Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Oct 1.
Published in final edited form as: Am J Perinatol. 2021 Nov 16;40(14):1585–1589. doi: 10.1055/s-0041-1739354

A Possible Mechanism of Action of 17α-Hydroxyprogesterone Caproate: Enhanced IL-10 Production

Christina J Megli 1, Alisse Hauspurg 1, Raman Venkataramanan 1, Steve N Caritis 1
PMCID: PMC9879021  NIHMSID: NIHMS1866383  PMID: 34784615

Abstract

Objective

The rate of recurrent spontaneous preterm birth (PTB) was reduced by 33% in the Maternal-Fetal Medicine Unit (MFMU) Network trial of 17α-hydroxyprogesterone caproate (17-OHPC), but the mechanism of action, 17 years later, remains elusive. The robustness of the interleukin-10 (IL-10) response to lipopolysaccharide (LPS) stimulation of leukocytes in pregnant women with a prior PTB correlates with gestational age at delivery. This study sought to determine if there is a relationship between the concentration of 17-OHPC and response to LPS stimulation.

Study Design

We performed a secondary analysis of data from the Omega-3 MFMU trial which evaluated the effectiveness of omega-3 fatty acid supplementation in reducing recurrent PTB. We utilized previously characterized data from a subanalyses of the Omega-3 trial of IL-10 and tumor necrosis factor alpha (TNF-α) levels from peripheral blood mononuclear cells stimulated with LPS. Blood was obtained from enrolled women at 16 to 22 weeks’ gestation (baseline) and 25 to 28 weeks’ gestation (posttreatment). All women received 17-OHPC and plasma 17-OHPC concentrations were measured at 25 to 28 weeks’ gestation. We analyzed these data to determine if there was a relationship between 17-OHPC concentration and cytokine production. We then performed an in vitro study to determine if 17-OHPC could directly alter cytokine production by THP-1-derived macrophages.

Results

In the clinical samples, we found that 17-OHPC plasma concentrations were correlated with the quantity of the LPS-stimulated production of IL-10. TNF-α production after LPS stimulation was unrelated to 17-OHPC concentration. In the in vitro study, we demonstrate a 17-OHPC concentration dependent increase in IL-10 production.

Conclusion

In women receiving 17-OHPC for PTB prevention, we demonstrate a relationship between plasma 17-OHPC and LPS-stimulated IL-10 production by circulating leukocytes. We also demonstrate that, in vitro, 17-OHPC treatment affects IL-10 production by LPS-stimulated macrophages. Collectively, these findings support an immunomodulatory mechanism of action of 17-OHPC in the prevention of recurrent PTB.

Keywords: progesterone, preterm birth, immune, pregnancy

In a landmark trial by the Maternal-Fetal Medicine Unit (MFMU), 17α-hydroxyprogesterone caproate (17-OHPC) reduced the rate of recurrent preterm birth (PTB) by ~33%.1 The mechanism of action remains unknown. A more recent study in a lower risk patient population was unable to demonstrate efficacy.2 As the efficacy of 17-OHPC remains under debate, it is important to elucidate a target mechanism by which the drug may function.

Inflammation and alteration in immune modulation have been implicated in the pathogenesis of spontaneous and recurrent PTB.3,4 This has been demonstrated in in vitro work from patient samples5 as well as in animal models.6,7 Ascending infection with intrauterine inflammation is associated with ~25% of PTB.8 Noninfection-based inflammation has also been shown to be associated with PTB. Introduction of systemic cytokines to animal models can precipitate PTB.9-11 Thus, it is thought that a balance between pro- and anti-inflammatory states may regulate parturition. The immunomodulatory cytokine interleukin-10 (IL-10) is produced by antigen-presenting cells in response to inflammatory stimuli as a mechanism to regulate the inflammatory response.12 Serum IL-10 increases across pregnancy and declines at the time of parturition in matched samples,13 suggesting that it may be involved in parturition. Moreover, low IL-10 and IL-10 polymorphisms have been associated with PTB in humans.14,15 In vitro models of bacterial or inflammatory stimuli of PTB have also implicated aberrant IL-10 response in the pathogenesis of prematurity.16-18

Despite gaining conditional Food and Drug Administration (FDA) approval, limited pharmacokinetic and pharmacodynamic data are available for 17-OHPC. Pharmacodynamic data to ascertain the proper dose have not been performed and a mechanism of action remains elusive. We have previously demonstrated that plasma levels of 17-OHPC correlate with efficacy and that women who have low plasma levels have a greater risk of recurrent PTB than women with higher concentrations of 17-OHPC.19 There is no clear molecular target or mechanism by which 17-OHPC might function to reduce PTB. As there have been questions regarding the efficacy of 17-OHPC with the recent PROLONG trial,2 it is crucial that a mechanism of action be defined which will also enable determination of an effective and safe dose and dosing frequency.

