A FLAT CAPILLARY TUBE SYSTEM FOR ASSESSMENT OF SPERM MOVEMENT IN CERVICAL MUCUS*

Transcription

1 FERTILITY AND STERILITY Copyright 1978 The American Fertility Society Vol. 29, No. I, January 1978 Printed in U.S.A. A FLAT CAPILLARY TUBE SYSTEM FOR ASSESSMENT OF SPERM MOVEMENT IN CERVICAL MUCUS* ROSS N. MILLS, PH.D.t DAVID F. KATZ, PH.D. Department of Mechanical Engineering, University of California, Berkeley, California 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 swimming speed of human sperm does not differ in flat capillary tubes of200-flln and 400-flln depth. Assessment of spermatozoan movement within cervical mucus is of value in the clinical diagnosis of male and female infertility and in the fundamental study of the mechanisms of in vivo sperm transport. I, 2 In vivo tests focus upon mucus collected postcoitally, while in vitro tests introduce spermatozoa to the mucus externally. The former tests generally utilize preparations in which the mucus is placed upon a microscope slide and enclosed by a cover glass, while the latter tests employ either plane slides or circular capillary tubes containing mucus. Slide preparations are optically convenient but are difficult to standardize with regard to mucus depth and volume. The use of such preparations for in vitro tests involves the added problem of achieving a welldefined interface between the sperm suspension and the mucus without layering one medium over the other. Circular capillary tube methods permit reliable determination of the penetration distance of vanguard spermatozoa in in vitro tests, which can be achieved at low magnification, e.g., X Observations have also been reported Received June 27, 1977; revised August 31, 1977; accepted September 1, *Supported by Contract from the World Health Organization Task Force on the Regulation of Sperm Migration. tto whom reprint requests should be addressed. of the percentage motility and grade of progressive sperm motility observed in circular tubes. 6 7 Such tubes, however, even if submerged in immersion oil, impose significant optical limitations. The curvature of the tube wall distorts the image of spermatozoa in all but a small region within the tube cross-section. Consequently, the detailed shapes and movements of individual spermatozoa, including flagellar undulations and even the precise location of spermatozoa within the tube crosssection, cannot be accurately determined.s There is considerable scientific motivation for measuring such details in the study of sperm movement in cervical mucus.9 The microstructure of cervical mucus consists of a mesh of elongated, interconnected microfilaments When the whole mucus is stretched the microstructure deforms, and the unidirectionality of sperm swimming in stretched mucus apparently is mediated by such deformation. The extent of the deformation experienced locally by the mucus microstructure is due not just to t.he history of the stretching of the mucus, but also to the resistance offered by nearby solid surfaces. The standardized assessment of sperm movement within the mucus should therefore incorporate a fixed rate of mucus shearing, a fixed size of the chamber in which the mucus is contained, and a fixed plane or planes of focus within the cham- 43

2 44 MILLS AND KATZ January 1978 b er. I n thi' s regard, a.capillary tube possessing a flat, rectangular cross-section represents a significant improvement over a plane slide or a tube of drcular cross-section. Such benefits of a flat tube have been appreciated for some time,s but a practical system employing inexpensive, commercially available tubes and a simple, controllable means of loading the tubes with mucus has not previously been described. We have developed such a system, and report here on its construction and application and upon the results of preliminary studies designed to determine standardized procedures for its use in the assessment of sperm movement in cervical mucus. MATERIALS AND METHODS The Flat Capillary Tube System. The complete system consists of the following components (cf. Fig. 1): (1) commercially available flat capillary tubes (Vitro Dynamics, Rockaway, N. J.) made of Pyrex; (2) a loading manifold for drawing cervical mucus or other highly viscous media into a tube; (3) a 3-ml disposable syringe connected to the circular port of the loading manifold by 3 cm of polyethylene tubing (O.2-cm inner diameter); (4) a microscope slide containing a reservoir for sperm suspensions, to which the loaded tube is attached. We fabricate the manifolds routinely, using specially built brass molds. Figure 2 is an engineering drawing for mold construction. The molding medium is RTV (room temperature vulcanization) standard molding rubber (TAP Plastics, San Leandro, Calif.) A standard mold release is applied to the mold prior to filling with a 2: 1 (v:v) mixture of molding