Danish Diabetes Birth Registry 2: a study protocol of a national prospective cohort study to monitor outcomes of pregnancies of women with pre-existing diabetes.

INTRODUCTION: Despite technological developments and intensified care, pregnancies in women with pre-existing diabetes are still considered high-risk pregnancies. The rate of adverse outcomes in pregnancies affected by diabetes in Denmark is currently unknown, and there is a limited understanding of mechanisms contributing to this elevated risk. To address these gaps, the Danish Diabetes Birth Registry 2 (DDBR2) was established. The aims of this registry are to evaluate maternal and fetal-neonatal outcomes based on 5 years cohort data, and to identify pathophysiology and risk factors associated with short-term and long-term outcomes of pregnancies in women with pre-existing diabetes. METHODS AND ANALYSIS: The DDBR2 registry is a nationwide 5-year prospective cohort with an inclusion period from February 2023 to February 2028 of pregnancies in women with all types of pre-existing diabetes and includes registry, clinical and questionnaire data and biological samples of mother-partner-child trios. Eligible families (parents age ≥18 years and sufficient proficiency in Danish or English) can participate by either (1) basic level data obtained from medical records (mother and child) and questionnaires (partner) or (2) basic level data and additional data which includes questionnaires (mother and partner) and blood samples (all). The primary maternal outcome is Hemoglobin A1c (HbA1c) levels at the end of pregnancy and the primary offspring endpoint is the birth weight SD score. The DDBR2 registry will be complemented by genetic, epigenetic and metabolomic data as well as a biobank for future research, and the cohort will be followed through data from national databases to illuminate possible mechanisms that link maternal diabetes and other parental factors to a possible increased risk of adverse long-term child outcomes. ETHICS AND DISSEMINATION: Approval from the Ethical Committee is obtained (S-20220039). Findings will be sought published in international scientific journals and shared among the participating hospitals and policymakers. TRIAL REGISTRATION NUMBER: NCT05678543.

Pre-analytical diagnostic differences despite high adherence to guidelines for gestational diabetes mellitus.

Regional variations in the prevalence of gestational diabetes mellitus (GDM) have been found across Denmark. The objectives of this exploratory survey were to evaluate adherence to the national guideline for screening and diagnosing GDM and to identify variations in pre-analytical or analytical factors, which could potentially contribute to variations in GDM prevalence across regions. In a national interview-based survey, obstetric departments and laboratories throughout Denmark handling GDM screening or diagnostic testing were invited to participate. Survey questionnaires were completed through personal interviews. In total, 21 of 22 identified obstetric departments and 44 of 45 identified laboratories participated. Adherence to guideline among obstetric departments ranged 67-100% and uniformity in laboratory procedures was high. However, the gestational age at the time of late diagnostic testing with oral glucose tolerance test (OGTT) varied considerably, with 48% (10/21) of departments testing outside the recommended 24-28 weeks' gestation. Procedural heterogeneity was most pronounced for the parts not described in current guidelines, with choice of laboratory equipment being the most diverse factor ranging 3-39% nationally. In conclusion, the overall adherence to the national guidelines was high across regions, and obstetric departments and laboratories had high uniformity in the procedures for screening and diagnosing GDM. Uniformity was generally high for procedures included in the guideline and low if not included. However, a high proportion of GDM testing was performed outside the recommended gestational window in late pregnancy, which may be a pre-analytical contributor to regional differences in GDM prevalence.

Highly Responsive Bioassay for Quantification of Glucocorticoids.

Measurement of total cortisol levels in serum samples is currently based on immunoassays or liquid chromatography-mass spectrometry (LC-MS/MS). However, measurement of bioavailable cortisol is laborious, unreliable, and inconvenient for the patient. Therefore, a new versatile assay with the ability to measure both total and bioavailable cortisol from serum represents an important supplement to the current methods. We have generated a cell-based glucocorticoid reporter assay (HEK293F-GRE). The assay was validated for cell line stability, accuracy by dilution, precision, repeatability, reproducibility, and specificity. Additionally, the assay was tested for measuring both total and bioavailable cortisol in serum. The assay showed linearity at five dilution levels with R2 = 0.98 and an accuracy between 0.8 and 1.2. Precision (CV < 20%) was validated down to 3-6 nM dexamethasone, and estimation of the total cortisol concentration was comparable to cortisol immunoassay and LC-MS/MS in most serum samples. Moreover, the assay estimated the bioavailable cortisol fraction in serum samples to a level that agreed with the literature. The HEK293F-GRE assay holds the potential to be a complementary method for estimating cortisol in clinical practice. The ability to quantify bioavailable cortisol directly from serum samples is alluring and provides an opportunity for monitored and personal dose regimens of exogenous glucocorticoids.

