Levels of prostaglandins F2α and E2 in human endometrium during the menstrual cycle

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Levels of prostaglandins F2a and E2 in human endometrium during the menstrual cycle ERIC J. SINGH, PH.D. IRACEMA M. BACCARINI, M.D. FREDERICK P. ZUSPAN, M.D. Chicago, Illinois

A comparison between the prostaglandins found in proliferative and secretory endometrium in normal and tumor-bearing tissues is reported. The content of prostaglandins F 2 a and E 2 is related to the three phases of the menstrual cycle and correlates with cyclic ovarian hormone variations. The menstrual phase is characterized by a high content of PGF 2 a. High prostaglandin levels are found in tissues from patients with pathologic diseases such as adenocarcinoma of the endometrium when compared to normal tissues.

p R 0 S TAG L A N D I N S are a family of biologically potent lipid substances which exert multiple pharmacologic actions. 1 Pickles and associates, 2 in 1965, reported the presence of prostaglandin F2a (PGF 2 a) and prostaglandin E 2 (PGE 2) in both menstrual fluid and the human endometrium. The present study details the levels of PGE 2 and PGF 2a during a normal human menstrual cycle and also in tumor-bearing endometrium.

Material and methods Thirty women of reproductive age who were undergoing surgery for benign gynecologic disease were studied. Each uterus showed no pathologic or histologic disease and was the source of our normal endometrial study tissue. Another group of 12 patients with known gynecologic malignancy (adenocarcinoma of the endometrium) served as the source of tumor-bearing endometrial tissue which was taken from the extirpated uteri. No tumor-bearing endometrial tissues were irradiated before operation. The specimens were obtained from women in the early and late proliferative phases and early and late luteal phases of the menstrual cycle as well as at menstruation. The weights of the endometrium biopsy specimens were in the range of 1 to 3 grams.

From the Department of Obstetrics and Gynecology, The University of Chicago. Supjlorted in part by Ford Foundation Grant No. 690-0108.

Reprint requests: Dr. Frederick P. Zuspan, The Ohio State University, 410 W. lOth Ave., Columbus, Ohio 43210.

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1004 Singh, Baccarini, and Zuspan

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April I, 197'> Obstt:>t. Gynrcnl

Table I. Content of prostaglandins in human endometrium during the menstrual cycle (amounts in nanograms per gram of wet weight of the tissue + standard deviation) I Proliferative phase Prostaglandin

Luteal

I

I

1

Ph~-

Late Early Late Early Group (a) Group (b) Group (c) Group (d)

110 ± 5*

125 ±

4+

!50± 7t 165 ± 8*

125 ± 5+

210 ± 9t

185 ± 9t

400 ± 12+

I

1--

M enstrual Group(e)

:H5 + 10*

545 ± 8*

Comparison of means for significance

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40§ 40§

45§

25§

60[[ 190![

!60ii 145[[

275[[

7511

*Data average of 5 samples. tData average of 4 samples. +Data average of 3 samples. §Not significant. If Significant.

Table II. Prostaglandin levels in human tumor endometrium (adenocarcinoma) during the menstrual cycle (amounts in nanograms per gram of wet weight of the tissue ± standard deviation)

Prost agland in

Comparison or means ror Slgmpcance

Luteal phase*

Proliferative phase*

I

I

Menstrual* Late Early Late Early Group (a) Group (b) Group (c) Group (d) Group (e)

150 ± 3 175 ± 5

176 ± 5 195 ± 4

165 ± 6 350 ± 7

340 ± 8 495 ± 9

375 ± 9

590 ± 7

Group a to b

I

b to c

llt

155t

I c to

d

175+

145t

Id

to e

35t

95t

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d

190t

320t

*Data average of 3 determinations. tNot significant. tSignificant.

The samples of fresh endometrial tissues were ~<:a l;no. tn. T"Arnn.uo r;TH."orl ..... ,;th J.t..I.J. o.:JitA- ... .1.,_&\_., ......... .1. V.I..I...L.._, y V

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dipped into chilled ethanol and· homogenized. A nitrogen atmosphere was used for various procedures 3 to minimize oxidation. All solvents were of analytic reagent grade and redistilled before use. Five milliliters of ethanol was added to 1 Gm. of tissue and filtered. The filtrate was acidified with 1N hydrochloric acid to pH 3, extracted with 10 ml. of petroleum ether, which was discarded, and then extracted three times with 10 ml. of ether. The ether solution was washed once with water, dried with anhydrous sodium suifate, and evaporated at a low temperature with nitrogen. The residue was dissolved in the least amount of ethanol and kept at 4° C. The methods for the separation of prostaglandins by silica gel-impregnated glass fiber paper chromatography and quantitation by transmission densitometry after charring with concentrated sulfuric acid have been described earlier. 4 • 5 The silica gel-impregnated glass fiber papers were prepared as follows. Glass fiber paper* was impregnated with a supersaturated silicic acid solution which subse*No. 934 AH, obtained from H. Reeve Angel Co., Clifton, New Jersey.

quently gels. The supersaturated silicic acid solu~ tion was prepared by mixing 30 m!. of ammonium chloride solution (5 per cent wjv in water) with 100 ml. of potassium silicate solution (approximately 2 per cent aqueous, prepared by making a 15 to 1 dilution of "Potassium Silicate-Electronics 200"*). Glass fiber paper was cut to 20 by 20 em. and dipped singly into the coating mixture for about 15 seconds. A clean glass rod was passed across both surfaces to drain excess fluid. Small binder clips were used to suspend the paper vertically over a hot plate for 60 minutes to dry, and then the papers were hung in a furnace at 400° C. for 60 minutes. The papers were stored in a clean covered container until used. Standard solutions of PGE 2 and PGF za, each containing 5 ng. per microgram, were prepared in ethanol and kept at 4° C. The standard mixtures nrmt:w·l::!nrlins wPrP nf ... - o eaual -- bv usimr ··--- nrPnared -- r------o--·------volumes. A 5 ,ul aliquot of standard mixture and unknown was applied to the silica gel-impregnated glass fiber paper with a 10 ,u.l microsyringe. t The ~---..~.----

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*Electrochemicals Department, E. I. DuPont de Nemours & Co., Wilmington, Delaware 19898. tHamilton Co., P. 0. Box 307, Whittier, California

90608.

