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1.  Return to fertility after extended chemical castration with a GnRH antagonist 
BMC Cancer  2001;1:18.
Background
Antagonistic analogues of GnRH for the treatment of prostate cancer may be used clinically in persons for whom return to fertility after such treatment is important or desirable. The purpose of this study was, therefore, to evaluate the effects of a long term treatment with orntide, a GnRH antagonist, on testosterone levels and fertility in male rats.
Methods
Two groups of male rats received either 120-day orntide microspheres (8.8 mg orntide/kg/120 days) or vehicle alone (control group). Serum orntide and testosterone levels in both groups were monitored at certain intervals for 9 months from the initiation of treatment. After recovery of normal serum testosterone levels in the treated animals, each rat was housed with two proven breeder, but drug-naive, females.
Results
All mates of treated rats achieved pregnancy as rapidly as the mates of control rats although two of the control rats did not sire a litter with either female and one sired only one litter. The mean size of the litters of treated (12.3 offspring per litter) and control (10.6 offspring per litter) were similar. All offspring were grossly normal morphologically and behaviorally during the time to weaning.
Conclusions
These results suggest that lack of fertility due to testosterone suppression is reversible after cessation of treatment with this GnRH antagonist.
doi:10.1186/1471-2407-1-18
PMCID: PMC59839  PMID: 11710965
2.  Evaluation of orntide microspheres in a rat animal model and correlation to in vitro release profiles 
AAPS PharmSciTech  2000;1(4):4-16.
Orntide acetate, a novel luteinizing hormone-releasing hormone (LHRH) antagonist, was prepared and evaluated in vivo in 30-day and 120-day sustained delivery formulations using a rat animal model. Orntide poly(d,l- lactide-co-glycolide) (PLGA) and poly(d,l- lactide) (PLA) microspheres were prepared by a dispersion method and administered subcutaneously in a liquid vehicle to rats at 2.2 mg Orntide/kg of body weight (30-day forms) or 8.8 mg Orntide/kg (120-day forms). Serum levels of Orntide and testosterone were monitored by radioimmunoassays, and a dose-response study at 4 closes (3, 2.25, 1.5, and 1.75 mg Orntide/kg) was conducted to determine the effective dose of Orntide. Microspheres with diameters between 3.9 and 14 μ were prepared. The onset and duration of testosterone suppression varied for different microsphere formulations and were influenced both by polymer properties and by microsphere characteristics. Microspheres prepared with 50∶50 and 75∶25 copolymers effectively sustained peptide release for 14 to 28 days, whereas an 85∶15 copolymer and the PLA microspheres extended the pharmacological response for more than 120 days. Increase in drug load generally accelerated peptide release from the microspheres, resulting in higher initial serum levels of Orntide and shorter duration of the release: In general, apparent release was faster in vivo than under in vitro conditions. Orntide microspheres effectively suppressed testosterone in rats, providing rapid onset of release and extended periods of chemical castration. Testosterone suppression occurred immediately after microsphere administration without the initial elevation seen with LHRH superagonists.
doi:10.1208/pt010427
PMCID: PMC2750451  PMID: 14727892
LHRH antagonist; Orntide acetate; Peptide controlled delivery; PLGA microspheres; Prostate cancer
3.  A novelin vitro release technique for peptide-containing biodegradable microspheres 
AAPS PharmSciTech  2000;1(1):30-40.
The purpose of this study was to develop and evaluate a dialysisin vitro release technique for peptide-containing poly(d, 1-lactide-coglycolide) (PLGA) microspheres (ms) that would correlate within vitro data. Using a luteinizing hormone- releasing hormone analogue (LHRH), Orntide acetate, solubility and stability were determined in 0.1 M phosphate buffer (PB), pH 7.4, and in 0.1 M acetate buffer (AB), pH 4.0, with highperformance liquid chromotography (HPLC), and peptide permeability through a dialysis membrane (molecular weight cut-off 300,000) was determined. Orntide ms were prepared by a dispersion/solvent extraction/evaporation method and characterized for drug content (HPLC), particle size distribution (laser diffraction method), and surface morphology (scanning electron microscopy).In vitro release was studied in PB using a conventional extraction method and with a new dialysis method in AB. Gravimetric analyses of polymer mass loss and matrix hydration, and peptide adsorption to blank PLGA ms (50∶50, Mw 28 022) were carried out in PB and AB upon incubation at 37°C. Serum Orntide and testosterone levels in rats after administration of Orntide ms were determined by radioimmunoassay. Orntide acetate solubility was influenced by pH; approximately 2.3 mg/mL dissolved in PB and >18 mg/mL in AB. Stability was pH- and temperature-dependent. The peptide was very stable at pH 4.0, 4°C, but degraded rapidly at pH 7.4,37°C. Peptide permeability through the dialysis membrane was accelerated by agitation and>95% equilibrium was reached within 48 hours. The overall release rate was higher with the dialysis method. Mass loss of the Orntide ms was faster in AB (50% loss in 3 weeks: 95% in 35 days) than in PB (65% in 35 days). In contrast, hydration after 35 days was 4-fold higher in PB. The nonspecific adsorption to blank ms was greater in PB (128 μ g Orntide/10 mg PLGA) compared with AB (<5 μ g Orntide/10 mg PLGA). Administration of 30-day Orntide PLGA ms to rats resulted in an initial serum Orntide level of 21 ng/mL after 6 hours and a Cmax of 87 ng/mL after 6 days. Testesterone levels were suppressed immediately after ms administration (3 mg Orntide/Kg) from 5.2 ng/mL to 0.3 ng/mL (after 24 hours) and remained suppressed for 38 days. Orntide acetate solubility and degradation kinetics were markedly influenced by pH of the buffer systems and mass loss; matrix hydration, as well as the nonspecific adsorption to blank ms, was pH-dependent. Thein vitro release profile obtained with the dialysis method in AB correlated well with thein vivo data, therepy providing a more reliable prediction ofin vivo performance.
doi:10.1208/pt010104
PMCID: PMC2784831  PMID: 14727853
Orntide Microspheres; Solubility; Stability; In vitro release; Dialysis, PLGA
4.  Regulation of pituitary gonadotropin-releasing hormone receptors by pulsatile gonadotropin-releasing hormone injections in male rats. Modulation by testosterone. 
