of Chlormadinone on Amount of Human Cervical Mucus and Its Glycogen Content
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1 " Effect, of Chlormadinone on Amount of Human Cervical Mucus and Its Glycogen Content A. T. GREGOIRE, PHD, and K. USTAY, MD* THE MODE OF ACTION of orally administered steroids in contraceptive therapy generally is believed to be a suppression of pituitary secretion. It has been suggested, however, that production of cervical mucus which inhibits sperm transport is a major factor in the inhibition of fertility,14 with the exogenous steroids perhaps exerting a direct action on the endocervical glands. 4 The daily administration of 2.0 mg of chlormadinone inhibited ovulation, while ovulation occurred with lower doses without ensuing pregnancy. In further investigations, the administration of chlormadinone either cyclically or continuously resulted in elevated progesterone levels indicative of ovulation. With cyclic administration, ovulation occurred in 27% of the women, and 90% of the postcoital tests contained minimal numbers of spermatozoa. 10 The daily administration of increasing amounts of chlormadinone, p.g, showed that the contraceptive effectiveness was correlated positively with dose level, and the best results obtained with levels between 300 and 500 p.g daily.ll The majority of recent investigations of the steroids utilized for contraception have been limited mostly to evaluation of clinical results following their administration. Some reports, however, have considered the effect of progestagens on biochemical components of the genital tract. There is, for example, a linear increase in the carbonic anhydrase of human endometrium following the administration of either 3,-desoxy-6-methyl-17- acetoxyprogesterone or norethindronep While the a-amylase content of Suppo1'letl by Ford Foundutioll Grunl :17-182, UIIU It GrulIl-in-Aid from lh.. SYIIlt'X Corporation. From the Department of Obstetrics unu Gynecology, University of Michigun Medical School, Center for Research in Reproductive Biology, Ann Arbor. *Ford Foundation Fellow, University of Michigan Medical School, Center for Research in Reproductive Biology. Present address: Department of Obstetrics and Gynecology, Hacettepe School of Medicine, Ankara, Turkey. 938
2 VOL. 20, No.6, 1969 CHLORMADINONE AND CERVICAL Mucus 939 cervical mucus increases with the administration of progestagens in either combined or sequential therapy, II the ectocervical tissue glycogen is not influenced by either exogenous or endogenous progesterone nor does it exhibit cyclic or menopausal changes. 6 MATERIAL AND METHODS A group of 11 volunteers exhibiting normal menstrual cycles were utilized. The volunteers first completed a control cycle with no medication and during the subsequent five cycles received 0.4 mg of chlormadinone acetate* orally daily with no interruption for menstrual flow. At 4-day intervals during the first, third, and fifth cycles, cervical mucus was obtained by aspiration with a 6-in., 15-gauge, blunt-end needle. The mucus was weighed to the nearest milligram and immediately frozen. At the time of chemical determination the mucus was thawed, hydrolyzed with 30% KOH, and the glycogen precipitated with alcohol and hydrolyzed with sulfuric acid. Thc resultant color reaction with anthrone was read at 620 mp..15 Daily vaginal smcars and hasal hody temperatures (BBTs) were obtained during four cycles. The vaginal smears were stained by the Papanicolaou method and thc karyopyknotic index determined by a certified cytologist. Ovulation was determined clinically by evaluation of both the temperature charts and the vaginal smears. The amount of mucus (milligrams) and glycogen (micrograms/ioo mg tissue wet weight) obtained during the control and treated cycles was subjected to an analysis of variance appropriate for unequal numbers of observation. RESULTS AND DISCUSSION Of the 11 individuals who began the experiment, 1 became pregnant and 2 others discontinued because of breakthrough bleeding. The length of the cycle did not differ significantly between the treated and untreated groups. The ovulation rate as determined by BBT and pyknotic index was variable but demonstrated that ovulation occurred in both treated and control cycles. The average amounts of cervical mucus obtained during the control cycle and during administration of chlormadinone are seen in Fig 1 and Table 1. During the control cycle, maximum amounts of mucus were obtained between Days 14 and 17, with the greatest secretion occurring on *Syntex Laboratories, Inc., Palo Alto, Calif.