The aim of this study was to evaluate the relationship between 17-OHPC concentration and IL-10 production in response to an inflammatory stimulus using both clinical samples and in vitro proof of principal analysis.

Materials and Methods

Retrospective Clinical Sample Data Analysis

We performed an analysis of data collected during the MFMU Omega-3 study which evaluated the efficacy of omega-3 fatty acids in reducing recurrent PTB in women with a prior PTB. In this trial, at randomization (16–22 weeks), all women received 17-OHPC and were randomized to placebo or omega-3 fatty acid supplementation. There was no effect of omega-3 fatty acid supplementation on the rate of recurrent PTB. During this trial, blood samples were collected at the time of randomization (baseline) and at 25 to 28 weeks. An ancillary study utilized the peripheral blood mononuclear cells (PBMCs) that were collected at both time points. These cells were stimulated by lipopolysaccharide (LPS) and IL-10, and tumor necrosis factor alpha (TNF-α) production was determined by enzyme-linked immunosorbent assay (ELISA).20 We related the baseline and LPS-stimulated measurements of IL-10 and TNF-α to the concentration of 17-OHPC.20,21 We then stratified the cytokine results by 17-OHPC concentration-dependent quartiles.

THP-1-Derived Macrophage Stimulation and IL-10 Production

We performed an in vitro study to further determine if 17-OHPC could modulate LPS-stimulated IL-10 production by THP-1-derived macrophages (American Type Culture Collection). THP-1 cells (a human monocytic cell line) were cultured in RPMI 1640 (HyClone) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies), sodium pyruvate (Life Technologies), and penicillin/streptomycin (Lonza). They were maintained in an incubator at densities 1 × 104 to 1 × 106 at 37 degrees with 5% supplemental oxygen. Activation to macrophages was performed as previously described. Briefly, 2 × 105 cells were plated in each well of a 12 well place. Phorbol 12-myristate 13-acetate (PMA) (Bio-Rad) 100 ng/mL was added and the cells were incubated for 24 hours. Cells were confirmed to be adherent at this time and the culture media were removed and RPMI media with 10% FBS were added for 24 hours. Following the 24-hour PMA washout, LPS (100 ng/mL) and varying concentrations of progesterone or 17-OHPC were added. Cells were incubated for 48 hours and supernatants were collected. IL-10 and TNF-α ELISA duo kit (R&D Systems) were used to determine cytokine concentration in collected supernatants. All experiments were performed at least three times and data from each biologic replicate were used.

Statistical Analysis

Statistical analysis was performed in GraphPad Prism. Student’s t-test was used to compare cytokine production by17-OHPC quartile. For looking at dose dependence in the in vitro analysis, a one-way analysis of variance with multiple comparisons analysis was used to test for a linear trend. A p < 0.05 was considered statistically significant.

Results

Retrospective Clinical Sample Data Analysis

We first evaluated the relationship between plasma 17-OHPC concentrations and the in vitro LPS stimulation of macrophages from subjects who received 17-OHPC in the Omega-3 study. We stratified 17-OHPC concentrations by quartiles and related the 17-OHPC concentration to the LPS-stimulated cytokine production by PBMCs. We have previously shown that higher concentrations of 17-OHPC (quartiles 2–4) were correlated with a rate of recurrent PTB of 25%, whereas the PTB rate was 42% in those women with the lower plasma concentrations.21 The quartile of 17-OHPC plasma concentrations was also correlated with LPS-stimulated cytokine production by PBMCs, which was significantly greater in women with the highest quartiles of plasma 17-OHPC concentration (p < 0.001) (Fig. 1). There was no association of 17-OHPC plasma concentrations with IL-10 in the absence of LPS stimulation of the leukocytes (data not shown). There was no association between LPS-stimulated TNF-α concentrations and 17-OHPC concentrations (data not shown).

