The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-upprepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 mL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 mL of spermatozoa vitrified in 50-mL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P , .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P , .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P . .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.
Two hundred and sixty-five unfertilized human metaphase II (M II) oocytes from an in vitro fertilization program were studied cytogenetically using our chromosomal technique, a gradual fixation-air drying method. Of the 265 oocytes, 185 (70%) were successfully karyotyped. There were 21 aneuploids (11.4%) consisting of 8 hyperhaploids (4.3%), 11 hypohaploids (5.9%) and 2 complex cases (1.1%). There were also 9 structural anomalies (4.9%) and 18 diploids (9.7%). In aneuploidy, the loss or gain of dyads (so-called nondisjunction) occurred more frequently than the loss or gain of monads (so-called predivision). The frequency of abnormally behaved chromosomes (segregation errors) due to nondisjunction, anaphase lag and predivision was studied among the seven chromosomal groups (A-G) and compared with the frequency expected from an equal probability of segregation errors in each of the 23 chromosomes. The observed frequency was somewhat higher than the expected frequency in groups E and G but the difference was not statistically significant in either group. These results were discussed in relation to previous studies on human M II oocyte chromosomes.
The effect of in-vitro treatment by polyvinylpyrrolidone (PVP) on the ultrastructure of human spermatozoa has been tested previously with the statistical analysis of B. Baccetti et al. (1995, J. Androl., 16, 356-371). PVP had a primary detrimental action on the plasma membrane, as well as on acrosomal and mitochondrial membranes. Furthermore, membrane damage induces deterioration of the chromatin, axonemal tubules, fibrous sheath, and accessory fibres.
The effects of follicle stimulating hormone (FSH) treatment on the quality of human spermatozoa were assessed by examining the ultrastructure and the function of infertile human spermatozoa using a previously-defined formula. Using the spermatozoa as an andrological monitor shows that the therapeutic effect of FSH depends on the type of sperm defect. The response to FSH is, in many cases, positive and can be evaluated by examining the state of the ejaculated spermatozoa. From an initial group of 81 patients, 15 were placebo-treated controls, and 19 were non-responders (mainly with microbially infected semen). Out of 47 responders, after therapy nine achieved improved sperm quality which approached the natural fertility threshold. These responders all had spermatozoa affected by immaturity or apoptosis (n = 27). The 20 microbially-infected responders also had immature spermatozoa and never achieved the quality level of natural fertility. Thus, a natural fertility level was only achieved by nine responders out of 27 (three with immature spermatozoa, and six with apoptotic spermatozoa). Using our method of sperm analysis, these patients' spermatozoa were clearly categorized before treatment as either immature or apoptotic. In consequence, the success of the therapy was predictable. The response of individual organelles to therapy was examined. Certain qualities of the acrosome, the chromatin, the mitochondria, and the axoneme appear to be sensitive to FSH. Most of the previous conflicting results reported in the literature may be due to a lack of relevant discrimination between the different defects present in the spermatozoa of the patients, without assessing the likelihood of their response.
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