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Dr. Tadros is assistant professor and director of male infertility and sexual health at Southern Illinois University, Springfield, and Dr. Agarwal is professor and director of the Andrology Center at Cleveland Clinic’s American Center for Reproductive Medicine and on staff in the Glickman Urological and Kidney Institute, Cleveland. Section Editor Steven A. Kaplan, MD, is professor of urology at the Icahn School of Medicine at Mount Sinai and director of benign urologic diseases, Mount Sinai Health System, New York. Follow him on Twitter at @MaleHealthDoc.
Despite the ongoing controversies regarding the accuracy and predictive power of routine semen analysis, it continues to be used by many clinicians worldwide as the de facto test for male infertility (Int Braz J Urol 2014; 40:443-53). In its best practice statement for the evaluation of the infertile male, the AUA has proposed the use of advanced tests of sperm function in certain patients to enhance the diagnostic accuracy of semen analysis, specifically in cases of unexplained infertility, recurrent pregnancy loss, or failure of intrauterine insemination (IUI) and in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) (The Optimal Evaluation of the Infertile Male. AUA Best Practice Statement, 2010).
As explained in this article, these new tests have the potential to improve our ability to better diagnose and treat complicated male infertility patients.
Oxidative stress is thought to contribute to 40%-80% of male infertility (Fertil Steril 2003; 79:829-43) and arises as a consequence of excessive production of reactive oxygen species (ROS) and impaired antioxidant defense mechanisms (figure 1) (Curr Med Chem 2001; 8:851-62). Although small amounts of ROS are important for normal sperm function, an excess of these highly reactive molecules can cause damage to the lipid-rich plasma membranes and the integrity of DNA within the sperm nucleus, as well as impaired motility and spermatozoa apoptosis (Fertil Steril 2003; 79:829-43).
Antioxidants such as some vitamins and minerals combat these overproduced ROS. In addition to excess ROS, oxidative stress can be due to insufficient concentrations of antioxidants as well. Over the last decade, research has provided growing support for the fact that oxidative stress leads to abnormal semen parameters. In addition, more and more laboratory tests are now available to measure this oxidative stress. Therefore, it would be reasonable to potentially screen all infertile men for the presence of increased ROS levels. Specialized training and equipment, the lack of cost-effective and efficient assays, and, perhaps most importantly, the lack of a universally accepted analytical methods have prevented ROS testing from being included as part of the routine infertility workup.
Next: "ROS can be measured both directly and indirectly"
ROS can be measured both directly and indirectly. Chemiluminescence is probably the most common way to directly measure ROS in sperm currently and can quantify both intracellular and extracellular ROS. It uses a luminometer in conjunction with a chemiluminescent probe such as luminol, which can also be used to measure a total antioxidant capacity (TAC). The results can be expressed as a ROS-TAC score, which can give an indication of the combined oxidant and antioxidant activities of seminal constituents. Unfortunately, this test requires special equipment, training, and is costly for widespread clinical use.
Due to these limitations, nitroblue tetrazolium (NBT) has been put forth as a cost-effective alternative. NBT interacts with free radicals and is converted to a blue pigment that can then be measured with light microscopy. This test suffers from a lack of standardization and low inter- and intra-observer reliability.
The new MiOXSYS Analyzer (figure 2) used at our institution measures the so-called oxidation-reduction potential (ORP). ORP measures the balance of all oxidants and antioxidants in the specimen and gives a complete picture of the oxidative stress. This test can be performed in less than 5 minutes. It requires no specialized training and may possibly replace the more complex and traditional oxidative stress tests without sacrificing the reliability. Elevated ORP levels correlate well with infertility, with a significantly higher ORP seen in infertile patients than in fertile controls.
In a recent study, ORP was able to detect at least one abnormal sperm parameter with a sensitivity of 70.4% and a specificity of 88.1%. It had an 88% sensitivity and 91.2% specificity when detecting oligozoospermia (Urology 2017; 104:84-89). Given the increased recognition that oxidative stress plays an important role in male infertility, development of reproducible and cost-effective techniques in measuring oxidative stress may help in tailoring our treatments for infertile couples.
Next: DNA fragmentation
Researchers have turned their attention to the genetic contents of sperm, as embryo development depends in part on the inherent integrity of sperm DNA. DNA integrity testing is relatively new to the armamentarium for fertility specialists. While originally described in 1993, it failed to gain traction as a clinical test due to lack of availability and standardization. Sperm DNA is highly compacted by binding tightly to protamine. A certain degree of sperm DNA damage can often be repaired by the oocyte’s antioxidant enzymes. When damage exceeds the repair capability of the oocyte, deleterious effects of sperm DNA fragmentation (SDF) may result, such as miscarriage and poor embryo development (Hum Reprod 1999; 14:1039-49).
Because of this, more attention has turned to testing for SDF. This test does not evaluate the actual genetic code of the DNA within the sperm, but rather the overall superstructure of the DNA strands. There are many different ways to test for SDF, each with pros and cons. We will focus our discussion on the most commonly used tests as well as what is considered the gold standard, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Asian J Androl 2016; 18:205-12).
The acridine orange (AO) test uses a fluorescent dye that emits a different wavelength of light based on whether it is bound to double-strand DNA (normal) or single-strand DNA (abnormal). This test is fast, simple, and inexpensive but limited by inter-laboratory variations and lack of reproducibility. Another relatively simple test, the “halo” test, has similar pros and cons as the AO test, but evaluates the characteristic halo of dispersed DNA loops around sperm with non-fragmented DNA.
The latest technology using TUNEL detects “nicks” or free ends of DNA by utilizing fluorescent nucleotides with a flow cytometer (or fluorescence microscopy) to quantify the incorporation of dUTP into DNA breaks. This test is sensitive, reliable, and has minimal inter-observer variability. It can also be performed on samples with severe oligozoospermia. Many now consider this the gold standard for DNA fragmentation testing. More importantly, SDF can provide valuable information for both the infertile couple and fertility specialists in guiding treatment.
In men with infertility and varicoceles, some will not see any improvement in semen parameters after varicocelectomy, and SDF testing may help to identify those in whom varicocele ligation would be of most benefit. SDF can also be used in couples with recurrent pregnancy loss or who have failed IUI as a predictive tool to identify and effectively stratify patients based on elevated SDF and potentially offer IVF or ICSI sooner rather than later. In patients who have already failed IVF or ICSI with ejaculated sperm, SDF testing can help determine the next steps in treatment.
Because DNA fragmentation increases during sperm transit through the epididymis, these patients may benefit from IVF/ICSI with sperm extracted from the testis instead of repeated IVF cycles with ejaculated or epididymal sperm. SDF may also help identify environmental exposures and lifestyle choices that affect male fertility. Factors like smoking, obesity, and occupational exposures can all worsen SDF. In these patients, SDF testing can help predict their fertility potential and monitor response to lifestyle modification.
Both oxidative stress testing and DNA fragmentation testing are relatively new technologies that will help clinicians choose the most appropriate therapies for their patients. While no large-scale randomized studies have been performed yet, emerging evidence on both are promising. We believe they will eventually become commonly used in clinical practice for the management of male infertility.
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