Nandrolone Decanoate and Physical EffortHistological and Morphometrical Assessment in Adult Rat Uterus.код для вставкиСкачать
THE ANATOMICAL RECORD 294:335–341 (2011) Nandrolone Decanoate and Physical Effort: Histological and Morphometrical Assessment in Adult Rat Uterus LUIZ GUSTAVO DE ALMEIDA CHUFFA,1* ROBERTA BARREIROS DE SOUZA,1 FERNANDO FREI,2 SUZANA DE FÁTIMA PACCOLA MESQUITA,3 1 AND ISABEL CRISTINA CHERICI CAMARGO 1 Department of Biological Sciences, Faculty of Sciences and Letters, Paulista State University (UNESP), Assis, São Paulo, Brazil 2 Department of Experimental Psychology, Faculty of Sciences and Letters, Paulista State University (UNESP), Assis, São Paulo, Brazil 3 Department of General Biology, State University of Londrina (UEL), Londrina, Paraná, Brazil ABSTRACT In the past decades, the therapeutic use of anabolic androgenic steroids (AAS) has been overshadowed by illicit abuse of these drugs by athletes and non-athletes. Since that AAS can affect the reproductive tract, resulting in reproduction and fertilization damages, the purpose of this study was to investigate the nandrolone decanoate (ND) effects, associated or not with physical effort, on the uterine histomorphometric parameters. Female Wistar rats, sedentary or not, were exposed to treatment with ND by intraperitoneal injection (5 mg/kg/day, once a week) during four consecutive weeks. Control animals, sedentary or not, received vehicle alone (propylene glycol) in the same manner. The physical activity was forced swimming (20 min/ day). During the experiment, all animals were monitored by daily vaginal smears. After 30 days of treatment, the females were sacriﬁced and their uteri collected and examined under light microscopy techniques. The NDtreated females showed estrus acyclicity and decreased thickness of both the epithelium and endometrial stroma. A reduction in the number and size of blood vessels was also found in ND-treated rats submitted to physical effort when compared to ND sedentary rats. ND-treated rats, regardless of exercise, exhibited stromal ﬁbrosis and reduced gland ducts that displayed high mitotic activity. A remarkable widespread presence of leukocytes occurred in rats receiving ND and submitted to exercise. These results suggest that ND associated or not with physical effort causes histomorphometric changes to C 2010 Wiley-Liss, Inc. the rat uterus. Anat Rec, 294:335–341, 2011. V Key words: nandrolone decanoate; steroids; uterus; histology; exercise; rat Anabolic androgenic steroids (AAS) can be useful adjuvant therapy for refractory anemia, hereditary angioedema, breast cancer, and starvation status (Clark et al., 1997), as well as for the treatment of hypogonadism, as they stimulate sexual development (Wilson and Grifﬁn, 1980). AAS, including testosterone and its synthetic analogues have also been used by healthy adolescents and adults, athletes and nonathletes to increase muscle mass, strength and physical dexterity (Iriart and Andrade, 2002). It is estimated that there are over 45 C 2010 WILEY-LISS, INC. V Grant sponsor: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil. *Correspondence to: Luiz Gustavo de Almeida Chuffa, Department of Biological Sciences, Paulista State University (UNESP), P.O. Box 19.806.900, Assis, SP, Brazil. Fax: (18) 33025848. E-mail: email@example.com Received 22 July 2009; Accepted 9 October 2010 DOI 10.1002/ar.21314 Published online 16 December 2010 in Wiley Online Library (wileyonlinelibrary.com). 336 DE ALMEIDA CHUFFA ET AL. anabolic steroid compounds available for abusive consumption by athletes, and in the past few years, their use has increased among male and female subjects (Lane and Connor, 1994; Bishop, 2005; Biden, 2006). Recently, a comparative study involving American teenager girls has suggested that several reports about AAS administration have been inﬂated by false-positive responses because of confusion with other supplementary compounds (Kanayama et al., 2007). According to Hickson et al. (1989), steroids use is associated with temporary or permanent adverse effects, which may appear within weeks (e.g., altered reproductive function) or