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Evaluation of male infertility with an in vitro cervical mucus penetration test*
Vol. 36, No.2, August 1981 Printed in U.8A.
FERTILITY AND STERILITY Copyright 0 1981 The American Fertility Society
EVALUATION OF MALE INFERTILITY WITH AN IN VITRO CERVICAL MUCUS PENETRATION TEST*
NANCY J. ALEXANDER, PH.D. Department of Reproductive Physiology, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, and Departments of Anatomy, Obstetrics-Gynecology, and Urology, University of Oregon Health Sciences Center, Portland, Oregon 972{)1
The use of an in vitro bovine cervical mucus penetration test (CMPT) provides unique data for fertility assessment. Flat capillary tubes were filled, kept frozen until use, exposed to a sub-sample of semen for 90 minutes, and microscopically evaluated. Adequate penetration was based on results of semen specimens from donors used for artificial insemination. Of 161 patients being evaluated for infertility, 37% had semen that penetrated the CMPT inadequately. Of the patients with inadequate penetration, 70% had sperm densities of greater than 21 x lO6/ ml, and 62% had over 50% motility. Thus neither evaluation of count nor motility provided the same information as the CMPT. Human spermatozoa had a similar swimming pattern in human and bovine mucus. Spermatozoa that exhibited poor in vitro penetration of human mucus also failed to penetrate bovine mucus. Comparison of the CMPT with postcoital tests of 42 couples revealed a good correlation. When the incidence of pregnancy for individuals with adequate and inadequate penetration was compared, more individuals with a good CMPT caused a pregnancy. It appears that the CMPT, an easy office test, allows greater discrimination of sperm function than semen analysis alone and is a useful tool for the diagnosis and management of infertility. Fertil Steril36:201, 1981
lack of uniformity in how this test is performed and wide variation in the interpretation of the results, partially because of the lack of specific criteria used for quantification. 2 In vitro cervical mucus penetration is assessed by either a slide or a capillary tube assay. In the first method drops of semen and cervical mucus are placed next to each other on a microscope slide, and sperm penetration of the mucus is subjectively determined. 3 , 4 Microscopic evaluation reveals the development of phalanges with a single sperm seeming to lead the cervical mucus penetration. The size and shape of the semen-mucus interface and local mucus shearing caused by coverslipping limit the quantitative application of this test and its reproducibility.5 With capillary tubes one can evaluate cervical mucus penetration more objectively. When tests of this kind are conducted, mucus is drawn into a capillary tube, which is then sealed at one end. The unsealed end is placed in a small amount of semen.
An essential part of the investigation of an infertile couple is evaluation of the interaction between spermatozoa and cervical mucus. In fact, measurement of sperm penetration and survival in cervical mucus is considered to be one of the most important tests of human sperm function. 1 Sperm penetration is usually assessed in (1) cervical mucus collected after coitus or (2) mucus collected and tested in vitro. In the most widely used test of sperm penetration, the postcoital test (peT), or Sims-Huhner test, after the couple have intercourse, a sample of mucus is collected and examined 2 to 10 hours later. However, there is a Received February 25,1981; revised and accepted April 10, 1981. *Publication No. 1134 of the Oregon Regional Primate Research Center, supported by NIH grant RR-00163 and by the Syva Company. Reprint requests: Nancy J. Alexander, Ph.D., Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006.
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202
August 1981
ALEXANDER
After a specified time interval the distance traveled by the spermatozoa is recorded, the density of spermatozoa in various segments of the tube is determined, and motility is evaluated. 6 , 7 Using capillary tubes, one can estimate the swimming speed of the fastest spermatozoa and qualitatively evaluate the flagellar beat. In the past, the use of round tubes made the optical resolution difficult, but recently flat tubes have been used for observations of sperm in cervical mucus. 8 The results of cervical mucus penetration tests have been closely correlated with fertility9, 10; the depth of penetration is correlated more closely than any other variable. 11 Such tests have a unique diagnostic value in the determination of male infertility because they allow evaluation of the interaction between spermatozoa and cervical mucus. One drawback, however, is that they do not differentiate between the separate functional characteristics of the spermatozoa and mucus. Use of an alternative standardized source ofmucus avoids this problem and allows more thorough examination of the spermatozoa as the only variable. The midcycle mucus of cows is similar to that of women. Laser light-scattering spectroscopy has revealed that mucus from both species consists of entangled, randomly coiled macromolecules. Furthermore, the viscoelastic properties of the two are remarkably similar. 12 Human spermatozoa rapidly penetrate cow mucus in a concerted manner and exhibit changes in their pattern of movement identical to those displayed by human spermatozoa penetrating human midcycle cervical mucus. 13 Furthermore, up to 100 ml of mucus (enough for 1000 tests) can be obtained from one cow in a single collection. Thus, bovine mucus may allow greater standardization of in vitro test conditions and also enable investigators to evaluate the interaction of spermatozoa and mucus in the absence of variables associated with the wife's mucus. Comparison of the data from such an in vitro test with those from postcoital tests could provide ~dditional cogent information for infertility evaluation. In this article we present evidence that an in vitro bovine cervical mucus penetration assay is a useful adjunct in the evaluation of infertility.
or. Care was taken to avoid fecal contamination, but no attempt was made to avoid contamination with vaginal secretions. Assay Method. Flat capillary tubes were filled with cervical mucus, sealed, and stored at - 20° C until used. Each capillary tube was thawed at room temperature for several minutes, scored with a diamond pencil several millimeters above the mucus meniscus, and broken at the score. The cut end was inserted in 150 j.LI of semen and incubated at room temperature for 90 minutes. The tubes were removed from the semen reservoir, wiped clean of any residual specimen, and placed on a glass slide on a microscope stage. The distance (in millimeters) covered by the vanguard spermatozoa was noted. A 250 x to 400 x magnification was used; phase optics are preferred. If it was obvious that an air bubble across the tube had blocked sperm penetration, that value was discarded. Each assay was run in duplicate. Semen. Semen samples were collected by masturbation after sexual abstinence of at least 48 hours and evaluated for volume, count per milliliter, percentage of motility based on a count of 200, and activity (determined on a scale of 0 [no movement] to 4 [active forward progression] after a 30-minute liquefaction period. Sperm with no movement were not used for CMPT). Semen specimens from fertile men (donors from the artificial insemination program) and patients attending an infertility laboratory were evaluated. Comparison with Human Cervical Mucus. Sperm-free cervical mucus was collected from several different women during the 3-day interval before the expected onset of ovulation. The mucus samples were used fresh or within 5 days of freezing. Bovine cervical mucus samples were tested concomitantly with human samples. For morphologic evaluation, samples of mucus were dried on coverslips, coated with gold palladium in a Technics sputter coater, and viewed with an AMR-lOOO scanning electron microscope. Statistical Evaluation. Significance was determined with Student's t-test and X2 analysis. Data are presented as means ± standard errors. RESULTS
Development of Test Criteria MATERIALS AND METHODS
Collection of Bovine Mucus. Mucus was collected from estrous cows by means of rectal palpation of the cervix and extrusion of mucus to the exteri-
Initially, round hematocrit capillary tubes were used14; but because of the poor optical clarity, in later testing we used flat tubes. In compar.ative and simultaneously run tests, cervical mu-
Vol. 36, No.2
203
IN VITRO CERVICAL MUCUS PENETRATION TEST
TABLE 1. In Vitro Bovine Cervical Mucus Penetration Values for Fertile Men and Patients a Group
Fertile men Patients
Motility
n
Count x 11f!Imi
%
1-4
%
n
%
56 161
144 ± 12.8 73.8 ± 4.5
67.9 ± 1.0 60.9 ± 0.9
3.2 2.8
80.4 ± 0.7 78.8 ± 0.6
56 101
100 63
Progression grade
Normal forms
Good cervical mucus penetration
aValues for count, motility, and normal forms are expressed as the mean ± standard error. Values from patients and fertile men are all significantly different (P < 0.05).
cus penetration was more extensive in the flat than in the round tubes. When semen specimens from 56 fertile donors were tested, penetration within 90 minutes at room temperature was 7.6 ± 1.0 mm further in the flat capillary tubes. No fertile donor had spermatozoa that traveled less than 15 mm in round tubes or less than 21 mm in flat tubes. Migration appeared to be a linear function of time times temperature. All further comparisons were based on the results of these known fertile men. In order to allow room for error, based on the results found in fertile men, it was arbitrarily decided that samples with spermatozoa that did not penetrate more than 15 mm within 90 minutes at room temperature were inadequate.
