Cyclic Changes of Cervical Mucus in Normal and Progestin-Treated Women

Cyclic Changes of Cervical Mucus in Normal and Progestin-Treated Women KAMRAN S. MOGHISSI, M.D.

CERVICAL MUCUS appears to have an important function in the process of human reproduction. In response to stimulation by estrogen, cervical glands produce increasing amounts of a characteristic mucoid secretion. 16 At the peak of this secretory activity, prior to ovulation, these glands produce copious amounts of a thin, isotonic mucus which is easily penetrated by the sperm. 5 , 7, 15, 16 Progesterone, on the other hand, is known to bring about both quantitative and qualitative alterations in the cervical secretion. During the luteal phase of the menstrual cycle, cervical mucus has been shown to become scanty in amount, as well as viscous and cellular. During the progestational phase also, such properties as spinnbarkeit and crystallization of the cervical mucus, which characterize estrogen stimulation, are markedly reduced or absent and consequently sperm migration is inhibited. Since endogenous progesterone causes an inhibition of sperm migration through cervical mucus, exogenously administered progestins, as prescribed for oral contraception, might be expected to have a similar effect. The purpose of this report is to present our data on sperm penetrability and the cyclic variations of certain properties of cervical mucus in a group of women during normal menstrual cycles and cycles in which oral progestogens were administered.

MATERIAL AND METHODS

Four healthy, fertile, parous women without clinical evidence of pelvic disease or abnormality were selected for this study. Table 1 shows the From the Department of Obstetrics and Gynecology, Wayne State University School of Medicine and Hutzel (Woman's) Hospital, Detroit, Mich. Supported in part by Grant AM 06705 from the U. S. Public Health Service and aided by a grant from Parke, Davis & Company, Detroit, Mich. I am indebted to Dr. O. W. Neuhaus for his generous help and advice, to Geraldine Habhab for her technical assistance, and to Dr. H. C. Mack for his critical review of the manuscript.

663

FERTILITY & STERILITY

MOGHISSI

664

TABLE 1. Clinical Data of Study Group Jl ell •• '""" dnrn ~--~-

p,,(-itml

No.

Init'ials

Age (yr.)

1 2 3 4

E.E. E.W.

20 22 39 24

LM.

E.G.

Parity

1 2 3 4

Age at menarche (yr.)

12 13 12 12

Cycle length (days)

Menstrual days

28 30 27 28

6 5 3 6

clinical data for these subjects. Samples of cervical mucus were obtained from each woman every 1-3 days (averaging every 2 days) throughout 1 menstrual cycle, beginning with termination of menses. In each instance, clean unlubricated speculum was introduced into the vagina. The cervix was gently cleansed with a dry cotton ball and the mucus aspirated from the cervical canal with a tuberculin syringe (without needle). Each sample of cervical mucus was tested as follows: 1. Quantity Quantity was graded as 1+ to 4+: "1+" indicated approximately 0.1 m!. or less and "4+" denoted 0.4 m!. or more of cervical mucus. 2. Viscosity The viscosity was evaluated in terms of 1+ to 4+ because of the small quantities involved: "1+" was indicative of normal midcycle mucus and "4+" usually denoted a thick, viscous, premenstrual mucus. 3. pH The pH was determined with pHydrion* paper which was introduced into the cervical canal and allowed to become moist. The color change was then read against the standard chart provided by the manufacturer. 4. F ern test Crystallization was recorded in tenns of 1+ to 4+, depending on its intensity: a "1+" rating indicated an atypical fern, while "4+" represented a strong preovulatory ferning pattern. 5. Spinnbarkeit The length of the mucus thread was measured in centimeters and was determined immediately upon collection of the mucus. 6. Cellularity An estimate of the number of leukocytes and other cells was made at the time of sperm penetration studies. When no cells or only occasional cells were observed in each high power field (HPF), the result was recorded as "0"; "1+" indicated not less than 5 cellsjHPF, while "4+" denoted innumerable cells. 7. Sperm penetration test The sperm penetration test was performed

a

*MicroEssential Laboratory, Brooklyn, N. Y.

