Screening Of Causative Genes In Chinese Patients With Idiopathic Hypogonadotropic Hypogonadism (IHH) And The Reproductive Performance Of Women With IHH During In Vitro Fertilization And Embryo Transfer Treatment

Part ⅠMutation screening of KISS1 and KISS1R genes in Chinese patients with IHHBackgroundIdiopathic hypogonadotropic hypogonadism (IHH), also termed as congenital hypogonadotropic hypogonadism, is a well-known cause of pubertal failure in both genders. The prevalence of IHH is about 1-10/100,000, and it is approximately 2-5 times less frequent in females than in males. IHH is usually caused by defective GnRH secretion or action, which cannot motivate sufficient secretion of pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Insufficient FSH and LH may fail to stimulate gonads to secrete sex hormones and drive normal gametogenesis. IHH is usually categried into Kallmann syndrome (with anosmia or hyposmia) and normosmic IHH (with normal smell).The cause of IHH is highly heterogeneous and genetic mutations are generally considered to have a prominent contribution to this disease. Genes that regulate secretion or activation of GnRH have been investigated and causative mutations have been found.The KISS1 gene encodes kisspeptins. In hypothalamus, kisspeptin acts as an upstream of GnRH and is sensitive to estradiol (E2)-mediated feedback. Kisspeptin is the ligand of the orphan G-protein coupled with receptor 54 (GPR54, also called KISS1R), which is expressed in GnRH neurons. Upon binding to KISS1R, kisspeptin mediates GnRH release from GnRH neurons, which in turn triggers the secretion of FSH and LH from the anterior pituitary in human. KISS1-null mice showed a phenotype of failure in pubertal maturation, sterility and hypogonadotropic hypogonadism. KISS1and KISS1R mutation should be a cause of IHH.ObjectiveTo investigate whether mutations in the KISS1and KISS1R gene are present in 170 Chinese patients with idiopathic hypogonadotropic hypogonadism (IHH).Methods170 Chinese patients with IHH (133 male cases and 37 female cases) and 187 matched controls (94 males and 93 females) were recruited in the Center for Reproductive Medicine, Provincial Hospital affiliated to Shandong University between March 2008 and November 2013. Genome DNA was extracted from peripheral blood and sequenced to scan for gene variants of KISS1 and KISS1R.ResultsTwo known single-nucleotide polymorphisms (SNP), rs12998:c.58G>A in exon 1 and rs4889:c.242C>G in exon 2, were identified in KISS1. Genotype and allelic frequencies showed no significant differences between the IHH cases and the controls in males and females. A new heterozygous variant-54C → A was found in 5’UTR of KISS1R gene and was not present in controls.ConclusionsA new heterozygous variant-54C → A was found in 5’UTR of KISS1R gene. Variants in the coding regions of KISS1 and KISSIR genes were not common in Chinese patients with IHH.Part IIThe role of TACR3 variants in the pathogenesis of Chinese patients with IHHBackgroundThe cause of IHH is highly heterogeneous and genetic mutations are generally considered to have a prominent contribution to this disease. Genes that regulate secretion or activation of GnRH have been investigated and causative mutations have been found. Kisspeptin, Neurokinin B (NKB) and Dynorphin A (Dyn), also termed as KNDy neuropeptides, have been implicated in regulating pulsatile LH secretion. NKB preferentially binds and activtes the neurokin 3 receptor (NK3R), then regulates the secretion of kisspeptins. Kisspeptins directly trigger pulsatile GnRH release.The human gene encoding the NK3R is TACR3, localized on chromosome 4 (4q25). NK3R is a member of the tachykinin family of G-protein-coupled receptors which also includes NK1 and NK2 receptors. The NK3R is predominantly expressed in the CNS (hippocampus, hypothalamus, substantia nigra, dorsal horn of the spinal cord) with limited expression in the periphery. NK3 receptors are thought to mediate afferent neuron transmission and intestinal motility and secretion. TACR3 gene mutation should be a cause of IHH.ObjectiveTo determine whether TACR3 mutations account for IHH in Chinese patients.Methods170 Chinese patients with IHH (133 male cases and 37 female cases) and 400 matched controls (200 males and 200 females) were recruited to scan for gene variants of TACR3. Polyphen-2, Mutation Taster and SIFT were performed to predict the function of novel missense mutation. Senktide was added to HEK293 