Steven M. Yellon

In brief: With an automated image analysis approach using artificial intelligence and machine learning, cytoarchitectural and spatial characteristics of prepartum cervix remodeling occur before term and preterm birth with loss of response to progesterone. Evaluation of local features of inflammation associated with prepartum cervix remodeling may distinguish pathophysiological from physiological processes to reduce risk of adverse pregnancy outcomes. The cervix functions as a gatekeeper barrier to maintain pregnancy and virtually vanishes for birth. Cervix remodeling occurs well before term while progesterone (P4) is at or near peak in maternal circulation. The inflammatory ripening process is associated with reduced cell nuclei (CN) density, increased density of resident macrophages (Mφs), and reduced cross-linked collagen in stroma. Whether a functional or actual loss of response to P4 regulates cytomorphological characteristics associated with prepartum ripening at term was the focus of the present study. In a murine model, ovariectomy on pregnancy day 16 produced preterm birth (<24 h, Ovx) in mice with an oil-filled implant, while P4 treatment (Ovx + P4) and controls (PP) birthed at term. Cytoarchitecture of cervix remodeling (reduced CN/area and cross-linked collagen degradation) was advanced in Ovx compared to control mice, but forestalled by sustained plasma P4 in Ovx + P4 mice. Loss of response to P4 was also evident at term by increased resident Mφs in controls and Ovx + P4 PP mice from initial responses. By contrast, births were delayed or did not occur in ovary-intact mice given P4. Adverse pregnancy outcomes and dystocia were associated with arrest of prepartum cervix ripening. A novel spatial morphometric approach found Mφ-stain area/Mφ/CN increased during prepartum ripening versus that on preterm or term birth. This biomarker for local inflammation was blocked in intact + P4 mice. Mφ area, as indicative of alternative activation versus smaller-sized inflammatory phenotypes, collectively suggests loss of prepartum cervix P4 responses in the final common pathway for term and preterm parturition.

The cervix functions both as gatekeeper barrier to maintain pregnancy and virtually vanish for birth at term in mammals. The period of remodeling well-before term is characterized by an inflammatory process associated with reduced cell nuclei density and cross-linked collagen, as well as increased density of resident macrophages in cervix stroma. Contemporarily, progesterone (P4) is at or near peak concentrations in maternal circulation. The functional or actual loss of response to P4 is thought to drive the process that enhances uterine contractile activity for labor and parturition at term. The objective of the present study was to determine if actual or functional loss of P4 regulated cytomorphological characteristics associated with prepartum cervix ripening at term and with preterm birth. On day 16 of pregnancy. Ovaries were removed to eliminate the main source of P4 production and a silastic capsule implanted (with vehicle or P4, Ovx or Ovx+P4, respectively). Controls received a vehicle-filled capsule, while a P4 capsule was implanted into an addition group of Intact mice to ensure sustained concentrations throughout pregnancy (Intact+P4). Pups were born in controls at term (days 19-20 postbreeding), but deliveries were preterm in Ovx mice within 24h (day 17). In the Ovx+P4 group, births were delayed to term and post-term in most Intact+P4 mice with adverse pregnancy outcomes commonplace. Characteristics of cell nuclei and degradation of cross-linked collagen were advanced with preterm birth in Ovx mice compared to controls that gave birth by at term. Treatment of Ovx mice with P4 blocked preterm birth, but parturition was complicated by dystocia. In addition, P4 given to ovary-intact mice sustained peak pregnancy concentrations, but had minimal effects on cytoarchitecture of the prepartum cervix stroma except term birth was forestalled with dystocia and fetal morbidity. Density of resident macrophages in the cervix stroma in term Ovx+P4 mice was reduced along with area of macrophage stain versus postpartum controls. Thus, analyses of cervix cellular cytoarchitecture provided useful biomarkers of local inflammation to assessment the ripening process for preterm and term parturition. Collectively, findings suggest a functional loss of prepartum cervix responses to progesterone are part of a final common mechanism for parturition across mammals. Summary Loss of response to progesterone withdrawal is associated with cervix ripening while some cytoarchitectural characteristics of remodeling are regulated to block preterm birth and dystocia at term

Preterm birth (PTB) is a leading cause of infant morbidity and mortality in the world. In 2020, 1 in 10 infants were born prematurely in the United States. The World Health Organization estimates that a total of 15 million infants are born prematurely every year. Current therapeutic interventions for PTB have had limited replicable success. Recent advancements in the field of nanomedicine have made it possible to utilize the vaginal administration route to effectively and locally deliver drugs to the female reproductive tract. Additionally, studies using murine models have provided important insights about the cervix as a gatekeeper for pregnancy and parturition. With these recent developments, the field of reproductive biology is on the cusp of a paradigm shift in the context of treating PTB. The present review focuses on the complexities associated with treating the condition and novel therapeutics that have produced promising results in preclinical studies.

