Suppression Of Hypothalamic-Pituitary-Gonadal Axis Function By Lipopolysaccharide Reducing Hypothalamic Kisspeptin-GPR54 System Expression And Spermatogenensis In Male Idiopathic Hypogonadotropic Hypogonadism Treated With Pulsatile Gonadotropin-Releasing

In the last ten years, researchers have illuminated that Kisspeptin neuropeptide secreted by hypothalamic Kisspeptin neurons stimulate GPR54 on Gonadotropin-releasing hormone(GnRH) neurons to synthetise GnRH regulating pituitary-gonad function. Hypothalamic Kisspeptin-GPR54 system has been hot spot for study on pathophysiology and clinical use of drugs in reproductive endocrinology disease.In part one, we study whether Lipopolysaccharide(LPS) have an effect on Kisspeptin-GPR54 system or not. And to explain the pathophysiology of LPS inducing hypogonadism. In the second part, we compare the effects of GnRH pulsatile infusion and Human Chorionic Gonadotropin/Human Menopausal Gonadotropin(HCG/HMG) intramuscular injection on spermatogenesis in male idiopathic hypogonadotropic hypogonadism(IHH). And analyze influencing factors on spermatogenesis.Part 1:Suppression of Hypothalamic-Pituitary-Gonadal Axis Function by Lipopolysaccharide Reducing Hypothalamic Kisspeptin-GPR54 System ExpressionBackgroud:LPS may suppress the function of hypothalamic-pituitary-gonadal axis. But the exact pathophysiological mechanism remains unclear. LPS induced stress accompanied by elevated glucocorticoids also playing a role in suppression of gonadal function by LPS. Kisspeptin-GPR54 system is a gatekeeper of hypothalamic-pituitary-gonadal axis. It may play a role in pathophysiological regulation of LPS and it inducing stress leading to hypogonadism.Objective:To investigate the express of Kisspeptin-GPR54 system treated by LPS and dexamethasone (Dex).Methods:1. MCF7 cell lines were cultured and were treated with different concentrations of LPS (10 μg/ml and 20 μg/ml) and Dex (10-6mol/L and 10-7mol/L). Kisspeptin and GPR54 mRNA and protein were measured by real-time PCR and Western blot, respectively.2. Serum LH levels, hypothalamic preoptic area Kisspeptin and GPR54 immunohistochemistry were measured at baseline and after 4 weeks in gonadectomized adult male mice. Then they were treated with LPS(100 μ g/Kg) and Dex(lmg/Kg) for 4 weeks. And serum LH levels, hypothalamic preoptic area Kisspeptin and GPR54 immunohistochemistry were measured before and after intervention.Results:1. In MCF7 cell lines, compared with control groups, LPS (10 μg/ml and 20 μg/ml) and Dex (10-6 mol/L and 10-7 mol/L) both reduced Kisspeptin mRNA(P<0.05) and protein. LPS (10 μg/ml and 20 μg/ml) and Dex (10-6 mol/L and 10-7 mol/L) both significantly increased the expression of GPR54 mRNA (P<0.05) and protein.2. Compared with normal adult male mice, in 4-week gonadectomized adult male mice, LH levels increased significantly(P< 0.01), Kisspeptin immunohistochemistry in hypothalamic preoptic area increased significantly(P< 0.05), GPR54 immunohistochemistry in hypothalamic preoptic area was not changed(P>0.05). In gonadectomized adult male mice, compared with control groups, LPS and Dex both significantly reduced serum LH levels in gonadectomized adult male mice(P<0.01); LPS(P<0.05), not Dex(P>0.05), significantly reduced the expression of Kisspeptin and GPR54 immunohistochemistry in hypothalamic preoptic area in gonadectomized adult male mice, respectively.Conclusion:LPS may reduce the expression of Kisspeptin-GPR54 system at the level of hypothalamus. It plays a role in pathophysiology of LPS inducing hypogoandism. Kisspeptin-GPR54 system may not take part in dexamethasone inducing hypogonadism. We support that kisspeptin neuron is target for negative feedback regulation of testosterone in adult male mice. concentrations of LPS (10 μg/ml and 20 μg/ml) and Dex (10-6mol/L and 10-7mol/L). Kisspeptin and GPR54 mRNA and protein were measured by real-time PCR and Western blot, respectively.2. Serum LH levels, hypothalamic preoptic area Kisspeptin and GPR54 immunohistochemistry were measured at baseline and after 4 weeks in gonadectomized adult male mice. Then they were treated with LPS(100 μ g/Kg) and Dex(lmg/Kg) for 4 weeks. And serum LH