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Male Infertility Research Projects

Oxidation Reduction Potential (ORP) – A New Measure of Oxidative Stress in Male Infertility

ORP as a new Measure of Oxidative Stress

We examined the effect of ascorbic acid (AA) in reducing redox potential in heat induced oxidative stress. The efficacy of 400 and 600 µmole/L concentration of AA was examined to study the heat and heat + hydrogen peroxide induced oxidative stress in sperm suspensions incubated for 2 and 4 h at three different temperatures. Ascorbic acid at 600 µmol/L was more effective at reducing heat induced oxidative stress in sperm preparations in vitro compared to 400 mol/L (P=0.01). Supplementation with AA may be advantageous for sperm preparations in IUI, IVF and ICSI.

ORP as a potential marker of male infertility

In another study, we evaluated the clinical utility of ORP as a potential marker of male infertility. ORP was measured in 293 patients and 15 proven fertile controls. Semen samples were categorized as normozoospermic; oligozoospermic; asthenozoospermic; teratozoospermic and oligoasthenoteratozoospermic (WHO 2010 criteria). Semen parameters and ORP levels were measured. ROC curves were generated to differentiate these categories. Significant differences were observed in ORP levels between control and normozoospermic group. When grouped based on concentration, motility and morphology, significant differences were observed between area under ROC curve, sensitivity, specificity, PPV, NPV and cutoff values. These differences were significant when ORP was combined with any two sperm abnormalities. ORP is a quick, easy, cost-effective and reliable marker of semen quality as well as oxidative stress for use in a clinical setting.

ORP as a method to predict poor sperm quality

Similarly in another study, 194 infertile men with 29 patients who had repeat samples for semen analysis and ORP measurement were examined to evaluate the 1) relationship between ORP and abnormal sperm quality, and 2) changes over time to identify if ORP can be used as a surrogate marker of poor semen quality that may help in the diagnosis of oxidative stress-related male infertility. ORP levels >1.57 mV/106 sperm/mL was able to detect at least 1 abnormal sperm parameter with a sensitivity of 70.4% and specificity of 88.1%. ORP at a cutoff of 2.59 mV/106 sperm/mL had the highest predictive value of detecting oligozoospermia with 88% sensitivity, and 91.2% specificity. With repeat samples, increase in sperm concentration and motility at 2 consecutive time intervals was associated with a decline in ORP levels. Thus, we have demonstrated that ORP is a reliable, rapid method in predicting poor sperm quality. It can overcome the high technical variability associated with semen analysis and assist in the diagnosis of oxidative stress-related male infertility.

ORP as an indicator of semen quality

In another study, 365 infertile men and 50 fertile men were examined for ORP and its association with semen parameters. Similarly we identified an ORP cut-off of 1.38 x 106 mV/mL that was able to differentiate normal from abnormal semen samples whereas a cut-off of 1.41 mV/106 sperm/mL was able to differentiate between fertile and infertile semen samples.

MiOXSYS as a novel method to determine ORP levels in semen and Seminal Plasma

In our initial studies, we demonstrated the ability of MiOXSYS system to measure ORP in fresh and frozen semen or seminal plasma samples. These measurements are stable up to 120 minutes. ORP measurement is rapid (<4 min) and requires only 30 µL of sample. ORP is negatively correlated with semen concentration/total sperm count, despite the fertility status.

ORP as a novel clinical test for assessment of sperm quality

We established elevated levels of ORP in infertile men (6.22 ±1.10 mV/106 sperm/mL) compared with controls (1.59 ± 0.29 mV/106/mL) (P=0.004). A cut-off of 1.36 mV/106 sperm/mL distinguishes normal and abnormal semen with a sensitivity 69.6%, specificity 83.1%, positive predictive value (PPV) 85.3% and negative predictive value (NPV) of 65.9%. We then demonstrated that ORP levels negatively correlate with semen parameters (concentration, total sperm count, motility and morphology) and therefore ORP can be used as a stand-alone test or as a surrogate marker of poor sperm quality.

