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  • Cited by 16
Cambridge University Press
Online publication date:
July 2010
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Book description

Protecting the reproductive potential of young patients undergoing cancer therapy is increasingly important. With modern treatment protocols, 80% of patients can be expected to survive. It has been estimated that up to one in 250 young adults will be a survivor of childhood cancer in the future; infertility, however, may be a consequence. As a wide range of fertility preservation methods are increasingly offered by clinicians, this systematic and comprehensive textbook dealing with the cryobiology, technology and clinical approach to this therapy will be essential reading to infertility specialists, embryologists, oncologists, cryobiologists, ObGyns, andrologists, and urologists with an interest in fertility preservation. Fertility Cryopreservation reviews all the techniques of this increasingly important field within reproductive medicine. It covers the basic principles of pertinent cryobiology, and contains major sections on the different therapies available, written by international specialists combining experience from both academic centers and commercial industries.

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  • 8 - Testicular tissue cryopreservation
    pp 57-66
  • View abstract


    Cryobiology is the core of fertility cryopreservation. The principal application for human fertility cryopreservation began with sperm freezing, and then developed to include embryo and oocyte as well as gonadal cryopreservation. This chapter briefly discusses the scientific background and the current basic knowledge of cryobiology. Aqueous solutions are important for cryobiology since the freezing of biological systems always involves solutions containing substances such as electrolytes, non-electrolytes, polymers, and so on. Some of the classic papers in the field of cryobiology describe the theories and the mechanisms of cryoinjury during cell freezing and thawing. Some cryoprotectants reduce the injury of cells during freezing and thawing. Today, the most commonly used cryoprotectants in the field are glycerol, dimethyl sulfoxide, ethylene glycol, and propylene glycol. Cryoprotectants can interact with each other in a mixture, or with crucial cell molecules, thereby producing effects other than those that would occur with an individual cryoprotectant.
  • 10 - Cryopreservation of pronuclear stage human embryos
    pp 76-88
  • View abstract


    For several decades, attempts to cryopreserve human oocytes have been performed in many in vitro fertilization (IVF) centers worldwide, with variable results. Ice-free cryopreservation is an attempt to circumvent the hazards of water crystallization as ice. Three key factors influence the probability of successful vitrification: cooling and warming rates, the composition of the cryoprotectants (CPA) solution, and the sample volume. Pressure is another factor that increases the chance of vitrification but this has had very little, if any, application in the clinical assisted reproductive technology (ART) arena. The osmotic stress during removal of CPAs was initially reduced in slow cooling by a stepwise dilution (using reduced concentration of CPAs progressively), allowing enough time for the cell to return to an equilibrium volume. Two of the dangers of cryopreservation are solution effects and intracellular ice formation. Other factors causing damage are extracellular ice and intracellular dehydration.
  • 11 - Cryopreservation of day two and day three embryos
    pp 89-94
  • View abstract


    The principal pathway for the movement of water and cryoprotectants in oocytes/embryos at various stages helps in the selection of suitable cryoprotectant(s) and optimal conditions for cryopreservation. The permeability to water and cryoprotectants of mammalian oocytes/embryos can be measured as changes in volume in hypertonic solutions containing sucrose or cryoprotectant. A longer exposure to the cryoprotectant solution(s) would be necessary to dehydrate the cells and allow the cryoprotectant(s) to permeate sufficiently. For vitrification of oocytes/early embryos, a two-step treatment would be effective, in which oocytes/embryos are first pretreated in a solution containing a low concentration of cryoprotectant for permeation in less toxic conditions, and then in the vitrification solution for a short time to cause the embryos to shrink by rapid dehydration. Conditions suitable for the cryopreservation of mammalian oocytes and embryos differ among maturational/developmental stages even in the same species.
  • 12 - Cryopreservation of blastocysts
    pp 95-105
  • View abstract