Journal of Clinical Investigation  1984;74(3):920-928.
The pattern of the gonadotropin-releasing hormone (GnRH) stimulus is critically important in the regulation of pituitary gonadotropin secretion and continuous infusions down-regulate secretion while intermittent pulses maintain luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responsiveness. We examined the effects of pulsatile GnRH administration on pituitary GnRH receptors (GnRH-R) and gonadotropin secretion in the presence of physiological concentrations of testosterone (T) to elucidate the mechanisms and sites of action of GnRH and T on the pituitary gonadotroph. Castrate male rats received one, two, or four testosterone (T) implants (serum T concentrations of 1.1, 2.4, and 5.2 ng/ml, respectively) to suppress endogenous GnRH secretion. Subsequently, intracarotid pulse injections of GnRH (5-250 ng/pulse) or saline in controls were given every 30 min for 48 h, after which gonadotropin responses and pituitary GnRH-R were measured. In control rats, the T implants prevented the rise in GnRH-R that was seen in castrates (empty implant--600 fmol/mg protein) and maintained receptors at the level that was present in intact animals (300 fmol/mg). Pulsatile GnRH administration increased GnRH-R in castrate T-implanted rats, but the response was dependent on the serum T concentration. With one T implant, increasing GnRH doses per pulse stimulated GnRH-R in a linear manner and the maximum receptor concentration (703 +/- 99 fmol/mg) was seen after the 250 ng GnRH dose. In the presence of two T implants, GnRH-R was maximal (705 +/- 45 fmol/mg) after the 25-ng dose and higher doses did not increase receptors above control values. With four T implants, GnRH doses of 5 ng induced a maximum response, 17-50 ng/pulse did not increase GnRH-R, but receptors were again increased by the 250-ng dose (633 +/- 86 fmol/mg). After 48 h of pulsatile GnRH administration there was no correlation between the number of GnRH-R and LH responses to GnRH. In rats with one or two T implants, LH responses were absent after all but the 250-ng doses. In contrast, LH responsiveness was not impaired in the presence of four implants. Thus, low dose GnRH pulses down-regulate LH secretion by an action at a post GnRH-R site, and this effect is regulated by testosterone. The results show that GnRH, given in a pulsatile manner, regulates its own receptor, and physiological increases in serum T produce a 50-fold increase in the sensitivity of GnRH-R stimulation by GnRH.
Images
PMCID: PMC425248  PMID: 6088587
5.  Pituitary gonadotropin-releasing hormone receptors. Effects of castration, steroid replacement, and the role of gonadotropin-releasing hormone in modulating receptors in the rat. 
Journal of Clinical Investigation  1981;67(3):615-623.
To study the role of gonadotropin-releasing hormone (GnRH) receptors in the regulation of gonadotropin secretion, we used D-125I-alanine6 des glycyl10 GnRH ethylamide (D-125I-Ala analog), a nondegradable, superagonist GnRH analog to assess GnRH receptors on rat pituitary membranes. Receptor affinity in intact adult rats was 5.0 X 10(9) M-1 and was unchanged after castration in both sexes. Castration of adult male and female rats produced a twofold increase in GnRH binding capacity by 7 d and binding capacity remained elevated for the subsequent 14 d. GnRH receptor number rose more rapidly after castration in males than females, and the time-course of receptor rise was similar to the increase in serum gonadotropin levels. The increase in GnRH binding capacity was prevented by gonadal steroid replacement at the time of castration in both sexes. Injections of the GnRH analog, D-Ser6 (TBu) des Gly10 GnRH ethylamide for 4 d produced a 70% increase in GnRH receptor number in intact male rats and testosterone-replaced castrates. The same regimen, however, failed to increase the elevated receptor numbers present after castration. Administration of rabbit anti-GnRH serum concomitant with castration inhibited the rise in both GnRH receptor number and luteinizing hormone. The changes in pituitary GnRH receptors parallel previously demonstrated changes in hypothalamic secretion of GnRH. Thus, GnRH probably regulates its own receptor in vivo and gonadal steroids may influence pituitary GnRH receptors by changing hypothalamic GnRH secretion.
PMCID: PMC370609  PMID: 6259205
6.  Evaluation of possible toxic effects of spearmint (Mentha spicata) on the reproductive system, fertility and number of offspring in adult male rats 
Objective: In this study we investigated the effects of spearmint (Mentha spicata Labiatae) on the reproductive system, fertility and number of offspring in adult male rats.
Materials and Methods: Adult Wistar male rats in one control (C) and three experimental groups (I, II and III) received 0, 10, 20 and 40 mg/kg spearmint extract orally for 45 days, respectively. Following this treatment, the animals’ weights, and the standard weight of reproductive tissues, sperm count, sperm motility and serum testosterone concentration were measured, and reproductive tissues were examined histopathologically. To evaluate the effects of spearmint on fertility of male rats and growth of their offspring, male rats of the control and experimental groups mated with untreated female rats.
Results: Results showed that spearmint did not affect the rats’ body and reproductive tissue weights. The sperm count, fast and slow progressive motility of sperm and serum testosterone concentration decreased while number of non-progressive sperm and immotile sperm increased in the experimental groups compared to the control group, but none of these changes were statistically significant. Histopathological studies showed no severe changes in reproductive tissues between control and experimental groups. Number and growth of offspring born from mating of male rats with untreated female rats showed no difference.
Conclusion: We concluded that spearmint has no significant toxic effect on the reproductive system, fertility and number of offspring in adult male rats at the above mentioned dose levels. However high levels of this extract may have adverse effects on male fertility.
