The line of work reviewed here uses an in vitro model system that combines biochemical/histologic with mechanical/engineering approaches. The model highlights potential mechanistic reasons for lack of efficacy of currently clinically used agents to prevent preterm birth. Likewise, it can be used to screen agents with potential for preventing pPROM and preterm birth. The cumulative results of these studies are illustrated in Fig. 13. These studies provide a unique perspective and information about the physiology of FM weakening and rupture.

Preterm premature rupture of membranes (PPROM) and thrombin generation by decidual cell-expressed tissue factor often accompany abruptions. Underlying mechanisms remain unclear. We hypothesized that thrombin-induced colony-stimulating factor-2 (CSF-2) in decidual cells triggers paracrine signaling via its receptor (CSF2R) in trophoblasts, promoting fetal membrane weakening and abruption-associated PPROM. Decidua basalis sections from term (n = 10), idiopathic preterm birth (PTB; n = 8), and abruption-complicated pregnancies (n = 8) were immunostained for CSF-2. Real-time quantitative PCR measured CSF2 and CSF2R mRNA levels. Term decidual cell (TDC) monolayers were treated with 10-8 mol/L estradiol ± 10-7 mol/L medroxyprogesterone acetate (MPA) ± 1 IU/mL thrombin pretreatment for 4 hours, washed, and then incubated in control medium with estradiol ± MPA. TDC-derived conditioned media supernatant effects on fetal membrane weakening were analyzed. Immunostaining localized CSF-2 primarily to decidual cell cytoplasm and cytotrophoblast cell membranes. CSF-2 immunoreactivity was higher in abruption-complicated or idiopathic PTB specimens versus normal term specimens (P < 0.001). CSF2 mRNA was higher in TDCs versus cytotrophoblasts (P < 0.05), whereas CSF2R mRNA was 1.3 × 104-fold higher in cytotrophoblasts versus TDCs (P < 0.001). Thrombin enhanced CSF-2 secretion in TDC cultures fourfold (P < 0.05); MPA reduced this effect. Thrombin-pretreated TDC-derived conditioned media supernatant weakened fetal membranes (P < 0.05), which MPA inhibited. TDC-derived CSF-2, acting via trophoblast-expressed CSFR2, contributes to thrombin-induced fetal membrane weakening, eliciting abruption-related PPROM and PTB.

Background Respiratory syncytial virus (RSV) is capable of transient viremia and extrapulmonary dissemination. Recently, this virus has been identified in fetal cord blood, suggesting the possibility of in utero acquisition in humans. Here, we assess permissivity and kinetics of RSV replication in primary human placental cells, examine their potential to transfer this infection to neighboring cells, and measure the inflammatory response evoked by the virus. Methods and findings Human placental villus tissue was collected immediately upon delivery and processed for isolation of placental cytotrophoblast, fibroblast, and macrophage (Hofbauer) cells. Isolated cells were infected with a recombinant RSV-A2 strain (rrRSV) expressing red fluorescent protein (RFP) and analyzed by fluorescence microscopy, Western blot, and quantitative PCR (qPCR). Based on RFP expression, rrRSV exhibited differential tropism for the three major placental cell types. Placental fibroblasts and Hofbauer cells were permissive and supported productive rrRSV replication. While infected cytotrophoblast cells expressed viral glycoprotein (G protein), only limited RSV replication was detected. Importantly, qPCR and fluorescence-focused unit assay revealed that the viral progeny remains trapped within infected Hofbauer cells for up to 30 days, with no release into surrounding media. Yet, Hofbauer cells passed the infection onto overlaid naïve 16HBE cells, suggesting contact-dependent trans-infection. Lastly, a significant increase in proinflammatory cytokines, particularly IL-6, TNF-alpha, and IFN-gamma was measured in the supernatant of infected Hofbauer cells by multiplex cytokine assay and conventional ELISA. Conclusions This study demonstrates that RSV can replicate in human placenta, exhibits differential tropism for distinct placental cell types, can be stored and transferred to neighboring cells by Hofbauer cells, and elicits an inflammatory response. It also supports the hypothesis that this respiratory virus can be vertically transferred to the fetus and potentially affect its development and the outcome of pregnancies.

