Research Overview

One of the major goals of our Center is to better understand the causes of infertility and to design studies aimed at understanding and improving semen quality. We hope this line of research will eventually result in strategies that will enable us to use the healthiest spermatozoa for fertilization to improve the chances of pregnancy. The fertilizing potential of sperm depends on the shape of the spermatozoa, the ability of the spermatozoa to perform the functions necessary for fertilizing an egg, and finally, the transfer of intact genetic material (DNA) to the egg at the time of fertilization. Abnormal spermatozoa are not fully mature, and when in the presence of white blood cells, they can produce harmful substances. These substances are very unstable and reactive molecules that are called free radicals. Other substances called antioxidants, which are present in normal healthy seminal ejaculates, reduce the harmful effects of free radicals by neutralizing them. However, infertile patients who have abnormal semen often have antioxidants that are ineffective neutralizers. This results in a preponderance of free radicals, which in turn results in a situation called oxidative stress. Oxidative stress can damage the DNA of the spermatozoa and prevent them from fertilizing an egg.

We are currently involved in projects evaluating oxidative stress-induced nuclear DNA damage and its effects on sperm quality and pregnancy outcome. Studies designed to understand how free radicals cause DNA damage and the possible methods to counteract them are currently being conducted. We are currently evaluating the role of nutritional supplements in reducing oxidative stress in infertile men and increasing the number of successful pregnancies.

It is now well documented that spermatozoa of infertile men have higher levels of DNA damage than their fertile counterparts. The DNA damage is seen in the form of single and double strand breaks, interstrand cross-links, modification of bases etc. When the male partner has >28-30% DFI, there is a 6-10X decreased probability of full term pregnancy. There is also a significantly decreased probability with this DFI value for an IUI pregnancy, as well as an increased probability of spontaneous miscarriage. The odds are reduced for routine IVF but much less so for ICSI when the DFI >30%.

The reason behind this DNA damage is not well understood. There are two prominent factors implicated in various studies - oxidative stress (OS) and dysregulated apoptosis. OS is the imbalance between the production of reactive oxygen species (ROS) by the spermatozoa and leukocytes, and the antioxidant capacity of the seminal plasma. The prime source of ROS production in infertile patients is the immature spermatozoa having residual cytoplasm. The excess nicotinamide adenine diphosphate hydrogen generated via the glucose 6 phosphate dehydrogenase in this cytoplasm triggers ROS production.

Apoptosis is an essential part of spermatogenesis that keeps a check on the number of proliferating germ cells so that they are within the supportive limit of the Sertoli cells. Recent reports suggest that a dysregulated apoptotic pathway can result in poor semen quality in terms of a low sperm concentration, malformed spermatozoa and higher levels of DNA damage.

We are examining whether or not there is a connection between OS and dysregulated apoptosis factors. There have been many suggestions that OS may be responsible for this dysregulated apoptosis and hence cause the DNA damage. This hypothesis is being tested via a series of experiments that would expose subsets of ejaculated spermatozoa to induce OS and determine whether this will induce any apoptotic activity in them. The extent of apoptotic activity hence visualized and the resulting DNA damage could help illustrate a complete molecular pathogenesis of sperm DNA damage and may provide clues to possible strategies for its prevention and treatment.

Motility is one of the important factors necessary for natural pregnancy to occur. It is a prerequisite for delivery of the genomic material for fertilization. In asthenozoospermic patients (<30% motility), natural pregnancy rate is very poor. Sperm motility is governed by motor and cytoskeleton proteins. These proteins are regulated by a group of small G proteins called RhoGTPase (Ras homologue) (RhoA-B & Rac). These proteins control actin/tubulin cytoskeleton assembly & vesicle transport (e.g. translocation of phospholipase C from the cytosol to the sperm plasma membrane). Defects in these motor/cytoskeleton proteins and their regulators lead to morphological abnormalities (teratozoospermia) and poor motility (asthenozoospermia). Defects in the tail region in these proteins might be associated with sperm immotility while head region specific RhoGTPase could be linked to failure of sperm to undergo hyperactivation (capacitation) acrosome reaction and sperm oocyte fusion. This may be because of improper sperm head structural organization as a result of abnormal cytoskeleton network proteins or their regulators (RhoGTPase). As these events are regulated by RhoGTPase, aberrant expression of these proteins could be associated with alterations either at the gene expression level or during sperm maturation process. Our aim is to investigate the signaling mechanism of RhoGTPase family proteins and their gene expression in relation to asthenozoopermic and teratozoospermic sperm abnormalities and study how they affect sperm functions (e.g. motility, capitation & acrosome reaction). This study will provide insight into the regulation of sperm motility and issues with sperm oocyte fusion failure.

