Severity of COVID-19 Respiratory Complications during Pregnancy are Associated with Degree of Lymphopenia and Neutrophil to Lymphocyte Ratio on Presentation: A Multicenter Cohort Study.

OBJECTIVE: This study aimed to determine if laboratory inflammatory markers can predict critical disease in symptomatic COVID-19 pregnant women. STUDY DESIGN: Multicenter, retrospective cohort study of all pregnant women presenting to New York City Health + Hospitals emergency departments from March 1 to May 30, 2020. We assessed all symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive pregnant women with room air oxygen saturation <95% on presentation. Logistic regression modeled the relationship of inflammatory markers to outcomes. Area under receiver operating characteristic (ROC) curve and maximum Youden index determined prognostic ability and optimal predictive cut-off values. RESULTS: A total of 498 of 5,002 pregnant women were SARS-CoV-2 RT-PCR positive of which 77 presented with hypoxemia. The absolute lymphocyte count (ALC) and neutrophil to lymphocyte ratio (NLR) were highly sensitive for progression to severe illness. ROC curve analysis identified predictive cutoffs: ALC < 1.49 × 109/L (96% sensitivity, 52% specificity, area under the receiver operating characteristic curve [AUC] = 0.80 (95% confidence interval [CI]: 0.70-0.90) and NLR >8.1 (100% sensitivity, 70% specificity, AUC = 0.86 (95% CI: [0.76-0.96]). CONCLUSION: ALC and NLR on presentation are sensitive markers of progression to critical COVID-19 disease in symptomatic pregnant women. This finding provides a practical, rapid method for assessment and can assist clinicians with decision-making regarding triage, level of care, and patient management. KEY POINTS: · Few tools exist to gauge risk of severe COVID-19 disease in pregnancy.. · ALC and NLR are sensitive predictive markers of disease progression in symptomatic women.. · Cut-off values for ALC and NLR will help direct patient triage and management..

Mechanism of Human Fetal Membrane Biomechanical Weakening, Rupture and Potential Targets for Therapeutic Intervention.

Using a novel in vitro model system combining biochemical/histologic with bioengineering approaches has provided significant insights into the physiology of fetal membrane weakening and rupture along with potential mechanistic reasons for lack of efficacy of currently clinically used agents to prevent preterm premature rupture of the membranes (pPROM) and preterm births. Likewise, the model has also facilitated screening of agents with potential for preventing pPROM and preterm birth.

Granulocyte macrophage colony stimulating factor (GM-CSF), the critical intermediate of inflammation-induced fetal membrane weakening, primarily exerts its weakening effect on the choriodecidua rather than the amnion.

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.

In an in-vitro model using human fetal membranes, α-lipoic acid inhibits inflammation induced fetal membrane weakening.

INTRODUCTION: We 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. OBJECTIVE: To determine the point of action of LA inhibition of FM weakening. METHODS: FM 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. RESULTS: TNF 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. CONCLUSIONS: LA 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.

Clinical yield of fetal echocardiography for suboptimal cardiac visualization on obstetric ultrasound.

OBJECTIVE: Suboptimal cardiac imaging on obstetric ultrasound is a frequent referral indication for fetal echocardiography, even in the absence of typical risk factors for fetal cardiac disease. The clinical profile of patients and findings of examinations performed for such an indication are not well defined. Given the increased cost, time and resource utilization of fetal echocardiography, we sought to determine the clinical findings of such referrals. STUDY DESIGN: We performed a single-center review of such referrals from January 2010 to June 2016. Patients with commonly accepted indications for fetal echocardiography were excluded. Demographic variables and echocardiogram findings were collected. Findings were classified as (1) "normal," (2) "probably normal," if minor pathology could not confidently be excluded, or if minor findings were noted that were expected to resolve, or (3) "abnormal." Rates of pathology were determined with comparison of nonobese and obese populations. RESULTS: A total of 583 gestations in 562 women were included (median gestational age 23.3 weeks, range 19.0-38.4). The median body mass index (BMI) was 34.6 kg/m2 (range 17.2-66.3 kg/m2 ). The majority of women were obese (BMI ≥ 30 kg/m2 in 74.6%). Overall, 574 of 583 examinations (98.5%) were normal or "probably normal." Pathology was noted in 9 fetuses (1.5%), 3 of whom required intervention (0.5%). No ductal dependent lesions were diagnosed. There was no significant difference in pathology rates between nonobese and obese mothers. CONCLUSIONS: We found a low fetal cardiac anomaly rate in studies performed for suboptimal views on obstetric ultrasound. The majority of women referred for this indication were obese. The practice of routine referral for this indication deserves further evaluation.