require up to several years (e.g., liver carcinoma). Administration of AAS by women is associated with certain androgenic effects such as facial hair growth, deepening of the voice, clitoral enlargement, and menstrual irregularities (Strauss et al., 1985; Korkia and Stimson, 1997). Moreover, some disturbances in gonadal function such as delayed puberty, luteal phase deﬁciency, oligo-amenorrhea, or anovulation may occur in girls and women participating in strenuous physical exercise (Cannavò et al., 2001). In fact, the short-term effects of AAS consumption, associated or not with physical effort, on the female reproduction is still an unsolved issue. Some studies have reported the steroids effects on behavior (Gruber and Pope, 2000; Elliot et al., 2007) and physiological characteristics such as sexual receptivity and vaginal cytology (Clark et al., 1998). Previous studies in our laboratory have shown that AAS treatment suppresses estral cyclicity, leading to follicular atresia and absence of corpora lutea (Gerez et al., 2005; Camargo et al., 2009). It has also been observed some vacuolated epithelial cells and stromal ﬁbrosis. In other experimental study, administration of AAS to cynomolgus macaques revealed increase in uterine weight and endometrial thickness, as well as adenomyosis-like alterations and incidence of mucometra (Obasanjo et al., 1998). Recently, Kramer and McDonald (2006) proposed a causal relationship between athletic activity and sexual dysfunction, and it was ascribed to aerobic exercise and its adverse effects on pregnancy outcomes and hypothalamic disturbances, which may result in cyclicity disruption (Warren and Perlroth, 2001). Additionally, maternal exercise seems to be associated with the onset of hypertension or intrauterine growth restriction (Clapp, 2003; Davies et al., 2003), which can be detrimental to uterine function. Accordingly, the aim of this study was to verify the effects of nandrolone decanoate (ND), associated or not with physical effort, on morphologic and morphometric parameters in adult rat uterus. MATERIALS AND METHODS Animals and Experimental Design Twenty adult female Wistar rats (Rattus norvegicus albinus), 90 days old, were obtained from the Universidade Estadual Paulista (UNESP, Botucatu, SP, Brazil) and kept at the Faculty of Sciences and Letters (UNESP, Assis, SP, Brazil). The females were weighed and randomly divided into four experimental groups (N ¼ 5/ group): (1) C þ S: sedentary animals that received only vehicle; (2) C þ E: animals that received vehicle and submitted to physical effort; (3) ND þ S: sedentary animals treated with anabolic steroid; (4) ND þ E: animals treated with anabolic steroid and submitted to physical effort. All animals were housed in polypropylene cages (43 cm 30 cm 15 cm) with laboratory-grade pine shavings as bedding and also maintained under controlled room temperature (23 C 1 C) and lighting conditions (12L, 12D photoperiod, lights switched on at 6 AM). ND (4-estren-17B-ol-3-one 17-decanoate) was purchased from Organon Industry (São Paulo, Brazil) as an injectable solution, containing 50 mg of the androgen. The treated females received doses of ND (5 mg/kg BW of Deca Durabolin), available as oily solution (Pope and Katz, 1988; Marqueti et al., 2010). The doses were given once a week via intraperitoneal injection (KarbalayDoust and Noorafshan, 2009), during four consecutive weeks. In this study, this dosage and administration schedule deals the same condition of AAS users found at ﬁtness centers. Thus, ND injection was administrated on the same day (Wednesday) and at the same time (11:30 AM) to keep the estrous condition. The control groups received 0.5 mL of the propylene glycol as vehicle solution, according to the same procedure applied to the treated groups. All animals received tap water and commercial NuvitalV chow ad libitum. As forced-exercise can cause forefoot and toe injuries because of the impact and stress arising from the exercise models, aerobic swimming was chosen as the method of physical effort, in accordance with Peres and Luciano (1995). The rats were conditioned to the exercise