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.

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.

Freezing of spermatozoa from infertile men may affect sperm motility, morphology, DNA integrity, mitochondrial activity, and viability. Different studies have demonstrated that cryopreservation of spermatozoa induces reactive oxygen species (ROS) production. Increase in ROS due to the freeze-thaw procedure may upregulate the apoptosis cascade leading to induction of sperm DNA fragmentation. Optimizing cryopreservation techniques is important in order to minimize the anticipated damage to the spermatozoa. Supplementing sperm freezing media with different antioxidants (Vitamin C, vitamin E, and pentoxifylline) have been suggested to optimize the existing freezing and thawing protocol for human spermatozoa in an effort to increase the efficiency of ART. Utilizing different combinations of antioxidants we will evaluate DNA damage, apoptotic changes, ROS levels and fertilization potentials in the spermatozoa obtained from normal donors and infertile men before and after cryopreservation and examine the potential benefit of utilizing different antioxidants alone as well as in combination. Improving the post-thaw quality of frozen spermatozoa from infertile male patients may help result in higher success rates per ART cycles, reducing the number of ART cycles and consequently reducing the overall costs to patients.

One of the goals of our study is to understand how the lack of a major antioxidant gene (glutathione-S-transferase, GST5mu) in the testis predisposes a subpopulation of male infertility patients to damaging effects of oxidative stress. Identifying the genetic and environmental factors that can be increased over a period due to increased production of reactive oxygen species (ROS) may be associated with secondary male infertility. We are utilizing a knockout mouse model deficient for one of the isozymes glutathione-S-transferase mu (GST5mu), which is reported to be similar in human, rats and mouse. Lack of the major antioxidant system in the testis may result, in increased susceptibility to oxidative stress, which increases over time compared to the wild type. This may be analogous to men who may be more susceptible to oxidative stress and present with secondary infertility.

We will validate GST5mu knockout model that may duplicate the testicular alterations seen after exogenous (environmental) or endogenous (genetic changes) exposure to oxidative stress. By localizing GST5mu in the wild type mouse testis and study how alterations in oxidative stress markers (histological, biochemical, immunohistochemical, DNA fragmentation, lipid peroxidation, apoptosis and antioxant status) are different in the knockout mouse. This study also attempts to identify the cell types (post-meiotic germs cells, spermatids, spermatzoa with excessive residual cytoplasm) in the testis where alterations in oxidative stress biomarkers occur. By establishing a useful knockout mouse model for study of GST5mu gene, this may be critical in preventing oxidative stress and conferring protection to the developing germ cells, and postmeiotic cells (spermatids and spermatozoa).

Hurricane Katrina hit the Gulf coast on August 29, 2005 and had a devastating effect, altering the ecosystem of the entire region. It caused extensive flooding and associated deposition of 6 to 8 inches of new soil in the adjoining residential and commercial areas, schools and parks in most of New Orleans. The levels of pollutants, mainly organophosphates, heavy-metals (e.g., lead, cadmium, cobalt, mercury, arsenic, etc.), pesticides (DDT and metabolites) and other contaminants that were already present in New Orleans soil and environment have increased. The long term effect of these pollutants on the general health, although not known at present, has raised serious concern. Many of these pollutants (e.g. organophosphates, heavy metals etc) have been shown to affect the reproductive system, induce cancer, and other toxic effects to lungs, kidney, liver etc. We are investigating the immediate effects of such pollutants, especially heavy metals (lead, arsenic and cadmium), present in New Orleans soil following hurricane Katrina on the reproductive system i.e. testis and alterations in sperm maturation affecting fertilizing ability in the epididymis. The focus is to elucidate possible mechanism(s) of such toxic effects utilizing rodents, both mice and rat as study model.

Sperm preparation is critical in assisted reproductive techniques. Samples have to be centrifuged for sperm processing and frozen for long term storage. Both of these procedures affect sperm quality. Review of the current literature reveals that carnitines have antiapoptotic, antioxidant, and anti TNF-alpha effects. We are investigating the effect of L-carnitine on sperm quality in terms of both motility improvement as well as reduction in the amount of DNA damage during centrifugation as well as freezing. Addition of L-carnitine may have a protective effect before centrifugation of a semen sample or prior to freezing of sperm.