Cervical Mucus Evaluation Many batches of bovine cervical mucus were used in these tests. There were batch-to-batch variations that will be thoroughly described in a subsequent communication, but any clear sample allowed human sperm penetration. No overt bacterial contamination was observed in any of the bovine cervical mucus samples.
Evaluation of Patient Semen Samples Semen from 161 patients referred to the infertility laboratory was tested by means of the bovine cervical mucus penetration test (CMPT). For many of the patients, semen analysis and the in vitro cervical mucus penetration tests were the first steps in their fertility evaluation. The results indicate that 37% of these men had semen that did not penetrate ;>- 15 mm. The mean sperm density (expressed as count x 106/m D for the en-
tire infertile group was 73.8 ± 4.5; the mean percentage of motility was 60.9 ± 0.9 (Table 1). The differences between the patient and the donor values were statistically significant. An important question is whether the CMPT provides additional information beyond that obtained in a semen analysis. The mean sperm density for patients whose sperm exhibited inadequate penetration was 53.8 ± 6.8 x 106/m l. Although this count is lower than that of either the fertile or the infertile group with adequate penetration, it would, by most standards, be considered within the normal range (Table 2). Sperm counts are compared with cervical mucus penetration in Table 3. The range of sperm densities was from 1 to 313 x 106/m l; 70% of the patients had counts over 21 x 106/m l. Thus the majority of the patients had what is considered to be normal sperm counts, but nonetheless, many of the spermatozoa had a reduced ability to penetrate cervical mucus. Evaluation ofthe percentage of motile sperm (Table 4) provided similar information. The mean percentage of motility was 51.9 ± 1.9, and the range was 9% to 80%. Motility of over 50% was found in 62% of the patients. Some of the individuals had normally motile sperm; yet their spermatozoa could not penetrate bovine cervical mucus.
Comparison with Postcoital Test Results Postcoital test results were obtained from 12 gynecologists practicing in Oregon. In most cases, if the first postcoital test was poor, additional tests were performed, and as many as eight postcoital tests were done for certain individuals. The mean number of postcoital tests was two. All tests were done within 8 hours after coitus. Of the 42
TABLE 2. Semen Analysis Values from Patients with Adequate and Inadequate Bovine Cervical Mucus Penetrationa Patients
Inadequate (n = 60) Adequate (n = 101)
Motility
Count
Form
Grade of activity
Mucus penetration
x 11f!Imi
%
% normal
1-4
mm
53.8 ± 6.8 85.7 ± 6.0
51.9 ± 1.9 66.3 ± 0.9
76.2 ± 1.0 80.4 ± 0.7
2.3 ± 0.08 3.1 ± 0.05
11.2 ± 0.5 23.2 ± 0.6
"Values are expressed as the mean ± standard error.
ALEXANDER
204
TABLE 3. Sperm Counts in Patients with Adequate and Inadequate Bovine Cervical Mucus Penetration Percentage (and number) of patients in each sperm count range Patients
Inadequate (n = 60) Adequate (n = 101) All patients (n = 161)
0--20·
21-40·
30 (18)
20 (12)
38 (23)
11.6 (7)
9.9 (10)
13.8 (14)
46.5 (47)
29.7 (30)
13 (28)
16.1 (26)
43.4 (70)
22.9 (37)
41-100-
101-300·
"'Counts expressed as sperm x 106 /ml.
couples evaluated, 81% had good postcoital test results, and 74% of the men had a good in vitro cervical mucus penetration test. We found no significant differences in sperm count per milliliter, percentage of motility, or forward progression when semen analyses of husbands with good postcoital tests were compared with analyses of husbands with a good in vitro bovine cervical mucus test (Table 5). Those 7 couples (17%) with a poor postcoital test but an adequate in vitro penetration had a mean sperm density of 86.8 ± 29.3 and a mean percentage of motility of 59.2 ± 1.8, values not very different from those listed in Table 5. All but four men from couples with good postcoital results had positive CMPT results. These data indicate a good correlation between the in vitro cervical mucus penetration assay and the postcoital test.
Comparison of Human and Bovine Cervical Mucus Ferning patterns of bovine and human samples were very similar (Figs. 1 and 2). When human and bovine mucus were run in parallel in the in vitro cervical mucus test, comparable results were observed (Table 6). Poor penetration of human mucus may indicate inadequacies in the women's mucus and provide no information regarding the husband's spermatozoa, a finding suggested in patients 8 to 13, who had spermatozoa that adequately penetrated bovine, but not human, mucus. Although the mean sperm density of patients 1 to 7 (195.3 ± 42.4) was signifi-
August 1981 cantly higher than patients 8 to 13 (84.2 ± 34.3), there were no significant differences between the groups when motility (60.6 ± 5.8 versus 66.5 ± 4.2), progression (3.1 ± 0.2 versus 2.8 ± 0.1), normal morphology (78.0 ± 2.0 versus 75.2 ± 3.3), or bovine cervical mucus penetration were compared (28.4 ± 4.1 versus 25.2 ± 2.2). Some semen samples (patients 14 to 20) exhibited inadequate mucus penetration in both bovine and human mucus. Although the mean count (57.28 ± 15.8) was not, the percentage of motility (38 ± 7.9) and progression (2.0 ± 0.3) were significantly different from those values for patients 1 to 13, who had adequate penetration of bovine cervical mucus. Good mucus penetration is dependent on the number of motile and progressive spermatozoa. Of paramount importance is the fact that no semen samples were found to exhibit only penetration of human and not bovine mucus. Such a finding supports the concept that bovine and human mucus are similar and that penetration of bovine mucus is a good indication of sperm function.
Correlation of the CMPT with Subsequent Pregnancy The incidence of pregnancy in patients who had good or poor CMPT in the 6 months following the test was compared. Couples were eliminated from the data base when a female problem that would obviously prevent conception (bilaterally blocked tubes or anovulation) was found. Of 21 patient couples in which the husband had good penetration, 9 could be evaluated for fertility, since no obvious female factor was observed. Of the group, 6 of the wives had conceived. There was no significant difference in the sperm density or percentage of motility of the specimens from those husbands whose wives did and did not conceive. Of 18 couples in which the husband had poor cervical mucus penetration, 4 wives conceived in the subsequent 6 months. Again, there were no significant differences in the sperm density or percentage of motility of sperm among men who did and did not impregnate their wives. When the
TABLE 4. Percentages of Motile Sperm in Patients with Adequate and Inadequate Bovine Cervical Mucus Penetration Percentage (and number) of patients in each sperm motility catsgory
Patients 0--20·
Inadequate (n = 60) Adequate (n = 101) All patients (n = 161) "'Percentage of motile sperm.