Introduction: We have previously demonstrated two associations of PPROM, (1) inflammation/infection (modeled by tumor necrosis factor (TNF)) and (2) decidual bleeding (modeled by thrombin), both decrease fetal membrane (FM) rupture strength in-vitro. Furthermore, Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) induced by both TNF and thrombin is a critical intermediate, necessary and sufficient for weakening by either agent. The amnion is the strength component of FM and must weaken for FM to rupture. It is unclear whether GM-CSF weakens amnion (AM) directly, or initially targets choriodecidua (CD) which secondarily releases agents to act on amnion. Methods: Full thickness FM fragments were treated with/without GM-CSF. Some were preincubated with alpha-lipoic acid (LA), a known inhibitor of FM weakening. The FM fragments were then strength-tested. Separately, FM fragments were initially separated to AM and CD. AM fragments were cultured with Medium +/- GM-CSF and then strength-tested. In other experiments, CD fragments were cultured with Medium, GM-CSF, LA, or LA + GM-CSF. Conditioned medium from each group was then incubated with AM. AM was then strength-tested. Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) were analyzed by Mutiplex Elisa. Results: GM-CSF weakened intact FM which was blocked by LA. GM-CSF did not weaken isolated AM. However, GM-CSF conditioned CD media weakened AM and this weakening was inhibited by LA. GM-CSF treatment of CD increased MMPs 2, 9, and 10, and decreased TIMPs 1-3. LA reversed these effects. Conclusions: GM-CSF does not weaken amnion directly; GM-CSF acts on CD to increase proteases and decrease anti-proteases which secondarily weaken the amnion.

IntroductionWe established an in-vitro model for the study of human fetal membrane (FM) weakening leading to pPROM. In this model, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both tumor necrosis factor-α (TNF; modeling infection/inflammation) and thrombin (modeling decidual bleeding/abruption)-induced weakening. Thus, inhibitors of FM weakening can be categorized as targeting GM-CSF production, GM-CSF downstream action, or both. Most progestogens inhibit both, except 17-α hydroxyprogesterone caproate which inhibits FM weakening at only one point, GM-CSF production. α-lipoic acid (LA), an over-the-counter dietary supplement, has also been previously shown to inhibit TNF and thrombin induced FM weakening.ObjectiveTo determine the point of action of LA inhibition of FM weakening.MethodsFM fragments were mounted in Transwell inserts and preincubated with/without LA/24 h, then with/without addition of TNF, thrombin or GM-CSF. After 48 h, medium was assayed for GM-CSF, and FM fragments were rupture-strength tested.ResultsTNF and thrombin both weakened FM and increased GM-CSF levels. GM-CSF also weakened FM. LA inhibited both TNF and thrombin induced FM weakening and concomitantly inhibited the increase in GM-CSF in a concentration-dependent manner. In addition, LA inhibited GM-CSF induced FM weakening in a concentration dependent manner.ConclusionsLA blocks TNF and thrombin induced FM weakening at two points, inhibiting both GM-CSF production and downstream action. Thus, we speculate that LA may be a potential standalone therapeutic agent, or supplement to current therapy for prevention of pPROM related spontaneous preterm birth, if preclinical studies to examine feasibility and safety during pregnancy are successfully accomplished.

BACKGROUND: The progestogen 17-alpha hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-alpha (TNF-alpha) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-alpha and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-alpha and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-alpha- and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor. OBJECTIVE: The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-alpha- and thrombin-induced fetal membrane weakening in vitro. STUDY DESIGN: Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5 cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10(-9) to 10(-7) M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony-stimulating factor and the fetal membrane fragments were rupture strength tested. RESULTS: Tumor necrosis factor-alpha and thrombin both weakened fetal membranes (43% and 62%, respectively) and increased granulocyte-macrophage colony-stimulating factor levels (3.7- and 5.9-fold, respectively). Pretreatment with 17-alpha hydroxyprogesterone caproate inhibited both tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening and concomitantly inhibited the induced increase in granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner. However, contrary to our prior reports regarding progesterone and other progestogens, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced fetal membrane weakening. CONCLUSION: 17-Alpha hydroxyprogesterone caproate blocks tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening by inhibiting the production of granulocyte-macrophage colony-stimulating factor. However, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced weakening. We speculate that progestogens other than 17-alpha hydroxyprogesterone caproate may be more efficacious in preventing preterm premature rupture of the fetal membranes-related spontaneous preterm birth.