In an in-vitro model using human fetal membranes, 17-α hydroxyprogesterone caproate is not an optimal progestogen for inhibition of fetal membrane weakening.

BACKGROUND: The progestogen 17-α 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-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α 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-α 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-α- 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-α- 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.

The physiology of fetal membrane weakening and rupture: Insights gained from the determination of physical properties revisited.

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.

Progesterone inhibits in vitro fetal membrane weakening.

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-α- (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 17α-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°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

Spontaneous forniceal rupture in pregnancy.

Forniceal rupture is a rare event in pregnancy. We report a case of a 26-year-old primigravid woman who experienced a forniceal rupture at 23 weeks of gestation with no inciting cause except for pregnancy. Pregnancy is associated with ureteral compression due to increase in pelvic vasculature with the right ureter more dilated due to anatomic reasons. Hormones such as prostaglandins and progesterone render the ureter more distensible to allow for pressure build-up and an obstructive picture at the collecting system. We will discuss physiologic changes in pregnancies that predispose to this uncommon phenomenon and the most up-to-date management strategies.

A comparison of podocyturia, albuminuria and nephrinuria in predicting the development of preeclampsia: a prospective study.

Preeclampsia, a hypertensive multisystem disease that complicates 5-8% of all pregnancy, is a major cause for maternal and fetal mortality and morbidity. The disease is associated with increased spontaneous and evoked preterm birth and remote cardio-renal disorders in the mother and offspring. Thus the ability to predict the disease should lead to earlier care and decreased morbidity. This has led to fervent attempts to identify early predictive biomarkers and research endeavors that have expanded as we learn more regarding possible causes of the disease. As preeclampsia is associated with specific renal pathology including podocyte injury, early urinary podocyte (podocyturia), or the podocyte specific proteinuria nephrin in the urine (nephrinuria), as well as the more easily measured urinary albumin (albuminuria), have all been suggested as predictive markers. We performed a prospective study recruiting 91 pregnant women (78 of whom were high risk) and studied the predictive ability of these three urinary biomarkers. The subjects were recruited between 15-38 weeks of gestation. Fourteen patients, all in the high-risk obstetric group, developed preeclampsia. The levels of podocyturia, nephrinuria, and albuminuria were variably higher in the high-risk pregnant patients who developed preeclampsia. The sensitivities and specificities for podocyturia were 70% and 43%, for albuminuria were 36% and 96%, and for nephrinuria were 57% and 58%, respectively. Also, abnormal nephrinuria (69%) and podocyturia (38%) were detected in low risk women who had uncomplicated gestations; none of these women exhibited albuminuria. In our study, none of the three urinary markers achieved the minimum predictive values required for clinical testing. The lack of excessive albuminuria, however, may indicate a preeclampsia-free gestation. Given a discrepant literature, further studies with larger sample size should be considered.

The impact of vitamin C supplementation in pregnancy and in vitro upon fetal membrane strength and remodeling.

Generation of reactive oxygen species (ROS) has been suggested as a mechanism of fetal membrane (FM) weakening leading to rupture, particularly with preterm premature rupture of the fetal membranes (PROM). In vitro, FM incubation with tumor necrosis factor (TNF) mimics physiological FM weakening, concomitant with generation of ROS and collagen remodeling. Proinflammatory cytokines are also postulated to have a role in the development of the FM physiological weak zone where rupture normally initiates in-term gestations. We hypothesized that antioxidant treatment may block ROS development and resultant FM weakening. Two studies examining antioxidant effects upon FM strength were conducted, one in vivo and the other in vitro. Fetal membrane of patients enrolled in a multicenter placebo-controlled trial to determine the effect of vitamin C (1 g/day) and vitamin E (400 IU/day) upon complications of pre-eclampsia were examined for FM biomechanical properties and biochemical remodeling at birth. Separately, biomechanics and biochemical markers of remodeling were determined in FM fragments incubated with TNF with or without vitamin C preincubation. Supplemental dietary vitamin C in combination with vitamin E did not modify rupture strength, work to rupture, or matrix metalloproteinase-9 (MMP9; protein or activity) either within or outside the term FM physiological weak zone. In vitro, TNF decreased FM rupture strength by 50% while increasing MMP9 protein. Vitamin C did not inhibit these TNF-induced effects. Vitamin C alone had a weakening effect on FM in vitro. We speculate that vitamin C supplementation during pregnancy will not be useful in the prevention of preterm PROM.

