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.

Human embryos generated from in vitro fertilization may exhibit varying degrees of cytoplasmic fragmentation, and increasing evidence demonstrates that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. Review of the current literature reveals that carnitines have antiapoptotic, antioxidant, and anti TNF-a effects. Administration of acetyl-L-carnitine (ALC) to mouse fibroblasts in culture media has shown a reduction in apoptosis through the mitochondrial pathway. L-carnitine (LC) was observed to promote neuronal survival and mitochondrial activity in a concentration-dependent manner in primary cultured neurons from cerebral cortex of rat embryos. LC has been investigated for its role in antagonizing the effect of TNF-a in conditions such as liver cell damage, tumors, and inflammatory diseases. The main objective of our study is to optimize embryo quality and enhance ART procedure outcomes. This can be achieved by supplementation of in vitro culture media with LC. Supplementation with LC is a novel approach due to its antioxidant, anti-TNF-a, and antiapoptotic effects.

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.

In a large prospective study, we are assessing the effects of EMW emitted by cell phones (900 - 1900 MHz) on various markers of sperm quality such as count, motility, morphology, viability etc. We will correlate the semen characteristics and exposure to electromagnetic waves from cell phones adjusted by such covariates as patient demographics, daily environment and lifestyles. The results of this study will allow us to estimate the degree of complication and the type of disturbances in male reproductive system caused by EMW. This in turn may help demonstrate the need for protective measures to prevent or reduce the effect of EMW in reproductive system.

• 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
• 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

** 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.