Biomedical subjects

D Mortimer

Publications and source records attributed to D Mortimer.

36 records · Page 2Linked to original sources

PubMed1988

Influence of system parameter settings on human sperm motility analysis using CellSoft.

The effect of changing CellSoft system parameter settings on measurements of motile sperm concentration and sperm movement characteristics (curvilinear and linear velocities, amplitude of lateral head displacement and beat/cross frequency) was investigated in semen. Specifically, the use of either four or 15 frames to distinguish motile from immotile cells was used in conjunction with a threshold curvilinear velocity of either 10 or 20 micron/s (recommended values: 4 and 10). In addition, a threshold for progressive motility based on linear velocity was also used by manual re-analysis of CellSoft Individual Cell Data printouts. Known reliable semen analysis methods or the analysis of manually reconstructed trajectories from videotape were used as standard methods for comparison. A curvilinear velocity threshold of 20 micron/s gave improved correspondence between CellSoft and standard methods for all characteristics evaluated. Whether a four or 15 frame setting was better varied with the measurement being made. However, using a linear velocity threshold to define the progressively motile sperm population provided the most acceptable values for sperm movement characteristics. Consequently, while substantial improvements in the measurements made by CellSoft can be obtained using different system parameters to those usually recommended, more reliable and certainly more useful information could probably be obtained if all analyses were performed on a defined population of progressively motile, and therefore potentially functional, spermatozoa.

Humans↗

PubMed1987

Prevalence of sperm surface antibodies in the male partners of infertile couples as determined by immunobead screening.

A single simple laboratory test for the detection of sperm surface antibodies is described. Spermatozoa from 300 men who were investigated for infertility were tested for sperm surface antibodies using the Immunobead test (IBT). IBTs for IgG, IgA, and IgM were performed on each sample as well as an IBT using an Immunobead with activity against both light and heavy immunoglobulin chains and therefore capable of detecting any of the three isotypes ("GAM-IBT"). An excellent correspondence was found between GAM-IBT-positive samples and those positive by one or more of the isotype-specific Immunobeads. In 29 of 32 positive tests, the GAM bead binding was within 5% of the most positive isotype-specific bead's binding. There were no false-positive or false-negative tests comparing the GAM-IBT with the isotype-specific IBTs.

Antigens, Surface↗

PubMed1987

Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon.

Experiments to bind fluorescein-conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) to washed human spermatozoa demonstrated that this lectin binds to the acrosome region in air-dried preparations. Since there was no binding when labelling was performed in suspension, and comparable labelling to that seen in air-dried preparations was seen when spermatozoa treated with saponin (to lyse the plasma membrane) were labelled in suspension, the lectin must bind to an intracellular structure, probably the outer acrosomal membrane. This was confirmed by ultrastructural localization of colloidal gold-conjugated lectin in saponin-treated spermatozoa. Treatment of spermatozoa with the detergent Nonidet P-40 caused a marked change in the binding pattern: more spermatozoa showed binding in the equatorial segment of the acrosome with no binding in the anterior cap region. A comparable, less marked, change was seen when spermatozoa were incubated overnight under conditions known to support the capacitation and spontaneous acrosome reactions. Treatment with the calcium ionophore A23187 for 1 h to induce acrosome reactions artificially in uncapacitated spermatozoa resulted in the appearance of patchy acrosome fluorescence. From these experiments it is concluded that PNA binds specifically to the outer acrosomal membrane, and that FITC-PNA-labelling may be used to monitor the human sperm acrosome reaction.

Acrosome↗

PubMed1986

Comparison of the fertilizing ability of human spermatozoa preincubated in calcium- and strontium-containing media.

Human spermatozoa freed from seminal plasma using a discontinuous Percoll gradient procedure were incubated overnight in modified Tyrode's media containing either CaCl2 (CA medium) or with SrCl2 instead of CaCl2 (SR medium). The following morning these spermatozoa were washed by centrifugation, resuspended in fresh CA medium and incubated for either 2 or 3 h with zona-free hamster eggs to test their fertilizing ability. Penetration rates were higher after 3 h of sperm-egg contact than after 2 h, and the spermatozoa preincubated in SR medium penetrated significantly more hamster oocytes than those preincubated in CA medium.

Animals↗

PubMed1986

An evaluation of different media for the zona-free hamster egg penetration test.

Hypertonic high-salt Biggers, Whitten, and Whittingham medium was compared with either a complex culture medium (synthetic tubal fluid) or a variant of Tyrode's medium for capacitating donor human spermatozoa to be used in the zona-free hamster egg penetration test. Five sperm preparations showed a slightly higher penetration rate in synthetic tubal fluid than in high-salt Biggers, Whitten, and Whittingham medium, five showed equivalent results, and only two had slightly lower penetration rates. However, in modified Tyrode's medium only one sample showed a lower penetration, while six showed comparable results and five showed higher rates of penetration. This last group included two samples that had demonstrated zero penetration in high-salt Biggers, Whitten, and Whittingham medium. Although no overall significant differences between media in the two series of experiments could be discerned with paired t tests, in view of the lower incidence of false negative results in modified Tyrode's medium and the absence of osmotic stress, this medium may represent a better choice for routine performance of the hamster egg penetration test.

Animals↗

PubMed1986

The use of strontium-substituted media for capacitating human spermatozoa: an improved sperm preparation method for the zona-free hamster egg penetration test.

An improved procedure for human sperm capacitation based upon calcium deprivation using a modified Tyrode's medium is described. Replacement of CaCl2 by SrCl2 significantly increased the penetration of zona-free hamster eggs after 20 hours of sperm preincubation, compared with parallel sperm aliquots incubated in normal medium. The effect of an extra 4 hours' sperm preincubation produced no significant shift in fertilizing ability. Addition of 0.1 mM ethyleneglycol-bis-(beta-amino-ethyl ether)-N,N,N',N'- tetraacetic acid (EGTA) to the strontium-substituted medium resulted in a further significant increase in the penetration rate as well as a significant increase in polyspermy. The average increase in the penetration capacity (penetration rate X polyspermy) of the strontium/EGTA-preincubated spermatozoa was approximately tenfold, with no significant shift as a result of an extra 4 hours' preincubation.

Animals↗

PubMed1986

Elaboration of a new culture medium for physiological studies on human sperm motility and capacitation.

The formulation of a new medium based upon published data on human tubal fluid and blood plasma is described. Sperm motility was well maintained for periods of up to 6 h in this 'synthetic tubal fluid' (STF), and movement characteristics (velocity of progression and amplitude of lateral head displacement) were quantitatively and qualitatively similar to values previously reported for other complex media. STF also supported human sperm capacitation and the spontaneous acrosome reaction as determined using the zona-free hamster egg penetration test. Spermatozoa pre-incubated in STF containing blood plasma levels of taurine (86 microM) for 3 h penetrated significantly more oocytes than parallel sperm populations pre-incubate in STF lacking taurine (P less than 0.001). This difference was no longer significant after 5 h of pre-incubation. These findings indicate a possible role for taurine in human sperm capacitation, and demonstrate the potential value of STF for performing more physiological invitro studies on human sperm function.

Culture Media↗

PubMed1986

Standardization and quality control of sperm concentration and sperm motility counts in semen analysis.

The paper reports a study of standardization and quality control of sperm concentration counts and visual motility assessments in human semen analyses performed for infertility investigations and from internal quality control procedures. Sperm concentration determinations were performed in Improved Neubauer haemocytometers on volumetric dilutions made using a positive displacement pipettor for sampling the liquefied semen. In addition to a standard 1 + 19 dilution a second dilution of either 1 + 9, 1 + 19 or 1 + 49 was made according to whether the estimated sperm concentration was less than 20, 20-100 or greater than 100 X 10(6)/ml respectively. The duplicate determinations of sperm concentration were highly significantly correlated (P much less than 0.001) with less than 5% variability. Parallel visual sperm motility assessments were made by two pairs of technicians and showed highly significant correlations (P much less than 0.001) between technicians in the determination of the percentages of motile and progressive spermatozoa as well as the subjective rating of sperm progressivity. When these values were incorporated into a calculated motility index which gave added weight to the progressive spermatozoa and to their quality of progression the correlations between technicians remained highly significant (P much less than 0.001) with average differences of the order of 1.0%. Therefore, provided that sufficient attention is paid to technician training, regular standardization checks and the use of only proven reliable procedures, quantitatively accurate values for sperm concentration and motility can be obtained in routine semen analyses.

Humans↗

PubMed1986

Relationship between human sperm motility characteristics and sperm penetration into human cervical mucus in vitro.

