SOX4 exerts contrasting regulatory effects on labor-associated gene promoters in myometrial cells.

The uterine muscular layer, or myometrium, undergoes profound changes in global gene expression during its progression from a quiescent state during pregnancy to a contractile state at the onset of labor. In this study, we investigate the role of SOX family transcription factors in myometrial cells and provide evidence for the role of SOX4 in regulating labor-associated genes. We show that Sox4 has elevated expression in the murine myometrium during a term laboring process and in two mouse models of preterm labor. Additionally, SOX4 differentially affects labor-associated gene promoter activity in cooperation with activator protein 1 (AP-1) dimers. SOX4 exerted no effect on the Gja1 promoter; a JUND-specific activation effect at the Fos promoter; a positive activation effect on the Mmp11 promoter with the AP-1 dimers; and surprisingly, we noted that the reporter expression of the Ptgs2 promoter in the presence of JUND and FOSL2 was repressed by the addition of SOX4. Our data indicate SOX4 may play a diverse role in regulating gene expression in the laboring myometrium in cooperation with AP-1 factors. This study enhances our current understanding of the regulatory network that governs the transcriptional changes associated with the onset of labor and highlights a new molecular player that may contribute to the labor transcriptional program.

The Future of Basic Science: Development of the Next Generation of Mechanistic Researchers in Female Pelvic Medicine.

INTRODUCTION AND HYPOTHESIS: The International Urogynecological Association (IUGA) brought together senior and junior members actively engaged in scholarly and educational activities for a consensus conference centered on developing a strategy for sustainable training of the next generation of mechanistic researchers in female pelvic medicine. METHODS: Four a priori identified major foci were explored in a half-day virtual consensus conference. Participants included representatives from various countries and disciplines with diverse backgrounds-clinicians, physician-scientists, and basic scientists in the fields of urogynecology, biomechanical engineering, and molecular biology. Following a keynote address, each focus area was first tackled by a dedicated breakout group, led by the Chair(s) of the most relevant IUGA committees. The break-out sessions were followed by an iterative discussion among all attendees to identify mitigating strategies to address the shortage of mechanistic researchers in the field of female pelvic medicine. RESULTS: The major focus areas included: research priorities for IUGA basic science scholar program; viable strategies for sustainable basic science mentorship; core competencies in basic science training; and the challenges of conducting complex mechanistic experiments in low-resource countries. Key gaps in knowledge and core competencies that should be incorporated into fellowship/graduate training were identified, and existing training modalities were discussed. Recommendations were made for pragmatic approaches to increasing the exposure of trainees to learning tools to enable sustainable training of the next generation of basic science researchers in female pelvic medicine worldwide. CONCLUSIONS: The attendees presented multiple perspectives to gain consensus regarding critical areas of need for training future generations of mechanistic researchers. Recommendations for a sustainable Basic Science Scholar Program were developed using IUGA as a platform. The overarching goal of such a program is to ensure a successful bench-to-bedside-and-back circuit in Urogynecology and Pelvic Reconstructive Surgery, ultimately improving lives of millions of women worldwide through scientifically rational effective preventative and therapeutic interventions.

Progesterone control of myometrial contractility.

During pregnancy, the primary function of the uterus is to be quiescent and not contract, which allows the growing fetus to develop and mature. A uterine muscle layer, myometrium, is composed of smooth muscle cells (SMCs). Before the onset of labor contractions, the uterine SMCs experience a complex biochemical and molecular transformation involving the expression of contraction-associated proteins. Labor is initiated when genes in SMCs are activated in response to a combination of hormonal, inflammatory and mechanical signals. In this review, we provide an overview of molecular mechanisms regulating the process of parturition in humans, focusing on the hormonal control of the myometrium, particularly the steroid hormone progesterone. The primary reason for discussing the regulation of myometrial contractility by progesterone is the importance of the clinical problem of preterm birth. It is thought that the hormonal mechanisms regulating premature uterine contractions represent an untimely triggering of the normal events occurring during term parturition. Yet, our knowledge of the complex and redundant hormonal pathways controlling uterine contractile activity leading to delivery of the neonate remains incomplete. Finally, we introduce recent animal studies using a novel class of drugs, Selective Progesterone Receptor Modulators, targeting progesterone signaling to prevent premature myometrial contractions.