Evaluating Sperm DNA Damage in Infertile Men

Intact genetic material, or DNA, is important for healthy fertilization. We studied the effect of paternal age on sperm DNA fragmentation to examine if men >40 years (y) of age have a higher risk for sperm DNA damage. Infertile men were divided into 4 groups based on age: ≤30y, 31y–40y, ≤40y, and >40y (i.e. advanced paternal age). Routine semen parameters (WHO 2010 criteria), ROS (chemiluminescence assay), TAC (colorimetric assay) and sperm DNA fragmentation (TUNEL assay utilizing flow cytometry; current cutoff for DNA fragmentation is <17%) were tested. Although no differences were seen in conventional semen parameters, ROS, and TAC, we identified a significant negative association between sperm DNA damage and advancing paternal age. Higher levels of sperm DNA damage was seen in men >40y (24.4 ± 18.5%) compared to men ≤30y (16.7 ± 11.7%; p <0.05). The findings of the study suggest that advanced paternal age >40y may increase the risk of sperm DNA damage.

Abstinence Time and Impact on Semen Parameters

Optimizing ejaculatory abstinence time is important to ensure both quantity and quality of sperm. We tested the effect of abstinence on basic and advanced semen parameters, thereby establishing the best abstinence time for maximizing semen quality for both diagnostic and therapeutic procedures. Semen samples were obtained after 1, 2, 5, 7, 9 and 11 days of abstinence from normozoospermic men. Comparison was made by grouping samples into short (1 day), recommended by WHO (2-7 days), and long (9-11 days) abstinence time. Each specimen was assessed for semen parameters, ROS (chemiluminescence assay) and sperm DNA fragmentation (TUNEL assay). Semen volume, sperm count and total sperm count increased with abstinence length. Similarly, sperm DNA fragmentation was associated with length of ejaculatory abstinence. Our results demonstrated that short abstinence of 1 to 2 days had the least amount of DNA fragmentation with no detrimental impact on semen characteristics (WHO 2010). This strategy is useful in optimizing sperm quality and the chances of natural and assisted conception.

Effect of Ascorbic Acid and Cumene Hydroperoxide on Sperm ORP

We examined the effect of ascorbic acid (AA) in reducing redox potential in heat induced oxidative stress. The efficacy of 400 and 600 µmole/L concentration of AA was examined to study the heat and heat + hydrogen peroxide induced oxidative stress in sperm suspensions incubated for 2 and 4 h at three different temperatures. Ascorbic acid at 600 µmol/L was more effective at reducing heat induced oxidative stress in sperm preparations in vitro compared to 400 mol/L (P=0.01). Supplementation with AA may be advantageous for sperm preparations in IUI, IVF and ICSI.

Similarly we examined the effect of exogenous induction of oxidative stress by cumene hydroperoxide on ORP in fresh and frozen semen samples. ORP was measured in 20 fresh and frozen samples incubated with 5 and 50 µmoles/L of cumene hydroperoxide. Both concentrations of cumene hydroperoxide resulted in a significant decline in sperm motility in prefreeze samples compared to controls. The increase in both prefreeze and post-thaw semen samples for ORP was higher in controls compared with 50 cumene hydroperoxide. We have demonstrated in this study that ORP as measured by the MiOXSYS system is simple, sensitive and a dynamic tool to measure the impact of ORP in fresh and frozen semen specimens.

Developing a Sperm Banking Kit to Preserve Sperm Parameters during Shipment

Standardization of a novel home sperm banking kit

In the first study, our goal was to develop and optimize the contents of the specialized sperm collection and transport kit designed for overnight delivery of semen sample. A total of 12 semen samples collected onsite and 9 collected at home and shipped overnight were tested. Two transport media (refrigeration media and human tubal fluid) and five different combinations of ice packs were tested for maintaining optimal shipping temperature (40oC, 37oC and 24oC) of semen samples. Even though motility and membrane integrity declined in samples shipped overnight using refrigeration medium and ice packs, adequate sperm motility was maintained using the home sperm banking kit and therefore these samples can used for procreation purposes.


