**4.1 Referring patients to a sperm bank**

Human semen cryobanking can be divided into two broad categories: autologous banking for personal fertility preservation and donor sperm banking. Semen banking is useful in many situations and can be considered a safeguard against unforeseen future circumstances. These may include: prior to chemotherapy or radiation therapy; pre- vasectomy; before certain types of pelvic or testicular surgery; in cases of degenerative illnesses such as diabetes or multiple sclerosis, spinal cord disease or injury; high risk occupations or sports;

Cryopreservation of Human Spermatozoa

additional children are desired.

**4.3 Long term storage** 

by Vitrification *vs.* Slow Freezing: Canadian Experience 89

Surgical sperm retrieval should be the last option in patients who can not produce a sample or patients who are diagnosed with azoospermia. Several methods of sperm retrieval are available depending on the etiology of the problem. To retrieve epididymal spermatozoa in cases of obstruction, percutaneous epididymal sperm aspiration (PESA) can be performed without surgical scrotal exploration, it is repeated easily, and does not require an operating microscope or expertise in microsurgery. Microsurgical epididymal sperm aspiration (MESA) is performed under the operating microscope and general anaesthesia. Individual tubules of the epydidymis are isolated and aspirated. Testicular sperm aspiration (TESA) is a needle biopsy of the testicle. It is an office procedure performed under local anaesthesia. Testicular sperm extraction (TESE) is the process of making a small incision and removing a small portion of tissue from the testicle under sedation or local anaesthesia. Microdissection (or sometimes referred to as microscopic or microsurgical) testicular sperm extraction (MicroTESE) is a very rigorous search for sperm under high magnification in cases of azoospermia or extremely low sperm production. MicroTESE is usually performed in the operating room under general anaesthesia utilizing an operating microscope. Cryopreservation of surgically retrieved epididymal and testicular spermatozoa is a valuable component in the effective treatment and management of these patients and it reduces the necessity of repeat surgeries when the intial procedure is unsuccessful or if

Proper long term storage is usually achieved by placing specimens in LN2 freezers, which have been safely used since the 1970s. Some automated systems are available and are capable of LN2 autofilling, supplied with alarms and data recordings for all activities and are designed to minimize the chances of loss or damage of samples. Despite automation, quality control procedures must be implemented by sperm banks to ensure proper monitoring and safety of samples and staff. The LN2 itself can be a source of microbial contamination so every available practical step has to be considered to reduce the risk of transmission (Fountain et al., 1997). In general cross-contamination of frozen samples by pathogens are extremely rare, and have not been reported in the setting of sperm banks. It is, however, theoretically possible, as often patients are not fully screened prior to freezing, as in cases of autologous sample cryopreservation or due to time constraints for oncology banking (Clarke, 1999). "Quarantine'" tanks are often used to separate samples with pending, unclear laboratory results or unscreened patients. However, contamination is still possible, as released samples that are moved to long term storage could have acquired pathogenic contamination from one of other "pending" samples in the quarantine tank. When samples are cryopreserved for patients with known infections such as HIV or hepatitis B and C carriers, separate tanks for each type of infection are required (Tomlinson & Sakkas, 2000). Cross-contamination can also be avoided by storage of samples in nitrogen vapour. However, in contrast to liquid nitrogen, there are some concerns that vapour has poor heat transfer rates, lower thermal capacity, and significant temperature fluctuation may exist within the vapour(Tomlinson & Saakas, 2000; Wood, 1999). Some older types of vapour storage systems could only guarantee the maintenance of temperature around - 100°C and were not acceptable for long term sperm storage. Storage temperatures have to be maintained below -135°C to ensure a glass-like condition of frozen water and for secure long

