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.

Markers of protein synthesis are increased in fetal membranes and myometrium after human labour and delivery.

Preterm birth remains one of the leading causes of neonatal death. Inflammation and maternal infection are two of the leading aetiological factors for preterm birth. Labour is associated with increased production of proinflammatory cytokines, chemokines and prolabour mediators in human gestational tissues. In non-gestational tissues, synthesis of proinflammatory and prolabour mediators is regulated by components of the protein synthesis machinery. Therefore, in the present study we investigated the effect of human labour on the expression of three protein synthesis markers, namely eukaryotic elongation factor 2 kinase (EEF2K), mitogen-activated protein kinase interacting protein kinase 1 (MKNK1) and eukaryotic translation initiation factor 4E (EIF4E), and their role in regulating inflammation in human gestational tissues. In fetal membranes and myometrium, EEF2K expression was significantly lower, whereas MKNK1 expression was significantly higher withterm and preterm labourcompared to term nolabour. In contrast, EIF4E expression did not change in fetal membranes or myometrium with labour. In primary myometrial cells, loss-of-function studies using specific chemical inhibitors of EEF2K (A484954) and MKNK1 (CGP57380) demonstrated that MKNK1, but not EEF2K, was required for polyinosinic-polycytidylic acid (poly(I:C); a viral double-stranded RNA mimetic) and interleukin (IL)-1ß-induced production of IL6, C-X-C motif chemokine ligand 8 (CXCL8), prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin F2α. In conclusion, spontaneous term and preterm labour is associated with decreased EEF2K and increased MKNK1 expression in fetal membranes and myometrium. Moreover, MKNK1 is involved in the genesis of proinflammatory and prolabour mediators that is mediated by inflammation or infection. However, further studies are required to elucidate the role of EEF2K in human labour.

Retrospective comparison of perinatal outcomes following emergency cervical cerclage with or without prolapsed membranes.

OBJECTIVE: To compare perinatal outcomes following emergency cerclage between patients with singleton pregnancies with prolapsed and non-prolapsed membranes. METHODS: The present retrospective cohort study included data from women who underwent physical examination-indicated emergency cerclage at between 15 and 25 weeks of pregnancy at Saint Luc University Hospital, Brussels, Belgium, between January 1, 2000, and December 31, 2014. Outcomes were compared based on the presence of prolapsed or non-prolapsed membranes. The primary outcome measures were the duration of pregnancy at delivery and the interval between cerclage and delivery. Secondary outcomes included delivery weight, fetal or neonatal death, and neonatal morbidity, including neonatal intensive care unit admission. RESULTS: Data were included from 140 patients with cervical dilation of at least 1 cm; 85 women had non-prolapsed membranes and 55 women had prolapsed membranes. Among patients with non-prolapsed membranes, the mean duration of pregnancy at delivery was later (P<0.001), the latency between cerclage and delivery was longer (P<0.001), neonatal survival was higher (P=0.036), mean delivery weight was higher (P<0.001), the prevalence of preterm delivery was lower (P<0.001), and severe neonatal morbidity and neonatal intensive care unit admission were lower (P<0.001). CONCLUSION: Having non-prolapsed membranes was associated with improved perinatal outcomes following emergency cerclage.

The investigation of the role of proteoglycans and ADAMTS levels in fetal membranes in physiopathological process of gestational diabetes.

About 2-5% of all pregnant women develop gestational diabetes mellitus (GDM) during pregnancy and its prevalence has increased markedly within the last decade. GDM is a metabolic syndrome produced by various degrees of carbohydrate intolerance during pregnancy. Various risk factors such as obesity, genetics, environmental factors, and hypertension have been described previously. Maternal and fetal complications occur in around 7% of pregnant women with GDM. In these patients, a relation between proteoglycans and ADAMTS proteases located in extracellular matrix in fetal membranes (placenta, cord, amnion) and complicated pregnancies has already been determined by various animal experiments. Changes in expression, structure and function of ADAMTS proteases and proteoglycans in fetal membranes lead to alteration in the structure of extracellular matrix. If we can establish a balance between these proteoglycans and ADMTS proteases or determine the changes in their structure and functions, it will be possible to predict the risk in high risk pregnancies at early weeks and to initiate treatment early or to follow the target population regularly. In addition, prevention or reduction of maternal and fetal complications may be possible. For this purpose, ADAMTS and proteoglycans the synthesis of which is too much or less, may be targeted and if we would be able to determine and prevent the changes in their levels in the early period of pregnancy, the development of GDM and its complications may be prevented or decreased. Thus, we may identify a marker for early diagnosis and treatment and reduce prematurity, which is the most common cause of fetal death. Fetal and maternal complications, and especially treatment and care costs of prematurity, may also be decreased.

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.

Amniotic membrane sweeping.

