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Genetic associations of relaxin: preterm birth and premature rupture of fetal membranes

      Objective

      Relaxin H2 (RLN2) is a systemic hormone (sRLN) that is produced by the corpus luteum, whereas decidual RLN (dRLN) acts only locally. Elevated sRLN is associated with spontaneous preterm birth (sPTB) and elevated dRLN with preterm premature rupture of membranes (PPROM). Associations were sought between single nucleotide polymorphisms (SNPs) in the RLN2 promoter with levels of dRLN and sRLN in Filipino patients with sPTB, PPROM, or normal term delivery.

      Study Design

      Stringent selection of women with sPTB (n = 20) or PPROM (n = 20) and term control subjects (n = 20) was made from >8000 samples from Filipino patients who delivered at 34-36 weeks' gestation. Twelve SNPs were genotyped on maternal blood, with 9 excluded based on the high linkage disequilibrium or being the same as in the control population. Quantitative immunocytochemistry on parietal decidual tissue was performed (n = 60); sRLN was measured by enzyme-linked immunosorbent assay in a subset of patients (n = 21).

      Results

      SNP rs4742076 was associated significantly with PPROM (P < .001) and increased expression of dRLN (P < .001). The genotype TT had increased dRLN in PPROM (P < .05). SNP rs3758239 was associated significantly with both PPROM and sPTB (P < .01), and genotype AA had increased dRLN expression (P < .05). The sRLN showed a trend of higher levels in PPROM and sPTB, but was not significant.

      Conclusion

      SNP rs4742076 in the RLN2 promoter was associated with increased dRLN expression and PPROM; SNP rs3758239 was associated with both PPROM and sPTB in these Filipino patients. Specific homozygous genotypes were identified for both SNPs and were shown to be associated with increased dRLN tissue expression.

