Circulating SPINT1 is a biomarker of pregnancies with poor placental function and fetal growth restriction

Funder: RANZCOG Research Foundation (RANZCOG); doi: https://doi.org/10.13039/501100001104Funder: The Stillbirth FoundationFunder: Tommy's; doi: https://doi.org/10.13039/501100009324Funder: National Institute Health Research Manchester Academic Health Science CentreFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268Funder: Centre for Trophoblast ResearchAbstract: Placental insufficiency can cause fetal growth restriction and stillbirth. There are no reliable screening tests for placental insufficiency, especially near-term gestation when the risk of stillbirth rises. Here we show a strong association between low circulating plasma serine peptidase inhibitor Kunitz type-1 (SPINT1) concentrations at 36 weeks’ gestation and low birthweight, an indicator of placental insufficiency. We generate a 4-tier risk model based on SPINT1 concentrations, where the highest risk tier has approximately a 2-5 fold risk of birthing neonates with birthweights under the 3rd, 5th, 10th and 20th centiles, whereas the lowest risk tier has a 0-0.3 fold risk. Low SPINT1 is associated with antenatal ultrasound and neonatal anthropomorphic indicators of placental insufficiency. We validate the association between low circulating SPINT1 and placental insufficiency in two other cohorts. Low circulating SPINT1 is a marker of placental insufficiency and may identify pregnancies with an elevated risk of stillbirth

Changes in Brain Metallome/Metabolome Pattern due to a Single i.v. Injection of Manganese in Rats.

Exposure to high concentrations of Manganese (Mn) is known to potentially induce an accumulation in the brain, leading to a Parkinson related disease, called manganism. Versatile mechanisms of Mn-induced brain injury are discussed, with inactivation of mitochondrial defense against oxidative stress being a major one. So far, studies indicate that the main Mn-species entering the brain are low molecular mass (LMM) compounds such as Mn-citrate. Applying a single low dose MnCl2 injection in rats, we observed alterations in Mn-species pattern within the brain by analysis of aqueous brain extracts by size-exclusion chromatography--inductively coupled plasma mass spectrometry (SEC-ICP-MS). Additionally, electrospray ionization--ion cyclotron resonance-Fourier transform-mass spectrometry (ESI-ICR/FT-MS) measurement of methanolic brain extracts revealed a comprehensive analysis of changes in brain metabolisms after the single MnCl2 injection. Major alterations were observed for amino acid, fatty acid, glutathione, glucose and purine/pyrimidine metabolism. The power of this metabolomic approach is the broad and detailed overview of affected brain metabolisms. We also correlated results from the metallomic investigations (Mn concentrations and Mn-species in brain) with the findings from metabolomics. This strategy might help to unravel the role of different Mn-species during Mn-induced alterations in brain metabolism

SEC chromatogram of a brain extract with peak alignment.

<p>The figure shows exemplarily a SEC chromatogram of one brain extract. SEC fractions were determined according to retention times of respective Mn standards as shown by colored peaks. The final peak alignment and area calculations were carried out by application of PeakFit^TM software (green line, generated/aligned chromatogram).</p

Correlation analysis of different variables from metallomic approach with brain metabolites categorized to brain metabolisms as shown in Fig 4.

<p>Pearsons´ correlations were calculated for total Mn, Mn-species, total Fe as well as Fe(II)/(III) with detected brain metabolites for amino acid (A) and fatty acid (B) metabolism. Only the significant correlations are shown, represented as violet (positive correlation) or brown (negative correlation) circles. The different circle size shows the strength of correlation (the bigger the stronger).</p

Data elaboration from ESI-FT-ICR-MS measurement.

<p>(A) Score Scatter Plot of OPLS-DA of detected metabolites by ESI-ICR/FT-MS revealed a good separation between control (Co) and Mn exposed group (+Mn). (B) Distribution of VIP values (variables important in projection) of metabolites from OPLS-DA: metabolites with values ≥1.50 were chosen to be important for group separation (important metabolites, in grey). (C) Annotation of detected masses by MassTRIX webserver revealed in total 1332 known masses. According to multivariate analysis, 178 of the annotated/known metabolites were determined as important metabolites with 117 metabolites being characteristic for the control group and 61 metabolites being characteristic for +Mn group.</p

HCA of Mn-species dependent on brain metabolites.

<p>HCA was carried out on data from Mn-speciation to observe clustering behavior in relation to changes of brain metabolites (the list of included metabolites can be found in Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138270#pone.0138270.s002" target="_blank">S2 File</a>). Fractions A and D, E and F as well as B and C were found to cluster together in subgroups.</p

Changes in major brain metabolisms.

<p>(A) Alterations in metabolites of purine and pyrimidine metabolism. (B) Alterations in amino acid metabolism. (C) Reaction of glucose to ribose-5-phosphate for energy production with fold changes (+Mn/Co). (D) Changes in glutathione metabolism (GSH = glutathione, GSSG = glutathione disulfide, Cys-Gly = cysteinylglycine). (E) Changes in synthesis of fatty acids (15(S)-HETE = 15(S)-Hydroxyeicosatetraenoic acid, DHA = Docosahexaenoic acid, PGB1 = prostaglandinB1, g = gamma, DH = dihomo; UFA = unsaturated fatty acid). *p<0.05, **p<0.01, ***p<0.001 t-test between intensities of control and +Mn samples; arrow means that value exceeded the maximum of axis.</p

Concentrations of Mn from different species in brain extracts according to SEC-ICP-MS.

<p>(A) The figure shows by columns the respective Mn concentrations of SEC fractions A-F (MW in kDa) for control and +Mn samples as well at the fold-change in increase (n = 15 per group). The pie charts show the percentage of Mn in each group (HMM, LMM or inorganic Mn) compared to total Mn in the aqueous brain extracts. A slight decrease in brain HMM was accompanied by an increase of Mn at LMM carriers in Mn-exposed animals. (B) Interaction plot from two-way ANOVA of results from Mn-speciation. Fractions C, D, and E were shown to have significant effects compared to the control group, which is in line with the findings in the fold-changes from t-test. *p<0.05, **p<0.01, ***p<0.001.</p

Concentrations of Mn in brain extracts, pellets, and total brain as well as extraction efficiency (EE).

<p>Total Mn concentration in brain was elevated (+19%) due to the injection of MnCl₂ compared to control. Extraction efficiency was high enough to use the extracts for Mn-speciation.</p><p>*p<0.05,</p><p>***p<0.001.</p><p>Concentrations of Mn in brain extracts, pellets, and total brain as well as extraction efficiency (EE).</p