Amniotic membrane sweeping or stripping is a safe and effective method of labor induction supported by national obstetrical organizations. While its use dates back to antiquity by both midwives and physicians there are still areas that need further research to define its role in induction of labor. A review of the literature reveals that amniotic membrane sweeping is a safe, effective, and inexpensive method of labor induction. It can be done in the outpatient setting with minimal risks so long as it is avoided in patients with contraindications. Amniotic membrane sweeping can be performed in Group B Streptococcus-positive women with studies showing no increase in untoward outcomes. However, there is no data in women infected with HIV or hepatitis.

[Rupture of membranes: pathophysiology, diagnosis, consequences and management]. / Rupture des membranes : physiopathologie, diagnostic, conséquences et prise en charge.

Rupture of membranes (ROM) depends on mechanical stretch, extracellular matrix components imbalance and increased apoptosis. It occurs in 2 to 3% of all pregnancies before 37 weeks' gestation (WG) and in up to 10% at term. Main consequences are labor induction and risk of maternal-fetal infection. ROM is associated with one third of preterm births and about 20% of perinatal mortality. This review deals with recent knowledge concerning ROM including diagnosis and management. In many cases, ROM is easily identified by clinical examination. In other cases, the use of vaginal pH appears to be less efficient than the use of immunochromatographic strips based on IGFBP-1 or PAMG-1 detection. Before 34WG, conservative management consists in in utero transfer, antibioprophylaxis and corticosteroids. After 37WG, delivery is the most appropriate option. Between 34 and 37WG, recent studies demonstrate that induction of labour does not improve pregnancy outcomes. Therefore, expectant management can be the first option between 34 and 37WG when no active infection is suspected especially in case of unfavourable cervix.

Multiaxial mechanical behavior of human fetal membranes and its relationship to microstructure.

This study was directed to the measurement of the mechanical response of fetal membranes to physiologically relevant loading conditions. Characteristic mechanical parameters were determined and their relation to the microstructural constituents collagen and elastin as well as to the pyridinium cross-link concentrations analyzed. 51 samples from twelve fetal membranes were tested on a custom-built inflation device, which allows mechanical characterization within a multiaxial state of stress. Methods of nonlinear continuum mechanics were used to extract representative mechanical parameters. Established biochemical assays were applied for the determination of the collagen and elastin content. Collagen cross-link concentrations were determined by high-performance liquid chromatography measurements. The results indicate a distinct correlation between the mechanical parameters of high stretch stiffness and membrane tension at rupture and the biochemical data of collagen content and pyridinoline as well as deoxypyridinoline concentrations. No correlation was observed between the mechanical parameters and the elastin content. Moreover, the low stretch stiffness is, with a value of 105 ± 31 × 10(-3) N/ mm much higher for a biaxial state of stress compared to a uniaxial stress configuration. Determination of constitutive model equations leads to better predictive capabilities for a reduced polynomial hyperelastic model with only terms related to the second invariant, I 2, of the right Cauchy-Green deformation tensor. Relevant insights were obtained on the mechanical behavior of fetal membranes. Collagen and its cross-linking were shown to determine membrane's stiffness and strength for multiaxial stress states. Their nonlinear deformation behavior characterizes the fetal membranes as I 2 material.

Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.

Chemotactic activity of cotyledons for mononuclear leukocytes related to occurrence of retained placenta in dexamethasone induced parturition in cattle.

Induction of parturition with glucocorticosteroids in cattle is used for research purposes, in diseased or injured pregnant cows, and as a management tool to time parturition. A negative side effect of induction of parturition with glucocorticosteroids is the high incidence of retained placenta that occurs after these calvings. Reaction of the maternal immune system against the 'foreign' foetal membranes contributes to the breakdown of the foetal-maternal attachment. Several studies indicate that failure of this immune assisted detachment increases the occurrence of retained placenta. We hypothesized that retained placenta occurring after induction of parturition with glucocorticosteroids is caused by failure of immune assisted detachment of the foetal membranes. The chemotactic activity of cotyledons for mononuclear leukocytes was used as a parameter to see whether immune assisted detachment of the foetal membranes had occurred. Cotyledons were collected from spontaneously calving non-retained placenta cows and from dexamethasone induced non-retained placenta and retained placenta cows. The study showed that the chemotactic activity of cotyledons for mononuclear leukocytes was lower (P < 0.001) in cotyledons obtained from retained placenta cows in which parturition was induced with dexamethasone compared to the chemotactic activity of cotyledons obtained from spontaneously calving non-retained placenta cows, whereas the chemotactic activity of cotyledons obtained from induced non-retained placenta cows was not lower (P = 0.10) than the chemotactic activity of cotyledons obtained from spontaneously calving non-retained placenta cows. We concluded that induction of parturition with dexamethasone causes a failure of immune assisted detachment of the foetal membranes and the accompanying release of chemotactic factors. As a result, the chemotactic activity of cotyledons for mononuclear leukocytes is lower in induced retained placenta cows than in cotyledons from non-retained placenta cows in which successful immune assisted detachment of the foetal membranes occurs.

