Human Cervical Mucus. IV. Viscoelasticity and Sperm Penetrability During the Ovulatory Menstrual Cycle*

FERTILITY AND STERILITY Copyright © 1978 The American Fertility Society

Vol. 30, No. 2, August 1978 Printed in U.SA.

HUMAN CERVICAL MUCUS. IV. VISCOELASTICITY AND SPERM PENETRABILITY DURING THE OVULATORY MENSTRUAL CYCLE*

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DON P. WOLF, PH.D.t LUIS BLASCO, M.D. MOHAMMAD A. KHAN, PH.D.:J: MITCHELL LITT, D.ENG.Sc.

Department of Obstetrics and Gynecology, School of Medicine, and Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Quantitative viscoelastic and sperm penetration measurements were made on individual human cervial mucus samples collected from several ovulatory menstrual cycles. An inverse relationship was found to exist between these two mucus properties, with peak penetrability seen during the ovulatory phase of the cycle when viscoelasticity was lowest. Limited sperm penetration was also observed with follicular phase mucus. When mucus was collected three times daily, the first sample of the day showed reduced pH values and elevated percentage ofnondialyzable solids (NDS) and viscoelasticity. Rates of mucus production were calculated for two cycles; the highest rate was seen during the ovulatory phase, with the follicular and luteal phases showing approximately 50% and 30% of this rate, respectively. Limited disparities were noted between midcycle changes in percentage of mucus NDS and viscoelasticity and in blood levels of total estrogens. Fertil Steril30:163, 1978

In the human, cervical mucus plays an important role in controlling sperm and bacterial access to the upper reproductive tract. During most of the ovulatory menstrual cycle, the mucus is scanty, thick and viscous, and resistant to penetration. Immediately prior to ovulation, under estrogenic influence, the mucus thins and its penetrability is maximal. 1 These alterations in mucus properties reflect changes in the concentration and perhaps in the composition of cervical mucins, the major glycoprotein fraction in mucus which is responsible for its viscoelasticity. 2 •4 A method is presently available for the quantitative assessment of mucus viscoelasticity on in-

dividual mucus samples, 2 and time-dependent changes in viscoelasticity during the ovulatory cycle have been defined. 3 Capillary tube methods have been developed for the in vitro evaluation of mucus penetrability by sperm. s- 7 The present study was undertaken to examine the relationship between mucus viscoelasticity, biochemical characteristics, and penetrability. MATERIALS AND METHODS

Mucus was collected from ovulatory donors three times daily and handled as described previously.3 Blood was sampled at the time of mucus collection, and levels ofluteinizing hormone (LH), progesterone, and estrogen were determined by radioimmunoassay. Samples were normalized temporally to the day of ovulation, i.e., the day on which serum LH values were highest. Mucus penetrability was measured by the capillary tube penetration test performed with a sperm penetration meter as described by Ulstein. 7 Capillary tubes (0.7 mm inner diameter x 5 em) were

Received Februa,ry 24, 1978; revised April 3, 1978; accepted April 3, 1978. *Supported by National Institutes of Health Contract N01-HD-4-2838. tReprint requests: Don P. Wolf, Ph.D., Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pa. 19104. :J:Present address: Parke-Davis, Inc., Greenwood, S. C. 29646.

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loaded with mucus by inserting the capillary into the sample retained in its 1-ml collecting syringe, i.e., a sample .)Vas cored out. The height of the mucus column varied from 1 to 5 em and was generally proportional to the sample volume. Care was exercised to avoid air bubbles in the mucus column, as penetrability was severely inhibited by their presence. This was usually accomplished by first mixing the sample with the aid of the plunger in the collecting syringe and then compressing the sample gently to express trapped air. Loaded capillaries or mucus samples in collecting syringes were stored at 4° C (fresh) or frozen at -20° C until use. Semen samples were obtained from healthy donors and characterized by microscopic and turbidimetric analyses. 8 Only samples qualifying as normal were employed for subsequent penetrability tests. Ejaculates were allowed to liquefy for 30 minutes before characterization was initiated and were employed for 1 to 2 hours thereafter. Mucus viscoelasticity (as measured with a microrheometer); wet weights and nondialyzable solids (NDS); and mucus pH, cellularity, ferning, and spinnbarkeit were determined as described previously. 3 In the present study, G', the storage modulus in dynes per square centi9

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meter, was used as the quantitative indicator of mucus viscoelasticity. This measurement was made at 30 rads/second.