rubber and hardener. A curing time of 24 hours is sufficient to yield a manifold with firmness and elasticity suitable for sealing the flat capillary tube and suction tubing. The sperm reservoir can be made of plastic, as described by Kremer.3 Alternati~ely, it can consist of a ring of Vaseline, over which a cover glass is laid after the flat capillary tube is in place. This latter arrangement prevents any 3 cc syringe with 1/8" 00 polyethylene tubing attached oc1,l",,,, '"'''''''' ~ FIG. 1. Flat tube-loading system. A I r~=ii OUTER RING ( n THICK TUBING) INNER RING ( -fi THICK TUBING) TOP VIEW - ---=rl r --! I" I 11 SECTION i DIA HOLE FIG. 2. Engineering drawing of the mold for the tubeloading manifold. The slit is cut for the appropriate flat tube size. sperm suspension from running along the outside of the tube as a result of capillary action. The system is used for an in vitro test of sperm penetration of cervical mucus as follows: a small aliquot of mucus is expelled from the collection tube into a Petri dish. A flat capillary tube is inserted into its port of a manifold until it protrudes slightly into the viewing point. The polyethylene tubing, attached to the 3-ml disposable syringe, is then inserted into the opposing port. Each side of the viewing port is sealed by the thumb and forefinger of one hand, and the open end of the flat tube is submerged in the mucus aliquot. The tube is then slowly filled with mucus by applying back pressure via the syringe, which is held by the other hand. Periodic removal of the thumb and forefinger allows observation of the end of the flat tube to determine whether the tube has been filled. Once filled, the tube is slowly raised from the body of the mucus aliquot, and the mucus strand is cut with thin surgical scissors. The tube is then removed from the manifold, and the proximal (manifold) end is sealed by slowly forcing it into a block of Critoseal (Sherwood Medical Industries, Inc., St. Louis, Mo.) so that there is no air gap between the sealing material and the mucus and so that a small blob of mucus protrudes from the distal end of the tube. The tube is then attached to a microscope slide via a dot of Vaseline in such a manner that the distal end rests in a reservoir, which is then filled with sperm suspension. AA

3 Vol. 29, No. 1 CAPILLARY TUBE SYSTEM FOR ASSESSMENT OF SPERM MOVEMENT 45 Once loaded in this manner, the system is amenable to the measurements performed in contemporary circular tube systems. If the slide contains a ruler or transverse scribe marks, the penetration distance of vanguard spermatozoa is easily observed. The plane of focus is determined precisely by calibrating the focusing vernier for the objective lens of the microscope. Additionally, the transverse location of a spermatozoon with respect to the tube center line is easily determined with the aid of an eyepiece micrometer, stage vernier, or a mark on the slide. Thus the 3- dimensional location of a field of view, or an individual spermatozoon, can be determined with precision. This system may also be used to study sperm movement in media initially containing the gametes, e.g., postcoital cervical mucus. The tubeloading procedure is identical with that described previously. One end is sealed with Critoseal and the other end is covered with Vaseline, taking care not to pressurize the tube and not to entrap any air bubbles. Thus loaded, the tube is then attached to a slide for observation. Preliminary Experiments. Sperm movement was initially observed in tubes with cross-sectional depths of 50 to 400 /Lm. All such tubes possessed excellent optical resolution throughout the cross-section. Tubes of 200-/Lm and 400-/Lm depth and 5-cm length were chosen as suitable for standardized studies of sperm movement in various media, and preliminary experiments were conducted to determine whether there were differences in swimming speed at these two suspension depths. Three samples of human cervical mucus from three different. donors of unproven fertility were used. They were assessed as normal midcycle mucus according to the criteria of spinnbarkeit, ferning, and cellularity, and the advice ~fthe collecting physician. All mucus samples were collected in no. 14 polyethylene tubing by gentle aspiration. ll Each collection tube was sealed and stored at 4 C immediately after collection and was utilized for study within the subsequent 3 days. Spermatozoa from four semen samples from three different donors of unproven fertility were used. They were obtained by masturbation from healthy adult males and were utilized,within 30 minutes after reliquefaction. All semen samples contained at least 50 x 10 6 spermatozoa/ml, a percentage motility visually assessed at 50% or greater, and a grade of progressive motility visually ranked (on a scale of 0 to 4) at 3 or greater. : Flat '~ Standard cover : ~ ; slip filled with mucus ' ' ~,;: /;--Vaseline ring ~/( \\ -prj/ L~~-~::::=~~~rs.m" ".",,", Standard microscopic slide FIG. 3. Slide configuration for a side-by-side experiment. All experiments were performed at 37 ± 0.5 C. Samples, slides, tubes, and cover slips were preheated and maintained in a small incubator when not placed under the microscope. The microscope stage and contents were maintained at body temperature by means of a forced hot-air curtain. All preparations were allowed 5 minutes on the stage to ensure temperature equilibration before observation. In experiments 1 to 3, each mucus sample was paired with one semen sample without crosstesting. The flat tubes were loaded with mucus by using the manifolds and technique described in the previous section. The rates at which the mucus was drawn into the tubes were approximately 0.25 cm/second and 0.50 cm/second for the 200-/Lm and 400-/Lm deep tubes, respectively. A semen reservoir was created on a microscope slide by injecting a ring of Vaseline through a syringe. The tubes were then placed onto the slide so that the open end rested within the ring, and the ring was closed by injecting Vaseline over the tubes. Semen was pipetted into the ring, which was then sealed with a cover slip to form a closed reservoir (Fig. 3). Spermatozoa readily entered the tubes and swam along trajectories that were primarily undirectional and parallel to the axis of the tubes. Tubes of200-/lm and 400-/Lm depth were placed side by side on a microscope slide for each experiment. Measurements commenced after at least 15 minutes of semen-mucus contact. The swimming speeds of individual spermatozoa were visually determined with the aid of a calibrated eyepiece micrometer and a stopwatch. The magnification was x200, and dry phase-contrast optics were employed. Motile spermatozoa were randomly selected for measurement and allowed to swim at least 500 /Lm before an effective path length was visually determined. Since the swimming trajectories were relatively straight, such measurements were accurate to within approxi-

4 46 MILLS AND KA'IZ mately ± 10%. Spermatozoa whose swimming trajectories changed direction appreciably during the measurement interval are not included in the results presented here. The measurement sequence consisted of assessing five motile spermatozoa in one tube and then assessing the motility of five spermatozoa in the other tube, alternating until at least 25 motile spermatozoa per tube had been assessed. Experiment 4 utilized a second semen sample from the same donor as in experiment 3. This sample contained 80 x 10 6 spermatozoa/ml, a percentage motility of 80%, and a grade of progressive motility ranked at 4. The swimming trajectories of individual spermatozoa were relatively straight. An aliquot of the semen samples was diluted 3:1 with its own plasma. Flat capillary tubes of 200-lLm and 400-lLm depth were then filled, sealed, and placed under the microscope for observation. The tubes were filled by capillary action. Owing to the straightness of the swimming trajectories, it was possible to measure the swimming speeds of individual spermatozoa visually by using the eyepiece micrometer. Such measurements followed the same procedure utilized in experiments 1 to 3. Statistical analysis of the data employed Student's t-test to compare mean values of spermatozoan swimming speed between tubes in each experiment. 12 RESULTS The results of the four preliminary experiments are summarized in Table 1. Mean values of spermatozoan swimming speed did not differ significantly between tubes in any of the experiments (}' <0.05}. There was a slight, although insignificant, tendency for higher values in the larger tubes containing cervical mucus. DISCUSSION A standardized sperm-mucus in vitro penetration test procedure should take into account the TABLE 1. Spermatozoan Swimming Speeds in Human Cervical Mucus and Human Semen Tube depth Swimming speeds" for N spermatozoa in Human cervical mucus p.mlsec Human semen 200/Lm 45.3 ± ± ± ± /Lm 50.9 ± ± ± ± 0.99 "Values are means ± standard error of the mean. January 1978 following factors: the time of the test after ejaculation, the time of the test after mucus collection, the temperature of mucus storage between collection and the test, the size and shape of the container in which the mucus is stored, the length and internal dimensions of the capillary tube used in the test, the shearing rate of the mucus during tube loading, the temperature at which the test is performed, and the duration of the test. The experimental system for microscopic assessment of sperm movement should be easy to use, employ readily obtainable and replaceable components, possess good optical resolution, and be amenable to small sample sizes. When cervical mucus is a test medium, the system should be capable of subjecting the mucus to