Amino Acids in Cervical Mucus D. P. Pederson, A.B., and W. T. Pommerenke, Ph.D., M.D. DURING THE ovulatory phase of the menstrual cycle, the secretions of the cervix are abundant and fluid. At this time they possess rheologic properties of elasticity and spinnbarkeit, to a marked degree. 2 At other times the secretions are scanty and have a high viscosity. These physical attributes are believed to have a direct bearing on the sperm penetrability, which occurs most readily under the conditions of least resistance at mid-cycle. At this time the cervical secretions are richly supplied with carbohydrates/ 7 some of which have been shown to be utilizable by spermatozoa in vitro. 5 Nature thus provides in the female a new substrate for spermatozoa, which functions well to provide a favorable medium for sperm migration, as well as foodstuffs that can be used for their metabolism. We may conclude that the specific physical and chemical characteristics of the cervical mucus at the time of ovulation enhance the success of the fertilization process. MacLeod 6 has shown that the addition of amino acids to a suspension of washed spermatozoa is without effect on the recovery of motility. It was deemed desirable, nevertheless, to investigate the presence of amino acids, for they contribute to the total environment to which spermatozoa are subjected during their migration through the cervix. Methods The subjects who supplied the specimens for this investigation were 7 women, in good health, with normal menstrual cycles and pelvic findings, ranging in age from 20 to 33 years. As far as possible, specimens of cervical mucus were collected daily, From the Department of Obstetrics and Gynecology, University of Rochester. Aided by a grant from the Ortho Research Foundation, Raritan, New Jersey. 527
528 PEDERSON & POMMERENKE [Fertility & Sterility during a period extending from about seven days before to about four days after the phase of maximum secretion, which occurs at about the midinterval of the menstrual cycle. The cervix was exposed by a dry speculum, and the external os cleared of mucus contaminated by the vaginal contents. Mucus was then aspirated from the cervical canal by means of a glass cannula attached to a syringe. Specimens were promptly weighed and subsequently extracted in 80 per cent ethyl alcohol. Samples were stored at -22 c. Amino acids were determined on the alcohol extract, by the method of paper chromatography. 3 4 The alcohol extract was concentrated on a watch glass at room temperature, and transferred to filter paper. A two-dimensional chromatogram was developed, using phenol-water and collidine-lutidinewater as partition systems. Ninhydrin was employed to reveal the position of the amino acids on the paper. Controls of known amounts of various amino acids were chromatographed concurrently in order to aid identification and attempts at a quantitative estimation of the amino acids present. Variations exist in sensitivity of the individual amino acids to the ninhydrin reagent. For example, under the conditions of these experiments, glutamic acid, glycine, alanine, and valine were found to be detectable in amounts as little as 0.2 microgram. However, 3-4 micrograms of lysine or arginine were required to produce a visible spot by reaction with ninhydrin. These variations fall in the range of sensitivity as quoted by Pratt and Auclair. In certain instances the ethanol concentrate was divided into two parts. One aliquot was chromatographed without further treatment, while the other was subjected to hydrolysis in 6N HCl for twenty-four hours before chromatography. This latter procedure hydrolyzes any ninhydrin-reacting peptides. In another experiment, 18 specimens, from 2 subjects, were divided into two approximately equal portions. One was immediately extracted in 80 per cent alcohol and stored at -22 C. The other was incubated at 37 C., from twelve to forty-eight hours, before alcohol extraction. The two portions were then chromatographed concurrently and the resultant amino acid patterns compared. Observations One hundred and sixty-three specimens of mucus, collected from 5 subjects, during 17 menstrual cycles, were analyzed for amino acids. The amino Since cervical mucus is not homogeneous throughout, absolute equality of division is not possible.
