An estimated 15 million babies are born too early every year i.e., 1 in 10 babies. Approximately 1 million children die each year due to complications of preterm birth (PTB) [1]. Globally, prematurity is the leading cause of death in children under the age of 5 years [1]. Many survivors face a lifetime of disability, including learning disabilities, visual and hearing problems. PTB rates are increasing despite advancing knowledge of risk factors for preterm labor (PTL) and the introduction of public health and medical interventions [2, 3, 4, 5, 6].

Key treatments for PTL have focused on the prevention or inhibition of myometrial contractions, mainly to provide time to administer steroids to aid fetal lung maturation and transfer to a special neonatal care unit [7, 8].

Understanding more about the mechanisms of labor is essential to identify targets for novel and more effective therapies to stop or prevent PTL. The neurohypophysial hormone oxytocin (OXT) is named after the “quick birth” which it causes due to its uterotonic activity [9]. Oxytocin receptor (OXTR) gene expression is present in humans in the amnion, chorion, and decidua [10] OXT binding to OXTR significantly increases in human fetal membranes specially amnion with the onset of labor. Terzidou et al. [11] revealed that human OXTR expression increases spontaneously in post-labor amnion epithelial cells and that treatment with interleukin (IL) 1B stimulates OXTR expression in pre-labor amnion when cultured. Amnion is not a contractile tissue; therefore, the physiological role of the OXT/OXTR system in amnion must be in some respect different from its role in the myometrium. The amnion plays an important role in the onset of human labor. It is a major source of prostaglandins (PTGs) and inflammatory cytokines synthesis, which increase both before and during labor. Terzidou et al. [11] found that the release of PTGE2 by human amnion epithelial cells is significantly increased after OXT stimulation. This supports a complementary role for OXT/OXTR in the activation of the amnion that occurs at the time of labor. It is established that the PTGS2 (prostaglandin synthetase 2) enzyme, which mediates the committing and rate-limited step of PTG biosynthesis, generating a PTGH2 intermediate that is converted to the terminal PTGs, is central to increased PTGs synthesis in the human amnion at the time of labor [12, 13, 14].

OXT stimulates myometrial contractions through multiple signaling pathways. Binding of OXT to its receptor has been known to lead to G-protein coupling and subsequently, an increase in intracellular calcium levels to mediate the generation of force [15]. There is a role for the OXT-OXTR system in the onset of human labor, additional to the stimulation of myometrial contractions, that involves an increase in the expression of cyclooxygenase-2 (COX-2) and other inflammatory mediators known to be associated with the onset of labor [16, 17].

Kim et al. [16] in their study demonstrated that NF-$\kappa$B activation is required for OXT-induced COX-2 expression in human myometrium and amnion. They also showed that Cross-talk exists between the OXT induced activation of Mitogen-Activated Protein Kinases (MAPKs) and NF-$\kappa$B signaling cascades in the human amnion, but not myometrium. OXT-OXTR interaction leads to NF-$\kappa$B activation and subsequent upregulation of PTGs, inflammatory chemokines, and cytokines that are known to play roles in fetal membrane remodeling, cervical ripening, and myometrial activation. Amnion is an important site of PTG production within the human uterus and its activation is critical for fetal membrane remodeling, cervical ripening, and the stimulation of myometrial contraction [16].

Therefore, OXTR is commonly used as a target for the development of tocolytics. Atosiban, an OXTR antagonist acting on both myometrial & decidual OXTRs has been used to arrest premature uterine contraction but has failed to reduce the incidence of PTB or improve the neonatal outcome compared with placebo [18].

The role of OXT in PTG pathway activation leading to the onset of labor suggests that potential clinical use of OXTR antagonists requires re-evaluation. The study of molecular genetics involving the OXT-OXTR system in gestational tissues may prove fruitful in deciphering these complex mechanisms leading to PTB. The present study gains insight into the OXT-OXTR system in PTB to build the ground for future research for better-targeted interventions.

