Circulating levels of proglucagon-derived peptides are differentially regulated by the glucagon-like peptide-1 agonist liraglutide and the centrally acting naltrexone/bupropion and can predict future weight loss and metabolic improvements: A 6-month long interventional study.

AIM: To investigate the changes of circulating levels of all proglucagon-derived peptides (PGDPs) in individuals with overweight or obesity receiving liraglutide (3 mg) or naltrexone/bupropion (32/360 mg), and to explore the association between induced changes in postprandial PGDP levels and body composition, as well as metabolic variables, after 3 and 6 months on treatment. MATERIALS AND METHODS: Seventeen patients with obesity or with overweight and co-morbidities, but without diabetes, were assigned to receive once-daily oral naltrexone/bupropion 32/360 mg (n = 8) or once-daily subcutaneous liraglutide 3 mg (n = 9). Participants were assessed before treatment initiation and after 3 and 6 months on treatment. At the baseline and 3-month visits, participants underwent a 3-hour mixed meal tolerance test to measure fasting and postprandial levels of PGDPs, C-peptide, hunger and satiety. Clinical and biochemical indices of metabolic function, magnetic resonance-assessed liver steatosis and ultrasound-assessed liver stiffness were measured at each visit. RESULTS: Both medications improved body weight and composition, carbohydrate and lipid metabolism, and liver fat and function. Naltrexone/bupropion produced a weight-independent increase in the levels of proglucagon (P < .001) and decreases in glucagon-like peptide-2 (GLP-2), glucagon and the major proglucagon fragment (P ≤ .01), whereas liraglutide markedly upregulated total glucagon-like peptide-1 (GLP-1) levels in a weight-independent manner (P = .04), and similarly downregulated the major proglucagon fragment, GLP-2 and glucagon (P < .01). PGDP levels at the 3-month visit were positively and independently correlated with improvements in fat mass, glycaemia, lipaemia and liver function, and negatively with reductions in fat-free mass, at both the 3- and 6-month visits. CONCLUSIONS: PGDP levels in response to liraglutide and naltrexone/bupropion are associated with improvements in metabolism. Our study provides support for the administration of the downregulated members of the PGDP family as replacement therapy (e.g. glucagon), in addition to the medications currently in use that induced their downregulation (e.g. GLP-1), and future studies should explore whether the addition of other PGDPs (e.g. GLP-2) could offer additional benefits.

Concentrations of oocyte secreted GDF9 and BMP15 decrease with MII transition during human IVM.

BACKGROUND: The suggested effects of the oocyte secreted GDF9 and BMP15 growth factors on oocyte maturation are currently based on recombinant proteins, and little is known about native GDF9 and BMP15 in humans. METHODS: Human immature cumulus-oocyte complexes (COCs) obtained in connection with ovarian tissue cryopreservation (OTC) underwent in vitro maturation (IVM). Oocyte-produced GDF9 and BMP15 were detected in COCs using immunofluorescence, and in fresh GV oocytes and in GV and MII oocytes after IVM by western blot. Concentrations of GDF9, BMP15 homodimers, and GDF9/BMP15 heterodimer in spent media after IVM were measured by ELISA. The relative expression of seven genes from the GDF9 and BMP15 signaling pathways (BMPR2, ALK5, ALK6, SMAD1, SMAD2, SMAD3, and SMAD5) was evaluated in fresh cumulus cells (before IVM) and in cumulus cells from GV and MII oocytes after IVM by RT-qPCR. RESULTS: We detected native pro-mature GDF9 and BMP15 in human oocytes with molecular weights (Mw) of 47 kDa and 43 kDa, respectively. Concentrations of GDF9 and BMP15 in spent media after IVM were detected in 99% and 64% of the samples, respectively. The GDF9/BMP15 heterodimer was detected in 76% of the samples. Overall, the concentration of GDF9 was approximately 10-times higher than BMP15. The concentrations of both GDF9 and BMP15 were significantly lower in spent medium from MII oocytes than in media from oocytes that remained at the GV stage. Concentrations of the GDF9/BMP15 heterodimer did not differ between GV and MII oocytes. Furthermore, BMPR2, SMAD3, and SMAD5 were significantly upregulated in cumulus cells from MII oocytes, indicating that both GDF9 and BMP15 signaling were active during oocyte meiotic resumption in vitro. CONCLUSION: These data suggest that the driving mechanisms for oocyte nuclear maturation may involve both GDF9 and BMP15 homodimers, while the role of the GDF9/BMP15 heterodimer is questionable.

The Activin/Follistatin Axis Is Severely Deregulated in COVID-19 and Independently Associated With In-Hospital Mortality.