Sperm cryosurvival rates – home sperm banking kit vs onsite collection

In the second study, 19 infertile patients and 17 cancer patients provided samples onsite and offsite. Initial testing of semen parameters was done before vs. after freezing using test yolk buffer and standard freezing protocol. To evaluate the efficacy of the home shipping kit, we tested sperm motility, total motile sperm, cryosurvival and DNA fragmentation in onsite and offsite samples. We observed similar percent cryosurvival rates in infertile men with home banking kit and onsite collection. Similarly, in cancer patients the cryosurvival rates was comparable between onsite collection and offsite collection. Cancer patients can bank specimens using the sperm banking kit as effectively as men banking for infertility reasons.

ORP as a novel clinical test for assessment of sperm quality

Leukocytospermia

Leukocytospermia is defined as the presence of >1x106 white blood cells/mL (WBC/mL) in a semen sample (WHO 2010). Leukocytes contribute both directly and indirectly to ROS production. However, whether low level leukocytospermia (<1x106 WBC/mL) may still produce detectable levels of ROS, and impair sperm function is unclear. Therefore, we assessed the effect of low level leukocytospermia on semen quality, ROS levels and DNA damage in infertile men. Patient semen samples (n=211) were divided into 3 groups: group 1: no leukocytes; group 2: low level leukocytospermia (0.1-1.0 x106 WBC/mL) and group 3: frank leukocytospermia >1.0x106 WBC/mL). Semen analysis, leukoctyospermia, ROS and DNA fragmentation was tested. About 63.2% of patients had ROS levels >93 RLU/s/106 sperm. The incidence of leukocytospermia increased to 81.6% in group 2 and 92.43% in group 3. Group 2 patients had significantly higher levels of ROS and sperm DNA fragmentation compared with group 1. ROS levels were comparable between group 2 and group 3. Higher level of DNA damage was seen in patients with low level leukocytospermia. Patients with low level leukocytospermia may benefit from antibiotic treatment / antioxidant supplements to reduce ROS-induced sperm DNA damage and improve their fertility.

 

Oligozoospermia

Our second study aimed to 1) examine semen parameters such as concentration, motility, morphology and ROS levels in men with oligozoospermia compared with three control groups, i.e. unproven donors, donors with proven fertility <2 years and donors with fertility established >2 years, and 2) establish the cutoff, sensitivity, specificity for ROS, and study how these parameters are altered in each group. A total of 101 infertile men with oligozoospermia; oligoasthenozoospermia (OA); oligoteratozoospermia (OT) and oligoasthenoteratozoospermia (OAT) and 56 donors were included in the study. A significantly improved sperm motility and morphology was observed in fertile men who had established a pregnancy in the past or recently compared to the OAT and OT group. Oligozoospermic patients in these groups had significantly higher levels of ROS. The cutoff value in donors with <2 years of fertility was significantly lower with higher sensitivity and specificity compared to unproven donors and donors who had not established a pregnancy. A positive association between semen parameters and ROS indicates a common underlying mechanism in these infertile patient groups.

 

Teratozoospermia

In this third study, we examined the relationship between seminal ROS production and sperm morphology in infertile patients with teratozoospermia (WHO 2010) as well as healthy men of proven and unproven fertility. Semen samples were collected from 79 infertile men with teratozoospermia and 56 healthy controls of unproven fertility, proven fertility <2 years and proven fertility >2 years. We found that ROS levels: 1) were significantly higher in infertile men with teratozoospermia and 2) correlated negatively with sperm concentration in all donor groups and teratozoospermic group. These results demonstrate that incidence of teratozoospermia is directly related to ROS. Therefore, besides sperm concentration and motility, sperm morphology should be carefully evaluated and included in the overall assessment of male fertility.

 

Proteomics

Protein Expression in ROS – vs. ROS + semen samples

Utilizing proteomic analysis with two-dimensional differential in-gel electrophoresis (2-DIGE) and LC-MS/MS (liquid chromatography–mass spectrometry/mass spectrometry), protein expression in a variety of sperm population was recently examined. A total of 1,343 protein spots in gel 1 (ROS-) and 1,265 spots in gel 2 (ROS+) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROS- sample compared with the ROS+ sample. Significantly different expression of protective proteins against oxidative stress was found in ROS- compared with ROS+ sample.