before the beginning of the experimental period, starting with 5 min/day for the ﬁrst 2 days, then 10 min on the third and fourth days, followed by 15 min on the ﬁfth and sixth days, and 20 min on the seventh day. After this period, the scheme of 20 min to swimming was daily taken, during ﬁve consecutive days per week. Following 30 days and 24 hr after the last injection, all animals were killed by decapitation. The experimental protocol followed the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation. R Light Microscopy The estral cycle was daily monitored by vaginal smear cytology, as previously described by Marcondes et al. (2002) and Chuffa et al. (2009). The female rats exhibiting regular or irregular estrous cycle were killed, and their ovaries, uterus, and hypophysis were collected and weighed. The uterine horns were ﬁxed in Bouin’s solution and processed by the usual histological routine for parafﬁn embedding (Paraplast Labware-Oxford, St.Louis, MO). The blocks were sliced into 5-lm-thick sections in a LEICA microtome and stained with Hematoxilin-Eosin (H&E) and Mallory’s Tricromic. Histological samples of uterine horns were handled in a blinded fashion study. Finally, the slides were analyzed and captured with a digital photomicroscope. Morphometric Measurements For morphometric analysis of uterine layers, one section was selected and ﬁve consecutive others were discarded, until the last section of the organ, resulting in 10 repetitions/rat/group (50 measures/group). Five micrometer sections of uterine epithelium, endometrial stroma, myometrium, and perimetrium were evaluated in the proximal, middle, and distal portions of uterus, 337 STEROID AND HISTOMORPHOMETRY OF UTERUS TABLE 1. Data of body weight (g), ovaries and uterus weights (g/100 g body weight), and hypophysis weight (g) in female rats receiving ND and submitted or not to physical effort Groups CþS Parameters Body Ovaries Uterus Hypophysis 236 0.110 0.548 0.011 CþE 1.0 0.084 0.025 0.001 237 0.103 0.450 0.011 ND þ S 9.0 0.001 0.014 0.031 274 0.099 0.364 0.009 1.0 0.023 0.100a 0.095a ND þ E 247 0.099 0.445 0.008 1.0 0.020 0.052 0.109b Values are expressed as median interquartile deviation. In the same line, median with letters show statistical differences among the groups (P < 0.05). Kruskal-Wallis with posthoc Dunn test. a P < 0.05 Signiﬁcant difference from C þ S group. b P < 0.05 Signiﬁcant difference from C þ E group. TABLE 2. Morphometric parameters (Mean 6 SD) regarding to three distinct uterine layers among the groups Groups Parameters Thickness PP MP DP Thickness PP MP DP Thickness PP MP DP Thickness PP MP DP of uterine epithelium of endometrial stroma of myometrium of perimetrium CþS CþE ND þ S ND þ E 27.45 5.52 32.05 7.22 23.85 1.98 30.80 6.81 29.80 6.98 29.30 2.08a,c 19.80 1.27a 19.75 2.08a 20.10 3.70 18.30 1.77b 17.20 3.05b 17.95 1.85 532.85 48.75 579.30 112.20 584.05 132.07 661.70 59.05a 638.50 158.00 634.20 128.43 342.75 48.88a 402.15 79.55a 356.00 74.00a 335.20 37.04b 334.05 69.68b 400.45 91.95b 342.95 92.44 327.40 68.91 336.50 69.45 342.95 87.02 416.90 92.22a 422.20 48.70a 326.95 74.22 432.95 87.00a 380.40 74.55 259.10 95.97b,d 300.40 71.24b,d 332.50 40.40b 11.15 1.58 10.20 2.90 11.30 2.02 12.60 3.00 11.55 2.45 12.30 3.75 14.95 3.02a 15.05 1.40a 14.30 3.08a 14.40 2.81 14.30 2.05b 14.95 2.99b PP, MP, and DP ¼ proximal, middle, and distal portion of uterine horn were determined according to ovary position, respectively. Values are expressed using 10 repetitions (lm 10)/animal/group. Letters indicate statistical differences among the groups (P < 0.05). ANOVA test complemented by Newman-Keuls. a P < 0.05 versus C þ S group. b P < 0.05 versus C þ E group. c P < 0.05 versus ND þ E group. d P < 0.05 versus ND þ S group. relative to the position of the oviduct and ovary. The thickness of epithelium was measured on the endometrial surface, and the stromal thickness was obtained by tracing around the on-screen image with a cursor considering repeated measurements in both basal and functional layers (Rossi et al., 2002). Values were taken as the total uterine cross-sectional area. The endometrial glands