      Key words

      Preterm birth (PTB) is the leading cause of neonatal morbidity and death in the United States, and its cause is associated with ethnicity.
      • Hamilton B.E.
      • Martin J.A.
      • Ventura S.J.
      Births: preliminary data for 2011.
      Socioeconomic parameters are associated with ethnic disparity but are not the cause of a higher incidence of PTB.
      • Kistka Z.A.
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      • Lee K.A.
      • et al.
      Racial disparity in the frequency of recurrence of preterm birth.
      Genetic factors appear to contribute significantly to the complex gene-environment interactions that result in prematurity, with the maternal genetic component being substantially more important than the fetal genetic component.
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      Mother's genome or maternally-inherited genes acting in the fetus influence gestational age in familial preterm birth.
      Immigration to Hawaii over the past 2 hundred years has resulted in 1 of the most heterogeneous populations known. However, the Filipino subpopulation in Hawaii, while having access to similar health care, has a substantially higher rate of PTB (11.7%) than either white (7.2%) or other Asian populations (9.0%) of these islands.
      • Hayes D.
      • Shor R.
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      • Fuddy L.
      Premature birth fact sheet.
      A recent study identified ancestry informative markers, showed major variations in East-Asian Americans, and concluded that Filipino women could be distinguished genetically from other East-Asian Americans.
      • Tian C.
      • Kosoy R.
      • Lee A.
      • et al.
      Analysis of East Asia genetic substructure using genome-wide SNP arrays.
      There are 3 genes for human relaxin (RLN1, 2, and 3).
      • Bathgate R.A.
      • Samuel C.S.
      • Burazin T.C.
      • et al.
      Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene: novel members of the relaxin peptide family.
      • Crawford R.J.
      • Hudson P.
      • Shine J.
      • Niall H.D.
      • Eddy R.L.
      • Shows T.B.
      Two human relaxin genes are on chromosome 9.
      Both RLN1 and RLN2 are expressed in human decidua and placenta. However, RLN2 is the major form, with RLN1 being only minimally expressed by the decidua. RLN2 is also produced by the corpus luteum and enters the systemic circulation in pregnancy (sRLN). The action of RLN2 from the maternal decidua (dRLN) and fetal trophoblast is purely autocrine/paracrine; it does not enter the systemic circulation. This was determined from patients with ovum donation pregnancies and no corpora lutea who were shown to have undetectable sRLN levels.
      • Johnson M.R.
      • Abdalla H.
      • Allman A.C.
      • Wren M.E.
      • Kirkland A.
      • Lightman S.L.
      Relaxin levels in ovum donation pregnancies.
      A different pattern of serum RLN levels during gestation is associated with PTB compared with normal control subjects. Women with spontaneous PTB (sPTB) have lower RLN levels in early pregnancy but higher levels in later gestation, compared with women who deliver at term.
      • Vogel I.
      • Thorsen P.
      • Hundborg H.H.
      • Uldbjerg N.
      Prediction of preterm delivery using changes in serum relaxin in low risk pregnancies.
      On the other hand, increased expression of intrauterine RLN has been shown in patients with preterm premature rupture of membranes (PPROM) without infection.
      • Bryant-Greenwood G.D.
      • Kern A.
      • Yamamoto S.Y.
      • Sadowsky D.W.
      • Novy M.J.
      Relaxin and the human fetal membranes.
      RLN has been shown to cause a dose-dependent increase in the expression of specific genes and proteins and the activities of some regulatory matrix metalloproteinases that are involved in PPROM.
      • Qin X.
      • Garibay-Tupas J.
      • Chua P.K.
      • Cachola L.
      • Bryant-Greenwood G.D.
      An autocrine/paracrine role of human decidual relaxin: I, interstitial collagenase (matrix metalloproteinase-1) and tissue plasminogen activator.
      • Qin X.
      • Chua P.K.
      • Ohira R.H.
      • Bryant-Greenwood G.D.
      An autocrine/paracrine role of human decidual relaxin: II, stromelysin-1 (MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1).
      It can also modulate the production of the proinflammatory cytokines directly; interleukin 6 and 8 are produced by both the chorion and decidua.
      • Horton J.S.
      • Yamamoto S.Y.
      • Bryant-Greenwood G.D.
      Relaxin modulates proinflammatory cytokine secretion from human decidual macrophages.
      • Horton J.S.
      • Yamamoto S.Y.
      • Bryant-Greenwood G.D.
      Relaxin augments the inflammatory IL6 response in the choriodecidua.
      However, there has been no attempt to date to integrate the data on the systemic and intrauterine RLN in either normal gestation or in sPTB.
      A recent study with an homogeneous Danish population showed that women who are homozygous for specific single nucleotide polymorphisms (SNPs) in the promoter region of RLN2 have a genetic susceptibility for PTB.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      No distinction was made in this study between women who deliver at preterm because of sPTB or PPROM, but this may be important because different mechanisms likely are involved. Thus, the aims in this exploratory study were to confirm these genetic results in a different ethnic population with a known high incidence of sPTB and to investigate whether DNA polymorphisms in RLN2 are associated with sPTB of different causes. In addition, we examined whether such a genetic change might alter either maternal dRLN and/or ovarian RLN in the systemic circulation (sRLN).

      Materials and Methods

      Study population

      The study population was selected from 8000 patients for whom samples were available in the University of Hawaii Biospecimen Repository that had been collected between 2005 and 2011. The University of Hawaii Biospecimen Repository is approved by the Western Institutional Review Board and deemed our study proposal exempt from institutional review board review because all the data and samples were deidentified.
      There were 94 patients with Filipino ancestry up to and including all 4 grandparents (self-reported), with singleton pregnancies, sPTB between 34 weeks 0 days' and 36 weeks 6 days' gestation by documented estimated due date in the absence of medical complications of pregnancy and either PPROM (n = 20) or sPTB (n = 23) according to the diagnosis at admission. For the latter group, 20 patients were selected randomly. Preterm gestational age was selected based on results of RLN SNP analysis in a Danish population.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      Control subjects (n = 20) were selected randomly from 45 patients who met the same ethnicity and inclusion/exclusion criteria, with normal spontaneous vaginal term delivery between 39 weeks 0 days and 40 weeks 0 days gestation.
      Exclusion criteria were delivery because of medical complications during pregnancy (preeclampsia, fetal growth restriction, histologic and/or clinical chorioamnionitis, renal disease, HELLP [hemolysis, elevated liver enzymes, and low platelet count syndrome]), multiple gestations, pregnancies from infertility treatment, fetal anomaly, uterine anomaly, induced preterm delivery or placental conditions (placental abruption, polyhydramnios, positive urine drug screen for cocaine or amphetamines), and pregnancies with an estimated due date that was based on third-trimester ultrasound scanning.
      We genotyped maternal blood in all patients for 12 SNPs that were located in the promoter region of the RLN2 and quantitated the RLN protein in the maternal decidual cells (n = 60) and measured the maternal sRLN in a subset of the same patients (n = 21). All samples were blinded, with the disclosure of clinical characteristics/outcomes only after the analyses were completed.