Impact of precise modulation of ROS levels on sperm proteins

In another study, spermatozoa were separated from 42 infertile men and 17 fertile donors into three groups based on low (0-<93 RLU/s/106 sperm), medium (>93-500 RLU/s/106 sperm) and high ROS (>500 RLU/s/106 sperm). For proteomic analysis, each group consisted of pooled samples from 4 subjects. A total of 1035 proteins were identified in the 3 ROS groups by global proteomic analysis. 305 DEP were identified of which 51 were unique to low ROS group, 47 medium ROS group and 104 to the high ROS group. 6 DEPs (Calmegin, Tripeptidyl peptidase II, Dynein intermediate chain 2, axonemal, Heat shock 70 kDa protein 4L, Early endosome antigen 1, and Plasma serine protease inhibitor) were identified that are involved in distinct reproductive functions and were expressed only in the 3 ROS groups but not in the fertile group. These DEPs may serve as potential biomarkers of oxidative stress induced male infertility.

Proteomic analysis of sperm proteins with oxidative stress

We examined protein alterations in 32 infertile men and 20 normal donors. Samples were categorized as ROS+ (>92 RLU/s/106 sperm) and ROS-(<92 RLU/s/106 sperm). Of the 74 proteins identified, 10 proteins were overexpressed with a two-fold difference and 5 were underexpressed in the ROS+ group. The processes affected in the ROS+ group were energy metabolism and regulation, carbohydrate metabolism processes such as gluconeogenesis and glycolysis, protein modifications and oxidative stress regulators. These proteins may have a significant impact on the fertilization potential of the sperm.

Proteomic analysis of ejaculated sperm from fertile men

We examined proteins that may be altered during the process of sperm maturation and may be responsible for male infertility. Sperm were separated into 4 fractions (F1, F2, F3 and F4) on a three layer density gradient (40%, 60% and 80%). F1 contained the most immature sperm and F4 had the most mature, highly motile and morphologically normal sperm. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. 1469 proteins were identified in the four fractions of the spermatozoa. The number of proteins identified decreased with increasing maturation level of the sperm. During sperm maturation, proteins involved in gamete generation, cell motility, energy metabolism and oxidative stress showed increasing expression and those involved in protein biosynthesis, protein transport, protein ubiquitination, and response to oxidative stress precursors showed decreasing expression level. 4 proteins (HSP701A, clusterin, tektin 2 and tektin 3) were also validated by western blotting. We have demonstrated the proteins that are involved in different stages of sperm maturation that may be altered or modified in infertile men.

Sperm Proteome in males with primary or secondary infertility

The sperm proteome in infertile men was analyzed to identify the underlying mechanism and reliable diagnostic markers. 16 infertile men with primary or secondary infertility and 7 proven fertile men were analyzed using 5 pooled samples in each group. DEPs were used for functional enrichment analysis and key proteins involved in altered functions and testis-specific proteins were validated by western blot analysis. A total of 1305 were identified of which 102 were underexpressed and 15 were overexpressed in both infertile groups. BAG6, HSPA2, and SPA17 were underexpressed and related to post-translational modification and folding. BAG6 was underexpressed in infertile men. HIST1H2BA a testis-specific protein was overexpressed. Therefore both BAG6 and HIST1H2BA may be potential candidate markers for male infertility.

Differential proteomic profiling of sperm proteins of infertile men with unilateral or bilateral varicocele

In the first study, 33 infertile men with unilateral varicocele and 17 with bilateral varicocele were examined for to examine the differences in protein expression based on spectral abundance and bioinformatics analysis. 253 DEPs were identified and involved in metabolism, apoptosis and signal transduction. 64 proteins were unique to the bilateral group and 31 were unique to the unilateral group. Core functions of the top protein interactions were post-translational modification, protein folding, free-radical scavenging, cell death and survival. Top molecular and cellular functions were protein degradation, free radical scavenging and post-translational modifications and top pathways were protein ubiquitination and mitochondrial dysfunction. In this study, we identified the differential processes or pathways affected based on the nature of varicocele i.e. unilateral or bilateral.

