The Effect of an Intrauterine Device on Myometrial Glycogen in the Hamster*

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Vol. 32, No.1, July 1979 Printed in U.SA.

FERTILITY AND STERILITY Copyright © 1979 The American Fertility Society

THE EFFECT OF AN INTRAUTERINE DEVICE ON MYOMETRIAL GLYCOGEN IN THE HAMSTER*

PAMELA J. MOORE, PH.D.t THOMAS H. ROSENQUIST, PH.D.

Department of Anatomy, Medical College of Georgia, Augusta, Georgia 30912

The myometrial glycogen content of the uterine horns of cycling, castrated, and castrated hormone-treated hamsters was basically unaffected by the presence of an intrauterine device (IUD). The study also indicates that visual evaluation of histochemical localization of glycogen is not adequate to determine minor differences in staining intensity. The "estrogen-like" effect or stimulatory effect of the IUD on uterine glycogen in the rat is not duplicated in the hamster. Fertil Steril32:115, 1979

procedure destroys uterine anatomy and disallows specific localization. For this study we chose a method which allows specific localization and objective quantitation. 18 We report only on myometrial glycogen because changes in endometrial glycogen evoked by the device, which we and others have observed,4, 12 are thought to be mainly a reflection of an increased neutrophil population in the endometrium.

The mechanism of action of the intrauterine device (IUD) in preventing implantation is not known. Of the several modes of action that have been proposed for the IUD, the following three proposals seem to be the most attractive in our opinion: (1) that the IUD induces an inflammatory reaction that forms a hostile uterine environment l -3 ; (2) that the IUD may alter the response of the endometrium to hormones4-7; and (3) that the IUD may exert an "estrogen-like" effect upon the uterus. 8• 11 All of these proposals require further study; we chose to investigate the latter for this report. IUD-induced changes in uterine wet weight, edema, glucosaminoglycan, and glycogen content reportedly mimic the effects of estrogen in several species. 4, 8-12 Several investigators 4, 12-16 have noticed elevated glycogen levels in IUD-containing uteri of rats, rabbits, and hamsters. These glycogen levels have been reported either by direct visual evaluation (usually by grading the intensity of the periodic acid-Schiff (PAS) reaction on a 0 to + + + + + scale) or by biochemical determination via the anthrone procedureY The results of the visual comparison are semiquantitative at best, while the biochemical

MATERIALS AND METHODS

Fifty-six golden Syrian hamsters were examined during two consecutive estrous cycles to establish their regularity and sexual maturity. Twenty-six animals were bilaterally ovariectomized and an intrauterine device of sterile 3-0 silk thread suture was placed through the entire length of the right uterine horn and tied in place. The untouched contralateral horn served as a control. The animals received no further treatmen t for 3 weeks, at which time they were divided into five groups. One group received no treatment and another received only the peanut oil vehicle of the hormones (0.3 mllday). The other three groups received either progesterone (4 mg/day) or estrogen (10 ng/day), or estrogen (10 ng/day) plus progesterone (4 mg/day). Hormone or vehicle treatments were administered subcutaneously at 8:00 A.M. for 3 consecutive days and necropsy was performed 24 hours after the last injection.

Received August 10,1978; revised October 6,1978; accepted November 15, 1978. *Supported by Institutional Grant GRS-5-S01RR5365-15. tTo whom reprint requests should be addressed.

