Vitamin D supplementation during pregnancy inhibits the activation of fetal membrane NF-κB pathway.

OBJECTIVE: Nuclear Factor-κB (NF-κB) is an important member of the basic cellular inflammatory pathway that regulates inflammation and apoptosis in fetal membranes. Vitamin D (VD) exerts its anti-inflammatory and immunomodulatory effects via the NF-κB pathway. This study was designed to investigate amniotic fluid NF-κB levels in pregnant women undergoing VD replacement therapy. PATIENTS AND METHODS: Sixty patients who received antenatal vitamin D supplementation from the 14th week of pregnancy until delivery were included in the study. Participants were selected among those whose serum vitamin D levels were compatible with insufficiency (20-30 ng/mL), according to the Endocrine Society proposal. Participants were divided into three groups with 20 patients in each group and one of the cholecalciferol or placebo treatments was given. Patients in Group 1 were given 500 IU/day of cholecalciferol, while patients in Group 2 were given 1000 IU/day of cholecalciferol. Patients in group 3 were not given cholecalciferol treatment (placebo). Patients in all groups underwent elective cesarean section. Amniotic fluid samples were collected after the fetal membranes were cut and before the fetal parts were manually removed. RESULTS: The amniotic fluid NF-κB level of the control group who did not receive VD replacement was 9.33±2.02 ng/mL. The amniotic fluid NF-κB level of the 500 IU/day VD replacement group was found to be 6.12±1.23 ng/mL. Compared to the control group, NF-κB levels of pregnant women given 500 IU/day VD replacement were significantly lower (9.33±2.02 ng/mL vs. 6.12±1.23 ng/mL, p<0.03). The amniotic fluid NF-κB level of the 1000 IU/day VD replacement group was found to be 3.09±0.44 ng/mL. Compared to the control group, amniotic fluid NF-κB levels of pregnant women given 1000 IU/day VD replacement were significantly lower (9.33±2.02 ng/mL vs. 3.09±0.44 ng/mL, p<0.01). When the VD replacement groups were compared among themselves, the amniotic fluid NF-κB level decreased approximately twice as much in the 1000 IU/day replacement group compared to the 500 IU/day replacement group (3.09±0.44 ng/mL vs. 6.12±1.23 ng/mL, p<0.01). A negative correlation was found between amniotic fluid NF-κB level and VD dose (r=-0.789, p<0.04). CONCLUSIONS: The present study showed for the first time that amniotic fluid NF-κB levels decreased in pregnant women who underwent VD replacement dose dependent manner.

Potential sealing and repair of human FM defects after trauma with peptide amphiphiles and Cx43 antisense.

OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.

The management of paediatric burns with Burns and Wounds ointment and burdock leaves: a case series.

OBJECTIVE: In the Amish community, natural therapies, such as Burns and Wounds (B&W) ointment and burdock leaves, are preferred over modern medicine when treating burn wounds. The primary aim of this case series is to highlight the use and clinical outcomes of this treatment for paediatric Amish patients. METHOD: At the a paediatric burn centre, two patients were treated with B&W ointment and burdock leaves. The first patient was 11 months old with 17% total body surface area (TBSA) partial and full-thickness scald burns to her lower extremities. The second patient was 24 months old with 20% TBSA partial-thickness scald burns to the torso, bilateral upper extremities, neck and chin. RESULTS: Soon after presentation to the hospital, both patients developed positive wound cultures and required cessation of ointment and burdock leaf therapy. Both patients ultimately underwent surgical interventions. CONCLUSION: Managing burn wounds with B&W ointment and burdock leaves should be considered as an additional option for wound care in select cases. However, the efficacy of this therapy is limited and standard-of-care modern medical burn treatments should remain an option for these patients. It is critically important to build a mutually respectful relationship with Amish patients' community leaders, as this allows open communication and collaboration in patient care and increases the likelihood that Amish guardians will bring their children to a hospital when necessary.

In vitro selenium supplementation suppresses key mediators involved in myometrial activation and rupture of fetal membranes.