Protein alterations in sperm from infertile men with unilateral varicocele

In the next study, we examined the protein alterations in infertile men with unilateral varicocele and compared these with those in the fertile men. Pooled samples from 5 infertile men with unilateral varicocele and 5 fertile men were examined by global proteomic analysis and subjected to bioinformatics analysis. 369 DEP were identified of which 120 proteins were unique to the fertile group and 38 were unique to the unilateral group. Compared to the control group, 114 proteins were overexpressed while 97 were underexpressed in the unilateral varicocele group. 29 proteins were involved in spermatogenesis reproductive functions such as sperm maturation, acquisition of sperm motility, hyperactivation, capacitation and acrosome reaction and fertilization. The major pathways related to unilateral varicocele group involved metabolism, disease, immune system, gene expression, signal transduction and apoptosis. Functional annotations showed that unilateral varicocele mostly affected small molecule biochemistry and post-translational protein modifications. Cysteine-rich protein 2 precursor (CRISP2) and arginase-2 (ARG2) were the two proteins uniquely expressed in the unilateral varicocele group.

Sperm protein alterations in infertile men with bilateral varicocele

We also examined the protein expression in infertile men with bilateral varicocele and compared with fertile men. 17 infertile men with bilateral varicocele and 10 proven fertile men were enrolled. Pooled samples from 3 men with bilateral varicocele and 5 fertile men were used for proteomic analysis. 73 DEPs were identified of which 58 were DEP and 7 were unique to the bilateral varicocele and 8 were unique to the fertile group. Majority of the proteins were associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system. 7 of the DEP were involved in sperm functions such as capacitation, motility, sperm zona binding. Two proteins TEKT3 and TCP11 were validated by western blotting. Proteins with distinct reproductive function have been identified which are altered in infertile men with bilateral varicocele. These proteins may provide insight into bilateral varicocele associated male infertility.

Proteomic Signatures of infertile men with clinical varicocele

We compared the fertile group with both unilateral and bilateral group to identify the signature proteins related with varicocele-associated male infertility irrespective of the stage and laterality of their clinical varicocele. Ninety nine proteins were differentially expressed in the varicocele group. Over 87% of the DEPs were involved in energy metabolism and key sperm functions that were underexpressed in the varicocele group. Key proteins affected were spermatogenesis, sperm motility, and mitochondrial dysfunction. Expression level of 5 proteins (PKAR1A, AK7, CCT6B, HSPA2, and ODF2) involved in stress response and sperm function including molecular chaperons was validated by western blotting. We have demonstrated that varicocele is an essentially a state of energy deprivation, hypoxia and hyperthermia due to impaired blood supply. This is also demonstrated by the downregulation of lipid metabolism, mitochondrial electron transport chain and enzymes of Krebs cycle. This study therefore contributes towards establishing the proteomic signature of the biomarkers to assess the sperm quality in infertile men with clinical varicocele based on the molecular parameters.

CURRENT RESEARCH FOCUS - Evaluating Sperm DNA Damage in Infertile Men

DNA Damage

Intact genetic material, or DNA, is important for healthy fertilization. A morphologically normal looking motile sperm can have DNA damage, which can result in impaired fertilization, miscarriage or subsequent complications associated with pregnancy. Sperm can be frozen and batched for DNA testing. DNA damage can be examined by staining the DNA by utilizing a reaction catalyzed by exogenous terminal deoxynucleotidyltransferase (tdt) and is termed as ‘end labeling’ or “TUNEL” (terminal deoxynucleotidyltransferase dUTP nick end labeling) assay. It utilizes a robust technique called flow cytometery. This technique is sensitive and requires 2-5 X106 sperm compared to other microscopic methods which are very subjective.

Infertile patients, diagnosed with varicocele, prostatitis or with a history of smoking have been shown to have higher percentage of sperm DNA damage. Furthermore, evaluating sperm DNA damage in men with unexplained infertility or idiopathic infertility or those who have severe oxidative stress-related abnormal semen quality may also be good candidates for evaluating sperm DNA damage. Oxidative stress and sperm apoptosis is a major contributor of sperm DNA damage.

