PEROXIDASE
J.A.
IN
Blain,
HUMAN CERVICAL MTJCUS MENSTRUAL CYCLE
P. J. Heald,
A.E.
Mack*
DURING
and C.E.
THE
Shaw
Department of Biochemistry, University of Strathclyde, George Street, Glasgow, Gl lXW, Scotland and *Department of Gynaecology Family Planning Clinic, Royal Infirmary, Glasgow, G4 OSF, Scotland
ABSTRACT Peroxidase
was
found to be a normal
constituent
of human
cervical
mucus. Its activity was determined in cervical mucus from 48 women, not taking oral contraceptives, at different times of the menstrual cycle. No correlation of activity was observed with the stage of the cycle.
Accepted
JUNE
1975
for
publication
VOL.
11 NO. 6
April
2,
1975
677
CONTRACEPTION
INTRODUCTION Althoughethere is a considerable interest in the development of a simple method of detection of ovulation, as a means of determining the time of the “safe period” of the menstrual cycle, experimentation in this Any such approach to the problem requires a method area is limited. which is both extremely simple to use and easy to interpret. These considerations suggested that colourimetric methods, based on the presence or absence of enzymes in the cervical mucus, which could be readily adapted for use in “test paper” form, would be of considerable value. It is known that the mucus secreted from the columnar cells of the endocervix is subject to changes in physical properties and chemical composition during the menstrual cycle. These changes are largely determined by the presence or absence of estrogen and progesterone (1). It has been found that the uterine peroxidase of rats may be elevated by While peroxidase does not appear to have been injection of estrogen (2). human salivary peroxidase has been studied in human cervical mucus, shown to be of glandular origin (3) and thus it was of interest to determine whether peroxidase could be detected in human cervical mucus and whether levels of this enzyme would undergo cyclic changes which might be correlated with stages of the monthly cycle. Since it was not feasible to follow changes in enzyme level for individual subjects throughout the monthly cycle, it was assumed that any major pattern of change would emerge if samples were obtained from an adequate number of subjects at different
stages. METHODS
The 48 subjects tested were not taking oral contraceptives or had, after stopping oral contraception, established a normal cycle. Samples of cervical mucus were drawn by syringe into nylon tubing of 2.8 mm external diameter and a length of 4-8 cm containing the mucus was cut off, sealed in a container and deep-frozen to await assay. After thawing, samples of mucus were weighed and homogenised for 30 seconds in pH 6.5 buffer (27.3 ml 0.1 M citric acid, 72.7 ml 0.2 M disodium hydrogen phosphate), the final volume being 8 ml. The majority of the samples were in the weight range lo-50 mg, the largest being Those small enough to introduce likelihood of weighing error 143 mg. were: 6 days 0.8 mg; 9 days 0.2 mg; 13 days 0.4 mg; 22 days 1.5 mg; After centrifugation, 2 ml of the supernatant or of a 30 days 0.5 mg. suitable dilution of this (up to tenfold dilution) were mixed with 2 ml To this was added 0.1 ml of 1% hydrogen saturated guaiacol solution. peroxide solution and the optical density of the brown colour which developed was read in a 1 cm cell at 470 nm at 30-second intervals.
678
JUNE 1975
VOL. 11 NO. 6
CONTRACEPTION
Under these conditions, the rate of increase of optical density was linear with time and change in optical density between 2 minutes and 3 minutes was taken as a measure of peroxidase activity. One unit of activity was defined as that producing an optical density change of 0.25 uiiits under Samples were compared on a basis of units activity assay conditions. Samples were examined for signs of blood. The per gram wet weight. only indications found were slight pink colorations in samples of the twelfth and fifteenth days which were both, in fact, very low in peroxidase activities. RESULTS In all mucus samples examined, peroxidase was present in quantities adequate for assay. As can be seen from the Figure, no general pattern of peroxidase activity which correlates with the menstrual cycle was detected.
p’
l-
T
‘i
!
/
I j ~i
/ !
1/I 2 I:
5 II
I
c x
DAYS
Figure.
Peroxidase activity in cervical mucus of normal women at different days of the menstrual cycle, absence of oral contraceptives.
cycling in the
It is recognised that the results are from different women whose point of ovulation is likely to differ by several days, and, before it can be concluded that peroxidase activity of cervical mucus is not an
JUNE 1975 VOL. 11 NO. 6
679
CONTRACEPTION appropriate marker for detection of ovulation time, it would be necessary to carry out a study in individual subjects. The study is not open to us, on ethical grounds. What the present work shows is that cyclic differences, if they exist, are too low in relation to differences between individuals for any pattern to be detected by the method used here. ACKNOWLEDGEMZNTS We are indebted to Dr. Joan Brotherton for helpful discussion, Schering Chemicals Ltd. for financial assistance and to the Family Planning Association for supply of samples.
to
REFERENCES 1.
2.
Effect of steroid hormones on cervical mucus. In Weiss, G. Advances in Steroid Biochemistry and Pharmacology, (Ed: M. H. Briggs), p. 137, Academic Press, 1970. Lucas,
F. V.,
Neufeld,
H.A.,
Utterback,
J.G.,
Martin,
A. P.
and
Stotz, E. J. The effect of estrogen on the production of a peroxidase in rat uterus. Biol. Chemistry 214, 775, 1955. 3.
680
Wolfe, A.D. and Turner, activity. J. Dental Res.
Studies N.D. 2, 843, 1956.
on salivary
JUNE 1975
peroxidase
VOL. 11 NO. 6
J.A.