BACKGROUND: Activins are members of the transforming growth factor-ß superfamily implicated in the pathogenesis of several immunoinflammatory disorders. Based on our previous studies demonstrating that overexpression of activin-A in murine lung causes pathology sharing key features of coronavirus disease 2019 (COVID-19), we hypothesized that activins and their natural inhibitor follistatin might be particularly relevant to COVID-19 pathophysiology. METHODS: Activin-A, activin-B, and follistatin were retrospectively analyzed in 574 serum samples from 263 COVID-19 patients hospitalized in 3 independent centers, and compared with demographic, clinical, and laboratory parameters. Optimal scaling with ridge regression was used to screen variables and establish a prediction model. RESULT: The activin/follistatin axis was significantly deregulated during the course of COVID-19, correlated with severity and independently associated with mortality. FACT-CLINYCoD, a scoring system incorporating follistatin, activin-A, activin-B, C-reactive protein, lactate dehydrogenase, intensive care unit admission, neutrophil/lymphocyte ratio, age, comorbidities, and D-dimers, efficiently predicted fatal outcome (area under the curve [AUC], 0.951; 95% confidence interval, .919-.983; P <10-6). Two validation cohorts indicated similar AUC values. CONCLUSIONS: This study demonstrates a link between activin/follistatin axis and COVID-19 mortality and introduces FACT-CLINYCoD, a novel pathophysiology-based tool that allows dynamic prediction of disease outcome, supporting clinical decision making.

Effect of the glucagon-like peptide-1 analogue liraglutide versus placebo treatment on circulating proglucagon-derived peptides that mediate improvements in body weight, insulin secretion and action: A randomized controlled trial.

AIM: To examine how circulating glucagon-like peptide-1 (GLP-1) concentrations during liraglutide treatment relate to its therapeutic actions on glucose and weight, and to study the effects of liraglutide on other proglucagon-derived peptides (PGDPs), including endogenous GLP-1, glucagon-like peptide-2, glucagon, oxyntomodulin, glicentin and major proglucagon fragment, which also regulate metabolic and weight control. MATERIALS AND METHODS: Adults who were overweight/obese (body mass index 27-40 kg/m2 ) with prediabetes were randomized to liraglutide (1.8 mg/day) versus placebo for 14 weeks. We used specific assays to measure exogenous (liraglutide, GLP-1 agonist [GLP-1A]) and endogenous (GLP-1E) GLP-1, alongside five other PGDP concentrations during a mixed meal tolerance test (MMTT) completed at baseline and at week 14 (liraglutide, n = 16; placebo, n = 19). Glucose during MMTT, steady-state plasma glucose (SSPG) concentration for insulin resistance and insulin secretion rate (ISR) were previously measured. MMTT area-under-the-curve (AUC) was calculated for ISR, glucose and levels of PGDPs. RESULTS: Participants on liraglutide versus placebo had significantly (P ≤ .004) decreased weight (mean -3.6%, 95% CI [-5.2% to -2.1%]), SSPG (-32% [-43% to -22%]) and glucose AUC (-7.0% [-11.5% to -2.5%]) and increased ISR AUC (30% [16% to 44%]). GLP-1A AUC at study end was significantly (P ≤ .04) linearly associated with % decrease in weight (r = -0.54) and SSPG (r = -0.59) and increase in ISR AUC (r = 0.51) in the liraglutide group. Treatment with liraglutide significantly (P ≤ .005) increased exogenous GLP-1A AUC (median 310 vs. 262 pg/mL × 8 hours at baseline but decreased endogenous GLP-1E AUC [13.1 vs. 24.2 pmol/L × 8 hours at baseline]), as well as the five other PGDPs. Decreases in the PGDPs processed in the intestines are independent of weight loss, indicating a probable direct effect of GLP-1 receptor agonists to decrease their endogenous production in contrast to weight loss-dependent changes in glucagon and major proglucagon fragment that are processed in pancreatic alpha cells. CONCLUSIONS: Circulating GLP-1A concentrations, reflecting liraglutide levels, predict improvement in weight, insulin action and secretion in a linear manner. Importantly, liraglutide also downregulates other PGDPs, normalization of the levels of which may provide additional metabolic and weight loss benefits in the future.

GLYCOSYLATED FIBRONECTIN AND INHIBIN ARE LOWER AND ANTI-MÜLLERIAN HORMONE IS HIGHER IN UMBILICAL CORD BLOOD WHEN MOTHERS HAVE PREECLAMPSIA.

