Conference Paper

Artificial microcontainers for cryopreservation of solitary spermatozoa

Conference: 23th Annual Meeting of the ESHRE

ABSTRACT Human Reproduction. 2007. V. 22, Suppl. 1. P. i154-i155.
http://humrep.oxfordjournals.org/content/22/suppl_1/i153.full.pdf

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    ABSTRACT: Conventional freezing procedures are not appropriate for surgically retrieved spermatozoa from the epididymis or testis because of their low numbers. Techniques for the cryopreservation of small numbers of spermatozoa have not been fully established. We tried to develop a cryopreservation method for a single spermatozoon using Cryotop, which has a simple structure and is easy to handle. Different parameters influencing the freezing procedure, types of container, sources of spermatozoa, and cryoprotectants were evaluated. The sperm recovery rate after thawing was similar between the sperm frozen using Cryotop or zona pellucida as containers (98.0% vs. 88.0%). Freezing of motile single spermatozoa obtained from ejaculates and testes were evaluated for recovery rate (90.0% vs. 95.0%) and motility rate (44.4% vs. 42.1%), which were not significantly different. The survival rate was significantly higher when sperm were treated with sucrose rather than with SpermFreeze (65.3% vs. 37.3%, P < 0.01). Cryotop was a highly effective tool for the cryopreservation of a single spermatozoon, and sucrose was determined to be an efficient cryoprotectant.
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    ABSTRACT: The introduction of the technique of intracytoplasmic sperm injection to achieve fertilization, especially using surgically retrieved testicular or epididymal sperm from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction. The techniques for the retrieval of spermatozoa vary from relatively simple percutaneous sperm aspiration to open excision (testicular biopsy) and the more invasive Micro-TESE. The probability of retrieving spermatozoa can be as high as 100% in men with obstructive azoospermia (congenital bilateral absence of the vas deferens, status post-vasectomy). However, in nonobstructive azoospermia, successful sperm retrieval has been reported in 10-100% of cases by various investigators. The surgical retrieval and cryopreservation of sperm, especially in men with non-obstructive azoospermia, to some extent ensures the availability of sperm at the time of intracytoplasmic sperm injection. In addition, this strategy can avoid unnecessary ovarian stimulation in those patients intending to undergo in vitro fertilization-intracytoplasmic sperm injection with freshly retrieved testicular sperm when an absolute absence of sperm in the testis is identified. Several different methods for the cryopreservation of testicular and epididymal sperm are available. The choice of the container or carrier may be an important consideration and should take into account the number or concentration of the sperm in the final preparation. When the number of sperm in a testicular biopsy sample is extremely low (e.g., 1-20 total sperm available), the use of an evacuated zona pellucida to store the cryopreserved sperm has been shown to be an effective approach.
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    ABSTRACT: The purpose of this study is to develop a novel method for the cryopreservation and efficient post-thaw recovery of individual or small numbers of human spermatozoa. Spermatozoa equilibrated in cryoprotectant buffer were injected with an intracytoplasmic sperm injection (ICSI) needle into a droplet of cryoprotectant on a homemade cryoleaf. The droplet was of cryoprotectant and seminal plasma at a ratio of 1:1. The sperm-loaded cryoleaf was slowly lowered over and stored in liquid nitrogen. Spermatozoa were thawed in a 37°C oil bath without dilution and centrifugation. To test the fertilizing ability of these spermatozoa, the recovered spermatozoa were injected by ICSI into 1-d-old or in vitro-matured human oocytes. Fresh spermatozoa from the same semen samples served as controls. The trials were performed in two separate experiments. In the first set of experiments, 92 spermatozoa were thawed and carefully investigated. The spermatozoa from percutaneous epididymal sperm aspiration had a motility recovery of 92.9% (13/14); ejaculated spermatozoa had a motility recovery of 61.5% (48/78), and only 1.3% (1/78) was lost. Together in the first and second set of experiments, the fertilization rates for the fresh and frozen-thawed spermatozoa were 67.6% (25/37) and 60.6% (40/66), respectively (P = 0.052). The mean embryo cleavage rates in the fresh and frozen-thawed groups were 88% (22/25) and 85% (34/40), respectively (P = 0.990). This cryopreservation method for individual or small numbers of human spermatozoa was efficient and simple. These findings make this method a promising technique for the clinical application of ejaculated sperm from oligozoospermic patients.
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