MYB and ELF3 differentially modulate labor-inducing gene expression in myometrial cells.

Spontaneous uterine contractions are initiated when smooth muscle cells (SMCs) within the uterine muscle, or myometrium, transition from a functionally dormant to an actively contractile phenotype at the end of the pregnancy period. We know that this process is accompanied by gestational time point-specific differences in the SMC transcriptome, which can be modulated by the activator protein 1 (AP-1), nuclear factor kappa beta (NF-κß), estrogen receptor (ER), and progesterone receptor (PR) transcription factors. Less is known, however, about the additional proteins that might assist these factors in conferring the transcriptional changes observed at labor onset. Here, we present functional evidence for the roles of two proteins previously understudied in the SMC context-MYB and ELF3-which can contribute to the regulation of labor-driving gene transcription. We show that the MYB and ELF3 genes exhibit elevated transcript expression levels in mouse and human myometrial tissues during spontaneous term labor. The expression of both genes was also significantly increased in mouse myometrium during preterm labor induced by the progesterone antagonist mifepristone (RU486), but not during infection-simulating preterm labor induced by intrauterine infusion of lipopolysaccharide (LPS). Furthermore, both MYB and ELF3 proteins affect labor-driving gene promoter activity, although in surprisingly opposing ways: Gja1 and Fos promoter activation increases in the presence of MYB and decreases in the presence of ELF3. Collectively, our study adds to the current understanding of the transcription factor network that defines the transcriptomes of SMCs during late gestation and implicates two new players in the control of labor timing.

International Urogynecological Consultation (IUC): pathophysiology of pelvic organ prolapse (POP).

INTRODUCTION AND HYPOTHESIS: This manuscript is the International Urogynecology Consultation (IUC) on pelvic organ prolapse (POP) chapter one, committee three, on the Pathophysiology of Pelvic Organ Prolapse assessing genetics, pregnancy, labor and delivery, age and menopause and animal models. MATERIALS AND METHODS: An international group of urogynecologists and basic scientists performed comprehensive literature searches using pre-specified terms in selected biomedical databases to summarize the current knowledge on the pathophysiology of the development of POP, exploring specifically factors including (1) genetics, (2) pregnancy, labor and delivery, (3) age and menopause and (4) non-genetic animal models. This manuscript represents the summary of three systematic reviews with meta-analyses and one narrative review, to which a basic scientific comment on the current understanding of pathophysiologic mechanisms was added. RESULTS: The original searches revealed over 15,000 manuscripts and abstracts which were screened, resulting in 202 manuscripts that were ultimately used. In the area of genetics the DNA polymorphisms rs2228480 at the ESR1 gene, rs12589592 at the FBLN5 gene, rs1036819 at the PGR gene and rs1800215 at the COL1A1 gene are significantly associated to POP. In the area of pregnancy, labor and delivery, the analysis confirmed a strong etiologic link between vaginal birth and symptoms of POP, with the first vaginal delivery (OR: 2.65; 95% CI: 1.81-3.88) and forceps delivery (OR: 2.51; 95% CI: 1.24-3.83) being the main determinants. Regarding age and menopause, only age was identified as a risk factor (OR : 1.102; 95% CI: 1.02-1.19) but current data do not identify postmenopausal status as being statistically associated with POP. In several animal models, there are measurable effects of pregnancy, delivery and iatrogenic menopause on the structure/function of vaginal support components, though not on the development of POP. CONCLUSIONS: Genetics, vaginal birth and age all have a strong etiologic link to the development of POP, to which other factors may add or protect against the risk.

The selective progesterone receptor modulator-promegestone-delays term parturition and prevents systemic inflammation-mediated preterm birth in mice.