PMCID: PMC4224956  PMID: 25386406
Fertility; Male; Spearmint; Sperm; Toxicity
7.  Effects of altered prenatal hormonal environment on expression of autoimmune disease in NZB/NZW mice. 
Environmental Health Perspectives  1996;104(Suppl 4):815-821.
F1 hybrid New Zealand Black (NZB) x New Zealand White (NZM) (NZB/NZW) mice spontaneously develop an autoimmune disease analogous to systemic lupus erythematosus (SLE). Testosterone experts a powerful suppressive effect on this disorder in adult NZB/NZW mice. A series of experiments was designed to determine if disease would also be suppressed by exposing fetal NZB/NZW mice to increased testosterone. A model was developed in which NZB dams carrying NZB/NZW fetuses were treated with testosterone in a dose adequate to masculinize the external genitalia in female fetuses. NZB/NZW mice that were derived from testosterone-treated dams and control NZB/NZW offspring were followed in a longevity study and had serial assays to assess development of SLE. Additional experiments were carried out to measure lymphocyte subsets and responses to mitogens. Results were compared with F1 hybrid offspring of C57BL/6 dams crossed with DBA/2 males, which are not autoimmune and do not develop SLE. Spleen cells from these groups were tested for Thy 1.2, CD4, CD8, and IgM receptors, and for responses to the mitogens Concanavalin A (ConA) and lipopolysaccharide. Control male NZB/NZW fetuses had unexpectedly high serum estradiol, which decreased significantly with maternal testosterone treatment. The testosterone-exposed male NZB/NZW fetuses developed into adults that lived longer than male NZB/NZW controls. Testosterone treatment of the dam was associated with elevated terminal anti-DNA levels but did not alter markers of renal diseases in adult NZB/NZW mice of either sex. Testosterone-exposed NZB/NZW females had altered T-lymphocyte subsets and testosterone-exposed males had increased response to ConA compared to controls. In male NZB/NZW fetuses whose mothers were administered testosterone, the naturally high level of circulating estradiol observed in untreated male fetuses was decreased significantly. This decrease was associated with an increase in longevity. This unique observation has important implications for fetal exposure to endocrine disruptors in the environment.
PMCID: PMC1469666  PMID: 8880004
8.  The Effect of Amantadine on Clomipramine Induced Sexual Dysfunction in Male Rats 
Oman Medical Journal  2011;26(6):404-409.
Objective
Several studies have reported that Clomipramine has the ability to suppress male rat sexual behavior. Literature indicates that the activation of brain D2 receptors causes facilitation of penile erection, and a number of reports have indicated dopamine’s involvement in sexual function. Hence this study was undertaken to investigate the effect of Amantadine, a dopamine agonists on the Clomipramine induced sexual dysfunction.
Methods
The study subjects involved a total of 48 males and 48 females, 4 months old Sprague-Dawley albino rats, all housed in a group of six males and females separately in plexi glass cages in an acclimatized colony room (25±0.50C) maintained on a 12/12 hr light/dark cycle. The male rats were randomly divided into four groups of 12 male rats each. Group I served as controls. Group II, III, and IV were treated with Amantadine (9 mg/kg body weight, p.o) 30 min, prior to the treatment with 13.5 mg/kg, 27 mg/Kg and 54 mg/Kg bodyweight p.o of Clomipramine respectively for 60 days. The control group received vehicle 1 ml / kg p.o. The sexual behavior of the male rats was observed to determine the following parameters: mount latency, intromission latency, ejaculation latency, post ejaculatory pause, and intromission frequency. As well as the sexual behavior; serum testosterone and histopathology of the testes were also investigated in this study.
Results
The results indicate that Amantadine in all aspects failed to antagonize Clomipramine induced sexual dysfunction in male rats. Even the sexual competence of male rats treated with ½ therapeutic dose (TD) of Clomipramine failed to regain their sexual competence in the presence of Amantadine. Testicular damage and decline in testosterone levels continued in the presence of Amantadine.
Conclusion
Overall, the results suggest that Amantadine could not be a safe antidote to antagonize Clomipramine induced sexual dysfunction.
doi:10.5001/omj.2011.104
PMCID: PMC3251195  PMID: 22253948
Clomipramine; Amantadine; Male rat sexual dysfunction; Testosterone; Testes
9.  TESTOSTERONE AND SOCIAL ISOLATION INFLUENCE ADULT NEUROGENESIS IN THE DENTATE GYRUS OF MALE RATS 
Neuroscience  2011;195:180-190.
Testosterone has been previously shown to enhance adult neurogenesis within the dentate gyrus of adult male rats, whereas social isolation has been shown to cause a decrease in adult neurogenesis under some conditions. The current study tested the combined effects of testosterone and social isolation upon adult neurogenesis using two experiments involving adult male rats. For both experiments, half of the subjects were pair-housed and half were housed individually for the duration of the experiments (34 days). For experiment 1, the subjects were divided into four groups (n=8/group): 1) sham/pair-housed, 2) sham/isolated, 3) castrate/pair-housed, and 4) castrate/isolated. Rats in the castrate groups were bilaterally castrated, and rats in the sham groups were sham castrated. For experiment 2, all rats were castrated and the effects of testosterone were tested using daily injections of testosterone propionate (0.500 mg/rat for 15 days) or the oil vehicle. Subjects were divided into four groups (n =8/group): 1) oil/pair-housed, 2) oil/isolated, 3) testosterone/pair-housed, and 4) testosterone/isolated. All rats were injected with 5-Bromo-2’-deoxyuridine (BrdU, 200 mg/kg body mass) and immunohistochemistry was used to determine levels of neurogenesis following a 16-day cell survival period. For experiment 1, castrated subjects had significantly fewer BrdU-labeled cells along the granule cell layer and sub-granular zone (GCL+SGZ) of the dentate gyrus than did intact subjects, and this effect was mainly due to low levels of neurogenesis in the castrate/isolated group. For experiment 2, social isolation caused a significant decrease in neurogenesis within the GCL+SGZ relative to the pair-housed groups. Testosterone injections did not buffer against this effect but instead tended to cause a decrease in neurogenesis. Thus, social isolation reduced hippocampal neurogenesis, but the effects of testosterone were inconsistent. This suggests that normal circulating levels of testosterone may buffer against the neurogenesis-impairing effects of isolation, whereas high doses of testosterone do not.