a well-defined, controllable, and repeatable rate of shear deformation. The system described herein possesses these characteristics, the principal new features being the excellent optical qualities associated with a flat capillary tube and the mechanical means of loading such a tube with cervical mucus at a controllable shear rate. For purposes of standardization, this rate can be defined as the speed at which the mucus column is advanced into a tube divided by the depth of the tube. Minimization of the rate of mucus shearing and of the manipulation of the mucus in general would seem desirable. The extent to which these factors influence the results of an in vitro sperm-mucus penetration test is unclear at this time and is the subject of a study now in progress. The small amounts of human cervical mucus commonly collectable necessitate a capillary tube system with minimal internal volume. In this regard there is a trade-off between tube crosssectional area and length. The flat capillary tubes of 5-cm length and internal dimensions of 200 ILm x 2 mm and 400 ILm x 4 mm have volumes of 20 ILl and 80 ILl, respectively. The use of shorter tubes limits the duration of the existence of a sperm vanguard and therefore measurement ofits penetration depth. For example, a vanguard migrating unidirectionally at a constant speed of 33.3 llm/second (2 mm/minute), which is comparable to that reported for circular tube systems,3-5 traverses a 5-cm tube in 25 minutes, thereby precluding measurements of its penetration depth over extended periods of time. It should be appreciated that the vanguard comprises the fastest swimming spermatozoa, although some of these may be recent arrivals. Consequently, the rate of vanguard penetration per unit time does not necessarily represent the maximal swimming

5 Vol. 29, No.1 CAPILLARY TUBE SYSTEM FOR ASSESSMENT OF SPERM MOVEMENT 47 speed in the suspension. In contrast, measurement of individual swimming speeds via the technique described here, which does not require the presence of a sperm vanguard, provides a more comprehensive description of within-sample variability. In this regard, the flat capillary tubes are easily and accurately cut to shorter lengths with a scribe. It should be emphasized, however, that such a quantitative visual method is amenable only to spermatozoa whose swimming trajectories are relatively straight. This is usually the case in normal midcycle cervical mucus, but would not apply in general to semen. The clinical significance of measuring vanguard penetration as opposed to the distribution of swimming speeds awaits further study. The results presented here suggest that the in vitro spermatozoan swimming speed in cervical mucus does not differ significantly in tubes of 200-lLm and 400-lLm depth. However, such results are preliminary and have been included primarily to demonstrate the use of the new flat tube system. In situations where the sperm trajectories are not straight, determination of the swimming speed requires other methods. 13 Acknowledgments. We appreciate the assistance of Dr. G. Buchanan, Department of Reproductive Endocrinology, Naval Regional Medical Center, Oakland, Calif.; Dr. R. H. Glass, Department of Obstetrics and Gynecology, University of California, San Francisco, Calif.; and Mr. W. Flom. REFERENCES 1. Davajan V, Nakamura RM, Kharma K: Spermatozoan transport in cervical mucus. Obstet Gynecol Survey 25:1, Elstein M, Moghissi KS, Borth R (Editors): Cervical Mucus in Human Reproduction. Copenhagen, Scriptor, Kremer J: A simple sperm penetration test. Int J Fertil 10:209, Carlborg L: Determination of sperm migration rate in small samples of cervical mucus. Endocrinology 62:732, Ulstein M: Evaluation of a capillary tube sperm penetration method for fertility investigations. Acta Obstet Gynecol Scand 51:287, Kesseru E, Larranaga A: In vitro sperm migration in the human cervical mucus with different contraceptive methods. Contraception 3:195, Ulstein M, Fjallbrant B: Interrelation of different parameters of cervical mucus penetration of spermatozoa. Acta Obstet Gynecol Scand 52:295, Kremer J, Kroeks MV AM: Modification of the in vitro spermatozoal penetration test by means of the sperm penetration meter. Acta Eur Fertil 6:377, Overstreet JW, Katz DF: Sperm transport and selection in the female genital tract. In Development in Mammals, Vol 2, Edited by MH Johnson. Amsterdam, Elsevier/ North Holland, 1977, p Blandau RJ, Moghissi KS (Editors): The Biology of the Cervix. Chicago, University of Chicago Press, Davajan V, Kunitake GM: Fractional in vivo and in vitro examination of postcoital cervical mucus in the human. Fertil Steril 20:197, Snedecor GW, Cockran WG: Statistical Methods, Sixth Edition. Ames Iowa, Iowa State University Press, Katz DF, Dott HM: Methods of measuring swimming speed of spermatozoa. J Reprod Fertil 45:263, 1975