Rupture of the fetal membranes (FM) is precipitated by stretch forces acting upon biochemically mediated, pre-weakened tissue. Term FM develop a para-cervical weak zone, characterized by collagen remodeling and apoptosis, within which FM rupture is thought to initiate. Preterm FM also have a weak region but are stronger overall than term FM. Inflammation/infection and decidual bleeding/abruption are strongly associated with preterm premature FM rupture (pPROM), but the specific mechanisms causing FM weakening-rupture in pPROM are unknown. There are no animal models for study of FM weakening and rupture. Over a decade ago we developed equipment and methodology to test human FM strength and incorporated it into a FM explant system to create an in-vitro human FM weakening model system. Within this model TNF (modeling inflammation) and Thrombin (modeling bleeding) both weaken human FM with concomitant up regulation of MMP9 and cellular apoptosis, mimicking the characteristics of the spontaneous FM rupture site. The model has been enhanced so that test agents can be applied directionally to the choriodecidual side of the FM explant consistent with the in-vivo situation. With this enhanced system we have demonstrated that the pathways involving inflammation/TNF and bleeding/Thrombin induced FM weakening overlap. Furthermore GM-CSF production was demonstrated to be a critical common intermediate step in both the TNF and the Thrombin induced FM weakening pathways. This model system has also been used to test potential inhibitors of FM weakening and therefore pPROM. The dietary supplement α-lipoic acid and progestogens (P4, MPA and 17α-hydroxyprogesterone) have been shown to inhibit both TNF and Thrombin induced FM weakening. The progestogens act at multiple points by inhibiting both GM-CSF production and GM-CSF action. The use of a combined biomechanical/biochemical in-vitro human FM weakening model system has allowed the pathways of fetal membrane weakening to be delineated, and agents that may be of clinical use in inhibiting these pathways to be tested.

OBJECTIVE: Inflammation/infection and abruption are leading causes of preterm premature rupture of the membranes. Recently, we identified granulocyte-macrophage colony-stimulating factor (GM-CSF) as a critical mediator of both tumor necrosis factor-alpha- (TNF; modeling inflammation) and thrombin-induced (modeling abruption) weakening of the fetal membranes. We found that (1) TNF and thrombin both induced GM-CSF in the choriodecidua, (2) blockade of GM-CSF action with neutralizing antibodies inhibited both TNF- and thrombin-induced fetal membrane weakening, and (3) GM-CSF alone induced fetal membrane weakening. GM-CSF is thus part of an overlap of the inflammation and abruption-induced fetal membrane weakening pathways. The effects of progesterone analogs on the pathways by which fetal membranes are weakened have not been investigated. We examined the effects of progesterone, medroxyprogesterone acetate (MPA) and 17 alpha-hydroxyprogesterone (HP) on TNF- and thrombin-induced fetal membrane weakening.STUDY DESIGN: Full-thickness fetal membranes from uncomplicated term repeat cesarean deliveries were mounted in Transwell inserts in Minimum Essential Medium alpha and incubated at 37 degrees C in 5% CO2. The choriodecidua side of the fetal membrane fragments were preincubated with progesterone, MPA, HP, or vehicle for 24 hours. Fetal membranes were then exposed to TNF, thrombin, or GM-CSF on the choriodecidua side for an additional 48 hours. The fetal membrane tissues were then strength tested, and medium from the choriodecidua and amnion compartments was assayed for GM-CSF content.RESULTS: TNF and thrombin both weakened fetal membranes and elevated media GM-CSF levels on the choriodecidua side of the fetal membrane. Pretreatment with progesterone, MPA, or HP inhibited both TNF- and thrombin-induced fetal membrane weakening and also inhibited the induced increase in GM-CSF. GM-CSF decreased fetal membrane rupture strength by 68%, which was inhibited by progestogen pretreatment with a potency order: progesterone < MPA < HP.CONCLUSION: Progestogen pretreatment blocks TNF-and thrombin-induced fetal membrane weakening by inhibiting both the production and action of GM-CSF. These findings are consistent with the administration of progestogens in the prevention of preterm premature rupture of the membranes.