Applying WHO2013 diagnostic criteria for gestational diabetes mellitus reveals currently untreated women at increased risk.

AIMS: To estimate the prevalence of gestational diabetes mellitus (GDM) in a Danish cohort comparing the current Danish versus the WHO2013 diagnostic criteria, and to evaluate adverse pregnancy outcomes among currently untreated women in the gap between the diagnostic thresholds. METHODS: Diagnostic testing was performed by a 75 g oral glucose tolerance test (OGTT) at 24-28 weeks' gestation in a cohort of pregnant women. GDM diagnosis was based on the current Danish criterion (2-h glucose ≥ 9.0 mmol/L, GDMDK) and on the WHO2013 criteria (fasting ≥ 5.1, 1 h ≥ 10.0 or 2 h glucose ≥ 8.5 mmol/L, GDMWHO2013). Currently untreated women fulfilling the WHO2013 but not the Danish diagnostic criteria were defined as New-GDM-women (GDMWHO2013-positive and GDMDK-negative). Adverse outcomes risks were calculated using logistic regression. RESULTS: OGTT was completed by 465 women at a median of 25.7 weeks' gestation. GDMDK prevalence was 2.2% (N = 10) and GDMWHO2013 21.5% (N = 100). New-GDM was present in 19.4% (N = 90), of whom 90.0% had elevated fasting glucose. Pregnancies complicated by New-GDM had higher frequencies of pregnancy-induced hypertension (13.3% vs 4.1%, p = 0.002), large-for-gestational-age infants (22.2% vs 9.9%, p = 0.004), neonatal hypoglycaemia (8.9% vs 1.9%, p = 0.004) and neonatal intensive care unit admission (16.7% vs 5.8%, p = 0.002) compared to pregnancies without GDM. CONCLUSIONS: GDM prevalence increased tenfold when applying WHO2013 criteria in a Danish population, mainly driven by higher fasting glucose levels. Untreated GDM in the gap between the current Danish and the WHO2013 diagnostic criteria resulted in higher risks of adverse pregnancy outcomes.

Non-cardiac causes of troponin elevation.

With the increased sensitivity of the newest cardiac troponin assays, the risk of false positive cardiac troponin measurements has also increased. As summarised in this review, there are multiple possible causes of cardiac troponin release including several non-cardiac illnesses, particularly kidney disease. Further, there is a risk of analytical interference in which case repeated measurements with a different assay is a good tool. When there is a discrepancy between troponin measurement and clinical presentation of the patient, the clinician should consider the possibility of analytical interference.

The impact of inter-laboratory glucose bias on the diagnosis of gestational diabetes mellitus: Comparison of common automated central laboratory methods.

BACKGROUND AND AIMS: The diagnosis of gestational diabetes mellitus (GDM) is based exclusively on glucose measurements, which are highly influenced by pre-analytical and analytical factors. Therefore, poor agreement across laboratories may affect the prevalence of GDM. We aimed to determine the inter-laboratory bias of glucose measurements and the impact on GDM prevalence. MATERIAL AND METHODS: A prospective cohort study of women (n = 110) referred for second-trimester GDM diagnostics using a 75 g oral glucose tolerance test. Maternal glucose was assessed from venous plasma at fasting, 1 h and 2 h. Venous blood were collected in Fluoride Citrate tubes and frozen. Samples were analyzed at five central laboratories using four different automated glucose Hexokinase methods and GDM prevalence was evaluated according to WHO2013 diagnostic criteria. RESULTS: Maximum inter-laboratory bias was 0.19, 0.30 and 0.27 mmol/L in fasting, 1 h and 2 h samples, respectively. GDM prevalence ranged 30.0-41.1% across laboratories. CONCLUSION: Inter-laboratory bias for mean venous glucose was low and within desirable limits. Nonetheless, the impact on GDM prevalence was considerable, which may inappropriately affect clinical practice.

Identification of Highly Sensitive Pleural Effusion Protein Biomarkers for Malignant Pleural Mesothelioma by Affinity-Based Quantitative Proteomics.

