Nestorone, a potent progestin, exhibits anti-inflammatory activity and prevents preterm birth in murine models of premature labor.

Results from the human PR reporter assay showed that Nestorone had the highest progestational activity among the progestins evaluated (Nestorone > Promegestone > progesterone > 17-OHPC > hydrocortisone) ( Figure 2A ). In GR reporter assays, Nestorone and 17-OHPC showed significant glucocorticoid-like activity but substantially lower than that of the potent agonist dexamethasone. Both progesterone and Promegestone showed minimal activity ( Figure 2B ). EC 50 (half maximal effective concentration) values for progesterone and GR transactivation are summarized in Table 1 . Consistent with these findings, Nestorone required approximately ∼30-fold lower concentrations than progesterone to achieve half-maximal PR activation, highlighting its greater progestational potency at the receptor level. Endotoxin (LPS) stimulation of human peripheral blood mononuclear cells increased proinflammatory cytokines, including TNF- α , IL-1 β , IL-6, and IL-12 ( P< .01). Nestorone (20 μ M) significantly reduced cytokine secretion, including an approximate 30% reduction in IL-6 and 3-fold reduction in TNF- α compared with endotoxin-treated controls, and increased IL-10 and IL-2R α concentrations (approximately 100% and 80%, respectively), with effects similar to hydrocortisone ( Figure 3 ). At the mRNA level, endotoxin induced 5-fold to 15-fold increases in proinflammatory cytokines ( P< .001), but Nestorone treatment reduced their expression toward baseline ( P< .005 for IL-8; P< .001 for others) and increased IL-10 expression (∼2-fold). Although progesterone exhibited anti-inflammatory effects, Nestorone produced a greater reduction in IL-6 and a greater increase in IL-10 ( P< .05 vs LPS) ( Figure 4 ). Endotoxin stimulation of immortalized human USMCs and BEAS-2B lung epithelial cells induced a 2-fold to 8-fold increase in proinflammatory cytokines ( P< .001). Nestorone suppressed endotoxin-induced IL-6, IL-8, TNF- α , and toll-like receptor 4 expression ( P< .05—.005) and increased IL-10 expression ( P< .005) in both cell lines ( Figure 5 ). While progesterone also reduced proinflammatory cytokines, Nestorone produced greater suppression of TNF- α and IL-6 in USMCs ( P< .001 vs LPS). In addition, Nestorone substantially increased endotoxin-induced IL-10 expression, indicating enhanced anti-inflammatory effects within USMCs, without implying generalized superiority across all tissues. Systemic inflammation model: In vehicle-treated controls without endotoxin, all mice delivered healthy pups at term (day ∼20 of pregnancy) with comparable litter sizes ( Table 2 ). Endotoxin injection alone (25 μ g/mouse, intraperitoneally) on day 16 of pregnancy induced PTB (day < 18.5) in all mice. Subcutaneous administration of Nestorone (0.02 mg/mouse) or progesterone (2 mg/mouse) on days 15 and 16 of pregnancy prevented endotoxin-induced PTB in 92% of mice ( P< .01 vs endotoxin alone; Table 2 ). While both Nestorone and progesterone prevented premature delivery, progesterone treatment resulted in more post-term deliveries or no deliveries by day 20 of pregnancy ( P< .005). Maternal health was unaffected by treatment and there were no major differences in average litter size between groups. All groups showed high viability except for the endotoxin-only group, which had a viability of approximately 50%, compared to > 95% for all other groups ( P< .005). Local uterine inflammation model: Local intrauterine endotoxin injection resulted in PTB in all untreated mice. Treatment with Nestorone or progesterone prevented PTB in 100% of animals ( P< .01 vs endotoxin [lipopolysaccharide] alone; Table 3 ). In this model, only 20% of Nestorone-treated and progesterone-treated mothers maintained pregnancy post-term; the majority delivered at full term. These findings likely reflect the reduced systemic inflammatory burden in this model, which more closely mimics isolated uterine inflammation. Both Nestorone and progesterone significantly reduced PTB compared with endotoxin-treated controls. Nestorone achieved comparable efficacy at a substantially lower administered dose, consistent with its greater apparent progestational potency in this experimental model.