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The Qther 30 animals were not castrated but received an IUD similar to that described above, and the IUD-containing uterine horn .served as a· control. These animals were followed for four complete cycles to ensure that cycling was re-established after the surgery. The animals were killed at one of eleven different times during the 4-day estrous cycle; day 1 represented the day of ovulation. 19 At necropsy each uterine horn was trimmed of excess tissue and the IUD was carefully removed. One half of each uterine horn was fixed for 24 hours in cold 80% pitric acid-alcohol-formalin (PAF), dehydrated in alcohols, embedded in paraffin, and sectioned at 7 pm. Adjacent sections from several different portions of each uterus were mounted on individual slides. Two adjacent slides from each area were used for glycogen localization: one slide was treated with PAS regent, using a modification of the McManus technique,20 while the second slide was treated with a 1.0% solution of malt diastase before the PAS procedure was applied in order to remove glycogen from the tissue. Each slide was studied without knowledge of the time of the cycle or treatment of the animal. The glycogen content of the circular and longitudinal layers of the myometrium was graded on a scale of oto + + + + + according to the number of glycogen granules in each area. To determine more specifically whether glycogen of the myometrium was altered by the experimental treatments, the intensity of the .PAS reaction was determined for both IUD-containing and control uterine horns from tissue sections. Photographs (a maximum of four for each uterine horn) of the myometrium of each control and experimental uterine horn from each animal were obtained with a Nikon SKe photomicroscope with constant light and a Kodak no. 11 geen filter, recording with Panatomic-X film and using x 4 objective. Circular and longitudinal muscle negative images were separated and the intensity of the dye reaction was determined for each negative by the photographic densitometry procedure of Troyer and Rosenquist. 1s Each piece of negative was weighed on a Cahn electronic balance, placed in a Nalgene test tube, and 2 ml of35% nitric acid were added to each tube to dissolve the silver. The tubes were agitated periodically for exactly 2 hours. The concentration ·of silver was then determined with a Perkin-Elmer 103 atomic absorption spectrophotometer attached to a digital multimeter. Photographs were also taken of malt diastase-PAS-treated tissue and

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the negatives were subjected to the above procedure. The amount of silver per negative weight was then calculated. The raw readings were standardized for each group when they were divided by the difference between the reading for a sample taken from an unexposed, developed section of film, and the reading from a section offilm exposed to daylight (completely exposed). The raw read~ ings from the malt diastase-PAS-treated tissue served as a control factor to prove that glycogen had been removed from the myometrium of companion sections. All densitometric data were statistically analyzed by a Student's t-test and are presented in tabular form. A one-way analysis of variance was also applied to the data and the results for the cycling animals are presented in the text. The difference of the means was considered to be statistically significant if P was less than 0.05. The oneway analysis of variance with a Tukey's multiplerange test was applied to the data from the castrated animals and the differences were declared significant (J> > 0.05) if the confidence intervals did not overlap. RESULTS

The results of the densitometric and visual evaluation of uterine myometrial glycogen in castrated animals are shown in Table 1. In only circular muscle of the estrogen plus progesterone~ treated animals did the IUD significantly increase glycogen over control levels. Table 2 demonstrates that the hormonal treatments significantly increased deposition of myometrial glycogen over the levels seen in castrated and castrated-peanut oil-treated animals. The IUD appeared to have an over-all depressive effect on total myometrial glycogen. In only the peanut oil-treated animals did the device have a stimulatory effect on total myometrial glycogen. The results of densitometric and visual evaluation of uterine myometrial glycogen in cycling animals are shown in Table 3. The IUD appears to have a slightly stimulatory effect on myometrial glycogen at most times during the cycle, but the differences are not significant. Only at diestrus I and diestrus IV did the device have· an over-all depressive effect on myometrial glycogen. When the densitometric data were contrasted by an analysis of variance according to the days of the cycle (i.e., day 1 = late estrus and metestrus; day 2 = diestrus I and II; day 3 = diestrus III and IV; day 4 = early proestrus through estrus), a distinct pat-

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EFFECT OF AN IUD ON MYOMETRIAL GLYCOGEN

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TABLE 1. Intensity of the PAS Reaction with Glycogen: IUD Compared with Control Uterine Horns in Castrated Hormone-Treated Hamsters a Effect of IUD (% of control level)

