Review: In vitro spermicidal tests

ELSEVIER

Review: In Vitro SDermicidal Tests Chi-Hyun

Lee

This review analyzes in vitro spermicidal tests that have been used to evaluate the spermicidal activity of contraceptive products. Special requirements and processes in numerous technologies were also reviewed. Nonoxynol-9 (N-9) was used as an example of a vaginally delivered spermicidal agent, and its spermicidal potency was compared by various spermicidal tests, such as Sander-Cramer, computer-assisted semen analysis, hypoosmotic swelling test, cervical mucus penetration test, and flow cytometry, and advantages and disadvantages of each test were specified. This provides an insight into the different aspects of sperm functionality on which each spermicidal agent exerts its activity. A rationale of the best combination of in vitro spermicidal tests, with particular emphasis on a simple and efficient strategy that targets the complete fertility 0 1996 Elsevier Science Inc. All control, was explained. rights reserved. CONTRACEPTTON 1996;54:131-147 KEY WORDS: spermicides, sperm motility,

sperm functionality, diagnostic technique, fertility control

Introduction undreds of spermicidal agents have been formulated into commercially available vaginal contraceptives. The surface active agents, such as nonoxynol-9 (N-9) and benzylchonium chloride, are the most commonly used spermicidal agents.14 Spermicides exert an anti-fertility effect upon spermatozoa as it passes through the female genital tract. To be an effective contraceptive agent, a compound must meet the following requirements. It must act rapidly and efficiently to kill or immobilize sperm on contact, or render the sperm incapable of fertilization. It also must be non-irritating to the vaginal and penile mucosa, should not have any adverse effect on the developing embryo or fetus, and must be free of long-term toxicity. Moreover, it should be systemically non-toxic.

H

Department of Pediatrics and Communicable Drsease, University of Michigan Medical School, Ann Arbor, MI USA Name and address for correspondence: Chi-Hyun Lee, Ph.D., Department of Pediatrics and Communicable Disease, Kresge II, Rm 5014, Medical School, University of Michigan, Ann Arbor, MI 48105-0576, USA. Tel: 313-764-4576; Fax: 313-747-3270 Submitted for publication May 17, 1996 Revised June 11, 1996 Accepted for publication June 11, 1996

0 1996 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

Testing of spermicides depends on a series of in vitro screening methods incorporating human sperm with sperrnicides and utilizing immobility or loss of functionality as an end-point. The special requirements in testing spermicides includes the screening process, toxicity studies, clinical testing, and new animal models for testing long-term usage of the contraceptive system. Spermicidal tests mainly focus on the screening process of spermicidal agents. The effectiveness of vaginally delivered spermicidal agents may be controlled by several factors, such as distribution of the spermicide in the vagina, removal or displacement of the spermicide by the coital act, and effects of vaginal fluids on the spermicide. These factors cannot be successfully evaluated by conventional visual observation tests, such as the Sander-Cramer method.5 The visual observation methods assessed the percentage of sperm cells that were motile, but did not evaluate the quality of that motility.6 It would seem essential to perform valid in vitro studies to evaluate the changes in various aspects of spermatozoa upon exposure to the contraceptive agents. Continuous research to define the parameters of sperm motility or function has led to the development of various technologies in the evaluation of a contraceptive’s efficacy as well as infertility management for men. Several photo-based7-‘i and mechanically oriented tests12-17 have been developed. In particular, advances in computer technology offer various computer-assisted semen analysis (CASA) for evaluation of sperm charcteristics. ‘s-z3 The requirement of an intact and functionally active sperm membrane for successful union of sperm, capacitation process, and subsequent acrosome reaction has led to the development of the hypoosmotic swelling test (HOS), which determines morphological changes in the sperm membrane. 24125 The measurement of sperm penetration and survival in cervical mucus is an important factor in evaluating human sperm function upon exposure to spermicides.26127 This principle has led to the employment of a cervical mucus penetration test. Recently, flow cytometry has been extensively used for the analysis of sperm functionality, sperm motility,28”1 and characterizing morphological aspects of spermatozoa.32 Flow cytometry was also employed to evaluate the potency of spermicidal agents. 33 In addition, several biochemical methods ISSN 0010.7824/96/$15.00 PII SOOlO-7824(96)00168-O

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have been introduced to substitute for complex bioassays. Such information leads to understanding of the biochemical basis of sperm fertilizing ability and consequently to an approach for successful fertility contro1.34f35 This review analyzes in vitro spermicidal assays used for demonstrating the spermicidal activity of contraceptive products. The spermicidal potency of N-9, used as a typical example of a vaginally delivered spermicidal agent, was evaluated by various spermitidal tests. Special requirements in numerous technologies, working conditions and merits of each experiment were also reviewed.

Semen Manipulation Since viscosity of semen makes it difficult to rapidly obtain a uniform mix of semen and spermicidal agent released or delivered from a contraceptive system, and the spermicidal agent in use is supposed to be diluted with vaginal and cervical secretions, a dilution of the spermicidal agent has been justified to facilitate intermixing and uniformity. An advantage of using in vitro systems for assessing the diagnostic value of sperm assays is that various confounding variables can be avoided; however, the disadvantage is that natural conditions are not maintained, thus the clinical interpretations of the results are questionable.36 In addition, the more the semen is diluted or modified before it is brought into contact with a spermicidal agent, the less reliable the results are likely to be related with that of in vivo test. A common error involved in in vitro systems is that sperm tests are performed on the original semen sample after a population of sperm is selected and this sperm population is subsequently used with the oocytes. Since the compositions of the selected sperm populations and that of the ejaculate are different, the results of sperm assay obtained from the ejaculate and the outcome of penetration or fertilization may not be comparable. From this point, non-modified semen, in which only motile or functionally active sperm are counted as a number of control sperm targeted by spermicidal agents, has the potential to offer reliable and objective results. But the more dilute the sample of semen was, the more activity of semen was shown from results acquired by cervical mucus penetration test.37 In some cases, the overestimation of sperm count or sperm motility has been reported from nonmanipulated sperm samples. For example, in a CASA test, cell count was overestimated and consequently the percentage of motility was reduced.‘l When semen specimens have been filtered or sperm was allowed to swim up into culture media, as occurred in a variety of experimental conditions, no overestima-