Various socio-demographic features in the two groups were comparable. The mean serum OXT level in active labor in PTB cases was 48.56 $\pm$ 6.2 pg/mL which was significantly higher (P 0.001) than those with term labor (43.00 $\pm$ 3.96 pg/mL) (Table 1). In the subgroup analysis of early v/s late preterm, the serum OXT levels continued to be higher in preterm than term but there was no statistically significant difference. The values of OXTR $\mathrm{\Delta }$Cq in maternal blood of cases and controls are compared in Table 1.

The maternal blood OXTR $\mathrm{\Delta }$Cq value in preterm cases was significantly lower (i.e., expression of OXTR gene in maternal blood was significantly higher) in comparison to controls (P 0.001). Also, the Fold change (FC) was $>$ 2.44 which means its expression was 2.44 times in PTL than that in term labor (Fig. 2). OXTR gene expression in amnion was significantly higher in PTL (FC $>$ 3.44) as compared to term labor (P 0.001) (Fig. 2). However, the value of OXTR $\mathrm{\Delta }$Cq in maternal blood as well in amnion was not statistically significantly different in early and late preterm subgroups.

Fig. 2.

OXTR gene expression in maternal blood and Amnion expressed in terms of Fold Change.

In correlation studies, a negative correlation was observed between maternal serum OXT levels and OXTR $\mathrm{\Delta }$Cq in maternal blood (i.e., positive correlation between maternal serum OXT levels and OXTR gene expression in maternal blood) in PTL (Table 2). Similarly, a negative correlation was seen between maternal serum OXT levels and OXTR $\mathrm{\Delta }$Cq in amnion in PTL (i.e., positive correlation between maternal serum OXT levels and OXTR gene expression in amnion) (Table 2).

A negative correlation was observed between maternal serum OXT levels and amnionic OXTR $\mathrm{\Delta }$Cq (i.e., positive correlation between maternal serum OXT levels and amnionic OXTRs) when all the study subjects (preterm & term) were combined into a single group (Table 3 and Fig. 3). There was a significant proportionate rise in maternal serum OXT levels with increased OXTR gene expression in amnion (P 0.025) in all laboring subjects irrespective of the period of gestation [POG].

Fig. 3.

Scatter dot plot graph showing a negative correlation between OXTR $\mathrm{\Delta }$Cq amnion and maternal serum OXT levels in the whole study population.

Similarly, a negative correlation was seen between maternal serum OXT levels with the period of gestation, birth weight, and placental weight in the whole study population (Table 3, Fig. 4 and Fig. 5). So, as maternal serum OXT levels increased; there is a corresponding significant reduction in POG, birth weight, placental weight.

Fig. 4.

Scatter dot plot graph showing a positive correlation between OXTR delta Ct in Amnion and period of gestation in preterm & term subjects combined.

Fig. 5.

Scatter dot plot graph showing a positive correlation between OXTR delta Ct in maternal blood and period of gestation in preterm & term subjects combined.

The mean serum OXT level in PTB cases was 48.56 $\pm$ 6.97 pg/mL; significantly higher than in controls (43.00 $\pm$ 3.96 pg/mL), P 0.001 similar to earlier studies. OXTR gene expression in maternal blood was significantly higher (2.44 folds) in the PTB cases as compared to term controls (P 0.001). There is also an increase in OXTR mRNA in human amniotic cells up to 3.44 folds in preterm. OXTR mRNA concentrations increased significantly after the initiation of the active phase of labor both in blood and amnion which was significant. Chibbar et al. [20] compared prelabour and post-labor tissues which revealed OXT mRNA levels were three to fourfold higher after the spontaneous labor. The second change is that the cervical connective tissue and smooth muscle must be capable of dilatation to allow the passage of the fetus from the uterus. These changes are accompanied by a shift from progesterone to estrogen dominance, increased responsiveness to oxytocin through up-regulation of the myometrial oxytocin receptor, increased PTG synthesis in the uterus, increased myometrial gap junction formation, decreased nitric oxide (NO) activity, and an increased influx of calcium into myocytes [21, 22] with ATP dependent binding of myosin to actin [23], increased endothelin leading to augmented uterine blood flow and myometrial activity [24]. The local increase in the estrogen/progesterone ratio in late pregnancy may activate OXT gene transcription and increase the number of OXTRs in the myometrium and other gestational tissues. The latter may sensitize the myometrium to any local OXT, thereby leading to increased contractile activity and production of stimulatory PTGs. The increased PTGs would directly stimulate the myometrium and potentially lead to a further increase in the production of OXT and its receptor. OXT binding to OXTR significantly increases in human fetal membranes specially amnion with the onset of labor [25]. Studies in several species have shown OXT to stimulate uterine prostaglandin (PTG) synthesis when administered near term. Amnion is a major site of PTG production in human pregnancy and its activation is critical for cervical ripening and the stimulation of myometrial contractions. Just prior to the onset of labor, there is an increase in inflammatory cytokine release from the amnion [26, 27] as well as increased PTG synthesis, particularly PTGE2 [28, 29]. Collectively these changes promote cervical ripening, lower uterine segment remodeling, and initiation of myometrial contractions [29, 30, 31, 32, 33].