    This chapter focuses on various cryoprotectants and their application in preserving gametes and embryos. Penetrating cryoprotectants provide buffering against salt-induced stress by acting as solvent, reducing the solute concentration in the remaining water fraction inside the cell until the system is cooled to a low enough temperature. Various penetrating cryoprotectants have been used successfully in in vitro fertilization (IVF) to preserve sperm, embryos, and oocytes. Non-penetrating cryoprotectants are often included in media used for warming/thawing of cells to avoid osmotic shock. Most toxicity concerns exist with penetrating cryoprotectants rather than the non-penetrating cryoprotectants. In regard to reproductive biology and assessing suitability/toxicity of a cryoprotective agent, certain endpoint measures are commonly assessed. Cryoprotectants serve to prevent damage induced by the cooling/freezing process, in part by reducing ice crystal formation and by reducing stress resulting from osmotic shock.
  • 13 - Aseptic vitrification of human blastocysts:
    pp 106-113
  • protocol development and clinical application
  • View abstract


    Sperm cryopreservation is a widely used and established method in humans, animals, fish, and insects. In humans, sperm cryopreservation is a widely used technique in assisted reproductive technologies (ART) and fertility preservation in patients with cancer. Sperm cryopreservation describes a complex multistep process for preserving male gametes. The process involves collecting a sperm sample, then gradually cooling the sample in the presence of a cryoprotective agent, followed by storage of the sample for future use. Cryoprotectants such as glycerol revolutionized cryopreservation techniques and paved the way for storing sperm samples for up to several years. As new cryoprotectants were discovered, the main issue was the degree of protection that they could provide for a sperm from damage caused by rapid freezing. Future studies are expected to concentrate on advancing technology to achieve the goal of damage-free sperm after cryopreservation.
  • Chapter 15 - Cryopreservation of oocytes by slow cooling
    pp 120-130
  • View abstract


    Sperm cryopreservation plays an important role in the field of male infertility and reproductive medicine. A donor sperm cryopreservation program has been developed and improved in mainland China. The conventional approach to sperm cryopreservation is to simply dilute semen with cryoprotectant and cryopreserve in liquid nitrogen until the sperm samples are thawed for use. Patients with spinal cord injuries often have a problem with poor sperm production as well as ejaculation after the damage. Electroejaculation is usually performed under a general anesthesia while the patient is placed in lateral decubitus. With the advance of new approaches for sperm vitrification, treatment of male infertility will become more effective without using sperm donors. Using vitrification for cryopreservation of sperm obtained from testicular biopsy, epididymal fluid, or a semen sample after electroejaculation could create new hope for infertile men.
  • 16 - Vitrification of human oocytes with different tools
    pp 131-143
  • View abstract


    This chapter focuses on various issues in the cryopreservation of surgically retrieved sperm. When the male partner is unable to produce an adequate amount of ejaculated sperm with good quality for intracytoplasmic sperm injection (ICSI), surgical sperm retrieval is indicated. Various techniques have been described and modified for sperm retrieval since the introduction of ICSI. The three principal sources of sperm are epididymides, testes, and ejaculation. The quantity of sperm retrievable surgically from the epididymides and testes is generally small compared with the amount of ejaculated sperm. With epididymal sperm, there are reductions in the concentration of viable spermatozoa as well as a significant decrease (by 50%) in the percentage of motile spermatozoa. For men with azoospermia, cryopreservation of sperm retrieved surgically in advance of the fertilization stage can allow better planning for the ICSI cycle and minimize unnecessary ovarian stimulation.
  • 17 - Vitrification of human oocytes using the McGill Cryoleaf protocol
    pp 144-156
  • View abstract


    Advances in assisted reproductive technology (ART) have created opportunities for preservation of the reproductive potential of young males with cancer. Semen cryopreservation is possible in most adolescents with cancer regardless of age or diagnosis. Awareness by physicians is even more essential when dealing with younger populations. Theoretically, testicular tissue from prepubertal boys facing gonadotoxic treatment could be banked for many years for spermatogonial stem cell transplantation. Male germline stem cells are the only cells in postnatal mammals that undergo self-renewal and transmit genes to subsequent generations, since all female germline stem cells cease their proliferation before birth. Future possible methods of restoring fertility might include the derivation of mature sperm cells from human embryonic stem cells. Embryoid bodies were shown to support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes round off the somatic diploid chromosome complement and develop into blastocysts.

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