      Genotyping and analysis

      DNA was extracted from maternal blood with the Autopure system (Qiagen, Valencia, CA); 10 ng of each sample was quality checked and used for genotyping with custom and predesigned TaqMan SNP Genotyping Assay (Life Technologies, Carlsbad, CA). Polymerase chain reactions were performed on an ABI GeneAmp 9700 thermocycler; allelic determination was carried out on an ABI 7900HT fast real-time polymerase chain reaction system (version 2.4; Applied Biosystems, Carlsbad, CA). In-house quality controls (in duplicate) were used as references to identify genotype cluster locations. Genotype data were uploaded into a database (Progeny Software, South Bend, IN) that contained deidentified demographic and clinical information for statistical analysis.
      SNPs were chosen to cover the promoter of the RLN2 from circa 2.2-6.6 kb before the 5 prime untranslated region, avoiding the region with highest homology to RLN1. Selection was biased toward either the functionality or the conservation of the particular SNP. A list of these SNPs, with their SNP database identification (rs) numbers and chromosome 9 base location, is shown in Table 1. Allele and genotype frequencies were calculated for each SNP. Of the 12 that were genotyped, 7 matched the reference allele and were therefore excluded from further analysis. The remaining 5 SNPs met Hardy-Weinberg equilibrium at the 5% alpha level.
      Table 1Single nucleotide polymorphisms selected from the promoter region of RLN2
      Single nucleotide polymorphismPublic location
      Genome Reference Consortium Human genome build 37
      on chromosome 9
      AllelesFunction or previously published association
      rs10115467
      Included in the genotyping analysis.
      5311171A/GPrematurity
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      rs1133904295310338C/G
      rs132934105308584A/G
      rs1833125575311086G/T
      rs3758239
      Included in the genotyping analysis.
      5306824A/G
      rs4742076
      Included in the genotyping analysis.
      5309831C/TPrematurity
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      rs625576885308318A/G
      rs70294005309793G/T
      rs78562375309087A/G
      rs78757355309322A/T
      rs793248645307823A/G
      rs1500324535311191C/G
      Rocha. Genetic associations of relaxin with preterm birth. Am J Obstet Gynecol 2013.
      a Genome Reference Consortium Human genome build 37
      b Included in the genotyping analysis.
      Association studies were performed with the use of the Fisher exact test (permutation test) and logistic regression with the control subjects as the reference group and neonatal sex as the covariate. The data were tested for linkage disequilibrium by calculating R2. Three SNPs (rs13293410, rs3758239, rs7029400) were in high linkage disequilibrium (R
      • Kistka Z.A.
      • Palomar L.
      • Lee K.A.
      • et al.
      Racial disparity in the frequency of recurrence of preterm birth.
      >0.9). Because this indicates redundancy, 2 SNPs were excluded, and only rs3758239 along with rs4742076 and rs10116567 remained for individual SNP association analyses.
      Additive, codominant, dominant, and recessive inheritance models were considered. The outcome group-genotype interaction was considered significant at a probability value of < .05. The inheritance model with the smallest probability value was considered to be the best-fitting model for the respective SNP and was used to calculate odds ratios. Because of small sample size, interaction between RLN genotype and dRLN expression was evaluated by bootstrap analysis, with tissue RLN-genotype interaction being considered significant at a probability value of < .05. Because this was an exploratory study, no adjustment to the alpha level was made for multiple comparisons, and all comparisons are reported. SAS software (SAS Institute, Cary, NC) and R (www.r-project.org) were used for the statistical analysis.