Fig. 1. Posttreatment stimulated IL-10 and TNF-α in correlation with 17-OHPC serum concentration.

Fig. 1

Open in a new tab

PBMC cytokine production in response to LPS stimulation was stratified by 17-OHPC plasma quartile. The dark boxes represent the results from patients with the first quartile of 17-OHPC concentration, whereas the gray boxes represent results from patients with higher quartiles of 17-OHPC (2–4) which coincide with clinical efficacy in timing of delivery. IL-10, interleukin-10; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cells; TNF-α, tumor necrosis factor alpha; 17-OHPC, 17α-hydroxyprogesterone caproate.

We then looked at the change in LPS-stimulated IL-10 production by the PBMCs from these women from the time of randomization (pretreatment) to the 25- to 28-week time point. The change in LPS-stimulated IL-10 concentration between the two gestational time points was correlated with the higher 17-OHPC concentration quartiles (p = 0.046) (Fig. 2). There was no relationship between 17-OHPC concentration and the magnitude of change of LPS-stimulated production of TNF-α by PBMC (data not shown). This suggested that 17-OHPC may mediate an immunomodulatory effect through its effect on IL-10 production.

Fig. 2. 17-OHPC concentration is associated with an increased change in IL-10 production in response to LPS.

Fig. 2

Open in a new tab

Posttreatment (25–28 weeks) and baseline IL-10 production were used to determine the percent change in IL-10 production in response to LPS after initiation of 17-OHPC. The change in IL-10 was then compared by 17-OHPC quartiles and was increased in women with higher quartiles (2–4) of 17-OHPC plasma concentrations. IL-10, interleukin-10; LPS, lipopolysaccharide; 17-OHPC, 17α-hydroxyprogesterone caproate.

THP-1-Derived Macrophage Stimulation and IL-10 Production

To determine if 17-OHPC has a direct effect on IL-10 secretion, we performed an in vitro analysis using a well-described model of macrophages using THP-1 cells. We exposed THP-1-derived macrophages, stimulated with LPS to clinically relevant concentrations of 17-OHPC or progesterone to determine if we could recapitulate the increased IL-10 production seen with PBMCs in clinical samples. We found a relationship between 17-OHPC concentrations and LPS-stimulated IL-10 secretion by macrophages (p = 0.0056) (Fig. 3A). This concentration response was evident over a concentration range of 25 to 250 ng/mL in the medium. We were unable to demonstrate an effect of 17-OHPC on THP-1 monocytes or macrophages in the absence of additional inflammatory stimulus (data not shown). Importantly, progesterone did not alter IL-10 production under the same experimental conditions (Fig. 3B).

Fig. 3. 17-OHPC enhances LPS-mediated IL-10 production in THP-1-derived macrophages in vitro.

Fig. 3

Open in a new tab

17-OHPC and progesterone were exposed to THP-1-derived macrophages with LPS in varying concentrations. Supernatant IL-10 production by ELISA is recorded. One way analysis of variance with multiple comparisons and test for a linear trend was performed. The p-value represents the test for linear trend with variation of progesterone and 17-OHPC dose. R2 for 17-OHPC (A) is 0.746 and for progesterone (B) is 0.254. ELISA, enzyme-linked immunosorbent assay; IL-10, interleukin-10; LPS, lipopolysaccharide; 17-OHPC, 17α-hydroxyprogesterone caproate.

Discussion

This investigation demonstrates a relationship between 17-OHPC concentration and IL-10 production using both in vivo and in vitro methodologies. Since IL-10 is a potent anti-inflammatory cytokine, it is possible that the mechanism of action of 17-OHPC in reducing PTB is mediated through changing the balance between pro- and anti-inflammatory cytokines in response to inflammatory stimulus. Elevations in proinflammatory cytokines are predictive of PTB and inhibition of proinflammatory cytokines can prevent PTB in primates supporting the idea that a subset of PTB is an inflammatory response. We propose that the immunoinhibitory effect of IL-10 could provide protection from inflammation-induced PTB (Fig. 4).

Fig. 4. Schematic of proposed mechanism of 17-OHPC.

Fig. 4

Open in a new tab

17-OHPC injections modulate the IL-10 leukocyte immune response to inflammatory stimulus which allows for mitigation of inflammation-induced preterm birth. IL-10, interleukin-10; TNF-α, tumor necrosis factor alpha; 17-OHPC, 17α-hydroxyprogesterone caproate. Created with BioRender.com.

