Spermatozoa as a transport system of large unilamellar lipid vesicles into the oocyte.

In addition to their role as man-made membranes, vesicles continue to be investigated as carriers for drug delivery. While most research focuses on their injectable properties, here a new delivery strategy is proposed. It is shown that spermatozoa can transport vesicles of variable composition. For human spermatozoa, the vesicles started to show binding after 20 mol% of the nonbinding vesicle backbone lipids were substituted with positive, negative, cerebroside or ganglioside lipids. Vesicle binding is a dynamic process with constant 'on' and 'off' binding. The physiological and motility attributes of the spermatozoa are not affected by the attached vesicles. Sperm swimming characteristics changed only marginally. Also, the activation status of the acrosomal membrane, tested with the fluorescent probe Pisum sativum agglutinin, was not affected by vesicle binding. Moreover, the hyaluronic acid-binding test showed that viable, fully developed spermatozoa will attach and remain bound to hyaluronic acid-coated slides regardless of vesicle binding. Therefore a new 'hybrid' delivery system was created with human spermatozoa, and tested with a mouse IVF system. Large unilamellar vesicles physisorbed to mouse spermatozoa can not only penetrate the mouse oocytes in these proof-of-principle experiments, but also deliver the cargo placed within the vesicles.

Optimal utilization of cryopreserved human semen for assisted reproduction: recovery and maintenance of sperm motility and viability.

PURPOSE: Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. METHODS: Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. RESULTS: Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. CONCLUSIONS: Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.

Hyaluronic acid substantially increases the retention of motility in cryopreserved/thawed human spermatozoa.

We have demonstrated previously that hyaluronic acid (HA) improves the velocity and the retention of motility in freshly ejaculated human spermatozoa. In the present work, we examined the effect of HA on cryopreserved/ thawed spermatozoa in four paradigms: (i) effect of HA on sperm motility and velocity in semen; (ii) stabilizing effect of HA after 4 h of incubation when the decline of sperm motility is already detectable; (iii) the duration of improved motility after the separation of spermatozoa from HA by Percoll gradient centrifugation; and (iv) motility of sperm cryopreserved in the presence of HA. HA improved the retention of sperm motility in thawed spermatozoa. Indeed, the motility values after 30 h were approximately 100% higher in the HA compared with the control samples. This effect of HA was also evident in the stabilization of spermatozoa with already declining motility. After removal of the HA from the incubation medium, significantly increased motility in the HA-exposed spermatozoa was still detectable for at least 4 h. Cryopreservation of spermatozoa in the presence of HA did not improve the recovery of motility. The data indicate that HA improves the retention of motility of cryopreserved/thawed spermatozoa, even after the removal of HA from the incubation medium. The utilization of HA will probably prove beneficial in assisted reproduction: in intrauterine insemination and in in-vitro fertilization (IVF), the extended sperm motility and velocity will enhance the fertilizing efficiency; in intracytoplasmic sperm injection (ICSI), the improved motility will facilitate the identification of viable spermatozoa. Because HA is a physiological component of the cumulus and of the female and male reproductive tracts, administration of HA should not cause ethical concerns.

Fertilization and cleavage rates of heparin-exposed human oocytes in vitro, and the effect of heparin on the acrosome reaction.

Fertilization and cleavage rates were compared in 1024 heparin-exposed and nonexposed human oocytes recovered from 183 consecutive in vitro fertilization (IVF) cycles. Heparinized Ham's F-10 medium, (Gibco, Grand Island, NY) 1.0 ml (2.0 mg heparin/ml) was added to bloody follicular fluid; clear follicular aspirates did not receive heparin. Fertilization and cleavage rates for heparin-exposed (n = 714) and nonexposed (n = 310) oocytes were not significantly different: 63.9 versus 61.6% fertilized (chi 2 = 0.472); 89.3 versus 87.4% of fertilized eggs cleaved (chi 2 = 0.445). A subset of 100 patients, each contributing both heparin-exposed and nonexposed oocytes, also was evaluated. Fertilization and cleavage rates were not significantly different: 59.1 versus 60.8% fertilized (chi 2 = 0.192); 87.6 versus 87.2% of fertilized oocytes cleaved (chi 2 = 0.014). A modified triple stain was used to evaluate the acrosome reaction rate of sperm that had been coincubated with 76 oocytes from ten patients. There was no significant difference in the proportion of viable acrosome-reacted sperm following incubation with heparin-exposed (1.9 +/- 1.0%) versus nonexposed (2.3 +/- 1.2%) (mean +/- standard deviation [SD]) oocytes. The addition of heparin to follicular fluid at the time of oocyte recovery for IVF has no apparent effect on fertilization or cleavage in vitro, nor any influence on the acrosome reaction.