      Quantitative immunocytochemistry

      Slides of formalin-fixed fetal membrane rolls (n = 60) were treated in sodium citrate buffer (10 mmol/L; pH 6.0) for 30 minutes for antigen retrieval, followed by 0.3% hydrogen peroxide then 2.5% horse serum. They were incubated for 1 hour with rabbit polyclonal antibody (immunoglobulin G) to human RLN (8 μg/mL; Calbiochem/EMD Biosciences, San Diego, CA). Negative controls were nonimmune immunoglobulin (DAKO North American Inc, Carpenteria, CA). Color was developed with the ImmPRESS reagent (Vector Laboratories, Burlingame, CA) for 30 minutes and DAB (3, 3-diaminobenzidine; Sigma-Aldrich, St. Louis, MO) for 5 minutes.
      A series of brightfield images were acquired between 420 and 700 nm at 20-nm intervals with the use of a multispectral imaging system comprised of an Olympus BX51 microscope (Olympus America Inc, Melville, NY) and a CRI Nuance spectral analyzer (Caliper Life Sciences, Hopkinton, MA). The images were stacked to create a 3-dimensional image cube. Quantitative image analysis of the unmixed data was performed with Inform software (version 1.4.0; PerkinElmer, Waltham, MA). The average signal intensity per pixel from 5 different fields of decidua from each patient was collected; results were expressed as mean ± SD. Immunocytochemical data were analyzed by nonparametric analysis (GraphPad Software Inc, San Diego, CA); the Kruskal-Wallis analysis of variance used to compare differences in protein expression.
      sRLN was measured in a subset of patients (n = 21) by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions; results were analyzed as described earlier.

