New Technic for Sperm-Mucus Penetration Tests Using a Hemocytometer. H. R. Guard, M.D. *

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1 New Technic for Sperm-Mucus Penetration Tests Using a Hemocytometer H. R. Guard, M.D. * LIE CERVICAL MU~US and its penetration by spermatozoa is the first and the most important barrier in the process of fertilization. It has been shown that cervical mucus varies in its consistency and chemical composition at different times in a menstrual cycle. At the time of ovulation the consistency is thin and the composition favorable for immediate penetration. At other times, the mucus is so viscous that it forms a plug in the cervical canal which is impenetrable by spermatozoa. The subsequent mode of travel of spermatozoa in the reproductive tract is not certain, and that cannot be studied as easily in vitro as the process of sperm penetration of mucus. Since this barrier is an important factor in the study of infertility problems, it is necessary to have a method that will give quantitative information in relation to the variations in receptive quality of the cervical mucus and sperm penetrability. Kurzrok and Miller 1 have described a method for studying the invasion of human cervical mucus by spermatozoa. In their technic a drop of cervical mucus is placed on a slide and a drop of seminal fluid placed near it, without mixing the two. A coverslip is then carefully laid over the two fluid pools and the edge of the mucus is observed microscopically for penetration by active spermatozoa. This method has several disadvantages which make the results From the Worcester Foundation for Experimental Biology, Shrewsbury, Mass. This work was aided by a research grant from the Population Council, Inc. The Rockefeller Institute, New York 21, N. Y. The author is grateful to Dr. Gregory Pincus of the Worcester Foundation for Experimental Biology, Shrewsbury, Mass., and Dr. John Rock of the Rock Reproductive Study Center, Brookline, Mass., for providing facilities for research. o Present address: 805 Vincent Road, Bombay 14, India. 392

2 ., Vol. 11, No.4, 1960 SPERM PENETRATION TEST 393 of observation not comparable from one test to another. The mucus and the seminal fluid sometimes mix and produce an artefact in which the spermatozoa seem to show penetration at the edges, whereas actually they have been splashed over by the lowering of the coverslip. In the Kurzrok-Miller test, the space between the slide and the overlying coverslip depends on the viscosity of the cervical mucus. If the mucus is thin and watery the coverslip lies close to the slide and there is only a capillary space. If the mucus is thick, the weight of the coverslip is not sufficient to Hatten it. Therefore the edge of the mucus has a thickness depending upon its consistency and the depth between the slide and the coverslip. The result is that areas of the mucus edge available for spermatozoal invasion differ from one preparation to another. The numbers of spermatozoa invading and penetrating therefore depend on what may be called the "mucus front." This is the actual surface area of mucus available for contact with seminal fluid. It must be remembered that, in vivo, the cervical mucus front is not merely a capillary space. Depending on the diameter of the external os of the cervix, the mucus front offers a variable area which is quite large, and innumerable spermatozoa can penetrate it almost immediately on contact. The mucus is not very homogeneous in its viscosity; hence the wide area at cervical os offers greater chances for spermatozoa to choose suitable sites for penetration. This is very easily seen in the in vitro tests when spermatozoa are seen conglomerating at particular sites along the edge of a mucus specimen. The method of Kurzrok and Miller therefore does not give consistent results because the area of the mucus front is not constant. The new technic described here strives to attain uniform conditions for all tests, so that the results may be comparable. METHOD The Levy hemocytometer* is used, with a little redesigning of the coverslip. The standard coverslip supplied with the Levy chamber is divided into two parts by a straight scratch with a diamond pencil to give a piece measuring 12 X 20 mm. The modified coverslip covers only one of the two ruled areas on the Levy hemocytometer, in such a manner that the moat dividing the two rulings is not covered, as shown in Fig. lao The cervical mucus is collected in a pipet with a rubber suction bulb. The coverslip is placed on one of the ruled areas as shown in Fig. la, and the " Manufactured by A. S. Aloe Co., St. Louis, Mo.