Objective: Preeclampsia is a common disorder of pregnancy, causing significant morbidity and mortality for mothers and infants. Several molecules, including glycosylated fibronectin (GlyFn), the inhibin-related proteins, anti-müllerian hormone (AMH), and the insulin-like growth factor axis, are altered in maternal plasma in the setting of preeclampsia; however, these molecules have not been previously measured in cord blood of infants born to mothers with preeclampsia, which may represent changes in fetal physiology. We evaluated potential biomarkers of preeclampsia in umbilical cord blood to fill the gap in knowledge. Methods: This is a case-control study of 196 neonates born at a tertiary teaching hospital in Boston from 2010-2017. Forty-nine neonates born to mothers with preeclampsia were matched 1:3 by gestational age, sex, and birth weight z-score with 147 controls. Eleven analytes were measured in cord blood by enzyme-linked immunosorbent assay. Binary logistic regression analyses were performed to evaluate associations between preeclampsia and analytes. Results: Mean cord blood levels of GlyFn and total inhibin were significantly lower in neonates born to mothers with preeclampsia compared to controls, and AMH levels were significantly higher in males born to mothers with preeclampsia than male controls. Associations remained significant after controlling for maternal and neonatal characteristics. Conclusion: Cord blood levels of GlyFn and inhibin are decreased and AMH (male) levels are increased in infants of preeclamptic mothers, which is opposite the pattern these biomarkers show in serum of mothers with preeclampsia. These molecules may be important in the pathophysiology and long-term effects of preeclampsia on the developing fetus. Abbreviations: AMH = anti-müllerian hormone; ELISA = enzyme-linked immunosorbent assay; GlyFn = glycosylated fibronectin; IGF = insulin-like growth factor; IGFBP5 = insulin-like growth factor binding protein 5; LOD = limit of detection; PAPP-A = pregnancy-associated plasma protein A; PAPP-A2 = pregnancy-associated plasma protein A2.

Roles of human epididymis protein 4, carbohydrate antigen 125, inhibin B and anti-Müllerian hormone in the differential diagnosis and follow-up of ovarian granulosa cell tumors.

OBJECTIVE: Evaluation of circulating tumor markers in ovarian cancer is crucial for optimal patient care. The goal of this study was to verify the most accurate circulating tumor markers for the diagnosis and follow-up of adult-type granulosa cell tumors (AGCTs). METHODS: The levels of circulating human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125), together with AGCT markers inhibin B and anti-Müllerian hormone (AMH), were measured in 135 samples from AGCT patients, 37 epithelial ovarian carcinoma (EOC) patients, and 40 endometrioma (ENDO) patients. The levels were plotted with receiver operating characteristic (ROC) graphs, and the area under the curves (AUC) of the different markers were calculated and compared. RESULTS: HE4 levels were significantly lower in AGCTs than in EOCs (p<0.0001). CA125 levels were above 35IU/l in 25% of AGCT patients and 47.5% of ENDO patients, whereas inhibin B and AMH levels were elevated only in patients with AGCTs. In the AUC comparison analyses, inhibin B alone was sufficient to differentiate AGCT from EOC. In differentiating AGCT from ENDO, inhibin B and AMH performed similarly, and the combination of inhibin B and AMH increased the accuracy compared to either marker alone (sensitivity, 100%; specificity, 93%). Among AGCT patients, inhibin B was the best marker for detecting the presence of AGCT. CONCLUSIONS: HE4 and CA125 levels were low in AGCTs, and inhibin B was the most accurate circulating biomarker in distinguishing AGCTs from EOCs and from ENDOs. Inhibin B was also the best single marker for AGCT follow-up.

Activin inhibition limits early innate immune response in rat kidney allografts-a pilot study.

Activins are members of the transforming growth factor-beta (TGF-ß) superfamily of cytokines. They play critical roles in the onset of acute and chronic inflammatory responses. The aim of this study was to investigate how activin inhibition affects acute kidney injury and inflammation after transplantation. The study was carried out in kidney transplantation and renal ischemia-reperfusion models in the rat. Soluble activin type 2 receptor (sActRIIB-Fc) was used to inhibit activin signaling. Transplantation groups were as follows: (i) cyclosporine A (CsA) (ii) CsA + sActRIIB-Fc, (iii) CsA+ inactive protein control Fc-G1. IRI groups were as follows: (i) no treatment, (ii) sActRIIB-Fc. Serum activin B concentration was significantly elevated after transplantation and IRI, whereas activin A was produced locally in renal allografts. Activin inhibition efficiently limited neutrophil, macrophage, and dendritic cell infiltration to the allografts measured 72 h after transplantation. In addition, sActRIIB-Fc treatment modulated serum cytokine response after transplantation and reduced the early accumulation of fibroblasts in the graft interstitium. In conclusion activin inhibition reduces the innate immune response early after renal transplantation in the rat. It also limits the accumulation of fibroblasts in the graft suggesting that activins may be involved in the fibrogenic signaling already early after kidney transplantation.

