Abstract
ObjectiveTo describe a new technique for freezing individually isolated spermatazoa from testicular biopsies, epididymal aspirates and oligospermic semen samplesMethodsSamples were evaluated for the presence of motile sperm before cryopreservation. Motile or twitching sperm were isolated with an ICSI needle for single sperm cryopreservation. Selected sperm were loaded on the High Security Straw (HSV; Irvine Scientific; Irvine,CA), in ~0.5 µl of fluid to facilitate recovery. The sample was also frozen using conventional methodology in cryovials (100–1000 µl aliquots). In both freezing techniques, the samples were slow cooled. Test-yolk buffer-glycerol (Irvine) was used as the cryoprotectant. Test-thaws were performed to assess sperm recovery and motility.ResultsSix men with azoospermia had single sperm cryopreservation, as well as freezing aliquots of their testicular or epididymal sperm in traditional cryovials. In addition, two men with oligospermia also had individual sperm selected and frozen. In all 8 cases, the ~0.5 µl of fluid containing sperm was quite easily unloaded from the HSV straw during thawing. The percent sperm recovery ranged from 33% to 100%. Motility was evident in all but one sample. In six cases, the sperm were used for intracytoplasmic sperm injection of mature oocytes. Fertilization occurred in all but one case. In this study, we report the first clinical pregnancy with this technique. This pregnancy was remarkable in that a single motile sperm identified and selected in the initial testicular preparation was successfully frozen. We were able to subsequently recover this sperm, fertilize an oocyte and the resultant embryo gave rise to a live birth. The methodology described in this preliminary report offers a new modality for sequestering small numbers of sperm. It may be particularly useful in cases involving severe impairment of spermatogenesis, where extensive screening may be necessary to find a few viable sperm.
Highlights
In vitro fertilization combined with intracytoplasmic sperm injection (ICSI) have provided new treatment options for azoospermic men through surgical or percutaneous isolation of sperm from the testis or epididymis
Two patients with severe oligospermia were offered single sperm freezing and these cases are included in this initial data set
The patient delivered a healthy baby girl who is presently 9 months old. This is the first description of a technique for freezing individually selected sperm in microquantities of fluid in a High Security Straw (HSV)
Summary
In vitro fertilization combined with intracytoplasmic sperm injection (ICSI) have provided new treatment options for azoospermic men through surgical or percutaneous isolation of sperm from the testis or epididymis. Oocyte injection with sperm isolated from testicular biopsies or epididymal aspirates have been quite effective in establishing pregnancies [9]. The first live births with epididymal and testicular sperm were reported by Silber et al [11, 12], respectively. To avoid the expense and complications resulting from repeated surgical sperm retrievals, cryopreservation of sperm is necessary. This has proven to be extremely challenging in cases of non-obstructive azoospermia due to very
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