BACKGROUND: Progesterone, acting via its nuclear receptors called progesterone receptors, promotes myometrial relaxation during pregnancy, and suspension of this activity triggers labor. We previously found that 20α-hydroxysteroid dehydrogenase causes a local withdrawal of progesterone in the term and preterm myometrium by converting the progesterone into an inactive form before it accesses the progesterone receptors. OBJECTIVE: We hypothesized that a selective progesterone receptor modulator called promegestone, which is not metabolized by 20α-hydroxysteroid dehydrogenase, would sustain progesterone receptor signaling and prevent/delay term labor and preterm labor in mice. STUDY DESIGN: In the term labor mouse model, promegestone (0.2 mg/dam) or a vehicle were administered subcutaneously in timed-pregnant CD-1 mice at gestational days 15, 16, and 17 (term gestational days, 19.5). In the inflammation preterm labor model, pregnant mice received promegestone or a vehicle on gestational days 15, 16, and 17, which was 24 hours before, immediately before, and 24 hours after systemic bacterial endotoxin (50 µg intraperitoneal; lipopolysaccharide group) or vehicle (saline) administration. The maternal and fetal tissues were collected on gestational day 16 6 hours after lipopolysaccharide±promegestone injection and at term gestational day 18.75. The protein levels of 10 cytokines were measured by multiplex immunoassay in maternal plasma and amniotic fluid. Myometrial, decidual, and placental messenger RNA levels of multiple cytokines and procontractile proteins were evaluated by real-time polymerase chain reaction and confirmed by immunoblotting. RESULTS: Promegestone prevented term labor and maintained mice pregnancy postterm >24 hours. The litter size and fetal weights were not different from the controls. Promegestone prevented systemic bacterial-endotoxin-induced preterm labor in 100% of the mice, blocked uterine contractions, significantly inhibited all systemic inflammation-induced myometrial cytokines, and partially inhibited decidual and placental inflammation. Promegestone did not prevent bacterial-endotoxin-induced fetal toxicity. CONCLUSION: Promegestone a selective progesterone receptor modulator that binds progesterone receptors with high affinity and is not metabolized by 20α-hydroxysteroid dehydrogenase could completely suppress term parturition and systemic bacterial-endotoxin-induced preterm birth in mice. We suggest that such selective progesterone receptor modulators may represent a potential therapeutic approach to the prevention of preterm labor in women at high risk of preterm birth.

Age and/or postmenopausal status as risk factors for pelvic organ prolapse development: systematic review with meta-analysis.

INTRODUCTION AND HYPOTHESIS: Age is named as a risk factor for pelvic organ prolapse (POP), despite not being the primary outcome for many observational studies. Postmenopausal status is another associated factor but has many confounders. We aimed to systematically review the role of age and/or postmenopausal status in POP development. METHODS: Systematic review addressing age and hormones, more specifically by postmenopausal status, from inception to March 2020 in four databases (PubMed, Embase, WOS, Cochrane Library). Quality of evidence was classified by the ROBINS-I classification for non-randomized studies. Experimental studies, animal studies, studies linking age with recurrent POP and case series were excluded. Effect estimates were collected from adjusted odds ratio plus 95% confidence intervals. Significance level was 5%. A discussion exploring mechanistic factors was also included. RESULTS: Nineteen studies (11 cross sectional, 6 cohort and 2 case control) were included for quantitative analysis. Only two studies presented a low overall risk of bias for age; most of the domains were of moderate risk. Every additional year was responsible for a 10% increase in the risk to develop POP (OR = 1.102 [1.021-1.190]; i2 = 80%, random analysis, p = 0.012). This trend was confirmed when age was dichotomized into a cutoff of 35 (p = 0.035) and 50 (p < 0.001) years. Although an increase in the risk for POP was noted in postmenopausal women, this did not reach statistical significance (OR = 2.080 [0.927-4.668], i2 = 0%, p = 0.076). CONCLUSION: Age is a risk factor for POP; postmenopausal status was not statistically associated with POP, prompting the need for further studies addressing this factor.

The effect of local estrogen therapy on the urinary microbiome composition of postmenopausal women with and without recurrent urinary tract infections.