Antimüllerian hormone level predicts ovulation in women with polycystic ovary syndrome treated with clomiphene and metformin.

OBJECTIVE: To describe the serum anti-Müllerian hormone (AMH) concentrations in a large, well-phenotyped cohort of women with polycystic ovary syndrome (PCOS) and evaluate whether AMH predicts successful ovulation induction in women treated with clomiphene and metformin. DESIGN: Secondary analysis of randomized controlled trial. SETTING: Not applicable. PATIENT(S): A total of 333 women with anovulatory infertility attributed to PCOS who participated in the double-blind randomized trial entitled the Pregnancy in Polycystic Ovary Syndrome I (PPCOS I) study (registration number, NCT00068861) who had serum samples from baseline laboratory testing available for further serum analysis were studied. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): The association between the baseline AMH levels in each of the 3 treatment groups and ovulation, pregnancy, and live birth rates were assessed. RESULT(S): A total of 322 individuals had a baseline AMH concentration available, of which the mean AMH was 11.7 ± 8.3 ng/mL (range 0.1-43.0 ng/mL). With each unit (1 ng/mL) increase in baseline AMH, the odds of ovulation decreased by 10% (odds ratio, 0.90; 95% confidence interval, 0.86-0.93); this effect did not differ by treatment group. Women with a high baseline AMH concentration (>8 ng/mL) were significantly less likely to ovulate compared with those with a normal baseline AMH concentration (<4 ng/mL) (odds ratio, 0.23; 95% confidence interval, 0.05-0.68). This remained statistically significant when controlling for confounders, including age, body mass index, time in study, and Homeostatic Model Assessment for Insulin Resistance score. Ovulation occurred even at very high AMH concentrations; there was no maximum level noted at which no ovulation events occurred. Baseline AMH concentration was not associated with pregnancy or live birth rates when controlling for confounders. CONCLUSION(S): These AMH values in well-phenotyped individuals with PCOS add to the literature and will aid in identifying AMH criteria for the diagnosis of PCOS. In women with infertility and PCOS, a higher AMH concentration was associated with reduced odds of ovulation with ovulation induction with clomiphene, clomiphene + metformin, and metformin. CLINICAL TRIAL REGISTRATION NUMBER: The original trial from which this analysis is derived was entitled "Pregnancy in Polycystic Ovary Syndrome: A 30 Week Double-Blind Randomized Trial of Clomiphene Citrate, Metformin XR, and Combined Clomiphene Citrate/Metformin XR For the Treatment of Infertility in Women With Polycystic Ovary Syndrome" and was registered on ClinicalTrials.gov as number NCT00068861. The URL for the trial is https://clinicaltrials.gov/study/NCT00068861. The first subject was enrolled in November 2002.

Analysis of Predictive Information From Biomarkers Added to Clinical Models of Preeclampsia: Consideration of PAPP-A2, Activin A, and sFlt-1:PlGF Ratio.

BACKGROUND: Preeclampsia remains a major cause of maternal and fetal adverse outcomes in pregnancy; however, accurate and universally acceptable predictive tools remain elusive. We investigated whether a panel of biomarkers could improve risk prediction for preeclampsia when measured at various pregnancy time points. METHODS: In this prospective cohort study, 192 women with first-trimester high-risk singleton pregnancies were consecutively recruited from tertiary obstetrics clinics in Montréal, Canada. Clinical information (height, pre-pregnancy weight, personal and family medical history, medication use) was collected at baseline. Blood pressure was measured and blood samples collected at each trimester to quantify soluble Fms-like tyrosine kinase 1 (sFlt-1), placental growth factor (PlGF), pregnancy-associated plasma protein A2 (PAPP-A2), PAPP-A, activin A, inhibin A, follistatin, and glycosylated fibronectin. A random-effects hierarchic logistic regression model was used to relate change in biomarker levels to incidence of preeclampsia. RESULTS: When added to a clinical model composed of maternal age, pre-pregnancy body mass index, race, and mean arterial pressure, a positive third-trimester result for both PAPP-A2 and activin A had a better positive predictive value than the sFlt-1:PlGF ratio added to the clinical model (91.67% [95% confidence interval (CI) 78.57%-100%] vs 66.67% [57.14%-100%]), while maintaining a comparable high negative predictive value (97.69% [95% CI 95.34%-100%] vs 96.00% [92.19%-99.21%]). CONCLUSIONS: Whereas the third-trimester sFlt-1:PlGF ratio can predict short-term absence of preeclampsia, PAPP-A2 and activin A had both high positive and negative predictive values and therefore could serve as biomarkers to predict the occurrence (and absence) of preeclampsia; these findings will be validated in future studies.

