- Email: [email protected]
Effects of metformin on the reproductive system of androgenized female rats
Effects of metformin on the reproductive system of androgenized female rats Metformin improved the glucose rate and the homeostasis model assessment–insulin resistance (HOMA-IR) index and caused partial reversion of ovaries and uterine morphology in female rats androgenized with testosterone. (Fertil Steril 2011;95:1507–9. 2011 by American Society for Reproductive Medicine.) Key Words: Androgenized rats, metformin, polycystic ovary syndrome, reproductive system
Research performed with animal models for studying polycystic ovary syndrome has enabled the assessment of different aspects of the disease pathogenesis. Administration of steroid hormones at the beginning of the lives of female rats led to chronic anovulation with absence of corpora lutea, cornification of the vaginal epithelium (an increase in steroid production), permanent estrus and closed vagina, and changes in hypophyseal cells (1–3). Our study, therefore, evaluated metformin treatment in female rats with permanent estrus. The EPM-1 Wistar rats (250 g) were mated by placing two female rats and one male rat in each cage. After delivery, the newborns were separated into female pups and male pups (4). Seventy-five female pups were selected and kept with their mothers. On their third day of life, 0.1 mL of testosterone propionate was administered diluted in castor oil (vehicle) to 50 female pups (1.25 mg/ animal) via the dorsal subcutaneous route (3). The 25 remaining female pups made up the control group, and they were administered vehicle only. Rats were handled in conformity with ethical principles for experimentation. This project was approved by the institutional review board of UNIFESP-EPM (no. 0302/07). All of the female newborns were kept together with their mothers for 90 days throughout nursing. Afterward, the animals Roberta Rassi Mahamed, M.D.a Carla Cristina Maganhin, M.D., Ph.D.a Ricardo Santos Simo˜es, M.D.b Manuel de Jesus Simo˜es, M.D., Ph.D.c Edmund Chada Baracat, M.D., Ph.D.a,b Jose´ Maria Soares Jr., M.D., Ph.D.a,b a Gynecology Department, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Brazil b Department of Obstetrics and Gynecology, School of Medicine, University of Sa˜o Paulo (USP), Sa˜o Paulo, Brazil c Department of Morphology and Genetics, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil Received June 17, 2010; revised July 20, 2010; accepted July 29, 2010; published online September 8, 2010. R.R.M. has nothing to disclose. C.C.M. has nothing to disclose. R.S.S. has nothing to disclose. M.d.J.S. has nothing to disclose. E.C.B. has nothing to disclose. J.M.S. has nothing to disclose. Reprint requests: Carla Cristina Maganhin, M.D. Ph.D., Av. do Cursino, 104–apt 82C, Sa˜o Paulo (SP), CEP 04132–000, Brazil (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.07.1093
were evaluated through vaginal smears collected for 4 days. The animals medicated with testosterone propionate had a closed vagina (1–3, 5). The animals were employed in two experiments. Thirty-six animals were selected and divided into three groups in experiment 1: the control group (CG) with 12 unmedicated rats receiving 0.5 mL of distilled water; the androgenized group (AG) with 10 testosterone propionate–treated rats in permanent estrus receiving 0.5 mL of distilled water; and the androgenized group associated with metformin (AGmet) with 14 testosterone propionate–treated rats in permanent estrus receiving 50 mg/kg metformin (6). The treatment was performed orally by gavage for 6 consecutive weeks. Vaginal smears were collected daily for 6 weeks after the first day of metformin administration to characterize the estrous cycle (7). The cycling animals were killed during the proestrus phase. Blood was collected for measuring fasting glucose and insulin after euthanasia and after a 12-hour fast before the procedure (8). Anesthesia was performed with 15 mg/kg of xylazine associated with 30 mg/kg of ketamine. Also, both ovaries and the middle third of the uterine horn were removed for analysis. Histomorphometric analysis was performed with AxioVision 4.6 REL images (Carl Zeiss, Jena, Germany) (9). Ten readings of each slide were done per animal. Two observers who did not know the groups performed the histomorphometric measurements separately. Thirty rats were set aside for experiment 2, and they were divided into groups like those in experiment 1: CG, AG, and AGmet. The animals were mated by putting together two male rats and one female rat in each cage. The following morning, the mating test was performed (10). All of the pregnant rats were killed after giving birth to their pups. The animals that did not test positive for spermatozoa or that did not deliver on day 22 of pregnancy were also killed (10). The results were evaluated by analysis of variance (ANOVA) and, subsequently, by Tukey’s multiple comparison test for analyzing variables to identify the groups with statistically significant differences in experiment 1. The comparison of the number of mated rats and of pregnancies was made using the chi-square test in experiment 2. In experiment 1, AG rats had higher levels of glucose and homeostasis model assessment–insulin resistance (HOMA IR) than
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
1507
TABLE 1 Mean and standard deviation of fasting glucose, insulin, homeostasis model assessment–insulin resistance (HOMA-IR), weight of animals, and histomorphometric parameters in each group.