Alpha-lipoic acid inhibits tumor necrosis factor-induced remodeling and weakening of human fetal membranes.

Untimely rupture of the fetal membranes (FMs) is a major precipitant of preterm birth. Although the mechanism of FM weakening leading to rupture is not completely understood, proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 beta (IL1B), have been shown to weaken FMs concomitant with the induction of reactive oxygen species, collagen remodeling, and prostaglandin release. We hypothesized that alpha-lipoic acid, a dietary antioxidant, may block the effect of inflammatory mediators and thereby inhibit FM weakening. Full-thickness FM fragments were incubated with control media or TNF, with or without alpha-lipoic acid pretreatment. Fetal membrane rupture strength and the release of matrix metalloproteinase 9 (MMP9) and prostaglandin E(2) (PGE(2)) from the full-thickness FM fragments were determined. The two constituent cell populations in amnion, the mechanically strongest FM component, were similarly examined. Amnion epithelial and mesenchymal cells were treated with TNF or IL1B, with or without alpha-lipoic acid pretreatment. MMP9 and PGE(2) were analyzed by ELISA, Western blot, and zymography. TNF decreased FM rupture strength 50% while increasing MMP9 and PGE(2) release. Lipoic acid inhibited these TNF-induced effects. Lipoic acid pretreatment also inhibited TNF- and IL1B-induced increases in MMP9 protein activity and release in amnion epithelial cells, as well as PGE(2) increases in both amnion epithelial and mesenchymal cells. In summary, lipoic acid pretreatment inhibited TNF-induced weakening of FM and cytokine-induced MMP9 and PGE(2) in both intact FM and amnion cells. We speculate that dietary supplementation with alpha-lipoic acid might prove clinically useful in prevention of preterm premature rupture of fetal membranes.

A photochemical source of methyl chloride in saline waters.

It is shown experimentallythatthe methoxy group in simple lignin-like molecules can be the source of the methyl group in CH3Cl produced by a photochemical reaction in an aqueous solution of chloride. Terrestrially derived colored dissolved organic matter (CDOM) in river water also yields CH3Cl through a photochemical process in a chloride solution. CDOM extracted from subsurface ocean waters showed some ability to enhance photochemical production of CH3Cl while CDOM from surface water showed no effect. Reactions of the kind described in this paper may be contributors to the marine source of methyl chloride and possibly other alkyl halides.

The force required to rupture fetal membranes paradoxically increases with acute in vitro repeated stretching.

OBJECTIVE: The purpose of this study was to determine whether acute repetitive stretching causes fetal membranes (FM) weakening. STUDY DESIGN: Cesarean or vaginally delivered FM were repeatedly stretched and thereafter subjected to rupture testing. Rupture strength (RS), work to rupture (WR), and stiffness were determined. Unstretched FM were compared with stretched FM. RESULTS: In the cesarean group, FM stretched to 50% or 75% of the baseline (unstretched) RS for 10-20 cycles of 10 seconds each paradoxically showed increased RS and stiffness. WR decreased compared with baseline. Detailed analysis revealed that even a single stretch cycle initiated these changes to physical properties. Vaginally delivered FM showed similar changes in physical properties, as did separated amnion. CONCLUSION: Acute stretch forces do not directly cause FM weakening.

Fetal membranes from term vaginal deliveries have a zone of weakness exhibiting characteristics of apoptosis and remodeling.

BACKGROUND: Recently we identified a weak zone in term, pre-labor (repeat Cesarean section) human fetal membranes (FM) overlying the cervix with biochemical characteristics suggestive of apoptosis and collagen remodeling. We suggested that this weak zone is the FM rupture initiation site. Vaginally delivered patients have a weak zone in their FM overlying the cervix; a comparable weak zone lies adjacent to the tear line in FM after spontaneous rupture (SROM). METHODS: FM from vaginally delivered patients with artificial rupture (AROM) and SROM were collected. FM of AROM patients were marked per vagina to identify the FM zone overlying the cervix. Postpartum FM were cut, strength tested, and piece strengths were remapped to their former location on a three-dimensional model. A 10-cm diameter zone centered on the marked area (AROM), or defined weak zone (SROM) was compared with the remaining FM. RESULTS: AROM FM exhibit a para-cervical weak zone. SROM FM exhibit a comparable zone on the tear line. The mean rupture strength within weak zones was 60% of the remaining membranes (P <.001). AROM and SROM FM weak zones both exhibit increased matrix metalloproteinase 9, increased poly (ADP-ribose) polymerase I cleavage, decreased tissue inhibitor of metalloproteinase 3 protein, and histology consistent with remodeling and apoptosis. CONCLUSION: Vaginally delivered AROM FM contain a weak zone overlying the cervix. Vaginally delivered SROM FM contain a weak zone adjacent to the tear line that exhibits biochemical and mechanical characteristics suggestive of collagen remodeling and apoptosis comparable to those of the AR FM weak zone.