This suggests a role for OXT/OXTR in the activation of the amnion that occurs at the time of labor. Present study results have confirmed the findings of previous studies where OXTR mRNA or OXT binding sites were identified in Amnion [11]. Our data have extended these findings by systematic determination of gestation and labor-associated effects on OXTR mRNA expression both in maternal blood and amnionic tissue samples. There was a significant effect of the labor process, with both blood and amnion OXTR mRNA concentrations increasing after the initiation of the active phase of labor. It indicates upregulation of OXTR concentrations seen at parturition is primarily regulated at the transcriptional level. This supports previous data from binding studies [25] and Northern blot analyses [34]. This coordinated interaction involving OXT, OXTRs, and PTGs in human fetal membranes could ultimately result in the onset of parturition. OXTRs are present in blood & amnion and show major regulatory changes at the onset of labor whether at term or preterm with increased expression in PTB cases.

Terzidou et al. [11] showed the increased synthesis of PTGs in human amnion between 2 and 6 hours following OXT treatment and was associated with increased prostaglandin synthetase 2 (PTGS2) expression. The increased ability of human amnion to produce PTGE2 in response to OXT treatment suggests a complementary role of the OXT/OXTR system in the activation of human amnion and the onset of labor. This suggests OXT-PTGs autocrine paracrine circuit system in decidua, amnion, and myometrium induce and facilitate labor in situ. It makes sense that plasma OXT levels do not alter before the onset of labor if this system is mainly regulated within decidua and fetal membranes in situ [20].

Terzidou V et al. [11], in their study, revealed that human OXTR expression increases in post-labor amnion epithelial cells and that treatment with interleukin IL1B stimulates OXTR expression before the onset of labor. The increased ability of human amnion to produce PTGE in response to OXT treatment suggests a complementary role of the OXT/OXTR system in the activation of the PTG pathway in the human amnion resulting in the onset of labor. In addition to mediating contractions, the role of OXT in biochemical processes i.e., PTG pathway that leads to the onset of labor suggests that the potential clinical use of OXTR antagonists requires re-evaluation. Szukiewicz et al. [35] concluded that upregulation of OXTR within placental trophoblast cells localized close or adherent to the uterine wall may play a crucial role in labor with efficient contractile activity leading to vaginal delivery. The present study further supports the concept that OXTR expression is regulated in a paracrine as well as endocrine fashion and OXTR expression is a limiting factor of parturition, which was proposed in the previous reports [25, 36]. They also explain how OXT may play a pivotal role in the onset of labor in the absence of significant changes in maternal plasma concentration.

Kim et al. [37] showed that OXT increases the expression of COX-2 and other inflammatory mediators known to be associated with the onset of labor in both the myometrium and amnion via activation of NF-$\kappa$B and MAPKs. OXT-OXTR interaction leads to Nuclear factor (NF-$\kappa$B) activation and subsequent upregulation of PTGs, inflammatory chemokines, and cytokines that are known to play a key role in fetal membrane remodeling, cervical ripening, and myometrial activation. It is well established that IL1B concentrations within the uterus increase at the time of both term and PTL, as does the expression of several “labor-associated proteins”, such as PTGS2 and IL8, each of which is up-regulated by IL1B [12, 38, 39, 40, 41]. OXTR expression is also upregulated at term, at a time when IL1B concentrations are high, and PTGS2 and IL8 expression increases. Increased mRNA levels encoding OXTR and other uterine activation proteins such as COX-2 or PTGF2$\alpha$ receptor in decidua after the onset of labor were observed in the uterine tissues during term and PTL [42], suggesting that the basic regulation for uterine activation was similar between term and PTL.