Spontaneous preterm birth, which can affect up to 20% of all pregnancies, is the greatest contributor to perinatal morbidity and mortality. Infection is the leading pathological cause of spontaneous preterm birth. Infection activates the maternal immune system, resulting in the upregulation of pro-inflammatory and pro-labor mediators that activate myometrial contractions and rupture of fetal membranes. Anti-inflammatory agents therefore have the potential for the prevention of spontaneous preterm birth. Selenium, an essential micronutrient, has been shown to be a potent anti-inflammatory regulator. Notably, clinical and epidemiological studies have suggested a link between selenium and preterm birth. Thus, the aim of this study was to assess the effect of selenite (an inorganic form of selenium) on the expression of pro-inflammatory and pro-labor mediators in human gestational tissues. Human fetal membranes and myometrium were pre-incubated with or without selenite before incubation with the bacterial product lipopolysaccharide (LPS) to stimulate inflammation associated with preterm birth. Selenite blocked LPS-induced expression of pro-inflammatory cytokines and chemokines and enzymes involved in remodelling of myometrium and degradation of fetal membranes. Of note, selenite also suppressed myometrial activation induced by inflammation as evidenced by a decrease in LPS-induced prostaglandin signalling and myometrial cell contractility. These effects of selenite were mediated by the MAPK protein ERK as selenite blunted LPS induced activation of ERK. In conclusion, selenite suppresses key mediators involved in inflammation induced activation of mediators involved in active labor in human fetal membranes and myometrium. These findings support recent clinical studies demonstrating selenium supplementation is associated with decreased incidence of spontaneous preterm birth.

Effect of n-3 PUFA-rich fish oil supplementation during late gestation on kidding, uterine involution and resumption of follicular activity in goat.

We have shown that dietary supplementation of n-3 polyunsaturated fatty acid (n-3 PUFA)-rich fish oil (FO) around the breeding time improved the utero-ovarian functions in the goat. Here, we investigated the effect of FO supplementation during the periparturient period on serum n-3 PUFA, prostaglandin F2α metabolite (PGFM), placental expulsion, uterine involution, resumption of oestrus and neonatal vigour. Rohilkhandi goat in advanced gestation (n = 16) was divided into two equal groups. One group was supplemented with FO containing 26% n-3 long-chain PUFA at the rate of 156 mg per kg body weight, while the control group was fed isocaloric palm oil (PO) from -3 to +3 week of kidding. Dietary FO increased serum concentration of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by 7.3- and 6.6-fold, respectively, after 6 weeks of supplementation. Goats in FO group expelled the foetal membranes 99.1 min earlier (p < .01) than those of PO group. Further, dietary FO significantly decreased the serum PGFM on day 7 post-partum. However, no difference was found on uterine involution, which was complete by day 20 post-partum in either group. Resumption of follicular activity by day 5 post-partum was 87.5% in the FO as compared to 25% in the PO group (p < .05). Similarly, occurrence of behavioural oestrus by day 90 post-partum was 57.1% in goats of the FO group while none of does was in the PO group (p < .01) expressed oestrus. It was concluded that feeding FO-rich diet during -3 to +3 weeks of kidding decreased the PGFM till day 7 post-partum, hastened the expulsion of foetal membranes and reduced the time from kidding to first post-partum oestrus in Rohilkhandi does.

Potent anti-inflammatory effects of honokiol in human fetal membranes and myometrium.

BACKGROUND: Preterm birth is the most prominent complication attributing to poor pregnancy and neonatal outcome. Infection is most commonly implicated in preterm birth; it initiates a cascade of inflammatory events that leads to the rupture of fetal membranes and spontaneous uterine contractions. Anti-inflammatory agents may thus be a therapeutic approach to prevent the premature rupture of fetal membranes and block contractions. In non-gestational tissues, the polyphenol honokiol has been shown to possess potent anti-inflammatory properties. PURPOSE: The aim of this study was to investigate the effect of honokiol on pro-inflammatory mediators in human gestational tissues. METHODS: Fetal membranes, myometrium and freshly isolated amnion cells and primary myometrial cells were treated with honokiol in the absence or presence of the products lipopolysaccharide (LPS) and fibroblast-stimulating lipopeptide-1 (fsl-1), the viral dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) or the pro-inflammatory cytokines TNF or IL1B. A luciferase assay was used to determine the effect of honokiol on nuclear factor kappa B (NF-κB) RelA transcriptional activity. RESULTS: Honokiol significantly decreased pro-inflammatory cytokine (IL1A, IL6) and chemokine (CXCL8, CXCL1, CCL2) mRNA expression and secretion from fetal membranes (amnion and choriodecidua) and myometrium stimulated with LPS, fsl-1 or poly(I:C). In amnion cells, honokiol also significantly decreased the expression and secretion of the extracellular matrix degrading enzyme MMP9. Moreover, in myometrium, honokiol significantly suppressed the expression of the contraction associated protein PTGFR, the secretion of the uterotonic prostaglandins PGE2 and PGF2α, and blocked TNF-induced myometrial cell contractility. Finally, honokiol significantly suppressed IL1B- and TNF-induced NF-κB RelA transcriptional activity in primary amnion and myometrial cells. CONCLUSIONS: Honokiol reduced the expression of pro-inflammatory and pro-labour mediators in human amnion, choriodecidua and myometrium and that this may be facilitated through the suppression of NF-κB activation. These results indicate that the polyphenol honokiol may be a potent therapeutic for the prevention of preterm birth.