This translational study used a stepwise experimental approach integrating receptor-based assays, human cell—based inflammatory models, and in vivo murine models of inflammation-induced PTB. Initial in vitro PR and GR transactivation assays were performed to define the receptor activity profile of Nestorone relative to progesterone and other reference compounds. In subsequent experiments, we evaluated the anti-inflammatory effects of Nestorone in endotoxin-stimulated human peripheral blood mononuclear cells and uterine smooth muscle and bronchial epithelial cell lines. Finally, the physiological relevance of these findings was assessed in timed-pregnant mice using systemic and intrauterine endotoxin-induced inflammation models to determine the efficacy of Nestorone in preventing PTB compared with progesterone. The transcriptional activity of Nestorone was evaluated using human progesterone and glucocorticoid receptor reporter assays (Indigo Biosciences, State College, PA) to assess its progestational and glucocorticoid-like activity compared to other progestins. Engineered mammalian reporter cell lines expressing full-length human receptors and a luciferase reporter under control of a receptor-responsive promoter were used. Stock solutions of Nestorone, Promegestone, progesterone, hydroxyprogesterone caproate, hydrocortisone, and dexamethasone were prepared in dimethyl sulfoxide and serially diluted in assay medium. For PR activation assays, cells were exposed to 8 concentrations of each progestin starting at 1000 nM with subsequent serial dilutions. For GR activation assays, 9 concentrations starting at 1000 nM were evaluated. The concentration range evaluated in the receptor transactivation assays (up to 1000 nM) was selected to characterize maximal receptor activation and comparative pharmacologic potency under standardized in vitro conditions. Direct correlation between in vitro assay concentrations and in vivo pharmacokinetics is limited because these systems do not account for protein binding, metabolism, or tissue distribution. After 24 hours of incubation at 37°C, luciferase substrate was added and luminescence was measured using a microplate luminometer (PerkinElmer, MA). Transactivation activity was quantified as relative light units, and assay performance was validated with reference agonist dose-response curves. Human peripheral blood mononuclear cells were isolated from leukocyte-enriched whole blood (New York Blood Center) using density gradient media (STEMCELL Technologies). Cells were washed, resuspended in RPMI-1640, and plated at ∼1×10 6 cells/well in 24-well plates. Human peripheral blood mononuclear cells were stimulated with endotoxin (100 ng/mL) for 30 minutes followed by treatment with Nestorone, progesterone, or hydrocortisone (20 μ M). The 20- μ M concentration of Nestorone and progesterone was selected based on previous results showing optimal efficacy with no cytotoxicity at 20 μ M, allowing direct comparative evaluation of anti-inflammatory activity under standardized conditions and ensuring measurable cytokine responses. These concentrations were not intended to directly reflect anticipated human plasma or tissue exposure levels, as in vitro cell-based assays do not account for pharmacokinetic factors such as protein binding, metabolism, or tissue distribution. After 24 hours at 37°C, culture supernatants and cell pellets were collected and stored at −80°C for protein and mRNA analyses. Cytokine protein concentrations in supernatants were measured using a human magnetic 25-plex cytokine panel (Invitrogen) analyzed on the Luminex 200 system. Total RNA was extracted from cell pellets using TRIzol (Invitrogen), and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time polymerase chain reaction using SYBR Green chemistry was performed on a QuantStudio 3 system. Gene expression levels of proinflammatory mediators, interleukin (IL) 6, and tumor necrosis factor α (TNF- α ) were selected as primary outcomes based on their role in inflammation-associated PTB, whereas the remaining analytes in the Luminex panel were considered exploratory. Cytokine expression levels were normalized to β -actin (human). All assays reported were tested n=3 times on unique blood samples and plated in triplicate. Hydrocortisone and progesterone were included as positive controls to confirm suppression of inflammation-driven responses within the experimental models. Primer sequences were selected using the PrimerQuest tool or from PrimerBank 62 and are provided in the supplementary material . Human uterine smooth muscle cells (USMCs) (ATCC) were chosen to investigate the efficacy of Nestorone at reducing local uterine inflammation associated with PTB. Human bronchial epithelial cells (BEAS-2B; ATCC) were selected to investigate the anti-inflammatory effects of Nestorone on lung