Earlier data show that lumbopelvic pain during pregnancy has a prevalence of more than 50% , . Such pain interferes with daily activities and accounts for most of the sick leave among pregnant women in Scandinavian countries . Often, the onset of pain occurs in early pregnancy and reaches peak intensity between the 24th and 36th gestation week , . Since the pain often disappears 3 months after birth , it has been suggested that lumbopelvic pain may be related to changes in the ligaments in the pelvic joints during pregnancy .
During pregnancy, high levels of relaxin is produced by the decidua and placenta . A previous study indicate that this hormone contributes to laxity of pelvic joints in pregnancy . Relaxin may through collagenases cause remodeling of the extracellular matrix (ECM) of the pelvic ligaments . One important collagenase that targets the ECM is matrix metalloproteinase 9 (MMP9). MMP9 is a zinc depended protease, which cleaves collagens of type I, III, IV, V and elastin , . This MMP9 zinc depended enzyme is expressed in active remodeling tissues , .
Relaxin has been shown to up-regulate MMP9 expression and to facilitate tissue remodeling both in-vivo and in-vitro , . The function of MMP9 may, however, also be influenced by genetic variability; for example, the single nucleotide polymorphism (SNP), rs17576 A>G in exon 6. Therefore, ECM remodeling may be influenced by this SNP. The MMP9 SNP, rs17576 A>G has previously been linked to conditions like pelvic organ prolapse, lumbar-disc herniation, endometrioses and metastases in cervical cancer , , , .
Genetic variability that influences tissue remodeling, degradation or inflammation – including the SNP emphasized above – may possibly also affect pain-sensitivity. Both reduced and increased MMP9 enzymatic activity could have undesirable effects on the tissue and cause pain. Hence, we hypothesized that the MMP9 SNP rs17576 A>G could affect lumbopelvic pain in pregnant woman. The aim of this study was to investigate whether the MMP9 SNP rs17576 A>G may be associated with lumbopelvic pain-intensity in pregnant woman during their pregnancy.
Pregnant women booking for routine ultrasound scans at Trondheim University Hospital and Stavanger University Hospital were recruited during the period from 2007 to 2009. This is a sub study of an earlier RCT, where a total of 855 pregnant women were randomized to an exercise intervention or a control group, for more details see . The two hospitals are serving large geographical catchment areas, and approximately 12,000 women had routine ultrasound scans in the inclusion period. However, only women from geographical areas within 30 min. drive from the hospitals were eligible. The inclusion criteria were age≥18 years, a singleton live fetus and a normal pregnancy with low risk for developing any complications. The characteristics of the subjects are shown in Table 1.
Exclusion criteria were: high-risk pregnancy, placenta previa, diabetes, any chronic disease, high blood pressure (>140/90 before week 20), drug misuse and alcoholism, non-European-Caucasian ethnicity or poor Norwegian language, missing blood samples, insufficient DNA isolated and nondetectable genotype.
In all, 875 women consented to participate, 23 women were excluded due to not meeting the inclusion criteria and five miscarried (Fig. 1). About 15% became dropouts during the follow up. The women received written information and signed informed consent forms. The study was performed in accordance with the Helsinki Declaration, approved by the Regional Committee for Medical and Health Research Ethics (REK 4.2007.81) and registered with ClinicalTrials.gov (NCT 00476567).
2.1 Clinical measures
Data collection was done at inclusion 18–22 weeks of pregnancy (T1), at follow-up 32–38 weeks of pregnancy (T2) and 3 months after delivery (T3). At each time point, the women were asked: “Do you have pain in the pelvic and/or lumbar area?” (Yes/No). Moreover, at each time point, the women were further asked to rate the worst pain in the evening on a 100 mm visual analog scale (VAS) with endpoints “no pain” and “worst pain”.
2.2 DNA extraction and SNP genotyping
Genomic DNA was extracted from whole blood cells using QIAamp 96 DNA Blood kit (Qiagen, Hilden, Germany). SNP genotyping was carried out using predesigned TaqMan SNP genotyping assays (Applied Biosystems) for MMP9 rs17576 A>G.