Glucose (mmol/L) Insulin (mIU/mL) HOMA IR Weight (g) Ovaries Degenerating follicles (n/slide) Corpora lutea (n/10 areas) Nonantral follicles (n/10 areas) Antral follicles (n/areas) Interstitial cells (n/area of 780 mm2) Epithelial thickness (mm) Endometrium Epithelial thickness (mm) Endometrial thickness (mm) Number of glands (n/area of 780 mm2) Number of eosinophils (n/area of 780 mm2)
CG (n [ 12)
AG (n [ 10)
AGmet (n [ 14)
5.28 0.52 10.35 4.11b 2.43 0.24 239.20 3.86a
6.86 0.86a 36.29 14.60 11.06 1.39a 263.20 18.28
5.72 0.49 26.16 14.34 6.66 0.57 264.50 23.09
3.10 1.15 7.16 2.71a 3.33 1.61 1.98 1.75 165.30 25.20 8.37 0.08
4.72 1.90a 0.0 0.0 4.60 4.78 2.88 1.77 310.10 27.30a 8.49 0.18
2.06 1.28 2.06 1.52 3.57 3.80 2.78 1.67 178.40 47.80 8.45 0.04
73.10 45.41 740.80 79.67 7.5 2.07 24.5 2.10
89.88 44.45 863.60 67.85c 5.51 1.61c 46.1 3.1a
78.42 33.24 770.80 77.42 6.01 1.51 30.2 6.10
Note: Ten slides per animal were analyzed. CG ¼ control group, AG ¼ androgenized group, AGmet ¼ androgenized group associated with metformin. The data are summed up as mean standard deviation for each group. Statistical tests performed were analysis of variance (ANOVA) test then Tukey’s test of multiple comparisons. a P< .01 in comparison with other groups. b P< .01 compared with other groups. c P< .05 compared with the CG. Mahamed. Correspondence. Fertil Steril 2011.
the other two groups, CG and AGmet (Table 1). The insulin levels of CG rats was lower than ones of other groups (P<0.01). The CG rats also weighed less than the other two groups, AG and AGmet. The surface epithelium of the AG rats had weak stratification in some areas. In the cortical region, all ovaries had voluminous follicles in several developmental stages, but corpora lutea were absent. In a few of the voluminous follicles, there was a reduction in the number of layers of the granulosa and a lack of oocytes as well as leukocyte infiltration or disarray of the cells in this region, suggesting the formation of ovarian cysts. In the ovarian stroma, there was a much higher concentration of interstitial cells than that evidenced in the CG and AGmet rats. In the AGMet rats, the outer covering of the ovary in this group was similar to the germinal epithelium on the outer surface of the ovary in the AG rats. The histomorphometric data of each group are displayed in Table 1. There was a large quantity of degenerating follicles and of interstitial cells in the AG rats compared with the CG and AGmet rats. There were no statistically significant differences between the number of antral and nonantral follicles or differences in the thickness of the ovarian surface epithelium among the three groups. Corpora lutea were absent in the AG rats but present in AGmet rats, though in a smaller quantity than in the CG rats. The endometrium of the AG rats was covered with welldeveloped and cornified simple columnar epithelium, whose cells, in some places, were keratinized. There was also intense leukocyte infiltration in this group. The stroma showed large clusters of collagen fibers and, in some places, a larger concentration of stromal cells. The endometrial glands could be seen in a comparatively smaller number than in the CG rats. The endometrium in the
1508
Mahamed et al.
