Low-molecular weight compounds in human seminal plasma as potential biomarkers of male infertility.

STUDY QUESTION: Is the determination of antioxidants, oxidative/nitrosative stress-related compounds, purines, pyrimidines and energy-related metabolites in human seminal plasma of utility to evidence biomarkers related to male infertility? SUMMARY ANSWER: The determination of 26 metabolites in seminal plasma allowed to evidence that 21/26 of them are biomarkers of male infertility, as well as to calculate a cumulative index, named Biomarker Score, that fully discriminates fertile controls from infertile patients and partially differentiates infertile without from infertile with spermiogram anomalies. WHAT IS KNOWN ALREADY: Epidemiological studies indicated that a male factor is involved in ~50% of cases of pregnancy failure, with a significant percentage of infertile males having no alterations in the spermiogram. Further laboratory analyses of male infertility are mainly dedicated only to gross evaluations of oxidative stress or total antioxidant capacity. STUDY DESIGN, SIZE, DURATION: Seminal plasma of 48 fertile controls and 96 infertile patients (master group), were collected from September 2016 to February 2018. A second group of 44 infertile patients (validation group) was recruited in a second, independent centre from September 2017 to March 2018. Samples were analysed in blind using a 'Redox Energy Test' to determine various low-molecular weight compounds, with the aim of finding metabolic profiles and biomarkers related to male infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS: In all seminal plasma, 26 water- and fat-soluble compounds (related to antioxidant defences, oxidative/nitrosative stress, purine, pyrimidine and energy metabolism) were analysed using high-performance liquid chromatographic methods. According to spermiogram, infertile patients of both groups were also categorized into normozoospermic (N, no anomalies in the spermiogram), or into the subgroup including all patients with anomalies in the spermiogram (asthenoteratooligozoospermic ATO + asthenozoospermic A + teratozoospermic T + oligozoospermic O). MAIN RESULTS AND THE ROLE OF CHANCE: In the master group, results indicated that 21/26 compounds assayed in seminal plasma of infertile males were significantly different from corresponding values determined in fertile controls. These 21 compounds constituted the male infertility biomarkers. Similar results were recorded in patients of the validation group. Using an index cumulating the biochemical seminal plasma anomalies (Biomarker Score), we found that fertile controls had mean Biomarker Score values of 2.01 ± 1.42, whilst infertile patients of the master and of the validation group had mean values of 12.27 ± 3.15 and of 11.41 ± 4.09, respectively (P < 0.001 compared to controls). The lack of statistical differences between the master and the validation groups, in both the metabolic profiles and the Biomarker Score values, allowed to pool patients into a single cohort of infertile males. The Biomarker Score values showed that fertile controls and infertile males clustered into two distinct groups. Infertile patients without (N, n = 42) or with (ATO + A + T + O, n = 98) spermiogram anomalies differed in some biomarkers (ascorbic acid, all-trans retinol, α-tocopherol, cytidine, uridine, guanine). These differences were reinforced by distribution frequencies and posterior probability curves of the Biomarker Score in the three groups. LIMITATIONS, REASONS FOR CAUTION: Results were obtained in relatively limited number of human seminal plasma samples. Using the 'Redox Energy Test' it was possible to associate specific metabolic profiles and values of the Biomarker Score to fertile controls or infertile males. However, it was not possible to evaluate whether the different anomalies of the spermiogram are associated with specific metabolic profiles and values of the Biomarker Score. WIDER IMPLICATIONS OF THE FINDINGS: The 'Redox Energy Test', coupled with the Biomarker Score that cumulates the biochemical characteristics of seminal plasma into a single index, evidenced a set of low-molecular weight biomarkers potentially useful in the laboratory management of male infertility. STUDY FUNDING/COMPETING INTEREST(S): The study was partly funded with research grants from the University of Catania. None of the authors have any conflicting interests to declare.

Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix.