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665

CHANGES

in vib·o according to a method previously describedY Only freshly ejaculated semen obtained from healthy donors was used. Each sample of semen was examined for adequacy (volume, population density, morphology, and motility) prior to being used for penetration studies. To quantitate the test, the following procedure was adopted: A small amount of mucus and a drop of semen were placed about 3 mm. apart on a microscope slide and were covered gently with a cover slip. Care was taken to prevent overlapping of the samples. The interface between the semen and the cervical mucus was observed vvith a phase-contrast microscope. The number of motile sperm in the first field bordering the interface between the semen and the mucus layer (F 1 ) were counted at 200X magnification 15 min. after the initiation of the test. Another count was made of the sperm in a second field adjacent to the first one. This field, indicated as F 2' was therefore one field removed from the interface; thus studies of it indicated depth of penetration. Usually, several F 1 and F 2 fields were counted and averaged (Fig. 1). Whenever the count exceeded 25jHPF, no attempt was made to continue counting since it could not be done with accuracy. Such results were recorded as "over 25." 8. Protein electrophoresis Samples of cervical mucus obtained at each examination were subjected to electrophoresis in a dilute agar-gel medium according to a technic described elsewhere. 13 The percentages of albumin, globulins, and nonmigrating mucoid were determined in this manner.

Fig. 1. Schematic representation of method of in-vitro sperm penetration test. eM indicates cervical mucus; Sp, sperm; I-F, interface; Ph, phalanges; F l , first microscopic field bordering interface; F 2 , second field adjacent to Fl.

eM /

Sp

\

\

" , ...

Each woman was instructed to record her oral basal body temperature (BBT) throughout the studied cycles. At the time of each pelvic examination, a smear was also obtained from the upper third of the lateral vaginal wall. The smear was stained by the Papanicolaou method and its karyo-

666

MOGHISSI

SPERM PENETRATION SPINNBARKEIT (em)

FERN TEST

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FERTILITY

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,

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; ;

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60

PERCENT PROTEINS

I

I

+ ill-il ~+

+

QUANTITY DAY OF CYCLE

#-

Ll

I

7.5

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7.5 7.57.5

i

!

& STERILITY

1

,

j

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8 10 12' 14 16 18 20 22 24 26 28 30

98_

BST

97

Fig. 2. Graphs representing various properties of cervical mucus dming normal (A) and progestin-treated (B) cycles in Subject 1. Arrows indicate probable day of ovulation dming normal cycle.

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17. No.5. 1966

CYCLIC

Mucus

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667

pyknotic index (KI) was determinedY Data obtained from the studies of cervical mucus were correlated with the BBT and KI for each woman. From Day 5 to Day 25 of the second cycle, each woman received one tablet containing 2.5 mg. of norethindrone acetate and 0.05 mg. of ethinyl estradiol (Norlestrin*) nightly before retiring. Withdrawal bleeding usually occurred 2-3 days after termination of this regimen. On the fifth day following the onset of bleeding a new cycle of 20 days of administration of Norlestrin was begun. In this manner, cyclic courses of therapy were continued for 3 cycles. Samples of cervical mucus were collected from each woman every 1-3 days throughout the third treated cycle and tested according to the technic outlined above. Karyopyknotic indices were determined from vaginal smears and BBT records were kept as in the control cycle. Endometrial biopsies were obtained from each woman at the onset of the menstntal period following the control cycle and on the last day of the progestin-treated cycle. RESULTS

Data obtained from cervical mucus and vaginal smear studies were plotted separately with the BBT for each woman during both the normal and the progestin-treated cycles (Fig. 2-5). Nonnal Cycles

Endometrial biopsies taken at the end of normal cycles in all 4 cases demonstrated advanced secretory changes, confirming the occurrence of ovulation. The peak of the KI for the vaginal smears usually preceded or coincided with the rise of the BBT (Fig. 6). Secretion of cervical mucus increased considerably at midcycle (A portions of Fig. 2-5). Although the amount of mucorrhea varied in the 4 women, its maximum in each case coincided with the apparent time of ovulation. A decline in the amount of mucus was usually noted after ovulation. Coincident with mucorrhea, there was a decrease in the cell content and viscosity of the mucus. This finding is in agreement with the observations of other investigators. 5 ,16 The pH of cervical mucus tended to increase at ovulation time. Contrary to the findings of Lamar et al., who noted alkalinity only at midcycle, in our subjects the pH remained alkaline throughout the cycle. A noteworthy difference of pH was usually observed between the cervical mucus pool at the portio and the endocervical mucus; invariably the latter was more alkaline. Since our measurements were performed on endocervical *Parke, Davis & Company.

668

MOGHISSI

SPERM PENETRATION SPINNBARKEIT

& STERILITY

L 20f-10

1ri ~

(em)

FERN TEST

FERTILITY

LT

.4 .2

pH

7.