Loudon et al. [39] have previously shown that PTGE2 production is lower in prelabour than in post-labor cells and that PTGE2 production in prelabour cells can be stimulated by IL1B to levels similar to that found in post-labor cells. This shows that the pathways for PTG synthesis are activated in post-labor cells, and our present study reinforces that upregulation of OXTR is a feature of amnion activation and is sensitive to inflammatory cytokines.

Szukiewicz D et al. [35] in their recent study concluded that upregulation of OXTR within placental trophoblast cells localized close or adherent to the uterine wall may play a crucial role in labor with efficient contractile activity leading to vaginal delivery.

There is some evidence for a premature activation of OXT secretion in PTB, suggesting a pathogenic role for it in PTL. Our study reinforces this fact as there is a significant rise in OXT levels in PTB cases with P value of 0.001.

OXTR antagonists are one class of treatments that have been developed as tocolytic agents for women in PTL. Kim SH et al. [37], in their study, found that Atosiban, a mixed OTR/V1a antagonist widely used as a tocolytic drug, acts as a biased ligand in amnion cells. It fails to inhibit the activation of pro-inflammatory mediators stimulated by oxytocin, and, in the absence of OXT, it activates the same pro-labor inflammatory pathways in amnion in a way similar to OXT and increases cytokine/chemokine and PTG secretion, which may have detrimental effects upon the fetus also in PTL at early gestational ages.

Amnion is a major site of prostaglandin and proinflammatory cytokine and chemokine release leading to cervical ripening and fetal membrane remodeling [16]. Persistent activation of pro-inflammatory pathways in human amnion including activation of NF-$\kappa$B, appears to precede the onset of labor. Atosiban has a low affinity for the OXTR and its antagonistic activities at the V${}_{1a}$R [37], has ignited interest in the development of other peptide and nonpeptide antagonists with greater specificity for the OXTR. It appears from the present study that a molecule with targeted intervention at amnionic OXTR gene may be a promising drug prospect to prevent inflammatory pathway cascade leading to PTL. The significant reduction in gestational length (PTL) with overexpression of the OXTR gene in amnion & blood could be used as a landmark for future studies on formulating preventive strategies for preterm birth.

Significantly higher maternal serum OXT levels seen in PTB cases suggest that the basic regulation for uterine activation was similar between term and PTL. The OXTR expression in blood & amnion is 2.44 folds & 3.44 folds higher respectively in women having PTB. Upregulation of OXTR expression in amnion seems to be crucial in the mechanisms involved in PTL leading to contractile activity and PTB. Amnion thus may be acting as a major site of prostaglandins production on stimulation of OXTRs and linking OXT-PTGs autocrine paracrine circuit system to facilitate PTL. This gives a new insight into the OXT/OXTR system in human parturition involving amnion and suggests that its therapeutic modulation could be a strategy for regulating both contractile and inflammatory pathways in the clinical context of PTB.

Future studies may be planned to unveil these local OXT/OXTR signaling to devise better OXTR antagonists preferably targeting amnionic OXTR receptors to prevent PTB.

All authors were involved in designing the research study. All authors contributed to the performance of the experimental work and analysis of data generated. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

All the participants gave informed written consent for participation in the study and also for public dissemination and future use of the data generated from the study for the welfare of mankind and research purposes. Ethical approval was taken from the Institutional Ethics Committee for Human Research (IEC-HR-3472), UCMS & GTB hospital, University of Delhi, India.

We thank two anonymous reviewers for the excellent criticism of the article.

The work has been carried out under the MD thesis program with the support of an extramural grant entitled “A study on gene environment interaction and high-risk phenotypes in the etiology of preterm birth in North Indian population (ref ID: 5/7/575/11-RHN)” sanctioned by Indian Council of Medical Research (ICMR), India for the duration from 2012–2016 to one of the authors Basu Dev Banerjee as Principal Investigator as project grant to Biochemistry department of UCMS institution for research purposes.

The authors declare no conflict of interest.