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Intrauterine growth restriction is one of the most common obstetric conditions, affecting 7-10% of fetuses. Affected fetuses are actually exposed in utero to an adverse environment during the highly critical time of development and may face life-long health consequences such as increased cardiovascular risk in adulthood. Already in utero, fetuses affected by growth restriction show remodeled hearts with signs of systolic and diastolic dysfunction. Cardiovascular remodeling persist into postnatal life, from the neonatal period to adolescence, suggesting a primary fetal cardiac programming that might explain the increased cardiovascular risk later in life. In this review we summarize the current evidence on fetal cardiovascular programming in fetuses affected by growth restriction, its consequences later and possible strategies from which they could benefit to reduce their cardiovascular risk. [ABSTRACT FROM AUTHOR]

Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA).Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63+ve and placental-type alkaline phosphatase (PLAP)+ve antibodies, respectively.Results: Maternal concentrations of CD63+ve and PLAP+ve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAP+ve ratio) and BW percentile, [rho = 0.77 (95% CI: 0.57 to 0.89); p = 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAP+ve ratios in FGR compared to controls and SGA cases.Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. [ABSTRACT FROM AUTHOR]

Aims: Myocardial structural and functional abnormalities are known to occur in fetuses of mothers with diabetes mellitus (FMDM). The main aim of this investigation was to explore the cardiovascular circulatory patterns in FMDM using a validated lumped computational model of the cardiovascular system.Methods: This was a multi-institutional study involving FMDM compared to fetuses of maternal controls (FC). Fetal echocardiographic Doppler data from left and right ventricular outflow tracts, aortic isthmus, middle cerebral and umbilical arteries were fitted into a validated fetal circulation computational model to estimate patient-specific placental and vascular properties. Non-parametric comparisons were made between resistances, compliances and flows in the brain and placenta in FMDM and FC.Results: Data from 23 FMDM and 31 FC were fitted into the model. In FMDM, compared to FC, placental relative resistance was lower (0.59 ± 0.50 versus 0.91 ± 0.41; p < .05) with higher brain relative resistance (2.36 ± 1.65 versus 1.60 ± 0.85; p < .05). Middle cerebral artery flow was lower in FMDM than FC (0.12 ± 0.14 vs. 0.27 ± 0.21 ml/min; p 0.04) with a lower cerebral-placental flow ratio. Combined stroke volume was lower in FMDM (3.65 ± 2.05 ml) than FC (4.97 ± 2.45 ml) (p 0.04).Conclusions: Blood flow is redistributed in FMDM to the placenta, away from the brain. This alteration may play a role in the postnatal health of these fetuses. [ABSTRACT FROM AUTHOR]

In the modern world, cardiovascular disease is a leading cause of death for both men and women. Epidemiologic studies consistently have suggested an association between low birthweight and/or fetal growth restriction and increased rate of cardiovascular mortality in adulthood. Furthermore, experimental and clinical studies have demonstrated that sustained nutrient and oxygen restriction that are associated with fetal growth restriction activate adaptive cardiovascular changes that might explain this association. Fetal growth restriction results in metabolic programming that may increase the risk of metabolic syndrome and, consequently, of cardiovascular morbidity in the adult. In addition, fetal growth restriction is strongly associated with fetal cardiac and arterial remodeling and a subclinical state of cardiovascular dysfunction. The cardiovascular effects ocurring in fetal life, includes cardiac morphology changes, subclinical myocardial dysfunction, arterial remodeling, and impaired endothelial function, persist into childhood and adolescence. Importantly, these changes have been described in all clinical presentations of fetal growth restriction, from severe early- to milder late-onset forms. In this review we summarize the current evidence on the cardiovascular effects of fetal growth restriction, from subcellular to organ structure and function as well as from fetal to early postnatal life. Future research needs to elucidate whether and how early life cardiovascular remodeling persists into adulthood and determines the increased cardiovascular mortality rate described in epidemiologic studies. [ABSTRACT FROM AUTHOR]