5

1~ 1

7.~ 7. 7.~

88

8

* ...... *

CELLS VISCOSITY QUANTITY DAY OF CYCLE

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DAY OF CYCLE

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SPERM PENETRATION 'TICI - 0 1 0 0 0 10 SPINNBARKEIT (em) f J. II II FERN TEST +2 I I,

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PERCENT PROTEINS ()oo ..... <) Albumin ........ Globulin

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024681012141618202224262830 DAY Of CYCLE

Fig. 3. Graphs representing various properties of cervical mucus during normal (A) and progestin-treated (B) cycles in Subject 2. Arrows indicate probable day of ovulation during normal cycle.

VOL.

17, No.5, 1966

CYCLIC

-~-

Mucus

669

CHANGES

------ ------

SPERM PENETRATION

20

,

I

I

IF, ~F,

10

SPIN NBARKE IT (em)

FERN TEST pH

IH

CELLS VISCOSITY QUANTITY DAY OF CYCLE

28

0

BBT PERCENT PROTEINS " .... <)

Albumin

60 50 40

®--4 Globulin

30

-

20

Mucoid (N.MF.)

10 0

CD

I

18 20 22 24 26 28

PERCENT PROTEINS 0---<>

Albumin

00---@

Globulin

_ _ _ Mucoid (N.M.F.l

B

DAY OF CYCLE

Fig. 4. Graphs representing various properties of cervical mucus during normal (A) and progestin-treated (B) cycles in Subject 3. Arrows indicate probable day of ovulation during normal cycle.

()70

FERTILITY

MOGHISSI

SPERM PENETRATION SPINNSARKEIT (em) FERN TEST

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PERCENT PROTEINS

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& STERILITY

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SPERM PENETRATION NSARKEIT(cm) SPIN_ ______

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98

60

PERCENT PROTEINS

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pH CELLS VISCOSITY QUANTJIY DAY OF CYCLE

SST

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,- --,--- --1o 0 o

ToTO 6O 0 6 --1- .T=-~ 2 15

--------~

li'tfr

Jnt1

8 10 12 14 16 18 20 22 24 26 28 30 DAY OF CYCLE

Fig. 5. Graphs representing various properties of cervical mucus during normal (A) and progestin-treated (B) cycles in Subject 4. AlTOWS indicate probable day of ovulation during normal cycle.

VOL.

17, No.5, 1966

CYCLIC

Mucus

671

CHANGES

mucus the chance of contamination of the mucus by vaginal fluid was eliminated. The crystallization pattern appeared at Day 9 or 10 of the cycle and reached its maximum at the time of ovulation. Immediately after ovula- -

- --r-

I\V

97 SST

98 97

......... l'

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8

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,-

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Fig. 6. Correlation of BBT and Karyopyknotic index of vaginal smears for normal and progestin-treated cycles in Case 4. Arrow indicates probable day of ovulation during normal cycle.

-

1-', '1\

"'..... 10 12

14

i"',~.f ..

16 18 20 22 24 26 28 30

DAY OF CYCLE

tion, the ferning became either atypical or disappeared completely. This is in accordance with the observations of Roland. Spinnbarkeit was usually maximal at the time of ovulation. Its decline thereafter was more gradual than the decrease in the other factors of the mucus which were studied. In-vitro sperm penetrability began at approximately the ninth day of the cycle and increased gradually to its peak value at ovulation. Mter ovulation the sperm penetrability persisted for several days for 3 subjects, but in 1 instance (Case 1), it was suddenly inhibited completely after ovulation. The optimal changes of cervical mucus, increase in quantity and pH, decrease in viscosity and cell content, maximal ferning and spinnbarkeit, and greatest sperm penetrability, coincided well with the rise of basal body temperature. They, also, paralleled the characteristic changes in proteins of the cervical mucus: the midcycle decrease of albumin and the increase of nonmigrating mucoid levels. Electrophoretic determination of the cervical mucus proteins showed a number of consistently demonstrable changes reported elsewhere,12 Briefly, the albumin level decreased markedly while the mucoid level increased, immediately before or coinciding with the time of ovulation. Shortly after ovulation, these changes were reversed; the albumin level increased while the mucoid level decreased. There was no significant change in the globulin content of the cervical mucus during the menstrual cycle. Of particular interest was the appearance of a second, less pronounced

672

MOGHISSI

FERTILITY & STERILITY

episode of outpouring of cervical mucus in 2 cases just prior to menstruatIOn. Associated with this mucorrhea was the appearance of atypical leming, some increase in spinnbarkeit, slight reduction in viscosity, and in 1 woman (Case 4), some degree of sperm penetration. Progestin-Treated Cycles