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tion was reported. In particular, in flow cytometry, to obtain reproducible results in detection and quantitation of sperm-bound antibodies, dead spermatozoa must be excluded from analysis because they can comprise lo-58% of the ejaculate in subfertile men and bind antibody nonspecifically.38 Therefore, various manipulating methods to select the optimum sperm and to obtain reproducible results in evaluation of spermicidal activity have been justified. The main purpose of sperm manipulation is to select sperm from the original ejaculate that have a significantly higher ferilizing capacity. The manipulation methods that have been frequently used are described in Table 1. The conventional sperm separation method (i.e., washing method) involved a simple washing procedure during which the semen was diluted with a suitable culture medium (such as Earle’s medium supplemented with 10% serum) and processed through multiple cycles of centrifugation and resuspension. For example, semen samples were washed four times in the original semen volume with buffered media and centrifuged at 400 g for 8 min. The sperm pellets were washed a second time and resuspended in buffered media to the proper concentration of sperm.39 This procedure serves as a conventional tool for separating the spermatozoa from seminal plasma, but it retains many cellular debris that may hinder the evaluation of sperm motility. Moreover, the reactive oxygen species generated by the spermatozoa in response to centrifugation was reported to initiate lipid peroxidation and cause the loss of membrane fluidity and integrity. 34~40Incorporation of antioxidant, such as vitamin E, as a protective measurement of lipid peroxidation into the culture medium during the centrifugation process has been tested and has shown a significant improvement of sperm function.41 Of the many different semen preparations for spermicidal test or insemination, the sperm swim-up procedure is simple and the most commonly employed technique.4244 The swim-up method can be further separated into two approaches: swim-up from semen and swim-up from pellet. Both methods used BiggersWhitten-Whittingham (BWW) medium as a diluting buffer.45 The swim-up from semen, in which centrifugation was applied to concentrate the motile cells after swim-up of sperm, was shown to be more effective than swim-up from pellet.46J47 Briefly, for swim-up from pellet, the mixture of liquefied semen and medium was centrifuged for 8 min at 24,000 rpm. The supernatant was discarded and the precipitate was washed again in 5 ml of medium, and the centrifugation procedure was repeated. One milliliter of BWW medium was added to the precipitate and the mixture was incubated at 37°C in 5% carbon dioxide in air for

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Table

Spermicidal Tests

133

1. Sperm selection method

Method

Number of Sperm/Motility

Washing Swim-up Filtration Density gradient separation

2 h. Then 1 ml of fluid at the surface of the supernatant was aspirated. This swim-up fraction usually contained 30-50 million sperm/ml with W-90% motility. 48 In the case of swim-up from semen, the BWW medium was gently layered over 1 ml of unprocessed semen. The tubes were inclined at 45” and incubated for 1 h, after which the upper-most 1 ml of medium was removed and the centrifugation procedure was repeated.43’44 Even though the swim-up method (i.e., swim-up from semen) yields a recovery of highly motile spermatozoa, the overall number of recovered spermatozoa is very poor. A variant of this method is a swimdown procedure, in which the semen is layered on top of a column of medium, the viscosity of which has been increased by the addition of extra albumin.47,49 The filtration of sperm through an aluminum49 or a glass wool column’~ was used to concentrate almost all of the viable sperm in an ejaculate. The filtration method is based on the fact that glass wool or an aluminum column prevents almost all non-motile spermatozoa with chemically inactive and physically damaged membranes from passing through. Furthermore, it was reported that almost all the viable spermatozoa from various quality semen were recovered by this procedure.51 Briefly, 1.5 mg of glass wool microfiber code 112 was gently packed to a depth of 3.5 mm in a l-ml disposable syringe barrel. The column was rinsed repeatedly with Ham’s F-10 medium to remove loose glass wool fibers prior to filtration. The spermatozoa were washed twice by centrifugation and the final sperm pellet was resuspended in 0.5 ml Ham’s F-10 medium. This sperm suspension was then layered over the wet glass wool column and allowed to filter by gravity. After the sperm suspension had filtered through, the filter was rinsed with 0.2 ml of Ham’s F-10 medium to flush any remaining viable spermatozoa trapped in the glass wool column. The number and viability of filtered spermatozoa were determined. The filtration of spermatozoa through glass wool by the above technique produces a higher yield of viable spermatozoa than swim-up procedure, especially from poor quality semen.50 Since the above techniques depend entirely on the vigorous motility of spermatozoa, they are not readily applicable to cases of defective sperm. Because successive pelleting and resuspension might damage

(%)

67-195 (x106)/51-74 12-50 (x106)/80-90 60-80 (x106)/70-85 5-10 (x106)/90-99

Reference 45 43,44,48,50 47,49,50 55,56,98

the spermatozoa and alter their motility characteristics, more gentle filtering techniques are required for considerable improvement in sperm motility. Isotonic continuous52’53 or discontinuous39,54 Percoll gradients were introduced to actively select the functionally intact spermatozoa. Some investigators used Nycodenz as an alternative media.55J56 The Percoll density gradient separation method has been found to improve the isolation of motile human spermatozoa, making the specimens free of the major contaminating constituents of seminal fluid.52 The requirement for a specialized centrifuge and rotor as well as the increased difficulty in maintaining sterility led to an adaptation of the discontinuous Percoll gradient system. For sperm isolation, Percoll was diluted with modified human tubule fluid and a discontinuous gradient was generated in a sterile polystyrene centrifuge tube by carefully pipetting 1.0 ml of the 90, 70, and 50% Percoll solutions, respectively. Approximately 2 ml of the semen sample was layered over the gradient and the preparation was centrifuged for 30 min at 300 g, at room temperature. After centrifugation, the bottom half of the 90% layer that contained the sperm was resuspended in 2 ml of Ham’s F- 10 media and centrifuged for 5 min (300 g, room temperature). The supernatant was removed and the resulting sperm pellet was resuspended in either Earle’s or Ham’s F-10 media. The specimen was analyzed to determine cell count using a computer-assisted semen analyzer. The specimen was then diluted to approximately 5 lo6 sperm/ml with Ham’s F-10 medium. The scale and composition of the Percoll gradient can be altered to meet the specific needs of individual samples. Percoll gradient procedure, like the swim-up from semen technique in which centrifugation is only applied after motile cells have been selected, seemed to be the optimal sperm preparation technique for selecting the highest quality of sperm suspension.47

Semen Condition Various parameters were used for evaluation of sperm status. The important criteria of human semen, used in most of the experiments, were sperm count, motility, and morphology. The various systems count or record the number of motile and non-motile sperm analyzed and compute their concentrations, which

