Tamer M. Said

Role of antioxidants in treatment of male infertility: an overview of the literature

Seminal oxidative stress in the male reproductive tract is known to result in peroxidative damage of the sperm plasma membrane and loss of its DNA integrity. Normally, a balance exists between concentrations of reactive oxygen species and antioxidant scavenging systems. One of the rational strategies to counteract the oxidative stress is to increase the scavenging capacity of seminal plasma. Numerous studies have evaluated the efficacy of antioxidants in male infertility. In this review, the results of different studies conducted have been analysed, and the evidence available to date is provided. It was found that although many clinical trials have demonstrated the beneficial effects of antioxidants in selected cases of male infertility, some studies failed to demonstrate the same benefit. The majority of the studies suffer from a lack of placebo-controlled, double-blind design, making it difficult to reach a definite conclusion. In addition, investigators have used different antioxidants in different combinations and dosages for varying durations. Pregnancy, the most relevant outcome parameter of fertility, was reported in only a few studies. Most studies failed to examine the effect of antioxidants on a specific group of infertile patients with high oxidative stress. Multicentre, double-blind studies with statistically accepted sample size are still needed to provide conclusive evidence on the benefit of antioxidants as a treatment modality for patients with male infertility.

Oxidative stress, DNA damage and apoptosis in male infertility: a clinical approach

clearly independent measures of sperm quality. Therefore, pathogenic ROS levels or poor-quality sperm chromatin structure may be considered indicative of male subfertility [1,2].IDIOPATHIC INFERTILITYMen with idiopathic infertility generally present with significantly higher seminal ROS levels and lower antioxidant properties than healthy controls [3]. Therefore, it appears that the presence of OS in infertile normozoospermic men may be the cause behind previously unexplained cases of infertility. Similarly, sperm DNA damage analysis may reveal hidden sperm DNA abnormalities in infertile men with normal standard sperm values who were diagnosed with idiopathic infertility. The increase in sperm DNA damage in these patients may be partly related to high levels of seminal OS.Finally and importantly, some conditions may pass unnoticed but still affect the sperm genomic integrity. In one case report [4], a fertile patient who had influenza and a 1-day fever of 39.9 ∞ C presented with a relatively high percentage of sperm with damaged DNA (36%) 18 days after the onset of his fever.GENITAL TRACT INFECTIONSGenital tract infections are usually associated with leukocytospermia and elevated ROS levels, as leukocytes represent the major source of ROS production in ejaculates. Although leukocytes are a constant component of human ejaculates and virtually no semen sample is free of them, if the prevalence of leukocytes exceeds normal values (1 ¥ 10

Evaluation of nuclear DNA damage in spermatozoa from infertile men with varicocele

OBJECTIVE To examine levels of sperm DNA damage and oxidative stress (OS) in infertile men with varicocele. DESIGN Prospective controlled study. SETTING Male infertility clinic, Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S) Thirty-one infertility patients and 16 fertile controls. INTERVENTION(S) Sperm DNA fragmentation index (DFI), levels of seminal reactive oxygen species (ROS), and total antioxidant capacity (TAC) were assessed using the sperm chromatin structure assay, chemiluminescence assay, and enhanced chemiluminescence assay, respectively. ROS-TAC score was calculated as a measure of OS. MAIN OUTCOME MEASURE(S) Median (interquartile range) DFI and ROS-TAC scores. RESULT(S) Sixteen of the 31 patients had left varicocele [grade I (n = 3), grade II (n = 10), and grade III(n = 3)], and the remaining 15 had normal genital examination. Patients with varicoceles had significantly higher percent DFI than controls (25%, range: 20%-35%; vs. 15%, range: 10%-22%). Patients with varicoceles had significantly lower ROS-TAC scores (21, range: 9.5-31) than the infertile patients with normal genital examination (34, range: 28-42) or the controls (40.3, range: 38-44). CONCLUSION(S) Infertile men with varicoceles showed significantly increased spermatozoal DNA damage that appears to be related to high levels of OS in semen.

Selection of Nonapoptotic Spermatozoa As a New Tool for Enhancing Assisted Reproduction Outcomes: An In Vitro Model

Abstract Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.

Carnitines and male infertility

L-Carnitine (LC) and acetyl-L-carnitine (ALC) are highly concentrated in the epididymis and play a crucial role in sperm metabolism and maturation. They are related to sperm motility and have antioxidant properties. The objective of this review is to summarize the multiple roles played by LC and ALC in male reproduction, and to highlight their limitations as well as their benefits in the treatment of male infertility. A variety of studies support the conclusion that LC and/or ALC at total daily amounts of at least 3 g per day can significantly improve both sperm concentration and total sperm counts among men with astheno- or oligoasthenozoospermia. Although many clinical trials have demonstrated the beneficial effects of LC and ALC in selected cases of male infertility, the majority of these studies suffer from a lack of placebo-controlled, double blind design, making it difficult to reach a definite conclusion. Additional, well-designed studies are necessary to further validate the use of carnitines in the treatment of patients with male infertility, specifically in men with poor semen quality.