doi:10.1016/j.neuroscience.2011.08.034
PMCID: PMC3198792  PMID: 21875652
adult neurogenesis; androgen; bromodeoxyuridine; hippocampus; social isolation; testosterone
10.  The Effect of Chronic Administration of Methylphenidate on Morphometric Parameters of Testes and Fertility in Male Mice 
Background
Due to common use of methylphenidate (MPH) for the treatment of Attention Deficit Hyperactivity Disorder (ADHD) and the role of the reproductive system in the production of gametes, studying the effects of this medication on the morphometry of testes, serum testosterone concentration, leydig cells function, and fertility rate was the aim of this study.
Methods
Twenty seven male mice (Balb/C), eight weeks old, were randomly divided into one control and two treated groups. After weighing the mice, the treated groups received MPH (produced in Novartis company) at the doses of 2 mg/kg and 10 mg/kg for 40 days. The control group received only normal saline. Subsequently, after weighing the animals, the weights of testes, dimensions of the testis, and the serum testosterone concentration were measured in six mice belonging to each group. After tissue processing, the samples were stained with hematoxylin and eosin, then the leydig cells were counted. In order to assess male fertility in each group, 3 male mice were chosen and each of them was kept with three female mice in a separate cage. After 10 days, the fertility rates of the male mice were determined by counting the number of embryos in uterus and the corpora lutea in their ovaries.
Results
The results of this study revealed that prescription of different doses of MPH can cause a significant decrease of the body weight. It reduces the number of leydig cells, too (p<0.01). Moreover, serum testosterone concentration (67.72±8.24 ng/ml in control group and 0.302±0.416 ng/ml after treatment with 2 mg/kg/day MPH) and fertility rate (95.42%±4.68% in control group and 64.96%±18.51% after treatment with 2 mg/kg/day MPH) of the male mice declined significantly in the treated groups compared with the control group (p<0.01), but it did not cause any changes in the weight or morphometric parameters of testes.
Conclusion
The results of this study confirmed that MPH can negatively affect serum testosterone concentration and fertility rate of the male mice by decreasing the number of leydig cells and reducing the body weight.
PMCID: PMC3719348  PMID: 23926551
Methylphenidate; Mice; Testis; Testosterone
11.  Effects of decreasing the frequency of gonadotropin-releasing hormone stimulation on gonadotropin secretion in gonadotropin-releasing hormone-deficient men and perifused rat pituitary cells. 
Journal of Clinical Investigation  1988;81(6):1725-1733.
The effects of decreasing the frequency of pulsatile gonadotropin-releasing hormone (GnRH) stimulation on pituitary responsiveness were studied in (a) men with isolated GnRH deficiency who had achieved normal sex steroid levels during prior long-term pulsatile GnRH replacement and (b) perifused dispersed pituitary cells from male rats in the absence of sex steroids. In three groups of four GnRH-deficient men, the frequency of GnRH stimulation was decreased at weekly intervals from (a) every 2-3-4 h (group I), (b) every 2-8 h without testosterone replacement (group II), or (c) every 2-8 h with testosterone replacement (group III). In three groups of three columns of perifused dispersed pituitary cells, pulses of GnRH were administered every 2, 4, or 8 h. In groups I and II, mean area under the luteinizing hormone (LH) curve increased (P less than 0.025) and serum testosterone levels fell (P less than 0.035) as the frequency of GnRH stimulation was decreased. In group III, the area under the LH curve also increased (P less than 0.01) although serum testosterone levels were constant, thereby demonstrating that the increase in pituitary responsiveness to slow frequencies of GnRH stimulation occurs independently of changes in the sex steroid hormonal milieu. The area under the LH curve also increased in the perifused dispersed rat pituitary cells when the frequency of GnRH administration was decreased to every 8 h (P less than 0.05), thus demonstrating that the enhanced pituitary responsiveness to slow frequencies of GnRH stimulation is maintained even in the complete absence of gonadal steroids. Nadir LH levels fell in all three groups (P less than 0.01) as the frequency of GnRH stimulation was decreased. In contrast, mean peak LH levels, the rate of LH rise, and the rate of endogenous LH decay were constant as the frequency of GnRH stimulation was decreased. Finally, as the GnRH interpulse interval increased, mean LH levels fell, and mean follicle-stimulating hormone levels were stable or fell. These results indicate that (a) pituitary responsiveness to GnRH increases at slower frequencies of GnRH stimulation in models both in vivo and in vitro, (b) these changes in pituitary responsiveness occur independently of changes in gonadal steroid secretion, and (c) the increases in LH pulse amplitude and area under the curve at slow frequencies of GnRH stimulation are due to decreases in nadir, but not peak, LH levels. Slowing of the frequency of GnRH secretion may be an important independent variable in the control of pituitary gonadotropin secretion.
Images
PMCID: PMC442617  PMID: 3290251
12.  Prenatal Testosterone Exposure Leads to Hypertension That Is Gonadal Hormone-Dependent in Adult Rat Male and Female Offspring1  
Biology of Reproduction  2012;86(5):137.