5 (3)
0(0) 1.8 (3)
21-40·
41-50·
51-60"
61-70"
71+"
16.6 (10) 0.9 (1) 6.8 (11)
16.6 (10) 2.9 (3) 8 (13)
41.6 (25) 18.8 (19) 27.3 (44)
15 (9) 47.5 (48) 35.4 (57)
5 (3) 29.7 (30) 20.4 (33)
IN VITRO CERVICAL MUCUS PENETRATION TEST
Vol. 36, No.2
TABLE 5. Comparison of the Bovine Cervical Mucus Penetration Assay and the Postcoital Tesfl' Semen characteristics Patients
n (of 42)
%
Sperm count
Adequate postcoital test Adequate cervical mucus penetration assay
34 31
81 74
Motility
Progression
x lo61ml
%
1-4
82.5 ± 10.9 82.9 ± 11.9
60.1 ± 3.1 66.3 ± 1.5
3.4 ± 0.7 2.9 ± 0.1
avalues are expressed as the mean ± standard error.
not have been considered to have a normal percentage of motility.
sperm density of the nonimpregnating men with good cervical mucu&. penetration were compared with that of samples from nonimpregnating men with poor penetration, the differences in sperm density were significantly different (100.5 ± 17 versus 55.0 ± 8.8, P ~ 0.05). A comparison of percent motility revealed similar findings (64.6 ± 3.3 versus 52.9 ± 3.0, P ~ 0.02). In those husbands whose wives conceived, the sperm density was not significantly different when good versus poor penetration was compared, but the motility of those specimens that had good penetration was significantly higher (P. ~ 0.05) than that of those that had a poor result (60 ± 1.8 versus 38.5 ± 10.4). Such findings of no differences in count or motility within the groups of poor or good penetrators but significant differences between the groups suggest · that the test does differentiate between the groups and that this differentiation is based more on motility than on sperm density. The CMPT, however, provides more information than an evaluation of motility, since half the men who had a good CMPT result and caused a pregnancy had less than 60% motility and thus would
Cervical mucus (1) protects sperm from the hostile vaginal environment, (2) filters out abnormal and poorly motile sperm while facilitating actively motile sperm of normal shape, (3) serves as a sperm reservoir, and (4) provides a likely site for capacitation. 15 Among most mammals the cervix and its secretion, cervical mucus, are the initial barrier that sperm must crossA The major component of cervical mucus is water, which constitutes from 85% to 98% of the total weight. This water is the vehicle for electrolytes, proteins, and other organic components. Sperm penetration is severely curtailed if the mucus water content drops below 95%. 16 The mucus is a hydrogel with nonuniform viscoelastic properties. 17 The highly viscous phase is composed of glycoproteins linked to peptide backbones. 18 Since bovine mucus can be frozen and stored without alteration of its viscoelastic properties, 13
FIG. 1. Scanning electron micrograph of human cervical mucus (original magnification x 700).
FIG. 2. Scanning electron micrograph of bovine cervical mucus (original magnification x 700).
DISCUSSION
Cervical Mucus
ALEXANDER
206
August 1981
TABLE 6. Comparison of Sperm Penetration in Human and Bovine Cervical Mucus'" Patient no.
Density
x 1rrlml
Motility
Progre88ion
Mucus penetration
Morphology
Human
Bovine
1-4
%
70 74 65 28 56 65 66
4.0 3.5 3.5 2.5 2.5 3.0 3.0
88 81 80 75 75 74 73
30 21 33 25 19 19 31
33 20 25 19 23 29 50
86 66 60
3.0 3.5 2.5 2.5 2.5 3.0
86 81 80 69 68 67
12 5 13 4 10 0
24 26 20 21 35
3.0 2.5 1.5 1.5 1.0 2.0 2.5
86 75 61 62 64 70 82
8 13 3 12 0 0 2
13 10 12 5 3 6 10
%
Adequate penetration in both human and bovine mucus
1 2 3 4 5 6 7
210 73 291 21 287 180 305
Adequate penetration in bovine mucus
8 9 10 11
12 13
26 242 32.5 54 24.5 55
64
57 66
25
Inadequate penetration in both human and bovine mucus
14 15 16 17 18 19 20
40 138 37 71
12 30 73
68 55 27 27 4 43 42
aNo sample could penetrate human but not bovine mucus.
it seems an excellent medium in which to test the penetration ability of sperm.
Use of Human or Bovine Cervical Mucus in an In Vitro Test It is difficult to fill capillary tubes with samples of human cervical mucus without including air bubbles, which block the passage of spermatozoa. With the greater quantity of bovine mucus, one can obtain excellent test vehicles in which to examine sperm penetration. The use of human mucus does not allow differentiation of the functional characteristics of the mucus and spermatozoa. With bovine mucus,evaluation of sperm penetration without the variable of the wife's mucus can be accomplished. Parallel test results of the effect of human and bovine mucus on the same semen specimen were similar; semen specimens with poor penetration of human samples exhibited poor penetration of bovine samples. Human and bovine mucus are rheologically similar. The glycoproteins. are markedly similar in sugar and amino acid composition, as well as in electrophoretic mobility, sedimentation rate, and banding density in a cesium chloride density gradient. 19 Ferning patterns, crystallization at right angles thought to be associated with levels of sodium and chloride, are a typical measurement of
the adequacy of mucus. 20 The patterns in bovine and human sampies are almost identical (Figs. 1 and 2). Data obtained through laser-scattering spectroscopy suggest both human and bovine cervical mucus are a mixture of entangled coiled macromolecules rather than a cross-linked macromolecular network and that penetration by spermatozoa is most probably by mechanical means. 21 Since the viscoelastic properties of bovine mucus are so similar to those of human midcycle inUCUS,21 investigators have used bovine mucus as a replacement for human mucus. Blandau and associates 13 have demonstrated that human sperm penetrated bovine cervical mucus in an unidirectional manner. The flagellar motion is sensitive to external forces; spermatozoa develop a characteristic swimming pattern in mucus. The flagellation pattern of the human spermatozoa in bovine mucus is identical to that in human muCUS. 22 The mucus often is almost bacteria-free. In fact, Blandau and associates13 could not develop significant bacterial growth in culture. Furthermore, since proteolysins such as those released by bacteria rapidly liquify mucus,23 the presence of bacteria can be readily gauged through the loss of viscoelasticity.
Vol. 36, No.2
IN VITRO CERVICAL MUCUS PENETRATION TEST
Evaluation of the Distance Traveled For simplicity, the test was developed to be done at room temperature for 90 minutes; 1 hour was adequate at 37° C. This time interval provided an adequate period for penetration, yet avoided a reduction of both motility and forward progression due to time. 24 The test evaluates the distance traveled by the vanguard sperm. One might argue that these sperm are not necessarily representative of the total sperm population. However, comparison of the vanguard sperm with sperm density revealed the same experimental results both in our studies14 and in reports by Ulstein and Fjallbrant. 25 A high density of sperm was found deep in the mucus in samples where the vanguard spermatozoa had penetrated a long distance.
Comparison of the Postcoital Test and the Cervical Mucus Penetration Test The value of the postcoital test has been frequently questioned. If there are spermatozoa in the cervical mucus, this finding certainly confirms that the couple's coital technique is adequate and that they are anatomically normal. But the results of the postcoital test depend upon various factors, including the interval since coitus, cervical mucus quality, semen quality, and the phase of the menstrual cycle. Investigators use different time intervals between coitus and collection. Danezis and associates 26 demonstrated that any interval up to 8 hours between coitus and mucus collection yields the same information. If the results are good, the test need not be repeated; nevertheless, poor test results are not necessarily indicative of a sperm-mucus problem. As one would expect, when good quality mucus and semen are involved, good results are often observed. The quality and quantity of mucus does not correlate with sperm counts or motility seen. 27 Postcoital test results have been found to be not significantly different in fertile and infertile couples. 26 On the basis of the incidence of pregnancy, the quality of the wife's cervical mucus seems more important, an indication that the postcoital test is not an important assay for determining the quality of human semen. The postcoital test is not a consistent predictor of pregnancy.28. 29 When the postcoital test results are in the intermediate range, they have no prognostic value.