RESULTS

Mucus was collected three times daily to obtain sufficient material for fractionation and subsequent chemical analyses, necessitating an examination of the daily variability in its chemical and viscoelastic properties (Fig. 1). Viscoelasticity, the percentage of NDS (% NDS), and pH data for the first sample of the day are circled, and the curves connect the mean values of the second and third samples. The first sample of the day was usually collected at 9 A.M. and subsequent specimens were obtained at 2- to 3-hour intervals thereafter. With few exceptions, the first sample collected was markedly different from the others in that its pH was lower and its % NDS or its viscoelasticity was higher; it failed even to show significant time-dependent changes in % NDS and in viscoelasticity. The reproducibility in pH and% NDS measurements of mucus derived from the second and third collections was good, with the range of the former usually ±0.15 pH unit and the latter ± 1%. Intersample viscoelasticity measurements were not as precise; however, in 18 of 22 cases, G' values from the second and third collections were not substantially different from each other and did reflect the % NDS of the sample. A second ovplatory cycle was analyzed as described here, and, although the over-all trends were similar, the first sample did not stand apart as markedly. Rate of Mucus Production. Second and third mucus samples were collected at timed intervals, allowing calculation of rates of mucus production assuming that the efficiency of collection was similar throughout the cycle. In Figure 2, the combined wet weight of the three daily samples and the daily rate of mucus production, expressed as wet weight per hour, are plotted versus time for two individual cycles. Although time-dependent changes in wet weight and in the rate of mucus production were similar, approximately a 2-fold difference was seen between the two cycles in the absolute values of these indices. Daily mucus production varied as much as 10-fold when individual luteal samples were compared with peak levels observed on day -1. For statistical purposes, cycles were divided into three phases: follicular phase (days -5 to -9), ovulatory phase (days -4 to 0), and luteal phase (days + 1 to + 11)

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MUCUS VISCOELASTICITY AND PENETRABll..ITY

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(Table 1). The highest rate of mucus production was seen during the ovulatory phase, with the follicular and luteal phases showing approximately 50% and 30% of this rate, respectively. Differences in the production of NDS were not as marked (..v2-fold), however, since ovulatory mucus contained the lowest NDS concentration. Mucus Dry Weight, Percentage ofN ondialyzable Solids, and Viscoelasticity. The results obtained for mucus collected from seven cycles are presented as mean values in Figure 3 (data points). The 95% confidence limits of the mean are included in the stippled areas. The number of observations in each case is listed with the data point. These results, extended timewise to cover

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more of the menstrual cycle, are in excellent agreement with those published by us previously (figs. 2 and 3 in reference 3). Thus, dry weights remained relatively constant, while the % NDS and viscoelasticity decreased to a nadir at or near midcycle. The % NDS and viscoelasticity of luteal phase samples (day + 1 to end) were higher than those of the follicular phase (day -5 to beginning) at means of 7.6% and 101 dynes/sq em versus 5.8% NDS and 83 dynes/sq em, respectively. However, individual variation was substantial, causing overlap in the confidence intervals of the means for the two phases. Estrogen values are also included in Figure 3 for consideration of the cause-and-effect relationships between changes in blood estrogen levels and mucus viscoelasticity (see below). Capillary Penetration Test. Although attention has centered on the use of the in vitro capillary tube test in evaluating semen quality, its application to defining mucus penetrability is obvious. Reproducibility of the test has been examined and found acceptable with midcycle mucus by others5· 7 and with both midcycle (low G ') and follicular or luteal phase mucus (high G') by us (for example, see duplicate values in Fig. 7). The test was also reproducible when different semen preparations were employed or when applied to fresh or frozen mucus. Maximal sperm velocities obtained here were in the range usually reported by others9 (2 to 3 mm/minute).

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Viscoelasticity and Penetrability. Mucus penetrability, quantitatively expressed as sperm velocity, was measured on samples obtained from four separate cycles and is plotted in Figure 4 as a function of viscoelasticity to illustrate the inverse

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FIG. 4. The relationship between viscoelasticity and sperm penetrability in cervical mucus from four ovulatory menstrual cycles. Data from individual cycles are represented by different symbols, and the arrows qualify data points as minimal estimates. In these cases, rapid sperm progression resulted in sperm at the top of the mucus column by the first timed interval.

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Vol. 30, No. 2

MUCUS VISCOELASTICITY AND PENETRABILITY

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relationship between these mucus characteristics. Data points from each cycle are illustrated by a different symbol. Sperm progression in many of the samples with low viscoelasticity was rapid, and only minimal estimates of velocity were obtained (indicated by arrows). It is clear from Figure 4 that occasionally mucus samples with high viscoelastic properties support sperm transport, and therefore factors in addition to viscoelasticity must be considered in future studies. Mucus from Individual Cycles. The quantitative characteristics of the mucus collected from three individual cycles are presented in Figures 5, 6, and 7. In all cycles, the inverse relationship between viscoelasticity and penetrability was evident, with maximal penetrability apparent during the ovulatory phase. In Figures 5 and 6, penetrability was measured on fresh mucus, whereas in Figure 7 the mucus had been frozen prior to analysis. Note the tendency for mucus samples from the follicular phase of the cycle to support penetration, especially those represented in Figure 6. The penetration of mucus collected on days -9, -10, and -11 ofthis cycle was also high (data not shown). In Figure 3, it is apparent that the precipitous decrease in mean viscoelasticity associated with ovulation preceded increases in blood estrogen

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concentrations, although the resolution of these parameters was limited by statistical considerations. A similar observation can be drawn from the data presented by Moghissil 0 (figs. 4 and 5 in reference 10). The relationship between initial changes in blood estrogen concentrations and in mucus characteristics can be examined on individual cycles. In Figure 5, estrogen data are incomplete; however, the largest daily decrease in G' and in% NDS occurred from day -3 to day -2 at a time when the estrogen level was probably just beginning to increase. In Figure 6, mucus viscoelasticity and % NDS decreased dramatically from day -6 to day-5, and this change was associated with a slight decrease in total blood estrogens. The latter was not elevated significantly until day -4. In the third cycle (Fig. 7), the decrease in G' observed from day -7 to day -6 was also associated with a decrease in estrogen levels; however, from day -6 through ovulation, changes in mucus G' and % NDS and in blood estrogens were gradual and mirrored each other until G' reached its nadir. With regard to this latter event, it is clear from these studies that the midcycle low point in mucus viscoelasticity is reached substantially before the attainment of the maximal preovulatory estrogen peak. It has been demonstrated that the extent to which mucus viscoelasticity varies over the normal cycle is characteristic for each donor; thus, in some cases, greater than 10-fold changes are seen whereas others show only a 2-fold difference. 3 This observation has important implications to the prediction of ovulation. In the present study, viscoelasticity properties were more uniform and the ovulatory phase was characterized by the production for at least 2 successive days of mucus with a G' ofless than 30 dynes/sq em (six or seven cycles). Moreover, although occasionally samples from the follicular phase or luteal phase displayed low values ofG ',such mucus was seldom collected on consecutive days outside the ovulatory phase (1 case in 14).

DISCUSSION

The present studies served to illustrate that the time and the sequence of sampling were important considerations in our quantitative characterization of human cervical mucus, since the first sample of the day had a lower pH and a higher % NDS and viscoelasticity than did specimens obtained subsequently. The lower pH presumably reflected enhanced contamination of mucus near

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the external os by acidic vaginal fluids. 11 Indeed, the greatest deviations in mucus pH occurred in the follicular and luteal phases of the cycle at a time when the mucus production rate, and consequently its turnover, was at its lowest. It is clear from this analysis that previous conclusions regarding changes in mucus pH at ovulation should be reconsidered. 1 The higher % NDS and viscoelasticity seen in the first daily sampling could result from limited desiccation of mucus in situ; however, it seems improbable that this represents the only consideration. While the timedependent changes in mucus characteristics were certainly obscured in the first sample data presented in Figure 1, mean values of mucus wet and dry weight, % NDS, and viscoelasticity were similar in this and in our previous study,3 which involved a single daily sampling. A major role for steroid hormones in controlling mucus production and quality is based on studies defining their endogenous concentrations throughout the menstrual cycle, as well as their effects when administered exogenously in high concentrations. The reported existence of estradiol and progesterone-specific binding activity in endocervical preparations is, of course, consistent with these observations. 12 • 13 Estimations of the rate of mucus production indicate that, on average, a 2- to 3-fold enhancement in total mucus wet weight occurred under the influence of the midcycle estrogen surge. Maximal total mucus production per day reached approximately 1700 gm, compared with values of up to 700 gm reported by others. 14 However, differences in the production of NDS were not as dramatic when the three phases were compared, because of compensatory changes in the state of hydration. The NDS in mucus represent blood proteins derived by transudation and glycoproteins and/or proteins secreted by the endocervical epithelium. Since steroid hormones undoubtedly affect both of these phenomena, it is obvious that further insight into the factors controlling mucus production will require separation of the two effects. Model in vitro animal systems that achieve this objective are currently in use. 15 It is generally conceded that mucus penetrability is maximal during the ovulatory phase of the menstrual cycle when, under estrogenic influence, the mucus thins and clears. Conversely, elevated progesterone levels during the luteal phase are associated with an inhibition of endocervical secretory activity and the presence of a thick, impenetrable mucus. During the follicular phase of

August 1978 the cycle, when circulating progesterone and total estrogen concentrations are low, the mucus is also relatively thick and impenetrable. 1. 16 The present quantitative measurements of mucus penetrability and viscoelasticity support these general concepts in that an inverse relationship was apparent between penetrability and viscoelasticity or % NDS. In this regard, it has been suggested that the protein content of mucus would serve as a useful index for assessing the sperm transport function of mucus, 17 and the present results support that suggestion. A tendency for follicular phase samples, in contrast to luteal phase samples, to support minimal penetration was observed here, which may reflect the lower mean Vt!-lues of mucus % NDS and viscoelasticity seen early in the menstrual cycle. Since mucins are responsible for the unique viscoelastic properties of mucus, time-dependent changes in their concentration or composition are critical to the sperm transport properties of the mucus. Mucin concentration changes, as reflected in% NDS values, are significant, whereas important compositional changes have yet to be demonstrated convincingly. With regard to mucin composition, most studies have focused on the possible existence of changes in the sialic acid-tofucose ratios, since the negative charge of sialic acid is thought to dictate, in large measure, mucin viscoelastic properties. 18 However, recently evidence was presented to show that removal of sialic acid from bovine cervical mucus 19 or canine tracheal mucin 20 had little effect on viscoelasticity. Two perhaps inter-related observations raised by the present study invite further comment: namely, the role of steroid hormones in controlling mucus production during the follicular phase of the cycle when endogenous concentrations are low and the apparently limited uncoupling observed between the preovulatory changes in endogenous estrogen concentration and in mucus NDS or viscoelasticity. One possible explanation that may apply to both of these situations is that the inhibitory influence of progesterone is longlived, requiring for its reversal the cumulative effects of prolonged exposure to low estrogen concentrations. Alternatively, these apparent incongruities may reflect the superimposition of changes in endocervical estrogen receptor levels on changes in estrogen concentration. Acknowledgments.

The authors express their appreciation

to Mr. Joseph Sokoloski and Ms. Sally Schweitzer for excellent technical assistance and to Ms. Pat Park for secretarial assistance.

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REFERENCES 1. Moghissi KS: Sperm migration through the human cervix. In The Biology of the Cervix, Edited by RJ Blandau, K Moghissi. Chicago, University of Chicago Press, 1973, p 305 2. Wolf DP, Blasco L, Khan MA, Litt M: Human cervical mucus. I. Rheologic characteristics. Fertil Steril 28:41, 1977 3. WolfDP, Sokoloski J, Khan MA, Litt M: Human cervical mucus. II. Changes in viscoelasticity during the ovulatory menstrual cycle. Fertil Steril 28:47, 1977 4. WolfDP, Sokoloski J, Khan MA, Litt M: Human cervical mucus. III. Isolation and characterization ofrheologically active mucin. Fertil Steril 28:53, 1977 5. Carlborg L: Determination of sperm migration rate in small samples of cervical mucus. Acta Endocrinol (Kbh) 62:732, 1969 6. Kremer J: A simple sperm penetration test. Int J Fertil 10:209, 1965 7. Ulstein M: Evaluation of a capillary tube sperm penetration method for fertility investigations. Acta Obstet Gynecol Scand 51:287, 1972 8. Sokoloski JE, Blasco L, Storey BT, Wolf DP: Turbidimetric analysis of human sperm motility. Fertil Steril 28:1337' 1977 9. Davajan V, Nakamura RM, Kharma K: Spermatozoan transport in cervical mucus. Obstet Gynecol Survey 25:1, 1970 10. Moghissi KS: The function of the cervix in fertility. Fertil Steril 23:295, 1972

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11. Pommerenke WT: Some biochemical aspects of the cervical secretions. Ann NY Acad Sci 97:581, 1962 12. Sanborn BM, Held B, Kuo HS: Specific estrogen binding proteins in human cervix. J Steroid Biochem 6:1107, 1975 13. Sanborn BM, Held B, Kuo HS: Hormonal action in human cervix-II. Specific progestogen binding proteins in human cervix. J Steroid Biochem 7:665, 1976 14. Pommerenke WT: Cyclic changes in physical and chemical properties of cervical mucus. J Obstet Gynecol 52:1023, 1946 15. Nicosia SV, Sokoloski J, Evangelista 1: Endocervical cells in tissue culture: an experimental model for the study of mucus production. Presented at the Ninth Annual Meeting of the Society for the Study of Reproduction, Philadelphia, 1976, p 66 16. Viergiver E, Pommerenke WT: Cyclic variations in the viscosity of cervical mucus and its correlation with amount of secretion and basal temperature. Am J Obstet Gynecol 51:192, 1946 17. Elstein M, MacDonald RR: The relation of cervical mucus proteins to sperm penetrability. J Obstet Gynaecol Br Cg_mmonw 77:1123, 1970 18. Doehr SA, Moghissi KS: Human and bovine cervical mucins. In The Biology of the Cervix, Edited by RJ Blandau, K Moghissi. Chicago, University of Chicago Press, 1973, p 125 19. Meyer FA: Mucus structure: relation to biological transport function. Biorheology 13:49, 1976 20. Litt M, Khan MA, Shih CK, Wolf DP: The role of sialic acid in determining rheological and transport properties of mucus secretions. Biorheology 14:127, 1977