The demonstration that type 2 transglutaminase (TG2) can incorporate polyamine into the E7 oncoprotein of human papillomavirus (HPV) type 18 has led to the hypothesis that TG2 can have a role in the host cellular response to HPV infection. The aim of this study was to investigate whether HPV-related pathology, in infected human cervical epithelium, was associated with modulation of TG2 expression. Normal controls and HPV-infected cervical biopsies were analyzed for the expression of TG2, and the findings were compared with lesion grade. The correlation between TG2 expression and p16, a marker for HPV-induced dysplasia, and the retinoblastoma protein (Rb), a target of the E7 protein of HPV, was also investigated. Results obtained showed that TG2 was absent in normal squamous mucosa, whereas it was present in 100% CIN I lesions. Low-grade lesions showed significantly higher TG2 expression than high-grade lesions (P<0.0001). In 94% of CIN I more than 50% of the cells were positive for TG2, with a strong staining intensity (+3), whereas a decreased staining intensity and a low number of positive cells were found in CIN II/III. In CIN I cases, both nuclear and cytoplasmic staining were found in cells exhibiting classical morphological features of HPV infection. In addition, during progression from low-grade squamous intraepithelial lesions to severe dysplasia, TG2 expression was inversely correlated with p16 (Pearson: -0.930), whereas a positive correlation was observed between the expression of TG2 and pRb (Pearson: 0.997). TG2 is expressed in HPV infection as an early phenomenon, not restricted to high-risk genotypes. TG2 upregulation is probably part of host cell reaction against HPV-induced tissue modification. It may act as a cellular antioxidant defense factor, playing an important role in counteracting oxidative damage in neoplastic disease.

Bioreactor Platform for Biomimetic Culture and in situ Monitoring of the Mechanical Response of in vitro Engineered Models of Cardiac Tissue.

In the past two decades, relevant advances have been made in the generation of engineered cardiac constructs to be used as functional in vitro models for cardiac research or drug testing, and with the ultimate but still challenging goal of repairing the damaged myocardium. To support cardiac tissue generation and maturation in vitro, the application of biomimetic physical stimuli within dedicated bioreactors is crucial. In particular, cardiac-like mechanical stimulation has been demonstrated to promote development and maturation of cardiac tissue models. Here, we developed an automated bioreactor platform for tunable cyclic stretch and in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. To demonstrate the bioreactor platform performance and to investigate the effects of cyclic stretch on construct maturation and contractility, we developed 3D annular cardiac tissue models based on neonatal rat cardiac cells embedded in fibrin hydrogel. The constructs were statically pre-cultured for 5 days and then exposed to 4 days of uniaxial cyclic stretch (sinusoidal waveform, 10% strain, 1 Hz) within the bioreactor. Explanatory biological tests showed that cyclic stretch promoted cardiomyocyte alignment, maintenance, and maturation, with enhanced expression of typical mature cardiac markers compared to static controls. Moreover, in situ monitoring showed increasing passive force of the constructs along the dynamic culture. Finally, only the stretched constructs were responsive to external electrical pacing with synchronous and regular contractile activity, further confirming that cyclic stretching was instrumental for their functional maturation. This study shows that the proposed bioreactor platform is a reliable device for cyclic stretch culture and in situ monitoring of the passive mechanical response of the cultured constructs. The innovative feature of acquiring passive force measurements in situ and along the culture allows monitoring the construct maturation trend without interrupting the culture, making the proposed device a powerful tool for in vitro investigation and ultimately production of functional engineered cardiac constructs.

Application of 3D Printing Technology for Design and Manufacturing of Customized Components for a Mechanical Stretching Bioreactor.

Three-dimensional (3D) printing represents a key technology for rapid prototyping, allowing easy, rapid, and low-cost fabrication. In this work, 3D printing was applied for the in-house production of customized components of a mechanical stretching bioreactor with potential application for cardiac tissue engineering and mechanobiology studies. The culture chamber housing and the motor housing were developed as functional permanent parts, aimed at fixing the culture chamber position and at guaranteeing motor watertightness, respectively. Innovative sample holder prototypes were specifically designed and 3D-printed for holding thin and soft biological samples during cyclic stretch culture. The manufactured components were tested in-house and in a cell biology laboratory. Moreover, tensile tests and finite element analysis were performed to investigate the gripping performance of the sample holder prototypes. All the components showed suitable performances in terms of design, ease of use, and functionality. Based on 3D printing, the bioreactor optimization was completely performed in-house, from design to fabrication, enabling customization freedom, strict design-to-prototype timing, and cost and time effective testing, finally boosting the bioreactor development process.

The combined role of sinuses of Valsalva and flow pulsatility improves energy loss of the aortic valve.