Correspondence
AGMet rats was well developed in comparison with that of the CG rats. There was a reduction in endometrial glands covered by simple cuboidal epithelium and an increase in eosinophilic leukocytes close to the myometrium in comparison with the CG rats. There were no statistically significant differences in the thickness of endometrial epithelium among the three groups (see Table 1). In the AG animals, there was an increase in the thickness of the endometrium and in the number of eosinophils in comparison with the other groups (CG and AGmet, P<.01). The endometrial gland number in the AG rats was reduced in comparison with that of the CG rats. In experiment 2, all of the CG (n ¼ 10) had spermatozoa in the vaginal lumen after mating. The animals were analyzed up to the end of pregnancy and delivery and each had 10 (11.14 1.04) pups. In AG, there had been no opening of the vaginal ostium. In AGmet, spermatozoa were found in the vaginal lumen of four animals, but only one rat carried its pregnancy to term, and it delivered one pup only. In the other animals, no signs of implantation or of fetal reabsorption were detected 20 days after coitus. Our data on androgenized female rats are consistent with those of previous studies (1–5, 11–12). Our findings suggest the animals had altered ovulation and even an absence of ovulation. The consequence of such a process was perhaps endocrine imbalance with an increase in androgens in the animals. Such characteristics, along with vaginas that did not open, may indicate a state of chronic anovulation in the animals. Also, testosterone may increase insulin resistance in vitro (13). However, metformin may improve ovulation in a few animals. The administered dose and the period of time the drug was used were not enough to reverse this situation, or this drug cannot completely inhibit the process of chronic anovulation initiated after birth. It must
Vol. 95, No. 4, March 15, 2011
be added that the choice of the concentration that was employed was based on Sander et al. (6). Physiologically, the decrease in the number of interstitial cells by the metformin treatment possibly mitigates the hyperandrogenic ovarian microenvironment, allowing for follicular growth, ovulation, and formation of corpora lutea. This may have happened in our animals. This was not evidenced, however, in another experimental model with dehydroepiandrosterone (DHEA). After treatment with metformin administered by way of the ration, the investigators observed that there had not been any morphologic improvement, and they reported the worsening of follicular atresia with troglitazone (14). It should be emphasized that the investigators did not inject the drugs or administer them orally by gavage. Hence, the administered quantities may not have been the same in each group, hindering data interpretation. Data analysis shows metformin may improve insulin resistance. These data corroborate those of Sander et al. (6). It is conceivable that improvement in insulin resistance leads to improvement in the hyperandrogenic microenvironment, helping to unblock the hypothalamus-hypophysis-gonad axis and promote
ovulation. Hence, the normalization of insulin action may partially justify the reduction of androgenic action in the ovaries, allowing for ovulation (15). Barraclough (2) described similar endometrial changes, with the resultant more compact mucous possibly hindering embryo penetration, which may also explain the infertility of the rats. Some investigators have studied the endometrium of female rats with testosterone propionate, and they noticed a stroma rich in collagen cells and a reduction in endometrial hydration (5, 11). As for women with polycystic ovary syndrome, the metformin treatment was associated with amelioration of the endometrial features. This improvement was related to an increase in ovulation with greater progesterone production (16). Our findings suggest that metformin treatment altogether reduced the area taken up by the degenerating ovarian follicles, the number of interstitial cells, and the thickness of the endometrium, and it increased the number of endometrial glands. However, further research into fertility following this model and using higher doses is necessary.
