lr IS WELL KNOWN that diabetes often is accompanied by a disturbance in

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1 Altered O,vul,ation Pattern in Experimental Diabetes RAM6N A. CHIERI, M.D., OMAR H. PIVETT A, M.D., and VIRGILIO G. FOGLIA, M.D. lr IS WELL KNOWN that diabetes often is accompanied by a disturbance in reproductive function. Sterility frequently is observed in diabetic women without treatment; 9 and abortion, fetal death, and premature delivery also have been reported Similar reproductive alterations have been described in animals with experimental diabetes After subtotal ( 95%) pancreatectomy in the white rat, three successive periods occur: ( 1) prediabetes, which lasts approximately 2-3 months, with normal fasting blood sugar level, aglycosuria, and normal growth; (2) incipient diabetes, which persists for approximately a month with gradual development of diabetic symptoms and glycosuria only after feeding; and ( 3) manifest diabetes, with all the characteristic signs of diabetes and which lasts approximately 6-7 months until the death of the animal. 6 Previous studies from our laboratory carried out in 95% pancreatectomized female rats showed a diminished pregnancy rate during diabetes and prediabetes.7 Several hypotheses could explain these findings; among them are impairment of ovulation andjor altered uterine physiology. Some evidence for the latter already has been demonstrated; during alloxan-induced diabetes in the rat, the estrogen-induced uterine water inhibition is diminished,8 and the lipid metabolism of the uterus altered. 3 Using the working hypothesis that some of the abnormalities observed in the physiology of reproduction during diabetes might be reflections of an altered pattern of ovulation, we studied the number of eggs at the third day of pregnancy in 95% pancreatectomized rats. MATERIALS AND METHODS Female white rats from our institution were used. Since it is known that some alterations in reproduction appear in the 95% pancreatectomized rats From the Institute of Physiology, Buenos Aires Medical School, Buenos Aires, Argentina. Supported by Grant M from the Population Council. 661

2 662 CHIERI ET AL. FERTILITY & STERILITY before they develop hyperglycemia,7 the experiments were designed to study the various influences that pancreatectomy, the time elapsed after operation, the degree of hyperglycemia, and insulin therapy had on ovulation. For this purpose the following groups of rats were studied: ( 1) 95% pancreatectomized rats, ( 2) sham-operated control, ( 3) 95% pancreatectomized rats who received injections of insulin, and ( 4) sham-operated control rats who received injections of saline. Pancreatectomy. The pancreatectomy was performed in rats with a body weight of 80 gm. ( 2-3 months of age) under ether anesthesia, following the technic previously described by one of us. 6 Mating. Vaginal smears were taken at 3 P.M. in all animals. Animals in proestrus were placed individually in a cage with a normal male at 6 P.M. The rats were examined for the presence of spermatozoa the next morning, and were considered to be at Day 1 of pregnancy when spermatozoa were found. The mating of the animals was performed at different times from 1.5 to 7 months after operation. Egg Counting. In order to study the eggs, the animals were sacrificed by ether anesthesia at 12:00 NOON on Day 3 of pregnancy. The eggs were collected by washing the fallopian tubes as described by Simpson and Williams. Eggs were counted with a dissecting microscope at a magnification of 50X. Control of Diabetes. After a 7-hr. fast, the blood sugar level was determined, by the method of Somogyi and Nelson/ 7 on samples withdrawn from the tip of the tail. On the pancreatectomized insulin-treated rats and their controls the blood sugar level was determined without previous fasting in order to prevent hypoglycemic shock. Insulin Treatment. Rats in Group 3 received subcutaneous injections of insulin zinc-protamine,* 4 U.jkg.jday for days. The last insulin injection was given to the animals 18 hr. before sacrifice. The control animals of this group were given injections once a day with the same volume of saline. RESULTS The results obtained with the rats studied at different times after operation, from 1.5 to 7 months, are given in Table 1. The pancreatectomized rats had a.significantly lower number of eggs in the fallopian tubes ( p < 0.001; t test). *Thanks are due to Eli Lilly Laboratories, Buenos Aires, for the provision of insulin.

3 VoL. 20, No.4, 1969 OvuLATION AND DIABETES 663 TABLE I. Effect of Various Factors on Number of Eggs in 95% Pancreatectomized Rats Measurement of eggs Measurement of blood glueose No. of No. of eggs No. of Glucose rats per rat* rats (mg./100 ml.)* Time after operation Total time (1.5 to 7 mo.) Sham op. control ± ± 3 Pancreatectomized ±0.4t ± 19t 1.5 to 3 mo. Sham op. control ±0.7 Pancreatectomized ± to 5 mo. Sham op. control ± 0.5 Pancreatectomized ± to 7 mo. Sham op. control ± 0.4 Pancreatectomized ± Effect of hyperglycemia Blood sugar level Sham op. control (120 mg./100 ml.) ± 0.3 Pancreatectomized 120 mg./100 ml ±0.9~ 12Q-180 mg./100 ml ± 0.6~ >180 mg./100 ml ± 0.4~ Duration of hyperglycemia 0 to 2 mo ± 0.6~ 2 to 6 mo ±0.4~ Effect of insulin treatment (3-5 mo. postop.) Sham op. control ± ± 6 Pancreatectomized, insulin treated ± ± 46** * Mean ± standard error of the mean. t Significantly lower than control (p <0.001, t test). t Significantly higher than control (p <0.001, t test). Significantly lower than control (p <0.001, t test). II Significantly lower than control (p <0.01, t test). ~Significantly lower than control (p <0.05, analysis of variance). **Significantly higher than control (p <0.01, t test).