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are used further to calculate sperm density. For semen specimens containing higher cell density, sperm concentration needed to be reduced before measuring motion characteristics.42’43 In a conventional visual observation test, the sperm motility was the only factor used to determine spermicidal potency.5 Percentage of motile sperm is expressed as motility. The term “spermicidal or kill” is used in a sense that spermicide prevents fertilization by ceasing sperm motility. Since seemingly motile sperm do not always have fertilizing capability, other parameters need to be investigated. Sperm morphology was evaluated from semen smears that were stained following the simplified Papanicolaou stain technique for sperm.57 Stained smears were coded and then evaluated using bright-field microscopy at 1,000 total magnification under oil. One hundred sperm on each smear were evaluated for head, mid-piece, tail, and cytoplasmic droplet morphology based on WHO guide1ines.s’ In most experiments, the criteria described in Table 2 were used. In vitro spermicidal activity cannot be explained by the alteration of any single sperm characteristic. Optimum discrimination appears to involve several variables of sperm morphology and motility. With the advance of computer technology, various parameters of sperm motion characteristics, such as straight line velocity, curve linear velocity, and angle of lateral head displacement, were defined.57!58 Among characteristics described in Table 2, the percentage of abnormal acrosomes and lateral displacement of the moving sperm head seemed to be the most significant factors. Straight line velocity is defined as time-average velocity of a sperm head along the straight line between its first detected position and its last position. Mean swimming speed is calculated by adding the distances between every two successive video frames analyzed on the cell’s track and dividing that sum by the total time interval for which the cell was tracked. This determination was influenced by the number of tracking points as well as by the concentration of Table

cells present in the counter chamber. More representative velocity estimations in semen were obtained when sperm concentration was less than 40 lo6 cells/ ml and 20 tracking points were used in the determination’l A lack of penetration of zona-free hamster eggs was found in sperm with a speed of ~30 m/sec.5g In a study of 2100 samples, a time-dependent decline in sperm speed in subfertile men, but not in normal fertile men, was demonstrated.60 Curve linear velocity is defined as time average velocity of a sperm head along its actual curve linear trajectory, as perceived in two dimensions under the microscope. Average path velocity is defined as time average velocity of a sperm head along its spatial average trajectory. This trajectory is computed by smoothing the actual trajectory according to algorithms in the CASA instrument; these algorithms vary among instruments. Mean linearity is a measure of the deviation of the actual sperm cell track from the straight line connecting the point of origin and end point of the cell’s track, and is expressed as a ratio and calculated by dividing the length of the straight line distance by the actual track distance. The more the actual track deviates from the straight line, the lower the linearity value. Angle of lateral head displacement is defined as magnitude of lateral displacement of a sperm head about its spatial average trajectory. It can be expressed as a maximum or an average of such displacement. The importance of the angle of lateral sperm head displacement in determining the ability of human spermatozoa to penetrate the mucus interface is described by Jeulin and co-workers.61 The positive correlation between this parameter and fertilization rate is attributed to the fact that sperm movement needed for the penetration of cervical mucus is also required for the egg investments and the zona pellucida. The particular emphasis on this parameter in evaluation of fertilization process or spermicidal test seems necessary. One important factor in evaluating fertilizing ability of sperm that has been neglected in most cases of

2. Sperm variables

Sperm Condition Number Motility Morphology Acrosine activity Vitality (excluding dye) Sperm motion characteristics Straight line velocity Curve linear velocity Mean linearity Angle of lateral head displacement

Normal Range

Reference

65-200 (~10~) 50-75 (%) 3141(%) 25-40 (uIu/106 sperm) >75 (%)

25,45,50,58,61,69,84,90,123

20,22,23,35,129 32.3-34.4 (um/sec) 52.0-53.6 (um/sec) 62.9-66.9 2.99-3.10 (urn)

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sample preparation is specific sterilizing defect. In domesticated animal species, several sterilizing defects have been described.62 Essentially all specific structural defects in sperm cause sperm dysfunction. This dysfunction cannot be detected by conventional or CASA. The presence of anti-sperm antibodies coating the spermatozoa is considered to be specific for immunological infertility.63 In vivo, the principal inhibition of fertility could be accomplished by the presence of immotile antibody-bound sperm that does not reach oocyte.62 Sperm antibodies in semen belong almost exclusively to two immunological classes: IgA and IgG. IgA antibodies might have a greater clinical importance than do IgG antibodies.64 The immunobead method and the mixed antiglobulin reaction (MAR) tests have been used as a screening test for antibodies. Immunobeads are polyacrylamide spheres with covalently bound rabbit anti-human immunoglobulins. The test is considered positive when 20% or more of motile sperm exhibit immunobinding. Another type of immunobead, originally intended for total-cell labeling, can also be used as a one-step screening test for IgG and IgA isotypes of sperm surface antibodies.65 The IgG MAR test was performed by mixing fresh, untreated semen with latex particles or sheep red blood cells coated with human IgG. To this mixture, a monospecific antihuman-IgG antiserum was added. The formation of mixed agglutinates between particles and motile spermatozoa proves the presence of IgG antibodies on the spermatozoa. The diagnosis of immunological infertility was probable when 50% or more of the motile sperm had adherent particles.66f67

Exposure Time It is well known that vaginal contraceptives have a relatively high failure rate due to mistakes in the application of spermicide rather than a poor contraceptive activity of the spermicide itself. Once sperm are deposited vaginally, they pass within minutes into the cervix at midcycle and begin their transport through the female genital tract. Therefore, available time for the vaginal contraceptives to exert their spermicidal activity in vivo is relatively short. In addition, decreased effectiveness may be due to a poor distribution of the spermicide in the vagina, removal or displacement of the spermicide by the coital act, inactivating effects of vaginal fluids on the spermitide, or an intercourse position that causes rather poor spermicide-sperm contact. Human semen normally undergoes liquefaction within 20 min of ejaculation because of the action of a protease secreted by the prostate, prostate-specific antigen. 68 Prostate-specific antigen induces proteolyt-

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ic fragmentation of semenogelin, the major protein constituent of the seminal coagulum that is produced and secreted by the seminal vesicles. Some spermitidal agents decreased the in vitro percentage of motile sperm in both dose- and exposure-time-dependent ways. The percentage of sperm killed by spermicidal agent kept changing with time, and was not changed significantly after some time of treatment. For this reason, in most experiments, an interval of that specific period was used for an appropriate exposure time for dose-response studies. The investigation of the relationship between sperm motility and duration of exposure of sperm to agent by CASA test, performed after 2-min, 30-min and 2-h incubations of semen with spermicidal agents, showed a temporal activity pattern.69’70 Sperm motility decreased as exposure time increased. This indicates that duration of exposure of sperm to agent should be strictly monitored to allow accurate comparison of the spermicidal action of agents. WHO updated and revised a manual, which provides an essential source of reference for all laboratories involved in the analysis of human semen and sets out the fundamental laboratory techniques that should be employed to diagnose male infertility and further usage of contraception?’ The duration of exposure of sperm to spermicidal agent should follow the protocol established by WHO.