were not measured. The blood vessels parameters were taken within the predetermined and ﬁxed 1 mm2 area. Thereafter, all the measures, including major or minor diameter as well as the respective areas, were performed randomly with the same slices and following the same criteria (cross-sections) used to determine the thickness of uterine layers. The trials were carried out using the computerized image analysis system Image R (Media Cybernetics) at 100 magniﬁcation. Pro-PlusV Statistical Analysis Body weight data and ovaries, uterus, and hypophysis weights were determined by the nonparametric Kruskal- Wallis test, complemented by Dunn, and the results were expressed as median interquartile deviation. All histomorphometric data were performed by ANOVA, complemented by Newman-Keuls or Tukey tests, and the results were given as mean SD. Statistical signiﬁcance was set at P < 0.05. The statistical software used was Sigma Plot version 11.0 and GraphPad Instat version 4. RESULTS After 4 weeks of treatment, there were no signiﬁcant differences (P > 0.05) in body weight and ovary weight among the experimental groups (Table 1), however, in animals treated with ND, the uterine horn weight was reduced compared to the sedentary control group. NDtreated rats, whether submitted or not to physical effort, presented signiﬁcantly (P < 0.05) lower hypophysis weight than the control groups (Table 1). Vaginal cytology examination revealed that both sedentary and trained control groups presented regular estrous cycles during the experimental period, while the ND-treated 338 DE ALMEIDA CHUFFA ET AL. TABLE 3. Morphometric analysis of blood vessels throughout the myometrial layer Groups Parameters Number of blood vessels (mm2) Major/minor diameter (mm2) Blood vessels area (mm2) CþS 21.11 46.92 45.15 22.46 2.73 1.29 1.89 1.44 CþE 19.20 44.29 42.47 21.11 0.74 1.46 1.70 1.28 ND þ S 14.30 26.13 23.18 15.97 1.70a,b 2.48a 1.98a 0.90a ND þ E 8.67 22.22 21.31 19.45 0.90c 1.43c 1.32c 0.77d Values are expressed as Mean SD. Letters indicate statistical differences among the groups (P < 0.05). ANOVA test complemented by Tukey-Krammer. a P < 0.01 implicate signiﬁcant difference from C þ S group. b P < 0.01 implicate signiﬁcant difference from ND þ E group. c P < 0.01 implicate signiﬁcant difference from C þ E group. d P < 0.01 implicate signiﬁcant difference from ND þ S group. groups showed persistent metaestrous phases, which was classiﬁed as the onset of anovulation. Morphometric data demonstrated that in ND-treated groups, the thickness of uterine epithelium and endometrial stroma were signiﬁcantly reduced (P < 0.05) in the proximal and middle portions (Table 2) compared to the control groups, regardless of exercise. The myometrial layer exhibited variations in thickness. In non-sedentary ND-treated rats, the myometrial layer was reduced in all portions. Although the exercise did not affect the perimetrium, this layer was signiﬁcantly (P < 0.05) thicker in the uterus of ND-treated rats (middle and distal portions) than in control groups. ND-treated rats, submitted or not to exercise, had a reduction in the number of blood vessels around the myometrium, with the most prominent effect (P < 0.001) being found in ND þ E group (Table 3). Regarding blood vessels properties, the major and minor diameter was signiﬁcantly lower in both ND-treated groups than nontreated groups, showing that this effect is because of treatment and not to exercise. Blood vessel area was more restricted in ND-treated rats than in control group, however, ND þ E rats evidenced a greater blood vessel area than those receiving only ND (Table 3). The females of control groups, submitted or not to exercise (Fig. 1A–C), presented a uterine lumen lining consisting of high columnar epithelial cells, with nuclei localized at different levels and no mitotic activity. Fragments of degenerating cells were more evident in C þ E than C þ S group, which were recognized in the epithelium by the presence of pyknotic nuclei and fragmented chromatin (Fig. 1C). Furthermore, stromal tissue presented a loose aspect, with slightly tortuous glands (Fig. 1B). In ND-treated