ABSTRACT
Prenatal testosterone exposure impacts postnatal reproductive and endocrine function, leading to alterations in sex steroid levels. Because gonadal steroids are key regulators of cardiovascular function, it is possible that alteration in sex steroid hormones may contribute to development of hypertension in prenatally testosterone-exposed adults. The objectives of this study were to evaluate whether prenatal testosterone exposure leads to development of hypertension in adult males and females and to assess the influence of gonadal hormones on arterial pressure in these animals. Offspring of pregnant rats treated with testosterone propionate or its vehicle (controls) were examined. Subsets of male and female offspring were gonadectomized at 7 wk of age, and some offspring from age 7 to 24 wk received hormone replacement, while others did not. Testosterone exposure during prenatal life significantly increased arterial pressure in both male and female adult offspring; however, the effect was greater in males. Prenatal androgen-exposed males and females had more circulating testosterone during adult life, with no change in estradiol levels. Gonadectomy prevented hyperandrogenism and also reversed hypertension in these rats. Testosterone replacement in orchiectomized males restored hypertension, while estradiol replacement in ovariectomized females was without effect. Steroidal changes were associated with defective expression of gonadal steroidogenic genes, with Star, Sf1, and Hsd17b1 upregulation in testes. In ovaries, Star and Cyp11a1 genes were upregulated, while Cyp19 was downregulated. This study showed that prenatal testosterone exposure led to development of gonad-dependent hypertension during adult life. Defective steroidogenesis may contribute in part to the observed steroidal changes.
Elevated maternal testosterone leads to hypertension that is associated with hyperandrogenism and defective gonadal steroidogenesis in adult offspring; peripubertal gonadectomy prevents hyperandrogenism and reverses hypertension in these animals.
doi:10.1095/biolreprod.111.097550
PMCID: PMC3364920  PMID: 22302690
gonadectomy; hypertension; prenatal testosterone; sex steroids; steroidogenesis
13.  Castration-induced testosterone deficiency increases fasting glucose associated with hepatic and extra-hepatic insulin resistance in adult male rats 
Background
Testosterone deficiency is associated with insulin resistance. However, how testosterone deficiency affects insulin actions remains unclear. The aim of this study was to investigate the influence of castration-induced testosterone deficiency on the metabolic kinetics of glucose and to evaluate the hepatic and extra-hepatic insulin sensitivity, in advanced-age male Sprague–Dawley (SD) rats.
Methods
Ten-week-old male SD rats were randomly divided into three groups: (1) a control group (n = 10) in which the rats underwent sham castration (2) a castrated group (TD group for testosterone deficiency, n = 10) in which the rats underwent bilateral orchidectomy surgery and (3) a castrated group given testosterone propionate via intraperitoneal injection (25 mg/kg/day) to supplement androgen (TD + TP group, n = 10). At ten weeks after castration in the noted groups, all rats were subjected to an oral glucose tolerance test (OGTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Twenty weeks following that treatment, all rats underwent a hyperinsulinemic-euglycemic clamp procedure in conjunction with isotope--labeled glucose and glycerol tracer infusions. The rate of appearance (Ra) of glucose, glycerol and gluconeogenesis (GNG), hepatic glucose production and the rate of glucose disappearance (Rd) were assessed. Glucose uptake was determined by measuring the 2-deoxy-D-14C-glucose in the gastrocnemius muscles.
Results
Ten weeks after castration in the TD group, the fasting blood glucose and insulin levels were significantly increased (p < 0.01), the glucose-- induced insulin secretion was impaired and ITT revealed a temporarily increased whole body insulin sensitivity compared with the control group; 30 weeks after castration, the Ra of glucose, Ra of glycerol, as well as the HGP and GNG were also increased (p < 0.01), while the exogenous glucose infusion rate and uptake glucose in the muscle markedly decreased (p < 0.01).
Conclusions
Castration-induced testosterone deficiency primarily increases fasting blood glucose levels. The clamp experiments revealed a clear insulin resistance both at the hepatic and extra-hepatic levels.
doi:10.1186/1477-7827-11-106
PMCID: PMC4225661  PMID: 24238614
Orchidectomy; Metabolic kinetics; Isotopic trace; Insulin resistance; Hyperinsulinemic-euglycemic clamp
14.  Effect of Phaleria macrocarpa on sexual function of rats 
Objectives: The purpose of current study was to determine the effect of Phaleria macrocarpa (P. macrocarpa) fruits aqueous extract on reproductive performance of adult male rats by assessing the serum testosterone level and evaluating their libido behavior.
Methods and Materials: Eighteen male adult Spraque Dawley rats were divided into three groups and designated as treatment (240 mg/kg P. macrocarpa aqueous extract), negative control (distilled water), and positive control (4 mg/kg testosterone) which were supplemented through intragastric gavage for seven weeks. On the seventh week of supplementation, each of the male rats was introduced to five female rats at five different days to allow mating and observed the libido behavior. The mounting latency and mounting frequency were recorded for each mating.
Results: P. macrocarpa aqueous extract significantly increased (p<0.05) the serum testosterone level and mounting frequency of male rats. However, there was no significant effect on mounting latency. Body weight was significantly lower in rats supplemented with P. macrocarpa aqueous extract compared with the control groups (p<0.05).
Conclusion: P. macrocarpa showed potential value as an alternative for improving the sexual strength by increasing the level of testosterone and libido behavior. Thus, it is suggested that P. macrocarpa can improve the fertility in man.
PMCID: PMC4075721  PMID: 25050295
Andropause; Llibido; Phaleria macrocarpa; Testosterone
15.  Diethylstilboestrol Exposure Does Not Reduce Testosterone Production in Human Fetal Testis Xenografts 
PLoS ONE  2013;8(4):e61726.
In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15–19 weeks’ gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5–e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus.
doi:10.1371/journal.pone.0061726
PMCID: PMC3631175  PMID: 23620786
16.  Administration of Gonadal Steroids to the Castrated Male Rat Prevents a Decrease in the Release of Gonadotropin-Releasing Hormone from the Incubated Hypothalamus 
Journal of Clinical Investigation  1979;63(2):262-267.
The influence of testosterone on gonadotropin-releasing hormone (GnRH) secretion was assessed indirectly by altering the serum testosterone concentration of male rats and measuring GnRH release from their incubated hypothalami 1 wk later.
GnRH release from hypothalami of castrated rats was 13.4±1.2 (SE) pg/h, compared to 35.3±3.8 pg/h from hypothalami of intact rats (P < 0.001). GnRH release from the hypothalami of castrated rats treated with testosterone propionate, 100 or 500 μg daily, was 25.0±3.4 pg/h and 27.9±3.6 pg/h, which is significantly greater (P < 0.05 and P < 0.01, respectively) than that from hypothalami of castrated rats treated only with sesame oil.