Mean ± SEM of densitometric values of myometriumb Treatment

Circular

Longitudinal

Longitudinal

Circular

No treatment Peanut oil Progesterone Estrogen Estrogen + progesterone

IUD

Control

0.9472 ± 0.055 NSc (±) 1.4630 ± 0.151 ( +) NS 1.5173 ± 0.100 (+++) NS 1.6582 ± 0.092 (+++) NS 2.1252 ± 0.214 (+++)P < 0.05

0.9203 ± 0.046 (0) 1.2032 ± 0.087 ( +) 1.5341 ± 0.067 (+++) 1.7894 ± 0.168 (+++) 1.4825 ± 0.107 (++)

IUD

1.0845 ± ( ++) 2.0707 ± (+++) 1.7706 ± (+++) 1.9474 ± (+++) 1.8217 ± (++++)

Control

1.118 ± 0.094 103% ( +) 1.4245 ± 0.118 122% (++) 1.7667 ± 0.076 99% (+ +) 2.3948± 0.324 93% (++++) 2.1180 ± 0.241143% (+++)

0.085 NS 0.402 NS 0.096 NS 0.177 NS 0.170 NS

(positive) (positive)

97% (negative) 145% (positive)

(negative) 100% (neutral) (negative)

81% (negative)

(positive)

86% (negative

"Data are expressed as the reciprocal ofthe average atomic absorption spectrophotometer reading for each group standardized to a scale of 0 to 1 in units of 10-4 • The raw readings are standardized for each group when they are divided by the difference between the reading for a sample taken from an unexposed, developed section of film and the reading from a section of film exposed to daylight (completely exposed). bBeneath each densitometric mean is the visual grading (0 to + + + +) of myometrial glycogen. eNS, No significant difference. dThe difference of the means was considered to be statistically significant if P was less than 0.05.

tern was seen. In the control uteri, the circular, longitudinal, and total myometrial glycogen content on day 4 was significantly different from that on days 1 and 2. Similarly significant differences were also seen between days 1 and 3 and between days 2 and 3. A similar analysis of myometrial glycogen in IUD-containing uteri indicates parallel significant differences between the above listed days. The device-bearing uterus apparently responds to cyclic fluctuation in myometrial glycogen, as does the control uterus, but at a generally higher level.

DISCUSSION

A previous study by one of US 12 indicated by a subjective grading technique that uterine glycogen in device-bearing hamsters compared favorably with that reported in other species, but the glycogen elevation evoked by the device in the hamster uterus did not appear to be as great as that seen in other animals. Since estrogen increases uterine glycogen in the hamster21. 22 and because the hamster uterus is not as sensitive to exogenous estrogen as is the rat uterus,23 it ap-

TABLE 2. The Effect" of Treatment on Densitometric Values of Myometrial Glycogen in Control and IUD-Containing Uteri of Castrated and Castrated Hormone-Treated Hamstersb Group

Longitudinal

Circular

< 0.001

Control Uteri No treatment Peanut oil Progesterone Estrogen Estrogen + progesterone

P 0.9203 ± 1.2032 ± 1.5341 ± 1.7894 ± 1.4825 ±

Experimental uteri No treatment Peanut oil Progesterone Estrogen Estrogen + progesterone

P < 0.001 0.9472 ± 0.055 a 1.4630 ± 0.151 1.5173 ± 0.100 1.6582 ± 0.092 2.1252 ± 0.214

0.046 a 0.087 0.067 0.168 0.107

b c c c

b b b b

Total

b b c c c

P < 0.001 2.0391 ± 0.1300 a 2.6277 ± 0.1737 a 3.3009 ± 0.1258 b b 4.1842 ± 0.4858 b 4.2432 ± 0.4243

b b b b

P < 0.001 2.0317 ± 0.1361 a 3.1170 ± 0.2596 3.2879 ± 0.1903 3.6055 ± 0.2631 3.3043 ± 0.2631