Preterm birth is a leading cause of infant morbidity and mortality in the world. In 2020, 1 in 10 infants were born prematurely in the United States. Globally, the World Health Or-ganization estimates 15 million infants are born prematurely. The efficacy of current ther-apeutic interventions for preterm birth have been called into question due to limited repli-cable success. Recent advancements in the field of nanomedicine have made it possible to utilize the vaginal administration route to effectively and locally deliver drugs to the fe-male reproductive tract. Also, studies using murine models have provided important in-sights about the cervix as a gatekeeper for pregnancy and parturition. It seems plausible that the field of reproductive biology is on the cusp of a paradigm shift in the context of treating preterm birth. The present review focuses on the complexities associated with treating the condition and novel therapeutics that have produced promising results in pre-clinical studies.

Key points: Nitric oxide (NO) is a gasotransmitter with important physiological and pathophysiological roles in pregnancy. There is limited information available about the sources and metabolism of NO and its bioactive metabolites (NOx) in both normal and complicated pregnancies. The present study characterized and quantified endogenous NOx in human and mouse placenta following determination of the stability of exogenous NOx in placental homogenates. NOx have differential stability in placental homogenates. NO and iron nitrosyl species (FeNOs), are relatively unstable in placental homogenates from normal placentas. Exogenous NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. FeNOs were also detected endogenously in mouse and human placenta. NOx levels in placental villous tissue are increased in fetal growth restriction vs. placentas from women with normal pregnancies, particularly in fetal growth restriction associated with pre‐eclampsia. Villitis was not associated, however, with an increase in NOx levels in either normotensive or pre‐eclamptic placentas. The results call for further investigation of FeNOs in normal and complicated pregnancies. Nitric oxide (NO) is a gasotransmitter with important roles in pregnancy under both physiological and pathophysiological conditions. Although products of NO metabolism (NOx) also have significant bioactivity, little is known about the role of NO and NOx under conditions of aberrant placental inflammation during pregnancy. An ozone‐based chemiluminescence approach was used to investigate the stability and metabolic fate of NOx in human placental homogenates from uncomplicated pregnancies in healthy mothers compared to that in placental tissue from normotensive and pre‐eclamptic pregnancies complicated with fetal growth restriction (FGR) with and without villitis of unknown aetiology. We hypothesized that placental NOx would be increased in FGR vs. normal tissue, and be further increased in villitis vs. non‐villitis placentas. Findings indicate that nitrate, nitrite and nitrosothiols, but not NO or iron nitrosyl species (FeNOs), are relatively stable in placental homogenates from normal placentas, and that NO, nitrite and nitrosothiols react with placental homogenates to form iron nitrosyl complexes. Furthermore, NOx levels in placental villous tissue are increased in FGR vs. placentas from women with normal pregnancies, particularly in FGR associated with pre‐eclampsia. However, in contrast to our hypothesis, villitis was not associated with an increase in NOx levels in either normotensive or pre‐eclamptic placentas. Our results also strongly support the involvement of FeNOs in both mouse and human placenta, and call for their further study as a critical mechanistic link between pre‐eclampsia and fetal growth restriction.

The cervix is the essential gatekeeper for birth. Incomplete cervix remodeling contributes to problems with delivery at or post-term while preterm birth is a major factor in perinatal morbidity and mortality in newborns. Lack of cervix biopsies from women during the period preceding term or preterm birth have led to use of rodent models to advanced understanding of the mechanism for prepartum cervix remodeling. The critical transition from a soft cervix to a compliant prepartum lower uterine segment has only recently been recognized to occur in various mammalian species when progesterone in circulation is at or near the peak of pregnancy in preparation for birth. In rodents, characterization of ripening resembles an inflammatory process with a temporal coincidence of decreased density of cell nuclei, decline in cross-linked extracellular collagen, and increased presence of macrophages in the cervix. Although a role for inflammation in parturition and cervix remodeling is not a new concept, a comprehensive examination of literature in this review reveals that many conclusions are drawn from comparisons before and after ripening has occurred, not during the process. The present review focuses on essential phenotypes and functions of resident myeloid and possibly other immune cells to bridge the gap with evidence that specific biomarkers may assess the progress of ripening both at term and with preterm birth. Moreover, use of endpoints to determine the effectiveness of various therapeutic approaches to forestall remodeling and reduce risks for preterm birth, or facilitate ripening to promote parturition will improve the postpartum well-being of mothers and newborns.