Tests of Spermicidal

Activity

Testing methods designed to screen or evaluate vaginally delivered spermicides require high efficiency and rapid action. As shown in Table 3, numerous in vitro tests have been devised for evaluation of sperm motility and functionality. Various technologies were tested to select optimum semen, dose of spermicide, and sperm-spermicide exposure time. Some methods were mainly developed for evaluation of male infertility, so certain conditions may not be suitable for the application of spermicidal tests. The conventional methods, such as Baker and coworkers,‘l Brown and Gamble,” Sander and Cramer,5 Millman, and International Planned Parenthood Foundation (IPPF),73 mainly depend on human visual observation. The Sander-Cramer method is regarded as the first well established, traditional semen analysis (TSA) test developed to examine the effect of spermicidal and pharmaceutical agents on sperm mobility.5 This method is simple and reproducible. The end point is death or immobility of the sperm. This method shows whether a spermicide immobilizes all spermatozoa in a semen sample, and whether the immobilization is reversible by addition of glucose. If any motility was observed, the dilution was regarded as failed for spermicidal activity. The detailed proce-

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3. Various methods for evaluation of snermicidal activity

Methods

References

Conventional visual microscopic method Baker and co-workers Brown and Gamble Sander and Cramer Millman International planned Parenthood Federation Objective photo-based (videomicrography) method Image capture Photography Spectrophotometry Videomicrography Time-exposure photomicrography Cinemicrography Mechanical method uv Laser light-scattering Turbidimetry Transmembrane migration Hemolytic potency Stripping technique Computer-assistedsemen analysis Hypoosmotic swelling test Sperm-cervical interaction test Slide test Sperm-cervical mucus contact test Cervical mucus penetration test Flow cytometry method Biochemical test

dure is described

in the WHO manual.” The IPPFsp er micide power was developed based on work by Harris.74 The IPPF test and Sander-Cramer methods were a step toward standardization in interpretation of spermicidal test. Although these methods provided a qualitative indication of relative sperm motility, they lacked accuracy in quantitative evaluation. To enhance the efficiency of evaluation of motile sperm, photo-based methods, which objectively determined sperm motility, were introduced. Multiple agreed test 73 for total

exposure photography

based methods,

(MEP), as one of the photo-

was developed

for periodical evaluaand percentage of motility.8 In this MEP method, each of two or three drops from a well-mixed specimen was placed in a

tion of spermatozoa1 velocity

special 10-m chamber, and 8-12 fields containing

200- 400 sperm/specimens were photographed at predetermined locations. Each film was exposed for 1 set, during which time the sample was illuminated by six light pulses. Images of photographed sperma-

tozoa, termed as Image Capture, were projected onto sheets of paper from which the percentage of motile spermatozoa and their velocity were calculated as described previously.’ Advanced photo-based methods, such as spectrophotometry,’

videomicrography,75

time-exposure

70 71 5 72 73 7 z ::, 77 78 11 10 12,13,82 14,15 16,17 18-23 24,25,36,85-87 27,28,37,65,94-97

2830,33,38,98-103,107-110 31,35,91,117,120-122,124

photomicrography,76 and cinemicrography” techniques, were introduced. Videomicrography shows a good resolution and efficiency75 and is further developed in combination with a computer software program. ” Time-exp o su re photomicrography provides detailed information on the movement of sperm head.76 Cinemicrography generates fomidable data on the movement of the head and flagellum.” These methods are expensive to perform and cannot provide any direct information on the characteristics of sperm movement. Mechanical methods, such as ultraviolet (UV),78 lamethod,” transser light-scattering, l1 turbidimetric membrane migration, l2 hemolytic potency, l4 and stripping technique, 16,” were introduced to increase the efficacy of evaluation of sperm motility by using advanced equipment and membrane or surface contacting strategies.

UV and laser light-scattering techniques give an overall indication of degree of movement exhibited by the spermatozoa. 78~79 With dynamic laser lightscattering, the concentration of progesterone,which would inhibit the swimming speed distribution of freshly washed human spermatozoa as expressed in the spectrum of scattered light, was determined.” According to the optical Doppler effect, the scattered light intensity spreading into various frequencies

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yields a spectrum that depends on the velocity profile of spermatozoa. The laser light-scattering technique can provide an objective, reliable, and rapid evaluation of density, percentage of motile sperm, and the swimming speed of spermatozoa present in the semen The disadvantages of laser methodology, sample.” like other photographic techniques, include the high cost of equipment and the slow speed at which the analysis can be performed. The turbidimetric method was used to quantitate the effects of biochemical and pharmacological agents on sperm motility.” This method consists of layering semen on the bottom of a specially constructed cuvette containing 0.6 ml of Lopata’s medium.‘l The semen is injected through an injection port that is open to the optical chamber at the bottom of the cuvette, thus allowing the semen to layer evenly at the bottom of the chamber. Sperm motility is measured spectrophotometrically as a change in absorbance as the sperm swim upward into the light path. Optical density was measured using a spectrophotometer equipped with a constant-temperature cell housing that was kept at 37°C. The spectrophotometer was connected to a linear strip chart recorder. Two parameters, lag time between the injection of the semen and the initial rise in absorbance, and the continuous change in absorbance that occurred as the sperm entered the light path, were measured. The transmembrane migration method was introduced by Hong and co-workers.” Like other tests, this method was also originally developed to aid in evaluating the effects of a drug on sperm motility and male fertility. This method has an apparatus consisting of two chambers separated by a membrane with 5 m-diameter capillary pores. The upper chamber is made from the plunger of a 2-ml Sabre syringe. A sheet of membrane (13 mm diameter] is bonded to the lower end. Fresh human semen (100 1) is pipetted into the upper chamber and then is placed into a siliconized glass bottle containing 2 ml of phosphatebuffered saline (pH 7.3). The drug solution can be mixed with semen at the ratio of 1:2 prior to placing 100 1 of the mixture into the upper chamber. The setup is placed in a water bath at 37°C. After 2 h, the upper chamber is removed and the spermatozoa in the lower chamber are killed with formalin. The number of spermatozoa in both chambers are then counted by a hemocytometer. The proportion of semen that moves across to the lower chamber is called the transmembrane migration ratio and is given in unit of percent. This method was further applied to the evaluation of the spermicidal potency of various agents.13rs2 Dolan proposed that the hemolysis of erythrocytes by surfactant-type spermicides follows the same mechanism as the interaction of these compounds

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137

with sperm.14 Therefore, a hemolytic test could be used as an alternative analysis over the use of human sperm samples in evaluating spermicidal potency. The study of hemolytic activity to evaluate a series of nonionic surfactants showed that the adsorption of surfactant resulted in the rearrangement of the membrane structure rather than the destruction of the membrane structure.” Brotherton used the Coulter counter to determine sperm number and size.16 The decrease in mean sperm size was measured against an increasing concentration of spermicide. The end point was taken as the point where the peripheral cytoplasm had been removed and only the sperm core of the nucleus and tail fibers remained. This method, also called a stripping method, was applied to the evaluation of sperm motility in various species.”