cells transfected with wild type and mutant TACR3, and intracellular free Ca2+ flow was observed by calcium imaging system and Dual-Luciferase Reporter Assay System with pGL4.30 (NFAT).ResultsA novel heterozygous mutation C.1108G>T (P.A370S) of TACR3 was identified in a male IHH patient with azoospermatism. This point was extremely conserved among orthologs, and functional prediction shows "probably disease damaging". However, in vitro analysis showed increase on intracelluar free Ca2+ flow after senktide activated mutant-type NK3R.ConclusionsA novel heterozygous mutation C.1108G>T (P.A370S) was identified in the large cohort of Chinese IHH patients. In vitro analysis indicated this mutation changed the function of NK3R as a receptor. More experiments should be done to clarify the relation between this TACR3 gene variant and IHH.Part ⅢScreening of causative genes in familial idiopathic hypogonado-tropic hypogonadismBackgroundIdiopathic hypogonadotropic hypogonadism (IHH) is usually caused by defective GnRH secretion or action, which cannot motivate sufficient secretion of pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Insufficient FSH and LH may fail to stimulate gonads to secrete sex hormones and drive normal gametogenesis. The cause of IHH is highly heterogeneous and genetic mutations are generally considered to have a prominent contribution to this disease. Genes that regulate secretion or activation of GnRH have been investigated and causative mutations have been found.A consanguineous family with 4 affected sisters was found in the clinics. We hope to find some novel genes related to IHH with pedigree analyse.ObjectiveTo investigate which genes are related with familial idiopathic hypogonadotropic hypogonadism (IHH).MethodsAll members of this consanguineous family and 400 matched controls (200 males and 200 females) were recruited. Genome DNA was extracted from peripheral blood. Whole exome sequencing was used to analyse the genome DNA of 4 affected sisters and their mother. The other members of this family and controls were sequenced to scan for new gene variants found in whole exome sequencing.ResultsA novel heterozygous missense variant c.37A>G(T13A) in ADHFE1 gene was identified in familial idiopathic hypogonadotropic hypogonadism. This point was conserved among orthologs, and functional prediction shows "disease damaging". This was the first time to relate ADHFE1 gene with IHH.ConclusionsA novel heterozygous missense variant c.37A>G (T13A) in ADHFE1 gene was identified in familial idiopathic hypogonadotropic hypogonadism. More experiments should be done to determine whether this variant of ADHFE1 gene participates in the development of IHH.Part IVThe reproductive performance of women with idiopathic hypogonadotropic hypogonadism during in vitro fertilization and embryo transfer treatmentObjectiveTo evaluate the reproductive performance of women with idiopathic hypogonadotropic hypogonadism (IHH) undergoing in vitro fertilization and embryo transfer (IVF-ET).Materials and Methods23 females with IHH and 196 infertile women with tubal factor (TF) who underwent IVF-ET treatment between 2009 and 2013 in the Reproductive Hospital Affiliated to Shandong University were recruited for retrospective study. Characteristics during IVF-ET cycle and pregnancy outcome were compared between the two groups by student’s t test and x2 test.ResultsFemales with IHH consumed significantly more doses of gonadotropins (5081.52±2372.14 vs 2045.21±707.33, p<0.01) and longer duration of ovulation induction (13.91±1.70 vs 11.08±1.70, p<0.01) than females with TF. There were no differences in the peak levels of E2, endometrial thickness and number of follicles>14mm on the day of hCG injection between two groups. No difference was observed in the number of oocytes retrieved, number of 2PN and grade A embryos in the 3rd day after oocyte retrieval between the two groups. There was no statistical significance in the percentage of pregnancy (60.87% vs 50.51%, p>0.05), clinical pregnancy rate (56.52% vs 45.91%, p>0.05), live birth rate (84.62% vs 81.11%, p>0.05). Until March 2015, accumulate clinical pregnancy rate is 100%, miscarriage rate is 13.04%(3/23).ConclusionsIn IVF cycles women with IHH need more Gn doses and longer Gn days than TF group, and the two groups had similar ovarian response, pregnant rate, clinical pregnant rate and live birth rate. Most women with IHH in IVF cycles could achieve the aim of ’take home baby’.