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.

The goal in assisted reproductive programs is to use viable sperm with minimum DNA damage. We aim to identify the best sperm fraction using 3 sperm preparation methods (density gradient centrifugation (DGC), magnetically activated cell sorting (MACS) and flow activated cell sorting (FACS) in terms of sperm motility, normal morphology, and percentage of sperm recovery. In addition we will examine the optimum sperm preparation method that will provide viable spermatozoa with minimal DNA damage and improve their fertilizing capacity. We will utilize markers of viability and early apoptosis (annexin V), mitochondrial membrane integrity (mitochondrial membrane potential), DNA damage and intracellular formation of specific reactive oxygen species (ROS).

The clinical importance of our study is that it will allow us to evaluate the sperm selection method based not only on motility, morphology, viability and recovery but also on the molecular markers indicative of mitochondrial membrane integrity, intracellular levels of reactive oxygen species formed, apoptosis as well as extent of DNA content. These selection criteria may help provide new therapeutic tools for the ART lab, and for the management of male infertility.

Separation of spermatozoa from the semen is an integral part of an andrology laboratory. Also, obtaining motile sperm from the semen is an important aspect of assisted reproductive techniques (ART). The bench-top centrifuges that are commonly used in the andrology labs and ART programs are not temperature regulated. This can possibly affect the quality of the spermatozoa. In addition we have demonstrated the damaging effects of centrifugation speed and time on the production of ROS. We are evaluating the effects of temperature controlled and non-temperature controlled human semen centrifugation on various parameters of the recovered spermatozoa. Temperature regulated centrifuge may prevent sperm damage which is linked to production of heat and ROS generation due to routine centrifugation.

Sperm cryopreservation is a major and essential component of any infertility service provided to patients. Sperm cryopreservation is currently done by slow freezing. After thawing, semen samples are usually compromised during the process of freezing and thawing as evident by a decrease in sperm viability and motility especially in infertility patients with poor semen parameters. One of the detrimental factor of decreased viability during freezing is the ice crystal formation.

Vitrification is the ultra-rapid method of freezing which avoids ice crystal formation by rapid transition from liquid to solid state. We have developed a new device "The Ohio-Cryo" that allows vitrification of larger sample volume suitable for freezing sperm. We are validating the use of this device in the vitrification of semen samples and comparing the outcome of slow freezing to vitrification by measuring sperm motility and effect on DNA damage. If sperm vitrification can be properly achieved, we would expect improvement in quality og post-thaw sperm parameters. This may be extremely beneficial, especially in preserving patient samples.