Problem: A breakdown of the cervical epithelial barrier has been associated with preterm cervical remodeling. It is unknown if Replens, the vehicle for vaginal progesterone, alters cervical epithelial junctional proteins impacting cervical remodeling and preterm birth.Method of study: E17 CD‐1 pregnant mice received an intrauterine injection of saline or lipopolysaccharide (LPS). Effect of intravaginal Replens given on day E16 and on E17 coincident with LPS was tested. A second experiment determined if an antibody to the interferon receptor (IFNaR) blocked the effects of LPS. Mice were killed after six hours, the preterm birth rate was recorded, and the serum and cervices were collected for analysis. Additionally, the epithelial cell barrier was assessed using an in vitro permeability assay.Results: Replens decreased the rate of LPS‐induced preterm birth within six hours, from 87.5% to 37.5% (P < .005). LPS + IFNaR antibody decreased the rate of preterm birth or vaginal bleeding compared to LPS + control antibody mice, 43.8% vs 87%, respectively (P < .01). E‐Cadherin in the mouse serum was increased by LPS, an effect mitigated by treatment with Replens (P < .0001) or the IFNaR antibody (P < .01). Replens + LPS decreased the expression of IFN‐β (P < .01). The anti‐IFNaR, as well as Replens, decreased the expression of MMP13 (P < .05) compared to LPS mice. Replens also prevented the LPS‐induced increase in permeability (P < .001).Conclusion: Replens prevents preterm birth by decreasing the interferon‐induced upregulation of MMP13 and the degradation of the cell adhesion protein E‐Cadherin. Further studies are needed to determine if Replens can be useful as treatment for preterm birth.

Preterm labor (PTL) is the predominant cause of childhood morbidity and mortality. It has several phenotypes, each with a distinct etiology often involving inflammation. Here, in samples of reproductive tissues obtained in early PTL from women with phenotypically defined PTL, we examined the presence and distribution of inflammation and its relationship with prolabor gene expression. In chorioamnionitis (CA-PTL), cytokine protein concentrations were increased across all tissues; in idiopathic (I-PTL), the inflammatory changes were limited to the choriodecidua; inflammation was not a feature of placental abruption (PA-PTL). CA-PTL was associated with activation of p65 in the myometrium and AP-1 in the choriodecidua, and PA-PTL with CREB in the choriodecidua. In the myometrium, PGHS-2 mRNA level was increased in CA- and I-PTL; in the amnion, PGHS-2 mRNA level was higher in PA- and I-PTL, while in CA-PTL, OT, OTR mRNA, and CX-43 expression were increased. In the choriodecidua, PGHS-2 mRNA level was unchanged, but in CA and I-PTL, OT mRNA level were increased and OTR was reduced. These data show that CA-PTL is associated with widespread inflammation and prolabor gene expression. In contrast, in I-PTL, inflammation is limited to the choriodecidua, with discrete increases in PGHS-2 in the amnion and OT in the choriodecidua. Inflammation is not a feature of PA-PTL, which is associated with increased OT and OTR in the amnion.

The prepartum transition from a soft to ripening cervix is an inflammatory process that occurs well before birth when systemic progesterone is near peak concentration. This 2-part study first determined that stromal fibroblasts but not macrophages in the cervix have progesterone receptors (PRs). Neither the number of PR cells in cervix sections nor the relative abundance or ratio of nuclear PR isoforms (PR-A/PR-B) were diminished in mice between day 15 of pregnancy and term. Second in mice lacking PR-B (Pgr^tm20mc), the number of cells that expressed the PR-A isoform were maintained during this period of prepartum cervix remodeling. Thus, progesterone effects to sustain pregnancy, as well as soften and ripen the cervix, are mediated by a stable stromal cell population that expresses PR-A and, through interactions with resident macrophages, are likely to mediate inflammatory ripening processes in preparation for birth.