INTRODUCTION AND HYPOTHESIS: Recurrent urinary tract infections (rUTIs) occur in 2-10% of postmenopausal women. Local estrogen therapy (LET) has been shown to reduce UTIs. This study aimed to compare the urinary microbiome between patients with and without a history of rUTIs and to examine whether treatment with LET influences the diversity and richness of microbiome species in two groups. METHODS: Postmenopausal women with and without rUTIs attending the urogynecology clinic between April 2019 and December 2020 were recruited. Participant baseline characteristics and demographics were recorded. Aseptic transurethral urine samples were collected at recruitment and at 3-6 months following treatment with LET. The V1-V2 and ITS regions of the 16S rRNA gene were sequenced to identify bacteria. RESULTS: A total of 37 women were recruited, 20 controls and 17 patients with rUTI. During follow-up, symptomatic UTIs occurred in 3/17 (17.6%) and 0/20 in the rUTI group and control group, respectively. Klebsiella aerogenes was present in 80% of rUTI samples and in 53.3% of control samples before LET. Abundance of Finegoldia magna was present in 33.3% of samples before LET, but only in 6.7% after LET. There was no change in relative abundance of lactobacillus species following LET in both groups. CONCLUSIONS: Treatment with vaginal LET altered the local hormonal environment of the urinary bladder and likely protected women from development of rUTI by decreasing the presence of F. magna. To confirm the significance of this bacterial species in rUTI symptomatology, our finding needs to be validated on a larger patient cohort.

Decidual Inflammation Drives Chemokine-Mediated Immune Infiltration Contributing to Term Labor.

Infiltration of maternal peripheral leukocytes into the uterine tissues is a critical event occurring before, during, and after term labor (TL). In this article, we investigate the contribution of uterine smooth muscle (myometrium) and pregnant endometrium (decidua) to the inflammatory process during human TL. We hypothesize that labor-related physiological inflammation is orchestrated by uterine-secreted cytokines, which dually activate the uterine vascular endothelium and maternal leukocytes to promote their adhesion and infiltration into the uterus. Using Luminex and ELISA assays, we examine a full range of cytokines (45 proteins) in media conditioned by primary decidual and myometrial cells from TL and term not in labor (TNL) women. The effect of conditioned media on the activation of human uterine microvascular endothelial cells was measured by qPCR and on peripheral leukocytes by flow cytometry. Transendothelial migration of calcein-labeled primary leukocytes toward media was assessed by fluorometry. Stromal decidual cells secrete significantly higher levels of multiple cytokines compared with myometrial cells (p < 0.05) and significantly more cytokines during TL than TNL. These cytokines activate uterine microvascular endothelial cells through the upregulation of cell adhesion molecule VCAM-1 and peripheral leukocytes by upregulation of CD11b. Furthermore, multiple cytokines secreted from the TL decidua and myometrium significantly increase migration of granulocytes, monocytes, and lymphocytes compared with TNL (p < 0.05), which was blocked by a broad-spectrum chemokine inhibitor (FX125L). These data reveal the critical role for decidual- and myometrial-secreted cytokines in the activation of inflammatory pathways leading to labor. We suggest that these pathways represent targets for therapeutic intervention during preterm labor.

Landscape of Preterm Birth Therapeutics and a Path Forward.

Preterm birth (PTB) remains the leading cause of infant morbidity and mortality. Despite 50 years of research, therapeutic options are limited and many lack clear efficacy. Tocolytic agents are drugs that briefly delay PTB, typically to allow antenatal corticosteroid administration for accelerating fetal lung maturity or to transfer patients to high-level care facilities. Globally, there is an unmet need for better tocolytic agents, particularly in low- and middle-income countries. Although most tocolytics, such as betamimetics and indomethacin, suppress downstream mediators of the parturition pathway, newer therapeutics are being designed to selectively target inflammatory checkpoints with the goal of providing broader and more effective tocolysis. However, the relatively small market for new PTB therapeutics and formidable regulatory hurdles have led to minimal pharmaceutical interest and a stagnant drug pipeline. In this review, we present the current landscape of PTB therapeutics, assessing the history of drug development, mechanisms of action, adverse effects, and the updated literature on drug efficacy. We also review the regulatory hurdles and other obstacles impairing novel tocolytic development. Ultimately, we present possible steps to expedite drug development and meet the growing need for effective preterm birth therapeutics.

Pro-inflammatory signals induce 20α-HSD expression in myometrial cells: A key mechanism for local progesterone withdrawal.