Distinct pathophysiologic pathways induced by in vitro infection and cigarette smoke in normal human fetal membranes.

OBJECTIVE: The purpose of this study was to document distinct pathways that are initiated by lipopolysaccharide and cigarette smoke stimulation of normal term fetal membranes. STUDY DESIGN: Fetal membranes from nonsmoking women at term, not in labor, from cesarean deliveries were placed in an organ explant system and stimulated with cigarette smoke extracts (CSEs), lipopolysaccharide, or lipopolysaccharide + CSE. Media were assayed for an interleukin (IL)-1beta, -1 receptor antagonist, -6, -8, -10, tumor necrosis factor alpha, soluble tumor necrosis factor receptors 1 and 2, and matrix metalloproteinases 1, 2, 3, 8, 9, and 12. Tissue homogenates were assayed for apoptotic markers (p53, caspase 3 activity, and cleaved poly [ADP-ribose] polymerase-1). RESULTS: Lipopolysaccharide stimulation resulted in higher cytokine and matrix metalloproteinase concentrations, whereas it was lower after CSE and CSE + lipopolysaccharide stimulations, compared with control specimens. Apoptotic factors were several folds higher after CSE or CSE + lipopolysaccharide stimulation, compared with control specimens or lipopolysaccharide stimulations. CONCLUSION: Cigarette smoke showed immunoinhibitory properties that potentially were mediated by apoptosis and lipopolysaccharide-induced proinflammatory response. This study demonstrated 2 independent pathophysiologic pathways that may alter pregnancy outcome.

Biomechanics of the fetal membrane prior to mechanical failure: review and implications.

Annually, premature birth is a major public health problem accounting for over 13,000 deaths and 30,000 surviving infants with life-long morbidity. Preterm premature rupture of the membranes is the initiating event leading to preterm birth of 40% of these premature infants. Fetal membrane (FM) rupture is a catastrophic tissue failure, a unique event in normal physiology; other tissue failures (bone breaks, aneurism ruptures) are pathological processes. The mechanisms which cause FM failure and thereby rupture are not understood. A full understanding of FM failure process requires a complete characterization of structural and biomechanical behavior at near/full term under sub-failure (forces well below that which induce rupture) and failure conditions as well as elucidating the biological factors which modulate its failure. The relatively, highly loaded state of the FM in vivo may also facilitate its susceptibility to enzymatic degradation, which was shown to be augmented with increased load in collagenous tissues. Indeed, this last observation may help to provide the link between biomechanical degradation and premature mechanical failure in the FM. This integrated approach will further the understanding of this unique physiological event and thereby provide insight into how to anticipate and when appropriate, intervene to prevent preterm FM rupture.

[Fetal compensatory and abnormal reactions in fetoplacental insufficiency].

The review of the data available in the literature traces the pathogenetic association of chronic fetoplacental insufficiency (CFPI) with the mechanisms responsible for cardioplacental circulation, hypoxic encephalopathy, and neonatal respiratory distress syndrome. Infectious abnormalities of the fetal membranes, placenta, and umbilical cord are of combined pathogenic importance in cases of CFPI.

Differential expression of microRNAs with progression of gestation and inflammation in the human chorioamniotic membranes.

OBJECTIVE: The aim of this study was to identify differential expression of microRNAs (miRNAs) in chorioamniotic membranes with advancing gestation, labor, and inflammation. STUDY DESIGN: Expression profiles of 157 miRNAs in the chorioamniotic membranes were obtained from patients in the following groups: 1) term not in labor (n = 10); 2) term in labor (n = 10); 3) preterm labor with histologic chorioamionitis (n = 9); and 4) without histologic chorioamnionitis (n = 10). RESULTS: More than 95% of the miRNAs screened were expressed. Gestational age-dependent changes in expression were observed for 13 miRNAs. No differences in miRNA expression were observed between women without labor and women in labor. Membranes with chorioamnionitis displayed increased expression of miR-223 and miR-338. Gene Ontology analysis of genes targeted by differentially expressed miRNAs revealed enrichment for specific biological process categories. CONCLUSION: Chorioamniotic membranes with advancing gestational age and chorioamnionitis are associated with the differential expression of a subset of miRNAs.

The role of matrix degrading enzymes and apoptosis in rupture of membranes.