Computer-Assisted

Method

With advances in computerized technology, semen analyzers based on videomicrography have been developed for the assessment of fertility as well as toxicology. l8 A CASA system evaluates semen samples rapidly, and thus permits large populations of sperm to be analyzed in a short period of time. This system also allows sperm motility to be measured objectively by removing the potential bias occurring from human visual observations. The Hamilton-Thorn motility analyzer was introduced to compare spermicidal activity of selected agents” and the fully automated CellSoft system followed. This system provides a greater number of end points for sperm motion than traditional motility analysis.20t21 The CASA definition of motile sperm is based on achievement of a threshold of forward velocity. By contrast, visual perception of a motile sperm is frequently influenced by the presence of flagellar activity, even though the cell is not swimming forward. Consequently, CASA and visual measures of percentage motility in semen are different. CASA seems to produce lower values.22 The CellSoft system consists of a phase-contrast microscope, a videocassette recorder, and a computer with CellSoft software for automated count and sperm motion analysis. Because the parameters of sperm motion are temperature-sensitive, the CASA system must include provision for the maintenance of specimen temperature at 37°C. The fully automated CASA requires a portion of the fresh semen specimen to determine sperm concentration, percent motility, and normal morphology along with various parameters of sperm motion characteristics described in Table 2. One of the drawbacks of this method is that values obtained for concentration, percentage of motile sperm, velocity, and linearity depend, to vary-

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ing degrees, on the settings of the device or computer. 23 For standard semen evaluation, an established procedure was presented in the WHO laboratory manua1.57

Hypoosmotic

Swelling

Test

A sperm HOS was introduced to find a more efficient method of diagnostic semen examination.24 This method is based on the assumption that an intact, functional sperm membrane is required for successful union of sperm and oocyte, and that an undamaged sperm tail membrane should permit free passage of fluid into the cell under hypoosmotic conditions. The HOS test reflects any change of sperm membrane during capacitation and the subsequent acrosome reaction.25 The HOS test is known to be a sensitive and efficient method for diagnosing semen functionality. The HOS test reflects any sperm membrane changes that occur during capacitation and the subsequent acrosomal reaction process. The plasma membrane around the tail fibers is more loosely attached than that around other sperm parts, and is particularly susceptible to abnormal conditions, such as exposure to hypoosmotic solution.83 The swelling of the sperm tail after exposure to hypoosmotic buffer implies that sperm is intact and functionally active.36 The function of the sperm membrane is of great importance for sperm capacitation, acrosomal reaction, and binding to and penetration of the egg investments during fertilization. 84 Therefore, assessment of sperm membrane function can help lead to a determination of the fertilizing capacity of sperm.85f86 A schematic illustration of human spermatozoa with various typical morphological changes after being subjected to hypoosmotic stress was classified.87 Under phase-contrast microscopy (400 ), sperm were examined upon exposure to spermicidal agent and all swollen sperm were regarded as functionally intact sperm. The resultant potencies of spermicides were compared with those acquired by traditional and computer-assisted semen analysis. The standard procedure of the HOS test is also recommended by the WHO manua1.57 To improve reliability of the HOS test, a modified method was introduced by Jeyendran.36 In this method, the first count of the swollen spermatozoa was done immediately upon mixing whole sperm with the hypoosmotic solution before incubation, and subsequent counts were done at 5 to lo-min intervals during an incubation period. Since the difference in tail configuration may reflect the degree of membrane permeability, the swollen spermatozoa were grouped according to shape of tail swelling described by Mordel and co-workers.87

Sperm-Cervical

Interaction

Test

Conventional sperm parameters used for semen evaluation, such as number, morphology, and motility of sperm, show wide individual variations and sometimes do not correlate with demonstrated fertility potency. Since sperm motility often does correlate well with the capability of penetration through cervical mucus, and the latter may be more representative of sperm function in vivo, a proper method to evaluate sperm functionality in cervical mucus seems necessary. Deficiencies in sperm function could be related to flagellar activity, surface properties, or enzyme content. A variety of factors affect sperm penetration and abnormal forms of spermatozoa supposedly cannot penetrate mucus. In most mammals, the cervix and cervical mucus comprise the initial barrier that sperm must cross. Cervical mucus protects sperm from the hostile vaginal environment, filters out abnormal and poorly motile sperm while facilitating actively motile sperm of normal shape, serves as a sperm reservoir, and provides a likely site for capacitation. Vaginal contraceptives exert their activities through the spermicidal effects of their active ingredients, as well as through a physical barrier to sperm penetration through the cervix. The measurement of sperm penetration and survival in cervical mucus is an important factor in evaluating human sperm function upon exposure to spermicides. Normally, ejaculation results in a pool of semen being deposited in the posterior vaginal fornix in close contact with the cervical mucus. Consequently, upon liquefaction of the seminal coagulum, it is possible for spermatozoa to pass rapidly into the mucus with little direct contact with the vaginal fluids containing spermicides. Therefore, it is important to know whether the domains within the cervical mucus through which sperm migration occurs are also accessible to the vaginally released spermicide. If passage of the vaginal spermicide into the mucus is impeded, or if activity of spermicides in the mucus is hindered, the contraceptive effects of spermicides will be limited. As shown in Table 3, the slide test, sperm-cervical mucus contact test (SCMC), and cervical mucus penetration test were used to evaluate sperm-cervical interaction. The mucus, the major components of which are water (about 97%) and mucoid (i.e., a carbohydrate-rich glycoprotein in which the proteins are tightly bound to polysaccarides containing sialic acid), is a hydrogel with non-uniform viscoelastic properties. *’ The highly viscous phase is composed of glycoproteins linked to peptide backbones.89 Sperm penetration is greatly reduced if the mucus water content drops below 95%, because water is the vehicle