Objective: To explore in women with late-onset preeclampsia (PE) the association between maternal levels of angiogenic/antiangiogenic factors in the first trimester of pregnancy and histological findings attributable to placental underperfusion (PUP).Methods: A nested case-control cohort study was conducted in 73 women with pregnancies complicated by late-onset PE (>34 weeks at delivery) matched with controls. First trimester uterine artery Doppler (UtA); maternal levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) were retrieved. Placentas were histologically evaluated using a hierarchical and standardized classification system. One-way ANOVA with linear polynomial contrast or linear-by-linear association test was performed to test the hypothesis of a linear association across study groups (controls, PE without PUP and PE with PUP).Results: In 54 (74%) placentas, 89 placental histological findings qualifying for PUP were found. Across study groups, significant values were observed in maternal levels of decreased PlGF (MoM values: 1.53, 1.41 and 1.37; p < 0.001), increased sFlt-1 (MoM values: 3.11, 3.11 and 3.22; p = 0.002), increased sFlt-1/PlGF ratio (MoM values: 2.3, 2.3 and 2.44; p < 0.001), abnormal UtA Doppler (MoM values: 1, 1.26 and 1.32; p < 0.001), and worse perinatal outcomes in terms of gestational age at delivery, cesarean section for not reassuring fetal status, birth weight and neonatal acidosis.Discussion: In late-onset PE an imbalance of circulating angiogenic and anti-angiogenic factors already present at 8-10 weeks of pregnancy was associated with histological findings reflecting placental insufficiency. An early first trimester screening by angiogenic factors might help to identify patients with placental involvement among late-onset PE cases.Conclusion: In late-onset preeclampsia, first-trimester uterine Doppler and circulating levels of angiogenic/antiangiogenic factors are associated with placental underperfusion. [ABSTRACT FROM AUTHOR]

Intrauterine Growth Restriction (IUGR) and premature birth are associated with higher risk of cardiovascular diseases throughout adulthood. The aim of this study was to evaluate the influence of these factors in ventricular electrical remodeling in preadolescents. Electrocardiography was performed in a cohort of 33-IUGR, 32-preterm with appropriate weight and 60 controls. Depolarization and repolarization processes were studied by means of the surface ECG, including loops and angles corresponding to QRS and T-waves. The angles between the dominant vector of QRS and the frontal plane XY were different among the study groups: controls [20.03°(10.11°-28.64°)], preterm [25.48°(19.79°-33.56°)], and IUGR [27.77°(16.59°-33.23°)]. When compared to controls, IUGR subjects also presented wider angles between the difference of QRS and T-wave dominant vectors and the XY-plane [5.28°±12.15° vs 0.49°±14.15°, p<0.05] while preterm ones showed smaller frontal QRS-T angle [4.68°(2.20°-12.89°) vs 6.57°(2.72°-11.31°), p<0.05]. Thus, electrical remodeling is present in IUGR and preterm preadolescents, and might predispose them to cardiovascular diseases in adulthood. Follow-up studies are warranted. [ABSTRACT FROM AUTHOR]

Introduction Intrauterine growth restriction (IUGR) due to placental insufficiency is associated with blood-flow redistribution in order to maintain perfusion to the brain. However, some hemodynamic parameters that might be more directly related to staging of the disease cannot be measured non-invasively in clinical practice. For this, we developed a patient-specific model of the fetal circulation to estimate vascular properties of each individual. Methods A lumped model of the fetal circulation was developed and personalized using measured echographic data from 37 normal and IUGR fetuses to automatically estimate model-based parameters. A multivariate regression analysis was performed to evaluate the association between the Doppler pulsatility indices (PI) and the model-based parameters. The correlation between model-based parameters and the placental lesions was analyzed in a set of 13 IUGR placentas. A logistic regression analysis was done to assess the added value of the model-based parameters relative to Doppler indices, for the detection of fetuses with adverse perinatal outcome. Results The estimated model-based placental and brain resistances were respectively increased and reduced in IUGR fetuses while placental compliance was increased in IUGR fetus. Umbilical and middle cerebral arteries PIs were most associated with both placental resistance and compliance, while uterine artery PI was more associated with the placental compliance. The logistic regression analysis showed that the model added significant information to the traditional analysis of Doppler waveforms for predicting adverse outcome in IUGR. Discussion The proposed patient-specific computational model seems to be a good approach to assess hemodynamic parameters than cannot be measured clinically. [ABSTRACT FROM AUTHOR]