Proinflammatory cytokines found in amniotic fluid induce collagen remodeling, apoptosis, and biophysical weakening of cultured human fetal membranes.

The mechanisms by which fetal membranes (FM) rupture during the birth process are unknown. We have recently reported that FM weaken, at least in part, because of a developmental process of extracellular matrix remodeling and apoptosis. We now hypothesize that cytokines that normally increase in amniotic fluid at term induce FM collagen remodeling and apoptosis with concomitant weakening. Full-thickness FM fragments were cultured with (0-100 ng/ml) or without tumor necrosis factor (TNF) or interleukin 1, beta (IL1B). Physical properties were then examined with specially adapted industrial rupture strength testing equipment. Cultured FM were also evaluated for evidence of collagen remodeling and apoptosis. Cytokine-treated FM exhibited a dose-dependent decrease in strength and work to rupture. Compared with controls, the highest TNF dose caused maximal decrease in FM rupture strength (13.2 +/- 1.2 N versus 3.8 +/- 1.5 N; P = 0.0003) and work to rupture (0.035 +/- 0.005 J versus 0.005 +/- 0.002 J; P < 0.0001). The highest IL1B dose also decreased rupture strength (12.9 +/- 3.2 versus 4.6 +/- 1.1 N; P = 0.0027) and work to rupture (0.018 +/- 0.005 J versus 0.005 +/- 0.002 J; P < 0.01). Matrix metalloproteinase 9 (MMP9) protein increased, tissue inhibitor of matrix metalloproteinase 3 (TIMP3) protein decreased, and poly (ADP-ribose) polymerase (PARP1) cleavage increased with increasing TNF or IL1B doses (all P < 0.05), suggesting collagen remodeling and apoptosis. TNF and IL1B cause significant weakening of cultured FM. Both cytokines induce biochemical markers in the FM in a manner characteristic of the weak zone of FM overlying the cervix. TNF and or IL1B may be involved in the development of the weak zone of the FM.

Lactosylceramide-induced apoptosis in primary amnion cells and amnion-derived WISH cells.

Amnion apoptosis is part of a programmed process of fetal membrane remodeling leading to weakening and rupture. The apoptotic agent lactosylceramide is elevated in amniotic fluid of premature infants with rupture of membranes. We have shown that apoptosis in WISH cells, induced by staurosporine, cycloheximide, or actinomycin D, can be blocked by cyclooxygenase inhibitors, suggesting a relationship between prostaglandin production and apoptosis. Cyclic adenosine monophosphate (cAMP) is known to inhibit prostaglandin release in amnion and WISH cells. This study was undertaken to determine the apoptotic potential of lactosylceramide and the effect of cyclooxygenase inhibitors and cAMP activators on lactosylceramide-induced apoptosis in primary amnion and WISH cells. Primary amnion cells and WISH cells were incubated with lactosylceramide to determine apoptosis and prostaglandin E(2) (PGE(2)) release. Apoptosis was confirmed by agarose gel electrophoretic DNA fragmentation analysis, nuclear matrix protein (NMP), and nucleosome enzyme-linked immunosorbent assay. In some studies, cells were preincubated with cyclooxygenase inhibitors or cAMP activators. Lactosylceramide induced a 20-fold increase in NMP (measure of cell death) in both cell types. Apoptosis was confirmed by the studies listed in methods. Lactosylceramide increased PGE(2) release in parallel with apoptosis. Cyclooxygenase inhibitors as well as cAMP activators inhibited both PGE(2) release and apoptosis. Lactosylceramide-induced apoptosis in both amnion and WISH cells. Parallel PGE(2) release was demonstrated with apoptosis. Cyclooxygenase inhibitors and cAMP activators blocked both processes.