STUDY QUESTION:Can gonadotrophin receptor variants separately or in combination, be used for the prediction of pregnancy chances in in vitro fertilization (IVF) trials?SUMMARY ANSWER:The luteinizing hormone/human chorionic gonadotrophin receptor (LHCGR) variant N312S and the follicle-stimulating hormone receptor (FSHR) variant N680S can be utilized for the prediction of pregnancy chances in women undergoing IVF.WHAT IS KNOWN ALREADY:The FSHR N680S polymorphism has been shown to affect the ovarian response in response to gonadotrophin treatment, while no information is currently available regarding variants of the LHCGR in this context.STUDY DESIGN, SIZE, DURATION:Cross-sectional study, duration from September 2010 to February 2015. Women undergoing IVF were consecutively enrolled and genetic variants compared between those who became pregnant and those who did not. The study was subsequently replicated in an independent sample. Granulosa cells from a subset of women were investigated regarding functionality of the genetic variants.PARTICIPANTS/MATERIALS, SETTING, METHODS:Women undergoing IVF (n = 384) were enrolled in the study and genotyped. Clinical variables were retrieved from medical records. For replication, an additional group of n = 233 women was utilized. Granulosa cells from n = 135 women were isolated by flow cytometry, stimulated with Follitropin alpha or Menotropin, and the downstream targets 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) measured with enzyme-linked immunosorbent assay.MAIN RESULTS AND THE ROLE OF CHANCE:Women homozygous for serine (S) in both polymorphisms displayed higher pregnancy rates than women homozygous asparagine (N) (OR = 14.4, 95% CI: [1.65, 126], P = 0.016). Higher pregnancy rates were also evident for women carrying LHCGR S312, regardless of FSHR variant (OR = 1.61, 95% CI: [1.13, 2.29], P = 0.008). These women required higher doses of FSH for follicle recruitment than women homozygous N (161 versus 148 IU, P = 0.030). When combining the study cohort with the replication cohort (n = 606), even stronger associations with pregnancy rates were noted for the combined genotypes (OR = 11.5, 95% CI: [1.86, 71.0], P = 0.009) and for women carrying LHCGR S312 (OR = 1.49, 95% CI: [1.14, 1.96], P = 0.004). A linear significant trend with pregnancy rate and increasing number of G alleles was also evident in the merged study population (OR = 1.34, 95% CI: [1.10, 1.64], P = 0.004). A lower cAMP response in granulosa cells was noted following Follitropin alpha stimulation for women homozygous N in both polymorphisms, compared with women with other genotypes (0.901 pmol cAMP/mg total protein versus 2.19 pmol cAMP/mg total protein, P = 0.035).LIMITATIONS, REASONS FOR CAUTION:Due to racial differences in LHCGR genotype distribution, these results may not be applicable for all populations.WIDER IMPLICATIONS OF THE FINDINGS:Despite that >250 000 cycles of gonadotrophin stimulations are performed annually worldwide prior to IVF, it has not been possible to predict neither the pregnancy outcome, nor the response to the hormone with accuracy. If LHCGR and FSHR variants are recognized as biomarkers for chance of pregnancy, more individualized and thereby more efficient treatment modalities can be developed.STUDY FUNDING, COMPETING INTERESTS:This work was supported by Interreg IV A, EU (grant 167158) and ALF governments grant (F2014/354). Merck-Serono (Darmstadt, Germany) supported the enrollment of the subjects. The authors declare no conflict of interest.