OBJECTIVES: Normal aortic valve opening and closing movement is a complex mechanism mainly regulated by the blood flow characteristics and the cyclic modifications of the aortic root. Our previous in vitro observations demonstrated that the presence of the Valsalva sinuses, independently from root compliance, is important in reducing systolic pressure drop across the aortic valve. This in vitro study was designed to ascertain if this effect is dependent on the flow characteristics. METHODS: Stentless 21, 23 and 25 mm aortic prostheses were sutured inside Dacron graft with and without sinuses. Hydrodynamic performance of the root models was investigated in steady-state (continuous) and unsteady-state (pulsatile) flow regimes. Aortic transvalvular pressure drop and effective orifice area (EOA) were evaluated. RESULTS: The continuous flow analysis revealed that no marked differences in pressure drop characterized the two root configurations at flow regimes lower than 15 l/min, independently of valve size. Conversely, at higher flow regimes (up to 30 l/min) a relatively low pressure drop continued to characterize grafts with sinuses, whereas marked increments in pressure drop were measured in straight grafts, especially in the smaller size (77.05 ± 4.58 vs 23.80 ± 2.44 mmHg; 18.40 ± 1.31 vs 7.66 ± 0.37 mmHg and 29.54 ± 0.17 vs 7.12 ± 0.07 mmHg, for 21, 23 and 25 mm valve, respectively). Under pulsatile conditions, the presence of sinuses clearly confirmed lower pressure drops also more evident in the smaller valve sizes (53.89 ± 1.06 vs 11.6 ± 0.24 mmHg at 7 l/min for 21 mm valve). EOA values were always lower in the absence of sinuses. In continuous flow regimes, at 30 l/min EOA of 25 mm valve size was 3.67 ± 0.02 cm(2) in the Valsalva model versus 1.79 ± 0.01 cm(2) for the Straight model. In pulsatile tests, at 7 l/min a 25-valve size demonstrated an EOA of 5.47 ± 0.60 in the Valsalva model versus 2.50 ± 0.02 cm(2) in the Straight model. CONCLUSIONS: These findings (i) confirm the hypothesis that the sinuses of Valsalva play a key role in optimizing the aortic haemodynamics during systole, minimizing energy losses; (ii) suggest that the sinuses of Valsalva are needed because of the complex nature of blood flow during ejection.

Role of the sinuses of Valsalva on the opening of the aortic valve.

OBJECTIVE: The present in vitro study was designed to ascertain whether the presence of sinuses of Valsalva in the aortic root were able to regulate the valve effective orifice area and modulate the gradient across the valve independently from root compliance. METHODS: Four different root configurations were prepared. Of the 4, 2 were silicon configurations with the same compliance, 1 with and 1 without sinuses of Valsalva, in which a 25-mm Solo stentless aortic valve was sutured inside. The other 2 configurations were obtained by substituting the upper part of the root with a straight Dacron graft or with a Valsalva graft in a remodeling fashion to reproduce the surgical situation. All roots were mounted in a pulse duplicator to measure the pressure decrease across the valve and effective orifice area at different cardiac outputs. RESULTS: With increasing cardiac output up to 7 L/min, an increase in the pressure decrease across the valve was evident in both configurations without sinuses of Valsalva (7.90 ± 1.7 and 11 mm Hg ± 0.1 mm Hg, respectively) but not in those with sinuses (2.87 ± 0.5 and 2.42 mm Hg ± 0.5 mm Hg). Similarly, with increasing cardiac output, the effective orifice area increased significantly only in the roots with sinuses (5.13 ± 0.5 and 5.47 ± 0.5 vs 3.06 ± 0.3 and 2.50 cm(2) ± 0.02 cm(2), respectively). CONCLUSIONS: When the cardiac output is increased to greater than the resting physiologic values, the presence of sinuses of Valsalva, independently of root compliance, prevents an increase in the pressure decrease across the valve by way of an increase of the effective orifice area.

Antiretroviral therapeutic drug monitoring in HIV-infected pregnant women: maternal immunovirological outcome at delivery and during the 18 month follow-up period.

No data are available on the long-term immunovirological outcome of HIV-positive pregnant women experiencing sub-therapeutic antiretroviral drug (ARV) concentrations during pregnancy. The objective of our study was to assess the long-term virological outcome in pregnant women treated with HAART. A prospective, multi-center study enrolled 60 HIV-infected pregnant women stratified into 3 groups according to the response to HAART. Group A, women successfully treated with HAART; Group B, women with confirmed virological failure during HAART; Group C, women successfully treated with HAART during pregnancy for prevention of vertical transmission only. Smoking, alcohol use, low adherence to therapy, and increased viral load at delivery were significantly associated to virological failure at univariate analysis. At multivariate regression analysis, only adherence to therapy was reported as an independent variable related to the virological response (p < 0.001). Virological failure during follow-up was reported in 2 (25.0%) of the 8 women with sub therapeutic Ctrough and in 4 of the 33 (12.1%) women with therapeutic Ctrough (p=0.33). In group C, the viro-immunological set points during follow-up did not differ from those observed before HAART initiation. No significantly increased rate of virological failure after delivery was reported in women with sub-therapeutic ARV concentrations during pregnancy and long-term follow-up. The long-term virological outcome was independently associated to reduced adherence to therapy. Evaluation of the clinical impact of the low plasma ARV concentrations during pregnancy on the long-term virological outcome deserves further larger studies.

