Reproducibility and usability assessment of the novel Fine Birth device for threatened preterm labor diagnosis.

BACKGROUND: Preterm delivery is considered the leading cause of mortality worldwide in children under 5 years old. Approximately 45 million pregnant women are hospitalized yearly for threatened preterm labor. However, only 50% of pregnancies complicated by threatened preterm labor end in delivery before the estimated date, classifying the rest as false threatened preterm labor. The ability of current diagnostic methods to predict threatened preterm labor is low (low positive predictive value), ranging between 8% and 30%. This highlights the need for a solution that accurately detects and differentiates between false and real threatened preterm labors in women who attend obstetrical clinics and hospital emergency departments with delivery symptoms. OBJECTIVE: Primarily, this aimed to assess the reproducibility and usability of a novel medical device, the Fine Birth, aimed at accurately diagnosing threatened preterm labor through the objective quantification of pregnant women's cervical consistency. Secondarily, this study aimed to evaluate the effect of training and the incorporation of a lateral microcamera on the device's reliability and usability outcomes. STUDY DESIGN: A total of 77 singleton pregnant women were recruited during their follow-up visits to the obstetrical and gynecologic departments at 5 Spanish hospitals. The eligibility criteria included pregnant women aged ≥18 years; women with a normal fetus and uncomplicated pregnancy; women without prolapse of membranes, uterine anomalies, previous cervical surgery, or latex allergy; and women signing the informed written consent. Cervical tissue stiffness was assessed using the Fine Birth device, whose technology is based on the propagation of torsional waves through the studied tissue. Cervical consistency measurements were taken for each woman until obtaining 2 valid measurements by 2 different operators. The intraobserver and interobserver reproducibilities of the Fine Birth measurements were assessed using the intraclass correlation coefficients with a 95% confidence interval and the Fisher test P value. The usability was evaluated on the basis of the clinicians' and participants' feedback. RESULTS: There was good intraobserver reproducibility (intraclass correlation coefficient, 0.88; 95% confidence interval, 0.84-0.95; Fisher test P value<.05). As the results obtained for the interobserver reproducibility did not reach the desired acceptable values (intraclass correlation coefficient of <0.75), a lateral microcamera was added to the Fine Birth intravaginal probe, and the operators involved in the clinical investigation received the corresponding training with the modified device. The analysis of 16 additional subjects demonstrated excellent interobserver reproducibility (intraclass correlation coefficient, 0.93; 95% confidence interval, 0.78-0.97) and an improvement after the intervention (P<.0001). CONCLUSION: The robust reproducibility and usability results obtained after the insertion of a lateral microcamera and the corresponding training make the Fine Birth a promising novel device to objectively quantify the patient's cervical consistency, diagnose threatened preterm labor, and, thus, predict the risk of spontaneous preterm birth. Further research is needed to demonstrate the clinical utility of the device.

Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia.

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2ß1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbß3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbß3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbß3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.

Down Regulation of the Munc18b-syntaxin-11 Complex and ß1-tubulin Impairs Secretion and Spreading in Neonatal Platelets.

Neonatal platelets are hyporeactive and show impaired agonist-induced secretion despite no obvious abnormalities in their granules. Here, we examined, for the first time, the ultrastructure of neonatal and adult platelets following agonist activation. Under resting conditions, neonatal and adult platelets appeared ultrastructurally identical. Following agonist stimulation, however, noticeable degranulation occurred in adult platelets, while granules in neonatal platelets remained clearly visible and apparently unable to centralize or fuse. To investigate the underlying mechanisms, we first examined the expression levels of the main SNARE proteins, which mediate the membrane fusion events required for exocytosis. Neonatal platelets showed significantly reduced levels of syntaxin-11 and its regulator, Munc18b. Since granule centralization depends on contraction of the microtubule ring, we also examined the expression of its main component, ß1-tubulin. Noteworthy, we found decreased TUBB1 mRNA and protein levels in neonatal platelets, while TUBB2A and TUBB isoforms were overexpressed, partially compensating for that deficiency. Finally, supporting the functional consequences of defective exocytosis, adhesion kinetic assays, performed in plasma-free medium, demonstrated delayed adhesion and spreading of neonatal platelets. This is the first report showing marked reductions of syntaxin-11-Munc18b complex and ß1-tubulin in neonatal platelets, indicating that these proteins, required for platelet degranulation, are developmentally regulated.

Comprehensive comparison of neonate and adult human platelet transcriptomes.