Endometrial biopsies in all 4 cases showed quiescent endometrium consisting of compact stroma containing scattered, small, tubular glands lined by columnar epithelium. There was no evidence of any characteristic secretory activity or decidual reaction. Comparison of the graphs of treated cycles with those of control cycles reveal a number of differences. Throughout the treated cycle, the BBT remained monophasic in all cases. The vaginal smears exhibited a low KI with no demonstrable peak at any time. The relationship of the KI with time was essentially linear (Fig. 6). Cervical mucus was scanty, viscous, and very cellular (B portions of Fig. 2-5). In treated cycles, the normal midcycle mucorrhea did not appear. Although the fem test was generally negative, atypical feming was occasionally observed (Day 11 in Case 3 and Days 5, 11, 22, and 26 in Case 4). The pH of the mucus remained alkaline; there was no tendency toward a midcycle increase in pH as observed during the normal cycle. Spinnbarkeit generally did not exceed 4-5 cm. In all 4 cases, there was a definite inhibition of sperm migration in cervical mucus. In Case 3, a few sluggish sperm were noticed to penetrate the mucus on Day 8 of the cycle, when the mucus showed atypical feming. Similar observations were made on Days 24 and 26 in Case 4. In progestin-treated cycles, the typical cyclic variation of proteins observed during the normal cycle did not appear. The sudden ovulatory decrease of albumin levels and increase of mucoid levels were absent. The postovulatory increase of albumin levels and the concomitant decrease in mucoid levels were also missing. The ratio of the proteins did not appear to follow any particular trend. The globulin content of the mucus, however, appeared to be increased. DISCUSSION

Cyclic variations in the amount and properties of normal cervical mucus has been described by several investigators. 5 , 7, 1r., 16 These observations have been confirmed by our present data. Although sperm penetration may occur several days prior to ovulation, it is maximal just prior to the time of ovulation and may persist to a lesser degree several days after

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CYCLIC

Mucus

CHANGES

673

ovulation. Our data support the original observation of Seguy and Vimeux, that cervical secretion remains alkaline throughout the menstrual cycle. The premenstrual mucorrhea reported by Hartman was observed in 2 cases. This has been attributed to a minor rise in estrogen levels 2-3 days before the menses. 4 It is also conceivable that the premenstrual fall of the progesterone level may be responsible for this phenomenon. The variation in the protein pattern-i.e., the preovulatory decrease in albumin and increase in mucoids, with postovulatory reversal of these changes, is of definite interest since it may be used clinically in addition to other criteria to determine the time of ovulation. Administration of norethindrone acetate combined with estrogen markedly altered the properties of the cervical mucus. Throughout this therapy, the mucus was scanty in amount, thick, viscous, and cellular. The crystallization property disappeared while the spinnbarkeit phenomenon was considerably reduced. Sperm migration through such cervical mucus was inhibited. The typical ovulatory protein changes did not occur. The absence of these protein changes during the treated cycle indicate that their appearance in the normal cycle might be connected in some way to the preovulatory rise in estrogen and/or ovulation itself. In a previous study in which a combination of medroxyprogesterone acetate and estrogen (Provest*) was used in cyclic fashion for contraception, we did not notice any consistent and significant alterations in several properties of cervical mucus in women at various times of their menstrual cycle. 10 Persistent ferning and sperm migration were noted in many cases, particularly at midcycle. According to Garcia and Pincus, the cervical mucus changes in women receiving oral contraceptives were inconsistent and did not seem an important mechanism for sterilization. Mears and Grant, however, found a negative postcoital test in women receiving a norethindrone and ethinyl estradiol combination. Zanartu made similar observations and found marked alteration of cervical mucus properties in women who received norethindrone acetate, 6 chloro-6 dehydro- 17a acetoxyprogesterone, norethindrone, or norethynodrel for contraception. Cohen and Perez-Palaez, studying the midcycle mucus, confirmed these findings and noted that dydrogesterone acted in a similar fashion while the sequential oral contraceptives did not bring about any change in the characteristics of cervical mucus. Data presented in this report support the finding that the oral contraceptives containing norethindrone cause profound alterations in the properties of human cervical secretion and interfere with the migration of sperm through the mucus during the entire conrse of the menstrual cycle. *The Upjohn Company, Kalamazoo, Mich.

674

MOGHISSI

FERTILITY & STERILITY

The effectiveness of oral contraceptives depends on several biological changes brought about in the female reproductive tract. Among these, inhibition of ovulation and endometrial changes have been well documented. a, 6, 9 Progestogens of the 19-nor-steroid group appear to produce, in addition, cervical mucus alterations which in themselves provide effective means of conception control. With lower dosage oral contraceptives, this added protection may be of considerable importance since "breakthrough ovulation" has been reported to occur in some cases. 3 ,6 Certain other oral progestins, such as medroxyprogesterone acetate, do not seem significantly to affect the quality of cervical secretion or to inhibit the migration of sperm in the mucus. 10 Their efficacy as a contraceptive agent depends, therefore, on their capacity to inhibit ovulation, produce endometrial changes, or alter other physiological reproductive processes in the female.