207
Factors Preventing Bovine Cervical Mucus Penetration A variety of factors affect sperm penetration. Deficiencies in sperm function could be related to flagellar activity, surface properties, or enzyme content. Abnormal forms of spermatozoa supposedly cannot penetrate mucus. 15 The percentage of abnormal forms did not seem to be an important factor in sperm penetration. Patients with adequate penetration and fertile men both had 80.4 ± 0.7% normal forms, as compared with 76.2 ± 1.0% in those patients with inadequate penetration (Tables 1 and 2). Whether samples that were severely morphologically abnormal but had good motility would be unable to adequately penetrate the mucus remains to be tested. In cases ofimmunologic infertility, antibodies to sperm can impede motility. In men with circulating antibodies to spermatozoa, such antibodies are not exposed to sperm antigens until ejaculation. At this time immunoglobulins, mainly from the prostate, meet epididymal sperm in the urethra. Such antibodies can coat the spermatozoa and reduce their ability to penetrate mucus. 30 This reduced ability was first described in 1976 by Kremer and Jager. 31 When sperm antibodies are present in either the semen sample or the cervical mucus, the motile sperm no longer exhibit forward progression, but instead become stationary and have a shaking motility pattern because of adherence to the mucus microstructure. There is a good correlation between circulating agglutinating or immobilizing antibodies and poor cervical mucus penetration. High titers of these antibodies adhere in the sperm surface, and although they do not kill or immobilize the sperm, they prevent them, even those found to be apparently normal in semen analyses, from effectively invading cervical mucus. In fact, when a small amount of serum from an individual with circulating antisperm antibodies is mixed with normal semen, the spermatozoa often no longer adequately penetrate the muCUS. 14 The bovine cervical mucus penetration test is easier to perform than more complicated serum antibody tests. Furthermore, this assay may provide more relevant information concerning immunologic infertility, since in some men levels of antisperm antibodies have been found in circulation but not in the seminal plasma. No reduction in fertility would be expected unless antibodies occurred in the seminal plasma. When immunologic infertility is suspected and a poor cervical mucus result is found, further evaluation of antisperm antibody levels is advisable.
ALEXANDER
208 Correlation with Fertility
The results of in vitro tests of sperm penetration are significantly correlated with pregnancy.10 In fact, penetration and duration of spetm motility in mucus is more closely associated with fertility than with any other semen variable among artificial insemination donorsY We have based our rating scale for the bovine cervical mucus assay on the results we have obtained with spermatozoa from known fertile men. We believe that observation of in vitro sperm penetration provides reliable data for fertility assessment, allows greater discrimination of sperm function than semen analysis alone, and is a useful tool for the diagnosis and management of infertility. Acknowledgments. I wish to thank the gynecologists who kindly provided information: Kenneth Burry, M.D., University of Oregon Health Sciences Center, Portland, Oregon; Allan Weiland, M.D., Kaiser, Vancouver, Washington; and Drs. John O. Bergstrom, Thomas Brugger, Raymond S. Corwin, John DeMaria, John Enbom, Thomas Hart, Herschel W. Lawson, Richard Thorne, Larry Veltman, and George Weghorst, all in private practice in Oregon. I also wish to thank David Fulgham, Marianne Joseph, Pam Judy, Barbara Mixon, and John Sampson for their excellent technical assistance and Patsy Kimzey for helping with the preparation of this manuscript.
REFERENCES 1. Davajan V, NakamuraRM, Kharma K: Spermatozoan transport in cervical mucus. Obstet Gynecol Surv 25:1, 1970 2. Moghissi KS: Current perspectives: the function of the cervix in infertility. Fertil Steril 23:295, 1972 3. Miller EG Jr, Kurzrok R: Biochemical studies of human semen: factors affecting migration of sperm through cervix. Am J Obstet Gynecol 24:19,1932 4. Moghissi KS, Dahich D, Levine J, Neuhaus OW: Mechanism of sperm migration. Fertil Steril 15:15, 1964 5. Katz DF, Overstreet JW, Hanson FW: A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 33:179, 1980 6. Lamar JK, Shettles LB, Delfs E: Cyclic penetrability of human .cervical mucus to spermatozoa in vitro. Am J Physiol 129:234, 1940 7. Kremer J: A simple sperm. penetration test. Int J Fertil 10:209, 1965 8. Mills RN, KatzDF: A flat capillary tube system for assessment of sperm movement in cervical mucus. Fertil Steril 29:43,1978 9. Tredway DR,. Settlage DS, Nakamura RM, Motoshima M, Umezaki CU, Mishell RR Jr: Significance of timing for the postcoital evaluation of cervical mucus. Am J Obstet GynecoI121:387, 1975
August 1981 10. Uistein M: Evaluation of a capillary tube sperm penetration method for fertility investigations. Acta Obstet Gynecol Scand 51:287, 1972 11. Ulstein M: Fertility of donors at heterologous insemination. Acta Obstet Gynecol Scand 52:97, 1973 12. Lee WI, Blandau RJ, Verdugo P: Laser light-scattering studies of cervical mucus. In The Uterine Cervix and Reproduction, Edited by V Insler, G Bettendorf. Stuttgart, Georg Thieme Verlag, 1977, p 68 13. Blandau RJ, Gaddum-Rosse P, Lee WI: Letter to the Editor. Fertil Steril 29:707, 1978 14. Alexander NJ, Fulgham DL: Antibodies to spermatozoa in male monkeys: Mode of action. Fertil Steril 30:334, 1978 15. Ulstein M: Functions and physical.properties of mucus in the female genital tract. Br Med Bull 34:83, 1978 16. Bergman P: Spermigration and cyclic changes in cervical mucus. Fertil Steril 4:183, 1953 17. Odeblad E, Rudolfsson C: Types of cervical secretions: Biophysical characteristics. In The Biology of the Cervix, Edited by RJ Blandau, K Moghissi. Chicago, The University of Chicago Press, 1973, p 267 18. Iacobelli S, Garcea N, Angeloni C: Biochemistry of cervical mucus: a comparative analysis of the secretion from preovulatory, postovulatory, and pregnancy periods. Fertil Steril 22:727, 1971 19. Meyer FA: Comparison of structural glycoproteins from mucus of different sources. Biochim Biophys Acta 493: 272, 1977 20. Kopitto LE, Kasasky JH, Sturgis SH, Lieberman BL, Shwachman H: Water and electrolytes in human cervical mucus. Fertil Steril 24:499, 1973 21. Lee WI, Verdugo P, Blandau RJ, Gaddum-Rosse P: Molecular arrangement of cervical mucus: a reevaluation based on laser light-scattering spectroscopy. Gynecol Invest 8:254,1977 22. Katz DF, Mills RN, Pritchett TR: The movement of human spermatozoa in cervical mucus. J ReprodFertil 53:259, 1978 23. Gibbons RA, Sellwood R: The macromolecular biochemistry of cervical secretions. In The Biology of the Cervix, Edited by RJ Blandau, K Moghissi. Chicago, The University of Chicago Press, 1973, p 251 24. Freund M: Interrelationships among characteristics of human semen and factors affecting semen-specimen quality. J Reprod Fertil 4:143, 1962 25. Ulstein M, Fjiillbrant B: Interrelation of different parameters at cervical mucus penetration of spermatozoa. Acta Obstet Gynecol Scand 52:295, 1973 26. Danezis J, Sujan S, Sobrero AJ: Evaluation of the postcoital test. Fertil Steril 13:559, 1962 27. Kovacs GT, Newman GB, Henson GL: The postcoital test: What is normal? Br Med J 1:818, 1978 28. Jette NT, Glass RH: Prognostic value of the postcoital test. Fertil Steril 23:29, 1972 29. Blasco L: Clinical approach to the evaluation of spermcervical mucus interactions. Fertil Steril 28:1133, 1977 30. Fjiillbrant B: Cervical mucus penetration by human spermatozoa treated with anti spermatozoal antibodies from rabbit and man. Acta .obstet Gynecol Scand 48:71, 1969 31. Kremer J, Jager S: The sperm-cervical mucus contact test: a preliminary report. Fertil Steril 27:335, 1976
FERTILITY AND STERILITY Copyright 0 1981 The American Fertility Society
EVALUATION OF MALE INFERTILITY WITH AN IN VITRO CERVICAL MUCUS PENETRATION TEST*
NANCY J. ALEXANDER, PH.D. Department of Reproductive Physiology, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, and Departments of Anatomy, Obstetrics-Gynecology, and Urology, University of Oregon Health Sciences Center, Portland, Oregon 972{)1