cells, and as a preliminary exploration of Nestorone’s potential to ameliorate fetal lung injuries associated with PTB. Cells were cultured in recommended basal media supplemented with growth factors. Cells were stimulated with endotoxin and treated with Nestorone or progesterone. All assays were performed n=3 times and plated in triplicate. Changes in proinflammatory gene expression were assessed by quantitative real-time polymerase chain reaction as described above. Timed pregnant C57BL/6 mice (gestation length ∼19—20 days) were used to model PTB. In this strain, term delivery typically occurs around gestational day ∼19.5±0.5 days. PTB was defined as delivery before gestational day 18.5, consistent with established definitions in murine models and prior guidelines 63 representing delivery more than 1 day prior to the expected term. Inflammation-induced PTB models were selected because inflammatory activation is a well-recognized mechanism contributing to premature activation of the common pathway of parturition and has been implicated in multiple etiologies of sPTB. Given the established anti-inflammatory actions of progesterone at the maternal‑fetal interface, these models provide a biologically relevant platform to evaluate whether enhanced PR activation by Nestorone can maintain pregnancy and suppress inflammatory signaling under conditions of inflammatory stress. Two endotoxin-induced inflammation models were employed: systemic (intraperitoneal injection) and local (intrauterine injection), simulating systemic inflammation and ascending vaginal infections, respectively ( Figure 1 ). Each in vivo model was repeated n=2 times using n=6 mice/group for a total of 12 mice/group for each experimental condition. Mice were housed under specific pathogen-free conditions with ad libitum access to food and water. The Rockefeller University Institutional Animal Care and Use Committee approved all experimental procedures (Protocol #21109-H). Bacterial endotoxin (lipopolysaccharide [LPS]) ( Escherichia coli O111:B4) was purchased from Sigma-Aldrich, Germany and dissolved in sterile saline at a dose of 1 mg/kg body weight in accordance with Hudalla et al. 64 Nestorone at a dose of 0.02 mg/mouse and progesterone at 2 mg/mouse 65 were administered subcutaneously in 100 μ L sesame oil/ethanol on days 15 and 16 of pregnancy, with endotoxin or saline given 30 minutes after the second Nestorone injection. The dose of progesterone was selected based on previous studies demonstrating efficacy in preventing PTB in similar inflammation-induced mouse models. 65 The approximately 100-fold lower dose of Nestorone (0.02 mg) was selected based on preclinical studies demonstrating substantially greater intrinsic progestational potency in the McPhail bioassay and other pharmacological models. 55 These doses were not intended to achieve pharmacokinetic or pharmacodynamic equivalence but rather to compare the biological activity of each compound within established biologically active dose ranges. For systemic inflammation, 25- μ g endotoxin in 100- μ L saline was injected intraperitoneally. For local inflammation, laparotomy was performed to inject 12.5- μ g endotoxin in 50- μ L saline into each uterine horn (25 μ g in total). Uteri were replaced, and incisions closed with sutures and staples. The technical success rate of the procedure was 88%, and mice that suffered surgical complications (mainly suture failure) were excluded from the reported statistics. Isoflurane and meloxicam were used for anesthesia and analgesia, respectively. Animals were monitored 3 times daily from day 16 until delivery. PTB was defined as delivery before day 18.5 of pregnancy. Mice delivering preterm were euthanized on the day of delivery; all other mice were euthanized on day 21. PTB outcomes, including gestational length, litter size, and pup viability, were recorded. Pup viability was defined as the presence of spontaneous movement and respiration at delivery (live at birth). Data are presented as mean±standard error of the mean unless otherwise indicated. Normality of data distribution was assessed prior to statistical testing by Kolmogorov-Smirnov test. Normally distributed data were analyzed using a 2-tailed Student t test, and non-normally distributed data were analyzed with a 2-tailed Mann-Whitney U test, both using Bonferroni corrections for multiple comparisons. Adjustments for multiple comparisons were applied where appropriate for cytokine and gene expression analyses. Luminex data were analyzed using the Kruskal-Wallis test. Statistical significance was defined as P< .05. Sample sizes for animal studies were selected based on prior experience with endotoxin-induced PTB models and published literature; formal a priori power calculations were not performed because these studies were exploratory preclinical investigations.