• The relationship between reactive oxygen species in semen and male infertility, RPC #4252, 1993
• Effect of motility stimulants on human sperm after cryopreservation, RPC #4925, IRB #4925, 1995
• Maximizing semen quality in spinal cord injured men, RPC #5269, IRB #5269, 1995
• Improving post-thaw semen quality in cancer patients, RPC #5489, IRB #1441, 1995
• Assessment of human sperm function after cryopreservation in processed and unprocessed semen, RPC #5490, IRB #1442, 1996
• Seminal reactive oxygen species in men with varicocele, and the effect of in vitro antioxidant supplementation, RPC #5549, IRB #1514, 1996
• Designing an algorithm to predict fertilizing capacity of spermatozoa using biochemical and functional assays, RPC #5765, IRB #1727, 1997
• The effects of mycoplasmas on the semen analysis and the function of spermatozoa, RPC #5825, IRB #1819, 1997
• The role of reactive oxygen species and total antioxidant capacity in persistent infertility following vasectomy reversal, RPC #6036, IRB #2155, 1997
• Impact of oxidative stress and IL-6 on fertility in varicocele patients, RPC #6206, 1998
• Predicting fertilization in an IVF program by use of mannose receptor assay, RPC #6238, IRB #2988, 1999
• Changes in frequency of sperm chromosomal anomalies after chemotherapy for testicular cancer and Hodgkin's disease: a longitudinal study, RPC #6265, IRB #2989,1999
• Role of reactive oxygen species in the pathophysiology of varicoceles and effects on antioxidant therapy alone or in combination with varicocele repair on sperm function and pregnancy outcome, AFUD, 1999-2001, IRB #3273
• The role of oxidative stress in assisted reproduction, RPC #6362, IRB #3244, 1999
• Role of oxidative stress and DNA damage in different subsets of ejaculated spermatozoa, RPC #6387, 1999
• The role of defective spermiogenesis in male infertility: relationship between sperm immaturity and oxidative stress, RPC #6509, IRB #3853, 2000
• Relevance of leukocytes on semen parameters, oxidative stress, and DNA damage in semen of infertile patients, RPC #6560, IRB #3841, 2000
• Mechanism of nuclear DNA damage in mature sperm from infertile men, RPC #6770, IRB #4716, 2001
• Levocarnitine fumerate and acetyl-l-carnitine combined supplementation in subfertile males with high reactive oxygen species, Sigma-Tau Health Sciences, Inc., Protocol STHS 01-01, IRB #4562, 2001
• Oxidative stress mediated sperm DNA damage in infertility patients - possible role of apoptosis, RPC #07025, IRB #5474, 2002
• Assessment of fertilizing potential of mouse germ cells matured in vitro, RPC #7531, ARC #07518, 2004
• Characterization of intracellular reactive oxygen species in human spermatozoa, RPC#07549, IRB #6967, 2004
• Assessing the quality of cryopreserved testicular biopsy tissue, it's relation to etiology of infertility and outcomes of assisted reproduction, IRB 7622, 2004
• Varying patterns of semen analysis in men evaluated for infertility: A retrospective review, IRB 7644, 2004
• Correlation of apoptotic markers with sperm fertilization potential, RPC #07812, IRB 7546
• Demographics of male patients seeking intervention for infertility, IRB 6519, 2004
• Comparison protocol for human semen samples: SQA-V versus microscopic assessment, IRB 8045, 2005
• A novel glutathione-S-transferase (GST5mu) knockout mouse model for studying oxidative stress, RPC#1066/ARC#08077
• Utility of PARP alterations following apoptotic and oxidative DNA damage in human spermatozoa, RPC #1067, IRB 8530
• Effect of post-Katrina contaminants from Lake Pontchartrain on the male reproductive system, RPC #1011/ARC #08110
• Antioxidant combinations to minimize human sperm apoptosis and DNA damage during cryopreservation, RPC #1097, IRB #425
• Optimizing human sperm preparation techniques for selecting reactive oxygen species (ROS)-based high quality spermatozoa in assisted reproductive procedures, RPC #2007-1057
• Effects of electromagnetic waves exposure from cell phones on semen quality, IRB #06-423
• Optimizing sperm preparation techniques for selecting high quality spermatozoa in assisted reproductive procedures, RPC #07-949
• Defining Sperm DNA Damage in Infertile Men with Varicocele and its Correlation with Oxidative Stress, RPC # 2007-1101 , IRB #08-095
• Is poor sperm motility associated with defects in RhoGTPase in infertile men? RPC# 2007-1024, IRB 205
• Effect of radiofrequency electromagnetic waves (RF-EMW) from the cell phone on human semen parameters, reactive oxygen species and DNA damage- an in vitro study, IRB # 08-231
• Efficacy of L Carnitine before sperm processing for assisted reproduction techniques in reducing sperm DNA damage, RPC # 2008-1007, IRB # 08-225
• Long term effects of electromagnetic waves on human semen, IRB #06-423
• Effects of Radiofrequency Electromagnetic Waves (RF-EMW) from Cellular Phones on Human Ejaculated Semen, RPC 2008-1025
• Smoking and semen parameters: Is the effect of smoking on male fertility a reversible process, RPC 2008-1033, IRB #304
• Antioxidants Combinations to Minimize Human Sperm Apoptosis and DNA Damage during Cryopreservation, IRB 06-425
• Comparison of temperature regulated vs. non-regulated bench-top centrifuge for sperm quality improvement, IRB 09-128
• Comparative study of sperm vitrification and slow freezing, IRB 09-127
• Characterizing pathophysiologic effects of radiofrequency electromagnetic waves (RF-EMW) emitted by cell phones on human spermatozoa, IRB 09-069
• Role of Curcumin in reducing oxidative stress-induced sperm damage, RPC #2009-1040
• Development and validation of a novel sperm vitrification system using the Ohio-Cryo vitrification device, RPC #2009-1038

** Click on the RPC # to view information about a Research Project; click on the IRB # to view information on Institutional Review Board approval. The above information will open as a PDF in Adobe Acrobat on your PC.