REFERENCES 1. Wrenn TR, Wood JR, Bitman J. Oestrogen responses of rats neonatally sterilized with steroids. J Endocrinol 1969;45:415–20. 2. Barraclough CA. Production of anovulatory sterile rats by single injections of testosterone propionate. Endocrinology 1961;68:62–7. 3. Barraclough CA, Gorski RA. Evidence tract the hypothalamus in responsible for androgen-induced sterility in the female rat. Endocrinology 1961;68:68–79. 4. Wolf CJ, Hotchkiss A, Ostby JS, LeBlanc GA, Gray LE Jr. Effects of prenatal testosterone propionate on the sexual development of male and female rats: a dose-response study. Toxicol Sci 2002;65:71–86. 5. Smaniotto S, Simo˜es MJ. Morphological aspects of the endometrial epithelium of persistent estrous rats under estrogenic action. Braz J Morphol Sci 1996;13:103–4. 6. Sander V, Luchetti CG, Solano ME, Elia E, Girolamo GD, Gonzalez C, et al. Role of the N, N’-dimethylbiguanide metformin in the treatment of female prepural BALB/c mice hyperadrogenized with dehydroepiandrosterone. Reproduction 2006;131:591–602.
Fertility and Sterility
7. Dair EL, Simoes RS, Simo˜es MJ, Romeu LR, Oliveira-Filho RM, Haidar MA, et al. Effects of melatonin on the endometrial morphology and embryo implantation in rats. Fertil Steril 2008;89:1299–305. 8. Geloneze B, Repetto EM, Geloneze SR, Tambascia MA, Ermetice MN. The threshold value for insulin resistance (HOMA-IR) in admixtured population IR in the Brazilian Metabolic Syndrome Study. Diabetes Res Clin Pract 2006;72:219–20. 9. Rossi AG, Soares- JM Jr., Motta EL, Simo˜es MJ, Oliveira-Filho RM, Haidar MA, et al. Metoclopramideinduced hyperprolactinemia affects mouse endometrial morphology. Gynecol Obstet Invest 2002; 54:185–90. 10. Panzan MQ, Ju´nior JM, da Motta EL, Haapalainen EF, de Jesus Simo˜es M, Baptista HA, et al. Metoclopramide-induced hyperprolactinaemia caused marked decline in pinopodes and pregnancy rates in mice. Hum Reprod 2006;21:2514–20. 11. Mahajan DK. Polycystic ovarian disease: animal models. Endocrinol Metab Clin North Am 1988;17:705–32.
12. Gerendai I, Wiessel O, Toth IE, Boldogkoi Z, Hornyak A, Halasz B. Occasional transsynaptic viral labeling in the central nervous system from the polycystic ovary induced by estradiol valerate. Microsc Res Tech 2005;66:186–92. 13. Zhang L, Liao Q. Effects of testosterone and metformin on glucose metabolism in endometrium. Fertil Steril 2010;93:2295–8. 14. Misugi T, Ozaki K, Beltagy KE, Tokuyama O, Honda K, Ishiko O. Insulin-lowering agents inhibit synthesis of testosterone in ovaries of DHEA-induced PCOS rats. Gynecol Obstet Invest 2006;61:208–15. 15. Allemand MC, Irving BA, Asmann YW, Klaus KA, Tatpati L, Coddington CC, et al. Effect of testosterone on insulin stimulated IRS1 Ser phosphorylation in primary rat myotubes— a potential model for PCOS-related insulin resistance. PLoS One 2009;4:e4274. 16. Legro RS, Zaino RJ, Demers LM, Kunselman AR, Gnatuk CL, Williams NI, et al. The effects of metformin and rosiglitazone, alone and in combination, on the ovary and endometrium in polycystic ovary syndrome. Am J Obstet Gynecol 2007;196:402.e1–11.