Malignant pleural mesothelioma (MPM) is an asbestos-associated, highly aggressive cancer characterized by late-stage diagnosis and poor prognosis. Gold standards for diagnosis are pleural biopsy and cytology of pleural effusion (PE), both of which are limited by low sensitivity and markedly inter-observer variations. Therefore, the assessment of PE biomarkers is considered a viable and objective diagnostic tool for MPM diagnosis. We applied a novel affinity-enrichment mass spectrometry-based proteomics method for explorative analysis of pleural effusions from a prospective cohort of 84 patients referred for thoracoscopy due to clinical suspicion of MPM. Protein biomarkers with a high capability to discriminate MPM from non-MPM patients were identified, and a Random Forest algorithm was applied for building classification models. Immunohistology of pleural biopsies confirmed MPM in 40 patients and ruled out MPM in 44 patients. Proteomic analysis of pleural effusions identified panels of proteins with excellent diagnostic properties (90-100% sensitivities, 89-98% specificities, and AUC 0.97-0.99) depending on the specific protein combination. Diagnostic proteins associated with cancer growth included galactin-3 binding protein, testican-2, haptoglobin, Beta ig-h3, and protein AMBP. Moreover, we also confirmed previously reported diagnostic accuracies of the MPM markers fibulin-3 and mesothelin measured by two complementary mass spectrometry-based methods. In conclusion, a novel affinity-enrichment mass spectrometry-based proteomics identified panels of proteins in pleural effusion with extraordinary diagnostic accuracies, which are described here for the first time as biomarkers for MPM.

First trimester serum apolipoproteins in the prediction of late-onset preeclampsia.

Late-onset preeclampsia occurring after 34 weeks of gestation is the most common form of preeclampsia, but little is known about either etiology or prevention. Current detection methods for preeclampsia in early pregnancy have not shown promising results in detecting late-onset preeclampsia. The aim of this study was to assess whether apolipoproteins in combination with maternal medical history and biophysical factors can be used as an early detection method for late-onset preeclampsia. This nested case-cohort study was based at Odense University Hospital, Denmark. Women attending their first trimester scan were invited to participate if they understood Danish or English, were above the age of 18, and had singleton pregnancies. Blood pressure, maternal medical history, uterine artery pulsatility indices, and blood samples were collected at inclusion. Outcome data were collected from participants' medical files postpartum, and cases were selected when preeclampsia diagnostics were present. Serum samples were analyzed by targeted mass spectrometry using a biomarker panel consisting of 12 apolipoproteins. Logistic regression analyses were performed and finally receiver operating curves were completed. The cohort consisted of 27 cases and 194 normotensive controls, randomized from 340 eligible participants. Significant differences were found between the two groups' baseline characteristics but none of the apolipoproteins showed significant difference (p < 0.05). The ROC-curve combining maternal characteristics, mean arterial pressure and two apolipoproteins showed the best sensitivity of 55.5% at a 10% false-positive rate and an area under the curve of 0.873. In conclusion, apolipoproteins did not improve the detection of late-onset preeclampsia in a combined screening model.

Regional divergence and time trends in the prevalence of gestational diabetes mellitus: a national Danish cohort study.

AIMS: To evaluate the prevalence and time trends of gestational diabetes mellitus (GDM) across the five regions of Denmark with uniform national guidelines for screening and diagnosing GDM. METHODS: This register-based national cohort study included 287,684 births from 2013 to 2017. Trends in GDM prevalence over time and differences between the five regions were evaluated. Crude and adjusted odd ratios (ORs) for GDM were calculated including potential confounding clinical risk factors as age, BMI, educational level, marital status, parity, country of origin and assisted reproduction. RESULTS: From 2013 to 2017, GDM prevalence in Denmark increased by 7% per year (OR 1.07, 95% CI 1.06-1.09, P < 0.001). GDM prevalence varied considerably between regions and ranged from 3.0 to 5.9% in 2017, corresponding to a maximal regional difference of 97%. In crude analyses, the risk of GDM in 2017 was significantly different in four of five regions compared to the remaining regions (OR ranging from 0.60 to 1.55), and these differences persisted after adjusting for confounding clinical risk factors (adjusted OR: 0.59-1.45). CONCLUSION: The prevalence of GDM increased over time in all Danish regions with substantial regional divergence. Up to a 97%, difference in GDM prevalence was observed between Danish regions, which was not explained by available clinical risk factors. This occurred despite national guidelines and raises the question of whether regional variations in screening efficacy, diagnostic procedures or inequality in clinical health care access may explain the observed differences.