      Results

      The demographics and clinical characteristics of the study population are shown in Table 2; none of the covariates were distributed unevenly among strata.
      Table 2Demographics and clinical characteristics of the study population
      CharacteristicPreterm premature rupture of membranes (n = 20)Spontaneous preterm births (n = 20)Term birth (n = 20)
      Gestational age, wk
      Data are given as mean (range)
      35.8 (34.6–36.5)36.12 (34.5–36.6)39.76 (39.5– 40)
      Neonatal sex (female/male)9/1110/1010/10
      Maternal age, y
      Data are given as mean (range)
      28 (28–37)27.85 (19–36)30.25 (20–40)
      Nulliparity, n/N8/207/204/20
      Previous preterm delivery, n/N3/207/201/20
      Length of rupture of membranes, h
      Data are given as mean ± SD.
      13.7 ± 15.12.4 ± 3.15.2 ± 6.0
      Birthweight, g
      Data are given as mean ± SD.
      2623 ± 3162522 ± 3443205.55 ± 345
      Length of baby, cm
      Data are given as mean ± SD.
      48.06 ± 1.7447.42 ± 1.8051.04 ± 2.58
      Rocha. Genetic associations of relaxin with preterm birth. Am J Obstet Gynecol 2013.
      a Data are given as mean (range)
      b Data are given as mean ± SD.
      Our results show that 1 SNP (rs4742076) was associated with only PPROM, whereas SNP rs3758239 was associated with both PPROM and sPTB. There was no significant association (P > .05) of rs10115467 in any patient group (Table 3). The TT genotype of SNP rs4742076 had a significant association (P < .001) with PPROM, with an odds ratio (OR) of 11.0 (95% confidence interval [CI], 2–60.5) when compared with both the control subjects and women with sPTB (Table 3). Genotype AA of SNP rs3758239 showed a significant association (P < .001) between PPROM (odds ratio, 12.5; 95% CI, 2.7–50) and the term controls (Table 3). It also had a significant association (P < .01) with sPTB (OR, 7.1; 95% CI, 1.7–25) when compared with the term controls (Table 3).
      Table 3Comparisons of genotype frequency between women with PPROM, sPTB, and term control subjects
      Single nucleotide polymorphism (genotype)PPROM/term, nPPROM/sPTB, nsPTB/term, nOdds ratio (95% CI)Best fitted modelOdds ratio (95% CI)P value
      rs10115467
       GG13/15ReferenceDom1.62 (0.41–6.34).48
       AG6/51.38 (0.34–5.62)
       AA1/0NA
      rs3758239
       AA17/6ReferenceDom12.5 (2.7–50).0002
       AG3/100.11 (0.02–0.52)
       GG0/4NA
      rs4742076
       CC4/6Reference
       CT5/120.63 (0.12–3.22)
       TT11/28.25 (1.15–59.00)Rec11.0 (2.0–60.5).001
      rs10115467
       GG13/10ReferenceDom0.6 (0.17–2.17).43
       AG6/80.58 (0.15–2.21)
       AA1/10.77 (0.04–13.87)
       Unable to be determined0/1
      rs3758239
       AA17/15ReferenceDom0.53 (0.11–2.6).42
       AG3/40.66 (0.13–3.45)
       GG0/1NA
      rs4742076
       CC4/2Reference
       CT5/160.16 (0.02–1.12)
       TT11/22.75 (0.28–26.61)Rec11.0 (2.0–60.5).001
      rs10115467
       GG10/15ReferenceDom0.6 (0.17–2.17).43
       AG8/52.4 (0.61–9.49)
       AA1/0NA
       Unable to be determined1/0
      rs3758239
       AA15/6ReferenceDom7.14 (1.7–25).003
       AG4/100.16 (0.04–0.71)
       GG1/40.1 (0.01–1.09)
      rs4742076
       CC2/6ReferenceDom3.86 (0.67–22.1).1
       CT16/124.0 (0.68–23.41)
       TT2/23.0 (0.24–37.67)
      CI, confidence interval; Dom, dominant; NA, not applicable; PPROM, preterm premature rupture of membranes; Rec, recessive; sPTB, spontaneous preterm birth.
