WHO laboratory manual for the examination and processing of human semen

Techniques for freezing bull sperm developed over the past 40 years have not yielded protocols for preserving sperm from other species. Recent advances in our understanding of cell membrane structure function and metabolism now permit alternative modes of investigation. These data will allow development of unique studies which should have a higher probability of yielding successful protocols for sperm from other species. In this review the authors will: (1) provide a general overview of cryopreservation; (2) review emerging concepts of membrane structure and the relationship of membrane composition to water and cryoprotectant movement; (3) emphasize how these parameters affect cell volume and surface areas; (4) focus attention on the concept that cryoprotectants will alter membrane structure and function in addition to their well-recognized effects on bulk solvent; and (5) emphasize the effect of the processing protocol on metabolic balance. These concepts are reintroduced in the context of the established and successful protocol for freezing bull sperm to illustrate the molecular responses that may be necessary to survive a freeze-thaw cycle.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.1990.tb01583.x

Cryopreservation of mammalian sperm: what we ask them to survive.

The onset of clinical assisted reproduction, a quarter of a century ago, required the isolation of motile spermatozoa. As the indication of assisted reproduction shifted from mere gynaecological indications to andrological indications during the years, this urged andrological research to understand the physiology of male germ cell better and develop more sophisticated techniques to separate functional spermatozoa from those that are immotile, have poor morphology or are not capable to fertilize oocytes. Initially, starting from simple washing of spermatozoa, separation techniques, based on different principles like migration, filtration or density gradient centrifugation evolved. The most simple and cheapest is the conventional swim-up procedure. A more sophisticated and most gentle migration method is migration-sedimentation. However, its yield is relatively small and the technique is therefore normally only limited to ejaculates with a high number of motile spermatozoa. Recently, however, the method was also successfully used to isolate spermatozoa for intracytoplasmic sperm injection (ICSI). Sperm separation methods that yield a higher number of motile spermatozoa are glass wool filtration or density gradient centrifugation with different media. Since Percoll® as a density medium was removed from the market in 1996 for clinical use in the human because of its risk of contamination with endotoxins, other media like IxaPrep®, Nycodenz, SilSelect®, PureSperm® or Isolate® were developed in order to replace Percoll®. Today, an array of different methods is available and the selection depends on the quality of the ejaculates, which also includes production of reactive oxygen species (ROS) by spermatozoa and leukocytes. Ejaculates with ROS production should not be separated by means of conventional swim-up, as this can severely damage the spermatozoa. In order to protect the male germ cells from the influence of ROS and to stimulate their motility to increase the yield, a number of substances can be added to the ejaculate or the separation medium. Caffeine, pentoxifylline and 2-deoxyadenosine are substances that were used to stimulate motility. Recent approaches to stimulate spermatozoa include bicarbonate, metal chelators or platelet-activating factor (PAF). While the use of PAF already resulted in pregnancies in intrauterine insemination, the suitability of the other substances for the clinical use still needs to be tested. Finally, the isolation of functional spermatozoa from highly viscous ejaculates is a special challenge and can be performed enzymatically to liquefy the ejaculate. The older method, by which the ejaculate is forcefully aspirated through a narrow-gauge needle, should be abandoned as it can severely damage spermatozoa, thus resulting in immotile sperm.

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Sperm preparation for ART.

The redox status of human spermatozoa was found to have a profound influence on the fertilizing potential of these cells in association with qualitative and quantitative changes in the patterns of tyrosine phosphorylation. In general, oxidizing conditions enhanced tyrosine phosphorylation and stimulated sperm function, whereas reducing conditions had the opposite effect. Unstimulated human spermatozoa exhibited low levels of spontaneous acrosomal exocytosis and sperm-oocyte fusion and minimal reactive oxygen species generation, while phosphotyrosine expression was largely confined to a single protein of 116 kDa. However, if the spermatozoa were exposed to oxidizing conditions through the addition of exogenous H2O2, or the stimulation of endogenous NADPH-dependent reactive oxygen species generation, then a dramatic increase in tyrosine phosphorylation was observed (major phosphotyrosyl bands at 222 kDa, 200 kDa, 159 kDa, 133 kDa, 116 kDa and 82 kDa) in concert with the functional activation of the spermatozoa. A causal association between reactive oxygen species generation, tyrosine phosphorylation and sperm function was indicated by studies with the ionophore, A23187, which induced high rates of spermoocyte fusion together with enhanced rates of reactive oxygen species production and the increased expression of phosphotyrosyl proteins. This functional response to A23187 could be abrogated, without any concomitant change in sperm motility or viability, by using membrane permeant thiols or catalase to suppress the reactive oxygen species-induced increase in phosphotyrosine expression. The fact that the biological responses of human spermatozoa to biological agonists (recombinant human ZP3 and progesterone) could also be inhibited by catalase indicated the general relevance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)

Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function

Effects of cryopreservation procedures on sperm membranes

The objective of this study was to develop a relatively simple test to evaluate the functional integrity of the membranes of human spermatozoa. As in some other species, human spermatozoa 'swell' under hypo-osmotic conditions due to the influx of water and the expansion of the membranes. A mixture of equal parts of fructose and sodium citrate (150 mosmol) with calculated ionic strength of 0.15 resulted in a maximal number of clearly identifiable swollen spermatozoa. Only small variations were seen when different aliquants of the same semen samples were separately evaluated. A high correlation (r = 0.94) was obtained between expected and observed values of swollen spermatozoa when known amounts of heat-treated spermatozoa, unable to undergo swelling, were added to untreated spermatozoa. A good correlation (r = 0.90) was also observed between the % spermatozoa in a semen sample that were capable of undergoing swelling and the % of denuded hamster oocytes that were penetrated by capacitated spermatozoa from the same semen sample. By contrast, the correlations between % sperm swelling in ejaculates and % normal sperm forms, % motile spermatozoa and % spermatozoa that do not stain with eosin-Y (supravital stain) in the same ejaculates were 0.30, 0.61 and 0.52, respectively. Therefore, the hypoosmotic swelling technique to evaluate the functional integrity of the sperm membrane appears to give high repeatability and accuracy and is closely correlated to the in-vitro fertilizing ability of spermatozoa. It may be a useful addition to the standard semen analysis.

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Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

Human ejaculates (n = 83) were analyzed for standard sperm parameters (concentration, motility, and morphology), as well as for the ability of the spermatozoa to react (swell) in a hypoosmotic medium (Jeyendran et al, 1984) Subsequently, the fertilizing capacity of the spermatozoa was tested by their ability to fertilize human oocytes in vitro Although the sperm concentration was adjusted for in vitro fertilization, no adjustments were made for sperm motility and morphology Correlation of the in vitro fertilizing capacity of the spermatozoa with the hypoosmotic swelling test (r = 056) was much higher than with standard sperm parameters (r varied from -004 to 025) Complete overlap was noted with standard semen parameters whether the ejaculate did or did not fertilize oocytes and ranged from very low to very high values in both cases By contrast, all the semen samples that fertilized oocytes showed a 60% or higher reaction in the hypoosmotic swelling test, whereas the majority of the "infertile" semen samples showed less than 60% swelling It therefore appears that, under the conditions of our studies, the hypoosmotic swelling test is a more accurate predictor of successful in vitro fertilization outcome than the conventional semen parameters A combination of all parameters, however, is likely to be most useful The hypoosmotic swelling test is simple and economical, and it is recommended that this test be further scrutinized for its value as an additional tool in the assessment of the in vivo fertilizing capacity of ejaculated spermatozoa

Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization.

Stained sperm morphology slides from an in-vitro fertilization programme were evaluated by the modified Williams system, the World Health Organization (WHO) system and morphometric size measurements (image analysis) to determine which system(s) were better predictors of fertility. Amorphous sperm head shape was the best predictor of fertility in both the modified Williams system and the WHO system, predicting the proper fertility status in 65% of the cases. Using only normal sperm in the statistical model, normal sperm became a correct predictor in 66% of the cases in the modified Williams system. Morphometric measurements were not useful in predicting the fertility status of these samples. These studies indicate that morphology can not be used as a sole predictor of fertilizing capacity of human spermatozoa.

M. Perez-Pelaez

Papers

Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics

Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization.

Association of the in-vitro fertilizing capacity of human spermatozoa with sperm morphology as assessed by three classification systems

Leucospermia and the fertilizing capacity of spermatozoa.

Concentration of viable spermatozoa for artificial insemination.