A targeted multiplex mass spectrometry method for quantitation of abundant matrix and cellular proteins in formalin-fixed paraffin embedded arterial tissue.

Assessment of proteins in formalin-fixed paraffin-embedded (FFPE) tissue traditionally hinges on immunohistochemistry and immunoblotting. These methods are far from optimal for quantitative studies and not suitable for large-scale testing of multiple protein panels. In this study, we developed and optimised a novel targeted isotope dilution mass spectrometry (MS)-based method for FFPE samples, designed to quantitate 17 matrix and cytosolic proteins abundantly present in arterial tissue. Our new method was developed on FFPE human tissue samples of the internal thoracic artery obtained from coronary artery bypass graft (CABG) operations. The workflow has a limit of 60 samples per day. Assay precision improved by normalisation to both beta-actin and smooth muscle actin with inter-assay coefficients of variation (CV) ranging from 5.3% to 31.9%. To demonstrate clinical utility of the assay we analysed 40 FFPE artery specimens from two groups of patients with or without type 2 diabetes. We observed increased levels of collagen type IV α1 and α2 in patients with diabetes. The assay is scalable for larger cohorts and advantageous for pathophysiological studies in diabetes and the method is easily convertible to analysis of other proteins in FFPE artery samples. SIGNIFICANCE: This article presents a novel robust and precise targeted mass spectrometry assay for relative quantitation of a panel of abundant matrix and cellular arterial proteins in archived formalin-fixed paraffin-embedded arterial samples. We demonstrate its utility in pathophysiological studies of cardiovascular disease in diabetes.

Profiling the Plasma Apolipoproteome of Normo- and Hyperlipidemic Mice by Targeted Mass Spectrometry.

Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modeling of atherosclerosis has been the mainstay for preclinical testing of genetic and pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity, and it is critical to monitor lipoprotein levels during preclinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during preclinical experiments limit analyses to measuring total cholesterol and triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple reaction monitoring (MRM) stable isotope dilution (SID) mass spectrometry assay for simultaneous relative quantification of nine apolipoproteins using a few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: the widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools, we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during preclinical investigations.

Selective inhibition of soluble tumor necrosis factor signaling reduces abdominal aortic aneurysm progression.

Background: Tumor necrosis factor (TNF) is pathologically elevated in human abdominal aortic aneurysms (AAA). Non-selective TNF inhibition-based therapeutics are approved for human use but have been linked to several side effects. Compounds that target the proinflammatory soluble form of TNF (solTNF) but preserve the immunomodulatory capabilities of the transmembrane form of TNF (tmTNF) may prevent these side effects. We hypothesize that inhibition of solTNF signaling prevents AAA expansion. Methods: The effect of the selective solTNF inhibitor, XPro1595, and the non-selective TNF inhibitor, Etanercept (ETN) was examined in porcine pancreatic elastase (PPE) induced AAA mice, and findings with XPro1595 was confirmed in angiotensin II (ANGII) induced AAA in hyperlipidemic apolipoprotein E (Apoe) -/- mice. Results: XPro1595 treatment significantly reduced AAA expansion in both models, and a similar trend (p = 0.06) was observed in PPE-induced AAA in ETN-treated mice. In the PPE aneurysm wall, XPro1595 improved elastin integrity scores. In aneurysms, mean TNFR1 levels reduced non-significantly (p = 0.07) by 50% after TNF inhibition, but the histological location in murine AAAs was unaffected and similar to that in human AAAs. Semi-quantification of infiltrating leucocytes, macrophages, T-cells, and neutrophils in the aneurysm wall were unaffected by TNF inhibition. XPro1595 increased systemic TNF levels, while ETN increased systemic IL-10 levels. In ANGII-induced AAA mice, XPro1595 increased systemic TNF and IL-5 levels. In early AAA development, proteomic analyses revealed that XPro1595 significantly upregulated ontology terms including "platelet aggregation" and "coagulation" related to the fibrinogen complex, from which several proteins were among the top regulated proteins. Downregulated ontology terms were associated with metabolic processes. Conclusion: In conclusion, selective inhibition of solTNF signaling reduced aneurysm expansion in mice, supporting its potential as an attractive treatment option for AAA patients.