Advantage of combining magnetic cell separation with sperm preparation techniques

The selection of vital, non-apoptotic spermatozoa is a prerequisite for achieving optimal conception rates in assisted reproductive techniques. Magnetic cell sorting using annexin-V microbeads can effectively separate apoptotic and non-apoptotic spermatozoa. The objective of the present study was to optimize the integration of magnetic cell sorting in standard sperm preparations and to correlate the effect of different sperm preparation procedures on apoptotic markers. Semen specimens collected from 15 healthy donors were prepared by either density gradient centrifugation or by one-step sperm wash technique separately and in combination with magnetic cell sorting. The preparation methods were evaluated by assessment of semen parameters (motility, viability and morphology) as well as markers of apoptosis (levels of active caspase-3, integrity of membrane mitochondrial potential and externalization of phosphatidylserine). The apoptotic markers were measured using fluorochrome dyes coupled with flow cytometry. The results showed that the combination of density gradient centrifugation and annexin-V magnetic cell sorting was superior to all other sperm preparation methods in terms of providing motile, viable and non-apoptotic spermatozoa. This study clearly shows the advantage of integrating magnetic cell sorting as a part of sperm preparation, which in turn may positively affect the success rates of assisted reproductive techniques.

Effects of advanced selection methods on sperm quality and ART outcome: a systematic review

BACKGROUND Current routine semen preparation techniques do not inclusively target all intrinsic sperm characteristics that may impact the fertilization potential. In order to address these characteristics, several methods have been recently developed and applied to sperm selection. The objective of this study was to systematically review the literature describing these advanced sperm selection methods focusing on their anticipated benefits on sperm quality and assisted reproductive technique (ART) outcome. METHODS Systematic literature review was conducted by means of a Medline literature search. Sperm quality parameters assessed included: motility, morphology, viability, DNA integrity, apoptosis and maturity. ART outcomes assessed included: fertilization, embryo quality, pregnancy, abortion and live birth rates. RESULTS A total of 44 studies were identified describing four advanced sperm selection methods based on: (i) surface charge (electrophoresis and zeta potential), (ii) apoptosis (magnetic cell sorting and glass wool), (iii) membrane maturity (hyaluronic acid binding) and (iv) ultramorphology (high magnification). Selection of high-quality sperm including improvements in DNA integrity, resulted from the application of these methods. Fertilization and pregnancy rates showed improvement following some of the advanced sperm selection techniques. CONCLUSIONS While some of the advanced sperm selection methods are of value in specific clinical ART settings, others are in need of further evaluation. More clinical studies on safety and efficacy are needed before the implementation of advanced sperm selection methods could be universally recommended in ART.

Implication of apoptosis in sperm cryoinjury

Apoptosis is an ongoing physiological phenomenon that has been documented to play a role in male infertility, if deregulated. Caspase activation, externalization of phosphatidylserine, alteration of mitochondrial membrane potential and DNA fragmentation are markers of apoptosis found in ejaculated human spermatozoa. These markers appear in excess in subfertile men and functionally incompetent spermatozoa. Sperm cryopreservation is a widely used procedure in the context of assisted reproductive techniques. Cryopreservation and thawing is a procedure that inflicts irreversible injury on human spermatozoa. The damage is manifested by a decrease in recovery of viable spermatozoa with optimum fertilization potential. This review describes the implication of apoptosis as one of the possible mechanisms involved in sperm cryoinjury. Evidence shows significant increase in some apoptosis markers following cryopreservation and thawing. On the other hand, the increase in sperm DNA fragmentation following cryopreservation and thawing requires further investigation. Specific technical measures should be applied to minimize the induction of apoptosis in human spermatozoa during cryopreservation and thawing. These include standardization of freezing protocols and cryoprotectant use. Selection of non-apoptotic spermatozoa may also prove to be of benefit.

Chemiluminescence technique for measuring reactive oxygen species

Accurate assessment of reactive oxygen species (ROS) concentrations may help in the diagnosis of infertility. The chemiluminescence technique, which uses a luminometer to measure ROS, is a common method of assessment. A better understanding of the chemiluminescence technique will allow its proper application in reproductive medicine. A wide range of luminometers are available in the market, and laboratories should select the instruments that suit their individual needs.

Evaluation of sperm recovery following annexin V magnetic-activated cell sorting separation

Magnetic-activated cell sorting (MACS) using paramagnetic annexin V-conjugated microbeads eliminates spermatozoa with externalized phosphatidylserine, which is considered one of the features of apoptosis. The objective of this study was to evaluate sperm recovery following the use of MACS as a sperm preparation technique. Mature spermatozoa were separated and divided into two fractions: the first was prepared by density gradient centrifugation (DGC) and MACS, while the second was prepared by DGC only. Following MACS, the percentage of cells collected in the annexin-negative fraction was significantly higher than the annexin-positive fraction and the sperm recovery rate was 73.8 +/- 12.1%. In conclusion, the integration of MACS with DGC can be considered as an effective sperm preparation technique that does not lead to significant cell loss. Separating a distinctive population of non-apoptotic spermatozoa with intact membranes may optimize the outcome of assisted reproduction.