< 0.001

P 1.1188 ± 1.4245 ± 1.7667 ± 2.3948 ± 2.1180 ±

0.094 a 0.118 a 0.076 0.324 0.241

P 1.0845 ± 2.0707 ± 1.7706 ± 1.9474 ± 1.8217 ±

< 0.001 0.085 a 0.402 0.096 0.177 0.170

b b b b

"Values are means ± standard error of the mean. bThe one-way analysis of variance with Tukey's multiple-range test segregates treatment groups into subsets, subset "a" being significantly different r.p < 0.05) from subsets "b" and "c" and subset "b" significantly different from subsets "a" and "c". Some adjacent subsets (a and b or b and c) within different areas ofthe uterus demonstrate an overlap of treatments (i.e., a treatment is found at the outer limit of one subset and at the inner limit of the next subset). If a treatment value is found in adjacent subsets it is not considered to be significantly different from the mean values directly preceding or following it, but it is included in both subsets because it separates values that are significantly different from each other. Exclusion of any of these overlapping values would present a false statement of the true ranges of each subset.

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TABLE 3. Intensity of the PAS Reaction with Glycogen: IUD Compared with Control Uterine Horns in Cycling Hamsters a Effect of IUD (% of control level)

Mean ± SEM of densitometric values of myometrium Treatment

Circular

Longitudinal Circular

Late estrus Metestrus Diestrus I Diestrus II Diestrus III Diestrus IV Early proestrus Mid-proestrus Late proestrus Early estrus Estrus

IUD

Control

IUD

1.4700 ± 0.017 (±) (NS) 1.4504 ± 0.124 (±) (NS) 2.2818 ± 0.089 (+) (NS) 2.3270 ± 0.142 (+) (NS) 3.1061 ± 0.234 (NS) (++) 3.3152 ± 0.230 (NS) (++) 3.9240 ± 0.367 (+++) (NS) 3.4225 ± 0.119 (+++) (NS) 3.4058 ± 0.305 (+++) (NS) 3.0332 ± 0.269 (NS) (++) 2.2483 ± 0.057 (+) (NS)

1.4088 ± 0.044 (0) 1.2380 ± 0.052 (0) 2.6936 ± 0.201 (+) 2.1617 ± 0.101 (+) 3.0281 ± 0.242 (+) 3.6114 ± 0.075 (++) 3.6783 ± 0.085 (++) 3.5315 ± 0.099 (+++) 3.0802 ± 0.173 (++) 1.8197 ± 0.187 (++) 2.1991 ± 0.050

1.5660 ± 0.070 (+) (NS) 1.6770 ± 0.163 (NS) ( +) 2.5122 ± 0.142 (+++) (NS) 2.5294 ± 0.143 (+++) (NS) 3.6184 ± 0.369 (++++)(NS) 3.5757 ± 0.277 (+++) (NS) 4.6479 ± 0.204 (++++)(NS) 4.2107 ± 0.328 (+++) (NS) 3.6271 ± 0.237 (+++) (NS) 3.6087 ± 0.263 (+++) (NS) 2.3827 ± 0.060 (NS) (++)

(±)

Longitudinal

Control

1.5682 ± 0.056 104% (positive)

100% (neutral)

(±) 1.4154 ± 0.093 117% (positive)

118% (positive)

(+) 3.0001 ± 0.302 (+) 2.3188 ± 0.120 (++) 3.4131 ± 0.340 (++) 4.2401 ± 0.116 (+++) 4.5848 ± 0.404 (+++) 4.0155 ± 0.199 (+++) 3.2900 ± 0.180 (+++) 3.1599 ± 0.221 (+++) 2.3751 ± 0.077 (++)

85% (negative)

84% (negative)

108% (positive)

109% (positive)

103% (positive)

106% (positive)

92% (negative) 107% (positive)

84% (negative) 101% (positive)

97% (negative) 105% (positive) 111% (positive)

110% (positive)

108% (positive)

114% (positive)

102% (positive)