First trimester serum matrix metalloproteinase-7 is a poor predictor of late-onset preeclampsia.

OBJECTIVES: This study aims to evaluate matrix metalloproteinase-7 as a first trimester biomarker for late-onset preeclampsia, both alone and in combination with mean arterial pressure, uterine artery pulsatility index, and maternal characteristics. STUDY DESIGN: We conducted a nested case-control study from a prospective cohort consisting of 416 pregnant women who attended a routine first trimester scan. Baseline variables were obtained at inclusion and analysed subsequently to formation of case and control groups. The study was designed to detect a mean difference of > 15% in matrix metalloproteinase-7 concentrations between groups with a statistical power of 80%. MAIN OUTCOME MEASURES: The primary outcome was preeclampsia with delivery after 34 weeks of pregnancy. RESULTS: The median matrix metalloproteinase-7 concentration in cases of late-onset preeclampsia (n = 27) was marginally lower compared to normotensive controls but this difference was not statistically significant. Matrix metalloproteinase-7 predicted 14.8% of cases at a 10% false-positive rate. Addition of matrix metalloproteinase-7 to any combination of variables did not significantly improve their performance. CONCLUSIONS: Matrix metalloproteinase-7 is not a useful biomarker for late-onset preeclampsia, neither alone nor in combination with mean arterial pressure, uterine artery pulsatility index, or maternal characteristics.

Apolipoprotein D and transthyretin are reduced in female adolescent offspring of women with type 1 diabetes: The EPICOM study.

AIMS: Adolescent offspring exposed to maternal diabetes during intrauterine life show a less favourable metabolic profile than the background population. Here, we hypothesize that offspring of women with type 1 diabetes (T1D), possess sex-specific alterations in the serum profile of proteins involved in lipid, metabolic and transport processes and that these alterations are associated with lipid profile and indices of insulin sensitivity and secretion. METHODS: A prospective nationwide follow-up study (EPICOM) in a Danish population. Blood samples were assessed from offspring of women with T1D (index offspring, n = 267, 13-20 years), and matched control offspring (n = 290). Serum proteins were analysed using a 25-plex cardio-metabolic targeted proteomics assay, which includes 12 apolipoproteins and 13 transport and inflammatory proteins. RESULTS: Apolipoprotein D (ApoD) and transthyretin (TTR) were reduced in index females as compared to female controls (-8.1%, p < 0.001 and -6.1%, p = 0.006 respectively), but not in index males (2.2%, p = 0.476 and -2.4%, p = 0.731 respectively). Sex-dependent inverse associations between exposure to maternal T1D in utero and ApoD and TTR were significant after adjusting for age, BMI-SDS and Tanner stage (OR = 0.252 [95% CI 0.085, 0.745], p = 0.013 and OR = 0.149 [95% CI 0.040, 0.553], p = 0.004). ApoD correlated to indices of insulin sensitivity and secretion in a similar sex-specific pattern in crude and adjusted analyses. CONCLUSIONS: Low ApoD may be regarded as an early risk marker of metabolic syndrome. A possible link between ApoD and cardiovascular disease needs further investigation.

Decision Threshold for Kryptor sFlt-1/PlGF Ratio in Women With Suspected Preeclampsia: Retrospective Study in a Routine Clinical Setting.

Background The objective was to evaluate predictive performance and optimal decision threshold of the Kryptor soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio when implemented for routine management of women presenting with symptoms of preeclampsia. Methods and Results Observational retrospective study of a cohort of 501 women with suspected preeclampsia after 20 weeks of gestation. Women referred to maternity ward for observation of preeclampsia had an sFlt-1/PlGF ratio test included in routine diagnostic workup. Maternal and offspring characteristic data included maternal risk factors, outcomes, delivery mode, and indication for suspected preeclampsia. Biochemical measurements to determine sFlt-1/PlGF ratio were performed using the BRAHMS/Kryptor sFlt-1/PlGF ratio immunoassays. Results were analyzed by area under receiver-operating characteristic curve. Preeclampsia occurred in 150 of 501 (30%) of symptomatic women with an sFlt-1/PlGF ratio determined before the time of diagnosis. Area under receiver-operating characteristic curve for diagnosis of early-onset preeclampsia within 1 and 4 weeks was 0.98 (95% CI, 0.96-1.00) and 0.95 (95% CI, 0.92-0.98), respectively. For late-onset preeclampsia, predictive performance within 1 and 4 weeks was lower: 0.90 (95% CI, 0.85-0.94) and 0.85 (95% CI, 0.80-0.90), respectively. The optimal single sFlt-1/PlGF ratio threshold for all preeclampsia and late-onset preeclampsia within 1 and 4 weeks was 66. The negative and positive predictive values for ruling out and ruling in developing preeclampsia within 1 week were 96% and 70%, respectively. Conclusions The Kryptor sFlt-1/PlGF ratio is a useful clinical tool ruling out and in preeclampsia within 1 week. Prediction within 4 weeks is superior for early-onset preeclampsia. A single decision threshold of 66 is indicated for use in clinical routine.