Summary: Selective intrauterine growth restriction (sIUGR) in monochorionic twins is associated with a substantial increase in perinatal mortality and morbidity for both twins. Clinical evolution depends on the combination of the effects of placental insufficiency in the IUGR twin with inter-twin blood transfer through placental anastomoses. Classification of sIUGR into types according to the characteristics of umbilical artery diastolic flow in the IUGR twin permits the differentiation of clinical and prognostic groups. sIUGR type I has normal diastolic flow and relatively good outcome. Type II is defined by persistently absent/reverse end-diastolic flow and is associated with a high risk of intrauterine demise of the IUGR twin and/or very preterm delivery. Type III is defined by the presence of intermittent absent/reverse end-diastolic flow (iAREDF), and is associated with 10–20% risk of unexpected fetal demise of the smaller twin and 10–20% risk of neurological injury in the larger twin. The management strategy for sIUGR with abnormal umbilical artery Doppler (types II and III) remains a challenge, and may include elective fetal therapy or close surveillance with fetal therapy or elective delivery in the presence of severe fetal deterioration. Small clinical series reporting the use of cord occlusion or laser therapy in severe cases suggest that the outcome of the larger twin might be improved. There is probably no single optimal strategy, since decisions will ultimately be influenced by the severity of IUGR, gestational age, parents’ wishes and technical issues. [ABSTRACT FROM AUTHOR]

Background: Preeclampsia and fetal growth restriction share some pathophysiologic features and are both associated with placental insufficiency. Fetal cardiac remodeling has been described extensively in fetal growth restriction, whereas little is known about preeclampsia with a normally grown fetus.Objective: To describe fetal cardiac structure and function in pregnancies complicated by preeclampsia and/or fetal growth restriction as compared with uncomplicated pregnancies.Study Design: This was a prospective, observational study including pregnancies complicated by normotensive fetal growth restriction (n=36), preeclampsia with a normally grown fetus (n=35), preeclampsia with fetal growth restriction (preeclampsia with a normally grown fetus-fetal growth restriction, n=42), and 111 uncomplicated pregnancies matched by gestational age at ultrasound. Fetal echocardiography was performed at diagnosis for cases and recruitment for uncomplicated pregnancies. Cord blood concentrations of B-type natriuretic peptide and troponin I were measured at delivery. Univariate and multiple regression analysis were conducted.Results: Pregnancies complicated by preeclampsia and/or fetal growth restriction showed similar patterns of fetal cardiac remodeling with larger hearts (cardiothoracic ratio, median [interquartile range]: uncomplicated pregnancies 0.27 [0.23-0.29], fetal growth restriction 0.31 [0.26-0.34], preeclampsia with a normally grown fetus 0.31 [0.29-0.33), and preeclampsia with fetal growth restriction 0.28 [0.26-0.33]; P<.001) and more spherical right ventricles (right ventricular sphericity index: uncomplicated pregnancies 1.42 [1.25-1.72], fetal growth restriction 1.29 [1.22-1.72], preeclampsia with a normally grown fetus 1.30 [1.33-1.51], and preeclampsia with fetal growth restriction 1.35 [1.27-1.46]; P=.04) and hypertrophic ventricles (relative wall thickness: uncomplicated pregnancies 0.55 [0.48-0.61], fetal growth restriction 0.67 [0.58-0.8], preeclampsia with a normally grown fetus 0.68 [0.61-0.76], and preeclampsia with fetal growth restriction 0.66 [0.58-0.77]; P<.001). Signs of myocardial dysfunction also were observed, with increased myocardial performance index (uncomplicated pregnancies 0.78 z scores [0.32-1.41], fetal growth restriction 1.48 [0.97-2.08], preeclampsia with a normally grown fetus 1.15 [0.75-2.17], and preeclampsia with fetal growth restriction 0.45 [0.54-1.94]; P<.001) and greater cord blood B-type natriuretic peptide (uncomplicated pregnancies 14.2 [8.4-30.9] pg/mL, fetal growth restriction 20.8 [13.1-33.5] pg/mL, preeclampsia with a normally grown fetus 31.8 [16.4-45.8] pg/mL and preeclampsia with fetal growth restriction 37.9 [15.7-105.4] pg/mL; P<.001) and troponin I as compared with uncomplicated pregnancies.Conclusion: Fetuses of preeclamptic mothers, independently of their growth patterns, presented cardiovascular remodeling and dysfunction in a similar fashion to what has been previously described for fetal growth restriction. Future research is warranted to better elucidate the mechanism(s) underlying fetal cardiac adaptation in these conditions. [ABSTRACT FROM AUTHOR]