1509
Research performed with animal models for studying polycystic ovary syndrome has enabled the assessment of different aspects of the disease pathogenesis. Administration of steroid hormones at the beginning of the lives of female rats led to chronic anovulation with absence of corpora lutea, cornification of the vaginal epithelium (an increase in steroid production), permanent estrus and closed vagina, and changes in hypophyseal cells (1–3). Our study, therefore, evaluated metformin treatment in female rats with permanent estrus. The EPM-1 Wistar rats (250 g) were mated by placing two female rats and one male rat in each cage. After delivery, the newborns were separated into female pups and male pups (4). Seventy-five female pups were selected and kept with their mothers. On their third day of life, 0.1 mL of testosterone propionate was administered diluted in castor oil (vehicle) to 50 female pups (1.25 mg/ animal) via the dorsal subcutaneous route (3). The 25 remaining female pups made up the control group, and they were administered vehicle only. Rats were handled in conformity with ethical principles for experimentation. This project was approved by the institutional review board of UNIFESP-EPM (no. 0302/07). All of the female newborns were kept together with their mothers for 90 days throughout nursing. Afterward, the animals Roberta Rassi Mahamed, M.D.a Carla Cristina Maganhin, M.D., Ph.D.a Ricardo Santos Simo˜es, M.D.b Manuel de Jesus Simo˜es, M.D., Ph.D.c Edmund Chada Baracat, M.D., Ph.D.a,b Jose´ Maria Soares Jr., M.D., Ph.D.a,b a Gynecology Department, Federal University of Sa˜o Paulo (UNIFESP), Sa˜o Paulo, Brazil b Department of Obstetrics and Gynecology, School of Medicine, University of Sa˜o Paulo (USP), Sa˜o Paulo, Brazil c Department of Morphology and Genetics, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil Received June 17, 2010; revised July 20, 2010; accepted July 29, 2010; published online September 8, 2010. R.R.M. has nothing to disclose. C.C.M. has nothing to disclose. R.S.S. has nothing to disclose. M.d.J.S. has nothing to disclose. E.C.B. has nothing to disclose. J.M.S. has nothing to disclose. Reprint requests: Carla Cristina Maganhin, M.D. Ph.D., Av. do Cursino, 104–apt 82C, Sa˜o Paulo (SP), CEP 04132–000, Brazil (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.07.1093
were evaluated through vaginal smears collected for 4 days. The animals medicated with testosterone propionate had a closed vagina (1–3, 5). The animals were employed in two experiments. Thirty-six animals were selected and divided into three groups in experiment 1: the control group (CG) with 12 unmedicated rats receiving 0.5 mL of distilled water; the androgenized group (AG) with 10 testosterone propionate–treated rats in permanent estrus receiving 0.5 mL of distilled water; and the androgenized group associated with metformin (AGmet) with 14 testosterone propionate–treated rats in permanent estrus receiving 50 mg/kg metformin (6). The treatment was performed orally by gavage for 6 consecutive weeks. Vaginal smears were collected daily for 6 weeks after the first day of metformin administration to characterize the estrous cycle (7). The cycling animals were killed during the proestrus phase. Blood was collected for measuring fasting glucose and insulin after euthanasia and after a 12-hour fast before the procedure (8). Anesthesia was performed with 15 mg/kg of xylazine associated with 30 mg/kg of ketamine. Also, both ovaries and the middle third of the uterine horn were removed for analysis. Histomorphometric analysis was performed with AxioVision 4.6 REL images (Carl Zeiss, Jena, Germany) (9). Ten readings of each slide were done per animal. Two observers who did not know the groups performed the histomorphometric measurements separately. Thirty rats were set aside for experiment 2, and they were divided into groups like those in experiment 1: CG, AG, and AGmet. The animals were mated by putting together two male rats and one female rat in each cage. The following morning, the mating test was performed (10). All of the pregnant rats were killed after giving birth to their pups. The animals that did not test positive for spermatozoa or that did not deliver on day 22 of pregnancy were also killed (10). The results were evaluated by analysis of variance (ANOVA) and, subsequently, by Tukey’s multiple comparison test for analyzing variables to identify the groups with statistically significant differences in experiment 1. The comparison of the number of mated rats and of pregnancies was made using the chi-square test in experiment 2. In experiment 1, AG rats had higher levels of glucose and homeostasis model assessment–insulin resistance (HOMA IR) than
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
1507
TABLE 1 Mean and standard deviation of fasting glucose, insulin, homeostasis model assessment–insulin resistance (HOMA-IR), weight of animals, and histomorphometric parameters in each group.