Vol. 1, No. 6, 1950] AMINO ACIDS IN CERVICAL MUCUS 529 acids most commonly found are presented in Figs. 1-, and Table 1. Concentrations are plotted against time before and after the phase of maximum secretion of mucus. This phase is objectively determinable, and characteris- 00 HO:;c-~;:l-C.ttz-~H-tG:JH 40 40 MG 20 8 BEFORE ; I.,.. FIGURE 1. Glutamic acid occurrence in cervical mucus; concentration was decreased at the phase of maximal secretion. 20.! - '.:.;. :1. it : io. I,, I t ' 8 6 4 2MAXIMUN2 4 6 BEFORE DAYS SECRETIOfl AFTER FIGURE 2. Taurine was the only amino acid found which is not a protein constituent. It is probably metabolically derived from cysteine. 14 ~H 2 -COOH., MG I() FIGURE 3. Note the low concentration of alanine as it occurred in cervical mucus. MU 2......... ' :'iq~;;a :: 8 6 4 2 MAXIMUM 2 BEFORE CAYS SECRETION 4 AFTER FIGURE 4. Glycine, the simplest amino acid, was found in small amounts throughout the cycle. tic of the period of ovulation. Aspargine, histidine, cysteine, threonine and tyrosine were found in occasional samples. Within any given cycle the concentrations of the different amino acids follow parallel patterns. Greater variation in pattern, however, is noted between different cycles, or between individual subjects. Frequency of
530 PEDERSON & POMMERENKE [Fertility & Sterility occurrence and quantitative degree of spread are indicated. All amino acids were found in decreased concentrations during the ovulatory phase, when total secretion of mucus is greatest. Average minimal concentration of each amino acid occurred from 0-2 days after the day of maximal secretion in all ICH3l2CH -crh-cooh ICHJ2 CH- CH 2-~H -COQH ' ~.. FIGURE 5. Valine, again, occurred in lowest concentration at mid-cycle. FIGURE 6. Leucine and isoleucine were not separately identified by chromatography. HOOC- CH2-9H-COOH 14 KzNOC- ot 2- CHf~H-COOH n 8 BEFORE 4 2MMUM2 4 6 DAYS SECRETICX\1 AFTER FIGURE 7. Aspartic acid was present in much lower concentration than its homologue glutamic acid... MU..-_ FIGURE 8. Glutamine, an amide, was found less frequently than its related glutamic acid. cases except taurine. In this case the minimal concentration occurred 3 days before the day of maximal secretion. Absolute quantitative determination of amino acids by the paper chromatographic method is not possible. The evaluation of area and intensity of color, factors determined by the amount of amino acid present, was sub-
Vol. 1, No. 6, 1950] AMINO ACIDS IN CERVICAL MUCUS 531 jective. A quantity of each amino acid is lost during the development of the chromatogram. Sodium chloride and other salts common to biologic materials may distort the migration of amino acids. The values as expressed, therefore, may have an accuracy no greater than + 30 per cent. ~ ~z-~h-cooh OH NH2 fh;ech2 ~H -COOH r. MG ~.,,. FIGURE 9. Serine, a common hydroxyamino acid, was detected in 20 per cent of the specimens. -8 4 2 MA Z 011 YS SECIIETIQN AFTEII FIGURE. Methionine, an essential sulfur-containing amino acid, was noted in irregular concentrations. LYSINE fh2 - (CH 2 ) 5 -~H -COOH NH2 NH2 HN-CH;rCHtCHfCH-COOH ARGININE ~-NH NHz 20 0 lefor I z z DAYS SECRETION AFTER FIGURE 11. The essential basic lysine was found in larger amounts before the phase of maximum secretion. BEFORE z MAXIMUM DAYS SECRETION FIGURE. There is wide variation in the concentration of arginine, detected in only 11 per cent of the specimens. Hydrolysis of the alcoholic concentrate before chromatography revealed that none of the ninhydrin-reacting spots, present before hydrolysis, were due to peptides. Hydrolysis of any peptides present would yield characteristic degradation products. The storage of the alcohol extracted mucus specimens at -22 C., between collection and chromatography, resulted in no significant effect on the amino acid pattern. z
532 PEDERSON & POMMERENKE [Fertility & Sterility TABLE 1. Occurrence of Amino Acids in 163 Specimens, from 17 Cycles, of 5 Subjects Glutamic Acid 156 Aspartic Acid 87 Taurine 154 Glutamine 57 Alanine 145 Serine 32 Glycine 1 Methionine 31 Valine 1 Lysine 27 Leu cines 93 Arginine 17 Following incubation of one aliquot of a mucus specimen, increased amounts of individual amino acids were noted, when compared with the nonincubated aliquot of the same specimen. It would appear probable that enzyme activity is responsible for this alteration. Further investigation of this hypothesis is in progress. Summary Free amino acids in human cervical mucus were determined by the method of paper chromatography. Seventeen amino acids were identified. A decrease in concentration of amino acids present during the ovulatory phase was noted. Enzyme activity probably contributes to the presence of the amino acids in cervical mucus. REFERENCES l. Breckenridge, M.A., and Pommerenke, W. T.: Fertility and Sterility. In press. 2. Clift, A. F.: Proc. Roy. Soc. Med. 39:1, 1945. 3. Consden, R., Gordon, A. H., and Martin, A. J.P.: Biochem. J. 38:224, 1944. 4. Dent, C. E.: Biochem. J. 43:169, 1948. 5. MacLeod, J.: Am. J. Physiol. 132:193, 1941. 6. MacLeod, J.: Endocrinology 29:583, 1941. 7. Pommerenke, W. T.: Am. J. Obst. & Gynec. 52:23, 1946. 8. Pratt, J. J., Jr., and Auclair, J. L.: Science 8:213, 1948.