Metabolism of progesterone (P4) by the enzyme 20α hydroxysteroid dehydrogenase (20α-HSD) in myometrial cells is postulated to be a mechanism for P4 withdrawal, which occurs concomitant to uterine inflammation (physiologic or infection-induced) and associated activation of transcription factors: NF-кB and AP-1, common to term and preterm labour. We found that 20α-HSD protein is significantly increased in human myometrium during term labour, and in mouse uterus during term and preterm labour. Treatment of human myometrial cells with the pro-inflammatory mediators, lipopolysaccharide (LPS, mimicking infection) and 12-O-tetradecanoylphorbol-13-acetate (TPA, mimicking inflammation), induced 20α-HSD gene expression and increased 20α-HSD protein abundance. LPS treatment decreased P4 release into the culture medium and resulted in up-regulation of GJA1 in the hTERT-HM cells. The NF-кB /AP-1 transcription factors mediated effects of LPS and TPA on 20α-HSD gene transcription. Both pro-inflammatory stimuli induced 20α-HSD promoter activity in LPS/TPA-treated cells which was significantly attenuated by inhibition of NF-кB (JSH: 20 µM) or AP-1 signalling (T5224: 10 µM). Deletion of NF-кB consensus sites abrogated LPS-mediated promoter induction, while removal of AP-1 sites reversed the TPA-mediated induction of 20α-HSD promoter. We conclude that inflammatory stimuli (physiologic or pathologic) that activate NF-кB or AP-1 induce 20α-HSD transcription and subsequent local P4 withdrawal resulting in up-regulation of GJA1 and activation of myometrium that precedes labour.

Transcriptional control of parturition: insights from gene regulation studies in the myometrium.

The onset of labour is a culmination of a series of highly coordinated and preparatory physiological events that take place throughout the gestational period. In order to produce the associated contractions needed for foetal delivery, smooth muscle cells in the muscular layer of the uterus (i.e. myometrium) undergo a transition from quiescent to contractile phenotypes. Here, we present the current understanding of the roles transcription factors play in critical labour-associated gene expression changes as part of the molecular mechanistic basis for this transition. Consideration is given to both transcription factors that have been well-studied in a myometrial context, i.e. activator protein 1, progesterone receptors, oestrogen receptors, and nuclear factor kappa B, as well as additional transcription factors whose gestational event-driving contributions have been demonstrated more recently. These transcription factors may form pregnancy- and labour-associated transcriptional regulatory networks in the myometrium to modulate the timing of labour onset. A more thorough understanding of the transcription factor-mediated, labour-promoting regulatory pathways holds promise for the development of new therapeutic treatments that can be used for the prevention of preterm labour in at-risk women.

Combination of histochemical analyses and micro-MRI reveals regional changes of the murine cervix in preparation for labor.

The cervix is responsible for maintaining pregnancy, and its timely remodeling is essential for the proper delivery of a baby. Cervical insufficiency, or "weakness", may lead to preterm birth, which causes infant morbidities and mortalities worldwide. We used a mouse model of pregnancy and term labor, to examine the cervical structure by histology (Masson Trichome and Picrosirius Red staining), immunohistochemistry (Hyaluronic Acid Binding Protein/HABP), and ex-vivo MRI (T2-weighted and diffusion tensor imaging), focusing on two regions of the cervix (i.e., endocervix and ectocervix). Our results show that mouse endocervix has a higher proportion of smooth muscle cells and collagen fibers per area, with more compact tissue structure, than the ectocervix. With advanced gestation, endocervical changes, indicative of impending delivery, are manifested in fewer smooth muscle cells, expansion of the extracellular space, and lower presence of collagen fibers. MRI detected three distinctive zones in pregnant mouse endocervix: (1) inner collagenous layer, (2) middle circular muscular layer, and (3) outer longitudinal muscular layer. Diffusion MRI images detected changes in tissue organization as gestation progressed suggesting the potential application of this technique to non-invasively monitor cervical changes that precede the onset of labor in women at risk for preterm delivery.

A Broad-Spectrum Chemokine Inhibitor Blocks Inflammation-Induced Myometrial Myocyte-Macrophage Crosstalk and Myometrial Contraction.