A series of 100 modified Kremer tests of human sperm penetration into human cervical mucus was carried out as part of the routine investigation of couples presenting with infertility. The outcome of these tests was significantly correlated with the concentration and progressive motility of the spermatozoa in the semen sample used for the test. Other semen characteristics significantly correlated with the test result were the mean velocity of progression (VP) and the amplitude of lateral head displacement about the axis of progression (AH) of the progressive spermatozoa. Normal sperm morphology was also correlated with the outcome. Using these semen characteristics as the independent variables to predict the test outcome in a discriminant analysis (normal vs abnormal tests), 34.2% of the variance was accounted for. From the discriminant function equation 75.0% of the test results could be predicted correctly. In the 30 cases in which the semen samples used for the tests showed greater than or equal to 25 X 10(6) progressively motile spermatozoa per ml, mean VP of greater than or equal to 25 microns/sec and mean AH of greater than or equal to 7.5 microns, 83.3% had normal test results. Conversely, all 13 cases for which the semen characteristics were below these limits had abnormal test results. Therefore, both the concentration of progressively motile spermatozoa and their movement characteristics are significant factors determining the outcome of homologous tests of human sperm-cervical mucus interaction.

Cervix Mucus↗

PubMed1986

A standardized approach for evaluating the penetration of human spermatozoa into cervical mucus in vitro.

Carefully timed and quantitated Kremer-type tests were performed for assessment of sperm-mucus interaction in 100 couples who underwent an infertility workup. A novel scoring system for these in vitro tests was elaborated and validated. The success of penetration of seminal spermatozoa into the mucus, and their migration within the mucus column, was related to both the sperm concentration and their motility, especially their progressivity. A calculated index of sperm motility quality gave a 70% accuracy rate in predicting the test result (normal versus abnormal), but this success rate was not significantly improved by incorporation of the sperm concentration into the index. No clear biologic relationship was found between this method for assessing Kremer-type tests (which correlated closely with accepted clinical reporting practices) and Katz's "percentage of successful collisions." This apparent discrepancy is considered to reflect basic differences in the approaches of the two scoring systems.

Cervix Mucus↗

PubMed1979

Functional anatomy of haploid and diploid rabbit spermatozoa.

Ultrastructural studies of surface replicas of haploid and diploid rabbit spermatozoa after treatment with various chemical media have shown that the various structural components of the diploid sperm head possess the same stabilities and labilities as those of the haploid. Therefore, at least in terms of functional anatomy, diploid rabbit spermatozoa should be capable of penetrating the egg investments and undergoing syngamy.

Acrosome↗

PubMed1979

Differential motility of diploid rabbit spermatozoa.

When rabbit spermatozoa were suspended in a fluid column and incubated for a period of 2.5 hr, there was a change in the relative distribution of morphologically normal, abnormal, and dead spermatozoa. Live, morphologically normal spermatozoa were concentrated in the lower levels of the column, whereas dead and most morphologically abnormal spermatozoa were distributed throughout the upper levels. It appears that the motility of various types of morphologically abnormal rabbit spermatozoa is impaired. These observations strengthen the proposal that the cervix plays an essentially passive role in the selection of spermatozoa during sperm transport in mammals with vaginal insemination.

Animals↗

PubMed1977

The survival and transport to the site of fertilization of diploid rabbit spermatozoa.

The possible involvement of diploid spermatozoa in the production of triploid zygotes in the rabbit has been investigated in terms of the success of diploids relative to haploids in reaching the site of fertilization, with special reference to any possible selectivity of sperm transport within the female tract. The observed large significant reduction in the incidence of diploid spermatozoa between the ejaculate and the oviducts would greatly reduce the chances of, but not necessarily prevent, a diploid spermatozoon fertilizing an egg. This reduction appears to be due to a combination of the reduced survival of diploids in the female tract and a selectivity of sperm transport dependent on sperm motility.

Animals↗

PubMed1976

Nuclear pores in the spermatozoon of the rat.

The paper describes a hexagonal array of nuclear pores in a non-redundant region of the nuclear envelope underlying the basal surface of the rat spermatozoon head. It is concluded that intranuclear material protruding through these pores is the cause of the characteristic rows of circular 'bumps' found in surface replicas of this region.

Animals↗

PubMedDate not supplied

Human sperm morphology and the outcome of modified Kremer tests.

The outcome of homologous in vitro tests of human sperm penetration into cervical mucus in couples undergoing infertility investigations have been shown to be correlated with the proportion of morphologically normal spermatozoa in the semen samples used for the tests. In addition, the ejaculates producing poor tests contained significantly more spermatozoa with defective tails than those semen samples producing normal or, surprisingly, negative tests.

Cervix Mucus↗

PubMedDate not supplied

Kinematics of human spermatozoa incubated under capacitating conditions.

Suspensions of seminal plasma-free human spermatozoa were prepared by swim-up from semen and studied using high magnification videomicrography after incubation under capacitating conditions for 1.5-2 h. Three subpopulations of capacitating spermatozoa showing different patterns of motility could be distinguished visually: forward progressive, transition phase, and hyperactivated motility. The purpose of this study was not to determine the relative proportions of spermatozoa in these three categories but to describe their movement characteristics. Manual track plotting and analysis allowed value derivation for the curvilinear, average path and straight-line velocities (VCL, VAP, and VSL respectively); for the three progression ratios of linearity (LIN = VSL divided by VCL X 100), straightness (STR = VSL divided by VAP X 100), and wobble (WOB = VAP divided by VCL X 100); and also for the amplitude of lateral head displacement (ALH) and the beat/cross frequency (BCF). Algorithms produced from these motion characteristics allowed distinctions to be made between cell motility patterns. Spermatozoa with straight-line velocity (VSL) greater than or equal to 40 microns/s, linearity (LIN) greater than or equal to 60% and amplitude of lateral head displacement (ALH) less than 5 microns were FP or non-hyperactivated. Tracks with curvilinear velocity (VCL) greater than or equal to 100 microns/s, linearity (LIN) less than 60% and amplitude of lateral head displacement (ALH) greater than or equal to 5 microns showed concomitants of hyperactivation. Classical hyperactivated tracks also showed straightness (STR) less than 60% and straight-line velocity (VSL) less than 30 microns/s.

Humans↗

PubMedDate not supplied

Glutamic-oxaloacetic transaminase isozymes in human seminal plasma and sperm extracts.

Leakage of glutamic-oxaloacetic transaminase (GOT) can be used as an indicator of cryo-damage for mammalian spermatozoa. However, two GOT isozymes exist: mitochondrial (m-GOT) and cytosolic or soluble (s-GOT), with acid and alkaline pH optima, respectively. The levels of these GOT isozymes were determined in samples of cell-free human seminal plasma and sperm extracts over a pH range of 3.8 to 9.6. Optimum GOT activities were found at pH 5.2 using cacodylate-HCl buffer (m-GOT) and pH 8.4 using barbital buffer (s-GOT). Differential inhibition studies with adipate (which inhibits s-GOT) were misleading because of the presence of another NADH-linked enzyme able to use adipate as a substrate. Only relatively low levels of m-GOT could be extracted from human spermatozoa (21.4 +/- 31.5 [SD] mU/10(8) cells vs. 202.3 +/- 49.4 mU/mL seminal plasma; n = 20). Consequently, future studies on GOT leakage should be carried out at the alkaline pH optimum, and will therefore measure s-GOT, making this a marker for plasma membrane integrity rather than a measure of mitochondrial damage.

Aspartate Aminotransferases↗

PubMedDate not supplied

Failure of hysteroscopic insemination of the fallopian tube in synchronized cycles.

The study consisted of a randomized, controlled trial of synchronized hysteroscopic insemination of the fallopian tube (SHIFT), in which selected infertile couples underwent hormonal delay of menstruation, ovulation induction, and insemination of a selected motile sperm population into the tubal isthmus above the utero-tubal junction. Of 40 couples recruited, only 2 became pregnant, both during a nonsynchronized control cycle. No conceptions were reported in either the 72 SHIFT or 73 non-SHIFT synchronized cycles. Hysteroscopic insemination was found to be a relatively easy procedure with a low rate of technical failure. There was low morbidity with no major clinical complications. However, 57% of tracked synchronized cycles showed a failed response to the follicular stimulation protocol; and 91% showed failure of follicular rupture greater than or equal to 52 hours after hCG induction. These failures of the ovulation synchronization/induction regimen were probably due to the use of medroxyprogesterone acetate for cycle synchronization, and should not preclude further evaluation of the technique of hysteroscopic insemination.

Clinical Trials as Topic↗