3 940 GREGOIUE & USTAY FERTILITY & STERILITY Day 16. Administration of chlormadinone resulted in a 68-75% significant decrease (p< 0.01) in the amount of mucus obtained during Cycles 3 and 5 with no peak secretion rates during the treatment cycles. The amount of cervical mucus which can be obtained during a normal cycle is tenfold :> u :> E g u "E.. u '0 ~ '" so 2S c:r" 0 0 \ \ 0 c:>-e ""'" A 28 cycle I contral cycle cycle 3 OAmg chioromaclinone daily A 28 cycles Fig 1. Amount of cervical mucus obtained during control and treated cycles. TABLE 1. Amount of Cervical Mucus and Glycogen During Control and Chlormadinone-Treated Cycles Control Cycle 3 Cycle 5 Ob$erv'litions (No.) Mucus (mg) ± ± ± 2.22 Glycogen (j.tg/ioo mg wet wt) ± ± ± Results are given as average values ± BE.
4 VOL. 20, No.6, 1969 CHLORMADINONE AND CERVICAL Mucus 941 greater during the ovulatory phase than in the pre- and postovulatory phases 13 but is decreased when obtained randomly from individuals receiving Norinyl.4 Our observations confirm both the maximum midcycle secretion and the. inhibition by administration of progestagens. Since ovulation occurred in both groups, whether the inhibition of mucus production is a function of pituitary inhibition or a direct action on the endocervical glands is unknown. The amount of cervical mucus glycogen during the control period was greatest in the latter part of the cycle. With the administration of chlormadinone, the amount of glycogen obtained during the two treatment cycles was almost identical and did not differ significantly from the control. This suggests that chlormadinone at this dose level inhibits the production of mucus but is without effect on the glycogen concentration. The origin of cervical glycogen is enigmatic. Glycogen could not be demonstrated histochemically in either the glandular surface epithelium or in the luminal mucus,1 and may be a product of the uterus 2 either as a result of uterine secretion or cellular destruction. The amount of glycogen in the uterine isthmus is less than that in the corpus,16 and although no cyclic differences were demonstrated, a decrease in concentration was believed to occur during maximum mucus secretion The glycogen content of ectocervical tissue does not vary significantly either during the cycle, with the onset of menopause, or following administration of progestagens for contraceptive therapy.6 Similar results were obtained with the glycogen-synthesizing enzymes in human squamous cervical epithelium. The enzymes responsible for glycogen synthesis, amylophosphorylase and amylo-l,4-1,6-transglucosidase did not exhibit cyclic activity or differences in concentration during the menopause. 5 The function of glycogen in the genital tract is unknown at present. Its presence in the uterus is believed to be a source of nutrition for the fertilized egg until proper anastomosis occurs. However, no implantation occurs in the cervix, and it has been suggested that the quantity of glycogen in cervical mucus is not great enough to support sperm nutrition.1s Investigation of animal cervical glycogen has demonstrated species differences. The hamster cervical glycogen exhibits an increase 10 hr preceding ovulation, with a rise maximum at 4:00 PM, and it rapidly decreases before actual ovulation takes place about 2:00 AM.s In the castrated rat, maximum cervical glycogen synthesis occurs with 25 ILg of estradiof with cervical tissue levels about half the amount that occurs in the uterus, but twice the amount found in vaginal tissue. Therefore, there are differences between the species; whereas both hamster and human cervical tissue contain large amounts of glycogen, in