High DNA damage in infertile men can lead to poor ART outcomes and increased miscarriage rates. Based on the extent of DNA damage found, certain assisted reproductive techniques may be recommended

Our goal is to identify key proteins that can serve as biomarkers in identifying the underlying pathology of male infertility and provide alternate approaches for treating these patients.

Characterization of Intracellular Reactive Oxygen Species In Human Spermatozoa

Lab2006

Free radicals such as superoxide anion (O2), hydrogen peroxide (H2O2), hydroxyl (OH-), and peroxyl radicals are involved in initiation and progression of oxidative damage to spermatozoa. If the levels of these reactive oxygen species (ROS) exceed the antioxidant capacity of the cell, oxidative stress will be induced, which reflect negatively on the male fertility potential.

Although chemiluminescence has been the standard method for measuring ROS in a given sample, the assay entails many limitations, such as: it fails to specifically target intracellular ROS, is not specific for any individual free radical species, and requires a large number of cells to perform. In contrast, assessment of ROS levels using flow cytometry offers the ability to identify specific radicals generated intracellularly in relatively low cell number. The main objective of our proposed study is to provide an accurate, easy to perform assay for the assessment of intracellular ROS. This method is used to characterize the different radicals present in human spermatozoa and correlate them with the pathogenesis of male infertility.

Enhancement of Sperm Quality by Magnetic Cell Separation

Lab

The externalization of the phospholipid phosphatidylserine (EPS) from the inner to the outer leaflet of the plasma membrane is a feature of the terminal phase of apoptosis and can be monitored by annexin V-binding. Colloidal superparamagnetic microbeads bind to annexin V label the dead and apoptotic spermatozoa and retain them within an external strong magnetic field provided by separation columns (magnetic cell separation, MACS). Utility of poly(ADP-ribose) polymerase (PARP), a nuclear enzyme that plays an important part in repairing damaged DNA is being investigated for its role in ejaculated spermatozoa. We are examining the ability of a new magnetic separation technique for its ability to separate PARP modified spermatozoa and to modulate its function by decreasing the incidence of DNA damage.

Effects of Electromagnetic Waves Exposure From Cell Phones on Semen Quality

Cell phones have become indispensable devices in our daily life. These emit electromagnetic waves (EMW) of different frequencies which have been linked to adverse effects in human beings. In United States cell phones operate at frequency 900 -1900 MHz, whereas in most other parts of world the cellular phones work at 900 -1800 MHz frequencies. Electromagnetic waves have been reported to affect neurological, cardiac and endocrine systems. There are preliminary reports that suggest that EMW can reduce the fertilizing potential of spermatozoa and cause sperm DNA damage. Some of the possible mechanisms by which EMW can alter reproductive function are non-thermal EMW-specific effect, a thermal molecular effect, or a combination of these mechanisms. While some authors have found little or no effect with the use of electronic devices on reproductive function, recent research by our group has shown damaging effects of these devices on semen variables in a study involving 361 men.

Free radicals called reactive oxygen species (ROS) have been shown to be detrimental to sperm and are involved in the pathophysiology of male infertility. Antioxidants present in the semen can neutralize ROS. Oxidative stress occurs when there is an imbalance in the formation of ROS and the ability of antioxidants to neutralize ROS.

In a large prospective study, we assessed the effects of EMW emitted by cell phones (900 - 1900 MHz) on various markers of sperm quality such as count, motility, morphology, viability etc. Pilot studies conducted by us have shown that exposure of ejaculated (neat) semen samples to commercially available cellular phones for one hour caused a significant decrease in sperm motility and viability, increased ROS levels and decreased ROS-TAC (reactive oxygen species-total antioxidant capacity) score when compared with neat semen from a non-exposed group.

It's important to note that many men carry their cell phone in a trouser pocket (or clipped to their belts on waist) while using Bluetooth. This technology exposes testes to high power cell phone density compared with the cell phone in the stand by mode. The phone and the male reproductive organs are separated by multiple tissue layers. The deleterious effects of RF-EMW exposure from cell phone use on functional markers of spermatozoa from fertile and infertile men are not clear. Furthermore, the effects on spermatozoa of frequency, distance of the phone from source and the talk time are not known.

