Spontaneous preterm birth (SPTB) is the leading cause of neonatal morbidity and mortality worldwide. Approximately 12.0% of all infants are born preterm in the USA; this figure is 6.0-12.0% in the European Union (EU) [1,2], and 7.6% in Slovenia [3].
Infants born preterm can suffer from lifelong morbidities such as developmental delay, lung disease, vision, and hearing deficits, as well as other neurosensory impairments [4,5]. They are also predisposed to hypertension and diabetes in adult life [6].
The etiology of SPTB is multifactorial. In addition to multiple gestations and assisted reproductive technologies, several environmental contributors to SPTB have been proposed, such as an infection [7,8], maternal smoking during pregnancy [9,10], maternal/fetal stress [11,12], adolescence or advanced maternal age [13], cervical dysfunction [14], decidual thrombosis [15], and metabolic enzyme variation [14]. In about 70.0% of SPTB cases no risk factor can be identified, which makes genetic contribution a likely cause to be considered [16]. The role of genetic predisposition to SPTB is based on epidemiological evidence showing that SPTB tends to recur in families. Mothers with previous preterm deliveries have a significantly increased risk of preterm delivery in subsequent pregnancies [17,18,19,20]. Spontaneous preterm birth also occurs across generations and sibships [18,21,22,23]. The heri-tability of SPTB has been estimated to be in the range of 27.0-36.0% [24,25].
Based on their potential role in pathogenesis, as many as 482 genes have already been tested for genetic association with SPTB (HuGENavigator), however, validation of study results remains challenging [26,27]. One of the most frequently investigated candidate genes is the β-2-adrenergic receptor (
β-2-Adrenergic receptors, members of the superfamily of G protein-coupled receptors, mediate the catecholamine-induced activation of the adenylate cyclase signaling cascade, a mechanism that plays an important role in smooth muscle relaxation [28]. They are ubiquitously expressed in numerous human tissues, including smooth muscle cells of the trachea, bronchi, vasculature and the uterus. Uterine contractility is modulated by stimulation of
Several association studies have linked
Participants of the study all signed a written informed consent. The Republic of Slovenia National Medical Ethics Committee approved the study.
We included healthy mothers with singleton pregnancies who delivered after a spontaneous onset of labor (SPTB) before completed 37 weeks’ gestation. Gestational age was determined by the last menstrual period and confirmed by an ultrasound examination in the first trimester. Cases with known risk factors for SPTB (
Characteristics of 98 mothers with SPTB and 135 controls. SPTB: spontaneous preterm birth.Parameters SPTB Controls Mean maternal age (years) 30.20 ± 4.59 30.10 ± 3.92 0.28 Smoking during pregnancy % ( 8.3 (8) 2.9 (4) 0.15 Previous SPTB % ( 12.4 (12) 4.3 (6) 0.08 4.1 (4) 1.4 (2) 0.31 Urogenital infections % ( 7.2 (7) 4.3 (6) 0.35 High blood pressure % ( 7.2 (7) 4.3 (6) 0.35 Stressful events during pregnancy % ( 3.1 (3) 4.3 (6) 0.50
We conducted a case-control study including 98 female patients with SPTB and 135 female controls who gave birth at the Division of Obstetrics and Gynecology, University Medical Centre in Ljubljana, Slovenia. Controls were age-matched healthy mothers who delivered after an uncomplicated pregnancy after 37 weeks and delivered a neonate with appropriate-for-gestational-age birth weight (Table 1).
Genomic DNA was isolated from peripheral blood leukocytes using standard procedures. Real-time polymerase chain reaction (PCR) method performed on a 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using KASPar SNP genotyping chemistry carried out genotyping of the single nucleotide polymorphism (SNP). The PCR reaction mix of 8 μL final volume consisted of 3 μL of DNA sample, 4 μL of reaction mix 2X, 0.11 μL assay mix and 0.89 μL H2O. The protocol for PCR amplification was as follows: initial denaturation step at 94 °C for 15 min., then 10 cycles of denaturation at 94 °C for 20 seconds, followed by 5 seconds at 57 °C or 61 °C, 10 seconds at 72 °C, 10 seconds at 94 °C, 20 seconds at 57 °C or 61 °C, and final extension at 72 °C for 40 seconds. The allelic discrimination analysis was performed using SDS Software Version 1.2 (Applied Biosystems). Genotype assignment was performed and interpreted independently by two investigators.
