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Human Sperm Cryopreservation 21 Human Sperm Cryopreservation Alex C Varghese, Parag Nandi, Reda Mahfouz, Kelly S Athayde, Ashok Agarwal ABSTRACT Human sperm cryopreservation has become widely accepted in our society. It has been proven to be a very successful method to keep the hope of a family alive for many men. In early 1940s cattle breeders started cryopreserving bull semen. The cryopreservation of human semen started in 1950s with more refined techniques designed for human sperm. Since then continuous ongoing research has developed more sophisticated techniques for human semen cryopreservation. This chapter is an introduction to the human sperm cryopreservation, the latest techniques of the sperm cryopreservation and its significance in assisted reproductive techniques. SPERM CRYOPRESERVATION: HISTORY Efforts to maintain the fertilizing capacity of mammalian spermatozoa for extended times have been made for at least 135 years. Having observed survival of human spermatozoa that had been cooled to –150°C in 1866, an Italian physician P. Mantegazza proposed the concept of a human sperm bank to store semen specimens.1 Since the birth of Louise Brown, the first test tube baby in the world on 25th July, 1978 by the pioneering work of Dr Robert Edwards and Dr Patrick Steptoe, it has been reported that three million babies have been born by assisted reproductive technology (ART). Human sperm cryopreservation has become a routine procedure since then in any assisted reproductive technology laboratories. SPERM CRYOPRESERVATION: SIGNIFICANCE Cryopreservation is widely used in many assisted reproductive programs to preserve male fertility, for example, before cytotoxic chemotherapy,2 radiotherapy, or certain surgical treatments that may lead to testicular HUMAN SPERM CRYOPRESERVATION 339 failure or ejaculatory dysfunction. Freezing of sperm before initiation of treatment provides patients “fertility insurance” and may allow them to father their own children through the use of intrauterine Insemination or in vitro Fertilization techniques such as intra cytoplasmic sperm injection (ICSI). Cryopreservation of sperm is mandatory in donor-insemination programs. The use of frozen semen allows screening of donors for infectious diseases such as HIV and hepatitis B before release for insemination.3-6 Cryopreservation is also widely used for storage of sperm retrieved from azoospermic patients who have undergone testicular sperm extraction or percutaneous epididymal sperm aspiration (PESA), avoiding the need of repeated biopsies or aspiration. SPERM CRYOPRESERVATION: PURPOSE At present, the most commonly used cryofreezing procedures are in propylene straws/vials of different sizes and volumes and it has been documented by several researchers.7-9 Several studies have aimed to find correlations between different parameters of frozen-thawed semen and fertility in several species including stallions. Unfortunately, so far, no single in vitro parameter has been identified to predict fertility. Sperm motility is the most commonly used criterion for evaluating quality of cryopreserved semen. It has been found that Computer Assisted Sperm Analysis (CASA) is sufficient for clinical use in crucial individual motility patterns.10-12 Nevertheless it may be inadequate for determining an individual’s fertility as only motility evaluation does not predict the fertility potential of the sperm cells. Different probes have been used to measure sperm viability and acrosome reaction in different species.13-15 It has been argued that such patients undergoing procedures that may lead to testicular failure or ejaculatory dysfunction should be offered cryopreservation of semen.16 Although in 1980 Braken and Smith reported that only 4% of patients had semen of suitable quality for a fertility program,17 the advent of ICSI overcame these limitations, it allows even low quality samples to be used successfully.18 Regarding the safety of the cryopreservation techniques, questions about the possibility of cross contamination through the liquid nitrogen could be raised. Hepatitis C virus has a low rate of sexual and intra-familial transmission,19 and till date there is not a single case report where it has been found to be transmitted by stored bone marrow or semen. Some studies of semen, saliva, and vaginal secretions in HCV-positive patients have failed to find HCV RNA.20,21 It is believed that semen samples may have very small viral load that would not survive even the liquefaction time. HCV is 340 ANDROLOGY LABORATORY MANUAL a RNA virus and only have RNA dependent RNA polymerase to reverse transcript to RNA strand only, which are not at all stable and can not be integrated to host DNA. A case of DNA virus hepatitis B acquired from stored bone marrow was recently