and preparation for future fertility treatment. Usually several steps are required prior to initiation of semen storage. Most sperm banks only accept patients referred by their physician, however sometimes self referral is possible. Patients have to be screened by collection of blood, urine and semen samples to ensure that their samples will not contaminate others with infectious diseases when placed into storage. Consent for freezing and storage of semen has to be signed and witnessed. It is important that patients have a clear understanding of the process of banking and that they provide clear instructions on who is responsible for the disposition of the specimens in the event of their demise. Similarly, costs associated with banking and long term storage need to be clearly defined and assigned in case the patient becomes incapacitated. When samples are either used for procreation, transported to another facility, or storage is to be discontinued, the patient or designate must complete specific consent forms allowing the bank to comply with their wishes. In most countries it is assumed or stated by law that the semen belongs to the individual who produced the sample, and it is this individual who must sign all consents, unless the sample was designated for donation (e.g., in the case of anonymous sperm donation). If possible, multiple donations are usually recommended to ensure an adequate sperm reserve for future procreation, depending on the quality of the sample, individual circumstances and reason for sperm banking.

#### **4.2 Methods of sperm collection and retrieval**

In most cases patients are able to produce an ejaculate by masturbation. Patients are given instructions to have minimum of 2 days and a maximum of 5 days of sexual abstinence, and to collect an entire ejaculate into a sterile specimen container. Condoms, creams or lubricants must not be used during collection as they can interfere with sperm motility and vitality due to the spermicidal properties of many products. Some patients would not be able to produce sample at the sperm bank by masturbation due to psychological, medical reasons or religious restrictions. In this case collection of samples by intercourse using a non-spermicidal condom, often referred to as a "semen collection device", is acceptable. Penile vibratory stimulation (PVS) can be helpful in spinal cord injury patients and those who are unable to produce semen by masturbation or intercourse (Brackett et al., 1998). However samples collected after PVS often exhibit relatively low motility (Hovav et al., 2002). PVS can help to produce a semen sample in most patients with spinal cord injuries (Brackett et al., 1998).

In some situations, collection of a retrograde sample is necessary. Retrograde ejaculation is a condition in which some or all of the sperm are not expelled through the urethra during ejaculation, but because of an incompetent bladder neck, the ejaculate refluxes back into the bladder. Reasons for this problem include organic conditions such as diabetes or multiple sclerosis, or pharmacological effects (eg. alpha adrenergic blocker use for hypertension). The general approach is to neutralize the urine pH and normalize the urine osmolarity by giving the patient sodium bicarbonate to alkalinize his urine. The urine sample is subsequently washed and used for insemination, in-vitro fertilization or cryopreservation. In patients with anejaculation, electro-ejaculation might be necessary to obtain semen, but this usually requires anaesthesia and has the associated risk of rectal injury. Additionally, samples produced by electro-ejaculation tend to be of relatively low quality (Denil et al., 1996).

Surgical sperm retrieval should be the last option in patients who can not produce a sample or patients who are diagnosed with azoospermia. Several methods of sperm retrieval are available depending on the etiology of the problem. To retrieve epididymal spermatozoa in cases of obstruction, percutaneous epididymal sperm aspiration (PESA) can be performed without surgical scrotal exploration, it is repeated easily, and does not require an operating microscope or expertise in microsurgery. Microsurgical epididymal sperm aspiration (MESA) is performed under the operating microscope and general anaesthesia. Individual tubules of the epydidymis are isolated and aspirated. Testicular sperm aspiration (TESA) is a needle biopsy of the testicle. It is an office procedure performed under local anaesthesia. Testicular sperm extraction (TESE) is the process of making a small incision and removing a small portion of tissue from the testicle under sedation or local anaesthesia. Microdissection (or sometimes referred to as microscopic or microsurgical) testicular sperm extraction (MicroTESE) is a very rigorous search for sperm under high magnification in cases of azoospermia or extremely low sperm production. MicroTESE is usually performed in the operating room under general anaesthesia utilizing an operating microscope. Cryopreservation of surgically retrieved epididymal and testicular spermatozoa is a valuable component in the effective treatment and management of these patients and it reduces the necessity of repeat surgeries when the intial procedure is unsuccessful or if additional children are desired.