IN
Blain,
HUMAN CERVICAL MTJCUS MENSTRUAL CYCLE
P. J. Heald,
A.E.
Mack*
DURING
and C.E.
THE
Shaw
Department of Biochemistry, University of Strathclyde, George Street, Glasgow, Gl lXW, Scotland and *Department of Gynaecology Family Planning Clinic, Royal Infirmary, Glasgow, G4 OSF, Scotland
ABSTRACT Peroxidase
was
found to be a normal
constituent
of human
cervical
mucus. Its activity was determined in cervical mucus from 48 women, not taking oral contraceptives, at different times of the menstrual cycle. No correlation of activity was observed with the stage of the cycle.
Accepted
JUNE
1975
for
publication
VOL.
11 NO. 6
April
2,
1975
677
CONTRACEPTION
INTRODUCTION Althoughethere is a considerable interest in the development of a simple method of detection of ovulation, as a means of determining the time of the “safe period” of the menstrual cycle, experimentation in this Any such approach to the problem requires a method area is limited. which is both extremely simple to use and easy to interpret. These considerations suggested that colourimetric methods, based on the presence or absence of enzymes in the cervical mucus, which could be readily adapted for use in “test paper” form, would be of considerable value. It is known that the mucus secreted from the columnar cells of the endocervix is subject to changes in physical properties and chemical composition during the menstrual cycle. These changes are largely determined by the presence or absence of estrogen and progesterone (1). It has been found that the uterine peroxidase of rats may be elevated by While peroxidase does not appear to have been injection of estrogen (2). human salivary peroxidase has been studied in human cervical mucus, shown to be of glandular origin (3) and thus it was of interest to determine whether peroxidase could be detected in human cervical mucus and whether levels of this enzyme would undergo cyclic changes which might be correlated with stages of the monthly cycle. Since it was not feasible to follow changes in enzyme level for individual subjects throughout the monthly cycle, it was assumed that any major pattern of change would emerge if samples were obtained from an adequate number of subjects at different
stages. METHODS
The 48 subjects tested were not taking oral contraceptives or had, after stopping oral contraception, established a normal cycle. Samples of cervical mucus were drawn by syringe into nylon tubing of 2.8 mm external diameter and a length of 4-8 cm containing the mucus was cut off, sealed in a container and deep-frozen to await assay. After thawing, samples of mucus were weighed and homogenised for 30 seconds in pH 6.5 buffer (27.3 ml 0.1 M citric acid, 72.7 ml 0.2 M disodium hydrogen phosphate), the final volume being 8 ml. The majority of the samples were in the weight range lo-50 mg, the largest being Those small enough to introduce likelihood of weighing error 143 mg. were: 6 days 0.8 mg; 9 days 0.2 mg; 13 days 0.4 mg; 22 days 1.5 mg; After centrifugation, 2 ml of the supernatant or of a 30 days 0.5 mg. suitable dilution of this (up to tenfold dilution) were mixed with 2 ml To this was added 0.1 ml of 1% hydrogen saturated guaiacol solution. peroxide solution and the optical density of the brown colour which developed was read in a 1 cm cell at 470 nm at 30-second intervals.
678
JUNE 1975
VOL. 11 NO. 6
CONTRACEPTION
Under these conditions, the rate of increase of optical density was linear with time and change in optical density between 2 minutes and 3 minutes was taken as a measure of peroxidase activity. One unit of activity was defined as that producing an optical density change of 0.25 uiiits under Samples were compared on a basis of units activity assay conditions. Samples were examined for signs of blood. The per gram wet weight. only indications found were slight pink colorations in samples of the twelfth and fifteenth days which were both, in fact, very low in peroxidase activities. RESULTS In all mucus samples examined, peroxidase was present in quantities adequate for assay. As can be seen from the Figure, no general pattern of peroxidase activity which correlates with the menstrual cycle was detected.
p’
l-
T
‘i
!
/
I j ~i
/ !
1/I 2 I:
5 II
I
c x
DAYS
Figure.
Peroxidase activity in cervical mucus of normal women at different days of the menstrual cycle, absence of oral contraceptives.
cycling in the
It is recognised that the results are from different women whose point of ovulation is likely to differ by several days, and, before it can be concluded that peroxidase activity of cervical mucus is not an
JUNE 1975 VOL. 11 NO. 6
679
CONTRACEPTION appropriate marker for detection of ovulation time, it would be necessary to carry out a study in individual subjects. The study is not open to us, on ethical grounds. What the present work shows is that cyclic differences, if they exist, are too low in relation to differences between individuals for any pattern to be detected by the method used here. ACKNOWLEDGEMZNTS We are indebted to Dr. Joan Brotherton for helpful discussion, Schering Chemicals Ltd. for financial assistance and to the Family Planning Association for supply of samples.
to
REFERENCES 1.
2.
Effect of steroid hormones on cervical mucus. In Weiss, G. Advances in Steroid Biochemistry and Pharmacology, (Ed: M. H. Briggs), p. 137, Academic Press, 1970. Lucas,
F. V.,
Neufeld,
H.A.,
Utterback,
J.G.,
Martin,
A. P.
and
Stotz, E. J. The effect of estrogen on the production of a peroxidase in rat uterus. Biol. Chemistry 214, 775, 1955. 3.
680
Wolfe, A.D. and Turner, activity. J. Dental Res.
Studies N.D. 2, 843, 1956.
on salivary
JUNE 1975
peroxidase
VOL. 11 NO. 6