described22 and hence awareness is required for all potentially transmissible agents. With isolated storage facilities now provided, all patients who are positive for antibody to hepatitis C virus should be able to preserve their long term fertility. Within the many reasons for sperm cryopreservation, this technique could assist the male partner in an IVF couple in the following situations: concern about the stress of the collection of semen on demand, any history of poor specimens collected for IVF, traveling husband (not available at the particular time of the insemination) or in case of oligozoospermic samples, when collection of more than one ejaculate are pooled for IUI/ IVF. Spermatozoa cryopre-servation is also of interest for maintaining genetic diversity in endangered species, especially those in zoological parks which are candidate species for captive breeding programs.23 CRYOPROTECTANTS: INDICATIONS In 1938, the survival of human spermatozoa stored at the temperature of solid carbon dioxide was reported. Despite this finding, it was not possible to start using cryopreserved semen in breeding programs until a considerable observation taken up by Parkes in 1945.24 The single most important development came with the significant discovery by Polge, Smith, and Parkes in 194925 that glycerol could act as a cryoprotectant for spermatozoa. The initial experiments with glycerol were performed using fowl spermatozoa but were quickly followed by the successful preservation of bull spermatozoa26 with the first calf from Artificial Insemination using frozen/thawed spermatozoa reported in 195l.27 The use of glycerol had allowed bull spermatozoa to survive at the comparatively slow freezing rates obtained using methanol cooled with solid CO2, and with semen contained in large glass-freezing ampoules. Effective cryoprotectants are preferentially excluded from proteins; thereby it is more stabilized and avoids unfolding or denaturation.28 Such cryoprotectants are hydrophobic enough to pass cell membranes easily and would be hydrophilic enough to readily mix with water. If the cryoprotectants are toxic then it may act more by hydrogen-bonding with water than by colligative interference with ice formation. It implies that understanding the molecular mechanisms of the toxicity of cryoprotective agents would be an important advance step toward finding means of reducing toxicity. Cryoprotectant toxicity and destabilization of polypeptides in cell membranes have been documented.29 HUMAN SPERM CRYOPRESERVATION 341 In general, cryoprotectant toxicities are lower at lower temperature, and may even become negligible if the cryoprotectants can be introduced at a low enough temperature. The challenges that cells have to overcome to survive after freeze-thaw are not their ability to endure storage at low temperature; rather, it is the lethality of an intermediate zone of temperature (– 15 to – 60°C) that cells must pass through during cooling as well as during warming.30 As the cells approach the freezing temperature, the water outside the cell freezes first, removing liquid solvent from the extracellular broth and resulting in increased intracellular osmolarity. There are two groups of cryoprotectants used in routine cryogenic work. One is cell membrane permeable and another one is membrane non- permeable. Cell membrane permeable cryoprotectants viz. glycerol, dimethyl sulfoxide (DMSO) and membrane non-permeable cryoprotectants viz. polyhydroxyethyl starch, glycine are being used randomly. At the present time different sugar molecules like glucose, sucrose, raffinose, etc. have also been frequently used as cryoprotectant in routine work.31 Most of these cryoprotectants are used at a concentration of 5 to 10%.32 Among these, glycerol is the most commonly used cryoprotectant for mammalian as well as human spermatozoa.33, 34 Conversely, Jeyendran35 showed that spermatozoa might become dependent on glycerol so that once it is removed, motility rapidly decreases. For the better recovery of the motile cells along with the cryoprotectants several other natural extenders viz. egg yolk, or synthetic extenders viz. Zwitterion buffers containing TRIS [hydroxy-methylaminomethane], TES [N-TRIS (hydroxymethyl) methyleaminoethanesulfonic acid], or combination of these are generally being used routinely.36, 37 Sodium citrate, another substance added to extending buffers, may give better result.38, 39 Nowadays HSPM (Human Sperm Preservation Medium) has become very popular in many laboratories.40, 41 It has been reported that TYB over HSPM gives better result in motility recovery both containing 7% (v/v) glycerol.42 But in a comparative study, TYB in contrast with HSPM can be recommended as a first choice cryoprotectant for semen preservation in order to avoid extra
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