Decreasing quality of semen.
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Fertility↗
Biomedical subjects
D Mortimer
Publications and source records attributed to D Mortimer.
Methods of sperm preparation for assisted reproduction.
Because seminal plasma contains factors which inhibit the fertilising ability of spermatozoa, it is essential that spermatozoa be separated from it quickly and efficiently. Four basic approaches for this exist: (1) simple dilution and washing; (2) sperm migration (either direct from liquefied semen, from a suspension of washed spermatozoa or from a washed sperm pellet); (3) selective washing procedures (mainly Percoll and Nycodenz density gradients); and (4) adherence methods for the elimination of debris and dead spermatozoa (glass wool, glass beads and Sephadex columns). While the success of a sperm preparation method is often assessed by its yield of motile spermatozoa, it is also vital that sperm preparations for clinical use should be free of any microbiological contaminants present in semen. Other relevant considerations in choosing a method include its technical complexity as well as its material, apparatus and time costs. Any possible exposure of spermatozoa during preparation to deleterious influences that may cause iatrogenic sperm dysfunction must obviously be avoided at all costs. Consequently, methods involving centrifugal washing steps prior to the selection of motile spermatozoa should be discontinued. Direct swim-up from semen remains the simplest way to obtain populations of highly motile spermatozoa and, dependent upon the absolute yield required, can be a very rapid procedure with normal semen samples. Abnormal samples, especially those with increased viscosity, may benefit from a prior filtration on a glass bead or Sephadex column. The initial motile sperm preparation should be washed once (perhaps twice) to minimise seminal plasma contamination of the final preparation. Several rapid, simple discontinuous Percoll gradients giving excellent yields are available.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Separation↗
Seminal plasma gamma-aminobutyric acid (GABA) levels in normospermic men.
Serum and seminal gamma aminobutyric (GABA) concentrations were documented in 10 healthy volunteers. Mean (+/- S.E.M.) GABA concentrations were 0.39 +/- 0.03 microM in serum and 103.3 +/- 28.2 microM in seminal fluid. These differences are in keeping with data reported for the rat and rabbit. A regulatory role for GABA in the reproductive tract is postulated.
Adult↗
Sperm preparation techniques and iatrogenic failures of in-vitro fertilization.
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Animals↗
Therapeutic donor insemination with frozen semen.
Although it is now accepted that cryopreserved semen must, on ethical and medicolegal grounds, be used for donor insemination many clinicians still believe that it has an unacceptably reduced fecundability rate as compared with fresh semen. We studied the outcome of 81 recipients who started therapeutic donor insemination (TDI) treatment during 1986 in a program that used exclusively cryopreserved semen; 55 had never undergone TDI and were receiving the first series (six cycles), 6 were receiving the second series (also six cycles), and 20 had achieved pregnancy through TDI previously and were starting the treatment again. Insemination with semen stored in 0.5-ml French straws was performed daily during the periovulatory period while the modified Insler score was 10 or greater out of 15. A total of 42 (52%) of the recipients became pregnant within six TDI cycles; 4 (10%) had a spontaneous abortion. An average of 4.8 straws were used per cycle among those who became pregnant and 5.1 per cycle among those who did not. On average 2.6 cycles were required to achieve pregnancy. The overall fecundability rate was 14.6%. We conclude that a TDI program involving exclusively frozen semen can be operated with a success rate comparable to rates achieved with fresh semen if a simple, established cryopreservation method and an uncomplicated clinical management protocol are used.
Adult↗
Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa.
Fluorescein-conjugated peanut agglutinin (PNA) lectin-labelling is an established procedure for assessing the status of the human sperm acrosome. However, unlike the triple-stain technique, PNA-labelling does not provide a simultaneous assessment of cellular vitality. We have therefore evaluated the use of the fluorescent dye Hoechst 33258 (H33258) as a vital stain for use in combination with PNA-labelling. Human sperm populations were stained for 1 min with 1 microgram/ml H33258 in culture medium and then washed through 2.0 (w/v) polyvinylpyrollidone columns and air-dried onto microscope slides. H33258 was found to provide vitality assessments comparable to those obtained using the standard eosin-exclusion method. However, best results were obtained with an ethanol fixation step between air-drying and PNA-labelling. This vitality assessment was found to be more reliable than that provided by Trypan blue staining under conditions equivalent to the triple-stain technique. There was no alteration of PNA-labelling due to the H33258 although ethanol fixation actually provided more uniform PNA-labelling than previously obtained without ethanol fixation. Consequently, we have stopped using the triple-stain technique for assessing human sperm acrosome reactions and now use the H33258/PNA procedure routinely.