females (Fig. 1D,E) were found a thin uterine epithelium shaped by cubic cells with marked mitotic activity, especially in sedentary rats (Fig. 1G). In addition, short and narrow glands were evidenced in a predominantly ﬁbrous compact stroma (Fig. 1D–F) showing a large amount of widespread leucocytes (Fig. 1E). The ﬁbrous stroma was remarkable in NDtreated rats (Fig. 2B), in contrast to the edematous cellular stroma of the sedentary control group (Fig. 2A). On the basis of transition layer from endometrium to myometrium, it was noted that large and small blood vessels area were lower in ND-treated rats submitted to physical effort (Fig. 2C) than in ND þ S group (Fig. 2D), showing only the effects of exercise. Finally, there was no evidence of morphological impairment in the myometrium or perimetrium of ND-treated rats. DISCUSSION Exercise training decreases fat mass storages in male and female rats (Estadella et al., 2004) as well as in humans (Aull et al., 2008). In this regard, the exercise model utilized in this experiment was not efﬁcient to decrease body weight. Despite of the well known anabolic effect of ND, these rats showed decreased body weight, although it was not statistically signiﬁcant. The results are in accordance with previous studies reported by Blasberg et al. (1997) and Clark et al. (2003). Studies involving female rats (Gerez et al., 2005) and mice (Gao and Short, 1993) have found no differences among reproductive organ weights in ND-treated animals; however, in the present study, the uterine horn weights were reduced when compared to controls (Table 1). One possible explanation is the direct or indirect ND effect on the uterus via hormonal imbalances, and not the role of physical effort by itself. Similarly, Bronson et al. (1996) reported that the ovary and uterus weights are affected by steroid replacement. The reduction of hypophysis weight in ND-treated rats are in agreement with results obtained by Gao and Short (1993), the only extant study reporting the role of ND and its interaction with the hypophysis gland. However, to better understand these effects, further experiments involving rat strains and different doses of AAS are required. To the best of our knowledge, AAS are typically administrated under supraphysiological dose (megadoses) ranging from 3 mg/kg BW to 25 mg/kg BW to increase muscle mass. These concentrations are reported to be 10- to 100fold higher than the therapeutic dose (Brower, 1993; Clark and Fast, 1996). For example, according to Far et al. (2007), a dose of 15 mg/kg BW corresponds to 40fold, the dose used to anaemia treatment. Thus, it should be borne in mind that our dose of 5 mg/kg is almost 15fold higher than those used for such therapies, and it is sufﬁcient to produce failures in rat uterus. According to Feinberg et al. (1997), the synthetic steroids alter the function of hypothalamic-pituitary-gonadal axis, thus indirectly affecting the reproductive tissues. In this condition, the estrous cycle disruptions in ND-treated females may be attributed to hormone interference caused by ND. Our ﬁndings are similar to the results obtained by Howe and Morello (1985); Gao and Short (1993); and Bronson et al. (1996). It should be emphasized that regular physical activity is related to higher levels of anabolic hormones in adults (Copeland, 2004). Blasberg et al. (1997) found that rats receiving 5.6 mg/kg of ND exhibited lordosis behavior accompanied by interrupted vaginal cyclicity because of STEROID AND HISTOMORPHOMETRY OF UTERUS 339 Fig. 1. Photomicrographs of uterine morphology (middle portion). In C þ S group (A) and C þ E group (B), note the high columnar epithelium (ep) and the stroma (st) with loose aspect. In (B), a slightly tortuous gland (*) in edematous stromal tissue is also evidenced; Bar ¼ 30 lm. (C) Fragmented chromatin (arrow) surrounding the epithelium was remarkable in C þ E rats; Bar ¼ 30 lm. In ND þ S and ND þ E groups (D–F), the reduced uterine epithelium (ep) with a thin aspect is shown. Stromal ﬁbrosis (st) was only prominent in those rats that received both ND and exercise. In (E), scarce leucocytes were present