We have designed a two dimensional anatomical model of the tissue to extrapolate the effects seen in "in vitro" condition to real-life conditions. We aim to examine the effect of specific cell phone RF, distance and talk time on functional markers of oxidative stress in immature and mature spermatozoa. In addition we are also evaluating DNA damage and apoptosis.

Results from our study may help us understand the mechanism of action of RF-EMW from cell phones on sperm quality in infertile men - a population who may already have sperm cells that are susceptible to oxidative stress and, therefore, be more susceptible to the negative cell phone effects. Such knowledge may help modify/ revise guidelines for reducing the adverse effects of EMW in men who may be at increased risk of sperm damage and subsequent infertility.

CellPhone Effects

Proteomic analysis – A New Tool in Unraveling the Molecular Mechanism of Sperm Dysfunction and Male Infertility

Proteomic Analysis

Semen analysis does not provide information on the underlying molecular alterations in the seminal ejaculates of infertile men. Oxidative stress (OS) can affect sperm function and results in modification of proteins in the spermatozoa. Proteomics is the study of the protein profile of spermatozoa or seminal plasma and utilizes MALDI-TOF (matrix assisted laser desorption ionization – time of flight) and LC-MS/MS (liquid chromatography –mass spectrometry/mass spectrometry).

Utilizing proteomic analysis with Two-dimensional differential in-gel electrophoresis (2-DIGE) and liquid chromatography –mass spectrometry/mass spectrometry (LC-MS/MS) protein expression in a variety of sperm population was recently examined in difference in intensity were excised from the preparatory gel and identified by liquid chromatography–mass spectrometry. A total of 1,343 protein spots in gel 1 (ROS-) and 1,265 spots in gel 2 (ROS+) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROS- sample compared with the ROS+ sample. Significantly different expression of protective proteins against oxidative stress was found in ROS- compared with ROS+ sample.

Utilizing Mascot and Sequest programs, further identification of proteins is done. The results from these SEQUEST searches are used to determine the spectral counts. Normalization of the spectral count is done using the total number of spectral counts for all proteins in the sample and the number of amino acids present in the protein. A 2-fold change in protein expression is considered significant since the precision of the proteomic analysis has an average error of 10-20%.

We have identified spermatozoal or seminal plasma proteins that are overexpressed or underexpressed in ROS – samples compared to ROS + samples. In addition we have studied proteins that are differentially expressed in patients with abnormal sperm count and sperm morphology compared with those that have normal sperm count and morphology.

Differentially affected processes pathways and cellular distribution as well as protein–protein interactions can be identified utilizing functional bioinformatics analysis available (Gene Ontology (GO) annotations from GO Term Finder and GO Term Mapper, UNIPROT, STRAP, BioGPS and proprietary software packages such as Ingenuity Pathway Analysis, Metacore™ and STRING database and Cytoscape to identify the differentially affected processes, pathways, cellular distribution, and protein-protein interactions amongst proteins in the two study groups as well as for data integration.

In these preliminary studies, we have established a platform to utilize proteomic tools and examine other etiologies in an effort to unravel the underlying mechanisms of male infertility and identify develop appropriate antioxidant therapy to alleviate oxidative stress related infertility.

Further validation through Western Blot is necessary to identify the biomarker status of the proteins in various pathological conditions attributed to oxidative stress or in patients with other etiologies.

The current focus is to study the alterations of major proteins that are overexpressed or underexpressed in the seminal plasma or spermatozoa of infertile men with various clinical diagnoses and compare with those present in normal healthy men who have established pregnancy.

Previous proteomic projects were:

1). Fertile versus infertile subjects, 2) primary versus secondary infertility, 3). patients who are presenting with non-obstructive azoospermia, 4) patients diagnosed with testicular cancer, 5) patients before and after varicocele repair, 6) idiopathic and ptients with 7) unexplained male infertility, 8) advanced paternal age (>40y) and 9). obese patients (>25 BMI).

In addition we are also evaluating a subset of patients who present with the following:

1). hyperviscosity, 2). leukocytospermia as one of the underlying characteristics of seminal ejaculate. Another study is aimed at examining the effect of abstinence time on the sperm proteome.