Circulating levels of all proglucagon-derived peptides are differentially regulated postprandially by obesity status and in response to high-fat meals vs. high-carbohydrate meals.

BACKGROUND & AIMS: We measured all proglucagon-derived peptides (PGDPs) levels in response to administration of three mixed meal tolerance tests (MMTs), examining differences in postprandial PGDP responses in subjects with leanness and obesity or between high-fat vs. high carbohydrate meals. METHODS: We designed three physiology interventional studies, administering MMTs over a 180-min period to individuals without diabetes after an overnight fast. In Study 1, a 450 kcal MMT was administered to n = 4 normal weight and n = 9 individuals with obesity. In Study 2, a 600 kcal high-fat MMT was administered to n = 15 normal-weight and n = 15 individuals with obesity. In Study 3, n = 32 participants with obesity were assigned to receive a 600-kcal high-fat (n = 15) or an isocaloric high-carbohydrate MMT (n = 17). Fasting and postprandial levels of c-peptide and PGDPs (proglucagon, GLP-1, GLP-2, glicentin, oxyntomodulin, glucagon, major proglucagon fragment [MPGF]) were assessed. RESULTS: In study 1, individuals with normal weight displayed elevated glicentin postprandial secretion compared with people with obesity (p = 0.002). Following a high-fat MMT with 33% higher energy content in study 2, all postprandial PGDPs levels were elevated (p-time<0.001), irrespective of weight status. In study 3, a prolonged postprandial upregulation of PGDPs during the high-fat MMT was observed in contrast with the acute, short-term (max 60 min) PGDP responses to a high-carbohydrate MMT (p-time∗meal<0.001). Across both studies 2 and 3, the postprandial responses of glucagon and MPGF were higher in subjects with male sex whereas glicentin was higher in subjects with female sex. CONCLUSIONS: Fat and carbohydrate content of a meal can substantially affect the postprandial levels of PGDPs. Circulating levels of PGDPs are influenced by the energy content of the meal, and additionally, the presence of leanness or obesity affects circulating levels of select PGDPs. These results, which are to be confirmed by additional studies, expand our understanding of PGDP physiology in leanness and obesity. CLINICALTRIALS: GOV REGISTRATION NUMBERS: (NCT04170010, NCT04430946, NCT04575194).

Activins, follistatins and inhibins in postmenopausal osteoporosis: A proof of concept, case-control study.

BACKGROUND: Bone metabolism has been proposed to be affected by the activins-follistatins-inhibins (AFI) hormonal system. We aimed to evaluate AFI in patients with osteoporosis and osteopenia compared with postmenopausal and premenopausal controls. METHODS: In this case-control study, circulating levels of the AFI system were evaluated, individually and jointly, between postmenopausal women with osteoporosis (BMD T-score ≤-2.5; n = 25) or osteopenia (BMD T-score >-2.5 and ≤-1; n = 25) and postmenopausal women with normal BMD (T-score >-1.0; n = 25) or premenopausal women with normal BMD (Z-score >-1.0; n = 25), with and without adjustment for potential confounders. RESULTS: In the sum of participants, AFI molecules and their ratios followed an opposite pattern of correlations for age and BMI vs. BMD. In unadjusted models, FSTL3 concentrations were higher, whereas activin B, inhibin A and inhibin B and the ratios of activin B/follistatin and activin B/FSTL3 were lower in the three postmenopausal groups compared with the premenopausal group. Activin A/follistatin and activin AB/follistatin ratios were lower in the osteoporosis group than the other three groups. After adjustment for BMI and age, inhibin B (p = 0.005), and the ratios of activin A/follistatin (p = 0.009), activin B/follistatin (p = 0.040) and activin AB/follistatin (p = 0.003) were lower in the osteoporotic group compared with the other groups. In fully adjusted logistic regression analysis log(inhibin B) (p = 0.041), log(activinA/follistatin) (p = 0.014), log(activinB/follistatin) (p = 0.025) and log(activinAB/follistatin) (p = 0.021), but not FSTL3, remained independently associated with the presence of osteoporosis. CONCLUSIONS: Lower inhibin B and higher ratios of activins A, B, and AB to follistatin are associated with lumbar spine BMD and the presence of osteoporosis independently from age or BMI.

Development of a quantitative fluorescence lateral flow immunoassay (LFIA) prototype for point-of-need detection of anti-Müllerian hormone.

