Sperm banking, consequences of its use in animal and clinical practice

Sperm banking, more formally referred to as sperm cryopreservation, is a process intended to preserve sperm function by freezing and storage at ultra-low temperature. Upon thawing, sperm are introduced into a suitable recipient female by insemination into either the endocervical canal or the intrauterine cavity, or are used to inseminate oocytes during in vitro fertilization. Sperm freezing originated in the late eighteenth century, but the widespread uses of sperm cryopreservation began after 1950. The discovery that glycerol had cryoprotectant properties, and the availability of liquid gases, especially liquid nitrogen, to achieve ultra low temperatures for freezing and storage, stimulated the development of many sperm banking applications. The advantages of frozen sperm over fresh sperm include the following: they can be stored almost indefinitely (at least for decades), allowing preservation of genetic characteristics that would be lost due to onset of disease, infertility, or death; they can be readily shipped anywhere in the world using small liquid nitrogen containers which can withstand the rigors of transport; and they can be placed in frozen ``quarantine'', while the human or animal donor can be tested for semen-borne infections or genetic problems.

Human Clinical Applications of Sperm Banking
An important medical use of sperm banking is patient autologous sperm cryopreservation, called client depositor sperm banking. Client depositor sperm banking is used in the following medical situations:

1) Medical disorders which inherently, or through the treatment used to cure or stabilize the disease, can impair fertility by causing decreased sperm count and function, early fetal loss, genetic mutation, or impotence. Examples include testicular cancer, Hodgkin's Disease and other lymphomas, leukemia, nephrotic syndrome, diabetes, and multiple sclerosis.

2) Prior to elective sterilization or exposure to hazardous environments. Occupational exposure to radiation, pesticides, and chemicals can affect sperm function or genetic integrity. Men engaging in military operations where risks of death or exposure to sperm toxicants exist also are candidates for sperm storage.

3) Before participating in fertility treatments which require semen collection at a specific time. For men who develop anxiety-related impotency or emission failure, sperm banking ensures that treatment cycles can proceed as planned. Patients whose occupations require unscheduled travel also find that sperm banking reduces the risk of cancelled treatment cycles.

One of the concerns often expressed by physicians about referring patients with systemic diseases for sperm banking is whether the patient's sperm are of sufficient quality and number to achieve a pregnancy. Although sperm count, motility, and physiology may be impaired before treatment is initiated, the technological advances in assisted reproduction, such as direct sperm injection into the ooplasm, often can, at the present time, or will, in the near future, be able to overcome many abnormalities present. Having many sperm stored is definitely an advantage, since it may reduce the need for in vitro fertilization and increase the chance for a successful pregnancy outcome, but the desire to bank multiple ejaculates must be balanced against the necessity of treatment initiation and financial constraints.

The use of cryopreserved sperm obtained from anonymous donors as a treatment for infertility caused by absent or defective sperm is the other major medical application of sperm banking. In a 1987 survey, the United States Office of Technology Assessment estimated that 30,000 births resulted from artificial insemination of donor sperm, with approximately 11,000 physicians providing the treatment to about 86,000 women. The practice has probably increased and the demand for fertile and safe sperm remains high. It is virtually impossible to adequately screen donors for infectious diseases with long incubation periods such as human immunodeficiency virus and hepatitis viruses or which are detected with tests that require more than a few minutes to perform, i.e., most infectious diseases. If the sperm are quarantined in the freezer, however, the donor can be examined repeatedly for disease exposure over months or years before the sperm are used. The Centers for Disease Control has cautioned that fresh anonymous donor sperm should not be used for artificial insemination, and frozen anonymous donor sperm should be used only if the donor tests negative for human immunodeficiency viruses after a minimum of 180 days quarantine.

The ability to store sperm from men with many different phenotypes and genotypes increases the selection that patients have in choosing a donor, and reduces excessive use of a donor within a limited geographic area. Population statistics can allow determination of the number of pregnancies that can be achieved without increasing the risk of consanguinity in future generations. Generally, sperm from a single individual are used to achieve no more than 10-15 pregnancies in a medium-sized city (500,000 to 1,000,000 inhabitants) in the United States. In other countries where ethnic diversity and ethnic intermarriage are not as common, the number could be smaller, but depends in any case on the live birthrate and number of inhabitants.

Usually, sperm banks attempt to package donor sperm in plastic vials or straws containing at least 10 million motile sperm post-thaw, which has been suggested as the minimum adequate insemination dose. Since frozen-thawed sperm have shorter longevity than fresh sperm, the route and timing of insemination is critically important to achieving a successful pregnancy. Using qualitative urinary luteinizing hormone (LH) measurement to determine ovulation, and one or two intrauterine inseminations within 20 to 40 hours of the LH surge, approximately 70% of patients who elect donor sperm insemination conceive, the majority within six insemination cycles.