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.

Concentrations of mercury (Hg) and selenium (Se) in afterbirth and their relations with various factors.

The aim of the study was to evaluate Hg and Se concentrations and Se:Hg molar ratios in the placenta, umbilical cord and fetal membranes, and to examine the relationship between the concentrations of the elements and selected factors. The study material consisted of the placenta, umbilical cord and fetal membranes obtained from 91 healthy women from northwestern and central Poland. In our study mean Hg and Se concentrations in afterbirth were ~ 0.01 mg/kg dry weight (dw) and ≤ 0.5 mg/kg dw, respectively. Correlation analysis showed negative relationships between placenta weight and Se concentration in the placenta and umbilical cord, as well as between placenta length and Se levels in the umbilical cord. We found negative correlations between THg concentration in the placenta and birth weight and between Se concentration in the placenta and umbilical cord and the morphological parameters of the placenta. Furthermore, we noted new types of interactions in specific parts of the afterbirth. In our study, Se:THg molar ratios ranged from 5 to 626; these values indicate protection against Hg toxicity.

Restored in vivo-like membrane lipidomics positively influence in vitro features of cultured mesenchymal stromal/stem cells derived from human placenta.

BACKGROUND: The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). METHODS: Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. RESULTS: A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. CONCLUSIONS: Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients.

All-trans retinoic acid promotes wound healing of primary amniocytes through the induction of LOXL4, a member of the lysyl oxidase family.

Thirty percent of preterm births directly result from preterm premature rupture of fetal membranes (PPROM). Clinical management currently proposes using a collagen plug to mechanically stop loss of amniotic fluid. Vitamin A and its active metabolite (retinoic acid) have well-known pro-healing properties and could thus make good candidates as a proposable adjuvant to this mechanical approach. Here we investigate the molecular mechanisms involved in the pro-healing properties of all-trans retinoic acid (atRA) in fetal membranes via an approach using an in vitro primary amniocyte wound model and transcriptomics. The results demonstrate that atRA promotes migration in primary amniocytes, improving wound healing in vitro by up to 90%. This effect is mediated by the induction of LOXL4, which plays a crucial role in the dynamics of the extracellular matrix by regulating collagen reticulation. This new insight into how atRA exerts its pro-healing properties prompts us to propose using atRA as a candidate strategy to help prevent future PPROM.

Effect of oleic acid supplementation on prostaglandin production in maternal endometrial and fetal allantochorion cells isolated from late gestation ewes.