levels, hypothalamic preoptic area Kisspeptin and GPR54 immunohistochemistry were measured before and after intervention.Results:1. In MCF7 cell lines, compared with control groups, LPS (10 μg/ml and 20 μg/ml) and Dex (10-6 mol/L and 10-7 mol/L) both reduced Kisspeptin mRNA(P<0.05) and protein. LPS (10 μg/ml and 20 μg/ml) and Dex (10-6 mol/L and 10-7 mol/L) both significantly increased the expression of GPR54 mRNA (P<0.05) and protein.2. Compared with normal adult male mice, in 4-week gonadectomized adult male mice, LH levels increased significantly(P< 0.01), Kisspeptin immunohistochemistry in hypothalamic preoptic area increased significantly(P< 0.05), GPR54 immunohistochemistry in hypothalamic preoptic area was not changed(P>0.05). In gonadectomized adult male mice, compared with control groups, LPS and Dex both significantly reduced serum LH levels in gonadectomized adult male mice(P<0.01); LPS(P<0.05), not Dex(P>0.05), significantly reduced the expression of Kisspeptin and GPR54 immunohistochemistry in hypothalamic preoptic area in gonadectomized adult male mice, respectively.Conclusion:LPS may reduce the expression of Kisspeptin-GPR54 system at the level of hypothalamus. It plays a role in pathophysiology of LPS inducing hypogoandism. Kisspeptin-GPR54 system may not take part in dexamethasone inducing hypogonadism. We support that kisspeptin neuron is target for negative feedback regulation of testosterone in adult male mice.Part 2:Comparison of Spermatogenesis and Analysis of Influencing Factors in Male Idiopathic Hypogonadotropic Hypogonadism Treated with Pulsatile Gonadotropin-Releasing Hormone Infusion and Gonadotropin TherapyObjective:To compare the effects of GnRH and HCG/HMG on spermatogenesis in male IHH. And analyze influencing factors on spermatogenesis.Methods:This retrospective study included a total of 92 male IHH patients in out-patient department of endocrinology in Peking Union Medical College Hospital from May 2010 to October 2014, who chose one therapy voluntarily and were categorized into GnRH group (n=40) and HCG/HMG group (n=52). Gonadotropin levels were measured every other day in the first week and every month after treatment in GnRH group; serum total testosterone (TT) levels, testicular volume (TV) and spermatogenesis were observed every month after the treatment in two groups. Spermatogenesis, time for initiation of spermatogenesis, TT levels and TV were compared between two groups. And influencing factors on spermatogenesis were analyzed.Results:1. All IHH patients were treated more than 3 months. The median follow-up period in GnRH and HCG/HMG group was 8.2 (3.0-18.4) months and 9.2 (3.0-18.6) months(P=0.413), respectively.2. In GnRH group, LH(0.5±0.6 vs.1.6±1.1 IU/L, P<0.05) and FSH(1.3±1.1 vs.4.3±3.9 IU/L, P<0.05) increased after a three-day treatment. TT (1.0±1.0 vs.8,2±7.3 nmol/L, P<0.01) and TV (2.3±1.5 vs.6.0±2.5 ml, .P=0.001) significantly increased after 2-month treatment. In the GnRH group, at the end of follow-up, TT (1.0±1.0 vs.7.4±5.2 nmol/L, P<0.01) and TV (2.3±1.5 vs.8.1±4.0 ml, P<0.01) significantly increased compared to the baseline. In HCG/HMG group, TT (0.8±0.6 vs.14.4±8.0 nmol/L, P<0.01) and TV (2.3±2.1 vs.7.6±4.2 ml, P<0.01) significantly increased after therapy. The success rate of spermatogenesis is 50.0% (20/40) in GnRH group and 28.8%(15/52) in the HCG/HMG group(P=0.038). GnRH group needs a shorter treatment time (6.5±3.1 months) for initial sperm appearance than HCG/HMG group (10.8±3.7 months)(P=0.001). TT levels of initial sperm appearance in GnRH and HCG/HMG group were (9.0±5.1)nmol/L and (14.8±8.8)nmol/L(P=0.034), respectively.3. In GnRH and HCG/HMG group, there is no significance about comparison of basic TV in male IHH patients who have sperm or have not(P> 0.05),respectively. In GnRH and HCG/HMG group, Logistic regression to basic TV on spermatogenesis in male IHH patients did not have significance(P>0.05), respectively.Conclusion:Pulsatile GnRH requires shorter period of time for initiation of spermatogenesis than gonadotropins therapy in IHH male patients. Basic TV do not influence spermatogenesis in male IHH patients treated with pulsatile GnRH and gonadotropins therapy.