Acrosome↗
Treatment of spermagglutination with proteolytic enzymes. I. Sperm motility, vitality, longevity and successful disagglutination.
The treatment of antibody-mediated spermagglutination by corticosteroid therapy has a high incidence of side-effects and sperm washing is often followed by re-agglutination. The possibility of enzymatic disagglutination was therefore investigated. In the first part of the study the effects of four proteases on sperm motility, vitality and longevity were evaluated. Subtilisin had prohibitively detrimental effects even at 10 U/ml. However, chymotrypsin (less than or equal to 500 U/ml), trypsin (less than or equal to 500 U/ml) and papain (less than or equal to 50 U/ml) had no adverse effects. In the second series of experiments one or more of these latter three enzymes was found to disagglutinate spermatozoa which had previously been incubated with sperm-agglutinating antibody-positive sera in 87% of cases. Although further investigation is required, enzymatic disagglutination may be beneficial for the treatment of immunologically mediated spermagglutination.
Cell Membrane↗
Treatment of spermagglutination with proteolytic enzymes. II. Sperm function after enzymatic disagglutination.
Antibody-mediated spermagglutination is responsible for infertility in some couples and fertilization in vitro can also be impaired by these antibodies. Having previously demonstrated the possibility of enzymatic disagglutination in such situations, the functional potential of disagglutinated spermatozoa has now been assessed. Chymotrypsin (500 U/ml) and papain (50 U/ml) resulted in impairment of oocyte penetration in the zona-free hamster egg penetration test. Trypsin (500 U/ml), while having no effect on egg penetration of normal spermatozoa, significantly improved oocyte penetration of spermatozoa which had been previously incubated with spermagglutinating antibody-positive sera. Sperm-mucus interaction was not improved, however, by trypsin treatment of agglutinated spermatozoa. This technique may be of value in conjunction with in-vitro fertilization in situations where spermagglutination exists, and also possibly with intra-uterine insemination if improved fertilizing ability can be confirmed in vitro.
Animals↗
A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test.
Fifty-one comparisons were made of human sperm migration into capillary tubes containing either human cervical mucus ('Kremer test') or a synthetic mucus substitute consisting of a 5 mg/ml solution of sodium hyaluronate (average mol. wt 2 x 10(6)) in a phosphate-buffered medium. The results of these two tests were highly significantly correlated and dependent upon the same sperm characteristics reflecting sperm progressive ability (including the specific movement characteristic of lateral head displacement amplitude), morphological normality and cellular vitality as well as the concentration of these more functional cells in the semen. The result of the hyaluronate migration test, in conjunction with the mucus quality measures of Insler score and pH, allowed a 92.2% correct prediction of the Kremer test outcome (90.9% of normal tests and 93.1% of abnormal tests). In this data set, these values also corresponded to the sensitivity and specificity of the analysis, respectively. From these studies, we propose the hyaluronate migration test as a useful adjunct to routine semen analysis, sperm movement analysis and the more traditional in-vitro tests of sperm-cervical mucus interaction in the diagnostic investigation of infertile couples. It effectively assesses the mucus-penetrating potential of a semen sample without the need for relatively large quantities of midcycle cervical mucus; it will therefore augment (as an internal control), although not necessarily replace, the homologous Kremer test and reduce the quantity of both patient and donor mucus needed for comprehensive crossed-hostility format testing of sperm-mucus interaction.
Cervix Mucus↗
The spontaneous acrosome reaction of human spermatozoa incubated in vitro.
Motile human sperm populations were prepared from liquefied semen (10 donors x 3 replicates) using Percoll density gradients at 30-60 min post-ejaculation. Sperm suspensions were incubated in a complex 'synthetic tubal fluid' culture medium (STF) at 37 degrees C under 5% CO2 in air for up to 36 h. Parallel aliquots were incubated with 50 microM A23187 to induce maximum acrosome loss (ARMAX). Acrosome reactions were assessed using both the triple-stain (TS) technique and fluorescent peanut agglutinin (PNA) lectin-labelling. During incubation, the proportion of TS acrosome reacted spermatozoa increased from 9.1 to 54.3% with ARMAX being 68.3%. Spermatozoa showing intact acrosomes by PNA labelling decreased from 68.4 to 26.1% over 36 h of incubation (ARMAX = 13.8%). Simultaneously, spermatozoa showing complete acrosomal loss (no PNA labelling) increased from 8.1 to 27.0% (ARMAX = 46.3%). Therefore, while only 23.5% of cells were actually undergoing acrosomal changes at the start of incubation, this had increased to 46.9% after 36 h (ARMAX = 40.7%). These experiments clearly show that even in selected populations, not all human spermatozoa are capable of undergoing an acrosome reaction. However, the incidence of acrosomal changes after 36 h of incubation did approach the ARMAX. These levels of spontaneous occurrence of the human sperm acrosome reaction were higher than those reported in many other in-vitro incubation studies: an improvement that may be attributable to the more physiological nature of the STF culture medium.
