Ahmet Ayaz

Impact of precise modulation of reactive oxygen species levels on spermatozoa proteins in infertile men

BackgroundElevated levels of reactive oxygen species (ROS) are detected in 25% to 80% of infertile men. They are involved in the pathology of male infertility. Understanding the effect of increasing levels of ROS on the differential expression of sperm proteins is important to understand the cellular processes and or/pathways that may be implicated in male infertility. The aim of this study was to examine differentially expressed proteins (DEPs) in spermatozoa from patients with low, medium and high ROS levels.MethodsA total of 42 infertile men presenting for infertility and 17 proven fertile men were enrolled in the study. ROS levels were measured by chemiluminescence assay. Infertile men were divided into Low (0- < 93 RLU/s/106 sperm) (n = 11), Medium (>93-500 RLU/s/106 sperm) (n = 17) and High ROS (>500 RLU/s/106 sperm) group (n = 14). All fertile men had ROS levels between 4-50 RLU/s/106 sperm. 4 subjects from fertile group and 4 each from the Low, Medium and High ROS were pooled. Protein extraction, protein estimation, gel separation of the proteins, in-gel digestion, LTQ-orbitrap elite hybrid mass spectrometry system was conducted. The DEPs, the cellular localization and pathways of DEPs involved were examined utilizing bioinformatics tools.Results1035 proteins were identified in the 3 groups by global proteomic analysis. Of these, 305 were DEPs. 51 were unique to the Low ROS group, 47 Medium ROS group and 104 were unique to the High ROS group. 6 DEPs were identified by Uniprot and DAVID that had distinct reproductive functions and they were expressed only in 3 ROS groups but not in the control.ConclusionsWe have for the first time demonstrated the presence of 6 DEPs with distinct reproductive functions only in men with low, medium or high ROS levels. These DEPs can serve as potential biomarkers of oxidative stress induced male infertility.

Infertile men older than 40 years are at higher risk of sperm DNA damage

BackgroundThe effect of paternal age on semen quality is controversial. In this retrospective study, the aim was to investigate the effects of advancing age on sperm parameters including reactive oxygen species (ROS), total antioxidant capacity (TAC) and sperm DNA damage in infertile men. We also examined whether paternal age >40 y is associated with higher risk of sperm DNA damage.MethodsA total of 472 infertile men presenting for infertility were divided into 4 age groups: group A: patients ≤ 30 y; group B: patients 31- 40 y, group C: ≤ 40 y and group D: patients >40 y. The following tests were performed - semen analysis according to WHO 2010 criteria, seminal ROS by chemiluminescence, TAC by colorimetric assay and sperm DNA damage by TUNEL assay - and the results were compared amongst the 4 age groups.ResultsThere was no statistical difference in conventional semen parameters, TAC and ROS with advancing paternal age as well as between different age groups. However, a significant negative association was noted between sperm DNA damage and advancing paternal age. Men >40 y showed higher levels of sperm DNA damage (24.4 ± 18.5%) compared to younger men (<30 y; 16.7 ± 11.2%; p <0.05).ConclusionsInfertile men over the age of 40 y have a greater percentage of sperm DNA fragmentation compared to infertile men aged 40 y and below. Advanced paternal age (>40 y) may increase the risk of sperm DNA damage in infertile men.

Major protein alterations in spermatozoa from infertile men with unilateral varicocele

BackgroundThe etiology of varicocele, a common cause of male factor infertility, remains unclear. Proteomic changes responsible for the underlying pathology of unilateral varicocele have not been evaluated. The objective of this prospective study was to employ proteomic techniques and bioinformatic tools to identify and analyze proteins of interest in infertile men with unilateral varicocele.MethodsSpermatozoa from infertile men with unilateral varicocele (n = 5) and from fertile men (control; n = 5) were pooled in two groups respectively. Proteins were extracted and separated by 1-D SDS-PAGE. Bands were digested and identified on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Bioinformatic analysis identified the pathways and functions of the differentially expressed proteins (DEP).ResultsSperm concentration, motility and morphology were lower, and reactive oxygen species levels were higher in unilateral varicocele patients compared to healthy controls. The total number of proteins identified were 1055, 1010 and 1042 in the fertile group, and 795, 713 and 763 proteins in the unilateral varicocele group. Of the 369 DEP between both groups, 120 proteins were unique to the fertile group and 38 proteins were unique to the unilateral varicocele group. Compared to the control group, 114 proteins were overexpressed while 97 proteins were underexpressed in the unilateral varicocele group. We have identified 29 proteins of interest that are involved in spermatogenesis and other fundamental reproductive events such as sperm maturation, acquisition of sperm motility, hyperactivation, capacitation, acrosome reaction and fertilization. The major functional pathways of the 359 DEP related to the unilateral varicocele group involve metabolism, disease, immune system, gene expression, signal transduction and apoptosis. Functional annotations showed that unilateral varicocele mostly affected small molecule biochemistry and post-translational modification proteins. Proteins expressed uniquely in the unilateral varicocele group were cysteine-rich secretory protein 2 precursor (CRISP2) and arginase-2 (ARG2).ConclusionsThe expression of these proteins of interest are altered and possibly functionally compromised in infertile men with unilateral varicocele. If validated, these proteins may lead to potential biomarker(s) and help better understand the mechanism involved in the pathophysiology of unilateral varicocele in infertile men.