Objective: Anti-Müllerian Hormone (AMH) is a quantitative marker for ovarian reserve and is used to predict response during ovarian stimulation. Streamlining testing to the clinic or even to the physician's office would reduce inconvenience, turnaround time, patient stress and potentially also the total cost of testing, allowing for more frequent monitoring. In this paper, AMH is used as a model biomarker to describe the rational development and optimization of sensitive, quantitative, clinic-based rapid diagnostic tests. Design and Methods: We developed a one-step lateral-flow europium (III) chelate-based fluorescent immunoassay (LFIA) for the detection of AMH on a portable fluorescent reader, optimizing the capture/detection antibodies, running buffer, and reporter conjugates. Results: A panel of commercial calibrators was used to develop a standard curve to determine the analytical sensitivity (LOD = 0.41 ng/ml) and the analytical range (0.41-15.6 ng/ml) of the LFIA. Commercial controls were then tested to perform an initial evaluation of the prototype performance and showed a high degree of precision (Control I CV 2.18%; Control II CV 3.61%) and accuracy (Control I recovery 126%; Control II recovery 103%). Conclusions: This initial evaluation suggests that, in future clinical testing, the AMH LFIA will likely have the capability of distinguishing women with low ovarian reserve (<1 ng/ml AMH) from women with normal (1-4 ng/ml AMH) ovarian reserve. Furthermore, the LFIA demonstrated a wide linear range, indicating the assay's applicability to the detection of other health conditions such as PCOS, which requires AMH measurement at higher concentrations (>6 ng/ml).

Association of activins, follistatins and inhibins with incident hip fracture in women with postmenopausal osteoporosis: a proof of concept, case-control study.

PURPOSE: The activins-follistatins-inhibins (AFI) hormonal system is considered to regulate muscle and bone mass. We aimed to evaluate AFI in postmenopausal women with an incident hip fracture. METHODS: In this post-hoc analysis of a hospital based case-control study, we evaluated circulating levels of the AFI system in postmenopausal women with a low-energy hip fracture admitted for fixation compared with postmenopausal women with osteoarthritis scheduled for arthroplasty. RESULTS: Circulating levels of follistatin (p = 0.008), FSTL3 (p = 0.013), activin B and AB (both p < 0.001), as well as activin AB/follistatin and activin AB/FSTL3 ratios (p = 0.008 and p = 0.029, respectively) were higher in patients than controls in unadjusted models. Differences for activins B and AB remained after adjustment for age and BMI (p = 0.006 and p = 0.009, respectively) and for FRAX-based risk for hip fracture (p = 0.008 and p = 0.012, respectively) but were lost when 25OHD was added to the regression models. CONCLUSIONS: Our data indicate no major changes in the AFI system in postmenopausal women at the time of hip fracture compared to postmenopausal women with osteoarthritis except for higher activin B and AB levels, whose significance, however, was lost when 25OHD was added to the adjustment models. CLINICAL TRIALS: Clinical Trials identifier: NCT04206618.

Methods paper: Performance characteristics of novel assays for circulating levels of proglucagon-derived peptides and validation in a placebo controlled cross-over randomized clinical trial.

BACKGROUND: The measurement of proglucagon-derived peptides (PGDPs) is a challenging task mainly due to major overlaps in their molecular sequence in addition to their low circulating levels. Here, we present the technical characteristics of novel ELISA assays measuring C-peptide and all six PGDPs including, for the first time, major proglucagon fragment (MPGF), and we validate them by performing a pilot in vivo cross-over randomized clinical trial on whether coffee consumption may affect levels of circulating PGDPs. METHODS: The performance and technical characteristics of novel ELISA assays from Ansh measuring GLP-1, GLP-2, oxyntomodulin, glicentin, glucagon, MPGF and C-peptide were first evaluated in vitro in procured samples from a commercial vendor as well as in deidentified human samples from three previously performed clinical studies. Their performance was further evaluated in vivo in the context of a cross-over randomized controlled trial, in which 33 subjects consumed in random order and together with a standardized meal, 200 ml of either (a) instant coffee with 3 mg/kg caffeine, or (b) instant coffee with 6 mg/kg caffeine, (c) or water. RESULTS: All assays demonstrated high accuracy (spike and recovery and average linearity recovery ±15%), precision (inter-assay CV ≤ 6.4%), specificity (no significant cross-reactivities) and they were sensitive in low concentrations. Measurements of glicentin in archived random human samples using the Ansh assay correlated strongly with the glicentin measurements of Mercodia assay (r = 0.968) and of GLP-1 modestly with Millipore GLP-1 assay (r = 0.440). Oxyntomodulin, glicentin and glucagon concentrations were 2-5 fold higher in plasma compared to serum and serum concentrations correlated modestly (for oxyntomodulin and glicentin) or poorly (for glucagon) with the plasma concentrations. The evaluated assays detected a postprandial increase of gut-secreted PGDPs (GLP-1, GLP-2, oxyntomodulin and glicentin) and a postprandial decrease of pancreas-secreted PGDPs (glucagon, MPGF) in response to consuming coffee in comparison to consuming water with breakfast (enter here composition of breakfast). Only coffee consumption at the high dose alter levels of gut-secreted PGDPs and both at low and high dose to lower levels of pancreas-secreted PGDPs compared to water consumption during breakfast. CONCLUSION: Accurate, precise and specific measurement of six PGDPs is possible with novel assays. A randomized controlled trial demonstrated in vivo utility of those assays and supports the notion that coffee may exert part of its beneficial effects on glucose homeostasis in the short term through the regulation of PGDPs.