The use of an in vitro bovine cervical mucus penetration test (CMPT) provides unique data for fertility assessment. Flat capillary tubes were filled, kept frozen until use, exposed to a sub-sample of semen for 90 minutes, and microscopically evaluated. Adequate penetration was based on results of semen specimens from donors used for artificial insemination. Of 161 patients being evaluated for infertility, 37% had semen that penetrated the CMPT inadequately. Of the patients with inadequate penetration, 70% had sperm densities of greater than 21 x lO6/ ml, and 62% had over 50% motility. Thus neither evaluation of count nor motility provided the same information as the CMPT. Human spermatozoa had a similar swimming pattern in human and bovine mucus. Spermatozoa that exhibited poor in vitro penetration of human mucus also failed to penetrate bovine mucus. Comparison of the CMPT with postcoital tests of 42 couples revealed a good correlation. When the incidence of pregnancy for individuals with adequate and inadequate penetration was compared, more individuals with a good CMPT caused a pregnancy. It appears that the CMPT, an easy office test, allows greater discrimination of sperm function than semen analysis alone and is a useful tool for the diagnosis and management of infertility. Fertil Steril36:201, 1981
lack of uniformity in how this test is performed and wide variation in the interpretation of the results, partially because of the lack of specific criteria used for quantification. 2 In vitro cervical mucus penetration is assessed by either a slide or a capillary tube assay. In the first method drops of semen and cervical mucus are placed next to each other on a microscope slide, and sperm penetration of the mucus is subjectively determined. 3 , 4 Microscopic evaluation reveals the development of phalanges with a single sperm seeming to lead the cervical mucus penetration. The size and shape of the semen-mucus interface and local mucus shearing caused by coverslipping limit the quantitative application of this test and its reproducibility.5 With capillary tubes one can evaluate cervical mucus penetration more objectively. When tests of this kind are conducted, mucus is drawn into a capillary tube, which is then sealed at one end. The unsealed end is placed in a small amount of semen.
An essential part of the investigation of an infertile couple is evaluation of the interaction between spermatozoa and cervical mucus. In fact, measurement of sperm penetration and survival in cervical mucus is considered to be one of the most important tests of human sperm function. 1 Sperm penetration is usually assessed in (1) cervical mucus collected after coitus or (2) mucus collected and tested in vitro. In the most widely used test of sperm penetration, the postcoital test (peT), or Sims-Huhner test, after the couple have intercourse, a sample of mucus is collected and examined 2 to 10 hours later. However, there is a Received February 25,1981; revised and accepted April 10, 1981. *Publication No. 1134 of the Oregon Regional Primate Research Center, supported by NIH grant RR-00163 and by the Syva Company. Reprint requests: Nancy J. Alexander, Ph.D., Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006.
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August 1981
ALEXANDER
After a specified time interval the distance traveled by the spermatozoa is recorded, the density of spermatozoa in various segments of the tube is determined, and motility is evaluated. 6 , 7 Using capillary tubes, one can estimate the swimming speed of the fastest spermatozoa and qualitatively evaluate the flagellar beat. In the past, the use of round tubes made the optical resolution difficult, but recently flat tubes have been used for observations of sperm in cervical mucus. 8 The results of cervical mucus penetration tests have been closely correlated with fertility9, 10; the depth of penetration is correlated more closely than any other variable. 11 Such tests have a unique diagnostic value in the determination of male infertility because they allow evaluation of the interaction between spermatozoa and cervical mucus. One drawback, however, is that they do not differentiate between the separate functional characteristics of the spermatozoa and mucus. Use of an alternative standardized source ofmucus avoids this problem and allows more thorough examination of the spermatozoa as the only variable. The midcycle mucus of cows is similar to that of women. Laser light-scattering spectroscopy has revealed that mucus from both species consists of entangled, randomly coiled macromolecules. Furthermore, the viscoelastic properties of the two are remarkably similar. 12 Human spermatozoa rapidly penetrate cow mucus in a concerted manner and exhibit changes in their pattern of movement identical to those displayed by human spermatozoa penetrating human midcycle cervical mucus. 13 Furthermore, up to 100 ml of mucus (enough for 1000 tests) can be obtained from one cow in a single collection. Thus, bovine mucus may allow greater standardization of in vitro test conditions and also enable investigators to evaluate the interaction of spermatozoa and mucus in the absence of variables associated with the wife's mucus. Comparison of the data from such an in vitro test with those from postcoital tests could provide ~dditional cogent information for infertility evaluation. In this article we present evidence that an in vitro bovine cervical mucus penetration assay is a useful adjunct in the evaluation of infertility.
or. Care was taken to avoid fecal contamination, but no attempt was made to avoid contamination with vaginal secretions. Assay Method. Flat capillary tubes were filled with cervical mucus, sealed, and stored at - 20° C until used. Each capillary tube was thawed at room temperature for several minutes, scored with a diamond pencil several millimeters above the mucus meniscus, and broken at the score. The cut end was inserted in 150 j.LI of semen and incubated at room temperature for 90 minutes. The tubes were removed from the semen reservoir, wiped clean of any residual specimen, and placed on a glass slide on a microscope stage. The distance (in millimeters) covered by the vanguard spermatozoa was noted. A 250 x to 400 x magnification was used; phase optics are preferred. If it was obvious that an air bubble across the tube had blocked sperm penetration, that value was discarded. Each assay was run in duplicate. Semen. Semen samples were collected by masturbation after sexual abstinence of at least 48 hours and evaluated for volume, count per milliliter, percentage of motility based on a count of 200, and activity (determined on a scale of 0 [no movement] to 4 [active forward progression] after a 30-minute liquefaction period. Sperm with no movement were not used for CMPT). Semen specimens from fertile men (donors from the artificial insemination program) and patients attending an infertility laboratory were evaluated. Comparison with Human Cervical Mucus. Sperm-free cervical mucus was collected from several different women during the 3-day interval before the expected onset of ovulation. The mucus samples were used fresh or within 5 days of freezing. Bovine cervical mucus samples were tested concomitantly with human samples. For morphologic evaluation, samples of mucus were dried on coverslips, coated with gold palladium in a Technics sputter coater, and viewed with an AMR-lOOO scanning electron microscope. Statistical Evaluation. Significance was determined with Student's t-test and X2 analysis. Data are presented as means ± standard errors. RESULTS
Development of Test Criteria MATERIALS AND METHODS
Collection of Bovine Mucus. Mucus was collected from estrous cows by means of rectal palpation of the cervix and extrusion of mucus to the exteri-
Initially, round hematocrit capillary tubes were used14; but because of the poor optical clarity, in later testing we used flat tubes. In compar.ative and simultaneously run tests, cervical mu-
Vol. 36, No.2
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IN VITRO CERVICAL MUCUS PENETRATION TEST
TABLE 1. In Vitro Bovine Cervical Mucus Penetration Values for Fertile Men and Patients a Group
Fertile men Patients
Motility
n
Count x 11f!Imi
%
1-4
%
n
%
56 161
144 ± 12.8 73.8 ± 4.5
67.9 ± 1.0 60.9 ± 0.9
3.2 2.8
80.4 ± 0.7 78.8 ± 0.6
56 101
100 63
Progression grade
Normal forms
Good cervical mucus penetration
aValues for count, motility, and normal forms are expressed as the mean ± standard error. Values from patients and fertile men are all significantly different (P < 0.05).
cus penetration was more extensive in the flat than in the round tubes. When semen specimens from 56 fertile donors were tested, penetration within 90 minutes at room temperature was 7.6 ± 1.0 mm further in the flat capillary tubes. No fertile donor had spermatozoa that traveled less than 15 mm in round tubes or less than 21 mm in flat tubes. Migration appeared to be a linear function of time times temperature. All further comparisons were based on the results of these known fertile men. In order to allow room for error, based on the results found in fertile men, it was arbitrarily decided that samples with spermatozoa that did not penetrate more than 15 mm within 90 minutes at room temperature were inadequate.
Cervical Mucus Evaluation Many batches of bovine cervical mucus were used in these tests. There were batch-to-batch variations that will be thoroughly described in a subsequent communication, but any clear sample allowed human sperm penetration. No overt bacterial contamination was observed in any of the bovine cervical mucus samples.
Evaluation of Patient Semen Samples Semen from 161 patients referred to the infertility laboratory was tested by means of the bovine cervical mucus penetration test (CMPT). For many of the patients, semen analysis and the in vitro cervical mucus penetration tests were the first steps in their fertility evaluation. The results indicate that 37% of these men had semen that did not penetrate ;>- 15 mm. The mean sperm density (expressed as count x 106/m D for the en-
tire infertile group was 73.8 ± 4.5; the mean percentage of motility was 60.9 ± 0.9 (Table 1). The differences between the patient and the donor values were statistically significant. An important question is whether the CMPT provides additional information beyond that obtained in a semen analysis. The mean sperm density for patients whose sperm exhibited inadequate penetration was 53.8 ± 6.8 x 106/m l. Although this count is lower than that of either the fertile or the infertile group with adequate penetration, it would, by most standards, be considered within the normal range (Table 2). Sperm counts are compared with cervical mucus penetration in Table 3. The range of sperm densities was from 1 to 313 x 106/m l; 70% of the patients had counts over 21 x 106/m l. Thus the majority of the patients had what is considered to be normal sperm counts, but nonetheless, many of the spermatozoa had a reduced ability to penetrate cervical mucus. Evaluation ofthe percentage of motile sperm (Table 4) provided similar information. The mean percentage of motility was 51.9 ± 1.9, and the range was 9% to 80%. Motility of over 50% was found in 62% of the patients. Some of the individuals had normally motile sperm; yet their spermatozoa could not penetrate bovine cervical mucus.
Comparison with Postcoital Test Results Postcoital test results were obtained from 12 gynecologists practicing in Oregon. In most cases, if the first postcoital test was poor, additional tests were performed, and as many as eight postcoital tests were done for certain individuals. The mean number of postcoital tests was two. All tests were done within 8 hours after coitus. Of the 42
TABLE 2. Semen Analysis Values from Patients with Adequate and Inadequate Bovine Cervical Mucus Penetrationa Patients
Inadequate (n = 60) Adequate (n = 101)
Motility
Count
Form
Grade of activity
Mucus penetration
x 11f!Imi
%
% normal
1-4
mm
53.8 ± 6.8 85.7 ± 6.0
51.9 ± 1.9 66.3 ± 0.9
76.2 ± 1.0 80.4 ± 0.7
2.3 ± 0.08 3.1 ± 0.05
11.2 ± 0.5 23.2 ± 0.6
"Values are expressed as the mean ± standard error.
ALEXANDER
204
TABLE 3. Sperm Counts in Patients with Adequate and Inadequate Bovine Cervical Mucus Penetration Percentage (and number) of patients in each sperm count range Patients
Inadequate (n = 60) Adequate (n = 101) All patients (n = 161)
0--20·
21-40·
30 (18)
20 (12)
38 (23)
11.6 (7)
9.9 (10)
13.8 (14)
46.5 (47)
29.7 (30)
13 (28)
16.1 (26)
43.4 (70)
22.9 (37)
41-100-
101-300·
"'Counts expressed as sperm x 106 /ml.
couples evaluated, 81% had good postcoital test results, and 74% of the men had a good in vitro cervical mucus penetration test. We found no significant differences in sperm count per milliliter, percentage of motility, or forward progression when semen analyses of husbands with good postcoital tests were compared with analyses of husbands with a good in vitro bovine cervical mucus test (Table 5). Those 7 couples (17%) with a poor postcoital test but an adequate in vitro penetration had a mean sperm density of 86.8 ± 29.3 and a mean percentage of motility of 59.2 ± 1.8, values not very different from those listed in Table 5. All but four men from couples with good postcoital results had positive CMPT results. These data indicate a good correlation between the in vitro cervical mucus penetration assay and the postcoital test.
Comparison of Human and Bovine Cervical Mucus Ferning patterns of bovine and human samples were very similar (Figs. 1 and 2). When human and bovine mucus were run in parallel in the in vitro cervical mucus test, comparable results were observed (Table 6). Poor penetration of human mucus may indicate inadequacies in the women's mucus and provide no information regarding the husband's spermatozoa, a finding suggested in patients 8 to 13, who had spermatozoa that adequately penetrated bovine, but not human, mucus. Although the mean sperm density of patients 1 to 7 (195.3 ± 42.4) was signifi-
August 1981 cantly higher than patients 8 to 13 (84.2 ± 34.3), there were no significant differences between the groups when motility (60.6 ± 5.8 versus 66.5 ± 4.2), progression (3.1 ± 0.2 versus 2.8 ± 0.1), normal morphology (78.0 ± 2.0 versus 75.2 ± 3.3), or bovine cervical mucus penetration were compared (28.4 ± 4.1 versus 25.2 ± 2.2). Some semen samples (patients 14 to 20) exhibited inadequate mucus penetration in both bovine and human mucus. Although the mean count (57.28 ± 15.8) was not, the percentage of motility (38 ± 7.9) and progression (2.0 ± 0.3) were significantly different from those values for patients 1 to 13, who had adequate penetration of bovine cervical mucus. Good mucus penetration is dependent on the number of motile and progressive spermatozoa. Of paramount importance is the fact that no semen samples were found to exhibit only penetration of human and not bovine mucus. Such a finding supports the concept that bovine and human mucus are similar and that penetration of bovine mucus is a good indication of sperm function.
Correlation of the CMPT with Subsequent Pregnancy The incidence of pregnancy in patients who had good or poor CMPT in the 6 months following the test was compared. Couples were eliminated from the data base when a female problem that would obviously prevent conception (bilaterally blocked tubes or anovulation) was found. Of 21 patient couples in which the husband had good penetration, 9 could be evaluated for fertility, since no obvious female factor was observed. Of the group, 6 of the wives had conceived. There was no significant difference in the sperm density or percentage of motility of the specimens from those husbands whose wives did and did not conceive. Of 18 couples in which the husband had poor cervical mucus penetration, 4 wives conceived in the subsequent 6 months. Again, there were no significant differences in the sperm density or percentage of motility of sperm among men who did and did not impregnate their wives. When the
TABLE 4. Percentages of Motile Sperm in Patients with Adequate and Inadequate Bovine Cervical Mucus Penetration Percentage (and number) of patients in each sperm motility catsgory
Patients 0--20·
Inadequate (n = 60) Adequate (n = 101) All patients (n = 161) "'Percentage of motile sperm.
5 (3)
0(0) 1.8 (3)
21-40·
41-50·
51-60"
61-70"
71+"
16.6 (10) 0.9 (1) 6.8 (11)
16.6 (10) 2.9 (3) 8 (13)
41.6 (25) 18.8 (19) 27.3 (44)
15 (9) 47.5 (48) 35.4 (57)
5 (3) 29.7 (30) 20.4 (33)
IN VITRO CERVICAL MUCUS PENETRATION TEST
Vol. 36, No.2
TABLE 5. Comparison of the Bovine Cervical Mucus Penetration Assay and the Postcoital Tesfl' Semen characteristics Patients
n (of 42)
%
Sperm count
Adequate postcoital test Adequate cervical mucus penetration assay
34 31
81 74
Motility
Progression
x lo61ml
%
1-4
82.5 ± 10.9 82.9 ± 11.9
60.1 ± 3.1 66.3 ± 1.5
3.4 ± 0.7 2.9 ± 0.1
avalues are expressed as the mean ± standard error.
not have been considered to have a normal percentage of motility.
sperm density of the nonimpregnating men with good cervical mucu&. penetration were compared with that of samples from nonimpregnating men with poor penetration, the differences in sperm density were significantly different (100.5 ± 17 versus 55.0 ± 8.8, P ~ 0.05). A comparison of percent motility revealed similar findings (64.6 ± 3.3 versus 52.9 ± 3.0, P ~ 0.02). In those husbands whose wives conceived, the sperm density was not significantly different when good versus poor penetration was compared, but the motility of those specimens that had good penetration was significantly higher (P. ~ 0.05) than that of those that had a poor result (60 ± 1.8 versus 38.5 ± 10.4). Such findings of no differences in count or motility within the groups of poor or good penetrators but significant differences between the groups suggest · that the test does differentiate between the groups and that this differentiation is based more on motility than on sperm density. The CMPT, however, provides more information than an evaluation of motility, since half the men who had a good CMPT result and caused a pregnancy had less than 60% motility and thus would
Cervical mucus (1) protects sperm from the hostile vaginal environment, (2) filters out abnormal and poorly motile sperm while facilitating actively motile sperm of normal shape, (3) serves as a sperm reservoir, and (4) provides a likely site for capacitation. 15 Among most mammals the cervix and its secretion, cervical mucus, are the initial barrier that sperm must crossA The major component of cervical mucus is water, which constitutes from 85% to 98% of the total weight. This water is the vehicle for electrolytes, proteins, and other organic components. Sperm penetration is severely curtailed if the mucus water content drops below 95%. 16 The mucus is a hydrogel with nonuniform viscoelastic properties. 17 The highly viscous phase is composed of glycoproteins linked to peptide backbones. 18 Since bovine mucus can be frozen and stored without alteration of its viscoelastic properties, 13
FIG. 1. Scanning electron micrograph of human cervical mucus (original magnification x 700).
FIG. 2. Scanning electron micrograph of bovine cervical mucus (original magnification x 700).
DISCUSSION
Cervical Mucus
ALEXANDER
206
August 1981
TABLE 6. Comparison of Sperm Penetration in Human and Bovine Cervical Mucus'" Patient no.
Density
x 1rrlml
Motility
Progre88ion
Mucus penetration
Morphology
Human
Bovine
1-4
%
70 74 65 28 56 65 66
4.0 3.5 3.5 2.5 2.5 3.0 3.0
88 81 80 75 75 74 73
30 21 33 25 19 19 31
33 20 25 19 23 29 50
86 66 60
3.0 3.5 2.5 2.5 2.5 3.0
86 81 80 69 68 67
12 5 13 4 10 0
24 26 20 21 35
3.0 2.5 1.5 1.5 1.0 2.0 2.5
86 75 61 62 64 70 82
8 13 3 12 0 0 2
13 10 12 5 3 6 10
%
Adequate penetration in both human and bovine mucus
1 2 3 4 5 6 7
210 73 291 21 287 180 305
Adequate penetration in bovine mucus
8 9 10 11
12 13
26 242 32.5 54 24.5 55
64
57 66
25
Inadequate penetration in both human and bovine mucus
14 15 16 17 18 19 20
40 138 37 71
12 30 73
68 55 27 27 4 43 42
aNo sample could penetrate human but not bovine mucus.
it seems an excellent medium in which to test the penetration ability of sperm.
Use of Human or Bovine Cervical Mucus in an In Vitro Test It is difficult to fill capillary tubes with samples of human cervical mucus without including air bubbles, which block the passage of spermatozoa. With the greater quantity of bovine mucus, one can obtain excellent test vehicles in which to examine sperm penetration. The use of human mucus does not allow differentiation of the functional characteristics of the mucus and spermatozoa. With bovine mucus,evaluation of sperm penetration without the variable of the wife's mucus can be accomplished. Parallel test results of the effect of human and bovine mucus on the same semen specimen were similar; semen specimens with poor penetration of human samples exhibited poor penetration of bovine samples. Human and bovine mucus are rheologically similar. The glycoproteins. are markedly similar in sugar and amino acid composition, as well as in electrophoretic mobility, sedimentation rate, and banding density in a cesium chloride density gradient. 19 Ferning patterns, crystallization at right angles thought to be associated with levels of sodium and chloride, are a typical measurement of
the adequacy of mucus. 20 The patterns in bovine and human sampies are almost identical (Figs. 1 and 2). Data obtained through laser-scattering spectroscopy suggest both human and bovine cervical mucus are a mixture of entangled coiled macromolecules rather than a cross-linked macromolecular network and that penetration by spermatozoa is most probably by mechanical means. 21 Since the viscoelastic properties of bovine mucus are so similar to those of human midcycle inUCUS,21 investigators have used bovine mucus as a replacement for human mucus. Blandau and associates 13 have demonstrated that human sperm penetrated bovine cervical mucus in an unidirectional manner. The flagellar motion is sensitive to external forces; spermatozoa develop a characteristic swimming pattern in mucus. The flagellation pattern of the human spermatozoa in bovine mucus is identical to that in human muCUS. 22 The mucus often is almost bacteria-free. In fact, Blandau and associates13 could not develop significant bacterial growth in culture. Furthermore, since proteolysins such as those released by bacteria rapidly liquify mucus,23 the presence of bacteria can be readily gauged through the loss of viscoelasticity.
Vol. 36, No.2
IN VITRO CERVICAL MUCUS PENETRATION TEST
Evaluation of the Distance Traveled For simplicity, the test was developed to be done at room temperature for 90 minutes; 1 hour was adequate at 37° C. This time interval provided an adequate period for penetration, yet avoided a reduction of both motility and forward progression due to time. 24 The test evaluates the distance traveled by the vanguard sperm. One might argue that these sperm are not necessarily representative of the total sperm population. However, comparison of the vanguard sperm with sperm density revealed the same experimental results both in our studies14 and in reports by Ulstein and Fjallbrant. 25 A high density of sperm was found deep in the mucus in samples where the vanguard spermatozoa had penetrated a long distance.
Comparison of the Postcoital Test and the Cervical Mucus Penetration Test The value of the postcoital test has been frequently questioned. If there are spermatozoa in the cervical mucus, this finding certainly confirms that the couple's coital technique is adequate and that they are anatomically normal. But the results of the postcoital test depend upon various factors, including the interval since coitus, cervical mucus quality, semen quality, and the phase of the menstrual cycle. Investigators use different time intervals between coitus and collection. Danezis and associates 26 demonstrated that any interval up to 8 hours between coitus and mucus collection yields the same information. If the results are good, the test need not be repeated; nevertheless, poor test results are not necessarily indicative of a sperm-mucus problem. As one would expect, when good quality mucus and semen are involved, good results are often observed. The quality and quantity of mucus does not correlate with sperm counts or motility seen. 27 Postcoital test results have been found to be not significantly different in fertile and infertile couples. 26 On the basis of the incidence of pregnancy, the quality of the wife's cervical mucus seems more important, an indication that the postcoital test is not an important assay for determining the quality of human semen. The postcoital test is not a consistent predictor of pregnancy.28. 29 When the postcoital test results are in the intermediate range, they have no prognostic value.
207
Factors Preventing Bovine Cervical Mucus Penetration A variety of factors affect sperm penetration. Deficiencies in sperm function could be related to flagellar activity, surface properties, or enzyme content. Abnormal forms of spermatozoa supposedly cannot penetrate mucus. 15 The percentage of abnormal forms did not seem to be an important factor in sperm penetration. Patients with adequate penetration and fertile men both had 80.4 ± 0.7% normal forms, as compared with 76.2 ± 1.0% in those patients with inadequate penetration (Tables 1 and 2). Whether samples that were severely morphologically abnormal but had good motility would be unable to adequately penetrate the mucus remains to be tested. In cases ofimmunologic infertility, antibodies to sperm can impede motility. In men with circulating antibodies to spermatozoa, such antibodies are not exposed to sperm antigens until ejaculation. At this time immunoglobulins, mainly from the prostate, meet epididymal sperm in the urethra. Such antibodies can coat the spermatozoa and reduce their ability to penetrate mucus. 30 This reduced ability was first described in 1976 by Kremer and Jager. 31 When sperm antibodies are present in either the semen sample or the cervical mucus, the motile sperm no longer exhibit forward progression, but instead become stationary and have a shaking motility pattern because of adherence to the mucus microstructure. There is a good correlation between circulating agglutinating or immobilizing antibodies and poor cervical mucus penetration. High titers of these antibodies adhere in the sperm surface, and although they do not kill or immobilize the sperm, they prevent them, even those found to be apparently normal in semen analyses, from effectively invading cervical mucus. In fact, when a small amount of serum from an individual with circulating antisperm antibodies is mixed with normal semen, the spermatozoa often no longer adequately penetrate the muCUS. 14 The bovine cervical mucus penetration test is easier to perform than more complicated serum antibody tests. Furthermore, this assay may provide more relevant information concerning immunologic infertility, since in some men levels of antisperm antibodies have been found in circulation but not in the seminal plasma. No reduction in fertility would be expected unless antibodies occurred in the seminal plasma. When immunologic infertility is suspected and a poor cervical mucus result is found, further evaluation of antisperm antibody levels is advisable.
ALEXANDER
208 Correlation with Fertility
The results of in vitro tests of sperm penetration are significantly correlated with pregnancy.10 In fact, penetration and duration of spetm motility in mucus is more closely associated with fertility than with any other semen variable among artificial insemination donorsY We have based our rating scale for the bovine cervical mucus assay on the results we have obtained with spermatozoa from known fertile men. We believe that observation of in vitro sperm penetration provides reliable data for fertility assessment, allows greater discrimination of sperm function than semen analysis alone, and is a useful tool for the diagnosis and management of infertility. Acknowledgments. I wish to thank the gynecologists who kindly provided information: Kenneth Burry, M.D., University of Oregon Health Sciences Center, Portland, Oregon; Allan Weiland, M.D., Kaiser, Vancouver, Washington; and Drs. John O. Bergstrom, Thomas Brugger, Raymond S. Corwin, John DeMaria, John Enbom, Thomas Hart, Herschel W. Lawson, Richard Thorne, Larry Veltman, and George Weghorst, all in private practice in Oregon. I also wish to thank David Fulgham, Marianne Joseph, Pam Judy, Barbara Mixon, and John Sampson for their excellent technical assistance and Patsy Kimzey for helping with the preparation of this manuscript.
REFERENCES 1. Davajan V, NakamuraRM, Kharma K: Spermatozoan transport in cervical mucus. Obstet Gynecol Surv 25:1, 1970 2. Moghissi KS: Current perspectives: the function of the cervix in infertility. Fertil Steril 23:295, 1972 3. Miller EG Jr, Kurzrok R: Biochemical studies of human semen: factors affecting migration of sperm through cervix. Am J Obstet Gynecol 24:19,1932 4. Moghissi KS, Dahich D, Levine J, Neuhaus OW: Mechanism of sperm migration. Fertil Steril 15:15, 1964 5. Katz DF, Overstreet JW, Hanson FW: A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 33:179, 1980 6. Lamar JK, Shettles LB, Delfs E: Cyclic penetrability of human .cervical mucus to spermatozoa in vitro. Am J Physiol 129:234, 1940 7. Kremer J: A simple sperm. penetration test. Int J Fertil 10:209, 1965 8. Mills RN, KatzDF: A flat capillary tube system for assessment of sperm movement in cervical mucus. Fertil Steril 29:43,1978 9. Tredway DR,. Settlage DS, Nakamura RM, Motoshima M, Umezaki CU, Mishell RR Jr: Significance of timing for the postcoital evaluation of cervical mucus. Am J Obstet GynecoI121:387, 1975
August 1981 10. Uistein M: Evaluation of a capillary tube sperm penetration method for fertility investigations. Acta Obstet Gynecol Scand 51:287, 1972 11. Ulstein M: Fertility of donors at heterologous insemination. Acta Obstet Gynecol Scand 52:97, 1973 12. Lee WI, Blandau RJ, Verdugo P: Laser light-scattering studies of cervical mucus. In The Uterine Cervix and Reproduction, Edited by V Insler, G Bettendorf. Stuttgart, Georg Thieme Verlag, 1977, p 68 13. Blandau RJ, Gaddum-Rosse P, Lee WI: Letter to the Editor. Fertil Steril 29:707, 1978 14. Alexander NJ, Fulgham DL: Antibodies to spermatozoa in male monkeys: Mode of action. Fertil Steril 30:334, 1978 15. Ulstein M: Functions and physical.properties of mucus in the female genital tract. Br Med Bull 34:83, 1978 16. Bergman P: Spermigration and cyclic changes in cervical mucus. Fertil Steril 4:183, 1953 17. Odeblad E, Rudolfsson C: Types of cervical secretions: Biophysical characteristics. In The Biology of the Cervix, Edited by RJ Blandau, K Moghissi. Chicago, The University of Chicago Press, 1973, p 267 18. Iacobelli S, Garcea N, Angeloni C: Biochemistry of cervical mucus: a comparative analysis of the secretion from preovulatory, postovulatory, and pregnancy periods. Fertil Steril 22:727, 1971 19. Meyer FA: Comparison of structural glycoproteins from mucus of different sources. Biochim Biophys Acta 493: 272, 1977 20. Kopitto LE, Kasasky JH, Sturgis SH, Lieberman BL, Shwachman H: Water and electrolytes in human cervical mucus. Fertil Steril 24:499, 1973 21. Lee WI, Verdugo P, Blandau RJ, Gaddum-Rosse P: Molecular arrangement of cervical mucus: a reevaluation based on laser light-scattering spectroscopy. Gynecol Invest 8:254,1977 22. Katz DF, Mills RN, Pritchett TR: The movement of human spermatozoa in cervical mucus. J ReprodFertil 53:259, 1978 23. Gibbons RA, Sellwood R: The macromolecular biochemistry of cervical secretions. In The Biology of the Cervix, Edited by RJ Blandau, K Moghissi. Chicago, The University of Chicago Press, 1973, p 251 24. Freund M: Interrelationships among characteristics of human semen and factors affecting semen-specimen quality. J Reprod Fertil 4:143, 1962 25. Ulstein M, Fjiillbrant B: Interrelation of different parameters at cervical mucus penetration of spermatozoa. Acta Obstet Gynecol Scand 52:295, 1973 26. Danezis J, Sujan S, Sobrero AJ: Evaluation of the postcoital test. Fertil Steril 13:559, 1962 27. Kovacs GT, Newman GB, Henson GL: The postcoital test: What is normal? Br Med J 1:818, 1978 28. Jette NT, Glass RH: Prognostic value of the postcoital test. Fertil Steril 23:29, 1972 29. Blasco L: Clinical approach to the evaluation of spermcervical mucus interactions. Fertil Steril 28:1133, 1977 30. Fjiillbrant B: Cervical mucus penetration by human spermatozoa treated with anti spermatozoal antibodies from rabbit and man. Acta .obstet Gynecol Scand 48:71, 1969 31. Kremer J, Jager S: The sperm-cervical mucus contact test: a preliminary report. Fertil Steril 27:335, 1976