Prematurity is the third leading cause of perinatal death, and preterm premature rupture of the membranes (pPROM) is associated with approximately 20-50% of all preterm births. The etiologic factors described for pPROM and preterm labor (PTL) are the same, although the clinical presentation (pPROM vs PTL) differs among patients. The reason for this disparity is unknown and poses a therapeutic dilemma. Several etiologic factors have been described for PTL and pPROM. PTL and pPROM are associated with overwhelming host inflammatory response. Many of these pro-inflammatory factors (inflammatory cytokine release) are common in both conditions; however, the clinical presentation differs. The objective of this review is to explain the differential expression pattern of matrix metalloproteinases (MMPs) and pro-apoptotic elements in human fetal membranes in pPROM and PTL and how they interact to present different clinical outcomes during pregnancy.

Mechanical failure of human fetal membrane tissues.

Mechanical integrity of the chorioamnion membrane, and the component chorion and amnion layers, was assessed with biaxial puncture testing. Fetal membranes were obtained from term placentas following labored natural delivery or scheduled cesarean section. Preterm specimens were obtained from deliveries prior to 37 weeks gestation. Dividing and peripheral membranes were obtained from multiple gestation pregnancies. Specimens were gripped between parallel plates with circular openings and loaded with an instrumented, hand-held blunt probe until rupture occurred. Peak force was recorded and rupture sites were examined. Defects in multi-layered membranes differed in both size and shape in the individual layers. Compared with chorion and whole chorioamnion, amnion was more mechanically sensitive to different obstetrical conditions. Amnion varied in response at different physical locations within the same patient. Membrane and component puncture force data were used to calculate biaxial failure strength. Membrane stresses arising from amniotic fluid pressure were computed as a function of gestational age, and compared to membrane strength to examine the criterion for membrane failure in vivo. Possible mechanical conditions for preterm membrane rupture were examined.

Comparison of two protocols for the treatment of retained fetal membranes in dairy cattle.

Two protocols for the treatment of retained fetal membranes in dairy cattle were evaluated in a field trial. Cows that retained the fetal membranes for more than 12h were assigned to two treatment groups in an alternating order. In both groups rectal temperature was measured daily for 10 days after enrollment. In Group 1 (n=35) cows with a rectal temperature >or=39.5 degrees C received a systemic antibiotic treatment with 600mg ceftiofur intramuscularly on three consecutive days. No manual removal of the fetal membranes or intrauterine treatment was conducted. In case of elevated temperature of >or=39.5 degrees C on Day 3 treatment was conducted for another 2 days. In Group 2 (n=35) cows received a local antibiotic treatment (2500 mg ampicillin, 2500 mg cloxacillin) and an attempt was made to remove the fetal membranes manually. In case of a rectal temperature >or=39.5 degrees C 6000 mg of ampicillin were administered intramuscularly. Treatment was repeated on three consecutive days. If temperature did not decrease below 39.5 degrees C systemic treatment was extended for another 2 days. During 10 days of observation 33 and 34 cows showed fever, i.e. a body temperature >or=39.5 degrees C in Groups 1 and 2, respectively (94.3 versus 97.1%). The proportion of cows considered as cured (temperature <39.5 degrees C on Day 10 after enrollment) was 65.7 and 68.6% in Groups 1 and 2, respectively. All cows showed signs of chronic inflammation of the genital tract on Day 14 after calving. Within 4 weeks postpartum three (8.6%) and four (11.4%) cows were culled in Groups 1 and 2, respectively. Days to first service and days open did not differ significantly between the groups. Proportion of cows pregnant on Day 200 postpartum was 71.4 and 54.3% for Groups 1 and 2, respectively (P>0.05). Results indicate that treatment of retained fetal membranes without intrauterine manipulation and treatment can be as effective as conventional treatment including detachment and local antibiotic treatment.

HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma.

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.

Factors influencing the flow rate through a surgical defect in human fetal membranes.

OBJECTIVES: In order to determine factors influencing the flow rate trough a created defect in human fetal membranes, an ex vivo set-up was used with fetal membranes collected from patients undergoing Caesarean section at term. METHODS: The membranes were secured at the bottom of a plastic tube and traumatised with needles ranging from 14-26 Gauges (Ga), under a hydrostatic pressure of 10 to 20 cm H(2)O and an angle of 45 degrees or 90 degrees. The column was filled with amniotic fluid or Hartmann's solution. The duration of the puncture was 1 s or the time it takes to aspirate 10 ml through the needle. The flow rate through the defect in the fetal membranes and size of the defect were measured. RESULTS: The flow rate and defect size increased with increasing diameter of the needle. Increasing the pressure in the column resulted in a significant linear increase in the flow rate. Replacing the saline solution with amniotic fluid did not result in significant changes in the measured flow rates, except for the small needle size (24 Ga). Increasing the duration of the puncture did not result in increased flow rates, except for small needle size (24 Ga). CONCLUSION: These experiments suggest that needle diameter, angle of needle insertion, duration of the procedure, amniotic fluid pressure and composition could influence the incidence of amniotic fluid leakage following amniocentesis.