INTRODUCTION: Elevated circulating non-esterified fatty acids including oleic acid (OA) are associated with many pregnancy related complications. Prostaglandins (PGs) play crucial roles during parturition. We investigated the effect of OA supplementation on PG production using an in vitro model of ovine placenta. METHODS: Maternal endometrium (ME) and fetal allantochorion (FC) were collected in late pregnancy (day 135). Confluent cells were cultured in serum-free medium supplemented with 0, 20 or 100 µM OA and challenged with control medium, oxytocin (OT, 250 nM), lipopolysaccharide (LPS, 0.1 µg/ml) or dexamethasone (DEX, 5 µM). Spent medium was harvested at 2 and 24 h after challenge for quantifying PGs. RESULTS: In ME cells OA increased PGE2 production moderately but attenuated PGF2α production leading to a doubling of the PGE2:PGF2α ratio (E:F) (P < 0.01). Without OA, both OT and LPS stimulated PG production for about 3-fold (P < 0.01) without changing the E:F ratio. In the ME cells challenged with OT, OA decreased both PGE2 and PGF2α production by up to 70% (P < 0.01) whereas in LPS treated cells OA increased the E:F ratio. In FC cells PGE2 production at 2 h was stimulated by 100 µM OA (P < 0.05). In these cells LPS caused a 3-fold increase in PGE2 (P < 0.01), an effect which was completely inhibited by DEX. DISCUSSION: OA supplementation favours basal PGE2 production in both ME and FC. In ME OA increased E:F ratios and antagonized the stimulatory effect of OT on PG production. This suggests that raised circulating OA may affect both the initiation and progression of parturition.

Effect of injectable vitamin E on incidence of retained fetal membranes and reproductive performance of dairy cows.

The objectives were to evaluate the effects of injectable vitamin E during the last 3 wk prepartum on the incidence of retained fetal membranes (RFM) and reproductive performance. Dairy cows (n=890), 390 Holsteins (132 nulliparous and 258 parous) and 500 crossbred Holstein × Gyr (199 nulliparous and 301 parous), from 3 dairy farms in Brazil were assigned to the study. In all 3 farms, from October to March, prepartum cows grazed tropical grasses and received 2 kg/d of a mixture of finely ground corn, soybean meal, and minerals and vitamins. From April to September prepartum cows received a total mixed ration composed of corn silage, finely ground corn, soybean meal, and minerals and vitamins. During the prepartum period, cows were fed 280 (farm 1), 390 (farm 2), and 480 IU (farm 3) of supplemental vitamin E per day, and throughout postpartum, cows were fed 370 (farm 1), 500 (farm 2), and 600 (farm 3) IU of supplemental vitamin E. Within each farm, cows were randomly assigned to remain as untreated controls or to receive 3 i.m. injections of 1,000 IU each of dl-α-tocopherol administered at 19.2 ± 4.3, 12.9 ± 3.3, and 6.2 ± 2.9 d before calving (VitE). Blood was sampled from 141 cows immediately before enrollment to determine the α-tocopherol and cholesterol statuses. Blood was also sampled and analyzed for concentrations of cortisol and nonesterified fatty acids in the last 3 wk of gestation. The serum concentration of α-tocopherol or α-tocopherol:cholesterol ratio did not differ between treatments and averaged 2.97 ± 0.10 µg/mL and 4.46 ± 0.16 × 10(-3), respectively. In total, 53.2% of the cows had an inadequate concentration of serum α-tocopherol based on the 3.0 µg/mL cut-off for adequacy. The risk of RFM decreased as serum α-tocopherol increased. Milk production did not differ between controls and VitE cows. Treatment with injectable α-tocopherol decreased RFM from 20.1 to 13.5%, decreased incidence of stillbirth from 14.9 to 6.8%, and tended to decrease death by 200 d postpartum. VitE cows tended to have improved pregnancy per insemination at first AI (36.7 vs. 30.1%) because of decreased pregnancy loss from 31 to 62 d of gestation (12.5 vs. 20.5%). Despite a similar insemination rate, VitE cows had 22% greater pregnancy rate than control cows. Cows receiving vitamin E had decreased circulating cortisol and nonesterified fatty acids around calving. In summary, when cows were fed limited amounts of supplemental vitamin E, 28 to 48% of the recommendations, prepartum supplementation with injectable α-tocopherol decreased incidence of RFM and improved reproduction.

The intrauterine treatment of the retained foetal membrane in dairy goats by ozone: novel alternative to antibiotic therapy.

One of the major post-parturient complications in dairy goats is the retention of foetal membrane (RFM), which negatively influences their health, reproductive efficacy and welfare. The aim of this study was to compare the efficiency of intrauterine either ozone (OZ) or antibiotic (AB) treatments to establish the use of OZ as a novel and potential alternative to AB therapy in does with the RFM. The study was performed on 7 herds of dairy goats (n = 563) kept in the farms in Croatia. The conception rate was 563 of 641 total matings or 87.83%. The does from selected farms were observed during early puerperium and were divided into animals without the RFM (n = 522) and with the RFM (n = 41), treated either with foam spray OZ (n = 21) or with foaming AB oxytetracycline tablets (n = 20). The does with the RFM were mated successfully and became pregnant next kidding season, regardless of the treatment applied. Treatment with OZ attained similar results to the standard AB therapy, indicating that it could be novel potential alternative therapy of the RFM in dairy goats.

