history of the condition. Cases with a positive family history had an increased frequency of the TT genotype in the COL1A1 gene compared with cases with no family history (21.9 % compared with 6.7%, P=.04). There was no increased frequency of the TGF-ß polymorphism in those cases with a family history of cervical insufficiency compared with those without a family history.
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Fifty-four women had a "classic" history, defined as clinical evidence of dilation of the cervix without labor in the second trimester. Table 3 shows gene polymorphisms for cases with and those without a history of classic cervical insufficiency. There were no significant differences in genotype frequencies between these groups.
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Table 4 demonstrates the genotype frequencies for both genes in the 105 women who had at least one cerclage placement each. There was no difference in frequencies of polymorphisms in the COL1A1 and TGF-ß genes in cases where cerclage placement resulted in successful pregnancy outcome (liveborn infant at more than 34 weeks of gestation) compared with those without successful pregnancy outcome.
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DISCUSSION |
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Both of these polymorphisms are associated with abnormal connective tissue in humans. Cervical insufficiency traditionally has been thought to be due to abnormalities in cervical connective tissue. Indeed, several studies have shown abnormalities in cervical collagen, hyaluronic acid, and sulfated glycosaminoglycans in women with cervical insufficiency.1 Thus, it is biologically plausible that these polymorphisms may increase the risk for cervical insufficiency.
The G to T single nucleotide polymorphism affecting the Sp1 binding region of the COL1A1 gene alters collagen gene regulation. This polymorphism has been associated with abnormal production of the 
1 collagen chain in type I collagen leading to the formation of homotrimers instead of the normal heterotrimers. These homotrimers in bone have been correlated with decreased mechanical strength.6 In fact, mutations in the COL1A1 gene have been described in osteogenesis imperfecta, a well-known connective tissue disorder.
Most classes of cells in the body, including epithelium, endothelium, hematopoietic, and connective-tissue, produce and have receptors for TGF-ß. TGF-ß regulates proliferation and differentiation, which are involved in many functions, including embryonic development, healing, and angiogenesis. The essential role of the isoforms of TGF-ß signaling pathway in these processes has been demonstrated in knockout mouse models.8 The Arg25Pro polymorphism in exon 1 of the TGF-ß1 gene alters transport of protein product across the membrane of the endoplasmic reticulum, thus affecting its concentration.14 The known perturbations in the TGF-ß pathway leading to abnormalities in the extracellular matrix and the presence of receptors in cervical tissue led to our interest in this gene and its relation to cervical insufficiency.
In the past, cervical insufficiency was thought to be a dichotomous variable, meaning the cervix is either insufficient or it is not. More recent data, however, which incorporate sonographic assessments of cervical length, suggest that cervical sufficiency may function as a continuous variable, with cervical insufficiency occupying the extreme of this continuum.14 Iams and colleagues15 demonstrated that a shortened cervical length increases the relative risk of preterm birth compared with normal cervical length. Despite the relation between sonographic cervical length and preterm delivery, the relation between these findings and the clinical presentation is still unclear, and a shortened cervix does not always signify clinical cervical insufficiency.
There is controversy surrounding this disease process and its treatment because the pathophysiology remains uncertain. Shortening, prolapsing membranes, and dynamic changes of the cervix likely have multiple contributing factors, including uterine activity, abruption, inflammation, infection, and abnormal implantation, which lead to a final common pathway of a shortened or incompetent cervix.16 In addition to the potential for multiple causal mechanisms, cervical insufficiency is not always recurrent. These observations suggest that this condition may not be the result of a single gene defect but is likely a heterogeneous group of disorders.15 Our finding of an increased prevalence of the rarer genotypes of these polymorphisms, in some but not all women with cervical insufficiency, is consistent with this concept.
We hypothesized that cases with a classic history would be more likely to have connective tissue polymorphisms than those with less typical histories. However, the frequency of polymorphisms was the same in both of these groups. Likewise, gene frequencies were similar in women whose cerclage placement resulted in successful and complicated pregnancies. These observations underscore the heterogeneity, complexity, and incompletely understood pathophysiology of cervical insufficiency.
There were several weaknesses in our study. First, not all women had a classic history of cervical insufficiency. This likely increased the heterogeneity of our phenotype and may negatively effect genetic studies. However, some aspects of obstetric histories are subjective and vary among women and pregnancies, even in women with classic presentations of insufficiency. Also, we wanted to study the condition as it is currently being defined and addressed in the "clinical arena." Patients with prior cervical procedures were excluded to refine the case group. We used subset analyses to attempt to assess a purer population of women with classic cervical insufficiency. However, this subset may have had inadequate power to detect differences in gene frequencies among groups. We estimate that it would require 150 women with classic cervical insufficiency to confirm this negative finding. Other weaknesses included missing samples due to patient refusal of blood draw and missing information, including lack of specific knowledge about family history and items not routinely recorded in the medical record. Recall bias is another potential drawback of the study.