Spermicidal

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for electrolytes, proteins, and other organic compounds. Proteolytic enzymes such as trypsin and chymotrypsin were present in cervical mucus and semen to catalyze hydrolytic reactions of the mucoid of cervical mucus and to reduce the viscosity.37!88 In some cases, surrogate gels were used as an alternative for human cervical mucus.” Synthetic gels used for this purpose are hyaluronate’i and polyacrylamide.92 Hyaluronate, which is a naturally occurring polysaccharide, has been used for internal therapeutic application.93 In comparison to other proposed mucus substitutes, hyaluronate has several important advantages. It is easy to prepare in relatively large quantities, has less variability between batches, and requires neither serum supplementation nor special equipment. These gels do not duplicate all the features of human sperm-mucus interaction, but can serve as a useful adjunct to routine semen analysis. The slide and SCMC tests are simple to perform and provide useful information about routine components in the assessment of semen quality. In the slide test, a drop of cervical mucus is placed on a slide and flattened by a coverslip. The depth of the preparation can be standardized by supporting the coverslip with silicone grease containing 100-m glass beads. Interpretation of this test is subjective, because it is impossible to standardize the size and shape of the semen-mucus interface in a plain slide preparation.94 Consequently, it is recommended that the test be used only as a qualitative assessment of sperm-mucus interaction.37 The SCMC test is designed to detect antispermatozoa1 antibodies that may be present on spermatozoa and/or in cervical mucus. The result of the test also indicates to what extent the antibodies inhibit sperm penetration and migration.65,95 The SCMC test is performed by placing a small amount (lo-50 1) of pre-ovulatory cervical mucus and an approximately equal amount of fresh semen on one end of a microscope slide. After materials are mixed thoroughly, another drop of the same semen sample is placed on the other end of the slide. The semen mucus mixture and the semen drop are covered with coverslips. The preparation is stored in a moist Petri dish at room temperature for 30 min and the percentage of motile spermatozoa in the semen-mucus mixture after rapid shaking is determined. The semen alone serves as a control for sperm activity. The high percentage of shading means that most sperm cannot pass the cervical mucus and thus cannot reach the oocyte. The cervical mucus penetration test is a more sophisticated form of analysis that provides a better semiquantitative measure of sperm penetration into mucus than other sperm-cervical interaction tests.

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With capillary tubes, the objective evaluation of the cervical mucus penetration test was feasible.26T27 Sperm penetration has usually been assessed in cervical mucus collected after coitus. The specific procedure of the cervical mucus penetration test was recommended by the WHO manual,” which was simplified and modified from the methods previously described.96,97

Flow Cytometry Flow cytometry is a technique in which individual cells or components are examined on a single-file flow of cells (monodispersed dispension), passed through a fluid stream, and moved past sensors that measure the physical or chemical parameters of the cells. Physical parameters are obtained by the use of an analytical flow cytometer composed of a flow system, a light source, a detection system, electronics, and computer. Since the flow cytometer relies on the hydrodynamic focusing of a stream of monodispersed particles, biological generation of a single-file flow cytometry was first applied to hematologic samples. The flow cytometry method improves further upon development of the computer-assisted semen analysis method. Flow cytometry has been extensively employed to the analysis of acrosomal integrity, mitochondrial function, and motility of sperm to determine the fertilizing potential of human and animal sperm samples.2830 Its applications also include characterizing morphological aspects of spermatozoa,32 vitality and acrosomal status of human spermatozoa,98 and determining the effects of cigarette smoking on sperm quality.99 Antibody-bound sperm were detected and quantified using flow cytometry to evaluate other aspects of infertility.38~‘00-‘02 Sperm-specific antigens are of interest because they may be needed as potential contraceptive molecules.62 In comparative studies, the indirect flow cytometric immunofluorescence assay showed a highly sensitive diagnostic standard for the detection of various therapeutic modalities on sperm antibody levels. lo3 Numerous staining techniques have been used to study various aspects of viable sperm. The use of viability-specific dyes makes it feasible to evaluate the action of the spermicide on a cellular level and increase the sensitivity. These techniques include fluorescein isothiocyanate (FITC)-labeled heparin and lectins,31,‘04 monobromobimane, lo5 and 4’,6’-diamidone-2phenylindole. lo6 Auger and co-workers used Rhodamine 123 to correlate mitochondrial membrane potential of sperm with motility and used its fluorescence to separate highly motile sperm.‘07,108 Combinations of fluorescent dyes have also been uti-

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lized to acquire more information from the analysis of an individual semen sample. Dual fluorescent staining has been conducted using 5 (and 6-) carboxy-4’,5’dimethyl fluorescein diacetate (CFDA) and hydroethidine to study the viability and fertilizing potential of bovine sperm following cryogenic storage, although close correlation between flow cytometric indicators and motility was not observed.lo9 In their continuous work, the toxicity of dyes to sperm cells was evaluated. Hoechst 33343, hydroethidine, and a tracking dye decreased oxygen consumption, although R123 did not.15 When more than one dye is used to label one cell, signals can overlap in the detection range. To alleviate the problem, compensation for the color overlap can be achieved by subtracting a percentage of one signal from the other. A dual staining technique involving CFDA and propodium iodide (PI) was employed to determine various functional aspects of avian, ’ l1 bovine,‘12 and porcine113 spermatozoa. The use of a triple staining technique involving propidium iodide, FITC-labeled Pisum Stivum agglutin, and Rhodamine 123 was also developed to simultaneously evaluate viability, acrosomal integrity, and mitochondrial function, respectively.29 The procedure using the esterase-specific dye 6-carboxyfluorescein diacetate (Sigma Chemical Co, St. Louis, MO) to label samples was applied to evaluate the spermicidal potency of N-9.33 This nonfluorescent analog of fluorescein readily enters cells by passive diffusion. Once inside the cell, the functional groups are modified by cellular esterases, resulting in a molecule with intense green fluorescence when excited with 488 nm light. In a typical experiment, carboxyfluorescein was added to a sperm sample to a final concentration of 5 M and incubated at 35°C for 15 min. Aliquots (100 1) of the labeled spermatozoa were then pipetted into polypropylene culture tubes containing 400 1 of phosphate-buffered saline consisting of increasing concentrations of spermicidal agents. The isotonicity of the phosphate buffer was maintained at 300 mOsmo1 with NaCl and confirmed using a Osmet (Precision Systems, Natick, MA]. Immediately before analysis, propodium iodide (Sigma Chemical Co, St. Louis, MO) was added to each tube to a final concentration of 10 M. Stained samples were analyzed using an EPICX Profile flow cytometer (Coulter Electronics, Inc., Hialeah, FL) equipped with an Omnichrome 25 mW argon laser emitting at 488 nm with 15 mW power. Green fluorscence signals were collected using a 550-nm longpass dichroic and 525-nm band-pass filters and were processed through a linear amplifier. Red fluorescence signals were collected through a 630-nm longpass filter and processed through a four-decade loga-

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rithmic amplifier. Cellular debris were excluded from fluorescence analysis using forward angle and log 90” light-scatter gates. Electronic color compensation was used to correct the signal overlap, which was observed in samples containing both red (propidium iodide) and green (carboxyfluorescein) fluorescence. Since propodium iodide is known to stain the nuclei of cells in which the membrane has been disrupted, the increase in red fluorescence reflected loss of membrane integrity and was interpreted here as cell death. Using the filters and laser settings described above, a signal subtraction of 3% and 25% was used to correct the green and red fluorescence, respectively. A minimum of 10,000 gated events were collected for each sample. The histograms generated in these experiments were analyzed using a software program, such as EPICS CytoLogic Software, Version 2.0 (Coulter Electronics, Inc, Hialeah, FL).