100% (neutral)

aData are expressed as the reciprocal of the average atomic absorption spectrophotometer reading for each group standardized to a scale of 0 to 1 in units of 10-4 • The raw readings are standardized for each group when they are divided by the difference between the reading for a sample taken from an unexposed, developed section of film and the reading from a section of film exposed to daylight (completely exposed). bBeneath each densitometric mean is the visual grading (0 to + + + +) of myometrial glycogen. eNS, No significant difference.

pears plausible that the estrogen-like action ofthe device would not evoke as great an increase in uterine glycogen in the hamster uterus as in the rat. The results of this study confirm earlier reports21. 22 that in castrated and castrated hormone-treated hamsters, the amount and distribution of myometrial glycogen is affected by the different treatments (Table 1 and 2). It is clear (Table 1) that the IUD influences glycogen deposition in the circular and longitudinal muscle, but in only the circular muscle of the estrogen plus progesterone-treated animals does the device have a significant stimulatory effect over control levels. The analysis of variance (Table 2) indicates that the device has an over-all slightly depressi ve effect on total myometrial glycogen. In only the peanut oil-treated hamsters does the IUD have a stimulatory effect on total myometrial glycogen, and this is not a significant increase over the controllevel. The effect of the peanut oil on myometrial glycogen is puzzling, since pilot studies had shown peanut oil to be the least stimulatory of three vehicles on uterine wet weight and visual evaluation of glycogen. It appears that it is not a

totally inert vehicle and further studies will have to be performed on the effects of this and other vehicles on uterine parameters. Histochemical evaluation of uterin glycogen in bilaterally ovariectomized rats 4 has demonstrated that the greatest number of glycogen granules occurred at the site of the IUD in the circular layer of uterine myometrium. However, we found greater amounts in the longitudinal layer in most groups of hamsters. In this study, the myometrial glycogen content of the uterine horns of the cycling, castrated and castrated hormone-treated hamsters was basically unaffected by the presence ofthe IUD. In the castrated rat, however, administration of either estrogen or progesterone increased glycogen in the presence of an IUD.4 Others have reported as much as a 62% increase in the glycogen content of rat uteri with an IUD which was further inhanced by estrogen therapy. 24. 25 This is obviously not the case in the hamster, and our results concur with the report of Gregoire and Ansbacher26 on the effect of the device on total uterine glycogen. In the cycling animals the fluctuations we saw in myometrial glycogen are basically in agree-

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EFFECT OF AN IUD ON MYOMETRIAL GLYCOGEN

ment with the previous reports on total uterine glycogen 27 and myometrial glycogen 28 in hamsters. Our results indicate that visual evaluation alone of glycogen content is a helpful tool for noting changes in histochemical staining intensities but is not sufficient for distinguishing minor changes. Usually one grade higher (i.e., + to + +) is not significantly different from the preceding grade, but a two-grade difference (i.e., + to + + +) may indeed represent a significant difference. These data emphasize that the investigator must be extremely cautious in assigning visual values and that there is a need to quantitate these values to add validity to the study. In conclusion, the IUD was found to have very little effect on hamster myometrial glycogen, and certainly no overpowering estrogen-like action was noted. The device does appear to have a mild effect on the uterine response to exogenous hormones and a slightly stimulatory effect on the myometrial glycogen in the cycling hamster. Acknowledgments. The authors wish to express their thanks to Mr. Rollie Harp and Mr. Bob Haggard for their assistance in statistical evaluation of the data.