Prophylactic administration of the broad-spectrum chemokine inhibitor (BSCI) FX125L has been shown to suppress uterine contraction, prevent preterm birth (PTB) induced by Group B Streptococcus in nonhuman primates, and inhibit uterine cytokine/chemokine expression in a murine model of bacterial endotoxin (LPS)-induced PTB. This study aimed to determine the mechanism(s) of BSCI action on human myometrial smooth muscle cells. We hypothesized that BSCI prevents infection-induced contraction of uterine myocytes by inhibiting the secretion of pro-inflammatory cytokines, the expression of contraction-associated proteins and disruption of myocyte interaction with tissue macrophages. Myometrial biopsies and peripheral blood were collected from women at term (not in labour) undergoing an elective caesarean section. Myocytes were isolated and treated with LPS with/out BSCI; conditioned media was collected; cytokine secretion was analyzed by ELISA; and protein expression was detected by immunoblotting and immunocytochemistry. Functional gap junction formation was assessed by parachute assay. Collagen lattices were used to examine myocyte contraction with/out blood-derived macrophages and BSCI. We found that BSCI inhibited (1) LPS-induced activation of transcription factor NF-kB; (2) secretion of chemokines (MCP-1/CCL2 and IL-8/CXCL8); (3) Connexin43-mediated intercellular connectivity, thereby preventing myocyte-macrophage crosstalk; and (4) myocyte contraction. BSCI represents novel therapeutics for prevention of inflammation-induced PTB in women.

Low-intensity physical activity may protect pregnant women against spontaneous preterm labour: a prospective case-control study.

The innate immune system plays a significant role in onset of parturition. Maternal antenatal physical activity can influence immune function and timing of labour. We examined physical activity patterns and concentration of 19 cytokines at 16 and 27 weeks gestational age (GA), in peripheral plasma of 28 asymptomatic women who later had spontaneous preterm labour (SPTL, <37 weeks GA) and 52 women who later delivered at term (TL; ≥37 weeks GA). This nested case-control study used data from the Ontario Birth Study cohort. Exercise was assessed using the International Physical Activity Questionnaire, and cytokines were analyzed using Luminex assays. There was no significant difference in exercise patterns between SPTL and TL subjects. Plasma concentration of interleukin (IL)-10 was significantly higher in SPTL women at 16 and 27 weeks, while tumour necrosis factor alpha (TNF-α), IL-8, and monocyte chemoattractant protein (MCP)-1 concentrations were increased at 27 weeks GA (p < 0.05). Concentration of IL-10 was negatively correlated with the amount of reported walking (ρ = -0.264, p = 0.03). Women should be encouraged to partake in low-intensity exercise throughout pregnancy, as it may confer a protective effect against SPTL through IL-10-mediated pathways. Additionally, plasma cytokine analysis at 27 weeks GA may be useful for predicting SPTL in asymptomatic women. Novelty: In women that delivered preterm, plasma levels of anti-inflammatory cytokine IL-10 were significantly elevated at 16 and 27 weeks of gestation. Plasma levels of IL-10 were negatively correlated with the amount of reported walking. Concentration of IL-8, MCP-1 and TNF-α were increased in plasma of asymptomatic women that subsequently deliver preterm.

Reprint of: Myometrial activation: Novel concepts underlying labor.

Term labour is a state of physiological inflammation orchestrated by multiple uterine tissues (both fetal and maternal). This physiological inflammation preceding and accompanying labour onset is characterized by an increase in cytokine and chemokine secretion by the fetal membranes, as well as uterine tissues (i.e., decidua and myometrium). Pro-inflammatory cytokines and chemokines activate circulating maternal peripheral leukocytes as well as the uterine vascular endothelium to permit leukocyte infiltration into the uterus. This inflammatory milieu, in the absence of infection, is required for the initiation of labour as the uterine-infiltrated leukocytes secrete matrix metalloproteinases to induce fetal membrane rupture and cervical ripening as well as various labour mediators, which promote contractions of the myometrium. Myometrial activation at term and the onset of labour contractions are directly related to the changes in the ovarian/placental hormone progesterone and its downstream mediators (i.e., the progesterone receptors, PRA/B), which are also critical for maintenance of pregnancy. Our recent data provides direct evidence in support of local and functional P4 withdrawal in the uterine muscle (myometrium) via the activator protein-1 (AP-1) mediated pathway. This review outlines known mechanisms regulating activation of human labour, including progesterone and cytokine signaling. Understanding of the molecular mechanism of myometrial activation and labour onset could facilitate the development of new therapeutics for high-risk pregnant women to prevent premature uterine activation and preterm birth.

The pregnant myometrium is epigenetically activated at contractility-driving gene loci prior to the onset of labor in mice.