Glucose (mmol/L) Insulin (mIU/mL) HOMA IR Weight (g) Ovaries Degenerating follicles (n/slide) Corpora lutea (n/10 areas) Nonantral follicles (n/10 areas) Antral follicles (n/areas) Interstitial cells (n/area of 780 mm2) Epithelial thickness (mm) Endometrium Epithelial thickness (mm) Endometrial thickness (mm) Number of glands (n/area of 780 mm2) Number of eosinophils (n/area of 780 mm2)
CG (n [ 12)
AG (n [ 10)
AGmet (n [ 14)
5.28 0.52 10.35 4.11b 2.43 0.24 239.20 3.86a
6.86 0.86a 36.29 14.60 11.06 1.39a 263.20 18.28
5.72 0.49 26.16 14.34 6.66 0.57 264.50 23.09
3.10 1.15 7.16 2.71a 3.33 1.61 1.98 1.75 165.30 25.20 8.37 0.08
4.72 1.90a 0.0 0.0 4.60 4.78 2.88 1.77 310.10 27.30a 8.49 0.18
2.06 1.28 2.06 1.52 3.57 3.80 2.78 1.67 178.40 47.80 8.45 0.04
73.10 45.41 740.80 79.67 7.5 2.07 24.5 2.10
89.88 44.45 863.60 67.85c 5.51 1.61c 46.1 3.1a
78.42 33.24 770.80 77.42 6.01 1.51 30.2 6.10
Note: Ten slides per animal were analyzed. CG ¼ control group, AG ¼ androgenized group, AGmet ¼ androgenized group associated with metformin. The data are summed up as mean standard deviation for each group. Statistical tests performed were analysis of variance (ANOVA) test then Tukey’s test of multiple comparisons. a P< .01 in comparison with other groups. b P< .01 compared with other groups. c P< .05 compared with the CG. Mahamed. Correspondence. Fertil Steril 2011.
the other two groups, CG and AGmet (Table 1). The insulin levels of CG rats was lower than ones of other groups (P<0.01). The CG rats also weighed less than the other two groups, AG and AGmet. The surface epithelium of the AG rats had weak stratification in some areas. In the cortical region, all ovaries had voluminous follicles in several developmental stages, but corpora lutea were absent. In a few of the voluminous follicles, there was a reduction in the number of layers of the granulosa and a lack of oocytes as well as leukocyte infiltration or disarray of the cells in this region, suggesting the formation of ovarian cysts. In the ovarian stroma, there was a much higher concentration of interstitial cells than that evidenced in the CG and AGmet rats. In the AGMet rats, the outer covering of the ovary in this group was similar to the germinal epithelium on the outer surface of the ovary in the AG rats. The histomorphometric data of each group are displayed in Table 1. There was a large quantity of degenerating follicles and of interstitial cells in the AG rats compared with the CG and AGmet rats. There were no statistically significant differences between the number of antral and nonantral follicles or differences in the thickness of the ovarian surface epithelium among the three groups. Corpora lutea were absent in the AG rats but present in AGmet rats, though in a smaller quantity than in the CG rats. The endometrium of the AG rats was covered with welldeveloped and cornified simple columnar epithelium, whose cells, in some places, were keratinized. There was also intense leukocyte infiltration in this group. The stroma showed large clusters of collagen fibers and, in some places, a larger concentration of stromal cells. The endometrial glands could be seen in a comparatively smaller number than in the CG rats. The endometrium in the
1508
Mahamed et al.