around the blood vessels of endometrial stroma. The uterine epithelium featured several cell divisions (arrow) in ND-treated rats in the absence of exercise (G). Bar ¼ 30 lm. G: uterine glands, L: leucocytes, M: epithelial metaplasia. Hematoxylin-Eosin. For all details; Bar ¼ 10 lm. persistent diestrous phases. Knowing that the bioconversion of AAS to estrogens may inﬂuence estrous cyclicity by altering aromatase activity (Friedl and Yesalis, 1989), these enhanced estrogenic/antiestrogenic effects could, at least in part, explain the changes in cyclicity and uterine morphology observed in our study. Regarding uterine blood circulation, all trained and ND-treated rats presented a reduction in the number and diameter of blood vessels throughout the myometrium layer. It seems reasonable to consider that during exhaustive exercise, blood ﬂow is preferentially delivered to skeletal muscle, allowing only a small fraction available to visceral smooth muscles (Rowell, 1993), a phenomenon also observed in rats by Dowell and Kauer (1993). Thus, our ﬁndings can be partially supported by the strenuous exercise. Also, in pregnant women, long-term exercise at moderate/high intensity leads to decreased uterine blood ﬂow (Clapp et al., 2000; Jeffreys et al., 2006). 340 DE ALMEIDA CHUFFA ET AL. Fig. 2. Morphology of the uterine layer in control and ND-treated rats, submitted or not to physical effort. In (A), note the slightly edematous stroma (*) with scattered leucocytes. In ND þ E group (B), ﬁbrous stroma (*) with narrow uterine glands (G) and blood vessels (BV) are prominent. Regarding the inner muscle layer of the myometrium, the blood vessels (arrow) of ND þ E rats (C) displayed a reduction in both number and diameter in contrast to ND þ S rats (D); ep: uterine epithelium, L: leukocytes, M: myometrium, P: perimetrium. Mallory’s Trichrome. Bar ¼ 20 lm. As previously stated, histological and morphometric changes pointed to endometrial atrophy (Table 2, Fig. 1D,E), indicating that uterine layers are quite responsive to AAS treatment. Similarly, supraphysiological doses of ND (15 mg/kg) led to alterations in the uterine morphology, such as endometrial atrophy and glands with tortuous and irregular branching (Far et al., 2007). Taking into account that androgenic receptor (AR) is expressed in both endometrium and myometrium and regulates the proliferative response to ND (Mertens et al., 1996), we conclude that this atrophy was promoted by the maintenance of metaestrus phase (depletion of estrogen levels) induced by the treatment. The appearance of leukocytes inﬁltrated into the endometrial stroma of ND þ E group was also clearly observed. It is well known that leukocytes are recruited by the uterus and undergo changes depending on estrous phase, which in turn is regulated by levels of steroids such as estrogen and progesterone (Kaeoket et al., 2001). In this context, the ND treatment could change the steroidal levels resulting in estrous disruption, which is associated with the presence of these cells. In the current study, the thickening of the perimetrium layer suggests an attempt to restore the composition of the uterus and, conversely, AAS treatment had no effect on this parameter, regardless of physical effort. In ND-treated females, the most evident morphological changes were the presence of a thin luminal epithelium and the ﬁbrous stroma. There are several studies focusing uterus alterations and its consequences to reproduction; however, it is not a consensus when related to androgen responsiveness. Thereby, other results obtained by YuYahiro et al. (1989) reported that treatment with ND promoted epithelial vacuolization, stromal edema, and peliosis in rat uteri. In contrast, Gerez et al. (2005) has assigned substantial ﬁbrosis on the endometrial stroma and some epithelial vacuolization in females treated with ND. Curiously, a recent study has demonstrated that replacement therapy with estradiol (E2) is directly related to stromal ﬁbrosis (Wood et al., 2010). Thus, we could explain partially these ﬁndings, as a part of AAS is converted to estrogen by aromatase activity. Nevertheless, the exact