AIMS/HYPOTHESIS:Mutations in the hepatocyte nuclear factor 1-alpha gene (HNF-1alpha, now known as the transcription factor 1 gene [TCF1]) cause the most common monogenic form of diabetes, MODY3, but it is not known if common variants in HNF-1a are associated with decreased transcriptional activity or phenotypes related to type 2 diabetes, or whether they predict future type 2 diabetes.SUBJECTS AND METHODS:We studied the effect of four common polymorphisms (rs1920792, I27L, A98V and S487N) in and upstream of the HNF-1alpha gene on transcriptional activity in vitro, and their possible association with type 2 diabetes and insulin secretion in vivo.RESULTS:Certain combinations of the I27L and A98V polymorphisms in the HNF-1alpha gene showed decreased transcriptional activity on the target promoters glucose transporter 2 (now known as solute carrier family 2 [facilitated glucose transporter], member 2) and albumin in both HeLa and INS-1 cells. In vivo, these polymorphisms were associated with a modest but significant impairment in insulin secretion in response to oral glucose. Insulin secretion deteriorated over time in individuals carrying the V allele of the A98V polymorphism (n = 2,293; p = 0.003). In a new case-control (n = 1,511 and n = 2,225 respectively) data set, the I27L polymorphism was associated with increased risk of type 2 diabetes, odds ratio (OR) = 1.5 (p = 0.002; multiple logistic regression), particularly in elderly (age > 60 years) and overweight (BMI > 25 kg/m(2)) patients (OR = 2.3, p = 0.002).CONCLUSIONS/INTERPRETATION:This study provides in vitro and in vivo evidence that common variants in the MODY3 gene, HNF-1alpha, influence transcriptional activity and insulin secretion in vivo. These variants are associated with a modestly increased risk of late-onset type 2 diabetes in subsets of elderly overweight individuals.

Background: In a cohort study of preterm infants increased plasma concentrations of proinflammatory cytokines were associated with premature rupture of membranes, arterial hypotension and cerebral damage. Objective: To evaluate in a follow-up if early postnatal levels of cytokines in preterm infants predict neuro-cognitive outcome at 2 years of age. Methods: A prospective cohort study of 71 surviving infants <32 gestational weeks. Plasma concentrations of proinflammatory (TNF-alfa, IL-1beta, IFN-gamma, IL-2, IL-6, IL-8, IL-12) and modulatory (IL-4, IL-10) cytokines were analyzed in umbilical cord and at 6, 24 and 72 h age. Intraventricular hemorrhage (IVH) and white matter brain damage (WMD) was assessed with ultrasound. Neuro-cognitive outcome at 24 months corrected age was evaluated using Bayley Scales of Infant Development (Mental Developmental Index (MDI) and Psychomotor Developmental Index (PDI)) and a standardized neurological examination (Neurological Optimality Score (NOS)). Results: 64 infants (mean gestational age (GA) 27.1 wks) were assessed at 24 (0.5) months (mean, SD). NOS correlated with both MDI and PDI (r=0.60 p<0.001 and r=0.70 p<0.001 respectively). GA correlated with MDI, PDI and NOS (r=0.34 p=0.006, r=0.42 p=0.001 and r=0.40, p=0.001 respectively). Male gender had lower NOS (p=0.03). Cord levels of the proinflammatory cytokineTNF-alfa were inversely correlated with PDI (r=-0.33 p=0.014) and levels of the modulatory cytokine IL-10 at 24 h correlated positively with PDI and NOS (r=0.42 p=0.001 and r=0.32, p=0.015 respectively) after adjustment for GA and gender. Infants with IVH grade III/IV or WMD (n=8) had lower median (range) MDI and NOS, 74 (50–106) and 63 (36–76), respectively, as compared to infants without brain injury (n=56): 88 (50–116) and 71 (58–78), respectively. Differences remained after adjustment for GA, gender and parental education. Conclusion: An early inflammatory response in preterm infants is associated with perinatal brain injury and might be prognostic for adverse neuro-cognitive outcome at 2 years of age.