      Rocha. Genetic associations of relaxin with preterm birth. Am J Obstet Gynecol 2013.
      Examples of decidual cell staining for RLN show darker cytoplasmic stain in PPROM (Figure 1, A) compared with sPTB (Figure 1, B) or at term delivery (Figure 1, C). The control nonimmune immunoglobulin on a section from a PPROM patient showed very low background immunostaining (Figure 1, D). The method for quantitation of this staining with the Inform software program does not require the negative controls. Instead, it uses pattern recognition of stained cells by spectra in the stained cells (Figure 1, E). Thus, the program excludes the nuclei and the extracellular matrix and quantifies only the relevant cytoplasmic RLN. The quantitative results for all patients showed significantly increased cytoplasmic RLN expressed in the PPROM group compared with both the women with sPTB (P < .001) and term control subjects (P < .001; Figure 1, F).
      Figure thumbnail gr1
      Figure 1Quantitative immunocytochemistry of dRLN protein in tissue
      Examples of decidual cell staining for RLN (dRLN) show darker cytoplasmic stain in A, PPROM, compared with B, sPTB or at C, term delivery. D, The control nonimmune immunoglobulin on a section from a PPROM patient showed very low background immunostaining. The method for quantitation of this staining with the Inform software program does not require the negative controls. E, Instead, it uses pattern recognition of stained cells by spectra in the stained cells, where the nuclear membranes are outlined in green, the extracellular matrix (ECM) pink and the individual decidual cells delineated in different colors. Thus, the program excludes the nuclei and the ECM and quantifies only the relevant cytoplasmic RLN. F, The quantitative results for all patients showed significantly increased cytoplasmic RLN expressed in the PPROM group compared with both the sPTB (P < .001) and term controls (P < .001).
      PPROM, preterm premature rupture of membranes; RLN, relaxin; sPTB, spontaneous preterm birth.
      Rocha. Genetic associations of relaxin with preterm birth. Am J Obstet Gynecol 2013.
      Only a subset of samples was available for RLN measurement by enzyme-linked immunosorbent assay; therefore, only a subpopulation of each group could be studied: 5 samples for PPROM, 7 samples for sPTB, and 9 for term control samples. The results showed a trend for higher levels of RLN in PPROM and sPTB compared with term control samples, but this did not achieve statistical significance (Figure 2).
      Figure thumbnail gr2
      Figure 2Enzyme-linked immunosorbent assay of sRLN in maternal serum
      PPROM, preterm premature rupture of membranes; RLN, relaxin; sPTB, spontaneous preterm birth; sRLN, systemic hormone.
      Rocha. Genetic associations of relaxin with preterm birth. Am J Obstet Gynecol 2013.
      Because increased dRLN expression was highly significant in PPROM, we linked this with the genotype analysis using bootstrap analysis for pairwise comparisons. Thus, by comparing CC with TT for SNP rs4742076 within all subjects (n = 60), the presence of the genotype TT showed significantly increased (P < .05) dRLN tissue expression (95% CI, 0.000–0.007). For the SNP rs3758239, the genotype AA, when compared with AG, also demonstrated significantly higher dRLN expression (P < .05) within all subjects (95% CI, 0.00012–0.00619).