Lipid-rich histology in a basal-type immuno-profile breast carcinoma: a clinicopathological histochemical and immunohistochemical analysis of a case.

We describe the clinicopathological and morphological features of an unusual breast carcinoma classifiable as a lipid-rich variant of ductal invasive carcinoma, with a basal-type immunohistochemical profile. Basal-type breast cancers show no hormonal receptor expression, rarely over-express HER-2 but exhibit molecular high weight cytokeratins, EGFR and c-kit positivity. Special stains and histochemistry tests were used to elucidate the nature of vescicles in the neoplastic cells. Sudan IV was performed on formalin-fixed tissue. Commercially available antibodies tested were: ER, PgR, EGFR, HER2, c-kit, high molecular weight cytokeratins. Cytoplasmic lipids were highlighted as red-orange droplets on Sudan IV staining. As for immunohistochemistry, the tumor showed no reactivity to ER, PgR and HER2 (triple negative), and diffuse and strong positivity to high weight cytokeratins, EGFR and c-kit, such as a basal-type breast carcinoma. A basaloid phenotype in a lipid-rich carcinoma has not been previously reported.

Transplacental transfer of antiretroviral drugs and newborn birth weight in HIV-infected pregnant women.

Although it is well known that antiretroviral drugs (ARVs) across the placenta in different extents, few data are available concerning the impact of the transplacental passage of ARVs on newborn outcome. The aim of this study is to evaluate the transplacental diffusion of ARVs and the clinical assessment of the newborn. Mother and cord lopinavir, nelfinavir, atazanavir and nevirapine plasma levels were determined by high-performance liquid chromatography. Newborn gestational age, weight, and Apgar score were recorded. Cord-to-mother ratio (C:M) was calculated to estimate the placental passage of ARVs. Preterm birth was defined as delivery at <37 weeks of gestation and low birth weight was defined as a birth weight of <2500g. Twenty-six HIV-infected pregnant women were enrolled. Nevirapine presented the highest C:M ratio (0.60 +/- 0.19), the C:M ratio of nelfinavir and atazanavir was 0.37 +/- 0.38 and 0.20 +/- 0.14, respectively. The lopinavir level in the cord was undetectable. The observed prevalence rate of neonatal low birth weight and preterm delivery was 19,2% (n = 5) and 15.4% (n = 4), respectively. A significant linear regression analysis was reported between the C:M ratio and newborn birth weight (p = 0.01). Although the role of highly active antiretroviral therapy (HAART) in preventing mother-to-child transmission is indisputable, these data indicate a pharmacological rationale to the association between birth weight and highly active antiretroviral therapy during pregnancy.

Hepatocyte-conditioned medium sustains endothelial differentiation of human hematopoietic-endothelial progenitors.

UNLABELLED: Liver neo-angiogenesis plays a fundamental role in physiological and pathological processes such as regeneration, cirrhosis, autoimmune hepatitis, and alcoholic liver disease. How liver parenchymal cells influence angiogenesis is largely unknown. We studied the influence of soluble factors released by hepatocytes on hematopoietic and endothelial cell differentiation. Human CD34+ cells cultured for several weeks in a hepatocyte-conditioned medium gradually decrease the expression of CD34 and CD133 markers (i.e. after 4 weeks from 85% and 69%, respectively, to 6% and 3%, respectively), whereas expression of CD144 and CD14 cell markers increased (from 2% and 8%, respectively, to 54% and 55%, respectively). The cells' capacity to form hematopoietic colonies in methylcellulose declined with time, whereas they acquired endothelial morphology, expressed endothelial markers, and incorporated into newly forming vascular structures both in vitro and in vivo. Cultured single CD34+ cells formed colonies expressing both hematopoietic (CD45+) and endothelial (CD144+) markers, suggesting they constitute a bona fide hemangioblast population. CONCLUSION: This system allowed subsequent stages of differentiation of hematopoietic cells to endothelial cells to be defined, underlining the strict interrelationship between endothelial and hematopoietic cells in a hepatocyte environment.