We analyzed the significance of associations between allelic and genotype frequencies and disease status using the χ2 test. Odds ratios (ORs) and their respective 95% confidence intervals (95% CIs), were calculated to compare allelic and genotype distribution in patients and controls. To provide an additional quality step of the genotyping process we calculated the χ2 goodness-of-fit tests for deviation of genotype distribution from those predicted by Hardy-Weinberg equilibrium. The investigated associations were regarded as significant when they reached
To calculate the power of the study DSS Researcher’s Toolkit (
A literature search to find potential eligible studies of the association between
We included human studies meeting following criteria:
We classified subjects into three genotypes: AA, GA and GG. Then pooled effect was calculated for the dominant genetic model (GA+GG
Cases and controls did not differ in any demographic characteristic or recognized risk factor for SPTB. The history of a previous SPTB was more frequent in the SPTB group (12.4%) in comparison to controls (4.3%), however, the difference did not reach statistical significance (Table 1).
Genotype frequencies of investigated polymorphisms were in accordance with those predicted by the Hardy-Weinberg equilibrium in the group of patients and in the control group. Genotype and allelic distribution of the
Genotype and allelic distribution of the SPTB: spontaneous preterm birth.Parameters Genotype Frequencies Allele Frequencies ( AA GA GG χ2 A G χ2 SPTB cases 17 (17.3) 48 (49.0) 33 (33.7) 0.99 0.01 65 (42.0) 81 (42.0) 1.0 0 Controls 24 (18.0) 66 (49.0) 45 (33.0) 90 (58.0) 111 (58.0)
Association of SPTB: spontaneous preterm birth; OR: odds ratio; 95% CI: confidence interval; WSPTB: SPTB women; WC: control women.Genetic Model WSPTB OR (95% CI) χ2 Dominant GA+GG 1.03 (0.52-2.04) 0.92 0.01 Recessive AA+GA 0.98 (0.57-1.70) 0.92 0.01 Codominant AA+GG 0.99 (0.59-1.68) 0.92 0.01
The initial keyword search identified 17 articles (Figure 1). Four previously published case-control studies were included after a review together with added results of our case-control study based on characteristics summarized in Table 4. Therefore, five studies met inclusion criteria with a total of 404 SPTB cases and 878 term controls.
Characteristics of studies included in the meta analysis. PPROM: preterm premature rupture of the membranes; SPTB: spontaneous preterm birth; GA: gestational age.Refs. Year Country Population PPROM Excluded SPTB Cases Criteria Case Sample Size Control Criteria Control Group Size this study 2017 Slovenia Caucasian no GA <37 98 GA >37 135 [30] 2013 Korea Asian no GA <37 166 GA >37 289 [31] 2004 Hungary Caucasian no GA <37 32 GA >37 127 [33] 2002 Turkey Caucasian yes GA <37 80 GA >37 76 [32] 2002 USA Hispanic no GA <37 28 GA >37 251
Cochrane’s Q test and I2 test showed that there was no evidence of heterogeneity across all studies under the recessive genetic model, while moderate heterogeneity was present under the dominant genetic model. We found that there was no significant association of
In the case-control association study in the Slovenian population and meta-analysis of previous studies, we did not find any evidence of an association between SPTB and
This led us, and other authors, to investigate the association between the
The results of genetic association studies quite frequently fail to be reproduced in subsequent studies, either because the original findings are false-positive reports, or because the small genetic effects were not detectable [38]. Large sample sizes or meta-analysis are required in order to identify the small genetic effects of polymorphisms [39]. A meta analysis is a statistical tool that enables objective, quantitative synthesis of research findings, thus overcoming the problem of a small sample size and the inadequate statistical strength of genetic association studies [40].
To further investigate the role of the
Alternatively, the previously published meta analysis of three reports, including both studies that found association [31,32] and studies by Ozkur
In conclusion, both the association study in the Slovenian population and meta analysis showed no evidence of an association between