mechanism by which ND histomorphologically affects the uterus remains unclear. In conclusion, our ﬁndings suggest that ND has a different pattern of response in female rats, independent to the physical effort. Moreover, ND promotes histomorphometric changes to the uterine structure of adult female rats. ACKNOWLEDGMENTS The authors thank Mrs. Maria Isabel de Oliveira from the Department of Biological Sciences, for her technical assistance. STEROID AND HISTOMORPHOMETRY OF UTERUS LITERATURE CITED Aull JL, Rowe DA, Hickner RC, Malinauskas BM, Mahar MT. 2008. Energy expenditure of obese, overweight, and normal weight females during lifestyle physical activities. Int J Pediatr Obes 3:177–185. Biden JR, Jr. 2006. Steroid side effects. The Washington Times, February 21, p A13. Bishop G. 2005. Growing issue for women; getting a boost-steroid use has increased among highschool girls. The Seattle Times, October 10, p D5. Blasberg ME, Langan CJ, Clark AS. 1997. The effects of alphamethyltestosterone, methandrostenolone and nandrolone decanoate on the rat estrous cycle. Physiol Behav 61:265–272. Bronson FH, Nguyen KQ, De La Rosa J. 1996. Effect of anabolic steroid on physiological characteristics of female mice. Physiol Behav 59:49–55. Brower KJ. 1993. Anabolic steroids. Psychiatr Clin North Am 16:97–103. Camargo ICC, Souza RB, Paccola-Mesquita SF, Chuffa LG, Frei F. 2009. Ovarian histology and follicular score in female rats treated with nandrolone decanoate and submitted to physical effort. Acta Biol Hung 60:253–261. Cannavò S, Curtò L, Trimarchi F. 2001. Exercise-related female reproductive dysfunction. J Endocrinol Invest 24:823–832. Chuffa LGA, Padovani CR, Martinez FE. 2009. Ovarian structure and hormonal status of the UChA and UChB adult rats in response to ethanol. Maturitas 62:21–29. Clapp JF, III. 2003. The effects of maternal exercise on fetal oxygenation and feto-placental growth. Eur J Obstet Gynecol Reprod Biol 110:S80–S85. Clapp JF, III, Stepanchak W, Tomaselli J, Kortan M, Faneslow S. 2000. Portal vein blood ﬂow-effects of pregnancy, gravity, and exercise. Am J Obstet Gynecol 183:167–172. Clark AS, Blasberg ME, Brandling-Bennett EM. 1998. Stanozolol, oxymetholone, and testosterone cypionate effects on the rat estrous cycle. Physiol Behav 63:287–295. Clark AS, Fast AS. 1996. Comparison of the effects of 17 alphamethyltestosterone, methandrostenolone, and nandrolone decanoate on the sexual behavior of castrated male rats. Behav Neurosci 110: 1478–1486. Clark AS, Harrold EV, Fast AS. 1997. Anabolic-androgenic steroid effects on the sexual behavior of intact male rats. Horm Behav 31:35–46. Clark AS, Kelton MC, Whitney AC. 2003. Chronic administration of anabolic steroids disrupts pubertal onset and estrous cyclicity in rats. Biol Reprod 68:465–471. Copeland JL. 2004. Anabolic hormones in aging women: effects of supplementation vs. physical activity. Can J Appl Physiol 29:76–89. Davies GAL, Wolfe LA, Mottola MF, MacKinnon C. 2003. Clinical Practice Guideline: exercise in pregnancy and the postpartum period. Can J Appl Physiol 28:329–341. Dowell RT, Kauer CD. 1993. Uteroplacental blood ﬂow at rest and during exercise in late-gestation conscious rats. J Appl Physiol 74:2079–2085. Elliot DL, Cheong J, Moe EL, Goldberg L. 2007. Cross-sectional study of female students reporting anabolic steroid use. Arch Pediatr Adolesc Med 161:572–577. Estadella D, Oyama LM, Dâmaso AR, Ribeiro EB, Oller Do Nascimento CM. 2004. Effect of palatable hyperlipidic diet on lipid metabolism of sedentary and exercised rats. Nutrition 20:218–224. Far HRM, Agren G, Lindqvist AS, Marmendal M, Fahlke C, Thiblin I. 2007. Administration of the anabolic androgenic steroid nandrolone decanoate to female rats causes alterations in the morphology of their uterus and a reduction in reproductive capacity. Eur J Obstet Gynecol Reprod Biol 131:189–197. Feinberg MJ, Lumia AR, McGinnis MY. 1997. The effect of anabolic-androgenic steroids on sexual behavior and reproductive tissues in male rats. Physiol Behav 62:23–30. Friedl KE, Yesalis CE. 1989. Self-treatment of gynecomastia in bodybuilders who use anabolic steroids. Phys Sportsmed 17:67–79. Gao Y, Short RV. 1993. Use of an oestrogen, androgen or gestagen as a potential chemosterilant for control of rat and mouse populations. J Reprod Fertil 97:39–49. 