Nestorone is a potent, Food and Drug Administration‑approved progestin with an established safety profile for contraception. Based on our results, Nestorone demonstrated: (1) high PR activity and mild glucocorticoid-like activity, supporting its anti-inflammatory potential; (2) at least comparable and, in some respects, greater suppression of inflammatory cytokine levels than progesterone in human peripheral blood mononuclear cells and cell lines; and (3) at least comparable efficacy and, in some measures, greater potency than progesterone at preventing PTB in endotoxin-based mouse models. These results suggest that Nestorone may offer advantages over natural progesterone in terms of potency and anti-inflammatory activity. While compelling, the current data are limited to preclinical models, and conclusions regarding efficacy in humans cannot yet be drawn. Further translational and clinical studies are required to determine the safety and efficacy of Nestorone for the prevention of PTB in pregnant women.

The studies reported here were designed to evaluate the effects of the Nestorone on inflammation-associated PTB. We found that: (1) Nestorone demonstrated potent progestational activity with mild GR activity, as assessed by transcriptional reporter assays. (2) Nestorone attenuated endotoxin-induced proinflammatory cytokine secretion and gene expression in human peripheral blood mononuclear cells. In addition, Nestorone inhibited cytokine gene expression in endotoxin-treated uterine smooth muscle and lung epithelial cells, while increasing expression of the anti-inflammatory cytokine interleukin-10. (3) In mouse models, Nestorone prevented inflammation-induced PTB without evidence of maternal toxicity. Although the clinical applicability remains to be established, these findings suggest that a more potent progestin may enhance stabilization of pregnancy in inflammation-associated settings, potentially at lower doses than progesterone. In most mammals, labor is initiated by a decline in circulating progesterone levels. 10 — 12 In humans, parturition is thought to result from a functional withdrawal of progesterone signaling via PR desensitization and/or increased metabolism at the tissue level despite sustained systemic progesterone levels. 13 — 16 This difference may limit the full effectiveness of conventional progesterone-based interventions for sPTB. 66 Nestorone maintained pregnancy in ovariectomized rats at lower doses than progesterone. 55 Our current findings showed that Nestorone prevents endotoxin-induced PTB in mice at a much lower dose than progesterone. Furthermore, Nestorone suppresses endotoxin-induced proinflammatory cytokines in human immune, uterine, and lung cells, supporting a mechanism of action that integrates both anti-inflammatory activity and enhanced progestational signaling. These preclinical findings support the therapeutic promise of a better progestin for the prevention of PTB, especially in selected subtypes of at-risk women and at doses much lower than that of progesterone. PTB remains a leading cause of neonatal morbidity and mortality. Therapeutic options for the prevention of PTB remain limited, in part because of the heterogeneous etiology and incomplete understanding of risk factors associated with this condition. Current approaches, including cervical cerclage and vaginal progesterone, are constrained by invasiveness, variable effectiveness among patient subgroups, 34 — 39 and limited bioavailability of progesterone. 33 , 66 These limitations underscore the urgent need for additional, effective, and less invasive preventive strategies. 