Approximately 10 ng genomic DNA was amplified in a 5 μL reaction mixture in a 384-well plate containing 1× universal TaqMan master mix and 1× assay mix, the latter containing the respective primers and MGB-probes. The probes were labeled with the reporter dyes FAM or VIC at 5′ end to distinguish between the two alleles. The reactions were performed on an ABI 7900HT sequence detection system (Applied Biosystems) at the following program: After initial denaturation and enzyme activation at 95 °C for 10 min, the reaction mixture was subjected to 95 °C for 15 s and 60 °C for 1 min. Negative controls containing water instead of DNA were included in every run. Genotypes were determined using the SDS 2.2 software (Applied Biosystems). Approximately 10% of the samples were re-genotyped and the concordance rate was 100%.
2.3 Statistical analysis
Linear mixed models were used to estimate the influence of the MMP9 genotype on VAS pain score for each time point. An allele dependent model was assumed, such that the effect of the genotype G/G was expected to be twice the effect of the genotype A/G when compared to the genotype A/A (adjusted for covariates age, parity, BMI and smoking). Random intercepts were added for subjects. In addition, the effect of genotype for primiparae versus other subjects at T2 were analyzed by linear regression (adjusted for covariates age, BMI and smoking).
In total 838 healthy pregnant women were included to the present study (Fig. 1). As expected, an increase of lumbopelvic pain-intensity was observed from 18–22 (T1) to 32–36 (T2) weeks of pregnancy followed by an after-birth (T3) recovery. Still, at all time points, a G allele dependent effect of the SNP was observed. Thus the A>G polymorphism was associated with lumbopelvic pain-intensitiy (Table 2). The pain among G/G carriers was higher than among A/G carriers, which in turn was higher than among the A/A carriers (Fig. 2). No deviation from the Hardy-Weinberg equilibrium was observed.
In particular, at 32–36 (T2) weeks of pregnancy, the G allele seemed to be associated with pain-intensity. The most pronounced association between the MMP9 G allele and pain-intensity was observed in primiparae (Fig. 3). In this subgroup, the effect of the MMP9 genotype G/G was twice the effect of the genotype A/G when compared to the genotype A/A. The estimated regression beta coef. at 32–36 weeks of pregnancy revealed that the VAS effect-size of the G allele in primiparae was 4.04 (Table 3) indicating an AA versus GG difference in VAS of 8.08.
As in previous studies, an increase of lumbopelvic pain-intensity was observed during pregnancy. For the first time, however, we here show evidence that individual genetic variability, possibly important for remodeling of the ECM and increased joint laxity, may be associated with such pain.
In accordance with our hypothesis, the MMP9 rs17576 A>G polymorphism was associated with lumbopelvic pain-intensity during pregnancy. In particular, at week 32–36 of the first pregnancy, the G allele was associated with increased pain-intensity. An allele dependent effect, such that the effect of the genotype G/G was twice the effect of the genotype A/G when compared to the genotype A/A, was observed.
The MMP9 rs17576 A>G polymorphism resides within the catalytic domain of the enzyme. This domain is highly conserved, and consists of gelatinase-specific fibronectin type II repeats, which plays an important role in substrate binding and substrate cleavage . The MMP9 rs17576 A>G polymorphism leads to a substitution of an uncharged amino acid (glutamine) by a positive charged amino acid (arginine) in exon 6 . Therefore, the SNP may change the enzymatic properties.
Previous studies support that this SNP is functional and may affect tissue remodeling. For example, evidence exists that the MMP9 rs17576 A>G in a G allele dependent manner promote cancer invasion and metastasis , . However, the present results do not show any causal relationship between the MMP9 rs17576 A>G, the MMP9 enzymatic activity and tissue remodeling in the pelvic ligaments.
During pregnancy, high levels of relaxin are produced by the decidua and placenta . Thus, relaxin may through collagenases including MMP9 cause remodeling of the ECM of the pelvic ligaments . Although this may be a natural process important for increased joint laxity  before birth, it could also in some subjects have other less desirable effects – for example, induce tissue degradation that in turn activate nociceptors and cause pain.
In the synovial joint and pubic symphysis fibocartilaginous tissues, the relaxin-mediated alterations in ECM composition causing joint laxcity, appear to be caused by increased degradative responses and changes in the collagen content , . The induction of MMP9 by relaxin occurs via the relaxin receptor, RXFP1. The activation of the RXFP1 involves the P13K, ERK, Akt and PKC-ζ pathways, including the Elk-1 and c-fos transcriptional factors, which in turn leads to transcription of the gene encoding MMP9 .
