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Background: Specific heat shock proteins are associated with pregnancy complications, including spontaneous preterm birth (SPTB). Placental proteomics and whole exome sequencing recently suggested an association between heat shock protein HSPA5 and uncomplicated SPTB. In the present study, we investigated the localization of and possible roles for HSPA5 in SPTB. Methods: Western blot was performed to validate the result from the previously published proteomic analysis. We used qPCR to assess mRNA expression of genes and immunohistochemistry and immunoelectron microscopy to examine localization of HSPA5 in placental tissue. We silenced the HSPA5 gene in the HTR8/SVneo human trophoblast cell line to investigate possible functions of HSPA5. Results: HSPA5 was upregulated in placentas from SPTBs compared to spontaneous term births. We did not observe upregulation of HSPA5 mRNA in placental samples. The protein was localized in placental trophoblast in both spontaneous preterm and term placentas. Gene silencing of HSPA5 in human trophoblast cell culture affected the inflammatory response and decreased the expression of several proinflammatory genes. Conclusions: We suggest that upregulation of HSPA5 in the placenta is associated with spontaneous preterm labor. HSPA5 may promote the inflammatory response and alter the anti-inflammatory state of the placenta which could eventually lead to premature labor. Impact: -We validated upregulation of HSPA5 in placentas from spontaneous preterm birth. HSPA5 was not upregulated at transcriptional level which suggests that it may be regulated post-translationally. -Silencing HSPA5 in a human trophoblast–derived cell line suggested that HSPA5 promotes expression of proinflammatory cytokines. The emerging inflammation could lead to spontaneous preterm labor. -Identifying inflammatory pathways and factors associated with spontaneous preterm birth increases knowledge of the molecular mechanisms of premature labor. This could pro

Background: Preterm birth (<37 weeks of gestation) is a major cause of neonatal death and morbidity. Up to 40% of the variation in timing of birth results from genetic factors, mostly due to the maternal genome. Methods: We conducted a genome-wide meta-analysis of gestational duration and spontaneous preterm birth in 68,732 and 98,370 European mothers, respectively. Results: The meta-analysis detected 15 loci associated with gestational duration, and four loci associated with preterm birth. Seven of the associated loci were novel. The loci mapped to several biologically plausible genes, for example HAND2 whose expression was previously shown to decrease during gestation, associated with gestational duration, and GC (Vitamin D-binding protein), associated with preterm birth. Downstream in silico-analysis suggested regulatory roles as underlying mechanisms for the associated loci. LD score regression found birth weight measures as the most strongly correlated traits, highlighting the unique nature of spontaneous preterm birth phenotype. Tissue expression and colocalization analysis revealed reproductive tissues and immune cell types as the most relevant sites of action. Conclusions: We report novel genetic risk loci that associate with preterm birth or gestational duration, and reproduce findings from previous genome-wide association studies. Altogether, our findings provide new insight into the genetic background of preterm birth. Better characterization of the causal genetic mechanisms will be important to public health as it could suggest new strategies to treat and prevent preterm birth., Author summary Annually, more than 15 million pregnancies are affected by preterm births all over the world. There are no effective ways to prevent preterm birth, and premature babies suffer from neonatal mortality and lifelong morbidities. Genetic factors of mother and fetus explain a large proportion, approximately 30–40%, of the of the variation in gestational age at delivery. To date, there have been just a few unbiased genome-wide investigations set out to locate these genes. Better characterization of the causal genetic mechanisms could suggest new strategies to treat and prevent preterm birth. In the current study, we aimed to identify maternal genetic factors that contribute to the timing of birth by meta-analyzing genome-wide association studies from European populations. We detected 17 independent loci that were associated with gestational duration and/or the risk of preterm birth. Ten of the loci replicated associations from previous studies, and seven were novel. The replicated associations provide strong evidence for the importance of these loci in the timing of birth, although the exact genes and causal variants and pathways still require further functional analysis. The seven novel associations provide further intriguing candidates that may account for the risk of preterm birth. Bioinformatics analysis proposed the associated loci have regulatory functions predominantly in immune cell types and reproductive tissues. The analysis further highlighted the unique nature or preterm birth as a phenotype, since the only traits with strong correlations were birth weight measures that are closely linked to the studied phenotypes. Our findings complement the knowledge of the genetic factors of preterm birth.

Background: Preterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth. Methods: We analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas. Results: Exome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit–Robo signaling, and extracellular matrix organization. Conclusions: Alpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.