Macro-TSH: A Diagnostic Challenge.

INTRODUCTION: Analytical problems should be considered in case of a discrepancy between the results of biochemical tests and the clinical findings. Macro-hormones often artefactually elevate biochemical tests. CASE PRESENTATION: A young male was referred with persistently elevated TSH (148 mIU/L) measured by a sandwich electrochemiluminescence immunoassay, ECLIA (Cobas; Roche, Basel, Switzerland). The patient's complaints were unspecific, and he appeared clinically euthyroid. The plasma levels of free T4 and free T3 were within the normal range, thyroid autoantibodies were negative, and thyroid ultrasonography was normal. During a short trial of thyroid hormone substitution, the level of TSH decreased to near-normal levels, but hyperthyroid symptoms emerged. TSH analysed by a different immunoassay (Architect; Abbott, Chicago, IL, USA) yielded similar results. In addition, serial dilutions were performed showing linearity, without detection of heterophilic antibody interference. Gel filtration chromatography confirmed the presence of macro-TSH. CONCLUSION: The patient harboured macro-TSH, which is a rare condition. The complex binding of TSH to other plasma proteins, most often immunoglobulins, results in elevated plasma TSH. However, the biologically active fraction of TSH is normal, reflected by clinical and biochemical euthyroidism.

Maternal prolactin is associated with glucose status and PCOS in pregnancy: Odense Child Cohort.

OBJECTIVE: Low circulating prolactin is a potential marker of metabolic risk during pregnancy. We aimed to investigate associations between prolactin and glucose status in pregnant women with and without gestational diabetes mellitus (GDM) or polycystic ovary syndrome (PCOS). DESIGN: Prospective observational cohort study. From the Odense Child Cohort, 1497 pregnant women were included. METHODS: Blood samples were assessed during first, second (prolactin, hemoglobin A1c (HbA1c)) and third trimester (fasting prolactin, testosterone, HbA1c, insulin, glucose). Oral glucose tolerance test (OGTT) was performed around gestation week 28 in 350 women with risk factors for GDM and in 272 randomly included women. GDM was defined by 2-h plasma glucose ≥9.0 mmol/L. RESULTS: The median (IQR) prolactin increased from 633 (451-829) mIU/L in first-second trimester to 5223 (4151-6127) mIU/L at third trimester. Prolactin was inversely associated with HbA1c in first (r = -0.19, P < 0.001) and third trimester (r = -0.07, P = 0.014). In third trimester, women with GDM (n = 37; 6.0%) had lower prolactin compared to women without GDM (4269 vs 5072 mIU/L, P = 0.004). Third trimester prolactin multiple of the median (MoM) was inversely associated with risk of GDM in multivariate regression analysis (OR 0.30, P = 0.034). PCOS was diagnosed in 10.0% (n = 146). Early pregnancy prolactin MoM was positively associated to PCOS diagnosis (OR 1.38, P = 0.051). CONCLUSIONS: Low prolactin levels during pregnancy were associated with higher HbA1c and risk of GDM. A diagnosis of PCOS was associated with higher early pregnancy prolactin levels.