As described above, the relaxin – MMP9 – tissue remodeling mechanism is complex, and also involves many molecules important for activation of nociceptive primary afferent nerve fibers. Although the main effect may be increased joint laxity, a secondary effect may be pain. We conclude that the MMP9 rs17576 A>G polymorphism is associated with development of lumbopelvic pain-intensity during pregnancy.
5.1 Study limitations
The estimated regression beta coef. at 32–36 weeks of pregnancy revealed that the VAS effect-size of the MMP9 rs17576 G allele in primiparae was 4.04. This indicate an AA versus GG difference in VAS of 8.08. Moreover, other SNPs in linkage disequilibrium with MMP9 rs17576 G could also be involved. How the MMP9 genotype interferes with daily activities during pregnancy may be debated. Still, a clear G allele dependent effect, in particular in primiparae, was observed. Hence, the present data suggest that MMP9 genotype may be associated to the pain mechanism during pregnancy.
We thank Øivind Skare for help with the statistical analyses.
Gutke A, Ostgaard HC, Oberg B. Pelvic girdle pain and lumbar pain in pregnancy: a cohort study of the consequences in terms of health and functioning. Spine (Phila Pa 1976) 2006;31:E149–55. PubMedCrossrefGoogle Scholar
Sydsjo G, Sydsjo A. Newly delivered women’s evaluation of personal health status and attitudes towards sickness absence and social benefits. Acta Obstet Gynecol Scand 2002;81:104–11. PubMedCrossrefGoogle Scholar
Wu WH, Meijer OG, Uegaki K, Mens JM, van Dieen JH, Wuisman PI, Ostgaard HC. Pregnancy-related pelvic girdle pain (PPP), I: terminology, clinical presentation, and prevalence. Eur Spine J 2004;13:575–89. CrossrefPubMedGoogle Scholar
Vollestad NK, Torjesen PA, Robinson HS. Association between the serum levels of relaxin and responses to the active straight leg raise test in pregnancy. Man Ther 2012;17:225–30. PubMedCrossrefWeb of ScienceGoogle Scholar
Okada Y, Gonoji Y, Naka K, Tomita K, Nakanishi I, Iwata K, Yamashita K, Hayakawa T. Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. J Biol Chem 1992;267:21712–9. PubMedGoogle Scholar
Katsuda S, Okada Y, Okada Y, Imai K, Nakanishi I. Matrix metalloproteinase-9 (92-kd gelatinase/type IV collagenase equals gelatinase B) can degrade arterial elastin. Am J Pathol 1994;145:1208–18. PubMedGoogle Scholar
Reponen P, Sahlberg C, Munaut C, Thesleff I, Tryggvason K. High expression of 92-kDa type IV collagenase (gelatinase) in the osteoclast lineage during mouse development. Ann N Y Acad Sci 1994;732:472–5. PubMedCrossrefGoogle Scholar
Alexander CM, Hansell EJ, Behrendtsen O, Flannery ML, Kishnani NS, Hawkes SP, Werb Z. Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation. Development 1996;122:1723–36. PubMedGoogle Scholar
Lenhart JA, Ryan PL, Ohleth KM, Palmer SS, Bagnell CA. Relaxin increases secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 during uterine and cervical growth and remodeling in the pig. Endocrinology 2001;142:3941–9. PubMedCrossrefGoogle Scholar
Cao WH, Liu HM, Liu X, Li JG, Liang J, Liu M, Niu ZH. Relaxin enhances in-vitro invasiveness of breast cancer cell lines by upregulation of S100A4/MMPs signaling. Eur Rev Med Pharmacol Sci 2013;17:609–17. PubMedGoogle Scholar
Chen HY, Lin WY, Chen YH, Chen WC, Tsai FJ, Tsai CH. Matrix metalloproteinase-9 polymorphism and risk of pelvic organ prolapse in Taiwanese women. Eur J Obstet Gynecol Reprod Biol 2010;149:222–4. PubMedCrossrefWeb of ScienceGoogle Scholar