There was no effect of 17-OHPC on IL-10 in the absence of inflammatory stimulation in patient samples from the Omega-3 trial as well as in our in vitro model. This suggests that 17-OHPC exerts its primary effect by modulating a provoked inflammatory response rather than preventing an inflammatory response. We hypothesize that the mechanism of 17-OHPC may have remained elusive due to the requirement for an inflammatory stimulus before 17-OHPC can exert a beneficial effect. Interestingly, there are no data that 17-OHPC dampens the adaptive immune response or increases susceptibility to infections. Our study suggests that these areas could be a target of future research.

This study is limited by the retrospective analysis of data and samples previously collected during the Omega-3 trial. Unfortunately, despite the large sample size included in the population, only a small proportion of these women had blood withdrawn with complete paired samples at both time points that were used for cytokine analysis and analysis of 17-OHPC concentration. This substantially limited our ability to demonstrate that the 17-OHPC concentration dependent increase in LPS-stimulated IL-10 correlates with its clinical efficacy in the prevention of recurrent PTB.

A strength of this study is the use of both clinical and basic research methodologies to investigate the mechanism of action of 17-OHPC in immune modulation. The fact that the in vitro studies support and enhance the findings from the clinical trial provides confidence that 17-OHPC modulates IL-10 response. Our findings, however, do raise that possibility and suggest that further investigation of immune response and PTB merits further consideration.

The currently FDA-approved dose of 17-OHPC (250 mg) was not based on any relevant pharmacologic data. The dose utilized in the MFMU trial was based on the dose used in studies from the 1950s to 1970s to prevent first trimester miscarriage. No dose–response studies were ever performed. Thus, the correct dose to prevent recurrent PTB is unclear. In fact, we have previously demonstrated that the current dose appears to provide inadequate plasma concentrations and consequently, a higher recurrence rate in a large proportion of women. If the mechanism of 17-OHPC efficacy is through IL-10, then a dose–response study in women with a prior PTB is needed with the outcome of PTB and IL-10 response.

17-OHPC has only been found to be effective in recurrent PTB, and our data suggest that a dysregulated innate immune inflammatory response, rather than chronic inflammation, may be an important mechanism of premature delivery in this subset of patients. Further understanding of the innate immune response to infectious and other inflammatory stimuli in this patient population is warranted.

These data add to the collection of other studies demonstrating that IL-10 is important for protection from PTB. As 17–OHPC has not demonstrated efficacy in other high-risk conditions for PTB, out data suggest that IL-10-mediated immune modulation may be a primary mechanism in recurrent PTB. Moreover, we suggest that women with recurrent PTB may have an altered response to inflammatory stimulus with alteration in inflammatory and immune modulatory signals.

Key Points.

  • 17-OHPC plasma concentrations and LPS-stimulated IL-10 levels correlate in clinical samples in women at risk for recurrent preterm birth.

  • 17-OHPC can modulate the response of LPS-stimulated macrophages to increase IL-10 production.

  • There was no relationship between TNF-α and plasma concentration of 17-OHPC in clinical samples or in vitro.

Acknowledgments

CJM is supported from the grant K12 HD000849, awarded to the Reproductive Scientist Development Program by the Eunice Kennedy Shriver National Institute of Health & Human Development. This research was supported by the Burroughs Wellcome Fund as part of the Reproductive Scientist Development Program. We would like to acknowledge the assistance of the MFMU network and the Omega 3 and Progesterone subcommittees in making the database available for our research. We would also like to thank Carolyn Coyne PhD for her assistance and mentorship for the in vitro assays as well as Magee Womens Research Institute and Foundation for ongoing support (CJM, AH).

Footnotes

The findings were presented at the Society for Maternal-Fetal Medicine Meeting, Las Vegas, NV, February 2019.

Conflict of Interest

None declared.