By consensus, late fetal growth restriction is that diagnosed >32 weeks. This condition is mildly associated with a higher risk of perinatal hypoxic events and suboptimal neurodevelopment. Histologically, it is characterized by the presence of uteroplacental vascular lesions (especially infarcts), although the incidence of such lesions is lower than in preterm fetal growth restriction. Screening procedures for fetal growth restriction need to identify small babies and then differentiate between those who are healthy and those who are pathologically small. First- or second-trimester screening strategies provide detection rates for late smallness for gestational age <50% for 10% of false positives. Compared to clinically indicated ultrasonography in the third trimester, universal screening triples the detection rate of late smallness for gestational age. As opposed to early third-trimester ultrasound, scanning late in pregnancy (around 37 weeks) increases the detection rate for birthweight <3rd centile. Contrary to early fetal growth restriction, umbilical artery Doppler velocimetry alone does not provide good differentiation between late smallness for gestational age and fetal growth restriction. A combination of biometric parameters (with severe smallness usually defined as estimated fetal weight or abdominal circumference <3rd centile) with Doppler criteria of placental insufficiency (either in the maternal [uterine Doppler] or fetal [cerebroplacental ratio] compartments) offers a classification tool that correlates with the risk for adverse perinatal outcome. There is no evidence that induction of late fetal growth restriction at term improves perinatal outcomes nor is it a cost-effective strategy, and it may increase neonatal admission when performed <38 weeks. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to assess whether abnormal cardiac function in human fetuses with intrauterine growth restriction (IUGR) is associated with ultrastructural differences in the cardiomyocyte sarcomere. Study Design: Nine severe early-onset IUGR fetuses and 9 normally grown fetuses (appropriate growth for gestational age) who died in the perinatal period were included prospectively. Cardiac function was assessed by echocardiography and levels of B-type natriuretic peptide and troponin-I. Heart sections were imaged by second harmonic generation microscopy, which allowed unstained visualization of cardiomyocyte's sarcomere length. Results: Echocardiographic and biochemical markers showed signs of severe cardiac dysfunction in IUGR fetuses. Second harmonic generation microscopy demonstrated a significantly shorter sarcomere length in IUGR as compared with appropriate growth for gestational age fetuses. Conclusion: IUGR is associated with changes in the cardiomyocyte contractile machinery in the form of shorter sarcomere length, which could help to explain the cardiac dysfunction previously documented in IUGR. [ABSTRACT FROM AUTHOR]

Objective: We sought to assess vascular structure and function in early- and late-onset preeclampsia (PE) at the time of diagnosis. Study Design: We evaluated 100 PE cases subdivided into 50 early- and 50 late-onset cases according to gestational age at onset (34 weeks), and 100 controls paired by maternal age and gestational age at scan with cases. Carotid intima-media thickness (IMT), distensibility, and circumferential wall stress together with inferior vena cava (IVC) collapsibility were assessed by ultrasound. Results: Early PE was characterized by increased carotid IMT diameters, and arterial stiffness with no significant changes in IVC parameters as compared to normotensive pregnancies. Late PE was characterized by significantly increased carotid IMT and lumen diameters as compared to controls while arterial stiffness, as expressed by distensibility, did not provide pronounced changes. A significant decrease of IVC collapsibility index was also observed in late PE as compared to controls. Conclusion: The current data suggest that distinct vascular adaptations in early and late PE could reflect different pathophysiologic mechanisms. Future studies are warranted to further assess the complex etiologies and clinical expressions of the 2 entities. [Copyright &y& Elsevier]

