Proteomic biomarkers in seminal plasma as predictors of reproductive potential in azoospermic men

IntroductionAzoospermia, characterized by an absence of sperm in the ejaculate, represents the most severe form of male infertility. While surgical sperm retrieval in obstructive azoospermia (OA) is successful in the majority of cases, patients with non-obstructive azoospermia (NOA) show retrieval rates of only about 50% and thus frequently have unnecessary surgery. Surgical intervention could be avoided if patients without preserved spermatogenesis are identified preoperatively. This prospective study aimed to discover biomarkers in seminal plasma that could be employed for a non-invasive differential diagnosis of OA/NOA in order to rationalize surgery recommendations and improve success rates.MethodsAll patients signed written informed consent, underwent comprehensive andrological evaluation, received human genetics to exclude relevant pathologies, and patients with azoospermia underwent surgical sperm retrieval. Using label-free LC-MS/MS, we compared the proteomes of seminal plasma samples from fertile men (healthy controls (HC), n=8) and infertile men diagnosed with 1) OA (n=7), 2) NOA with successful sperm retrieval (mixed testicular atrophy (MTA), n=8), and 3) NOA without sperm retrieval (Sertoli cell-only phenotype (SCO), n=7). Relative abundance changes of two candidate markers of sperm retrieval, HSPA2 and LDHC, were confirmed by Western Blot.ResultsWe found the protein expression levels of 42 proteins to be significantly down-regulated (p ≤ 0.05) in seminal plasma from SCO NOA patients relative to HC whereas only one protein was down-regulated in seminal plasma from MTA patients. Analysis of tissue and cell expression suggested that the testis-specific proteins LDHC, PGK2, DPEP3, and germ-cell enriched heat-shock proteins HSPA2 and HSPA4L are promising biomarkers of spermatogenic function. Western blotting revealed a significantly lower abundance of LDHC and HSPA2 in the seminal plasma of men with NOA (SCO and MTA) compared to controls.DiscussionThe results indicate that certain testis-specific proteins when measured in seminal plasma, could serve as indicators of the presence of sperm in the testis and predict the success of sperm retrieval. Used in conjunction with conventional clinical assessments, these proteomic biomarkers may assist in the non-invasive diagnosis of idiopathic male infertility

Triose Phosphate Isomerase Deficiency Is Caused by Altered Dimerization–Not Catalytic Inactivity–of the Mutant Enzymes

Triosephosphate isomerase (TPI) deficiency is an autosomal recessive disorder caused by various mutations in the gene encoding the key glycolytic enzyme TPI. A drastic decrease in TPI activity and an increased level of its substrate, dihydroxyacetone phosphate, have been measured in unpurified cell extracts of affected individuals. These observations allowed concluding that the different mutations in the TPI alleles result in catalytically inactive enzymes. However, despite a high occurrence of TPI null alleles within several human populations, the frequency of this disorder is exceptionally rare. In order to address this apparent discrepancy, we generated a yeast model allowing us to perform comparative in vivo analyses of the enzymatic and functional properties of the different enzyme variants. We discovered that the majority of these variants exhibit no reduced catalytic activity per se. Instead, we observed, the dimerization behavior of TPI is influenced by the particular mutations investigated, and by the use of a potential alternative translation initiation site in the TPI gene. Additionally, we demonstrated that the overexpression of the most frequent TPI variant, Glu104Asp, which displays altered dimerization features, results in diminished endogenous TPI levels in mammalian cells. Thus, our results reveal that enzyme deregulation attributable to aberrant dimerization of TPI, rather than direct catalytic inactivation of the enzyme, underlies the pathogenesis of TPI deficiency. Finally, we discovered that yeast cells expressing a TPI variant exhibiting reduced catalytic activity are more resistant against oxidative stress caused by the thiol-oxidizing reagent diamide. This observed advantage might serve to explain the high allelic frequency of TPI null alleles detected among human populations

Modeling visual-based pitch, lift and speed control strategies in hoverflies

<div><p>To avoid crashing onto the floor, a free falling fly needs to trigger its wingbeats quickly and control the orientation of its thrust accurately and swiftly to stabilize its pitch and hence its speed. Behavioural data have suggested that the vertical optic flow produced by the fall and crossing the visual field plays a key role in this anti-crash response. Free fall behavior analyses have also suggested that flying insect may not rely on graviception to stabilize their flight. Based on these two assumptions, we have developed a model which accounts for hoverflies´ position and pitch orientation recorded in 3D with a fast stereo camera during experimental free falls. Our dynamic model shows that optic flow-based control combined with closed-loop control of the pitch suffice to stabilize the flight properly. In addition, our model sheds a new light on the visual-based feedback control of fly´s pitch, lift and thrust. Since graviceptive cues are possibly not used by flying insects, the use of a vertical reference to control the pitch is discussed, based on the results obtained on a complete dynamic model of a virtual fly falling in a textured corridor. This model would provide a useful tool for understanding more clearly how insects may or not estimate their absolute attitude.</p></div

Uropathogenic E. coli Induce Different Immune Response in Testicular and Peritoneal Macrophages: Implications for Testicular Immune Privilege

