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Couple infertility is gradually increasing, and couples unable to conceive naturally are dependent on assisted reproductive techniques (ART). Intracytoplasmic sperm injection (ICSI) bypasses the natural selection process of sperm in the female reproductive tract, hence sperm-handling techniques are used to select the most suitable sperm for oocyte fertilization. In this chapter we discuss the conventional sperm-processing methods such as swim-up and density gradient centrifugation. Furthermore, advanced techniques such as magnetic-activated cell sorting, microfluidic devices, motile sperm organelle morphology examination, flow cytometry, and zeta potential are presented. We provide an overview of the sperm-handling approach and its outcome in ART. Additionally, we highlight the future sperm-handling techniques such as Raman spectrometry, interferometric phase microscopy, confocal light absorption and scattering spectroscopy, proteomic analysis, and peptide-based selection of sperm.
Human sperm cryopreservation is a highly desirable technique for preserving fertility potential and future use in couples desiring to have a biological child. Slow cryopreservation of sperm has been the mainstay technique. The drawback of this technique is the inability to freeze extremely small numbers of sperm as in the case of surgical retrieval of testicular sperm. These sperm are extremely few in number and it is difficult to retrieve motile, viable sperm post-thaw. In the past decade, sperm vitrification has been introduced along with both biological and non-biological carriers to freeze extremely low numbers of sperm. Vitrification also allows the ability to freeze single spermatozoa. These techniques are in many ways more efficient and better than the older techniques. This chapter aims to provide a detailed introduction to various approaches used for preserving spermatozoa. It also discusses indications of sperm cryopreservation based on semen quality and summarizes the advantages and shortcomings of these techniques.
Spermatozoa are mature male gametes that are produced in the testes of a healthy man by spermatogenesis, with further maturation of sperm taking place during their transit through the epididymis. In the human, approximately 20 to 240 million sperm are produced per day . Unlike other somatic cells present in the human body, spermatozoa contain a head, neck, mid-piece and tail region. The head region contains the genetic material which is transferred to the oocyte during the fertilization process. Apart from DNA, spermatozoa also deliver additional subcellular materials such as oocyte activating factors, RNA, microRNAs and exosomal proteins that are essential for the development of the oocyte into a zygote.
Assessment of sperm vitality is an important component of semen analysis. It helps to distinguish spermatozoa that are alive and immotile from those that are dead. Sperm vitality can be assessed routinely on all semen samples by assessing the membrane integrity of the cell by identifying the spermatozoa with an intact cell membrane. This can be done by using 1) the dye exclusion test or 2) the hypotonic or hypoosmotic swelling test. Sperm vitality can therefore provide a good comparison with the motility of the sample. Eosin is used as a marker for dead cells because eosin can penetrate the cells when the membrane is damaged, while cells that have an intact membrane remain unstained. Nigrosin is a background stain that increases the contrast to the otherwise faintly stained cells [1, 2, 3, 4]. Both the single step and two-step staining using eosin and nigrosin have been used to assess sperm vitality. Both the wet preparation and the air-dried methods have been compared to study the correlation with motility [5, 6, 7]. The wet preparations evaluated by using either positive or negative phase-contrast microscopy consistently showed higher percentage of nonviable cells compared to the air-dried eosin-nigrosin smears. The air-dried smears have consistently shown that the sum of the motile (viable) and stained (presumed dead) preparations never exceeded 100 percent indicating that the air-dried method is the method of choice for determining vitality. In this chapter, we describe the staining protocols for vitality, the cut-off of motility when vitality must be tested, indications for poor motility and quality control recommended for performing sperm vitality in conjunction with basic semen analysis.
Oxidative stress (OS) is the consequence of an imbalance between reactive oxygen species (ROS) and the failure of antioxidants to neutralize excessive ROS production. Although many sperm functions require physiological levels of ROS, excessive levels of ROS are detrimental to the sperm . OS is one of the most common etiologies of male infertility affecting 30–80 percent of infertile men [2, 3]. The role of OS in men with unexplained infertility has been clearly established . OS affects sperm quality as a result of alterations in proteins, lipid peroxidation, DNA damage and apoptosis . Damage to sperm DNA can compromise the contribution of paternal genome to the embryo . Hence the advent of numerous tests to diagnose OS in the semen. There are several laboratory tests available to measure OS – both direct and indirect. Direct tests measure OS or free radicals such as ROS and reactive nitrogen species. These include chemiluminescence, nitroblue tetrazolium, cytochrome C reduction test, electron spin resonance, fluorescein isothiocynate (DFITC)-labeled lectins, and measurement of oxidation reduction potential. Indirect tests measure oxidized products resulting from ROS sources such as the oxidized form of nicotinamide adenine dinucleotide (NADPH)-oxidase in the sperm, the reduced form of NAD (NADH)-dependent oxidoreductase in mitochondria, or leukocytospermia. These include myeloperoxidase or Endtz test, antioxidants (both enzymatic and non-enzymatic), lipid peroxidation, and DNA damage. In this chapter we will discuss the indirect tests that are available to assess OS and also elaborate on the interpretation and their clinical significance [4, 5].
Male infertility affects men worldwide with about 20 percent of couples having male factor infertility . The routine semen analysis is the first step in the assessment of male infertility. However, conventional semen analysis does not provide a complete understanding of fertility potential, especially in patients with idiopathic infertility . In this scenario, DNA integrity is the most important feature to ensure normal fertilization, implantation, pregnancy and embryonic development. Sperm DNA fragmentation (SDF) can be due to several intrinsic factors such as varicocele, oxidative stress, apoptosis, and chromatin packaging defects. Further, SDF can be caused by extrinsic factors such as lifestyle alterations, infections, exposure to xenobiotics, etc. [3–7].