4 664 CHIERI ET AL. FERTILITY & STERILITY To determine if the time elapsed after surgical intervention had any effect on ovulation, we divided both groups (pancreatectomized and shamoperated control) in the following manner: months after operation, 3-5 months after operation, and 5-7 months after operation. The data (Table 1) show that during the first observation period ( months) there is no statistical difference in the number of eggs; during the next two periods (3--5 and 5-7 months) the number of eggs in pancreatectomized rats is diminished ( p < and p < 0.01, respectively; t test). To investigate the possibility that the blood sugar level had an effect on ovulation, we divided the pancreatectomized rats into three groups: blood sugar less than 120 mg.j100 mi., blood sugar between 120 and 180, and blood sugar greater than 180. Five additional pancreatectomized rats that were studied before 1.5 months or after 7 months were included. The results show that the diminution in the number of eggs at Day 3 of pregnancy becomes evident in the pancreatectomized rats before they develop hyperglycemia (prediabetic stage) as seen in Table 1. To continue the investigation of the effect of hyperglycemia, we divided the pancreatectomized rats into two groups according to the length of the time they had been hyperglycemic before they were studied: 0-2 and 2-6 months. There is no statistical difference in the number of eggs between these two groups (Table 1). Table 1 also shows the effect of insulin treatment on the ovulation pattern in pancreatectomized rats. It can be seen that insulin therapy restored to normal the number of eggs found in the pancreatectomized animals. DISCUSSION Partially pancreatectomized rats showed a diminished number of eggs in the fallopian tubes at Day 3 of pregnancy. To our knowledge there are no previous reports showing more direct evidence of altered ovulation during diabetes. A lower number of eggs was observed in pancreatectomized hyperglycemic rats and pancreatectomized normoglycemic (prediabetic) rats, suggesting that the blood sugar level is not an important factor in producing this impairment of ovulation. This impression is substantiated by the results obtained in the rats that had been hyperglycemic for different lengths of time; there was no difference in the number of eggs between rats that were hyperglycemic during 0-2 months and rats with a high blood sugar level during 2-6 months. Ovulation in the partially pancreatectomized rats probably depends on factors other than blood sugar level. Arachidonic acid deprivation altered the normal pregnancy rate in rats, 5 and arachidonic

5 VoL. 20, No.4, 1969 OvuLATION AND DIABETES 665 acid given to the male rats with alloxan-induced diabetes improved the altered spermatogenesis. 2 Insulin therapy was able to restore the number of eggs to a normal level, suggesting that the ovulatory disturbance was related critically to the insulin deficit. The diminished number of eggs in the fallopian tubes of pancreatectomized rats at Day 3 of pregnancy may be due to a lower number of ova shed at the time of ovulation. This could be a consequence of a lower level of endogenous gonadotropins or to a diminished ovarian sensitivity to them, or both. The work of Shipley and Danley suggests that endogenous gonadotropin levels are lower in rats with alloxan-induced diabetes, whereas Lawrence and Contopoulos demonstrated an alteration in ovarian structure during alloxan-induced diabetes in the rat. In connection with the hypothesis of diminished ovarian sensitivity to gonadotropins, we have unpublished data showing that the number of ova released by prepubertal rats following treatment with gonadotropins was lower in alloxan-induced diabetic rats than in the corresponding normal control. Altered mucosal or muscular activity, which would speed the passage of ova through the tubes, is another possibility in interpreting our findings. Unfortunately, we did not check the uteri for the presence of eggs. Further work is in progress to test these hypotheses. SUMMARY 1. The number of eggs in the fallopian tubes at the third day of pregnancy is significantly decreased in 95% pancreatectomized rats. 2. The diminution in the number of eggs was observed even in the normoglycemic pancreatectomized (prediabetic) rats. 3. The level and duration of hyperglycemia did not affect the differences. 4. The lower number of eggs becomes evident from the third month after operation. 5. Treatment of 95% pancreatectomized rats with insulin returned the number of eggs to normal levels even though hyperglycemia persisted. Institute of Physiology, Buenos Aires Medical School Paraguay Piso Buenos Aires, Argentina REFERENCES 1. BARNS, H. H., and MoRGANS, l\1. E. Pregnancy complicated by diabetes mellitus. Brit Med l 8:51, BRENNER, R. R., PELUFFO, R. 0., RESTELLI, M. A., MERCURI, 0., DANA, R., and!rusta, 0. "Acci6n Protectora del Acido Araquid6nico en la Degeneraci6n Tes-

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