Spermatozoa protein alterations in infertile men with bilateral varicocele.

Among infertile men, a diagnosis of unilateral varicocele is made in 90% of varicocele cases and bilateral in the remaining varicocele cases. However, there are reports of under-diagnosis of bilateral varicocele among infertile men and that its prevalence is greater than 10%. In this prospective study, we aimed to examine the differentially expressed proteins (DEP) extracted from spermatozoa cells of patients with bilateral varicocele and fertile donors. Subjects consisted of 17 men diagnosed with bilateral varicocele and 10 proven fertile men as healthy controls. Using the LTQ-orbitrap elite hybrid mass spectrometry system, proteomic analysis was done on pooled samples from 3 patients with bilateral varicocele and 5 fertile men. From these samples, 73 DEP were identified of which 58 proteins were differentially expressed, with 7 proteins unique to the bilateral varicocele group and 8 proteins to the fertile control group. Majority of the DEPs were observed to be associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system processes. Seven DEP were involved in sperm function such as capacitation, motility, and sperm-zona binding. Proteins TEKT3 and TCP11 were validated by Western blot analysis and may serve as potential biomarkers for bilateral varicocele. In this study, we have demonstrated for the first time the presence of DEP and identified proteins with distinct reproductive functions which are altered in infertile men with bilateral varicocele. Functional proteomic profiling provides insight into the mechanistic implications of bilateral varicocele-associated male infertility.

Differential Proteomic Profiling of Spermatozoal Proteins of Infertile Men With Unilateral or Bilateral Varicocele

OBJECTIVE To compare the sperm protein profile between infertile men with unilateral varicocele and infertile men with bilateral varicocele. METHODS This prospective study investigated 50 infertile patients with clinical varicocele (33 unilateral and 17 bilateral) seeking fertility workup between March 2012 and April 2014. Routine sperm parameters, reactive oxygen species, total antioxidant capacity, and sperm deoxyribonucleic acid fragmentation were assessed in their semen. Sperm protein profile was characterized only in pooled samples of 5 unilateral and 3 bilateral varicocele samples, respectively, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Linear Trap Quadrupole-Orbitrap Elite hybrid mass spectrophotometer system. Differences in protein expression were analyzed using gel analysis software, followed by protein identification using mass spectroscopy analysis. Differentially expressed proteins and their abundance were quantified by comparing spectral counts, followed by bioinformatics analysis. RESULTS Unique expression of 64 proteins in the bilateral group and 31 proteins in the unilateral group was obtained. Core functions of the top protein interaction networks were post-translational modification (∼122 proteins associated with acetylation), protein folding, free-radical scavenging, cell death, and survival. The top molecular and cellular functions were protein degradation, free radical scavenging, and post-translational modifications, whereas the top pathways were protein ubiquitination and mitochondrial dysfunction. Major biological pathways for the 253 differentially expressed proteins were metabolism, apoptosis, and signal transduction. CONCLUSION Functional proteomic profiling helps identify the differential processes or pathways that are affected based on the nature of varicocele (bilateral or unilateral) and provide insights into the mechanistic implications of varicocele-associated male infertility.

Post-Translational Modifications in sperm Proteome: The Chemistry of Proteome diversifications in the Pathophysiology of male factor infertility

BACKGROUND The spermatozoa undergo a series of changes in the epididymis to mature after their release from the testis and subsequently in the female reproductive tract after ejaculation to get capacitated and achieve fertilization potential. Despite having a silenced protein synthesis machinery, the dynamic change in protein profile of the spermatozoa is attributed either to acquisition of new proteins via vescicular transport or to several post-translational modifications (PTMs) occurring on the already expressed protein complement. SCOPE OF REVIEW In this review emphasis is given on the PTMs already reported on the human sperm proteins under normal and pathologic conditions with particular reference to sperm function such as motility and fertilization. An attempt has been made to summarize different protocols and methods used for analysis of PTMs on sperm proteins and the newer trends those were emerging. MAJOR CONCLUSIONS Deciphering the differential occurrence of PTM on protein at ultrastructural level would give us a better insight of structure-function relationship of the particular protein. Protein with multiple PTMs could be used to generate the complex interaction network involved in a physiological function of a sperm. It can be speculated that crosstalk between different PTMs occurring either on same/ other proteins actually regulate the protein stability and activity both in physiological and pathological states. GENERAL SIGNIFICANCE The analytical prospective of various PTMs reported in human spermatozoa and their relevance to sperm function particularly in various pathophysiological states, would pave way for development of biomarkers for diagnosis, prognosis and therapeutic intervention of male infertility.

