**4.3 Long term storage**

88 Current Frontiers in Cryobiology

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

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

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).

circumstances and reason for sperm banking.

(Brackett et al., 1998).

**4.2 Methods of sperm collection and retrieval** 

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

Cryopreservation of Human Spermatozoa

by Vitrification *vs.* Slow Freezing: Canadian Experience 91

sperm cryopreservation have created opportunities for many families to achieve pregnancies through therapeutic donor insemination or IVF with donor sperm. Pregnancy rates are estimated to be around 10-12% per unstimulated cycle and can be achieved when at least 5 × 106 progressively motile spermatozoa inseminated into the lower cervical canal on 2–3 occasions during the ovulatory phase of menstrual cycle (Scott et al., 1990). At present, some 30,000 births per year worldwide are attributable to frozen donor sperm inseminations (Mortimer, 2004). While this seems like a large number, it may fall in the future, as the recruitment of sperm donors is increasingly difficult due to complicated and

The screening process for donor sperm is quite rigorous and includes obtaining a complete medical and sexual history, physical examination, psychological assessment and laboratory work-up on blood, urine and semen specimens to screen for pathogens including Hepatitis B, C, Human Immunodeficiency Virus (HIV 1&2), Human T-cell Lymphotrophic Virus (HTLV 1&2), Treponema pallidum (Syphilis), Cytomegalovirus, Chlamydia trachomatis and Neisseria gonorrhoea. Sperm banks perform genetic screening for heritable diseases based on the ethnic background of sperm donors (eg. Cystic Fibrosis for Caucasians). Donors must be retested after the required quarantine interval, and specimens may be released only if the results of repeat testing are negative. Specimens can only be used after they have been quarantined for a minimum of 180 days to avoid the risk of HIV transmission. Donor eligibility restrictions apply to employees of sperm banks, poor donors' health or quality of the semen and in some countries by sexual orientation of the donor, as gay or bisexual men are considered at higher risk for HIV and prohibited from being sperm donors in some countries (including Canada and USA). Many countries have age restrictions for sperm donation. The minimum age is usually 18 and the maximum 40 years of age (Health Canada

Semen donors can be classified into two specific groups, anonymous and non-anonymous (known). Currently, with the establishment of many commercial sperm banks and the ability to safely transport samples even between continents, anonymous sperm donation is the method of choice for most recipients. The anonymity of the donor is maintained through the process. This is an important issue to both the recipient and the donor (Ernst et al., 2007). For fully anonymous sperm donation, the recipients would not be known to the donor and the donor offspring would have no future contact with the donor. The sperm donor gives up all legal rights over the biological children conceived from his samples donated to sperm bank. Anonymous donation allows parents, if they wish, to conceal the issue of infertility, or the fact of non-genetic parenting from the offspring. The motivation to hide this information most commonly is driven by pressure from other family members; fear of being rejected by the child or to protect children from the complicated psychosocial matters related to sperm donation. In many Western countries disclosure is encouraged by many counsellors, and if open disclosure is chosen by the parents, it is usually advised to disclose the method of conception to their children at an early age. Non-disclosure by parents of the biological origin of their children is viewed by some as misleading the child and could potentially affect trust between parents and their children, if their origin eventually becomes known to the child (Patrizio et al., 2001). However, it is ultimately the decision of the parents to

strict regulatory procedures, as well as lack of interest from potential donors.

2000, American Society for Reproductive Medicine (ASRM), 2004)

**5.2 Anonymous donors** 

disclose or not as in adoption cases.

term storage of semen samples (Clarke, 1999). Newer types of high efficiency LN2 vapour freezers and others that have a LN2 "jacket"' provide working environments of below - 160°C and are more suitable for sperm banks.