Volume 121 Number 7

chromatogram was developed by the ascending method in a tightly closed rectangular tank to a distance of 15 em. with the solvent. The solvent used was ethyl acetate:water:methanol ( 110:20: 1, v: v: v). The phases were equilibrated overnight before the organic layer was used. The chromatogram was allowed to dry 30 minutes in a forced air hood; then it was sprayed evenly with concentrated sulfuric acid and heated at 180° C. for 30 minutes. The sequence of separation was PGE 2 (Rf = 0.46) and then PGF 2 a (Rf == 0.21). The density of the spots was determined by a densitometer equipped with a recorder. A photovolt thin-layer chromatography densitometer, Model 52C, equipped with a collimating slit aperture of 0.1 by 55 mm. was used. The thin-layer chromatography stage was driven 1 inch per minute. The signal from the photometer was recorded at a chart speed of 2 inches per minute. Peak areas (spot densities) were measured with a planimeter. The unknown amounts were calculated through the help of the standards, and the results are recorded in the Tables I and II. The precision of the method was described earlier. 4 Results

Table I shows the endometrial prostaglandin contents of normal tissues at various phases of the menstn.1al cycle. The dominating fraction v;as PGF 2 (( during all phases of the menstrual cycle. Fig. 1 shows the content of prostaglandins in different phases of the cycle and underscores the cyclic variations of prostaglandins in relation to ovarian hormone changes. The PGF 2 ":PGE 2 ratio was higher in the luteal phase. The significantly higher content of PGF 2 ,. and PGE 2 was found during the period of progesterone secretion after ovulation. Note the rather lower levels of PGF 2 ,. and PGE 2 in the estrogenic phase of the cycle. At the time of menstruation, the prostaglandin vaiues increased stili higher to their highest levels as illustrated in Table I and Fig. 1. Table II shows the prostaglandin levels in human tumor-bearing endometrium during the menstrual cycle. It is noted that tumor-bearing tissues contained a large amount of prostaglandins as compared with normal tissues. The levels of PGF 2 a were significantly higher in the luteal phase as compared with the proliferative phase. Comment

The objective of this study was to obtain data on the amount of endometrial prostaglandins in

Endometrial PGF 2a and PGE2 during menstrual cycle 1005

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PGF2 PGE 2 ....-&

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400

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Fig. 1. Levels of PGF," and PGE, in human endometrium during the menstrual cycle.

both physiologic and pathologic states. To our knowledge, this is the first systemic, complete report of endometrial prostaglandin levels during the menstrual cycle. Our values are higher than the findings of Pickles and associates. 2 The most significant finding '\vas the high prosta-glandin content of the endometrium at menstruation and the dominance of PGF 2 a over PGE 2 • The high contents of PGF 2a that occur in the human endometrium at menstruation must have some clinical significance. The PGF 2 a may be needed to induce vaginal menstrual bleeding. Additionally, the high level of uterine activity noted during menstruation may be related to the elevated levels of PG. Tumor-bearing tissues contain large amounts of PGF 2 a and PGE2. It appears that prostaglandins are also involved in these cancerous lesions. It may be possible to predict preovulation by analyzing the PGE 2 content of endometrium since the level of PGE 2 rises before ovulation; however, the rise is not significant. A reversal occurs at ovulation since PGE 2 decreases and PGF 2 a increases in the endometrium. Similar observations are also seen in human cervical mucus prostaglandins. 6 Pickles and associates 2 found the PGF 2 a/PGE ratio to be higher in patients with primary dysmenorrhea when compared to those with no dysmenorrhea. It is now possible to block ovulation with oral contraceptives and find out whether the

1006

Singh, Baccarini, and Zuspan Am.

prostaglandin content of the endometrium is different. Studies such as these may indeed help us to answer questions for those individuals who have dysmenorrhea. It is our contention that prostaglandin plays a significant role in the human reproductive process.

J.

April I. 197.) Obstf't. Gyt~t·<·ol.

The authors thank Dr. George L. Wied for statistical analysis.

REFERENCES 1

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211, 1974. 2. Pickles, V. R., Hall, W. ]., Best, F. A., and Smith, G. N.: J. Obstet. Gynaecoi. Br. Commonw. i2: 185, 1965. 3. Singh, E. J., and Zuspan, F. P.: AM. J. 0BSTET. GYNECOL. 117: 919, 1973.

4. Singh, E. ]., and Zuspan, F. P.: Chromatographia 7: 200, 1974. 5. Singh, E. J., and Zuspan, F. P.: AM. J. 0BSTET. GYNECOL. i18: 358, 1974. 6. Singh, E. J.: Round Table on Ovulation Prediction, Rome, Italy, April 2-6, 1974. (Abst.)