Serum and urine profiles of TGF-ß superfamily members in reproductive aged women.

BACKGROUND: TGF-ß superfamily members are important biomarkers of reproductive health in women desiring fertility and during pregnancy. TGF-ß proteins derived from the ovary and/or placenta have been detected in serum in women, but there have been very few attempts to measure them non-invasively, such as in urinary samples, and to compare them to serum concentrations. METHODS: We measured inhibin A, inhibin B, total inhibin, AMH, activin A, activin B, activin AB, follistatin, the GDF-9/BMP-15 complex, and GDF-15 in paired serum and urine samples from healthy reproductive aged women and in pregnant (second trimester) women. RESULTS: We detected all hormones in serum in both pregnant and non-pregnant women. Inhibin A, total inhibin, activin A, activin AB, follistatin, and GDF-15 were significantly higher in pregnant than in non-pregnant women. GDF-15 was the only hormone consistently detected in urine. We also measured, for the first time, the GDF-9/BMP-15 functional heterodimer and the GDF-15 protein harboring the H202D polymorphism. CONCLUSIONS: We report the successful measurement of the GDF-9/BMP-15 heterodimer (its native form) in serum and the ability to measure GDF-15 non-invasively, in urinary samples. This novel GDF-15 assay also captures the antigen in the presence of a common genetic variant.

Intrafollicular Concentrations of the Oocyte-secreted Factors GDF9 and BMP15 Vary Inversely in Polycystic Ovaries.

CONTEXT: The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome. OBJECTIVE: Are there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)? DESIGN AND SETTING: Follicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation. PATIENTS: Twenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size. MAIN OUTCOME MEASURES: Intrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays. RESULTS: The detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ±â€…189 pg/mL [mean ±â€…SEM], n = 188; non-PCO: 3498 ±â€…199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ±â€…40 pg/mL, n = 125; non-PCO: 573 ±â€…55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO. CONCLUSIONS: Intrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO.

High Variability of Molecular Isoforms of AMH in Follicular Fluid and Granulosa Cells From Human Small Antral Follicles.

Anti-Müllerian hormone (AMH) is a member of the TGF-ß superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive life. AMH is thought to inhibit aromatase (i.e., CYP19) expression and decrease the conversion of androgens to oestrogens, especially in small antral follicles before dominance is achieved. Thus, AMH acts as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH has not been fully elucidated. The present study measured and determined AMH isoforms in human follicular fluid (FF) from small antral follicles and in human GCs using four ELISAs, western blot, and immunofluorescence analysis. We evaluated the presence of the following isoforms: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. A negative correlation between follicle diameter and the AMH forms was detected. Moreover, western blot analysis detected various AMH forms in both FFs and GCs, which did not match our consensus forms, suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size in the same woman. This study detected several AMH forms in FF and GCs obtained from human small antral follicles, which suggests that intrafollicular processing of AMH is complex and variable. Thus, it may be difficult to develop an antibody-based AMH assay that detects all AMH isoforms. Furthermore, the variability between follicles suggests that designing a recombinant AMH standard will be difficult.

Inhibin A-A Promising Predictive Parameter for Determination of Final Oocyte Maturation in Ovarian Stimulation for IVF/ICSI.