Hirose Y, Chiba K, Karasugi T, Nakajima M, Kawaguchi Y, Mikami Y, Furuichi T, Mio F, Miyake A, Miyamoto T, Ozaki K, Takahashi A, Mizuta H, Kubo T, Kimura T, Tanaka T, Toyama Y, Ikegawa S. A functional polymorphism in THBS2 that affects alternative splicing and MMP binding is associated with lumbar-disc herniation. Am J Hum Genet 2008;82:1122–9. CrossrefPubMedWeb of ScienceGoogle Scholar
Han YJ, Kim HN, Yoon JK, Yi SY, Moon HS, Ahn JJ, Kim HL, Chung HW. Haplotype analysis of the matrix metalloproteinase-9 gene associated with advanced-stage endometriosis. Fertil Steril 2009;91:2324–30. Web of ScienceCrossrefPubMedGoogle Scholar
Brooks R, Kizer N, Nguyen L, Jaishuen A, Wanat K, Nugent E, Grigsby P, Allsworth JE, Rader JS. Polymorphisms in MMP9 and SIPA1 are associated with increased risk of nodal metastases in early-stage cervical cancer. Gynecol Oncol 2010;116:539–43. Web of SciencePubMedCrossrefGoogle Scholar
Stafne SN, Salvesen KA, Romundstad PR, Stuge B, Morkved S. Does regular exercise during pregnancy influence lumbopelvic pain? A randomized controlled trial. Acta Obstet Gynecol Scand 2012;91:552–9. CrossrefWeb of ScienceGoogle Scholar
Shipley JM, Doyle GA, Fliszar CJ, Ye QZ, Johnson LL, Shapiro SD, Welgus HG, Senior RM. The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type II-like repeats. J Biol Chem 1996;271: 4335–41. CrossrefPubMedGoogle Scholar
Zhang B, Henney A, Eriksson P, Hamsten A, Watkins H, Ye S. Genetic variation at the matrix metalloproteinase-9 locus on chromosome 20q12.2-13.1. Hum Genet 1999;105:418–23. CrossrefPubMedGoogle Scholar
Hu Z, Huo X, Lu D, Qian J, Zhou J, Chen Y, Xu L, Ma H, Zhu J, Wei Q, Shen H. Functional polymorphisms of matrix metalloproteinase-9 are associated with risk of occurrence and metastasis of lung cancer. Clin Cancer Res 2005;11:5433–9. CrossrefPubMedGoogle Scholar
Naqvi T, Duong TT, Hashem G, Shiga M, Zhang Q, Kapila S. Relaxin’s induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants. Arthritis Res Ther 2005;7: R1–11. CrossrefPubMedGoogle Scholar
Ahmad N, Wang W, Nair R, Kapila S. Relaxin induces matrix-metalloproteinases-9 and -13 via RXFP1: induction of MMP-9 involves the PI3K, ERK, Akt and PKC-zeta pathways. Mol Cell Endocrinol 2012;363:46–61. PubMedCrossrefWeb of ScienceGoogle Scholar
About the article
Published Online: 2018-02-14
Published in Print: 2018-01-26
Research funding: The study was supported by a grant from the Central Norway Regional Health Authority.
Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could lead to potential conflicts of interest.
Informed consent: The participants received written information and signed informed consent forms.
Ethical approval: The study was performed in accordance with the Helsinki Declaration and approved by the Regional Committee for Medical and Health Research Ethics (REK 4.2007.81).
AKM, AM, SNS, SM and JG performed the research. AKM, AM, SNS, SM and JG analyzed the data. AKM, AM, SNS, SM, HSR, NKV, KS and JG drafted the manuscripts. AKM and JG wrote the paper. HSR, NKV, SM and JG designed the study. All authors read and approved the final manuscript.
Availability of data and materials
All data underlying the findings may be available upon request. Requests for the data should be addressed to Director General Pål Molander or Director of Communication Sture Bye at National Institute of Occupational Health (NIOH), Norway: email@example.com.
Citation Information: Scandinavian Journal of Pain, Volume 18, Issue 1, Pages 93–98, ISSN (Online) 1877-8879, ISSN (Print) 1877-8860, DOI: https://doi.org/10.1515/sjpain-2017-0168.