Acrosome↗
A technical note on diluting semen for the haemocytometric determination of sperm concentration.
A comparison was made of the use of either an SMI positive displacement pipette or an Eppendorf Varipette as the method of sampling 63 liquefied semen samples for volumetric dilution and haemocytometry to determine sperm concentration. The 95% range of the differences between the values obtained using the SMI and the Varipette with whole tips was from -46.9 to 63.8 x 10(6)/ml. With the Varipette tips cut off 12.5 mm from the end the 95% range was from -52.4 to 55.8 x 10(6)/ml. Previous work had shown that the 95% ranges of differences between duplicate determinations using the SMI pipette were from -7.2 to 6.9 x 10(6)/ml for two 1 + 19 dilutions, or from -16.0 to 12.1 x 10(6)/ml for 1 + 19 and 1 + 49 parallel dilutions. Therefore, since the Varipette had a far greater potential for error, a positive displacement pipette should be used when taking precise volume aliquots of human semen.
Humans↗
The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro.
Motile human sperm populations were prepared from liquefied semen (5 donors x 3 replicates) using Percoll gradients at 30-60 min post-ejaculation and preincubated in a complex 'synthetic tubal fluid' culture medium (STF) at 37 degrees C under 5% CO2 in air for 6 h. Aliquots of these suspensions were then incubated for a further 2 h in STF containing 0, 5, 10, 25, 50, 75 and 100% (v/v) pooled human follicular fluid (FF). Another aliquot was treated with 10 micron A23187 in STF for 20 min and then incubated in fresh STF medium for a further 2 h to induce maximal acrosome loss. Acrosome reactions were assessed using both the triple-stain technique and fluorescent peanut agglutinin lectin-labelling. Sperm motility and movement characteristics were assessed from videorecordings using digital image analysis (CellSoft). Exposure to FF caused only relatively small proportions of the preincubated spermatozoa to undergo acrosome reactions. The size of these responsive sub-populations was smaller than that capable of responding to a Ca2+ influx generated by A23187. Increased FF concentrations induced a progressive loss of motility and trends for changes in movement characteristics that may have been related to reduced intracellular Ca2+. This interpretation of these observations is that while FF may act to stimulate or promote the human sperm acrosome reaction it does not appear to be a specific inducer of it. Consequently, a precise role for FF at the relatively low concentrations that would be expected to be present in the tubal ampulla in the physiological regulation of human fertilization remains unproven.
Acrosome↗
Calcium dependence of human sperm fertilizing ability.
The Ca2+ dependency of human-sperm fertilizing ability was investigated using a modified Tyrode's medium either containing 2.4 mM CaCl2 (CA medium) or with the CaCl2 replaced by SrCl2 and 0.1 mM EGTA added to chelate any residual Ca2+ ions (SREG-medium). Ten washed sperm populations incubated in either medium for 0, 6, and 22 hours showed the same occurrence of acrosome reactions (by fluorescent lectin labelling and triple stain). A further 3-hour incubation after washing into fresh CA medium resulted in only a slight increase in acrosome reactions in both media. Eight sperm populations preincubated overnight in CA and SREG media were coincubated for 1 hour with previously salt-stored human zonae pellucidae also in the same media. Significantly more motile spermatozoa were bound to more of the zonae in CA medium (53.9% vs. 27.6% of zonae with 13.8 vs. 4.3 sperm/bound zona). In three hamster egg penetration test (HEPT) experiments, sperm populations preincubated overnight in either CA or SREG media were coincubated with hamster oocytes prepared in the same media. Only 2.1% of oocytes (1.0 polyspermy) were penetrated in SREG medium, cf., 30.9% of oocytes (1.3 polyspermy) in CA medium. These results demonstrate that while Sr2+ ions can substitute fully for Ca2+ in the capacitation and acrosome reaction of human spermatozoa, sperm-zona, and sperm-oolemma interaction seem to involve some more Ca2+-specific process(es). Furthermore, the increased HEPT fertilizing ability of human spermatozoa using overnight preincubation in SREG medium and CA medium for the test cannot be explained on the basis of differential kinetics of capacitation or the acrosome reaction.
Acrosome↗
Influence of image sampling frequency on the perceived movement characteristics of progressively motile human spermatozoa.