Background: Increased proinflammatory activity is associated with oxidative stress, hemodynamic impairment and tissue damage. Cystein is an essential amino acid in preterm infants and metabolized to glutathione which is one of the most important intracellular antioxidant systems. Plasma levels of isoprostane are considered a stable marker of oxidative stress. Aim: To evaluate if plasma levels of cystein and isoprostane at 6 h of age are associated with proinflammatory activity and with subsequent morbidity in preterm infants. Methods: A two year prospective cohort study including inborn infants delivered at < 32 +0 gestational weeks after antenatal informed consent and excluding infants with major anomalies. 74 infants were enrolled with a mean (SD) gestational age of 27.1 (1.9) weeks. Blood sampling for analysis of proinflammatory (TNFá, IL-1 â, IL-2, IL-6, IL-8, IL-12, IFN- ã) and modulatory (IL-4, IL-10) cytokines was performed from umbilical cord and at 6 h postnatal age. Plasma levels of cystein and isoprostane were determined at 6 h postnatal age. Continuous invasive measurement of arterial blood pressure (ABP) was digitally stored during the first 72 h. Ultrasound examinations of the brain were performed at day 1, 3 and 7, at 6 weeks and at term age. Results: Levels of cystein in plasma at 6 h of age were positively related to gestational age at birth (r=0.46, p=0.001). Increased levels of IFN-ã, TNFá, IL-1â, IL-6 and IL-12 at 6 h were associated with a decrease in level of cystein (r=−0.37, p=0.002; r= -0.25, p=0.04; r=−0.45, p=0.000; r=−0.29, p=0.02 and r=−0.28, p=0.02 respectively). Level of cystein at 6h was positively related to average of mean ABP (0– 6 h), r=0.38, p=0.004. Decreased levels of cystein at 6 h were associated with development of severe IVH (grade III + IV), OR (95 % CI) 0.93 (0.88– 0.97), p=0.000. All associations remained significant after adjustment for gestational age and gender. Levels of isoprostane at 6 h were neither associated with cytokine levels in umbilical cord nor with those at 6h of age. Conclusion: Increased proinflammatory activity, arterial hypotension and development of severe IVH are associated with low levels of cystein early after birth in preterm infants.

Background: Increased levels of umbilical cord proinflammatory cytokines are associated with early and late neonatal morbidity. Extremely preterm male infants need more circulatory and ventilatory support than female infants, and have worse outcome. Experimental interventions which include administration of interleukin-1 receptor antagonist (IL1-ra) improves cerebral outcome after neonatal brain injury, and stabilise arterial blood pressure after experimental sepsis. Furthermore, IL-1ra is increased in amniotic fluid and neonatal urine from female fetuses/newborns. We tested the hypothesis that umbilical cord IL-1ra is related to infant gender, postnatal blood pressure and neonatal morbidity.Method: Blood from the umbilical cord of 58 infants (33 male, 25 female) with gestational age <32 weeks was sampled at birth. Receiver operating characteristics (ROC-curve) were used for identifying IL-1ra values with high sensitivity and specificity for “poor outcome”. “Poor outcome” was defined as either death (n=3), grade 3–4 IVH or cystic PVL (n= 9). A cut-off for IL-1ra at 13500 pg/ml rendered an area under the curve (AUC) = 0.735, p-value= 0.013, with 67% sensitivity and 85% specificity for “poor outcome”.Results: There was no correlation between IL-1ra and newborn gender, blood pressure during the first 72 hours or need for inotrope support. There were significant correlations (p-value; rs) between IL-1ra and a) depression at birth, including Apgar at 1 minute (0.020; −0.305) and 10 minutes (0.020; − 0.313) and, b) age at intubation during the first 12 hours (0.011; −0.330). Besides the association with “poor outcome”, IL-1ra values above 13500 pg/ml were associated with other parameters of neonatal morbidity such as development of NEC (p= 0.015), sepsis with positive blood culture (p= 0.027), and need for extra oxygen at 36 weeks (p= 0.033). There were no significant correlations between IL1-ra >13500 pg/ml and development of ROP (p= 0.096), days with mechanical ventilation (p= 0.098), or clinical seizures (p= 0.220).Conclusion: IL-1ra levels above 13500 pg/ml in umbilical cord blood are associated with depression at birth, need for early intubation and severe neonatal morbidity.