Understanding the underlying mechanisms of the well-substantiated platelet hyporeactivity in neonates is of interest given their implications for the clinical management of newborns, a population at higher bleeding risk than adults (especially sick and preterm infants), as well as for gaining insight into the regulatory mechanisms of platelet biology. Transcriptome analysis is useful in identifying mRNA signatures affecting platelet function. However, human fetal/neonatal platelet transcriptome analysis has never before been reported. We have used mRNA expression array for the first time to compare platelet transcriptome changes during development. Microarray analysis was performed in pure platelet RNA obtained from adult and cord blood, using the same platform in two independent laboratories. A high correlation was obtained between array results for both adult and neonate platelet samples. There was also good agreement between results in our adult samples and outcomes previously reported in three different studies. Gene enrichment analysis showed that immunity- and platelet function-related genes are highly expressed at both developmental stages. Remarkably, 201 genes were found to be differentially expressed throughout development. In particular, neonatal platelets contain higher levels of mRNA that are associated with protein synthesis and processing, while carrying significantly lower levels of genes involved in calcium transport/metabolism and cell signaling (including GNAZ). Overall, our results point to variations in platelet transcriptome as possibly underlining the hypo-functional phenotype of neonatal platelets and provide further support for the role of platelets in cellular immune response. Better characterization of the platelet transcriptome throughout development can contribute to elucidate how transcriptome changes impact different pathological conditions.

A method for lipid droplet isolation from human placenta for further analyses in clinical trials.

We describe a method to isolate lipids droplets from human placental tissue for future lipid analyses. We collected placentas at term from healthy women (n=5) and tested three methods published for lipids droplets isolation in other tissues. Only one of the methods, when modified, isolated lipids droplets from placental tissue, whereas all three methods allowed lipids droplets isolation from rat liver (control tissue) and separation of lipids droplets from blood contamination of the tissue. The placental lipids droplets layer was characterized by the presence of adipophilin while no N+ /K+-ATPase as plasma membrane contamination was detected. Intraplacental triglyceride content showed a high coefficient of variation (~42%), whereas for cholesterol and phospholipids this was lower. One method was effective for isolation of placental lipids droplets. It is necessary to collect a pool of placental tissue pieces for placental lipids droplets analyses. Freezing in liquid nitrogen is recommended.

Influence of gestational diabetes on circadian rhythms of children and their association with fetal adiposity.

OBJECTIVE: To analyse the circadian rhythm maturation of temperature, activity and sleep during the first year of life in offspring of diabetic mothers (ODM) and its relationship with obesity markers. METHODS: A prospective analysis of the children of 63 pregnant women (23 controls, 21 gestational diabetes mellitus (GDM) controlled with diet and 19 GDM with insulin). Fetal abdominal circumference was evaluated ecographically during gestation. Skin temperature and rest-activity rhythms were monitored for 3 consecutive days in children at 15 days and 1, 3 and 6 months. Anthropometrical parameters of the children were evaluated during the first year of life. RESULTS: Children from the GDM groups tended to higher fetal abdominal circumference z-score than controls at the beginning of the last trimester (p = 0.077) and at delivery (p = 0.078). Mean skin temperature or activity was not different among the groups. The I < O sleep index pointed to increasing concordance with parental sleeping at 3 and 6 months but no significant GDM-dependent differences. However, some of the parameters that define temperature maturation and also the circadian function index from the temperature-activity variable were significantly lower at 6 months in the GDM + insulin group. Fetal abdominal circumference z-score, as a predictor of fetal adiposity, correlated negatively with parameters related to circadian rhythm maturation as the circadian/ultradian rhythm (P1 /Pult ratio). CONCLUSIONS: Fetal adiposity correlated with a worse circadian rhythm regulation in ODM. In addition, ODM insulin-treated showed a disturbed pattern of the circadian function index of temperature activity at 6 months of age.

Placental transfer of fatty acids and fetal implications.

Considerable amounts of long-chain polyunsaturated fatty acids (LC-PUFAs), particularly arachidonic acid and docosahexaenoic acid (DHA, 22:6n-3), are deposited in fetal tissues during pregnancy; and this process is facilitated by placental delivery. Nevertheless, the mechanisms involved in LC-PUFA placental transfer remain unclear. Stable isotope techniques have been used to study human placental fatty acid transfer in vivo. These studies have shown a significantly higher ratio of (13)C-DHA in cord to maternal plasma compared with other fatty acids, which reflects a higher placental DHA transfer. In addition, a selective DHA accumulation in placental tissue, relative to other fatty acids, has been reported. The materno-fetal transfer of fatty acids is a slow process that requires ≥12 h. A high incorporation of dietary (13)C-DHA into maternal plasma phospholipids appears to be important for placental uptake and transfer. DHA in cord blood lipids correlates with placental messenger RNA expression of fatty acid transport protein (FATP)-4, compatible with a role of FATP-4 in DHA transfer. Impaired materno-fetal LC-PUFA transport has been proposed in pregnancies complicated by abnormal placental function (eg, due to gestational diabetes mellitus or intrauterine growth restriction), which should be addressed in future studies. Given that placental DHA transfer is important for child outcomes, elucidation of its potential modulation by transport mechanisms, maternal diet, and disease appears to be important.