Low CLOCK and CRY2 in 2nd trimester human maternal blood and risk of preterm birth: a nested case-control study†

Abstract

Previous studies have observed an association between maternal circadian rhythm disruption and preterm birth (PTB). However, the underlying molecular mechanisms and the potential of circadian clock genes to serve as predictors of PTB remain unexplored. We examined the association of 10 core circadian transcripts in maternal blood with spontaneous PTB (sPTB) vs term births using a nested case–control study design. We used a public gene expression dataset (GSE59491), which was nested within the All Our Babies (AOB) study cohort in Canada. Maternal blood was sampled in trimesters 2–3 from women with sPTB (n = 51) and term births (n = 106), matched for 5 demographic variables. In 2nd trimester maternal blood, only CLOCK and CRY2 transcripts were significantly lower in sPTB vs term (p = 0.02 ~ 0.03, FDR < 0.20). A change of PER3 mRNA from trimesters 2 to 3 was significantly associated with sPTB (decline in sPTB, p = 0.02, FDR < 0.20). When CLOCK and CRY2 were modeled together in 2nd trimester blood, the odds ratio of being in the low level of both circadian gene transcripts was greater in sPTB vs term (OR = 4.86, 95%CI = (1.75,13.51), p < 0.01). Using GSVA and Pearson correlation, we identified 98 common pathways that were negatively or positively correlated with CLOCK and CRY2 expression (all p < 0.05, FDR < 0.10). The top three identified pathways were amyotrophic lateral sclerosis, degradation of extracellular matrix, and inwardly rectifying potassium channels. These three processes have previously been shown to be involved in neuron death, parturition, and uterine excitability during pregnancy, respectively.

Co-alterations of circadian clock gene transcripts in human placenta in preeclampsia

“…3 B). Interestingly, the Two-way ANOVA showed that gestation length trended towards a reduction in CLOCK [F(1,5) = 6.020, p = 0.0577], supporting our previous work that identified that PTB correlated with reduced maternal blood CLOCK mRNA 56 .

Figure 3 CLOCK protein is reduced in term PE placenta.

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“…Indeed, the fact that PF670462 upregulates PER1/2/3 might mask a specific effect of PER3 on HTR-8 cell migration; however, due to experimental constraints, we were unable to test whether PER3 downregulation would decrease HTR-8 migration. Another possibility is that PER3 might signal gestation length, as supported by our maternal blood study 56 , and preliminary GWAS study 74 , both of which suggest that deregulated or low PER3 levels might be linked to gestation length and predispose women to PTB. Mechanisms that PER3 may engage to signal gestation length remain unknown.…”

“…1 ). By referring to our previous method 56 , we linearly combined these common significant PE-associated circadian genes to generate a risk score of PE (Fig. 1 ).…”

“…Next, with our previous methods 56 , we conducted the Gene Set Variation Analysis (GSVA) using two independent datasets to explore the biological pathways that are involved in PE-clock gene relationship, respectively (Fig. 1 ).…”

Placental circadian lincRNAs and spontaneous preterm birth

“…Studies in mice have shown that loss of molecular clock function reduces reproductive success ( Miller et al, 2004 ; Kennaway et al, 2012 ; Sen and Hoffmann, 2020 ; Yaw et al, 2020 ; Hoffmann et al, 2021 ), and is associated with increased risk of mis-timed birth and dystocia ( Ratajczak et al, 2012 ; Miller and Takahashi, 2014 ). Our recent studies in human placenta indicate that co-alterations of the molecular clock mRNAs NR1D2 , CLOCK and PER3 are significantly associated with term preeclampsia ( Zhou et al, 2022 ), whereas co-alteration of CLOCK and CRY2 at the mRNA levels in second trimester maternal blood are significantly associated with an increased risk of sPTB ( Zhou et al, 2021a ). However, the expression pattern of core molecular clock genes in placentas of sPTB vs term births, as well as the underlying mechanism(s) are still unknown.…”