A similar decrease in GnRH release from hypothalami of hypophysectomized rats and prevention of this decrease by treating the hypophysectomized rats with testosterone propionate is evidence that the observed effects of testosterone are not mediated via luteinizing hormone and(or) follicle-stimulating hormone secretion. Treatment of castrated rats with either dihydrotestosterone propionate or estradiol benzoate also prevented the decrease in GnRH release from the hypothalami of castrated rats.
We conclude that testosterone, dihydrotestosterone, and estradiol all prevent the decrease in GnRH release from hypothalami of castrated rats treated with these steroids. The possibility exists that these steroids may also maintain GnRH secretion in vivo.
PMCID: PMC371948  PMID: 372239
17.  Prenatal Testosterone Exposure Worsen the Reproductive Performance of Male Rat at Adulthood 
PLoS ONE  2013;8(8):e71705.
The reproductive system is extremely susceptible to environmental insults, for example exogenous steroids during gestational development and differentiation. Experimental induction of androgen excess during prenatal life in female animal models reprograms their reproductive physiology, however the fetal programming of the male reproductive system by androgen excess has not been well studied. We aimed to determine the effect of prenatal exposure of two different doses of testosterone on different gestational days, on the male reproductive system using a rat model. Sixteen pregnant rats were randomly divided into two experimental groups and two control groups. Experimental group І were subcutaneously injected with 3 mg free testosterone on gestational days 16-19 and its controls received solvent for that time; experimental group П were subcutaneously injected with 20 mg free testosterone on day 20 of gestational period and its controls received solvent at the same time. The reproductive system morphology and function of 32 male offspring of these study groups were compared at days 6-30-60 of age and after puberty. The anogenital distance of the male offspring of both experimental groups had no significant differences on the different days of measurement, compared with controls. In the offspring of experimental group І, the testes weight, number of Sertoli, Spermatocyte and Spermatid cells, sperm count and motility and the serum concentration of testosterone after puberty were significantly decreased; except for reduction of sperm motility (p< 0.01), the other effects were not observed in the offspring of experimental group ІІ. In summary, our data show that prenatal exposure of male rat fetuses to excess testosterone disrupted reproductive function, an effect highly dependent on the time, duration and level of exposure. It seems that the reproductive system in individuals exposed to high levels of androgens during fetal life should be evaluated at puberty and likely to be treated.
doi:10.1371/journal.pone.0071705
PMCID: PMC3744450  PMID: 23967236
18.  Testosterone restoration using enclomiphene citrate in men with secondary hypogonadism: a pharmacodynamic and pharmacokinetic study 
Bju International  2013;112(8):1188-1200.
Objectives
To determine the pharmacodynamic profile of serum total testosterone and luteinizing hormone (LH) levels in men with secondary hypogonadism after initial and chronic daily oral doses of enclomiphene citrate vs transdermal testosterone.To determine the effects of daily oral doses of enclomiphene citrate in comparison with transdermal testosterone on other hormones and markers in men with secondary hypogonadism.
Patients and Methods
This was a randomized, single-blind, two-centre, phase II study to evaluate the effects of three different doses of enclomiphene citrate (6.25, 12.5 and 25 mg) vs transdermal testosterone on 24-h LH and total testosterone in otherwise normal healthy men with secondary hypogonadism.Forty-eight men were enrolled in the trial (the intent-to-treat population), but four men had testosterone levels >350 ng/dL at baseline. Forty-four men completed the study per protocol. All subjects enrolled in this trial had serum total testosterone in the low range (<350 ng/dL) and had low to normal LH (<12 IU/L) on at least two occasions.Total testosterone and LH levels were assessed each hour for 24 h to examine the effects at each of three treatment doses of enclomiphene citrate vs a standard dose (5 g) of transdermal testosterone. In the initial profile, total testosterone and LH were determined in a naïve population after a single initial oral or transdermal treatment (day 1). This was contrasted to that seen after 6 weeks of continuous daily oral or transdermal treatment (day 42).The pharmacokinetics of enclomiphene citrate were assessed in a select subpopulation.Serum samples were obtained over the course of the study to determine the levels of various hormones and lipids.
Results
After 6 weeks of continuous use, the mean (sd) concentration of total testosterone at day 42 was 604 (160) ng/dL for men taking the highest dose of enclomiphene citrate (enclomiphene citrate, 25 mg daily) and 500 (278) ng in those men treated with transdermal testosterone. These values were higher than day 1 values but not different from each other (P = 0.23, t-test).All three doses of enclomiphene citrate increased the testosterone concentration at time 0 of each 24-h sampling period, and the mean, maximum, minimum and range of testosterone concentrations over the 24-h sampling period. Transdermal testosterone also raised total testosterone, albeit with more variability, and with suppressed LH levels.The patterns of total testosterone over the 24-h period after 6 weeks of dosing could be fit to a nonlinear function with morning elevations, mid-day troughs, and rising night-time levels.Enclomiphene citrate and transdermal testosterone increased levels of total testosterone within 2 weeks, but they had opposite effects on FSH and LH.Treatment with enclomiphene citrate did not significantly affect levels of thyroid-stimulating hormone, adenocorticotropic hormone, cortisol, lipids or bone markers. Both transdermal testosterone and enclomiphene citrate decreased insulin-like growth factor-1 levels (P < 0.05) but suppression was greater in the enclomiphene citrate groups.
Conclusions
Enclomiphene citrate increased serum LH and total testosterone; however, there was not a temporal association between the peak drug levels and the maximum concentration levels of LH or total testosterone.Enclomiphene citrate consistently increased serum total testosterone into the normal range and increased LH and FSH above the normal range. The effects on LH and total testosterone persisted for at least 1 week after stopping treatment.
doi:10.1111/bju.12363
PMCID: PMC4155868  PMID: 23875626
serum testosterone; LH; secondary hypogonadism; transdermal testosterone; testosterone restoration
19.  Changes in Androgen Receptor, Estrogen Receptor alpha, and Sexual Behavior with Aging and Testosterone in Male Rats1 
Hormones and behavior  2010;58(2):306-316.
Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.
doi:10.1016/j.yhbeh.2010.03.001
PMCID: PMC2879440  PMID: 20223236
androgen receptor; estrogen receptor alpha; aging; male rat; anteroventral periventricular nucleus (AVPV); medial preoptic nucleus (MPN)
20.  Fetal programming of adult hypertension in female rat offspring exposed to androgens in utero 
Early human development  2011;87(6):407-414.
Aims
The influence of prenatal factors on the development of arterial hypertension has gained considerable interest in recent years. We examined the effects of prenatal testosterone treatment on blood pressure in adult female rats. Further, to define the mechanisms whereby blood pressure may be raised, we examined vascular endothelial function and nitric oxide synthesis.
Methods and Results
Testosterone propionate (0.5mg/kg/day;SC) or vehicle was administered to pregnant Sprague-Dawley rats from gestational day 15–19. Maternal feed intake and plasma levels of steroid hormones were measured in the dams. In the female offspring, birth weight, growth rate, blood pressure, vascular reactivity, eNOS expression, and nitric oxide production were examined. In the pregnant rats, testosterone-treatment increased plasma testosterone levels by 2-fold without any significant changes in 17β-estradiol, progesterone and corticosterone levels. Testosterone-treatment did not affect maternal feed intake. The pups born to testosterone mothers were smaller in size but exhibited catch-up growth. The blood pressure in the testosterone offspring at 6 months of age was significantly higher compared to controls. Endothelium-intact mesenteric arteries from testosterone group exhibited increased contractile responses to phenylephrine, decreased vasodilation to acetylcholine and unaltered responses to sodium nitroprusside in comparison to control rats. Testosterone rats demonstrated decreased expression for eNOS, and reduced nitric oxide production.
Conclusions
Our data show that elevated plasma maternal testosterone levels: (1) causes low birth weight followed by catch-up growth and hypertension in female offspring; (2) alters endothelium-dependent vascular responses. The endothelial dysfunction is associated with decreased activity/expression of eNOS.
doi:10.1016/j.earlhumdev.2011.03.001
PMCID: PMC3093104  PMID: 21450421
21.  The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker 
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like compounds are widely encountered toxic substances suspected of interfering with the endocrine systems of humans and wildlife, and of contributing to the loss of fertility. In this study, we determined the changes in testicular gene expression caused by in utero exposure to TCDD along with the intra-testicular testosterone levels, epididymal sperm reserves, daily sperm production (DSP) and testis histology. To this purpose, female pregnant Sprague–Dawley rats orally received TCDD (10, 100 or 200 ng/kg body weight) or vehicle at embryonic day 15, and the offspring was killed throughout development. Hepatic Cyp1a1 gene expression was measured in the offspring to confirm the exposure to TCDD. The gross histology of the testes and intra-testicular testosterone levels were normal among the studied groups. Sperm reserves were altered in 67-day-old rats of the TCDD-200 group, but not in 145-day-old animals or in the other TCDD-exposed groups. Nonetheless, fertility was not altered in males of the TCDD-200 group, and the F2 males generated had normal sperm reserves and DSP. Microarray analysis permitted the identification of eight differentially expressed genes in the 4-week-old testes of the TCDD-200 compared with that of the control group (cut-off value ± 1.40), including the down-regulated chemokine Ccl5/Rantes. Inhibition of Ccl5/Rantes gene expression was observed throughout development in the TCDD-200 group, and at 67 and 145 days in the TCDD-100 group (animals of younger ages were not examined). Ccl5/Rantes gene expression was mostly confined in Leydig cells. F2 males generated from males of the TCDD-200 group had normal levels of Ccl5/Rantes in testis and Cyp1a1 in liver, which might indicate that Ccl5/Rantes is a marker of TCDD exposure in testis such as Cyp1a1 in liver. In conclusion, we demonstrated a decrease in Ccl5/Rantes RNA levels and a transitory decline in sperm reserves in the testes of rats of TCDD-dosed dams.
doi:10.1111/j.1365-2605.2009.01020.x
PMCID: PMC2871170  PMID: 20059583
2,3,7,8-tetrachlorodibenzo-p-dioxin; Ccl5/Rantes; in utero exposure; sperm count; testis; transcriptomic analysis
22.  TESTOSTERONE-DOWNREGULATED AKT PATHWAY DURING CARDIAC ISCHEMIA/REPERFUSION: A MECHANISM INVOLVING BAD, BCL-2 AND FOXO3A 
The Journal of surgical research  2010;164(1):e1-e11.
Background
Lower levels of myocardial Akt activity in males are associated with a higher incidence of heart failure and worsened cardiac function after ischemia/reperfusion (I/R). While Akt activation by estrogen provides cardioprotection in females, no information exists regarding the effect of testosterone on the myocardial Akt pathway following I/R. We hypothesized that following I/R: 1) endogenous testosterone will decrease myocardial Akt activation in male hearts; 2) endogenous testosterone will mediate downstream signals of Akt, including Bad, Bcl-2 and FOXO3a; 3) administration of exogenous testosterone will recapitulate negative effects on the Akt pathway in castrated male hearts.
Methods and Results
Rat hearts from age-matched adult males, females, castrated males, males with androgen receptor blocker-flutamide, castrated males with chronic 5α-dihydrotestosterone (DHT) implantation or acute testosterone infusion (ATI) (n=9/group) were subjected to I/R (Langendorff). Castration or flutamide treatment significantly upregulated myocardial Akt activation, increased downstream apoptosis-regulatory molecules: p-Bad, Bcl-2, p-FOXO3a, but reduced Fas-L, consistent with decreased myocardial injury in male hearts following I/R. ATI administration, but not chronic DHT, reversed these effects on Akt signaling associated with further exacerbated cardiac dysfunction in castrated males. Notably, lower levels of MnSOD were observed in male hearts, and castration or flutamide treatment restored myocardial MnSOD expression to the levels of females in male hearts after I/R.