During gestation, uterine smooth muscle cells transition from a state of quiescence to one of contractility, but the molecular mechanisms underlying this transition at a genomic level are not well-known. To better understand these events, we evaluated the epigenetic landscape of the mouse myometrium during the pregnant, laboring, and postpartum stages. We generated gestational time point-specific enrichment profiles for histone H3 acetylation on lysine residue 27 (H3K27ac), histone H3 trimethylation of lysine residue 4 (H3K4me3), and RNA polymerase II (RNAPII) occupancy by chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq), as well as gene expression profiles by total RNA-sequencing (RNA-seq). Our findings reveal that 533 genes, including known contractility-driving genes (Gap junction alpha 1 [Gja1], FBJ osteosarcoma oncogene [Fos], Fos-like antigen 2 [Fosl2], Oxytocin receptor [Oxtr], and Prostaglandin G/H synthase 2 (Ptgs2), for example), are up-regulated at day 19 during active labor because of an increase in transcription at gene bodies. Labor-associated promoters and putative intergenic enhancers, however, are epigenetically activated as early as day 15, by which point the majority of genome-wide H3K27ac or H3K4me3 peaks present in term laboring tissue is already established. Despite this early exhibited histone signature, increased noncoding enhancer RNA (eRNA) production at putative intergenic enhancers and recruitment of RNAPII to the gene bodies of labor-associated loci were detected only during labor. Our findings indicate that epigenetic activation of the myometrial genome precedes active labor by at least 4 days in the mouse model, suggesting that the myometrium is poised for rapid activation of contraction-associated genes in order to exit the state of quiescence.

A Broad Spectrum Chemokine Inhibitor Prevents Preterm Labor but Not Microbial Invasion of the Amniotic Cavity or Neonatal Morbidity in a Non-human Primate Model.

Leukocyte activation within the chorioamniotic membranes is strongly associated with inflammation and preterm labor (PTL). We hypothesized that prophylaxis with a broad-spectrum chemokine inhibitor (BSCI) would downregulate the inflammatory microenvironment induced by Group B Streptococcus (GBS, Streptococcus agalactiae) to suppress PTL and microbial invasion of the amniotic cavity (MIAC). To correlate BSCI administration with PTL and MIAC, we used a unique chronically catheterized non-human primate model of Group B Streptococcus (GBS)-induced PTL. In the early third trimester (128-138 days gestation; ~29-32 weeks human pregnancy), animals received choriodecidual inoculations of either: (1) saline (N = 6), (2) GBS, 1-5 × 108 colony forming units (CFU)/ml; N = 5), or (3) pre-treatment and daily infusions of a BSCI (10 mg/kg intravenous and intra-amniotic) with GBS (1-5 × 108 CFU/ml; N = 4). We measured amniotic cavity pressure (uterine contraction strength) and sampled amniotic fluid (AF) and maternal blood serially and cord blood at delivery. Cesarean section was performed 3 days post-inoculation or earlier for PTL. Data analysis used Fisher's exact test, Wilcoxon rank sum and one-way ANOVA with Bonferroni correction. Saline inoculation did not induce PTL or infectious sequelae. In contrast, GBS inoculation typically induced PTL (4/5, 80%), MIAC and fetal bacteremia (3/5; 60%). Remarkably, PTL did not occur in the BSCI+GBS group (0/4, 0%; p = 0.02 vs. GBS), despite MIAC and fetal bacteremia in all cases (4/4; 100%). Compared to the GBS group, BSCI prophylaxis was associated with significantly lower cytokine levels including lower IL-8 in amniotic fluid (p = 0.03), TNF-α in fetal plasma (p < 0.05), IFN-α and IL-7 in the fetal lung (p = 0.02) and IL-18, IL-2, and IL-7 in the fetal brain (p = 0.03). Neutrophilic chorioamnionitis was common in the BSCI and GBS groups, but was more severe in the BSCI+GBS group with greater myeloperoxidase staining (granulocyte marker) in the amnion and chorion (p < 0.05 vs. GBS). Collectively, these observations indicate that blocking the chemokine response to infection powerfully suppressed uterine contractility, PTL and the cytokine response, but did not prevent MIAC and fetal pneumonia. Development of PTL immunotherapies should occur in tandem with evaluation for AF microbes and consideration for antibiotic therapy.