Objective: We sought to evaluate the effectiveness of an integrated first-trimester screening test to predict preeclampsia (PE). Study Design: A prospective cohort of singleton pregnancies underwent routine first-trimester screening from 2009 through 2011 (n = 5759). A logistic regression-based predictive model for early- and late-onset PE was constructed based on: maternal characteristics; levels of pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin at 8-12 weeks; and blood pressure and uterine artery Doppler at 11.0-13.6 weeks. Results: Of the 5170 enrolled participants, 136 (2.6%) developed PE (early PE: 26 [0.5%]; late PE: 110 [2.1%]). At 5% and 10% false-positive rates, detection rates were 69.2% and 80.8% for early PE (area under the curve, 0.95; 95% confidence interval, 0.94–0.98) and 29.4% and 39.6% for late PE (area under the curve, 0.71; 95% confidence interval, 0.66–0.76), respectively. Conclusion: First-trimester screening combining maternal factors with uterine artery Doppler, blood pressure, and pregnancy-associated plasma protein-A is useful to predict PE in a routine care setting. [ABSTRACT FROM AUTHOR]

Objective: The objective of the study was to evaluate cardiovascular function in children who were small-for-gestational-age (SGA) fetuses. Study Design: This was a prospective study including 100 controls and 50 children diagnosed in utero as SGA after 34 weeks subdivided into the following categories: SGA and intrauterine growth restriction (IUGR) according to the absence or presence, respectively, of weight centile less than 3 or abnormal cerebroplacental Doppler. Postnatal cardiovascular outcome was evaluated at 3-6 years of age by echocardiography, blood pressure, and carotid ultrasound. Results: Both SGA and IUGR presented in childhood more globular hearts, reduced longitudinal motion, and impaired relaxation with an increase in radial function. Both groups showed increased blood pressure and carotid intima-media thickness. There was a linear tendency to worse cardiovascular results in IUGR as compared with SGA. Conclusion: Fetal cardiovascular programming occurs in SGA, regardless of Doppler and weight centile. These findings challenge the concept of constitutionally small and warrant further investigation to identify predictors of cardiovascular outcome in SGA. [ABSTRACT FROM AUTHOR]

Objective: The objective of the study was to evaluate echocardiographic markers of cardiac dysfunction in small-for-gestational age (SGA) fetuses with normal umbilical artery Doppler. Study Design: Cardiac function was evaluated in 58 SGA (mean gestational age, 38 weeks) and 58 gestational-age matched normally grown fetuses by conventional echocardiography (peak early [E] and late [A] ratios and myocardial performance index [MPI]), and tissue Doppler imaging (TDI) (annular peak velocities and MPI''). Results: With conventional echocardiography, SGA fetuses had a nonsignificant trend to increased E/A ratios and left MPI compared with controls. TDI demonstrated that SGA fetuses had significantly lower right E'' and A'' peak velocities and higher MPI'' values. Conclusion: These findings further support that a proportion of SGA fetuses have true late-onset intrauterine growth restriction, which is associated with subclinical cardiac dysfunction, as previously described for early-onset intrauterine growth restriction. [Copyright &y& Elsevier]

Objective: The purpose of this study was to assess cardiac function and cell damage in intrauterine growth-restricted (IUGR) fetuses across clinical Doppler stages of deterioration. Study Design: One hundred twenty appropriate-for-gestational-age and 81 IUGR fetuses were classified in stages 1/2/3 according umbilical artery present/absent/reversed end-diastolic blood flow, respectively. Cardiac function was assessed by modified-myocardial performance index, early-to-late diastolic filling ratios, cardiac output, and cord blood B-type natriuretic peptide; myocardial cell damage was assessed by heart fatty acid–binding protein, troponin-I, and high-sensitivity C-reactive protein. Results: Modified-myocardial performance index, blood B-type natriuretic peptide, and early-to-late diastolic filling ratios were increased in a stage-dependent manner in IUGR fetuses, compared with appropriate-for-gestational-age fetuses. Heart fatty acid–binding protein levels were higher in IUGR fetuses at stage 3, compared with control fetuses. Cardiac output, troponin-I, and high-sensitivity C-reactive protein did not increase in IUGR fetuses at any stage. Conclusion: IUGR fetuses showed signs of cardiac dysfunction from early stages. Cardiac dysfunction deteriorates further with the progression of fetal compromise, together with the appearance of biochemical signs of cell damage. [Copyright &y& Elsevier]