Conclusion
Our study represents the initial evidence of testosterone-induced downregulation of the Akt pathway in male hearts following I/R, thereby mediating cardiac injury through decreased p-Bad, reduced ratio of Bcl-2/Bax in the cytoplasm, and increased FOXO3a in the nucleus.
doi:10.1016/j.jss.2010.07.041
PMCID: PMC2957547  PMID: 20850791
sex; myocardium; ischemia; signal transduction; apoptosis
23.  Aquaporin 9 expression in the developing rat epididymis is modulated by steroid hormones 
Reproduction (Cambridge, England)  2009;139(3):613-621.
Fluid and solute transport across the epithelium of the male excurrent duct is important for sperm maturation and storage. Aquaporin 9 (AQP9), which allows permeation of water and neutral solutes, is abundant throughout the male reproductive tract, where it is expressed at the apical membrane of rat epididymal principal cells as early as at 1 week of age. We evaluated the effect of neonatal exposure to: 1) a GNRH antagonist (GNRHa); 2) diethylstilbestrol (DES); 3) ethinyl estradiol (EE); 4) DES plus testosterone (DES+TE); and 5) the anti-androgen flutamide on AQP9 expression in the epididymis of peripubertal rats. Control groups received the vehicle alone. In 25-day-old rats, quantification of the mean pixel intensity of immunofluorescence-stained sections showed a significant decrease in AQP9 staining in the apical membrane of epididymal principal cells after treatments with GNRHa, DES, or flutamide, compared to controls. These results were confirmed by western blotting. While EE induced a marked decrease in AQP9 levels by western blotting, the decrease in AQP9-associated fluorescence was not significant compared to controls. DES+TE-treated rats showed levels of AQP9 protein similar to controls, indicating maintenance of AQP9 expression by testosterone treatment in the presence of DES. Our data show that expression of AQP9 in the developing rat epididymis is downregulated by neonatal DES, GNRHa, EE, and flutamide, and that the effects mediated by estrogens can be prevented by testosterone administration.
doi:10.1530/REP-09-0284
PMCID: PMC3773695  PMID: 19948840
24.  EVIDENCE FOR SUPPRESSANT EFFECTS OF TESTOSTERONE ON SEX-TYPICAL ETHANOL INTAKE IN MALE SPRAGUE-DAWLEY RATS 
Behavioural brain research  2011;224(2):403-407.
Previous studies have shown that adult female rats consume more ethanol than adult males. Castration of male rats has been found to increase their ethanol intake and preference to levels significantly elevated above their sham-gonadectomized counterparts and similar to levels observed in females. The purpose of the present experiment was to examine whether testosterone replacement in castrated adult male rats would be sufficient to restore the relatively low levels of ethanol drinking characteristic of intact adult male rats. Males were either gonadectomized and implanted with a testosterone propionate pellet (RPL), gonadectomized and implanted with a placebo pellet (GX), sham-gonadectomized and implanted with a placebo pellet (SH), or were left non-manipulated (NM). Voluntary ethanol intake was measured using a 2 hr limited-access drinking paradigm, with access to two bottles: one containing water, and the other a sweetened ethanol solution. Hormone replacement was sufficient to return ethanol intake and preference of castrates to levels comparable to both SH and NM control males. Ethanol preference of RPL males was also significantly suppressed compared to GX males by the end of the measurement period, whereas these group comparisons did not reach statistical significance for g/kg ethanol intake. These data suggest that testosterone serves to suppress ethanol preference in male rats, and may contribute to the sex differences in ethanol preference and consumption commonly reported in adult rats.
doi:10.1016/j.bbr.2011.06.020
PMCID: PMC3159851  PMID: 21726585
ethanol intake; gonadal hormones; testosterone; gonadectomy; male; rat
25.  The mechanism responsible for the supraphysiologic gonadotropin surge in GnRH-agonist-treated, GnRH-antagonist-primed females 
Fertility and sterility  2009;93(5):1668-1675.
Objective
To elucidate the physiologic mechanism responsible for the supraphysiologic gonadotropin release from the pituitary induced by GnRH-agonist in GnRH-antagonist-primed female rats.
Design
Controlled experimental intervention.
Setting
Government research facility.
Intervention
Forty oophrectomized rats were randomized into 4 groups of 10 and treated with: Group A) control vehicles; Group B) GnRH-a (leuprolide acetate; 1.7μg/kg BID) on day 4; Group C) GnRH-ant (Nal-Lys; 3mg/kg QD) days 1–4; or D) GnRH-ant (Nal-Lys; 3mg/kg QD) days 1–4 and GnRH-a (1.7μg/kg BID) on day 4.
Main Outcome Measure(s)
Immunohistochemical methods, Northern, and in situ hybridization to quantitate pituitary FSH-β, LH-β and GnRH-R mRNA and receptor protein levels in all treatment groups.
Results
Treatment with GnRH-ant was associated with increased storage of gonadotropin in the pituitary for FSH-β and LH-β, but mRNA levels were unchanged. GnRH-R mRNA decreased following GnRH-a treatment but remained stable in the GnRH-ant-treated groups. Levels of GnRH-R were decreased following GnRH-ant treatment.
Conclusions
These data indicate that the in vivo mechanism responsible for the exaggerated release of gonadotropins in the GnRH-ant primed, GnRH-a treated rat was an increase in releasable gonadotropin pools coupled with a reduction in GnRH-R, but receptor function was preserved.
doi:10.1016/j.fertnstert.2008.12.047
PMCID: PMC2858341  PMID: 19200975
LH-RH; GnRH receptor; ovulation induction; LH surge; OHSS; Ovarian Hyperstimulation syndrome; pituitary

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