Tracks of 30 progressively motile washed human spermatozoa were plotted manually from 200-Hz frame rate video recordings. Tracks at 100, 66.7, 50, 40, 33.3, 25, 20, 10, and 5 Hz were then constructed using every 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 20th, or 40th point. The 200-Hz tracks were analyzed by traditional manual methods, and all ten sets of tracks analyzed using a computer-assisted method ("Videomot," developed originally to analyze 30-Hz tracks) to eliminate observer bias. Progression velocity (VSL) remained constant under all analysis conditions. Average path velocity (VAP) also remained essentially constant, although Videomot was less reliable at high frame rates due to problems in determining the average path. Curvilinear velocity (VCL) was very frame rate dependent (the 25-Hz mean value was only 56.5% of that at 200 Hz), and Videomot was more accurate than manual analysis at 200 Hz. Values of the amplitude of lateral head displacement (ALH) were acceptable at most frame rates. At less than 66.7 Hz the inclusion of spurious curvilinear track deviations caused lower mean ALH values, and at 5 Hz ALH could not be measured since the track was essentially the average path. Beat/cross frequency (BCF) was also frame rate dependent; at high rates there was the same problem as with ALH measurements, while at less than or equal to 25 Hz the maximum BCF was restricted by the frame rate. We conclude that human sperm movement characteristics can be measured at frame rates ca. 30 Hz but only if the constraints affecting VCL and BCF values are understood and accepted. Finally, less than 10 Hz can only give values for VSL and, perhaps, VAP.
Humans↗
Role for fucose-sulfate-rich carbohydrates in the penetration of zona-pellucida-free hamster eggs by hamster spermatozoa.
The interaction of acrosome-reacted hamster spermatozoa and zona-pellucida-free hamster eggs was investigated by incubating gametes in the presence of a variety of simple and complex carbohydrates. Significant inhibition of gamete fusion was achieved only in the presence of fucoidan and ascophyllin, two algal polysaccharides containing fucose sulfate. These compounds did not interfere with sperm motility, capacitation, or acrosome reactions. It is concluded that these two compounds share common structural features with putative cell-surface carbohydrates involved in sperm-oolemmal interaction.
Animals↗
Computerized semen analysis.
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Autoanalysis↗
Evaluation of the CellSoft automated semen analysis system in a routine laboratory setting.
The sperm concentration and percentage motility values generated by version 3.2 of the CellSoft (Cryo Resources Ltd., New York, NY) automated semen analyzer on 200 ejaculates were compared with those obtained by standardized traditional methods. Overall, CellSoft gave mean concentrations that were 20.9 x 10(6)/ml lower (95% range of differences = -112.6 to +154.4 x 10(6)/ml). However, the difference between methods was not systematic. Below 50 x 10(6)/ml, CellSoft more often gave higher values, and above 100 x 10(6)/ml, it usually gave lower values. In the middle range, differences were randomly distributed. For motility, the CellSoft values were usually higher than those obtained by visual counting (mean difference = -17.5%, 95% range = -56.0% to +21.0%). Multiple regression analyses revealed a strong concentration dependency such that reliable values will probably be obtained only if all samples are diluted (with homologous seminal plasma) before CellSoft analysis. This upper concentration limit is of the order of 30 to 50 x 10(6)/ml. Without such dilution, this version of CellSoft will not provide sufficiently accurate values for basic semen characteristics and cannot be accepted as a routine diagnostic method.
Humans↗
Semi-automated analysis of manually reconstructed tracks of progressively motile human spermatozoa.
Several alternative algorithms for computer-assisted derivation of measurements of movement characteristics from manually reconstructed tracks of progressively motile human spermatozoa were compared. Fifty tracks were reconstructed at 30 Hz from video recordings and analysed using traditional manual methods and by four combinations of computer algorithms. The best algorithm set was identified ('Videomot.mdpt') and the values for the curvilinear, average path and linear velocities (VCVL, VAVE and VLIN respectively), the amplitude of lateral displacement of the sperm head about the axis of progression (AH) and the number of times the sperm head crossed the average path (the 'beat/cross frequency', BXF) obtained using it were compared to those obtained by manual analysis. There was a considerable time saving when the computer-assisted method was used and the values it gave for the various movement characteristics showed good correspondence with those obtained manually. In addition, repeated data entry and analysis was found to be highly reproducible. When the tracks were re-plotted at 6 Hz (as used by the multiple-exposure photomicrography method for sperm motility analysis) insufficient information remained in the tracks for reliable determination of anything other than VLIN. We conclude that the Videomot.mdpt program provides reliable values for the movement characteristics of progressively motile human spermatozoa, and believe it will be of great value in the validation of commercial systems providing automated sperm movement analysis and in laboratories which do not have access to such costly equipment.
Algorithms↗