REFERENCES

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1. Greenwald GS: Interruption of pregnancy in the rat by uterine suture. J Reprod Fertil 9:9, 1965 2. Parr E, Schaedler RW, Hirsch JG: The relationship of polymorphonuclear leukocytes to infertility in uteri containing foreign bodies. J Exp Med 126:523, 1967 3. Parr E: The role of inflammation in uterine weight increase caused by an IUD. J Reprod Fertil 18:221, 1969 4. Bo WJ, Moore PJ, Ashburn MJ: The effect ofa foreign body on the glycogen and sulfomucopolysaccharides of the uterus. Fertil Steril 20:351, 1969 5. Wynn R: Intrauterine devices: effects on ultrastructure of human endometrium. Science 156:1508, 1967 6. WHO Scientific Group: Intra-uterine devices: physiological and clinical aspects. WHO Tech Rep Ser 397:13,1968 7. Joshi S, Sujan-Tejuja S: Biochemistry of human endometrium in users of intrauterine contraceptive devices. Fertil Steril 20:98, 1969 8. Joshi S: Effect of an intrauterine foreign body on the response ofthe rat uterus to exogenous estrogen plus progesterone. Indian J Exp BioI 5:68, 1967 9. Brown-Grant K: Effect of an IUCD on an endometrial response to steroid hormones in the rat. J Reprod Fertil 18:475, 1969

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10. Laumas KR, Yadava HS: Metabolism of(U- 1'C)-glucose in the rat uterus in the presence of an intrauterine contraceptive device. J Reprod Fertil 20:389, 1969 11. Janakiraman K, Buch NC: Effect of intrauterine devices on uterine structure in goats: part I-endometrial characteristics. Indian J Exp BioI 11:149, 1973 12. Moore PJ: The influence of an intrauterine device on the hamster uterus. Diss Abstr Int B Sci Eng 32:5266B, 1972 13. Parr E: Glycogen accumulation in the rat uterus containing an intrauterine contraceptive device. Fertil Steril17: 797, 1966 , 14. Kar AB, Kamboj UP: Effect of an intrauterine contraceptive devices on histological and histochemical changes in rabbit uterus. Indian J Exp BioI 3:141, 1965 15. Gulati OP, Charavarti RN, Chaudhury RR: Effect of an intrauterine device on the water content and the weight of the rat uterus. Indian J Med Res 57:2267, 1969 16. Wrenn TR, Wood JR, Bitman J: Alterations in the uterine environment produced by IUDs. J Reprod Fertil 19:511, 1969 17. Seifter S, Dayton S, N ovie B, Muntwyler E: The estimation of glycogen with the anthrone reagent. Arch Biochem Biophys 25:191, 1950 18. Troyer H, Rosenquist TR: Atomic absorption spectrophotometry applied to photographic densitometry. J Histochem Cytochem 23:941, 1975 19. Greenwald GS: The effects of unilateral ovariectomy on follicular maturation in the hamster. Endocrinology 66:89, 1960 20. McManus JFA: The periodic acid routine applied to the kidney. Am J Pathol 24:643, 1948 21. Hall K, Khaligh HS: The action of ovarian steroids and relaxin on glycogen and some enzyme activities in the uterus of the hamster. J Endocrinol 40:353, 1968 22. Gregoire AT, Richardson DW: Glycogen and water responses to estrogen in the hamster reproductive tract. Endocrinology 87:1369, 1970 23. Giannina T, Butler M, Popick F, Steinetz B: Comparative effects of some steroidal and non steroidal antifertility agents in rats and hamsters. Contraception 3:347, 1971 24. Wrenn TR, Wood JR, Bitman J: IUD's and the biochemical responses of the uterus to estrogen in ovariectomized rats. BioI Reprod 1:234, 1969 25. Joshi SG: Effect of a foreign body on responses of the uterus to estrogen in ovariectomized rats. Contraception 2:137, 1970 26. Gregoire AT, Ansbacher R: The effect of an intrauterine device on uterine weight and water content and on genital tract glycogen of cycling ovariectomized, pseudopregnant or oestrogen-treated hamster. J Reprod FertiI31:341, 1972 27. Gregoire AT, Guinness BJ: Cyclic and pre-ovulatory changes in the glycogen content ofthe female hamster genital tract. J Reprod Fertil 17:427, 1968 28. Sype WE, Hilleman HH: Glycogen: distribution in the estrus and postpartum hamster uterus. Trans Am Microsc Soc 89:6, 1970