3 GUARD Fertility & Sterility a ~ ~ Fig. 1. (a) top and (b) side view of Levy double Neubauer hemocytometer. Number 1 represents special coverslip which leaves the central moat open; 2, the central moat from where cervical mucus is fed into the chamber; 3, cervical mucus; 4, zone of contact, the "mucus front" of 0.10 mm.; and 5, pool of seminal fluid. Only the Levy hemocytometer has a slope, as shown, which can form a pool. mucus is fed into the counting chamber from the side of the central moat which divides the two rulings. If the mucus is obtained during ovulation time, it is thin and clear and flows very easily into the chamber by capillary attraction. It is allowed to flow in until it covers the entire ruled area. The flow of mucus will automatically stop at the edge of the slope, as shown in Fig. lb, due to lack of capillary attraction. The chamber is then examined under the low power of the microscope to see the contents of the mucus, and its edge is kept in focus in the center of the field. While the chamber is still on the stage of the microscope, the seminal fluid is fed by a pipet from the opposite side, as shown in Fig. lb. It fills up the sloping area and forms a reservoir, one side of which comes into contact with the mucus edge which now has a uniform exposure front of 0.10 mm. The Levy counting chamber is specially recommended here because other chambers do not have a slope to form a reservoir of seminal fluid which can allow large numbers of spermatozoa to invade the mucus edge. As soon as the mucus and the seminal fluid come into contact, the spermatozoa start migrating. Normal spermatozoa with active progressive motility penetrate the mucus edge rapidly and travel almost straight toward the ruled area. The number of living spermatozoa lying on 1 sq. mm. is counted

4 Vol. II, No.4, 1960 SPERM PENETRATION TEST 395 after intervals of 5 and 15 minutes. The areas representing 1 sq. mm. each are the large squares in the four corners of the entire ruling used ordinarily for counting leukocytes. Since the depth of the counting chamber is 0.10 mm., the total number of spermatozoa per cubic millimeter can be calculated as follows: Total number of spermatozoa in 2 sq. mm. areas 2 X 10 = Number of spermatozoa per cubic millimeter Normally, within 5 minutes a fairly large number of spermatozoa migrate to the ruled area, and after 15 minutes the number is often too large for convenience in counting because of active motility. However, in oligospermic fluids an interval of 15 minutes is often necessary for the few spermatozoa to approach the edge of the mucus and penetrate and be available on the ruled area for counting. If no spermatozoa penetrate within 15 minutes it is no use observing any further because after that time the two fluids tend to mix gradually because of the action of mucolytic enzymes present in the seminal fluid. It is advisable to keep the hemocytometer in a moist chamber if the test is prolonged for 15 minutes. A convenient moist chamber is a petri dish of 100cm. diameter with a fitted filter-paper disk moistened with water. RESULTS The technic was utilized in determining spermatozoal migration into different physiologic media and various human body fluids. In order that the results shown in Table 1 may be comparable, tests 1 to 10 were carried out with the same sample of a normal seminal fluid and at the same time to avoid variations in spermatozoal counts and differences in active motility. In lowviscosity media, such as normal saline and lachrymal fluid, which did not contain any mucin, it was seen that a slight mixing at the line of contact did occur. The sperm migration could still be studied. In media such as watery clear nasal mucus and cervical mucus, the edge was sharply demarcated and invasion could be studied very well. Thick, viscid cervical mucus plugs were rather difficult to handle since they could not be made to flow into the counting chamber. However, such a specimen could be placed on the ruled area directly and the coverslip lowered until it rested on the platforms to give the constant depth of 0.10 mm. The approximate quantitation of results that can be obtained with this

5 396 GUARD Fertility & Sterility TABLE 1. Comparison of Sperm Penetration of Cervical Mucus and Other Physiologic Media Total No. spermatozoa per cu. mm. after exposure of Type of medium exposed to sperm migrationa 5 min. 15 min. 1. Nonnal saline Ringer-Locke's fluid Ringer-Locke's fluid deficient in calcium Lachrymal fluid Salivary mucus Nasal mucus, obtained after irritation with a swab Nasal mucus, catarrhal Ovulatory cervical mucus Ovulatory cervical mucus Ovulatory cervical mucus frozen for 1 month at 0 C. and thawed Ovulatory mucus of infertile wife exposed to 0 0 husband's spennatozoa in 3 successive cycles Same patient's mucus exposed to two different specimens of donor spennatozoa Same husband's spennatozoa exposed to two different specimens of donor mucus Cervical mucus from 5 patients on long-tenn therapy with Enovid 0 0 a To obtain comparable results, the same sample of seminal fluid was utilized for tests from 1 to 10 and test no. 14., method of testing shows the effect of viscosity and differences in chemical composition on sperm migration. Tests 11 and 12 were done on a case of infertility for 6 years (Mrs. J. S. from the Rock Reproductive Study Center, Brookline, Mass.). The cervical plucus at ovulation time was obtained on three occasions in three successive cycles from this patient and exposed to her husband's seminal fluid; no sperm penetration was noticed. The same patient's cervical mucus was, at the same time, also exposed to two normal donor seminal fluids on each occasion and positive sperm penetration was noted. This illustrates the type of approximate quantitation of results that can be recorded in cases of infertility. Test 14 was done on 5 patients on a long-term therapy with Enovid (Searle) for suppression of ovulation. The cervical mucus obtained at ex-

6 Vol. 11, No.4, 1960 SPERM PENETRATION TEST 397 pected ovulation time in each case was exposed to a nonnal seminal fluid and no spenn penetration was noted. The cervical mucus in all these cases was thick and viscid. COMMENTS A test of mucus penetration by sperm properly carried out under standardized conditions as described above is much more infonnative than the Kurzrok-Miller Test. It can evaluate (1) the "receptivity" of cervical mucus and (2) the capacity of spennatozoa to penetrate the mucus, which may be termed as the "penetrative capacity." The receptive capacity of cervical mucus depends upon various factors, among which the most important are cyclical variations in its consistency and chemical composition. In the case of infertility cited above, the mucus was nonreceptive to the husband's spermatozoa, whereas spennatozoa from other donors could easily penetrate the same mucus. The "penetrative capacity" of spennatozoa has not received much attention. In fact, what is given importance as active motility is merely a vibratile action of a cellular flagellum. Whether a particular spennatozoon in spite of its active motility can penetrate the cervical mucus must be demonstrated by a test of mucus penetration by spenn. In the infertility case discussed above, it was seen that the husband's spennatozoa could not penetrate his wife's cervical mucus. This penetrative inability was proved consistently in three successive cycles. To prove whether his spennatozoa did lack the nonnal "penetrative capacity," they were exposed to two other specimens of cervical mucus obtained from other donors. In both cases a positive spenn penetration was obtained. This showed that his spennatozoa did contain what is believed to be a mucolytic enzyme which is necessary for penetration of cervical mucus. In a routine seminal-fluid appraisal, the count on total, living, and dead spennatozoa seems to be of much less infonnative value if the most actively progressive spennatozoa do not possess any penetrative capacity. This can only be tested in sperm-mucus contact. In all cases of infertility, therefore, a routine procedure as suggested below could be adopted. A Huhner's postcoital test should be done 2 hours after intercourse. If spenn penetration of mucus in vivo is not detected, then an in vitro test as described by the author should be carried out. If there is no sperm penetration of mucus in vitro, it is necessary to do what may be tenned a "cervical mucus receptivity test" of the wife. This is done by an in

7 398 GUARD Fertility & Sterility vitro exposure of the cervical mucus to spermatozoa other than those of her husband. Similarly a "penetrative capacity test" of the husband's spermatozoa would have to be done by an in vitro exposure to cervical mucus from at least two donors other than the wife. It may sound difficult to obtain "mucus donors" and "seminal fluid donors" for such cross-compatibility tests, but in any regular infertility clinic there are usually many couples whose specimens could be utilized for such in vitro tests without their knowledge and without inconvenience to them. This method of investigation is likely to reveal several cases such as the one described above. The causes for such incompatibility in many instances have not been understood. J SUMMARY A new technique utilizing a modified hemocytometer chamber has been described for sperm-mucus penetration tests. The number of spermatozoa penetrating cervical mucus can be counted and would quantitatively indicate the receptivity of the mucus as well as the penetrative capacity of spermatozoa under test. A new procedure is suggested for investigating cases of infertility in which Huhner's postcoital test shows no mucus penetration by sperm. REFERENCE ~ 1. KURZROK, R., and MILLER, E. G. Biochemical studies of human semen and its relation to mucus of the cervix uteri. Am. /. Obat. & Gynec. 15:56, 1928.