5 942 GREGOIRE & USTAY FERTILITY & STERILITY the former it exhibits cyclic changes, while the human glycogen content is stable and not influenced by endogenolls or exogenolls progestagens. Chlormadinone administration resulted in an antiestrogenic effect by inhibiting cervical mucus production but did not affect the content of glycogen in the cervical mucus. These and the findings of others5 6 suggest that the glycogen content of the cervical tissue and its mucus is constant and not subjected to either endogenous or administered steroids. SUMMARY The daily administration of 0.4 mg chlormadinone caused a decrease in the amount of cervical mucus produced. The quantity of cervical mucus glycogen, however, did not differ significantly between the control or treated groups. The presence of glycogen in cervical tissue and its mucus, and the variation between species, is discussed. Center for Research in Reproductive Biology Department of Obstetrics and Gynecology University of Michigan Medical School Ann Arhor, Mic REFERENCES 1. ATKINSON, \V. fl., SHETTLES, L. B., and ENGLE, K T. Histochemical studies on the secretion of mucus by the human endocervix. Amer J Obstet Gynec 56:712, BERGMAN, P., and WERNER, 1. Analysis of carbohydrates in human cervical mucus by means of paper partition chromatography. Acta Obstet Gynec Scand 30:273, BRECKENRIDGE, M. A., and POMMERENKE, W. T. Analysis of carbohydrates in human cervical mucus. Fertil Steril 2:29, COHEN, M. R. Cervical mucorrhea and spinnbarkeit in patients taking norethindrone plus mestranol (Norinyl I-mg). Fertil Steril 19:405, FIENBERG, R., and COHEN, R. B. Enzymes of glycogen metabolism in the squamous epithelium of the cervix. A histochemical study. Obstet Gynec 31 :608, GREGOIRE, A. T., and LEDGER, W. J. The glycogen content of human ectocervical tissue of females receiving steroid contraceptive therapy. Fertil Steril 20:91, GREGOIRE, A. T., RAMSEY, H., and ADAMS, A. The effect of various doses of oestradiol 17 -B on glycogen deposition in the rat uterus, cervix and vagina. J Reprod Fertil14:231, GREGOIRE, A. T., and GmNNEss, B. J. Cyclic and preovulatory changes in the glycogen content of the female hamster genital tract. J Reprod Fertil17:427, GREGOIRE, A. T., RANKIN, J., JOHNSON, W. D., RAKOFF, A. K, and ADAMS, A. a-amylase content in cervical mucus of females receiving sequential, non-sequential, or no contraceptive therapy. Fertil Steril18:836, MARTINEZ-MANAUTOU, J., CORTEZ, V., GINER, J., AzNAR, R., CASASOLA, J., and RUDEL, H. Low doses of progestogen as an approach to fertility control. Fert;l Steril17:49, MARTINEZ-MANAUTOU, J., GINER-VELASQUEZ, J., and RUDEL, H. Continuous pro-
6 VOL. 20, No.6, 1969 CHLORMADINONE AND CERVICAL Mucus gestogen contraception: A dose relationship study with chlormadinone acetate. Fertil Steril18:57, NICHOLLS, R. A., and BOARD, J. A. Carbonic anhydrase concentration in endometrium after oral progestins. ArneI' 1 Obstet Cynec 99:829, POMMERENKE, W. T. Cyclic changes in the physical and chemical properties of cervical mucus. ArneI' I Obstet Cynec 52:1023, RUDEL, H., MARTINEZ-MANAUTOU, J., and MAQUEO-ToPETE, M. The role of progestogens in the hormonal control of fertility. Fertil Stel'il16:158, SEIFTER, S., DAYTON, S., NOVIC, B., and MUNTWYLER, E. The estimation of glycogen with the anthrone reagent. Arch Biochern 25: 191, SIMON, H. J. Glycogen production in the isthmus uteri. ArneI' I Obstet Cynec 27:284, VIERGlVER, E., and POMMERENKE, W. T. The determination of reducing substances in human cervical mucus. ArneI' 1 Obstet Cynec 54:459, WERNER, I. The chemistry of cervical mucus. Acta Obstet Cynec Scand.'38 (Suppl. 1) :39, 1959.
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