References

  • 1.Meis PJ, Klebanoff M, Thom E, et al. ; National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate. N Engl J Med 2003;348(24): 2379–2385 [DOI] [PubMed] [Google Scholar]
  • 2.Blackwell SC, Gyamfi-Bannerman C, Biggio JR Jr, et al. 17-OHPC to prevent recurrent preterm birth in singleton gestations (PROLONG study): a multicenter, international, randomized double-blind trial. Am J Perinatol 2020;37(02):127–136 [DOI] [PubMed] [Google Scholar]
  • 3.Romero R, Espinoza J, Gonçalves LF, Kusanovic JP, Friel L, Hassan S. The role of inflammation and infection in preterm birth. Semin Reprod Med 2007;25(01):21–39 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Romero R, Dey SK, Fisher SJ. Preterm labor: one syndrome, many causes. Science 2014;345(6198):760–765 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bonney EA, Johnson MR. The role of maternal T cell and macrophage activation in preterm birth: cause or consequence? Placenta 2019;79(March):53–61 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Coleman M, Orvis A, Wu TY, et al. 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. Front Immunol 2020;11:770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Rizzuto G, Tagliani E, Manandhar P, Erlebacher A, Bakardjiev AI. Limited colonization undermined by inadequate early immune responses defines the dynamics of decidual listeriosis. Infect Immun 2017;85(08):e00153–17 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Mendz GL, Kaakoush NO, Quinlivan JA. Bacterial aetiological agents of intra-amniotic infections and preterm birth in pregnant women. Front Cell Infect Microbiol 2013;3(OCT):58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Toothaker JM, Presicce P, Cappelletti M, et al. Immune cells in the placental villi contribute to intra-amniotic inflammation. Front Immunol 2020;11:866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gomez-Lopez N, Romero R, Garcia-Flores V, et al. Inhibition of the NLRP3 inflammasome can prevent sterile intra-amniotic inflammation, preterm labor/birth, and adverse neonatal outcomes. Biol Reprod 2019;100(05):1306–1318 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Payne MS, Bayatibojakhi S, Fang K, Shen B. Exploring preterm birth as a polymicrobial disease: an overview of the uterine microbiome. Front Immunol 2014;5:595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol 2010;10(03):170–181 [DOI] [PubMed] [Google Scholar]
  • 13.Reyes-Lagos JJ, Peña-Castillo MÁ, Echeverría JC, et al. Women serum concentrations of the IL-10 family of cytokines and IFN-γ decrease from the third trimester of pregnancy to active labor. Neuroimmunomodulation 2017;24(03):162–170 [DOI] [PubMed] [Google Scholar]
  • 14.Ruiz RJ, Jallo N, Murphey C, Marti CN, Godbold E, Pickler RH. Second trimester maternal plasma levels of cytokines IL-1Ra, Il-6 and IL-10 and preterm birth. J Perinatol 2012;32(07): 483–490 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Pandey M, Awasthi S, Singh U, Mahdi AA. Association of IL-10 gene polymorphism (−819C > T, −592C > A and −1082G > A) with preterm birth. Indian J Pediatr 2018;85(02):93–101 [DOI] [PubMed] [Google Scholar]
  • 16.Robertson SA, Care AS, Skinner RJ. Interleukin 10 regulates inflammatory cytokine synthesis to protect against lipopolysaccharide-induced abortion and fetal growth restriction in mice. Biol Reprod 2007;76(05):738–748 [DOI] [PubMed] [Google Scholar]
  • 17.Robertson SA, Skinner RJ, Care AS. Essential role for IL-10 in resistance to lipopolysaccharide-induced preterm labor in mice. J Immunol 2006;177(07):4888–4896 [DOI] [PubMed] [Google Scholar]
  • 18.Salminen A, Vuolteenaho R, Paananen R, Ojaniemi M, Hallman M. Surfactant protein D modulates levels of IL-10 and TNF-α in intrauterine compartments during lipopolysaccharide-induced preterm birth. Cytokine 2012;60(02):423–430 [DOI] [PubMed] [Google Scholar]
  • 19.Caritis SN, Venkataramanan R, Thom E, et al. ; Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network and Obstetric-Fetal Pharmacology Research Units Network. Relationship between 17-alpha hydroxyprogesterone caproate concentration and spontaneous preterm birth. Am J Obstet Gynecol 2014;210(02):128. e1–128.e6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Harper M, Li L, Zhao Y, et al. ; Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Maternal-Fetal Medicine Units (MFMU) Network* Change in mononuclear leukocyte responsiveness in midpregnancy and subsequent preterm birth. Obstet Gynecol 2013;121(04):805–811 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Caritis SN, Simhan HN, Zhao Y, et al. ; Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Relationship between 17-hydroxyprogesterone caproate concentrations and gestational age at delivery in twin gestation. Am J Obstet Gynecol 2012;207 (05):396.e1–396.e8 [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES