Vaginal deployment and tenofovir delivery by microbicide gels.

Gels are one of the soft material platforms being evaluated to deliver topically acting anti-HIV drugs (microbicides) to the vaginal environment. For each drug, its loaded concentration, gel properties and applied volume, and frequency of dosing can be designed to optimize PK and, thence, PD. These factors also impact user sensory perceptions and acceptability. Deterministic compartmental modeling of vaginal deployment and drug delivery achieved by test gels can help delineate how multiple parameters characterizing drug, vehicle, vaginal environment, and dosing govern details of PK and PD and also gel leakage from the canal. Such microbicide delivery is a transport process combining convection, e.g., from gel spreading along the vaginal canal, with drug diffusion in multiple compartments, including gel, mucosal epithelium, and stroma. The present work builds upon prior models of gel coating flows and drug diffusion (without convection) in the vaginal environment. It combines and extends these initial approaches in several key ways, including: (1) linking convective drug transport due to gel spreading with drug diffusion and (2) accounting for natural variations in dimensions of the canal and the site of gel placement therein. Results are obtained for a leading microbicide drug, tenofovir, delivered by three prototype microbicide gels, with a range of rheological properties. The model includes phosphorylation of tenofovir to tenofovir diphosphate (which manifests reverse transcriptase activity in host cells), the stromal concentration distributions of which are related to reference prophylactic values against HIV. This yields a computed summary measure related to gel protection ("percent protected"). Analyses illustrate tradeoffs amongst gel properties, drug loading, volume and site of placement, and vaginal dimensions, in the time and space history of gel distribution and tenofovir transport to sites of its anti-HIV action and concentrations and potential prophylactic actions of tenofovir diphosphate therein.

Spectrophotometric analysis of molecular transport in gels.

An automated spectrophotometric method has been developed for analyzing molecular transport out from and into gels. A Beckman DU7500 diode-array UV-visible spectrophotometer with gel scanner was modified to accept and longitudinally scan a quartz diffusion cell, 0.3x10x40 mm. Molecules of interest are identified and concentrations quantitated via analysis of spectrophotometric absorbance peaks relative to background absorbance of the gel. Thus, concentration profiles are obtained as functions of both position and time. Test data are fitted to a diffusion model via nonlinear least-squares regression. Precision and accuracy of the method were assessed via analysis of several test molecules and gels: (1) 30 mg/ml nonoxynol-9 (N9), contained in 1% sodium alginate gel cross-linked with 2.5 mM calcium chloride, permeating standardized, reconstituted bovine cervical mucus (BCM); (2) 2.5 mg/ml sodium fluorescein, contained in and permeating 10 mg/ml and 100 mg/ml gelatin gels; and (3) 1.0 mg/ml sodium ganciclovir, contained in and permeating 10 mg/ml sodium hyaluronate gel. Diffusion coefficients for (1) and (3) were 3.8x10-7 and 54.1x10-7 cm2/s, respectively. All measurements of diffusion coefficients, partition coefficients, and solute loads obtained in this study were highly repeatable (most C.V.'s<8%). The mean diffusion coefficient for (2) was within 3% of values predicted from theory for the 100 mg/ml gel; the mean partition coefficient for (3) was within 2% of the expected value. This new technique is simpler than many traditional ones in that it does not require labeling of test molecules nor changes in refractive index of target materials. It is particularly well-suited to situations in which the target material is a gel, because no stirring of the target is necessary.

Factors influencing nonoxynol-9 permeation and bioactivity in cervical mucus.

The effects of delivery gel pH and osmolarity on both the mass transport and 'biodiffusion' of the spermicide nonoxynol-9 (N9) in bovine cervical mucus were evaluated. Delivery gels were calcium chloride crosslinked alginate containing 3% N9, and were manufactured over a pH range of 3.4 to 5.9 and an osmolarity range of 300 to 900 mosmol. Mass transfer parameters (diffusion coefficients and total drug loading) were determined using a new UV spectrophotometric technique while biodiffusion (the diffusion distance into mucus at which sperm are killed) was assessed using the Double Ended Test. It was found that delivery gel pH had a significant effect on spermicidal efficacy of the alginate-N9 system; biodiffusion increased with decreasing pH. Actual N9 diffusion into mucus was found to be influenced by both the delivery gel pH and osmolarity. At high N9 concentration (near the gel/mucus interface), mass transport tended to decrease with decreasing pH at the highest osmolarity. At low concentration, mass transport tended to decrease with increasing osmolarity and decrease with increasing pH at the highest osmolarity. The difference between low and high concentration behavior can be attributed to N9 micelle formation. These findings are interpreted in the context of the design of intravaginal drug delivery vehicles for spermicides.

A flat capillary tube system for assessment of sperm movement in cervical mucus.

A new flat capillary tube system has been developed for studies of sperm movement in cervical mucus. The system is easy to use, employs readily obtainable components, and is amenable to small sample sizes characteristic of human cervical mucus. The system possesses good optical resolution and is suitable for visual assessment of sperm migration as well as more detailed studies of the movement characteristics of individual spermatozoa. The system subjects the mucus to a well-defined and controllable rate of shear deformation. Preliminary experiments have suggested that the swimmming speed of human sperm does not differ in flat capillary tubes of 200-micrometer and 400-micrometer depth.

Sperm motility assessment by videomicrography.

A technically simple, inexpensive method is described for measuring objective parameters of sperm motility. The instruments involved are commercially available, home-oriented videotape equipment. Quantitative measurements of sperm motility are made directly from the video image and are facilitated by use of an analysis transparency that is applied as an overlay to the screen of the television monitor. A protocol is given for describing the motility of a suspension of human spermatozoa in terms of percentage motility, mean swimming speed, and the percentage of progressive sperm. A complete analysis can be done in 20 minutes or less. Examples are presented of videomicrographic assessment of the motility of human and bull spermatozoa.

Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2 degrees C to 5 degrees C.

The motility of human spermatozoa and their ability to penetrate zona-free hamster eggs were examined after dilution of the semen with TES-Tris (TEST) yolk buffer and storage for 48 hours at 2 degrees C to 5 degrees C. Semen samples from 10 fertile donors and 19 infertility patients were studied. More than 65% of the spermatozoa which were initially motile in the TEST yolk buffer remained active after storage. During storage, the mean swimming speed of the sperm declined to approximately 60% of the prestorage value. The percentage of zona-free hamster eggs that were penetrated by spermatozoa from patients and donors increased significantly following 48 hours of storage at 2 degrees C to 5 degrees C. Normal semen and abnormal semen were equally preserved by this storage method. This procedure may be used to ship semen samples by commercial transportation to specialized laboratories for testing. Low temperature storage in the TEST yolk buffer appears to enhance the fertilizing capacity of human spermatozoa in vitro.

Improved motility recovery of human spermatozoa after freeze preservation via a new approach.

A new sperm isolation technique was studied in conjunction with the short-term freeze preservation of human spermatozoa. The isolation procedure yielded subpopulations of spermatozoa with very high percentage motility and progressive motility score, and which were virtually free of seminal debris. For 24 semen samples from 13 donors, the mean prefreeze values of percentage motility and motility score were 53% (2.8), 73% (3.1), and 88% (3.5) for the parent semen, 7.5% BSA (middle) fractions, and 17.5% BSA (bottom) fractions, respectively. These samples were used without a priori constraint on semen quality. Eleven samples were preselected on the basis of good motility in the semen. These yielded prefreeze motility values of 66% (3.2), 70% (3.4), and 87% (3.9) for the semen and two fractions, respectively. Sperm motility in the middle fractions was thus intermediate to that in the semen and in the bottom fractions, although closer to the former in these 11 cases. The sperm freeze preservation procedure involved dilution of the semen samples and separated sperm fractions with a cryoprotective semen extender, and freezing and thawing in a conventional manner. Post-thaw percentage motility, motility score, and percentage survival were substantially higher for the separated fractions than for the parent semen. For the 24 cases, the bottom fractions yielded mean values of 57% motility, 3.0 motility score, and 70% survival, in comparison with the respective values of 20%, 2.3, and 34% for the semen. In the 11 preselected cases, the bottom fractions yielded post-thaw mean values of 60% motility, 3.3 motility score, and 72% survival; the middle fractions yielded respective values of 45%, 3.1, and 64%; and the parent semen yielded respective values of 27%, 2.3, and 41%. It was concluded that the major factor in improving post-thaw motility recovery was the separation process as a whole, rather than the degree of separation.

A microliter oxygen electrode system for sperm suspensions.

A new polarographic oxygen electrode system is described which can accept sperm suspensions with volumes as low as 50 microliters. In this configuration, the electrode tip forms the base of the suspension reaction chamber, and there is no stirring. Consequently, structured fluids such as cervical mucus can be applied. Measurements can be performed in as little time as 10 minutes. The system reliably measures oxygen consumption rates as low as 0.1 mm Hg/minute, so that mammalian sperm suspensions with concentrations of the order 1 X 10(6)viable cells/ml can be applied. Sample experiments, comparing the oxygen uptake of human spermatozoa in semen and in Tyrode's solution, are described.

Mechanisms of filtration of morphologically abnormal human sperm by cervical mucus.

It is well known that cervical mucus restricts penetration of morphologically abnormal human sperm, both in vitro and in vivo. However, the mechanisms of such restriction are not well understood. Using videomicrography to simultaneously analyze the motions and morphology of individual human sperm, we analyzed differential penetration of normal and abnormal sperm into fresh human cervical mucus. Abnormal sperm swam slower in mucus than the normal sperm, but their flagellar beat parameters were not commensurately different. Multivariate statistical analysis of the relationship between individual sperm velocity and flagellar beat parameters indicated that the heads of the abnormal sperm experienced greater resistance from the mucus than did normal heads. Differential mucus resistance, more than altered motile vigor, appears to be responsible for the restriction of abnormal sperm during migration through mucus.

A new quantitative test for sperm penetration into cervical mucus.

A new experimental and theoretical procedure is described for characterizing the penetration of spermatozoa into cervical mucus in vitro. Semen is introduced to a prescribed volume of mucus contained in a flat capillary tube. Use of the tube enables the surface area of semen-mucus contact to be fixed, and provides excellent visualization of sperm movement in the mucus. The time interval of semen-mucus contact is also controlled. The number of motile sperm and their swimming speeds are determined in both the semen and mucus by hemocytometer counts and simple time-exposure photomicrography. The assay provides two new measures of the sperm-mucus interaction. The number of successful sperm entries into the mucus is compared with the number of original collisions between seminal sperm and the semen-mucus interface. The comparison is expressed as a ratio of the numbers of sperm in these two groups, viz., PSC = percentage of successful collisions. The vitality of spermatozoa that do succeed in entering the mucus is assessed by comparing their swimming speeds with those of sperm in the semen. This second comparison is also expressed as a ratio, viz., VR = velocity ratio = mean swimming speed in mucus/mean swimming speed in semen. The clinical application of the method to human semen and cervical mucus is described, and sample results are presented.

The importance of seminal plasma for sperm penetration of human cervical mucus.

Experiments were carried out in which semen samples were diluted 1:1 with Tyrode's solution or with their own seminal plasma (obtained by centrifuging another semen aliquot) as a control. Each experiment consisted of a paired comparison of these two sperm suspensions, using a quantitative cervical mucus penetration test with aliquots of the same mucus sample. Videomicrography was used to measure the swimming speeds of spermatozoa in the semen and in the mucus. The spermatozoa swam faster in Tyrode's diluted seminal plasma than in whole seminal plasma, but their swimming speeds in cervical mucus were similar after mucus penetration. Significantly more of the collisions between spermatozoa and the mucus resulted in successful penetration in tests where the sperm were suspended in whole seminal plasma than in tests where they were suspended in Tyrode's diluted seminal plasma. These observations indicate that components of the seminal plasma are important for efficient entry of human spermatozoa into cervical mucus.

What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs?

Human spermatozoa were capacitated in media containing either high concentrations (3.5%) of human serum albumin (HSA) or low concentrations (0.3%) of bovine serum albumin (BSA). The effects of both capacitation media were assessed immediately and after overnight preincubation (18 to 24 hours) by adding a mixture of nonliving human oocytes and living zona-free hamster eggs to the sperm suspension. Overnight preincubation of sperm in media containing 3.5% HSA enhanced sperm fusion with hamster vitelli, but the capacity for zona penetration was lost. These effects could be attributed to the concentration of HSA rather than a general effect of albumin concentration, because overnight preincubation in 3.5% BSA did not interfere with zona penetration. During overnight incubation in 3.5% HSA, the percentage of acrosome reactions increased, as did alterations in the equatorial segment of the acrosome-reacted sperm. The percentage of motile sperm remained high after overnight incubation in 3.5% HSA, but both the mean swimming speed and flagellar activity on the zona surface declined. The sperm also lost their ability for strong zona binding after overnight incubation in 3.5% HSA.

A simple inexpensive method for objective assessment of human sperm movement characteristics.

A simple, objective method is described for obtaining a well-defined and diversified set of human sperm movement characteristics. These data are suitable for comprehensive statistical comparisons of sperm suspensions. One-second time-exposure photomicrographs are taken with dark-field illumination of slides prepared from semen, cervical mucus, or artificial media. The negatives are projected as a filmstrip and are analyzed on a specially designed console. The filmstrips can be taken and developed in 15 minutes without darkroom facilities, and a complete analysis of the film requires an additional 15 to 20 minutes. Fifty spermatozoa are examined to determine percentage motility, and movement characteristics are recorded for 15 spermatozoa. With these data the swimming trajectories of individual spermatozoa can be reconstructed retrospectively. The sperm suspension as a whole is described in terms of percentage motility, mean swimming speed, percentage of progressive spermatozoa, mean swimming speed of progressive spermatozoa, percentage of straight-swimming spermatozoa, percentage of rolling spermatozoa, and percentage of yawing spermatozoa.

Assessment of a new spermicidal agent against ejaculated dog and human spermatozoa in vitro.

The spermatostatic potencies of a new vaginal contraceptive agent, RS-37367, and a standard surfactant compound, nonoxynol-9, have been compared by using ejaculated dog and human spermatozoa. RS-37367 was 25 to 50 times more potent than nonoxynol-9 against dog spermatozoa. Nonparallel concentration-response lines were obtained against human spermatozoa. Concentrations of RS-37367 causing immediate spermatostasis against dog spermatozoa resulted in vesiculation of the plasma and outer acrosomal membranes of spermatozoa; similarly, immediately spermatostatic concentrations of nonoxynol-9 were associated with the previously documented generalized membrane stripping. The activities of both RS-37367 and nonoxynol-9 were affected by the concentration of dog spermatozoa in semen-compound mixtures. Short-term (5-minute) exposure of spermatozoa to concentrations of RS-37367 not immediately spermatostatic resulted in progressive immobilization of spermatozoa. Extensive washing of the spermatozoa was not able to reverse this effect, in contrast to spermatozoa transiently exposed to nonoxynol-9.

Semen analysis.

The capability to analyze semen is not new but until recently was a clinical art practiced well by only a few experts. The development of objective, quantitative methods for semen evaluation has brought these diagnostic capabilities to every clinical laboratory. These methods are described, and an interpretation of semen parameters is presented for the reader.

Automatic analysis of human sperm motion.

A new computerized methodology is described in which sperm movement characteristics are analyzed automatically. Video fields containing images of spermatozoa are electronically digitized, and the centroid position of each sperm head is determined. Over time, strings of centroids identify the swimming paths of individual spermatozoa. Details of path acquisition are described for human spermatozoa in semen, and include a discussion of how individual cells are identified and distinguished from each other. A diversified set of movement characteristics is computed for each spermatozoon, including two new measures of path shape based on the instantaneous turning angle. The traditional and the new measures of vigor and swimming pattern are evaluated and compared for consistency and redundancy. Analysis of data from human semen indicates that the new angular measures may be particularly useful in discriminating between spermatozoa exhibiting widely different patterns of motion.

Standardization and comparability of CASA instruments.

Thirty human semen specimens were analyzed using a standard manual method, then videotaped and reanalyzed using two different computer-aided sperm analysis (CASA) instruments (the HTM system, Hamilton-Thorn Research, Danvers, MA, and the CTS system, Motion Analysis Corp., Santa Rosa, CA). Videotaped specimens were analyzed by CASA for 5 frames for sperm concentration (CON) and percent motility (MOT), and for 15 frames for kinematic variables (straight-line velocity, VSL; curvilinear velocity, VCL; linearity, LIN; and amplitude of lateral head displacement, ALH). Machine parameter settings for the two instruments were matched as closely as possible. CASA values were compared with each other for all measures and with manual results for sperm count and the percentage of motile sperm. Results show: 1) HTM and CTS average values for CON are not different from manual measures for the 5- or 15-frame analysis, but slight differences are seen between CTS and HTM; 2) average values for MOT for the 5-frame analysis are higher than the 15-frame analysis for both instruments, but the average manual measurement for percent motility is much higher than any CASA value; 3) average VSL and LIN are slightly higher for HTM than CTS, but pair-wise comparison shows a high degree of concordance between the instruments; and 4) the mean values for VCL and ALH are equal for the two instruments, and there is a close concordance for the pair-wise comparison for VCL; however, pair-wise comparison of ALH reveals significant differences between the instruments. Overall, the differences seen between these instruments are slight, and are probably not biologically or clinically significant.

Sampling factors influencing accuracy of sperm kinematic analysis.

Sampling conditions that influence the accuracy of experimental measurement of sperm head kinematics were studied by computer simulation methods. Several archetypal sperm trajectories were studied. First, mathematical models of typical flagellar beats were input to hydrodynamic equations of sperm motion. The instantaneous swimming velocities of such sperm were computed over sequences of flagellar beat cycles, from which the resulting trajectories were determined. In a second, idealized approach, direct mathematical models of trajectories were utilized, based upon similarities to the previous hydrodynamic constructs. In general, it was found that analyses of sampling factors produced similar results for the hydrodynamic and idealized trajectories. A number of experimental sampling factors were studied, including the number of sperm head positions measured per flagellar beat, and the time interval over which these measurements are taken. It was found that when one flagellar beat is sampled, values of amplitude of lateral head displacement (ALH) and linearity (LIN) approached their actual values when five or more sample points per beat were taken. Mean angular displacement (MAD) values, however, remained sensitive to sampling rate even when large sampling rates were used. Values of MAD were also much more sensitive to the initial starting point of the sampling procedure than were ALH or LIN. On the basis of these analyses of measurement accuracy for individual sperm, simulations were then performed of cumulative effects when studying entire populations of motile cells. It was found that substantial (double digit) errors occurred in the mean values of curvilinear velocity (VCL), LIN, and MAD under the conditions of 30 video frames per second and 0.5 seconds of analysis time. Increasing the analysis interval to 1 second did not appreciably improve the results. However, increasing the analysis rate to 60 frames per second significantly reduced the errors. These findings thus suggest that computer-aided sperm analysis (CASA) application at 60 frames per second will significantly improve the accuracy of kinematic analysis in most applications to human and other mammalian sperm.

Operational standards for CASA instruments.

Computer-aided sperm analysis (CASA) technology is 7 years old. Over 120 papers have been written that verify the technology or apply it in basic and clinical studies. Most of the technical problems with CASA, such as the dependence of velocity on video frame rate, inaccuracy of count and percent motility for low- and high-concentration specimens, parameter dependence on the number of frames analyzed, sensitivity of the subjective threshold setting, confusion over the presence of debris, and different implementations of algorithms across instruments, still persist. A critical review of the literature reveals that no standard practices are followed within or across instruments. Moreover, no standards have been embraced or recommended by professional societies. Despite its potential to provide objective measurements of specimen and individual sperm parameters, and to automate the laboratory semen analysis, the promise of CASA has not been fulfilled. Unless laboratory medicine defines instrument performance and laboratory standards and co-operates with industry to achieve these goals, CASA technology may remain a research curiosity. This outcome is especially worrisome in the context of increasing requirements for laboratory accuracy, precision, standardization, and accreditation under the Clinical Laboratory Improvement Act of 1988.

New measures of sperm motion. I. Adaptive smoothing and harmonic analysis.

Several kinematic measures from computer-aided sperm analysis (CASA) instruments depend critically upon the computation of the spatially averaged path of a sperm's curvilinear swimming trajectory. Presently available instruments compute the average path by smoothing the curvilinear trajectory using a fixed-length running average. We demonstrate that this method significantly distorts the spatially averaged path for irregularly swimming sperm, both within and between trajectories, resulting in inaccurate calculations for the velocity of the average path (VAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), wobble of the curvilinear trajectory (WOB), and straightness of the curvilinear trajectory (STR). The authors introduce an alternative approach, based on engineering signal processing techniques, where the width of the running average is adapted to the changing wavelengths of the major spatial oscillations of each curvilinear trajectory. How this adaptive method results in less distortion of the average path and produces more accurate characterizations of the above measures is shown. This method is implemented in a computer program developed by the authors, called PathTool. It is also demonstrated that the simple methods used to characterize the frequency and amplitude of the major oscillation in a sperm's curvilinear trajectory (ie, BCF and ALH) are only accurate for the most periodic, progressive, and symmetrical trajectories. Three new measures are introduced, based on mathematical harmonic analysis, that are more robust alternatives to the present methods. These new methods and measures are initially evaluated using prototypical sperm trajectories from human semen. Results suggest that adaptive smoothing and harmonic analysis produce more accurate estimates of the frequency and magnitude of oscillations in sperm trajectories than the method based on fixed-length smoothing, BCF, and ALH.