Correspondence
AGMet rats was well developed in comparison with that of the CG rats. There was a reduction in endometrial glands covered by simple cuboidal epithelium and an increase in eosinophilic leukocytes close to the myometrium in comparison with the CG rats. There were no statistically significant differences in the thickness of endometrial epithelium among the three groups (see Table 1). In the AG animals, there was an increase in the thickness of the endometrium and in the number of eosinophils in comparison with the other groups (CG and AGmet, P<.01). The endometrial gland number in the AG rats was reduced in comparison with that of the CG rats. In experiment 2, all of the CG (n ¼ 10) had spermatozoa in the vaginal lumen after mating. The animals were analyzed up to the end of pregnancy and delivery and each had 10 (11.14 1.04) pups. In AG, there had been no opening of the vaginal ostium. In AGmet, spermatozoa were found in the vaginal lumen of four animals, but only one rat carried its pregnancy to term, and it delivered one pup only. In the other animals, no signs of implantation or of fetal reabsorption were detected 20 days after coitus. Our data on androgenized female rats are consistent with those of previous studies (1–5, 11–12). Our findings suggest the animals had altered ovulation and even an absence of ovulation. The consequence of such a process was perhaps endocrine imbalance with an increase in androgens in the animals. Such characteristics, along with vaginas that did not open, may indicate a state of chronic anovulation in the animals. Also, testosterone may increase insulin resistance in vitro (13). However, metformin may improve ovulation in a few animals. The administered dose and the period of time the drug was used were not enough to reverse this situation, or this drug cannot completely inhibit the process of chronic anovulation initiated after birth. It must
Vol. 95, No. 4, March 15, 2011
be added that the choice of the concentration that was employed was based on Sander et al. (6). Physiologically, the decrease in the number of interstitial cells by the metformin treatment possibly mitigates the hyperandrogenic ovarian microenvironment, allowing for follicular growth, ovulation, and formation of corpora lutea. This may have happened in our animals. This was not evidenced, however, in another experimental model with dehydroepiandrosterone (DHEA). After treatment with metformin administered by way of the ration, the investigators observed that there had not been any morphologic improvement, and they reported the worsening of follicular atresia with troglitazone (14). It should be emphasized that the investigators did not inject the drugs or administer them orally by gavage. Hence, the administered quantities may not have been the same in each group, hindering data interpretation. Data analysis shows metformin may improve insulin resistance. These data corroborate those of Sander et al. (6). It is conceivable that improvement in insulin resistance leads to improvement in the hyperandrogenic microenvironment, helping to unblock the hypothalamus-hypophysis-gonad axis and promote
ovulation. Hence, the normalization of insulin action may partially justify the reduction of androgenic action in the ovaries, allowing for ovulation (15). Barraclough (2) described similar endometrial changes, with the resultant more compact mucous possibly hindering embryo penetration, which may also explain the infertility of the rats. Some investigators have studied the endometrium of female rats with testosterone propionate, and they noticed a stroma rich in collagen cells and a reduction in endometrial hydration (5, 11). As for women with polycystic ovary syndrome, the metformin treatment was associated with amelioration of the endometrial features. This improvement was related to an increase in ovulation with greater progesterone production (16). Our findings suggest that metformin treatment altogether reduced the area taken up by the degenerating ovarian follicles, the number of interstitial cells, and the thickness of the endometrium, and it increased the number of endometrial glands. However, further research into fertility following this model and using higher doses is necessary.