Biochemical

Tests

Because of the apparent deficiencies in application of traditional sperm parameters as standard criteria for the fertilization process in vivo, various attempts have been made to develop alternative methods based on sperm function. Egg phosphatidylcholine liposomes were used as a sperm cell membrane model to study spermicidal activity.“4J15 Carboxyfluorescein was incorporated into the interior of the liposomes as an indicator. The release of carboxyfluorescein was found to be correlated with the inhibition of fertilizing ability of sea urchin sperm. Such techniques have proven to be extremely useful in predicting the competence of human spermatozoa for fertilization in vitro, analyzing the influence of potential therapeutic drugs on human sperm function, and in determining the molecular basis of defective sperm function.‘16 Recent interest in understanding the mechanisms responsible for defective sperm function has led to development of simple biochemical tests for evaluation of sperm functionality. For example, peroxide damage due to generation of excessive reactive oxygen species and presence of high level of creatine phosphokinase contribute to abnormalities in the sperm. 34~35r117-119The human sperm plasma membrane is particularly susceptible to peroxide damage, because it is filled with unsaturated fatty acids.40 Based on this mechanism, biochemical tests can be further developed to find a simple spermicidal test. Several biochemical tests for evaluation of sperm functionality, such as the zona-free hamster oocyte test,34J35 human zona pellucida binding test,‘20-‘22 and acrosome reaction scoring test,311123J124 can substitute for complex bioassays used for evaluation of the fertilization process. It has been reported that de-

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fects in capacitation and sperm motion characteristics, binding of the spermatozoa to the zona pellucida, acrosome reaction, acrosin activity of the spermatozoa, and the ability of the spermatozoa to penetrate zona-free hamster oocytes cause male infertility. 125-127 The ability of sperm to fertilize an oocyte depends on previous events, that together are essential for fertilization. 128 Sperm capacitation, the acrosome reaction, and zona binding are part of the reqSperm-oocyte interactions uisites for fertilization.‘29 were studied using intact human oocytes13’ or under internally controlled conditions using hemizonae pellucidae from bisected human oocytes to record zona binding. l3 During sperm passage or storage in the cervix, there may be certain interactions between the mucus and sperm surface.132 Since these methods were originally developed for evaluation of male infertility, their application to spermicidal tests may not be suitable and have not been actually conducted. But the aid of such information provides an understanding of the biochemical basis of sperm fertilizing ability and consequent prevention. Ultimately, clinical trials will be the key determinant in assessing the effectiveness of dosage strengths, since in vitro assays cannot account for actual physiological conditions such as the drainage of spermicides before ejaculation, uneven distribution of spermicides in the vagina, and variation of vaginal fluid present at different times during the estrous cycle.

Example N-9 has been extensively used in spermicidal formulations currently marketed for barrier contraceptive devices.2l3 The spermicidal activity of N-9 has been investigated in numerous studies.2t’9,133-136 In this review, the results of quantitative potency of spermitidal activity of N-9 determined by various tests were compared and merits of each test were characterized. Some of the experimental conditions in tests are different, so absolute comparison of spermicidal potency may not be possible, but it can provide some insight in evaluating the merit of each test. To specifically investigate the mechanistic aspect of each test, the effects of pH, osmolarity, and ionic components were controlled.137-143 The pH of the buffer was not significantly changed upon addition of N-9 within the tested concentration range, and the osmolarity of the control buffer was maintained at 301 mOsmo1. The results of each test were compared with controls by using one-factor ANOVA and Student’s t-test. Data were normalized by expressing results as percent of control, because initial semen conditions, such as motility and morphology, varied among samples.

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The results of spermicidal potency of N-9 on sperm motility evaluated by various methods showed a significant difference in concentrations required to exert complete inhibition of sperm motility, as shown in Table 4. The spermicidal activity of N-9 is very potent, showing total loss of sperm motility at concentrations less than 200 g/ml in most tests except cervical mucus penetration method. Fifty percent of the sperm were immobile at concentrations of 60-125 g/ml of N-9, depending on test methods. The Sander-Cramer method is a pass-fail, all-ornone test, which means that upon exposure to the specific concentration of spermicidal agent (i.e., N-9), if at least one sperm has motility, the agent was regarded as non-spermicidal at that concentration. Thus, this method is not suitable for constructing a quantitative relationship between dose and spermitidal potency. The concentration of N-9 required for 100% inhibition of sperm motility is almost equivalent to that of CASA. But its projecting EC,, concentration of N-9 observed by the Sander-Cramer method was greater than that of CASA. As previously described, the CASA definition of motile sperm is based on achievement of a threshold of forward velocity, which is greatly influenced by flagellar activity. Consequently, the required dose of spermicidal agent to show spermical action measured by CASA seems lower than that measured by the Sander-Cramer method. The results of the CASA test showed that N-9 at 150 g/ml alone caused about 52% inhibition of sperm motility. The mean effects of N-9 on motility of human sperm measured by the CASA test and statistical values of sigmoidicity coefficient (S), affinity constants (l/Q) and spermicide potency calculated from SAS were previously reported.‘44 The cervical mucus penetration test was used to evaluate entry of agents into the domain of mucus by diffusion, the penetration of sperm in mucus against agents, and the agent’s spermicidal activity in the mucus on sperm motility. N-9 was known to disrupt

4. Concentration of N-9 required for EC,, and EC,,, spermicidal activity determined by various spermitidal tests Table

N-9 (w/ml) Methods

Sander and Cramer Computer-assisted semen analysis Hypoosmotic swelling test Cervical mucus penetration test Flow cytometry method ‘not determined. References: 19, 33, 69, 133,

136,

144,

150.