      Discussion

      We have demonstrated some significant differences in the frequency of specific SNPs in the RLN2 promoter in both PPROM and sPTB in a Filipino population with a high risk of sPTB. In addition, we have associated successfully 2 genotypes with increased phenotypic tissue expression of dRLN with PPROM and sPTB.
      Our study population was selected from a biorepository and ethnicity based on self-reported information. Our patient numbers were relatively small because of our stringent selection of patients; rigorous inclusion/exclusion criteria were used that included only late PTB (34-36 weeks, 6 days' gestation). Multiple studies have shown a high correlation between early gestational age sPTB and infection, which suggests that genetic influences in sPTB and PPROM may be more significant in late sPTB.
      • Goldenberg R.L.
      • Culhane J.F.
      • Iams J.D.
      • Romero R.
      Epidemiology and causes of preterm birth.
      • Andrews W.W.
      • Hauth J.C.
      • Goldenberg R.L.
      • Gomez R.
      • Romero R.
      • Cassell G.H.
      Amniotic fluid interleukin-6: correlation with upper genital tract microbial colonization and gestational age in women delivered after spontaneous labor versus indicated delivery.
      A study that sought associations between SNPs in the RLN2 promoter and sPTB that stratified the patients by early and late sPTB showed significant associations in patients only with late sPTB.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      There have been remarkably few genetic studies on RLN and sPTB, considering that altered levels in serum have long been associated with sPTB.
      • Vogel I.
      • Glavind-Kristensen M.
      • Thorsen P.
      • Armbruster F.P.
      • Uldbjerg N.
      S-relaxin as a predictor of preterm delivery in women with symptoms of preterm labour.
      • Vogel I.
      • Goepfert A.R.
      • Moller H.J.
      • Cliver S.
      • Thorsen P.
      • Andrews W.W.
      Early mid-trimester serum relaxin, soluble CD163, and cervical length in women at high risk for preterm delivery.
      It is clear that ovarian RLN enters the systemic circulation, whereas decidual/placental RLN has only autocrine/paracrine actions within the uterus. Thus, sRLN could target the decidua through its blood supply and influence both the structural properties of the fetal membranes and its proinflammatory milieu.
      • Bryant-Greenwood G.D.
      • Kern A.
      • Yamamoto S.Y.
      • Sadowsky D.W.
      • Novy M.J.
      Relaxin and the human fetal membranes.
      Although these actions have been shown for intrauterine RLN, to date there has been no plausible explanation for the alterations in sRLN being associated with sPTB.
      Our study shows a significant association between SNP rs4742076 and PPROM. In a homogeneous Danish population, the homozygous allele of this SNP also had a significant association with patients who had late PTB, in which PPROM was included but not distinguished.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      However, there were no tissue or serum RLN analyses performed. We have confirmed this same association in a genetically different population and extended it to show that the patients with the genotype TT of this SNP had an increased risk of PPROM. However, we found no association between rs4742076 and sPTB. This may be due to the small size of our patient population or the inclusion of PPROM together with sPTB in the Danish study, which caused them to identify this SNP with PTB in general, rather than specifically with PPROM.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      In addition, we show that dRLN was increased in these same PPROM patients, compared with the sPTB or the control subjects. Indeed, patients with the homozygous T allele showed a significantly increased dRLN, which suggests a potential genetic effect on decidual cell phenotype. We also attempted to link sRLN levels with the genetic analyses in a subset of patients, based on the previous finding of a midpregnancy increase in serum RLN in patients destined to have sPTB.
      • Vogel I.
      • Thorsen P.
      • Hundborg H.H.
      • Uldbjerg N.
      Prediction of preterm delivery using changes in serum relaxin in low risk pregnancies.
      We showed that patients with both PPROM and sPTB tended to have higher systemic RLN levels than patients with normal term deliveries, but in neither case was this significant.
      We showed that polymorphisms in SNP rs3758239 were associated with both PPROM and sPTB outcomes in Filipino women. This SNP was studied in a Danish population, and no associations with sPTB were found.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      On the other hand, these authors showed a significant association between SNP rs10115467 and sPTB; we failed to find this. These differences may be due to the very different populations or the relatively low numbers of patients, both here and in the Danish study.
      • Vogel I.
      • Hollegaard M.V.
      • Hougaard D.M.
      • Thorsen P.
      • Grove J.
      Polymorphisms in the promoter region of relaxin-2 and preterm birth: involvement of relaxin in the etiology of preterm birth.
      Certainly, there is a need for further studies that will use highly selected but larger patient cohorts.
      The association between carriers of genotype AA of SNP rs3758239 and both PPROM and sPTB may indicate the sharing of molecular pathways, such as activation of proinflammatory cytokines.
      • Bollapragada S.
      • Youssef R.
      • Jordan F.
      • Greer I.
      • Norman J.
      • Nelson S.
      Term labor is associated with a core inflammatory response in human fetal membranes, myometrium, and cervix.
      However, an increase in matrix metalloproteinases and apoptotic pathways predominate in PPROM and are either absent or minimally present in sPTB, which suggests that specific pathways before the common terminal pathway are also involved.
      • Menon R.
      • Fortunato S.J.
      Fetal membrane inflammatory cytokines: a switching mechanism between the preterm premature rupture of the membranes and preterm labor pathways.
      Thus, different RLN SNPs may be associated with PPROM and sPTB. The local action of RLN on the extracellular matrix of the fetal membranes that is caused by increased dRLN may be of greater consequence to PPROM than to preterm labor. This agrees with a recent study that showed that alterations in genes that are involved in the integrity of the maternal extracellular matrix are of special importance to PPROM.
      • Romero R.
      • Friel L.A.
      • Velez Edwards D.R.
      • et al.
      A genetic association study of maternal and fetal candidate genes that predispose to preterm prelabor rupture of membranes (PROM).
      In conclusion, complimentary positive results have been found by SNP analysis and immunocytochemical phenotypic expression of RLN in decidual cells. Thus, we have shown that patients with the genotype TT of rs4742076 in the RLN2 promoter have an increased risk of PPROM. In addition, patients who were carriers of genotype AA of rs3758239 had an increased risk of PPROM and sPTB. Therefore, the presence of these polymorphisms may allow the identification of pregnancies that are at high risk for PPROM and sPTB, which could lead to proper risk stratification and ideally improved outcomes for these patients.

      Acknowledgments

      We thank the University of Hawaii Biorepository team (Dr Timothy Dye, Tina Dean, and Skol Watanawongskul) and Ms Sandra Yamamoto and Ms Annette Lum Jones for technical help.

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