REFERENCES 1. Wrenn TR, Wood JR, Bitman J. Oestrogen responses of rats neonatally sterilized with steroids. J Endocrinol 1969;45:415–20. 2. Barraclough CA. Production of anovulatory sterile rats by single injections of testosterone propionate. Endocrinology 1961;68:62–7. 3. Barraclough CA, Gorski RA. Evidence tract the hypothalamus in responsible for androgen-induced sterility in the female rat. Endocrinology 1961;68:68–79. 4. Wolf CJ, Hotchkiss A, Ostby JS, LeBlanc GA, Gray LE Jr. Effects of prenatal testosterone propionate on the sexual development of male and female rats: a dose-response study. Toxicol Sci 2002;65:71–86. 5. Smaniotto S, Simo˜es MJ. Morphological aspects of the endometrial epithelium of persistent estrous rats under estrogenic action. Braz J Morphol Sci 1996;13:103–4. 6. Sander V, Luchetti CG, Solano ME, Elia E, Girolamo GD, Gonzalez C, et al. Role of the N, N’-dimethylbiguanide metformin in the treatment of female prepural BALB/c mice hyperadrogenized with dehydroepiandrosterone. Reproduction 2006;131:591–602.
Fertility and Sterility
7. Dair EL, Simoes RS, Simo˜es MJ, Romeu LR, Oliveira-Filho RM, Haidar MA, et al. Effects of melatonin on the endometrial morphology and embryo implantation in rats. Fertil Steril 2008;89:1299–305. 8. Geloneze B, Repetto EM, Geloneze SR, Tambascia MA, Ermetice MN. The threshold value for insulin resistance (HOMA-IR) in admixtured population IR in the Brazilian Metabolic Syndrome Study. Diabetes Res Clin Pract 2006;72:219–20. 9. Rossi AG, Soares- JM Jr., Motta EL, Simo˜es MJ, Oliveira-Filho RM, Haidar MA, et al. Metoclopramideinduced hyperprolactinemia affects mouse endometrial morphology. Gynecol Obstet Invest 2002; 54:185–90. 10. Panzan MQ, Ju´nior JM, da Motta EL, Haapalainen EF, de Jesus Simo˜es M, Baptista HA, et al. Metoclopramide-induced hyperprolactinaemia caused marked decline in pinopodes and pregnancy rates in mice. Hum Reprod 2006;21:2514–20. 11. Mahajan DK. Polycystic ovarian disease: animal models. Endocrinol Metab Clin North Am 1988;17:705–32.
12. Gerendai I, Wiessel O, Toth IE, Boldogkoi Z, Hornyak A, Halasz B. Occasional transsynaptic viral labeling in the central nervous system from the polycystic ovary induced by estradiol valerate. Microsc Res Tech 2005;66:186–92. 13. Zhang L, Liao Q. Effects of testosterone and metformin on glucose metabolism in endometrium. Fertil Steril 2010;93:2295–8. 14. Misugi T, Ozaki K, Beltagy KE, Tokuyama O, Honda K, Ishiko O. Insulin-lowering agents inhibit synthesis of testosterone in ovaries of DHEA-induced PCOS rats. Gynecol Obstet Invest 2006;61:208–15. 15. Allemand MC, Irving BA, Asmann YW, Klaus KA, Tatpati L, Coddington CC, et al. Effect of testosterone on insulin stimulated IRS1 Ser phosphorylation in primary rat myotubes— a potential model for PCOS-related insulin resistance. PLoS One 2009;4:e4274. 16. Legro RS, Zaino RJ, Demers LM, Kunselman AR, Gnatuk CL, Williams NI, et al. The effects of metformin and rosiglitazone, alone and in combination, on the ovary and endometrium in polycystic ovary syndrome. Am J Obstet Gynecol 2007;196:402.e1–11.
1509