Effect of silibinin in reducing inflammatory pathways in in vitro and in vivo models of infection-induced preterm birth.

Infection-induced preterm birth is the largest cause of infant death and of neurological disabilities in survivors. Silibinin, from milk thistle, exerts potent anti-inflammatory activities in non-gestational tissues. The aims of this study were to determine the effect of silibinin on pro-inflammatory mediators in (i) human fetal membranes and myometrium treated with bacterial endotoxin lipopolysaccharide (LPS) or the pro-inflammatory cytokine IL-1ß, and (ii) in preterm fetal membranes with active infection. The effect of silibinin on infection induced inflammation and brain injury in pregnant mice was also assessed. Fetal membranes and myometrium (tissue explants and primary cells) were treated with 200 µM silibinin in the presence or absence of 10 µg/ml LPS or 1 ng/ml IL-1ß. C57BL/6 mice were injected with 70 mg/kg silibinin with or without 50 µg LPS on embryonic day 16. Fetal brains were collected after 6 h. In human fetal membranes, silibinin significantly decreased LPS-stimulated expression of IL-6 and IL-8, COX-2, and prostaglandins PGE2 and PGF2α. In primary amnion and myometrial cells, silibinin also decreased IL-1ß-induced MMP-9 expression. Preterm fetal membranes with active infection treated with silibinin showed a decrease in IL-6, IL-8 and MMP-9 expression. Fetal brains from mice treated with silibinin showed a significant decrease in LPS-induced IL-8 and ninjurin, a marker of brain injury. Our study demonstrates that silibinin can reduce infection and inflammation-induced pro-labour mediators in human fetal membranes and myometrium. Excitingly, the in vivo results indicate a protective effect of silibinin on infection-induced brain injury in a mouse model of preterm birth.

Prevalence of periparturient diseases and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates.

The objectives were to characterize the prevalence of periparturient diseases and their effects on reproductive performance of dairy cows in seasonal grazing farms. A total of 957 multiparous cows in 2 farms (555 in farm A and 402 in farm B) were evaluated and diseases characterized. At calving, dystocia, twin birth, stillbirth, and retained fetal membranes were recorded and grouped as calving problems. On d 7±3 and 14±3 postpartum, cows were evaluated for metritis and on d 28±3 for clinical endometritis based on scoring of the vaginal discharge. From parturition to 30 d after artificial insemination (AI), prevalence of mastitis, lameness, and digestive and respiratory problems were recorded. For subclinical diseases, diagnosis was based on blood samples collected from 771 cows and analyzed for concentrations of Ca, nonesterified fatty acids (NEFA), and ß-hydroxybutyrate. Cows were considered as having elevated NEFA concentration if the concentration was ≥0.70 mM, subclinical ketosis if the ß-hydroxybutyrate concentration was ≥0.96 mM, and subclinical hypocalcemia if the Ca concentration was ≤2.14 mM. Ovaries were scanned on d 35±3 and 49±3 postpartum for determination of estrous cyclicity. All cows were enrolled in a timed AI program and inseminated on the first day of the breeding season: on average, 86 d postpartum. Overall, 37.5% (359/957) of the cows presented at least 1 clinical disease and 59.0% (455/771) had at least 1 subclinical health problem. Prevalence of individual diseases was 8.5% for calving problems, 5.3% for metritis, 15.0% for clinical endometritis, 13.4% for subclinical endometritis, 15.3% for mastitis, 2.5% for respiratory problems, 4.0% for digestive problems, 3.2% for lameness, 20.0% for elevated NEFA concentration, 35.4% for subclinical ketosis, and 43.3% for subclinical hypocalcemia. Clinical and subclinical diseases had additive negative effects on reproduction, delaying resumption of estrous cyclicity and reducing pregnancy per AI (P/AI). Occurrence of multiple diseases further reduced reproductive efficiency compared with a single disease. Individually, subclinical hypocalcemia, elevated NEFA concentration, metritis, and respiratory and digestive problems reduced estrous cyclicity by d 49 postpartum. Elevated NEFA concentration, calving problem, metritis, clinical and subclinical endometritis, and digestive problems reduced P/AI on d 65 after AI. Moreover, calving problems and clinical endometritis increased the risk of pregnancy loss between gestation d 30 and 65. Serum concentrations of Ca and NEFA were negatively correlated, and both were associated with prevalence of uterine diseases. In conclusion, periparturient diseases were highly prevalent in seasonally calving grazing dairies and affected cows had delayed resumption of estrous cyclicity, reduced P/AI, and increased risk of pregnancy loss.