First-trimester proteomic profiling identifies novel predictors of gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is a common pregnancy complication associated with adverse outcomes including preeclampsia, caesarean section, macrosomia, neonatal morbidity and future development of type 2 diabetes in both mother and child. Current selective screening strategies rely on clinical risk factors such as age, family history of diabetes, macrosomia or GDM in a previous pregnancy, and they possess a relatively low specificity. Here we hypothesize that novel first trimester protein predictors of GDM can contribute to the current selective screening strategies for early and accurate prediction of GDM, thus allowing for timely interventions. METHODS: A proteomics discovery approach was applied to first trimester sera from obese (BMI ≥27 kg/m2) women (n = 60) in a nested case-control study design, utilizing tandem mass tag labelling and tandem mass spectrometry. A subset of the identified protein markers was further validated in a second set of serum samples (n = 210) and evaluated for their contribution as predictors of GDM in relation to the maternal risk factors, by use of logistic regression and receiver operating characteristic analysis. RESULTS: Serum proteomic profiling identified 25 proteins with significantly different levels between cases and controls. Three proteins; afamin, serum amyloid P-component and vitronectin could be further confirmed as predictors of GDM in a validation set. Vitronectin was shown to contribute significantly to the predictive power of the maternal risk factors, indicating it as a novel independent predictor of GDM. CONCLUSIONS: Current selective screening strategies can potentially be improved by addition of protein predictors.

A Mild Inhibition of Cathepsin K Paradoxically Stimulates the Resorptive Activity of Osteoclasts in Culture.

Cathepsin K (CatK) inhibition allows reducing bone resorption with specific advantages compared to the existing anti-osteoporosis drugs. Its clinical use appears even more promising with the recent development of ectosteric inhibitors. A confusing observation, however, is that a low dose of the active site CatK inhibitor odanacatib (ODN) was reported to decrease bone mineral density and increase serum levels of the bone resorption marker carboxy-terminal collagen crosslinks (CTX). The present study provides a possible explanation for this paradox. The resorptive activity of human osteoclasts seeded on bone slices was inhibited when subjected to ODN at doses of 20 nM, but about 100-fold lower doses induced a significant increase in CTX levels and in eroded surface (12 repeats). This low-dose-induced stimulation was prevented by inhibition of non-CatK cysteine proteinases, thereby indicating that the stimulation results from an interplay between CatK and other cysteine proteinases. Effective interplay between these proteinases was also shown in enzymatic assays where the CatK-mediated degradation of collagen was enhanced upon addition of cathepsins B or L. Furthermore, extracts of osteoclasts subjected to a low dose of ODN showed higher levels of cathepsin B compared with extracts of control osteoclasts. In conclusion, the low-dose-induced stimulation of resorption observed in the clinical study can be reproduced in osteoclasts cultured in the absence of any other cell. Our data support an osteoclast-intrinsic mechanism where a mild inhibition of CatK results in increased levels of other proteinases contributing to the collagen degradation process.

Mid-Luteal 17-OH Progesterone Levels in 614 Women Undergoing IVF-Treatment and Fresh Embryo Transfer-Daytime Variation and Impact on Live Birth Rates.

Introduction: Corpus luteum (CL) produces progesterone (P4) and 17-OH progesterone (17-OH P4) during the luteal phase. Contrary to P4, 17-OH P4 is not supplied as part of the luteal phase support following IVF-treatment. Therefore, measuring endogenous serum 17-OH P4 levels may more accurately reflect the CL function compared to monitoring serum P4 concentrations. Objective: To explore the correlation between mid-luteal serum 17-OH P4 levels and live birth rates and to explore the possible daytime variations in mid-luteal serum 17-OH P4. Design: Prospective cohort study. Patients: 614 women undergoing IVF-treatment and fresh embryo transfer. Intervention: All patients had serum 17-OH P4 measured 7 days after oocyte pick-up (OPU+7). Furthermore, on OPU+7, seven patients underwent repeated blood sampling during daytime to clarify the endogenous daytime secretory pattern of 17-OH P4. Outcome measure: Live birth rate. Secondary outcome measure: Daytime variation in serum 17-OH P4 levels. Results: The highest chance of a live birth was seen with mid-luteal 17-OH P4 between 6.0 and 14.0 nmol/l. The chance of a live birth was reduced below (RD -10%, p = 0.07), but also above the optimal range for 17-OH P4 (RD -12%, p = 0.04). Patients with diminished CL-function (17-OH P4 < 6 nmol/l) displayed clinically stable 17-OH P4 values, whereas patients with 17-OH P4 levels >6 nmol/l showed random 17-OH P4 fluctuations during daytime. Conclusion: The association between 17-OH P4 and reproductive outcomes is non-linear, and the negative effect of excessive CL-secretion seems to be just as strong as the negative effect of a reduced CL-function during the peri-implantation period.