Background:: The fetal inflammatory response has been suggested causal in acute neonatal and chronic neurological morbidity. Insulin-like growth factor 1 (IGF-1) is essential for tissue growth and has protective properties after induced ischemia. Knowledge of the interaction between induced inflammation and the components of the IGF-1 system may be of benefit for therapeutic strategies aiming at promoting growth and tissue protection in preterm infants. Aim: To evaluate the effect of increased levels of proinflammatory cytokines and early nutrition on levels of IGF-1, binding protein –3 (BP-3) and high and low phosphorylated BP-1 (hp BP-1, lp BP-1) in umbilical cord blood and at 72 h of age in preterm infants. Methods:: A two year prospective cohort study including inborn infants delivered at <32 gestational weeks after antenatal informed consent and excluding infants with major anomalies. 74 infants were enrolled with a mean (SD) gestational age of 27.1 (1.9) weeks. Blood sampling for analysis of proinflammatory (TNF-alpha, IL-1, IL-2, IL-6, IL-8, IL-12, IFN-gamma) cytokines was performed from umbilical cord and at 6, 24 and 72 h postnatal age. Levels of IGF-1, BP-3, hp- and lp BP-1 were determined in cord blood and at 72 h of age. Enteral and parenteral protein- and caloric intake was prospectively registered during the first 3 days of life. Results:: Increased levels of IL-8 and IL-6 were associated with a decrease in IGF-1 in umbilical cord blood, r= −0.31 (p=0.017) and r = −0.25 (p= 0.03) respectively, and with an increase in hp BP-1, r = 0.39 (p= 0.001) and r= 0.49 (p= 0.000) and in lp BP-1, r = 0.38 (p= 0.001) and r= 0.39 (p= 0.002). These associations remained significant after adjustment for gestational age, gender and birthweight. Levels of IGF-1, BP-3 and lf BP-1 at 72 h were decreased by 34, 29 and 50 % (median) as compared to those in the umbilical cord (p<0.001 respectively) whereas hf BP-1 remained unchanged. Parenteral- and enteral protein and caloric intake (0–72 h) were not associated with levels of IGF-1, hp - or lp BP-1 at 72 h whereas BP-3 at 72 h was inversely associated with total caloric intake, r= −0.44, p= 0.000. Conclusion:: Fetal proinflammatory activity is associated with decreased circulating levels of IGF-1 and an increase in hp- and lp BP-1 which may reflect a decreased protective capacity of the endogenous IGF system. Early enteral or parenteral nutrition does not appear to modify the decrease in circulating levels of IGF-1 resulting from preterm birth.

Background: Antenatal inflammation elicits a fetal inflammatory response which has been suggested causal in acute neonatal and chronic neurological morbidity. Knowledge of temporal changes in levels of proinflammatory and modulatory cytokines during the transition from fetal to neonatal life may improve understanding of the possible relationship between inflammatory response, circulatory impairment and subsequent brain damage in preterm infants. Objective: To evaluate temporal profiles of proinflammatory and modulatory cytokines in fetal and neonatal blood and determine their relationship to arterial hypotension and morphological brain damage in preterm infants. Methods: A two year prospective cohort study including inborn infants delivered at <32 gestational weeks after antenatal informed consent and excluding infants with major anomalies. 74 infants were enrolled with a mean (SD) gestational age of 27.1 (1.9 ) weeks. Blood sampling for fluorocytometric analysis of proinflammatory (TNF-á, IL-1, IL-2, IL-6, IL-8, IL-12, IFN-ã) and modulatory (IL-4, IL-10) cytokines was performed from umbilical cord and at 6, 24 and 72 h postnatal age. Continuous invasive measurement of arterial blood pressure (ABP) was digitally stored during the first 72 h. Ultrasound examinations of the brain were performed at day 1, 3 and 7, at 6 weeks and at term age. Results: Increased levels of IL-6 at 6h and IL-8 at 24 h were associated with a decrease in mean ABP during the first 72 h (r2=0.28, p=0.002 and r2=0.36, p=0.001 respectively). Level of IL-8 > 85 pg/ml at 6 h was predictive of dopamine treatment for arterial hypotension during the first 72 h (sensitivity 79%, specificity 73%). Infants who developed white matter brain damage (WMD) on ultrasound had increased mean levels of IFN- in umbilical cord and at 6, 24 and 72 hours as compared to those without WMD (p= 0.016, 0.027, 0.003 and 0.022 respectively). MABP during the first 72 h was not associated with development of WMD. Conclusion: An early postnatal increase in levels of IL-6 and IL-8 was strongly associated with arterial hypotension requiring treatment in preterm infants. However, neither arterial hypotension nor increases in IL-6 or IL-8 were related to development of WMD as defined by ultrasound. Levels of IFN-ã were increased in cord blood and up to 72 h in infants who developed WMD. This suggests an early induction of inflammation with the main part of the inflammatory response taking place before birth in infants developing WMD.

Aims/hypothesis. Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1 α . In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1 α gene. Methods. Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments. Results. Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1 α in HeLa cells lacking HNF1 α . The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1 α suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1α protein showed normal nuclear targeting but impaired binding to an HNF1 α consensus sequence. Conclusion/interpretation. Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism.