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Conductivity of Cervical Mucus During the Menstrual Cycle and Pregnancy
Conductivity of Cervical l\lucus During the Menstrual Cycle and Pregnancy HERBERT A. PLATT, PH.D., ELIZABETH B. CONNELL, M.D., and MARTIN L. STONE, M.D.
THE IMPORTA...~CE of cervical mucus in human reproduction has been recognized for many years. As long ago as 1868 it was observed that cervical mucus exhibited particular characteristics, and that these underwent marked and cyclic variation in the human female during the reproductive cycle.4 The estrogen or proliferative phase prior to ovulation shows decreasing viscosity and increasing volume, spinnbarkeit, ferning, and sperm penetration, all maximum at midcycle. The reverse situation obtains in the progesterone or secretory phase of the ovulatory cycle (Fig. 1). In recent years multiple analysis of cervical mucus have been conducted using many clinical, some chemical, but relatively few physicochemical studies. Chemical assays have shown that the specific properties of cervical secretions are due primarily to glycoproteins. 5 These highly reactive macromolecular materials associate with the equally reactive albumin and globulins to form aggregates of varying size. It is probable that the molecular weight of these particles changes during the cycle since the osmotic pressure, which is directly related to molecular weight, has been demonstrated to fluctuate. 1 Although the rates of mucus secretion vary considerably throughout the cycle, the salt concentration of wet mucus, measured as sodium chloride, shows little variation, remaining constant near isotonicity.s This fact would support the belief that the salt concentration produces no large net effect on the molecular binding. In all probability, a changing sodium:potassium ratio is the significant variable rather than salt concentration per se. From the Department of Obstetrics and Gynecology, New York Medical College, New York, N. Y. Presented at the 23rd Annnal Meeting of The American Fertility Society, Washington, D. C., Apr. 14-16, 1967.
85
86
PLATT ET AL.
FERTILITY
&
STERILITY
Many physicochemical aspects of cervical mucus remain to be explored. The objective of the present report is to demonstrate the variability of one of these, conductivity, throughout the menstrual cycle and normal pregnancy. Fern
o
+1
+1
7
+2
+2
+3
+3
14 Cycle
+2
+2
21
+1
28
Day
Fig. 1. Cyclic changes in cervical mucus.
The basic constituents of cervical mucus are now fairly well determined: water, inorganic salts, low molecular weight compounds and fragments, and glycoproteins of high molecular weight. These substances are all electrolytes-that is, are ionic in solution and therefore conduct electric current. Electrolytic conductivity is a measure of the ability of a solution to carry an electric current; it is nonspecific, and all ions in the solution contribute. This is in contrast to the small influence of ions other than the hydrogen ion on a pH measurement. The equipment and methods used for measuring conductivity and pH are entirely different. Conductivity measurements involve resistance determination by an AC Wheatstone bridge, while pH measurements require a DC potential difference determination. Changes in electrolytic conduction reflect the degree of dissociation of the electrolyte molecules, the interaction of the ions formed both with one another and with the solvent, and the forces acting on the ions as they move through the solvent. 6 MATERIAL AND METHODS
Cervical mucus specimens were obtained from 2 groups of patients attending the Obstetric and the Family Planning Clinics at Metropolitan Hospital.
VOL.
19, No.1, 1968
CONDUCTIVITY OF CERVICAL
Mucus
87
The first group consisted of 159 nonpregnant and untreated patients, who had no evidence of pelvic disease of any type. The second group was made up of 140 pregnant women, whose pregnancies were uncomplicated, and who also exhibited no evidence of pelvic infection or other abnormality. A sterile unlubricated speculum was placed in the vagina, the cervix exposed, and a specimen of mucus removed, and placed on a tared cover slip. The slip was then reweighted, placed in a known volume of distilled water, and the entire sample frozen. Prior to the conductivity measurement, the specimen was allowed to come to room temperature, and the mucus was dispersed by agitation. The conductance was measured with a glass dip cell (cell constant = 10.0/cm.) and a conductivity meter (Model RC-16B2*). Since conductivity varies with temperature, a reading was taken at each determination, and the conductivity value corrected to 25.0° C. In his basic studies of electrolytes, Kohlrausch2 found that the equivalent conductance, a function of the specific conductance, is a linear function of the square root of the concentration for strong electrolytes at high dilutions. Since the equivalent conductance is directly related to the specific conductance, the original relationship may be simplified to: L
= Lo + b yC
where L is the specific conductance of a diluted mucus specimen at 25.0° c.; C, the concentration of the mucus specimen-that is, milligrams of mucus per 10 m!. diluent; b, a constant dependent only on the temperature and the solvent; and La' a constant, the intrinsic specific conductance of a diluted mucus specimen at 25.0° C.-that is, a specific characteristic of the mucus sample at infinite dilution. A measurement of the specific conductance was made for each sample. The data were grouped by cycle day for the nonpregnant group and by month of gestation for the pregnant group. Conductance was plotted against the square root of mucus concentration since there has been shown a linear relationship between these functions. Since this type of relationship describes many lines, the principle of "least squares" was applied to determine the line which best described the data. This principle states that the line of best fit may be computed from the sums of the squares of the value deviations, and that there can only be one line having this qualification. The intercept value thus obtained is the intrinsic specific conductance. The slope values, b, were also plotted to confirm their constancy throughout the menstrual cycle and pregnancy. *Beckman Instruments, Inc., Cedar Grove Operations, Cedar Grove, N. J.
88
PLATT ET AL.
FERTILITY
& STERILITY
RESULTS
Specimens from the untreated patients were grouped by time intervals of 3 days. Values from the pregnant patients were pooled in 30-day time segments throughout the period of gestation. Figures 2 and 3 show the changes observed in the intrinsic specific conductance of cervical mucus as seen in the normal cycle and pregnancy. 40 Ul (I)
(I)
8''::
..... ro
20
til:>
o ---------------------------________________________ _
. o ~
(I)
til 0
-20
I:
(J
ro
-,.; -I--'
~
-,.;
g -40 '1J
H I: 0 I:()
-I--'
H
-60
30
o
6
12
18 Cycle Day
24
30
Fig. 2. Changes in intrinsic specific conductance of cervical mucus, untreated nonpregnant patientc;. 40 Ul (I)
(I)
P,::S
~ ';ri 20 til:>
--
"-
. . . - - . - - ...... . -
...-
.......1--......... ,.-_ ..........
01---------------_____________________
.
(J
~ (1)-20 til 0
o
I:
~
g -40
30
12
III
'''; -I--'
'''; '1J
H I:
-I--'
0
~ ()-60--'---~----~--~----._--~----._--_P----__--~ o 60 120 180 240 Days of gestation
Fig. 3. Changes in intrinsic specific conductance of cervical mucus during pregnancy.
VOL.
19, No.1, 1968
CONDUCTIVITY OF CERViCAL
Mucus
89
Above each conductivity curve may be seen the line for the constant, b, which reaffirms the validity of the equation. Figure 2 show the curve obtained in the untreated cycle of the nonpregnant patient. The value appearing at each point indicates the number of samples entering into the computation of that particular point. A sharp rise in conductivity is noted late in the proliferative phase of the cycle. This is followed by a lower level during the early secretory and a second rise in the late secretory phase. Figure 3 shows the curve of conductivity during pregnancy. Again the number of specimens used to determine a single point is shown along the curve. An early rise is followed by a decrease near the end of the first trimester. The level again rises to about the same height, falling off near term. DISCUSSION
The exact significance of the variation in the intrinsic specific conductance of cervical mucus as seen in the untreated or the pregnant woman must remain, at the moment, a matter of conjecture. It would appear that conductivity is greatest in the untreated cycle when the estrogen level is high during the proliferative stage. It becomes less when the progesterone increase occurs, again rising as progesterone levels fall. A similar situation may obtain during pregnancy, owing to the relatively greater increase in the progesterone levels. In the pregnant patients less variation is seen in the range of conductivity than in the untreated patients, perhaps on the same basis. It is suggested that the physical structure of cervical mucus is equivalent to a dual-phased gel. During periods of unopposed estrogen, the mucus may hydrate, the over-all concentration of ionizable materials remaining constant per unit volume of mucus. Progesterone and/or its metabolites conversely may oppose estrogen, and may dehydrate the glycoprotein entities by serving as "bridges" between them and actually "squeezing out" water, yielding a greater concentration of ionizable materials per unit volume. During pregnancy this effect may be related to mucus plug formation. SUMMARY
The specific conductance of cervical mucus specimens obtained from 159 untreated, nonpregnant patients and from 140 pregnant patients was measured, grouped, and expressed as the intrinsic specific conductance. The intrinsic specific conductance variations of cervical mucus in the normal cycle and pregnancy were graphically expressed..
90
PLATT ET AL.
FERTlLITY & STERILITY
Changes in conductivity of cervical mucus would appear to reflect the estrogen:progesterone ratio. Additional specimens from patients receiving a variety of hormonal preparations are currently being analyzed in an attempt to elucidate further this possible relationship. New York Medical College 1249 Fifth Ave. New York, N. Y. 10029
REFERENCES 1. BERGMAN, P., and LUND, C. C. Acta Obstet Gynec Scand 30:267, 1951. 2. CLASSTONE, S. Textbook of Physical Chemistry (ed. 2). Van Nostrand, Princeton, N. J., 1956, 893 pp. 3. ODEBLAD, E., WESTIN, B., and MALMFORS, K. C. Acta Radiol 49: 137, 1958. 4. SIMS, J. M. Brit Med J 2:465, 492, 1868. 5. WERNER, 1. Acta Soc Med Upsal 58: 1, 1953. 6. PLATT, H. A. Ann NY Acad Sci 130:925, 1966.
Society for the Study of Reproduction The SOciety for the Study of Reproduction is organizing flights to attend the meeting of the Society for the Study of Fertility at Newcastle, England, on July 9-13, 1968, and the Sixth International Congress on Animal Reproduction and Artificial Insemination in Paris, July 21-26, 1968. Flight arrangements for either or both meetings are available at low group rates through the Conlin Travel Agency. For further information, please contact A. T. GREGOIRE, PH.D., Department of Obstetrics and Gynecology, 121-C, Women's Hospital, University of Michigan Medical Center, Ann Arbor, Mich. 48104.
THE IMPORTA...~CE of cervical mucus in human reproduction has been recognized for many years. As long ago as 1868 it was observed that cervical mucus exhibited particular characteristics, and that these underwent marked and cyclic variation in the human female during the reproductive cycle.4 The estrogen or proliferative phase prior to ovulation shows decreasing viscosity and increasing volume, spinnbarkeit, ferning, and sperm penetration, all maximum at midcycle. The reverse situation obtains in the progesterone or secretory phase of the ovulatory cycle (Fig. 1). In recent years multiple analysis of cervical mucus have been conducted using many clinical, some chemical, but relatively few physicochemical studies. Chemical assays have shown that the specific properties of cervical secretions are due primarily to glycoproteins. 5 These highly reactive macromolecular materials associate with the equally reactive albumin and globulins to form aggregates of varying size. It is probable that the molecular weight of these particles changes during the cycle since the osmotic pressure, which is directly related to molecular weight, has been demonstrated to fluctuate. 1 Although the rates of mucus secretion vary considerably throughout the cycle, the salt concentration of wet mucus, measured as sodium chloride, shows little variation, remaining constant near isotonicity.s This fact would support the belief that the salt concentration produces no large net effect on the molecular binding. In all probability, a changing sodium:potassium ratio is the significant variable rather than salt concentration per se. From the Department of Obstetrics and Gynecology, New York Medical College, New York, N. Y. Presented at the 23rd Annnal Meeting of The American Fertility Society, Washington, D. C., Apr. 14-16, 1967.
85
86
PLATT ET AL.
FERTILITY
&
STERILITY
Many physicochemical aspects of cervical mucus remain to be explored. The objective of the present report is to demonstrate the variability of one of these, conductivity, throughout the menstrual cycle and normal pregnancy. Fern
o
+1
+1
7
+2
+2
+3
+3
14 Cycle
+2
+2
21
+1
28
Day
Fig. 1. Cyclic changes in cervical mucus.
The basic constituents of cervical mucus are now fairly well determined: water, inorganic salts, low molecular weight compounds and fragments, and glycoproteins of high molecular weight. These substances are all electrolytes-that is, are ionic in solution and therefore conduct electric current. Electrolytic conductivity is a measure of the ability of a solution to carry an electric current; it is nonspecific, and all ions in the solution contribute. This is in contrast to the small influence of ions other than the hydrogen ion on a pH measurement. The equipment and methods used for measuring conductivity and pH are entirely different. Conductivity measurements involve resistance determination by an AC Wheatstone bridge, while pH measurements require a DC potential difference determination. Changes in electrolytic conduction reflect the degree of dissociation of the electrolyte molecules, the interaction of the ions formed both with one another and with the solvent, and the forces acting on the ions as they move through the solvent. 6 MATERIAL AND METHODS
Cervical mucus specimens were obtained from 2 groups of patients attending the Obstetric and the Family Planning Clinics at Metropolitan Hospital.
VOL.
19, No.1, 1968
CONDUCTIVITY OF CERVICAL
Mucus
87
The first group consisted of 159 nonpregnant and untreated patients, who had no evidence of pelvic disease of any type. The second group was made up of 140 pregnant women, whose pregnancies were uncomplicated, and who also exhibited no evidence of pelvic infection or other abnormality. A sterile unlubricated speculum was placed in the vagina, the cervix exposed, and a specimen of mucus removed, and placed on a tared cover slip. The slip was then reweighted, placed in a known volume of distilled water, and the entire sample frozen. Prior to the conductivity measurement, the specimen was allowed to come to room temperature, and the mucus was dispersed by agitation. The conductance was measured with a glass dip cell (cell constant = 10.0/cm.) and a conductivity meter (Model RC-16B2*). Since conductivity varies with temperature, a reading was taken at each determination, and the conductivity value corrected to 25.0° C. In his basic studies of electrolytes, Kohlrausch2 found that the equivalent conductance, a function of the specific conductance, is a linear function of the square root of the concentration for strong electrolytes at high dilutions. Since the equivalent conductance is directly related to the specific conductance, the original relationship may be simplified to: L
= Lo + b yC
where L is the specific conductance of a diluted mucus specimen at 25.0° c.; C, the concentration of the mucus specimen-that is, milligrams of mucus per 10 m!. diluent; b, a constant dependent only on the temperature and the solvent; and La' a constant, the intrinsic specific conductance of a diluted mucus specimen at 25.0° C.-that is, a specific characteristic of the mucus sample at infinite dilution. A measurement of the specific conductance was made for each sample. The data were grouped by cycle day for the nonpregnant group and by month of gestation for the pregnant group. Conductance was plotted against the square root of mucus concentration since there has been shown a linear relationship between these functions. Since this type of relationship describes many lines, the principle of "least squares" was applied to determine the line which best described the data. This principle states that the line of best fit may be computed from the sums of the squares of the value deviations, and that there can only be one line having this qualification. The intercept value thus obtained is the intrinsic specific conductance. The slope values, b, were also plotted to confirm their constancy throughout the menstrual cycle and pregnancy. *Beckman Instruments, Inc., Cedar Grove Operations, Cedar Grove, N. J.
88
PLATT ET AL.
FERTILITY
& STERILITY
RESULTS
Specimens from the untreated patients were grouped by time intervals of 3 days. Values from the pregnant patients were pooled in 30-day time segments throughout the period of gestation. Figures 2 and 3 show the changes observed in the intrinsic specific conductance of cervical mucus as seen in the normal cycle and pregnancy. 40 Ul (I)
(I)
8''::
..... ro
20
til:>
o ---------------------------________________________ _
. o ~
(I)
til 0
-20
I:
(J
ro
-,.; -I--'
~
-,.;
g -40 '1J
H I: 0 I:()
-I--'
H
-60
30
o
6
12
18 Cycle Day
24
30
Fig. 2. Changes in intrinsic specific conductance of cervical mucus, untreated nonpregnant patientc;. 40 Ul (I)
(I)
P,::S
~ ';ri 20 til:>
--
"-
. . . - - . - - ...... . -
...-
.......1--......... ,.-_ ..........
01---------------_____________________
.
(J
~ (1)-20 til 0
o
I:
~
g -40
30
12
III
'''; -I--'
'''; '1J
H I:
-I--'
0
~ ()-60--'---~----~--~----._--~----._--_P----__--~ o 60 120 180 240 Days of gestation
Fig. 3. Changes in intrinsic specific conductance of cervical mucus during pregnancy.
VOL.
19, No.1, 1968
CONDUCTIVITY OF CERViCAL
Mucus
89
Above each conductivity curve may be seen the line for the constant, b, which reaffirms the validity of the equation. Figure 2 show the curve obtained in the untreated cycle of the nonpregnant patient. The value appearing at each point indicates the number of samples entering into the computation of that particular point. A sharp rise in conductivity is noted late in the proliferative phase of the cycle. This is followed by a lower level during the early secretory and a second rise in the late secretory phase. Figure 3 shows the curve of conductivity during pregnancy. Again the number of specimens used to determine a single point is shown along the curve. An early rise is followed by a decrease near the end of the first trimester. The level again rises to about the same height, falling off near term. DISCUSSION
The exact significance of the variation in the intrinsic specific conductance of cervical mucus as seen in the untreated or the pregnant woman must remain, at the moment, a matter of conjecture. It would appear that conductivity is greatest in the untreated cycle when the estrogen level is high during the proliferative stage. It becomes less when the progesterone increase occurs, again rising as progesterone levels fall. A similar situation may obtain during pregnancy, owing to the relatively greater increase in the progesterone levels. In the pregnant patients less variation is seen in the range of conductivity than in the untreated patients, perhaps on the same basis. It is suggested that the physical structure of cervical mucus is equivalent to a dual-phased gel. During periods of unopposed estrogen, the mucus may hydrate, the over-all concentration of ionizable materials remaining constant per unit volume of mucus. Progesterone and/or its metabolites conversely may oppose estrogen, and may dehydrate the glycoprotein entities by serving as "bridges" between them and actually "squeezing out" water, yielding a greater concentration of ionizable materials per unit volume. During pregnancy this effect may be related to mucus plug formation. SUMMARY
The specific conductance of cervical mucus specimens obtained from 159 untreated, nonpregnant patients and from 140 pregnant patients was measured, grouped, and expressed as the intrinsic specific conductance. The intrinsic specific conductance variations of cervical mucus in the normal cycle and pregnancy were graphically expressed..
90
PLATT ET AL.
FERTlLITY & STERILITY
Changes in conductivity of cervical mucus would appear to reflect the estrogen:progesterone ratio. Additional specimens from patients receiving a variety of hormonal preparations are currently being analyzed in an attempt to elucidate further this possible relationship. New York Medical College 1249 Fifth Ave. New York, N. Y. 10029
REFERENCES 1. BERGMAN, P., and LUND, C. C. Acta Obstet Gynec Scand 30:267, 1951. 2. CLASSTONE, S. Textbook of Physical Chemistry (ed. 2). Van Nostrand, Princeton, N. J., 1956, 893 pp. 3. ODEBLAD, E., WESTIN, B., and MALMFORS, K. C. Acta Radiol 49: 137, 1958. 4. SIMS, J. M. Brit Med J 2:465, 492, 1868. 5. WERNER, 1. Acta Soc Med Upsal 58: 1, 1953. 6. PLATT, H. A. Ann NY Acad Sci 130:925, 1966.
Society for the Study of Reproduction The SOciety for the Study of Reproduction is organizing flights to attend the meeting of the Society for the Study of Fertility at Newcastle, England, on July 9-13, 1968, and the Sixth International Congress on Animal Reproduction and Artificial Insemination in Paris, July 21-26, 1968. Flight arrangements for either or both meetings are available at low group rates through the Conlin Travel Agency. For further information, please contact A. T. GREGOIRE, PH.D., Department of Obstetrics and Gynecology, 121-C, Women's Hospital, University of Michigan Medical Center, Ann Arbor, Mich. 48104.