Jill Stronk

Spermatozoa Bound to Solid State Hyaluronic Acid Show Chromatin Structure With High DNA Chain Integrity: An Acridine Orange Fluorescence Study

During human spermiogenesis, the elongated spermatids undergo a plasma membrane remodeling step that facilitates formation of the zona pellucida and hyaluronic acid (HA) binding sites. Various biochemical sperm markers indicated that human sperm bound to HA exhibit attributes similar to that of zona pellucida-bound sperm, including minimal DNA fragmentation, normal shape, and low frequency of chromosomal aneuploidies. In this work, we tested the hypothesis that HA-bound sperm would be enhanced in sperm of high DNA chain integrity and green acridine orange fluorescence (AOF) compared with the original sperm in semen. Sperm DNA integrity in semen and in their respective HA-bound sperm fractions was studied in 50 men tested for fertility. In the semen samples, the proportions of sperm with green AOF (high DNA integrity) and red AOF (DNA breaks) were 54.9% ± 2.0% and 45.0% ± 1.9%, whereas in the HA-bound sperm fraction, the respective proportions were 99% and 1.0%, respectively. The data indeed demonstrated that HA shows a high degree of selectivity for sperm with high DNA integrity. These findings are important from the points of view of human sperm DNA integrity, sperm function, and the potential efficacy of HA-mediated sperm selection for intracytoplasmic sperm injection.

Double probing individual human spermatozoa: aniline blue staining for persistent histones and fluorescence in situ hybridization for aneuploidies

OBJECTIVE To study the potential relationship between two sperm nuclear attributes: persistence of histones and occurrence of chromosomal aneuploidies. DESIGN The two variables were examined by double probing of the same spermatozoa. SETTING Academic Andrology Laboratory. PATIENT(S) Semen samples subjected for analyses were examined. INTERVENTION(S) We studied >58,000 spermatozoa, in seven men, first with aniline blue histone staining, graded as light (mature sperm), intermediate (moderately immature), and dark (severely arrested maturation). After recording the staining patterns and destaining, the same spermatozoa were subjected to fluorescence in situ hybridization (FISH), using centrometric X, Y, and 17 chromosome probes. MAIN OUTCOME MEASURE(S) Proportions of sperm with light, intermediate, and dark staining were assessed, and ploidy of these sperm was evaluated. RESULT(S) The aneuploidy frequencies in intermediate versus light (mature) spermatozoa were increased four- to sixfold. In addition, aneuploidy frequencies and proportions of intermediate sperm were related. There was no FISH signal detectable in the darkly stained, severely arrested mature sperm. CONCLUSION(S) The data suggest that in sperm with arrested maturity and DNA fragmentation, the binding of FISH probes is diminished. DNA damage is further aggravated by the decondensation and denaturation steps of FISH. Thus, there is a strong likelihood that in oligozoospermic men, with a higher proportion of sperm with arrested maturation, the sperm disomy frequencies are historically underestimated.

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.

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 Hams 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.