EC,,

EC,,,

-*

200-500 175

140-150 85-95 a500 60-65

110

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cellular and intracellular membranes, and disruption of membranes led to the loosening and detachment of the acrosome, nucleus, neck, and midpiece, and could be expected to result in cessation of sperm motility in semen-saline suspensions.133,‘34 The spermicidal potency of N-9 seems closely related to its mucus-water partition coefficient. 145-147 In contrast to the results obtained by conventional observation test and CASA, N-9 appeared to have a mild inhibitory effect on sperm penetration through mucus. The sperm penetrated more than one-half the distance achieved in the control mucus at concentrations that showed a total spermicidal effect on sperm motility in semen, and some were still mobile at the time of observation. The concentration of N-9 up to 1000 g/ml did not fully inhibit sperm penetration through cervical mucus. The spermicidal activity of N-9 seems decreased or hindered by cervical mucus. The reason for this phenomenon is not clear. The low partition coefficient of N-9 between aqueous phase and mucus may hinder the spermicidal effect of N-9 on sperm penetration and motility. The complex physical interaction of surfactant with water may result in micelle formation, generating macromolecular complexes that may not penetrate the domains of the mucus that the motile sperm could access.148 In evaluating the amount of N-9 diffused into the mucus, N-9 in the mucus was negligible, indicating either it was not diffused into mucus at all, or a very small amount was diffused. This result indicates that the cervical mucus penetration test is required to thoroughly test spermicidal activity of prospective agents in fertility control. Most of the spermicides on the market have not been tested by this method. The HOS test revealed that exposure of semen to N-9 decreased the percentage of functionally active sperm in a concentration-dependent manner, as shown previously.86~87~149~150 N-9 was found to inhibit human sperm functionality in semen by 99% at concentrations as low as 100 g/ml. Total loss of sperm functionality (ECloo) was shown at a concentration of about 110 g/ml of N-9. Reduction of the proportion of functionally active sperm at low concentrations was also greater for HOS than for CASA.15’ The HOS test tends to measure different factors from the standard semen parameters. Thus, the outcome of the HOS test has not been accurately predicted from the performance of the conventional observation-based methods. A higher correlation between hypoosmotic viability and sperm motility was observed.150,‘51 The major activity of N-9 on the sperm membrane greatly contributes to a close correlation between hypoosmotic viability and sperm motility. The previous study indicated that HOS could

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assist further in the evaluation of spermicidal activity of various agents, and that concentrations of spermitidal agent required for inhibition of sperm functionality are much lower than those for inhibition of sperm motility. This indicates that the concentration of spermicidal agent required to regulate fertility control can be lowered. The hypoosmotic swelling test is simple, rapid, and inexpensive, and offers promising results. Therefore, it can be employed as a complementary method to CASA and can greatly contribute to the quantitative assessment of spermicidal activity. In flow cytometry, concentration of N-9 greater than 100 g/ml were found to kill 100% of the sperm, as shown in Table 4. Low concentrations of N-9 in this test were less active as a spermicidal agent.145 Concentrations of N-9 below 40 g/ml were found to have no effect on sperm viability. At concentrations greater than 70 g/ml N-9, essentially all the sperm were non-viable after exposure to N-9. In comparison to the computer-assisted semen analysis results, overall results were similar, although the required dose used for flow cytometry was lower. This also indicates that the mechanism of spermitidal action of N-9 is concentration-dependent. This result suggests that the sperm may undergo disruption of membrane integrity on exposure to low concentrations of N-9, followed by total cessation of sperm motility on exposure to concentrations of more than 250 g/ml. Doses of N-9 corresponding to EC,, and ECloo are similar to those of HOS tests, indicating both methods evaluate morphological changes in the sperm membrane. The flow cytometry method measures fluorescence and light scattering on a cell-by-cell basis as the cells flow through a stream in single file. Samples are delivered via syringe into the flow cell apparatus, where the sample passes through a narrowing tube into a laminar-flowing sheath fluid. The efficiency of flow cytometry in evaluation of sperm motility improves as does the computer-oriented method. The use of specific dyes increases the sensitivity of this method, and further increases its capability. Flow cytometry can be used as a screening process aimed at comparison and preliminary evaluation at more dilute concentrations. The assay can provide information on the determination of an optimal therapeutic dosage, but, for evaluation at therapeutic strength, the analytical capacity of an assay would be hindered. Nevertheless, this method can provide an analytical tool for a sensitive spermicidal evaluation and elucidate part of the mechanism of spermicidal activity of contraceptive agents.

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Conclusion The conventional visual observation method has been largely used, mainly because this method is simple and rapid. The requirement of testing other sperm characteristics for complete evaluation of the fertilization process led to the development of various objective photo-based and mechanical methods. These photo-based and mechanical methods enhanced test efficiency, but most methods are quite expensive and time consuming. The addition of CASA mostly covered the drawbacks of visual observation methods. CASA measured various sperm characteristics, such as straight linear velocity and angle of lateral head displacement, which are considered as major factors in controlling fertilization. Further advances in computer programs make it much easier and faster to evaluate various sperm parameters. This computer-based system evaluates most of the processes in vitro fertility control except the effect of cervical mucus. Considering the route of a spermicidal agent and active site, interaction of a spermicidal agent with/ within cervical mucus should be investigated by the cervical mucus penetration test. Even though the cervical mucus penetration test is complex and shows relatively high variance, the results of this technique are very important. Both HOS and flow cytometry enhanced the efficacy of measurement of sperm functionality. The percent motility of sperm upon exposure to N-9 measured by flow cytometry is lower than that observed by other methods. Even in different sample preparations, flow cytometry had the lowest percentage motility of sperm. The continued presence of cytoplasmic vesicles after sample preparation seems a major factor affecting use of this system. The motility of sperm upon exposure to spermicidal agents measured by the HOS method was similar to that of flow cytometry. Considering the complexity of flow cytometry, HOS has a great advantage over flow cytometry. The close correlation between fertilization rate and sperm functionality shown by HOS supports the usefulness of the HOS test. Due to the deficiencies inherent in each test, one test alone may not serve as a complete tool for evaluating spermicidal agents. In view of the heterogeneity of sperm velocity in human semen samples and difficulty of evaluating the motility characteristics of each sample, various combinations of in vitro spermicidal tests may serve as an efficient tool to screen and evaluate the spermicidal potency of prospective agents. A combination of computer-assisted method and cervical penetration method seems an ideal approach to evaluate spermicidal activity of prospective

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agents. The sperm count, motility, morphology, and sperm motion characteristics determined by CASA will represent objective parameters of the sperm, while percentage or rate of sperm penetration will represent that of sperm functionality. For further accuracy, either the HOS test or flow cytometry can be used as a complementary tool. This review provides an insight into the different aspects of sperm functionality on which each spermicidal agent exerts its activity. This review also provides a rationale for the best combination of in vitro spermicidal tests, with particular emphasis on simple and efficient strategies, that aim for complete fertility control. With the aid of such information, attempts can be made to understand the biochemical basis of sperm fertilizing ability and consequent fertility control. This can lead to the further development of a new generation of diagnostic techniques, designed to give objective and efficient data on the functional competence of human spermatozoa.

Acknowledgment The author wishes to express his appreciation to Dr. Yie W. Chien for his help and support in the preparation of this work.

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46.

47.

48. 49.

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