The number of mature oocytes is a key factor in the success of Assisted Reproductive Techniques (ART). Exogenous gonadotropins are administered during ovarian stimulation in order to maximize the number of oocytes available for fertilization. During stimulation, monitoring is mandatory to evaluate individual response, to avoid treatment complications and assist in the determination of the optimal day for final oocyte maturation and oocyte retrieval. Routine monitoring during stimulation includes transvaginal ultrasound examinations and measurement of serum estradiol (E2). Due to multifollicular growth of follicles of varying size, serum E2 levels are commonly supraphysiological and often variable, rendering E2-measurement during ovarian stimulation unreliable as a determinant of oocyte maturity. In contrast to serum E2, serum Inhibin A levels increase once a minimum follicle size of 12-15 mm is achieved. Due to this fact, serum Inhibin A levels could present in combination with ultrasound monitoring a more reliable parameter to determine the optimal follicle size for final oocyte maturation, as only follicles with a size of 12 mm and beyond will contribute to the serum Inhibin A level. This prospective observational, cross-sectional study demonstrates, that on the day of final oocyte maturation serum Inhibin A is strongly correlated to the number of follicles ≥15 mm (0.72) and to the number of retrieved and mature oocytes (ρ 0.82/0.77, respectively), whereas serum E2 is moderately correlated to the parameters mentioned above (ρ 0.64/0.69/0.69, respectively). With an area under the curve (AUC) of 0.91 for Inhibin A, compared to an AUC of 0.84 for E2, Inhibin A can be regarded as a better predictor for the optimal timing of trigger medication with a threshold number of ≥10 mature oocytes. It can be concluded from this data that serum Inhibin A in combination with transvaginal ultrasound monitoring may be a more powerful tool in the decision making process on trigger timing as compared to E2.

Anthropometric and biochemical correlates of PAPP-A2, free IGF-I, and IGFBP-3 in childhood.

OBJECTIVE: Pregnancy-associated plasma protein-A2 (PAPP-A2) is a metalloproteinase that cleaves IGFBP-3 and IGFBP-5. Human mutations in PAPPA2 result in short stature with a low percentage of free IGF-I. Little is known about PAPP-A2 levels and the regulation of free IGF-I throughout childhood. We examined PAPP-A2 and intact IGFBP-3 levels in childhood and explored associations between PAPP-A2, free and total IGF-I, and total and intact IGFBP-3 and their relationship to the percentage of free to total IGF-I and anthropometric factors. DESIGN: Cross-sectional study at a single center. METHODS: PAPP-A2, free IGF-I, and intact IGFBP-3 levels were measured in childhood (3-18 years old) and an evaluation of the relationship between these proteins and anthropometric factors. RESULTS: In 838 children, PAPP-A2 consistently decreased throughout childhood. In contrast, free IGF-I increased. A pubertal peak in free IGF-I was present in females but was less evident in males. Intact and total IGFBP-3 increased throughout childhood; however, intact IGFBP-3 had a more marked rise than total IGFBP-3. Percent free IGF-I decreased with no distinct pubertal peak. PAPP-A2 levels positively correlated with the percent free IGF-I (Male, Female; r = 0.18, 0.38; P < 0.001) and negatively with intact IGFBP-3 (Male, Female; r = -0.58, -0.65; P < 0.0001). CONCLUSIONS: This is the first study to describe serum PAPP-A2 and intact IGFBP-3 in children between 3 and 18 years of age. Our correlative findings suggest that PAPP-A2 is an important regulator of the percent free IGF-I which can be a marker of perturbations in the GH/IGF-I axis.

PAPP-A2 and Inhibin A as Novel Predictors for Pregnancy Complications in Women With Suspected or Confirmed Preeclampsia.

Background We aimed to evaluate the value of inhibin A and PAPP-A2 (pregnancy-associated plasma protein-A2) as novel biomarkers in the prediction of preeclampsia-related complications and how they compare with angiogenic biomarkers. Methods and Results Making use of a secondary analysis of a prospective, multicenter, observational study, intended to evaluate the usefulness of sFlt-1 (soluble Fms-like tyrosine kinase-1)/PlGF (placental growth factor) ratio, we measured inhibin A and PAPP-A2 levels in 524 women with suspected/confirmed preeclampsia. Women had a median gestational age of 35 weeks (range, 20-41 weeks) while preeclampsia occurred in 170 (32%) women. Levels of inhibin A and PAPP-A2 were significantly increased in women with preeclampsia and in maternal perfusate of preeclamptic placentas. Inhibin A and PAPP-A2 (C-index = 0.73 and 0.75) significantly improved the prediction of maternal complications when added on top of the traditional criteria; gestational age, parity, proteinuria, and diastolic blood pressure (C-index = 0.60). PAPP-A2 was able to improve the C-index from 0.75 to 0.77 when added on top of the sFlt-1/PlGF ratio for the prediction of maternal complications. To discriminate fetal/neonatal complications on top of traditional criteria, inhibin A and PAPP-A2 showed additive value (C-index = 0.79 to 0.80 and 0.82, respectively) but their discriminative ability remained inferior to that of sFlt-1/PlGF ratio or PlGF. Interestingly, the PAPP-A2/PlGF ratio alone showed remarkable value to predict pregnancy complications, being superior to sFlt-1/PlGF ratio in the case of maternal complications. Conclusions Inhibin A and PAPP-A2 show significant potential to predict preeclampsia-related pregnancy complications and might prove beneficial on top of the angiogenic markers.