40 , 41 , 67 Nestorone’s high progestational potency, anti-inflammatory activity, and partial GR binding 55 support its continued evaluation as a potential therapeutic approach for sPTB. In the present study, Nestorone achieved comparable efficacy to progesterone at a substantially lower administered dose and demonstrated greater apparent potency in both systemic and local inflammation murine models. These findings suggest that Nestorone may modulate inflammatory processes involved in activation of the common pathway of parturition. However, because these observations were obtained in preclinical models, their clinical relevance remains uncertain and will require further evaluation through pharmacokinetic studies and appropriately designed clinical trials. These findings highlight the continued potential of hormonal strategies in preventing PTB despite the limitations of previous treatments. Nestorone prevented PTB at a substantially lower dose than progesterone, suggesting it may represent a promising alternative approach for further investigation. Additionally, Nestorone suppressed the secretion of proinflammatory cytokines in human immune, uterine and lung cells, addressing one of the key mechanisms involved in preterm labor. Given Nestorone’s high potency and favorable physicochemical properties, such as its lipophilic nature, it is well suited for sustained delivery via transdermal or intravaginal gel formulations. Notably, a transdermal Nestorone gel formulation is currently being evaluated in clinical trials for male contraception 68 and is also under consideration for clinical testing in patients with multiple sclerosis for the treatment of demyelinating disease. 58 Clinical formulations, such as intravaginal rings containing Nestorone, are already approved for contraception and could be repurposed for PTB prevention. The combination of strong preclinical efficacy, favorable pharmacologic properties, and an established clinical safety profile provides a compelling rationale for further exploration. A major limitation of this study is that the endotoxin-induced murine models used herein represent only one biologic pathway leading to sPTB and do not fully capture the complexity and heterogeneity of the human syndrome. 63 , 69 , 70 The purpose of these models is not to reproduce every pathway leading to sPTB, but rather to provide a reproducible biological stress test of inflammatory activation of the common pathway of parturition. Within this framework, the models offer a useful platform to evaluate whether candidate therapies can suppress inflammatory signaling and maintain pregnancy under conditions of inflammatory challenge. Using these models, we demonstrated that both Nestorone and progesterone effectively prevented endotoxin-induced PTB. A second limitation of our study is the limited scope of dosing, as only a single dose of Nestorone (0.02 mg) and progesterone (2 mg) was evaluated in both murine models. These doses were selected based on prior preclinical studies and established biologically active ranges, reflecting the substantially higher progestational potency of Nestorone relative to progesterone. However, these doses were not intended to represent pharmacokinetic or pharmacodynamic equivalence. It is possible that lower doses of either compound may be similarly effective, warranting further dose-response studies in pregnant mice. A third limitation is that neonatal outcomes were not evaluated in this study. Assessment of neonatal survival, neurodevelopment, and longer-term functional outcomes will be essential to determine the full clinical relevance of these findings. Future studies incorporating clinically relevant models, pharmacokinetic characterization, and neonatal outcome assessment will be critical to support translation of these findings. In addition, building on our previous findings demonstrating the neuroprotective and remyelinating effects of Nestorone in adult and neonatal animal models, 58 , 71 , 72 we will investigate whether Nestorone may also support neonatal brain development and protect against neurologic injury associated with PTB.