341 Gerez JR, Frei F, Camargo ICC. 2005. Histological assessment of ovaries and uterus of rats submitted to nandrolone decanoate treatment. Contraception 72:77–80. Gruber AJ, Pope HG, Jr. 2000. Psychiatric and medical effects of anabolicandrogenic steroid use in women. Psychother Psychosom 69:19–26. Hickson RC, Ball KL, Falduto MT. 1989. Adverse effects of anabolic steroids. Med Toxicol Adverse Drug Exp 4:254–271. Howe GR, Morello CJ. 1985. Effects of an anabolic steroid on reproduction in female rats. Steroids 45:495–501. Iriart JAB, Andrade TM. 2002. Body-building, steroid use, and risk perception among young body-builders from a low-income neighborhood in the city of Salvador, Bahia State, Brazil. Toxicol Appl Pharmacol 18:71–81. Jeffreys RM, Stepanchak W, Lopez B, Hardis J, Clapp JF,III. 2006. Uterine blood ﬂow during supine rest and exercise after 28 weeks of gestation. BJOG 113:1239–1247. Kaeoket K, Persson E, Dalin AM. 2001. The sow endometrium at different stages of the oestrous cycle: studies on morphological changes and inﬁltration by cells of the immune system. Anim Reprod Sci 65:95–114. Kanayama G, Boynes M, Hudson JI, Field AE, Pope HG, Jr. 2007. Anabolic steroid abuse among teenage girls: an illusory problem? Drug Alcohol Depend 88:156–162. Karbalay-Doust S, Noorafshan A. 2009. Stereological study of the effects of nandrolone decanoate on the mouse liver. Micron 40:471–475. Korkia P, Stimson GV. 1997. Indications of prevalence, practice and effects of anabolic steroid use in Great Britain. Int J Sports Med 18:557–562. Kramer MS, McDonald SW. 2006. Aerobic exercise for women during pregnancy. Cochrane Database Syst Rev 3:CD000180. DOI: 10.1002/14651858.CD000180.pub3. Lane JR, Connor JD. 1994. The inﬂuence of endogenous and exogenous sex hormones in adolescents with attention to oral contraceptives and anabolic steroids. J Adolesc Health 15:630–634. Marcondes FK, Bianchi FJ, Tanno AP. 2002. Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol 62:609–614. Marqueti RC, Prestes J, Wang CC, Ramos OH, Perez SE, Nakagaki WR, Carvalho HF, Selistre-de-Araujo HS. Biomechanical responses of different rat tendons to nandrolone decanoate and load exercise. Scand J Med Sci Sports. DOI: 10.1111/j.1600-0838.2010.01162.x. Mertens HJ, Heineman MJ, Koudstaal J, Theunissen P, Evers JL. 1996. Androgen receptor content in human endometrium. Eur J Obstet Gynecol Reprod Biol 70:11–13. Obasanjo IO, Cline JM, Schmotzer S, Weaver DS. 1998. Nandrolone decanoate causes pathologic changes in the uterus of surgically postmenopausal female cynomolgus macaques. Menopause 5:163–168. Peres SB, Luciano E. 1995. Inﬂuências de esteróide anabólico (Deca Durabolin) sobre o metabolismo de ratos submetidos ao treinamento fı́sico. Rev Paul Educ Phys 9:131–137. Pope HG, Jr, Katz DL. 1988. Affective and psychotics symptoms associated with anabolic steroids use. Am J Psychiatry 145:487–490. Rossi AG, Soares JM, Jr., Motta EL, Simões MJ, Oliveira-Filho RM, Haidar MA. 2002. Metoclopramide-induced hyperprolactinemia affects mouse endometrial morphology. Gynecol Obstet Invest 54:185–190. Rowell LB. 1993. Control of regional blood ﬂow during dynamic exercise. In: Rowell LB, editor. Human cardiovascular control. New York: Oxford University Press. p 209–218. Strauss RH, Liggett MT, Lanese RR. 1985. Anabolic steroid use and perceived effects in ten weight-trained women athletes. JAMA 253:2871–2873. Warren MP, Perlroth NE. 2001. The effects of intense exercise on the female reproductive system. J Endocrinol 170:3–11. Wilson JD, Grifﬁn JE. 1980. The use and misuse of androgens. Metabolism 29:1278–1295. Wood CE, Kaplan JR, Fontenot MB, Williams JK, Cline JM. 2010. Endometrial proﬁle of tamoxifen and low-dose estradiol combination therapy. Clin Cancer Res 16:946–956. Yu-Yahiro J, Michael RH, Nasralah DV, Schoﬁeld B. 1989. Morphologic and histologic abnormalities in female and male rats treated with anabolic steroids. Am J Sports Med 17:686–689.