SUMMARY Marked alteration of various properties of cervical mucus was found to occur in women receiving an oral contraceptive containing norethindrone acetate. Among the changes observed were a definite inhibition of sperm migration in cervical mucus, absence of crystallization, decreased spinnbarkeit, pronounced cellularity, and modification of the normal protein pattern. Wayne State University School of Medicine 1401 Rivard St. Detroit 7, Mich.

REFERENCES 1. COHEN, M. R, and PEREZ-PELAEZ, M. The effect of norethindrone acetate-ethinyl estradiol, clomiphene citrate, and dydrogesterone on spinnbarkeit. Fertil Steril 16: 141, 1965. 2. GARCIA, C. S., and PINCUS, G. Clinical considerations of oral hormonal control of human fertility. Glin Obstet Gynec 7:844, 1964. 3. GOLDZIEHER, J. W., MOSES, L. E., and ELLIS, L. T. Study of norethindrone in contraception. JAMA 180:359, 1962. 4. HARTMAN, C. G. Science and the Safe Period. Williams & Wilkins, Baltimore, 1962, p. 157. 5. HARVEY, C., LINN, R A., and JACKSON, M. H. Certain characteristics of cervical mucus in relation to the menstrual cycle. J Reprod F ertil 1: 157, 1960. 6. HOLMES, R L., and MANDL, A. M. Oral contraceptives: assessment of their mode of action. Lancet 1:1174, 1962. 7. LAMAR, J. K., SHETTLES, L. B., and DELFS, E. Cyclic penetrability of human cervical mucus to spermatozoa in vitro. Arner J PhysioI129:234, 1940. 8. MEARS, E., and GRANT, E. C. G. Anovlar as an oral contraceptive. Brit Med J 2:75, 1962.

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9. MOGIDSSI, K. S., ROSENTHAL, A., and Moss, N. Provera-ethinyl estradiol for control of conception. lnt J Fertil 8:703, 1963. 10. MOGHISSl, K. S. Symposium on Provest. lnt J FertiI8:746, 1963. 11. MOGIDSSl, K. S., DABlCH, D., LEVINE, J., and NEUHAUS, O. W. Mechanism of sperm migration. Fertil Steril 15: 15, 1964. 12. MOGIDSSI, K. S., and NEUHAUS, O. W. Cyclic changes of cervical mucus proteins. Amer J Obstet Gynec In press. 13. MOGIDSSI, K. S., NEUHAUS, O. W., and STEVENSON, C. S. Composition and properties of human cervical mucus. I. Electrophoretic separation and identification of proteins. J Clin Invest 39:1358, 1960. 14. ROLAND, M. The office application of fern test. Clin Obstet Gynec 5:218, 1962. 1.5. SEGUY, J., and VIMEUX, J. Contribution a l'etude des sterilites inexpliques: Etude de l'ascension des spermatozoides dans les voies genitales basses de la femme. Gynec Obstet (Paris) 27:346, 1933. 16. VIERGlVER, E., and POMMERENKE, W. T. Cyclic variations in the viscosity of cervical mucus and its correlation with amount of secretion and basal temperature. Amer J Obstet Gynec 51: 192, 1946. 17. WIED, G. I. Suggested standard for karyopykrosis. Fertil Steril 6:61, 1955. 18. ZANARTU, J. Effect of synthetic oral gestagens on cervical mucus and sperm penetration. lnt J Fertil 9:225, 1964.

Symposium on the Ovary The Departments of Gynecology and Obstetrics of Harper Hospital and Wayne State University School of Medicine will present a Symposium: The Ovary: Its Physiology and Pathology, Nov. 9 and 10, 1966. Participants include the following: Richard Blandau, Ph.D., M.D., Seattle, Wash., David H. Carr, M.B., Ch.B., Ph.D., Ontario, Canada, Prof. Carl Gemzell, Uppsala, Sweden, Joseph W. Goldzieher, M.D., San Antonio, Tex., Robert B. Greenblatt, M.D., Augusta, Ga., Herbert S. Kupperman, M.D., New York, N. Y., Somers H. Sturgis, M.D., Boston, Mass., and J. Donald Woodruff, M.D., Baltimore, Md. For information, write Harold C. Mack, M.D., Program Chairman, Harper Hospital, 3825 Brush St., Detroit, Mich. 48201.