The American Association of Tissue Banks (AATB) has standards for both donor and client depositor sperm banking, and accredits banks by peer inspection. The AATB also maintains a list of non-accredited sperm banks. Several states have certification programs and the Food and Drug Administration has recently begun to regulate tissue banking, including gametes.
Sperm Banking in Animals

Sperm cryopreservation has important uses in the livestock industry, especially in the breeding of cattle, pigs, sheep and poultry, and in animal husbandry for domesticated animals such as horses, cats and dogs. Sperm from genetically desirable or "prized'' animals can be used to inseminate many females to increase the number of offspring with the desired characteristics. The ability to easily transport sperm has permitted the improvement of existing herds or the establishment of new herds in regions of the world needing development of native food sources. Sperm banking has also become an important way to perpetuate endangered or exotic species in the wild and in zoological parks.

The ability to use sperm banking to preserve important research animal strains has been appreciated recently. Sperm cryopreservation could reduce the extraordinary cost of maintaining genetic lines that now must be preserved by continual breeding of the animals, increase the accessibility of various strains to researchers since frozen sperm are easier to transport than live animals, and reduce the risk of losing a valuable genetic line through catastrophic accident, impaired reproductive efficiency, genetic drift, or disease. Because the millions of sperm normally present in a single ejaculate represent millions of meiotic recombination events, cryopreserved sperm can be stored for future studies of gene recombination frequency and mapping of genetic loci when new DNA probes become available.

The Process of Sperm Cryopreservation
In spite of the important uses for cryopreserved sperm, little is known about the physical and biochemical events which occur during sperm freezing, storage, and thawing, or about methods for detecting cryogenic damage. Sperm from most species survive current cryopreservation protocols very poorly, and best efforts usually result in recovery of only about half of the original sperm motility. Sperm function is also impaired, as manifested after thawing by shorter longevity and reduced membrane stability.

The goal of any sperm freezing protocol is to prevent lethal intracellular ice crystal formation, control wide fluctuations in cell volume, and reduce membrane damage that accompanies temperature-induced phase changes. The process is complicated by the biochemically and physically diverse compartments of the sperm cell (acrosome, nucleus, mitochondrial-flagellar network), all of which may respond quite differently to freezing and thawing. The sperm also are subject to damaging oxygen radical exposure during their transit through wide temperature changes. Attempts to maximize post-thaw survival have led to the development of sperm cell diluents (extenders), cryoprotectants, and various rates of temperature change to control alterations in extracellular and intracellular solvents and solutes.

In most cryopreservation protocols, the ejaculated sperm are mixed with a buffered diluent that contains an energy source such as fructose or glucose, lipid, and a penetrating cryoprotectant such as glycerol. After dilution, the sperm initially undergo rapid shrinkage as intracellular water leaves the cell, then slowly return to their original volume as the glycerol enters. Rapid cooling is initiated at a rate of about -20 C per minute. Extracellular formation of ice crystals begins and, as water freezes, the solutes present in the liquid phase surrounding the sperm rapidly become concentrated. Glycerol lowers the intracellular water freezing point, thus the cells remain unfrozen and become supercooled well below their actual freezing point. In response to high extracellular solute concentration and the osmotic tendency of supercooled intracellular water to leave the cells, sperm undergo a second volume adjustment as water moves outward, and the cells become dehydrated. When extracellular water freezes and therefore solidifies, an exothermic reaction known as the ``heat of fusion'' occurs, which can cause serious disruption of the cells unless externally reduced by controlled cooling of the environment. Upon reaching the temperature of liquid nitrogen, -196 C, the sperm are placed in storage indefinitely, where they are presumed to reside in a quiescent state of minimal molecular motion.

During thawing, the sperm are subjected to similar rapid and dramatic changes in cell volume and membrane permeability. As the extracellular ice melts and becomes liquid, solute concentrations are rapidly diluted and water rushes into the sperm. As the temperature rises, and as glycerol leaves the cells, the sperm cell volume continues to expand. In order for function to be restored, the surface area and volume must return to normal, the membrane proteins and lipids must redistribute to restore molecular structure and mobility, and bioenergetic demands must be met. For maximum functional recovery to take place, both the freezing and the thawing protocols must be optimized, a very difficult task given the paucity of data available about the processes.

Research efforts to improve sperm banking techniques and post-thaw survival have intensified in the last decade and offer many career opportunities for basic and applied research. As protocols improve, the success of cryopreserved sperm applications will undoubtedly increase.

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