Infertility affects one in seven couples and ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male factor infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of importance. Whole genome expression profiling of TM and peritoneal macrophages (PM) infected with uropathogenic E. coli (UPEC) revealed major differences in regulated genes. However, a multitude of genes implicated in calcium signaling pathways was a common feature which indicated a role of calcium-dependent nuclear factor of activated T cells (NFAT) signaling. UPEC-dependent NFAT activation was confirmed in both cultured TM and in TM in an in vivo UPEC infectious rat orchitis model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by rapid influx of calcium, an activity delineated to the pore forming toxin alpha-hemolysin by bacterial mutant analysis. Alpha-hemolysin suppressed IL-6 and TNF-α cytokine release from PM and caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to reciprocal expression of key pro-inflammatory cytokines in PM (IL-1α, IL-1β, IL-6 downregulated) and TM (IL-1β, IL-6 upregulated). In addition, unlike PM, LPS-treated TM were refractory to NFκB activation shown by the absence of degradation of IκBα and lack of pro-inflammatory cytokine secretion (IL-6, TNF-α). Taken together, these results suggest a mechanism to the conundrum by which TM initiate immune responses to bacteria, while maintaining testicular immune privilege with its ability to tolerate neo-autoantigens expressed on developing spermatogenic cells

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Improved risk stratification of patients with atrial fibrillation: an integrated GARFIELD-AF tool for the prediction of mortality, stroke and bleed in patients with and without anticoagulation.

OBJECTIVES: To provide an accurate, web-based tool for stratifying patients with atrial fibrillation to facilitate decisions on the potential benefits/risks of anticoagulation, based on mortality, stroke and bleeding risks. DESIGN: The new tool was developed, using stepwise regression, for all and then applied to lower risk patients. C-statistics were compared with CHA2DS2-VASc using 30-fold cross-validation to control for overfitting. External validation was undertaken in an independent dataset, Outcome Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF). PARTICIPANTS: Data from 39 898 patients enrolled in the prospective GARFIELD-AF registry provided the basis for deriving and validating an integrated risk tool to predict stroke risk, mortality and bleeding risk. RESULTS: The discriminatory value of the GARFIELD-AF risk model was superior to CHA2DS2-VASc for patients with or without anticoagulation. C-statistics (95% CI) for all-cause mortality, ischaemic stroke/systemic embolism and haemorrhagic stroke/major bleeding (treated patients) were: 0.77 (0.76 to 0.78), 0.69 (0.67 to 0.71) and 0.66 (0.62 to 0.69), respectively, for the GARFIELD-AF risk models, and 0.66 (0.64-0.67), 0.64 (0.61-0.66) and 0.64 (0.61-0.68), respectively, for CHA2DS2-VASc (or HAS-BLED for bleeding). In very low to low risk patients (CHA2DS2-VASc 0 or 1 (men) and 1 or 2 (women)), the CHA2DS2-VASc and HAS-BLED (for bleeding) scores offered weak discriminatory value for mortality, stroke/systemic embolism and major bleeding. C-statistics for the GARFIELD-AF risk tool were 0.69 (0.64 to 0.75), 0.65 (0.56 to 0.73) and 0.60 (0.47 to 0.73) for each end point, respectively, versus 0.50 (0.45 to 0.55), 0.59 (0.50 to 0.67) and 0.55 (0.53 to 0.56) for CHA2DS2-VASc (or HAS-BLED for bleeding). Upon validation in the ORBIT-AF population, C-statistics showed that the GARFIELD-AF risk tool was effective for predicting 1-year all-cause mortality using the full and simplified model for all-cause mortality: C-statistics 0.75 (0.73 to 0.77) and 0.75 (0.73 to 0.77), respectively, and for predicting for any stroke or systemic embolism over 1 year, C-statistics 0.68 (0.62 to 0.74). CONCLUSIONS: Performance of the GARFIELD-AF risk tool was superior to CHA2DS2-VASc in predicting stroke and mortality and superior to HAS-BLED for bleeding, overall and in lower risk patients. The GARFIELD-AF tool has the potential for incorporation in routine electronic systems, and for the first time, permits simultaneous evaluation of ischaemic stroke, mortality and bleeding risks. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier for GARFIELD-AF (NCT01090362) and for ORBIT-AF (NCT01165710)

Two-year outcomes of patients with newly diagnosed atrial fibrillation: results from GARFIELD-AF.

AIMS: The relationship between outcomes and time after diagnosis for patients with non-valvular atrial fibrillation (NVAF) is poorly defined, especially beyond the first year. METHODS AND RESULTS: GARFIELD-AF is an ongoing, global observational study of adults with newly diagnosed NVAF. Two-year outcomes of 17 162 patients prospectively enrolled in GARFIELD-AF were analysed in light of baseline characteristics, risk profiles for stroke/systemic embolism (SE), and antithrombotic therapy. The mean (standard deviation) age was 69.8 (11.4) years, 43.8% were women, and the mean CHA2DS2-VASc score was 3.3 (1.6); 60.8% of patients were prescribed anticoagulant therapy with/without antiplatelet (AP) therapy, 27.4% AP monotherapy, and 11.8% no antithrombotic therapy. At 2-year follow-up, all-cause mortality, stroke/SE, and major bleeding had occurred at a rate (95% confidence interval) of 3.83 (3.62; 4.05), 1.25 (1.13; 1.38), and 0.70 (0.62; 0.81) per 100 person-years, respectively. Rates for all three major events were highest during the first 4 months. Congestive heart failure, acute coronary syndromes, sudden/unwitnessed death, malignancy, respiratory failure, and infection/sepsis accounted for 65% of all known causes of death and strokes for <10%. Anticoagulant treatment was associated with a 35% lower risk of death. CONCLUSION: The most frequent of the three major outcome measures was death, whose most common causes are not known to be significantly influenced by anticoagulation. This suggests that a more comprehensive approach to the management of NVAF may be needed to improve outcome. This could include, in addition to anticoagulation, interventions targeting modifiable, cause-specific risk factors for death. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362