Preterm birth (PTB), defined as delivery before 37 weeks of gestation, is the leading cause of mortality in children aged less than 5 years worldwide. Approximately 10% of neonates are born preterm, with an estimated 15 million PTBs globally in 2020. Disability among those who do survive is inversely related to gestational age at delivery, where those born at a younger age face the greatest risks of long-term morbidities, including cerebral palsy, autism spectrum disorders, and respiratory, visual, or auditory impairments. 1 — 5 Spontaneous preterm birth (sPTB) is increasingly recognized as a complex syndrome rather than a single disease entity. Multiple maternal, fetal, placental, and environmental factors can contribute to premature activation of the common pathway of parturition, which involves coordinated changes in the decidua, cervix, fetal membranes, and myometrium. A growing body of evidence suggests that several disorders associated with sPTB, including inflammatory conditions, adenomyosis, endometriosis, and other forms of impaired decidual function, may share common biological pathways characterized by altered progesterone responsiveness and inflammatory activation. 6 — 10 Progesterone plays a central role in maintaining decidual integrity, suppressing inflammatory signaling, preserving cervical competence, and promoting uterine quiescence throughout pregnancy. 11 , 12 Consequently, diminished progesterone action and sterile inflammation have emerged as important mechanisms linking diverse pathologic processes to premature activation of the common pathway of parturition. 13 — 19 Premature cervical ripening involves extensive extracellular matrix remodeling mediated by matrix metalloproteinases and shifts in cytokine signaling, while fetal inflammatory responses may independently trigger parturition. 20 — 24 Despite the high prevalence of sPTB, effective preventive therapies remain limited, supporting continued investigation of biologically targeted interventions. 25 , 26 Tocolytics, which inhibit uterine contractions during active preterm labor, are limited to short-term use, do not prevent fetal morbidities associated with sPTB, and carry significant maternal risks. 27 , 28 They are therefore not suitable for primary prevention, which requires sustained treatment initiated before the onset of labor in women at an elevated risk, such as those with a prior PTB. Historically, 17-hydroxyprogesterone caproate (17-OHPC) (Makena) and ritodrine were approved for sPTB prevention. Ritodrine was withdrawn in 1998 due to severe maternal cardiopulmonary adverse events, and Makena was withdrawn in 2023 after post-marketing studies failed to demonstrate efficacy. 29 , 30 The American College of Obstetricians and Gynecologists now advises that 17-OHPC is not recommended for general use in sPTB prevention. 31 — 33 Currently, vaginal progesterone is recommended to prevent sPTB risk in women with a mid-trimester sonographically measured short cervix, 34 — 37 but its efficacy in women at an elevated risk without cervical shortening is unproven. 38 , 39 Given the prevalence of sPTB and the lack of broadly effective therapies, there is a critical need for novel, safe, and effective preventive strategies. 40 — 42 Several new therapeutics are in development for PTB prevention. 43 — 45 Promegestone, a selective progesterone receptor (PR) modulator, inhibits inflammation-induced PTB in mice and is not metabolized by 20-hydroxysteroid dehydrogenase, conferring enhanced uterine stability relative to progesterone. 46 , 47 Other investigational agents include rytvela, a novel anti-inflammatory allosteric peptide; 48 , 49 resveratrol, a naturally occurring antioxidant; 50 , 51 and N,N-dimethylformamide, a common pharmaceutical excipient. 52 In addition, repurposing existing drugs as a therapeutic approach is also being considered for the prevention of PTB. 53 , 54 Nestorone is a synthetic progestin with high selectivity and potency, transactivating the PR 30 to 50 times more effectively than progesterone in vitro and in rabbit endometrial bioassays. 55 — 57 It exhibits anti-inflammatory activity in rodent models, lacks androgenic and estrogenic effects, and avoids the side effects associated with other androgenic progestins. 55 , 58 Results from prior studies demonstrated that Nestorone more effectively maintained pregnancy in estrone-primed, ovariectomized rats compared with progesterone. 55 Nestorone has been extensively studied in nonpregnant women as a hormonal contraceptive (Annovera) approved by the Food and Drug Administration and Health Canada and has been evaluated in multiple alternative drug delivery systems, including implants, vaginal rings, and transdermal gels. 59 — 61 We hypothesized that a highly potent PR agonist with anti-inflammatory properties would be more effective than progesterone in preventing inflammation-induced activation of the common pathway of parturition. Because this is a preclinical study, the patient populations most likely to benefit from Nestorone therapy remain to be determined and will require future clinical investigation. We evaluated (1) transcriptional activity of Nestorone compared to other progestins in human progesterone and glucocorticoid receptor (GR) reporter assays; (2) anti-inflammatory effects of Nestorone in bacterial endotoxin-stimulated human peripheral blood mononuclear cells and immortalized uterine and lung cell lines; and (3) its ability to prevent PTB in systemic and local inflammation models using endotoxin-treated timed-pregnant mice.