Protein kinases and phosphatases regulate cellular processes by reversible phosphorylation and dephosphorylation events. CPPED1 is a recently identified serine/threonine protein phosphatase that dephosphorylates AKT1 of the PI3K-AKT signalling pathway. We previously showed that CPPED1 levels are down-regulated in the human placenta during spontaneous term birth. In this study, based on sequence comparisons, we propose that CPPED1 is a member of the class III phosphodiesterase (PDE) subfamily within the calcineurin-like metallophosphoesterase (MPE) superfamily rather than a member of the phosphoprotein phosphatase (PPP) or metal-dependent protein phosphatase (PPM) protein families. We used a human proteome microarray to identify 36 proteins that putatively interact with CPPED1. Of these, GRB2, PAK4 and PIK3R2 are known to regulate the PI3K-AKT pathway. We further confirmed CPPED1 interactions with PAK4 and PIK3R2 by coimmunoprecipitation analyses. We characterized the effect of CPPED1 on phosphorylation of PAK4 and PIK3R2 in vitro by mass spectrometry. CPPED1 dephosphorylated specific serine residues in PAK4, while phosphorylation levels in PIK3R2 remained unchanged. Our findings indicate that CPPED1 may regulate PI3K-AKT pathway activity at multiple levels. Higher CPPED1 levels may inhibit PI3K-AKT pathway maintaining pregnancy. Consequences of decreased CPPED1 expression during labour remain to be elucidated.

MicroRNAs (miRNAs) are important regulators of gene expression, and their expression is associated with many physiological conditions. Here, we investigated potential associations between expression levels of miRNAs in human placenta and the onset of spontaneous term birth. Using RNA sequencing, we identified 54 miRNAs differentially expressed during spontaneous term labor compared to elective term births. Expression levels of 23 miRNAs were upregulated, whereas 31 were downregulated at least 1.5-fold. The upregulated miRNA miR-371a-5p putatively targets CPPED1, expression of which decreases during spontaneous birth. We used a luciferase reporter–based assay to test whether a miR-371a-5p mimic affected translation when it bound to the 3′ untranslated region of CPPED1. In this setting, the miR-371a-5p mimic resulted in lower luciferase activity, which suggests that miR-371a-5p regulates levels of CPPED1. In conclusion, inversely correlated levels of miR-371a-5p and CPPED1 suggest a role for both in spontaneous delivery.

Spontaneous preterm birth (SPTB) is the leading cause of neonatal death and morbidity worldwide. Both maternal and fetal genetic factors likely contribute to SPTB. We performed a genome-wide association study (GWAS) on a population of Finnish origin that included 247 infants with SPTB (gestational age [GA] < 36 weeks) and 419 term controls (GA 38–41 weeks). The strongest signal came within the gene encoding slit guidance ligand 2 (SLIT2; rs116461311, minor allele frequency 0.05, p = 1.6×10−6). Pathway analysis revealed the top-ranking pathway was axon guidance, which includes SLIT2. In 172 very preterm-born infants (GA <32 weeks), rs116461311 was clearly overrepresented (odds ratio 4.06, p = 1.55×10−7). SLIT2 variants were associated with SPTB in another European population that comprised 260 very preterm infants and 9,630 controls. To gain functional insight, we used immunohistochemistry to visualize SLIT2 and its receptor ROBO1 in placentas from spontaneous preterm and term births. Both SLIT2 and ROBO1 were located in villous and decidual trophoblasts of embryonic origin. Based on qRT-PCR, the mRNA levels of SLIT2 and ROBO1 were higher in the basal plate of SPTB placentas compared to those from term or elective preterm deliveries. In addition, in spontaneous term and preterm births, placental SLIT2 expression was correlated with variations in fetal growth. Knockdown of ROBO1 in trophoblast-derived HTR8/SVneo cells by siRNA indicated that it regulate expression of several pregnancy-specific beta-1-glycoprotein (PSG) genes and genes involved in inflammation. Our results show that the fetal SLIT2 variant and both SLIT2 and ROBO1 expression in placenta and trophoblast cells may be correlated with susceptibility to SPTB. SLIT2-ROBO1 signaling was linked with regulation of genes involved in inflammation, PSG genes, decidualization and fetal growth. We propose that this receptor-ligand couple is a component of the signaling network that promotes SPTB.

Understanding of timing of human parturition is incomplete. Therefore, we carried out proteomic analyses of full-term placentas from uncomplicated pregnancies to identify protein signatures associated with the onset of spontaneous delivery. We found quantitative associations of 10 proteins with spontaneous term birth, evident either in the basal or in the chorionic plates or in both. Additional 18 proteins were associated according to the location within placenta indicating local variations in protein amounts. Calcineurin-like phosphoesterase domain-containing 1 (CPPED1), a phosphatase previously suggested dephosphorylating AKT1/PKB, was one of the identified proteins. qRT-PCR revealed the mRNA level of CPPED1 was higher in elective caesarean deliveries than in spontaneous births, while immunohistochemistry showed CPPED1 in cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts. Noteworthy, phosphorylation status of AKT1 did not differ between placentas from elective caesarean and spontaneous deliveries. Additionally, analyses of samples from infants indicated that single-nucleotide polymorphisms rs11643593 and rs8048866 of CPPED1 were associated with duration of term pregnancy. Finally, post-transcriptional silencing of CPPED1 in cultured HTR8/SVneo cells by siRNAs affected gene expression in pathways associated with inflammation and blood vessel development. We postulate that functions regulated by CPPED1 in trophoblasts at choriodecidual interphase have a role in the induction of term labour, but it may be independent of AKT1.