Introduction

Recurrent spontaneous abortion (RSA) is clinically defined as the spontaneous loss of three or more pregnancies before the 24^th week of gestation. It affects approximately 0.5–3% of women and can be caused by different factors [1]. However, in about 50% of the cases the cause remains unknown [2]. Genetic contribution to idiopathic RSA (IRSA) has been extensively investigated and several studies suggest that genetic variants may predispose to RSA [3, 4].

There is growing evidence that circadian rhythms influence a large variety of physiological systems including reproduction [5, 6]. Recent evidence in animals links disruption of synchronous clock activity with the pathogenesis of pregnancy complications. Repeated shifting of the light-dark cycle, which disrupts endogenous circadian timekeeping, strongly reduces pregnancy success in mice [7]. Additionally, impaired reproductive capacities were associated with night shift work in humans as well [8]. Circadian clock genes are expressed rhythmically in the gravid uterus and placental tissue, suggesting that circadian clocks contribute to the regulation of rhythms in the developing fetus [9]. Despite the evidence of the importance of circadian rhythms in reproduction in animal models, there is very limited knowledge regarding the role of the circadian system in pregnancy in humans.

Circadian rhythms are controlled by an endogenous oscillator, the circadian clock, which consist of a network of transcriptional-translational feedback loops of many genes [10]. The CLOCK gene represents the central node in the network and is an essential activator of downstream elements in the pathway critical to the generation of circadian rhythms [11].

Genetic variants in the CLOCK gene have been previously associated with reproductive abnormalities, such as irregular menstrual cycles and increased risk of endometriosis [12, 13].

Therefore, we aimed to evaluate whether there is an association between the two genetic variants in the CLOCK gene and idiopathic recurrent spontaneous abortions. For that purpose, we performed a case-control genetic association study of polymorphic sites in the CLOCK gene on a sample of patients with IRSA in comparison with sample of women with two or more live births and no history of pathologic pregnancies.

Materials and methods

Results

Genotype frequencies of investigated polymorphisms were in accordance with those predicted by the Hardy-Weinberg equilibrium in the study and in the control group (p<0.05). Genotype distribution of the CLOCK polymorphisms of the 268 IRSA patients and 284 controls are shown in Table 1.

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Table 1. Genotype distribution of the CLOCK polymorphisms of the 268 IRSA patients and 284 controls.

https://doi.org/10.1371/journal.pone.0196345.t001

We found statistically significant differences in genotype distribution of rs6850524 (p = 4∙10⁻⁴, x² = 15.55) and rs11932595 (p = 0.03, x² = 6.76) in the CLOCK gene. Our analysis showed that G allele under dominant model (GG+GC/CC) for rs6850524 (p = 2∙10⁻⁴, x² = 13.58, OR = 2.28, 95%CI = 1.46–3.56) as well as G allele under dominant model (GG+GA/AA) for rs11932595 (p = 0.04, x² = 4.12 OR = 1.47, 95%CI = 1.01–2.04) might be risk factors against IRSA.

The statistical power to detect a significant result in the presence of the actual genotype-phenotype effect with genotype relative risk equal to at least 1.4, was 82%, when taking into account the sample size, the significance threshold of 0.05, the prevalence of IRSA in the general population, disease allele frequency and considering multiplicative model of genetic association.

Discussion

Progress in understanding the pathological mechanisms underlying recurrent spontaneous abortions has been greatly hampered by the complex nature of the disorder. Previous studies suggest that genetic factors play an important role in unexplained recurrent spontaneous abortions [3, 4]. In the case-control study we found evidence of an association between IRSA and gene variants of the CLOCK gene in a sample of 552 women. To the best of our knowledge, this is the first association study between genetic variability in the CLOCK gene and IRSA.

Several lines of evidence support our findings. In humans, it has been shown that night shift workers have an increased rate of reproductive abnormalities as well as adverse pregnancy outcomes in terms of miscarriage, low birth weight and preterm birth [17]. Similarly it has been seen in two other studies, where night shift work had influence on early spontaneous pregnancy loss, time to achieve pregnancy, menstrual cycle disturbances and endometriosis [18, 19]. Circadian disruption as potential reproductive hazard has been also noticed in flight attendants; where risk of a first-trimester miscarriage was increased in women flying during home-base sleep hours [20].

Genetic variants in the circadian rhythm genes ARNTL and NPAS2 have been suggested to contribute to fertility, and the genetic variants in the ARNTL gene has been associated with a higher number of miscarriages. Certain genotypes in the Npas2 gene have been associated with a decreased number of miscarriages [21]. Genetic variation in circadian rhythm genes ARNTL2, CRY2, DEC1, PER3 and RORA have also been associated with increased risk of placental abruption [22].

Furthermore, it has been demonstrated that low CLOCK expression level in pregnant women may lead to spontaneous abortion [23]. Significantly lower CLOCK gene expression has been detected in spontaneously aborted fetuses compared with fetuses from induced abortion [23].

In mice, mutations in the circadian CLOCK gene cause multiple reproductive abnormalities, including increased fetal reabsorption during pregnancy and an elevated rate of full-term pregnancy failure [24, 25].

Although functional studies on rs6850524 and rs11932595 were not performed in IRSA, the polymorphisms affect the expression of the CLOCK gene in a number of different tissues including nervous system, subcutaneous adipose tissue, skin, muscle, esophagus, thyroid (https://www.gtexportal.org/home/snp/rs6850524, https://www.gtexportal.org/home/snp/rs11932595).

However, this study has limitations. A relatively small sample might influence the statistical power and, consequently, the detection of the true effect size. Also, genetic association studies are prone to α (Type I) statistical error and population-specific genotype effects.

In conclusion, we provide evidence that genetic variants in the CLOCK gene might be associated with IRSA. Further confirmation and mechanistic investigation of circadian system in idiopathic recurrent spontaneous abortion is warranted.