Our study also had several strengths. Over 130 women with cervical insufficiency were included. Cases and controls were similar with regard to ethnicity, an important feature of any genetic study. We included only first-degree relatives with the condition to increase the likelihood of accurate results as well as the probability that genes are relevant. Finally, we had excellent obstetric detail in all participants, something that is often lacking from studies using large obstetric databases.
In summary, over one fourth of women with cervical insufficiency in our study had at least one first-degree relative with the condition. Also, the G to T polymorphism in the Sp1 binding site of the COL1A1 gene and the Arg25Pro polymorphism in the TGF-ß1 gene were both associated with cervical insufficiency. These findings indicate that cervical insufficiency is, in part, a genetic condition and that polymorphisms in genes relevant to connective tissue metabolism may play a role in some cases. Careful family histories will allow us to identify women at risk for cervical insufficiency and should facilitate future studies of the pathophysiology of the condition. Other genes, as well as environmental factors, are likely to play a role in this complex and heterogeneous disorder.
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Footnotes |
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Financial Disclosure The authors have no potential conflicts of interest to disclose.
doi:10.1097/01.AOG.0000277261.92756.1a
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REFERENCES |
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2. Rechberger T, Uldbjerg N, Oxlund H. Connective tissue changes during normal pregnancy and pregnancy complicated by cervical incompetence. Obstet Gynecol 1988;71:563–7.
3. Petersen LK, Uldbjerg N. Cervical collagen in non-pregnant women with previous cervical incompetence. Eur J Obstet Gynecol Reprod Biol 1996;67:41–5.[Medline]
4. Grant SF, Reid DM, Blake G, Herd R, Fogelman I, Ralston SH. Reduced bone density and osteoporosis associated with a polymorphic Sp1 binding site in the collagen type I alpha 1 gene. Nat Genet 1996;14:203–5.[Medline]
5. Mann V, Hobson EE, Li B, Stewart TL, Grant SF, Robins SP, et al. A COL1A1 Sp1 binding site polymorphism predisposes to osteoporotic fracture by affecting bone density and quality. J Clin Invest 2001;107:899–907.[Medline]
6. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor ß in human disease. N Engl J Med 2000;342:1350–8.
7. Singh KK, Rommel K, Mishra A, Karck M, Haverich A, Schmidtke J, et al. TGFBR1 and TGFBR2 mutations in patients with features of Marfan Syndrome and Loeys-Dietz Syndrome. Hum Mutat 2006;27:770–7.[Medline]
8. Mátyás G, Arnold E, Carrel T, Baumgartner D, Boileau C, Berger W, et al. Identification and in silico analyses of novel TGFBR1 and TGFBR2 mutations in Marfan Syndrome-related disorders. Hum Mutat 2006;27:760–9.[Medline]
9. Crilly A, Hamilton J, Clark CJ, Jardine A, Madhok R. Analysis of transforming growth factor ß1 gene polymorophisms in patients with systemic sclerosis. Ann Rheum Dis 2002;61:678–81.
10. Lewis CM. Genetic association studies: design, analysis, and interpretation. Brief Bioinform 2002;3:146–53.
11. D’Alessio M, Bernard M, Pretorius PJ, de Wet W, Ramirez F. Complete nucleotide sequence of the region encompassing the first twenty-five exons of the human pro alpha 1(I) collagen gene (COL1A1). Gene 1988;67:105–15.[Medline]
12. Ward LM, Lalic L, Roughley PJ, Glorieux FH. Thirty-three novel COL1A1 and COL1A2 mutations in patients with osteogenesis imperfecta types I-IV. Hum Mutat 2001;17:434.[Medline]
13. Pollitt R, McMahon R, Nunn J, Bamford R, Afifi A, Bishop N, et al. Mutation analysis of COL1A1 and COL1A2 in patients diagnosed with osteogenesis imperfecta types I-IV. Hum Mutat 2006;27:716.[Medline]
14. Stoll C, Mengsteab S, Stoll D, Riediger D, Gressner AM, Weiskirchen R. Analysis of polymorphic TGFB1 codons 10, 25, and 263 in a German patient group with non-syndromic cleft lip, alveolus, and palate compared with healthy adults. BMC Med Genet 2004;5:15.[Medline]
15. Iams JD, Goldenberg RL, Meis PJ, Mercer BM, Moawad A, Das A, et al. The length of the cervix and the risk of spontaneous preterm delivery. N Engl J Med 1996;334:567–72.
16. Rust OA, Atlas RO, Reed J, van Gaalen J, Balducci J. Revisiting the short cervix found by transvaginal ultrasound in the second trimester: why cerclage therapy may not help. Am J Obstet Gynecol 2001;185:1098–105.[Medline]
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