Effect of advancing paternal age on semen parameters and seminal oxidative stress markers in infertile men

BackgroundHigh rate of subfertility and adverse pregnancy related out-comes associated with childbearing are seen after age 40.In contrast to oogenesis, spermatogenesis continues inelderly men. Adult male germ cells pass through signifi-cantly more mitotic replications than the germ cells inadult female. In men, there is an age associated increase inthe incidence of breaks in sperm DNA, decrease in apopto-sis, and a higher frequency of point mutations. Advancedpaternal age is associated with an increased time to preg-nancy and decreased pregnancy rates. After adjusting forfemale age, conception during a 12-month period was > 30percent less likely for men over 40 years of age as com-pared to men 45years compared to men 40 years (n = 105). We evaluated the conven-tional semen parameters (WHO, 2010) [5] and OS mar-kers: seminal ROS (chemiluminescence assay), totalantioxidant capacity (TAC) and sperm DNA damage(TUNEL assay) in all patients.ResultsThe mean age of the study subjects was 36.8 ± 6.7 years.No age-related differences were seen in conventionalsemen parameters (volume, concentration, motility, andmorphology) (Table 1). ROS and antioxidant levels werecomparable in the 3 groups. Significantly higher levels ofsperm DNA damage (19.94 ± 15.30%) was seen in infertilemen >40 years compared to men in younger age groups(P = 0.028 and P = 0.027, respectively).ConclusionsSperm DNA damage increases with advancing paternalage. Evaluation of sperm DNA damage will help diagnosethe underlying cause of poor fertility in some men andassist the clinician in offering correct treatment modality.

MP66-05 OXIDATIVE STRESS PROTEINS IDENTIFIED IN HUMAN SPERMATOZOA BY PROTEOMIC AND BIOINFORMATIC ANALYSIS

predictive accuracy of identifying tubules with spermatogenesis vs. SCO tubules. RESULTS: Raman peak intensity changes were noted at 1000 cm-1 and 1690 cm-1 between tubules with spermatogenesis and SCO tubules. When wavelengths were utilized to predict whether seminferious tubules were SCO or had spermatogenesis, sensitivity and specificity were estimated as 96% and 100% respectively. The area under the receiver operator characteristic curve for predicting tubules with spermatogenesis was 0.98. CONCLUSIONS: RS is capable of identifying seminiferous tubules with spermatogenesis in a SCO ex-vivo rat model. Future ex-vivo studies on human testicular tissue will be necessary to confirm whether these findings can be translated into a clinical setting.

Impact of World Health Organization (WHO) new standards on the referral pattern of infertile men for assisted reproduction

Background New reference values for semen parameters are significantly lower in the WHO 5th edition (2010) than in the 4th edition [1,2]. Some of the highlights of the fifth edition were: 1) subjects included in this edition had < 12 months’ time to pregnancy, 2) semen analyses results were pooled and analyzed for reference values, 3) laboratories generating the data used standardized methods for semen analysis according to WHO manual available at the time of original studies and 4) one-sided lower reference limits (5th centile) were generated and proposed as lower cut-off limits for normal values. The new reference values were aimed at providing evidencebased thresholds to assist clinicians in calculating relative fertility of the patient. The goal of our study was to examine the impact of the new WHO reference values on the referral pattern of infertile men referred for assisted reproduction (IUI, IVF/ICSI).

The effect of low level leukocytospermia on oxidative stress markers in infertile men

Background Leukocytospermia is defined as presence of ≥1 x 106 WBC/mL of the seminal ejaculate. The World Health Organization (WHO) recommends peroxidase staining as the standard method for the detection of semen leukocytes [1,2]. The incidence of leukocytospermia ranges from 10 20% among infertile men. Both morphologically abnormal spermatozoa and leukocytes produce reactive oxygen species (ROS). The polymorphonuclear neutrophils and macrophages are the main components of seminal leukocytes which can generate significantly higher (>100-fold) quantities of ROS, overwhelming the ROS-scavenging mechanisms in seminal plasma and resulting in oxidative stress and damage to spermatozoa. The presence of very few activated leukocytes can produce a detectable amount of ROS [3,4]. Therefore, even a very low number of leukocytes in the sperm suspension may influence the integrity of sperm and, consequently, the outcome of assisted reproduction treatment [5]. Leukocytes contributed directly to ROS production and release and indirectly through the leukocyte-stimulated sperm. Such stimulation may be via direct contact or mediated by soluble products released by the leukocytes. The goal of our study was to assess the effect of low level leukocytospermia on semen quality and oxidative stress markers in infertile men. Materials and methods In this prospective study, 211 infertile patients with no history of genital tract infections or varicocele were included. Semen samples were examined for sperm concentration, motility, seminal leukocyte levels (Endtz test) [2], reactive oxygen species (ROS) by chemiluminescence assay, and sperm DNA damage by TUNEL test. Patients were divided into 3 groups based on their seminal leukocyte levels. Group 1: no seminal leukocytes (n = 153); group 2: low level leukocytospermia (0.1 -1 X 106 WBC/mL; n = 22); and group 3: leukocytospermia (>1 X 106 WBC/mL; n = 36).