Objective: To evaluate the brain venous circulation in fetuses with severe intrauterine growth restriction (IUGR) before 32 weeks of gestation. Study Design: Fifty fetuses with severe IUGR diagnosed between 27 and 32 weeks of gestation and 50 appropriate-for-gestational age (AGA) fetuses matched by gestational age were evaluated. IUGR fetuses were classified according to their hemodynamic deterioration pattern in relation to the Doppler examination of the umbilical artery (UA), middle cerebral artery (MCA) and ductus venosus (DV). The fetal venous brain blood flow was evaluated in the vein of Galen (VG), superior sagittal (SS), straight and transverse venous sinuses. Results: Only the transverse sinus (TS) showed a significant reduction in the pulsatility index (PI) values in IUGR fetuses. All other veins showed similar PI values between IUGR and AGA fetuses. All cerebral veins of IUGR fetuses showed significantly increased maximum and mean velocities. All these findings did not change in relation to the hemodynamic IUGR deterioration. In nearly all normal and all IUGR fetuses, a pulsatile blood flow pattern was observed in the straight and transverse sinuses, whereas an increased pulsatile pattern in the VG and in the SS was noted in IUGR fetuses. Conclusion: Brain venous blood flow in IUGR fetuses shows an increment in the maximum and mean velocities of all veins and a reduction in the PI in the transverse sinus. [ABSTRACT FROM AUTHOR]

Pregnancy is considered an important process, since different stressors during gestation could affect maternal mental health and fetal neurodevelopment. Stress is regulated by the steroid hormone cortisol. Although cortisol production increases along pregnancy, an excess has been associated with fetal neurotoxic complications. To study cortisol circadian rhythm changes throughout pregnancy and its association with sub-clinical maternal psychopathology. 112 healthy primiparous pregnant women collected four saliva samples throughout one day at 12th, 22th and 32th weeks of gestation to analyze cortisol. Stress perception and depressive symptomatology were evaluated following PSS and EPDS criteria, respectively. As expected, pregnant women showed increasing cortisol concentrations (AUCg) throughout the pregnancy (F=6.830,p=.002). However, women with risk for depression showed higher cortisol concentrations during pregnancy (F=7.566,p=.007) with a less pronounced decrease at the end of day at 12th and 22th weeks. Our study suggest that subclinical depressive symptoms at first half of pregnancy compromises maternal and fetal wellbeing due to excessive cortisol levels. [ABSTRACT FROM AUTHOR]

• A LC–MS/MS method for steroids quantification in alternative matrices is validated. • The steroids endogenous range in saliva, breast milk and amniotic fluid is reported. • Endogenous ranges of steroids in alternative matrices are reported for first time. The simultaneous determination of a broad panel of steroids provides more accurate information about the hormonal status than the detection of a single hormone. For that reason, the determination of the steroid profile, i.e. the endogenous steroid hormones and their main metabolites, has become the most powerful tool for the study of hormonal imbalances. The usefulness of the evaluation of the steroid profile in urine and plasma is widely accepted. However, despite its broad potential applicability, the evaluation of the whole steroid profile in alternative matrices such as amniotic fluid, saliva and breast milk remains almost unexplored. In this research we developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the quantification of several steroids and their metabolites in amniotic fluid (28 analytes), saliva (15) and breast milk (12). Sample preparation, chromatographic conditions and mass spectrometric conditions (e.g. ionization species or ion source parameters) were optimized. The method was shown to be linear in the range of endogenous concentrations for all studied metabolites. Intra- and inter-assay accuracies were between 80% and 120% while intra- and inter-precisions were below 20% for all analytes in all matrices. The applicability of the method was evaluated by the comparison between the concentration ranges obtained in healthy volunteers (n = 30 per matrix) and the scarce data previously reported in literature. The concentration ranges for several analytes are reported for the first time. The present methodology represents a useful tool for the comprehensive evaluation of the steroid profile in alternative matrices and can be applicable for different clinical purposes. [ABSTRACT FROM AUTHOR]