Acupuncture and/or sweeping of the fetal membranes before induction of labor: a prospective, randomized, controlled trial.

AIMS: To evaluate the efficacy of acupuncture, and sweeping of the fetal membranes, as methods for induction of labor. METHODS: Four hundred and seven pregnant women with normal singleton pregnancies and cephalic presentations were randomized at three delivery wards in Denmark at day 290 of gestation into groups of acupuncture, sweeping, acupuncture and sweeping and controls. The primary objective was to compare the proportion of women going into labor before induction of labor at 294 days in the four groups. The secondary objective was to compare the combined groups: with and without acupuncture, and with and without sweeping of the fetal membranes. The midwives, completing the forms for the trial at labor or induction, were blinded to group assessments. RESULTS: Four hundred and seventeen women were randomized. Ten were excluded after randomization. One hundred and four women were randomized to acupuncture, 103 to sweeping of the membranes, 100 to both acupuncture and sweeping, and 100 were randomized to the control group. Comparison of the four groups demonstrated no significant difference in the number of women achieving spontaneous labor before planned induction. No difference was demonstrated by comparing the combined groups treated with acupuncture with the groups not treated with acupuncture (P=0.76). However, significantly more women went into labor before planned induction (P=0.02) in the combined groups receiving sweeping, compared with the groups not treated with sweeping. CONCLUSIONS: Acupuncture at 41+ weeks of gestation did not reduce the need for induction. The study was of a sufficient size to demonstrate, in parallel, that sweeping of the fetal membranes significantly reduced the need of induction, sparing about 15% for formal induction of labor.

Dietary phytophenols curcumin, naringenin and apigenin reduce infection-induced inflammatory and contractile pathways in human placenta, foetal membranes and myometrium.

A tenet of contemporary obstetrics is that a significant proportion of preterm births involve bacterial infection. Bacterial endotoxin induces pro-inflammatory cytokines, prostaglandins and proteases via the pro-inflammatory pathway nuclear factor-κB (NF-κB), which plays a key role in initiating uterine contractions and rupture of foetal membranes. In non-gestational tissues, the phytophenols curcumin, naringenin and apigenin exert anti-inflammatory properties via inhibition of NF-κB. The aim of this study was to determine whether these treatments regulate pro-inflammatory and pro-labour mediators in human gestational tissues. Placenta, foetal membranes and myometrium were treated with curcumin, naringenin and apigenin in the presence of lipopolysaccharide (LPS) or interleukin (IL)-1ß. In placenta and foetal membranes, all treatments significantly reduced LPS-stimulated release and gene expression of pro-inflammatory cytokines IL-6 and IL-8; placenta decreased cyclooxygenase (COX-2) mRNA expression, subsequent release of prostaglandins PGE2 and PGF2α and expression and activity of matrix-degrading enzyme matrix metalloproteinase (MMP)-9. In myometrial cells, all treatments attenuated IL-1ß-induced COX-2 expression, release of PGE2 and PGF2α and expression and activity of MMP-9. Although naringenin significantly attenuated IL-1ß-induced IL-6 and IL-8 mRNA expression and release, there was no effect of curcumin and apigenin. LPS-stimulated release of 8-isoprostane, a marker of oxidative stress, was attenuated by all treatments. NF-κB p65 DNA-binding activity was also decreased using these treatments. In conclusion, curcumin, naringenin and apigenin exert anti-inflammatory properties in human gestational tissues by inhibiting the transcriptional activity of NF-κB. Further studies should be undertaken to define a possible implication of these natural spices in the management of preterm labour and delivery.

Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids, and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis.

The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, i.m., q96h); COMBO (n = 6; administered combination therapy of CCFA, altrenogest, and pentoxifylline); UNTREAT (n = 3, no treatment). Treatment was initiated at the onset of clinical signs. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur metabolites, were measured using high-performance liquid chromatography. Maximum and minimum serum concentrations of DCA at steady state in treated mares were 2.40±0.40 µg/mL and 1.06±0.29 µg/mL, respectively. Concentration of DCA in colostrum was 1.51±0.60 µg/mL. DCA concentrations in placenta and fetal tissues were very low (median = 0.03 µg/mL) and below the minimum inhibitory concentration of relevant pathogens. DCA was not detected in amniotic fluid or foal serum. Treatment did not appear to improve foal survival (CEFT: 0/3; COMBO: 2/6; UNTREAT: 2/3). Bacteria were recovered from the uterus of most mares postpartum and from blood cultures of most foals regardless of treatment.

Increased oxidative stress in human fetal membranes overlying the cervix from term non-labouring and post labour deliveries.

Enzymatic breakdown of the collagen-rich extracellular matrix (ECM) that connects the amnion and chorion layers of the fetal membranes is one of the key events leading to rupture of membranes. Oxidant stress caused by increased formation of reactive oxygen species and/or reduced antioxidant capacity may predispose to membrane rupture, a major cause of preterm birth. The aim of this study was to determine the effect of human labour and supracervical (SC) apposition on antioxidant enzymes and 8-isoprostane (a marker of lipid peroxidation). To determine the effect of human labour on oxidative stress status, fetal membranes from the SC site (SCS) were collected from women at term Caesarean section (no labour), and from the site of membrane rupture (SOR) after spontaneous labour onset and delivery (post labour). To determine the effect of SC apposition on oxidative stress status, amnion was collected from the SCS and a distal site (DS) in women at term Caesarean section in the absence of labour. The release of 8-isoprostane was significantly higher in amnion from the SCS compared to DS, and in fetal membranes from the SOR compared to the SCS. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were lower in amnion from the SC compared to DS. SOD gene expression and enzyme activity were lower in fetal membranes after labour. There was no difference in expression or activity in catalase, GPx and glutathione reductase (GSR) between no labour and post labour fetal membranes. In primary amnion cells, SOD supplementation significantly augmented IL-1ß induced MMP-9 expression and activity. In summary, non-labouring SC fetal membranes are characterised by reduced antioxidant enzyme activity when compared to distal membranes, and, as such, may be more susceptible to oxidative damage and thus membrane rupture.

The effects of homocysteine and folic acid on angiogenesis and VEGF expression during chicken vascular development.

Homocysteine (Hcy) has been implicated in the development of cardiovascular developmental defects. Additionally, in experimental studies, vasculotoxic properties of Hcy have been described. Although Hcy has been identified as a vascular pathogen, little is known about the direct effects Hcy exerts during early embryonic vascular development. Angiogenesis is a critical process involved in embryo survival and development. There are limited studies on the effects of Hcy on early embryonic vasculogenesis and angiogenesis. Folic acid (FA) is a B vitamin essential in embryo development, and FA supplementation may lead to reduced Hcy levels. Therefore, the purpose of our study was to explore the effects of Hcy and FA on early embryonic vascular development. Embryonic day (E) 3.5 chicken embryos were treated with a sham, Hcy or FA solution. We developed a computational program for systematic analysis of microscopic images obtained from the extra embryonic vascular beds. These results were combined with real-time PCR data on the expression of VEGF-A and its receptor in these vascular beds. Our data show that Hcy exposure inhibits early vascular development, displayed by a significant reduction of vessel area and altered composition of the vascular beds. Vascular beds of Hcy embryos for the greater part consisted of vessels of the smallest diameters, compared to middle size vessels in control and FA embryos. Hcy also reduced expression of VEGF-A and VEGFR-2. No significant effects of FA were found. We conclude that Hcy exposure causes impaired early extra embryonic vascular development, shown by altered composition of the vascular beds as well as reduced expression of VEGF-A and VEGFR-2. These effects of Hcy, and the consecutive cascade of events, may be involved in the development of cardiovascular developmental defects.