Identification Of Genetic Pathology For Patients With 46, XY Disorders Of Sex Development And Efficacy And Outcome Predictors Of Gonadotropin-Induced Spermatogenesis For Male Patients With Congenital Hypogonadotropic Hypogonadism

Development of reproductive system in humans is a dynamic process that begins at embryonic period and may complete after puberty with the development of secondary sexual characteristics and fertility. The whole course requires complex and precise interaction of multitude of genes, proteins, signaling molecules, and endocrine hormones. Factors which disturb and block this process would lead to gonadal dysgenesis, ambiguous genitalia and impaired fertility.Disorders of sex development (DSD) have always been difficult to be diagnosed and treated. In Part 1, the aim of our study is to make an accurate genetic diagnosis on patients with 46, XY DSD by using targeted gene capture with second-generation sequencing method. The relationship between genotype and phenotype was also analyzed, hoping to further understand the molecular basis of sex development.Congenital hypogonadotropic hypogonadism (CHH) is characterized by delayed puberty and infertility. Data on spermatogenesis of CHH patients is still very limited due to the low incidence. In Part 2, efficacy and outcome predictors of gonadotropin-induced spermatogenesis for male patients with congenital hypogonadotropic hypogonadism (CHH) were analyzed. Findings from the present study may provide evidence for better clinical diagnosis and treatment of CHH.Part 1 Identification of Genetic Pathology for Patients with 46, XY Disorders of Sex Development by using Targeted Gene Capture with Second-Generation SequencingIntroductionIn normal human embryo with 46, XY, bipotential gonadal ridge develops at 4-5 weeks post-fertilization and differentiates into testes at 7-8 weeks. Sertoli cell and Leydig cell formed sequentially and secret anti-Mullerian hormone (AMH) and androgens, insulin-like factor 3 (1NSL3), respectively. Testosterone and AMH cause the regression of Mullerian structures and differentiation of the Wolffian duct into the epididymis, vas deferens and seminal vesicles, whereas INSL3 is required for testicular descent. The testosterone is converted to dihydrotestosterone (DHT) by enzyme 5-a-reductase, both of which act on androgen receptors to generate biologic effect. Factors which disturb the testes differentiation, androgen synthesis and function would lead to 46, XY DSD.46, XY DSD is a congenital disorder characterized incomplete intrauterine masculinization caused by androgen deficiency or insensitivity, presenting with ambiguous or female external genitalia. The genetic pathology of the disease is complex, involving a wide range of genes. At least 30 genes related to 46, XY DSD have been identified. Clinical manifestations vary in severity and treatment would change accordingly. Even with the same gene mutation, the external genitalia phenotype of the patients may be different. Therefore, it is important to identify molecular diagnosis of 46, XY DSD for improvement of treatment, prognosis and prenatal counseling. Sanger sequencing of each candidate gene was too expensive and time-consuming. Targeted gene capture with second -generation sequencing provides a more sensitive, economic and effective way to make the final molecular diagnosis. To summary and analyze the genetic characteristics of 46, XY DSD in this study will enrich our understanding of this disease in the Chinese population.Objective1. To summarize clinical characteristics of patients with 46, XY DSD.2. To screen the pathogenic genes of 46, XY DSD.3. To analyze the relationship between genotype and phenotype of 46, XY DSD.Subjects and Methods1. Subjects:63 patients with 46, XY DSD admitted to Peking Union Medical College Hospital between 2014.3 and 2015.10 were included.2. Methods:(1) Clinical study:Clinical characteristics of 46, XY DSD patients were summarized.(2) Genetic study:Targeted gene capture with second-generation sequencing, including 299 genes involved gonadal differentiation and sex development, was conducted to identify the probable causative gene mutations in 46, XY DSD. Candidate gene mutations would be compared to known genetic database. SIFT, Polyphen-2 and Mutation taster were used to predict the impairment of function of mutated genes. Sanger sequencing was used to verify the mutation.(3) Relationship between genotype and phenotype:Summarize clinical characteristics of 46, XY DSD caused by different pathogenic genes. Genotype-phenotype correlation was analyzed.Results1. Of all the 63 patients with 46, XY DSD,49 (78%) were raised as females and 12 (19%) had a family history of DSD. Among 34 patients who were referred to hospital before puberty,24 were raised as females. Twelve patients visited doctors with main complaint of ambiguous genitalia,12 patients with main complaint of palpable mass located in labia or inguinal area. Another 10 patients who complained of ambiguous genitalia were raised as males. There were 29 patients referred to hospital during or after puberty,25 out of whom were raised as females. Nineteen patients manifested primary amenorrhea,5 patients experienced spontaneous virilization during puberty, and 1 patient was screened karyotype 46, XY because of short stature. Two patients who were raised as males complained of ambiguous genitalia and another 2 gynaecomastia. Images (B ultrasound and MRI) showed that 50/63 (79%) patients had testes or testis-like tissue,14/59 (24%) got a uterus. After clinical diagnosis, the rate of sex reassignment is 33%(8/24) before puberty and 8%(2/25) during or after puberty.2. Genetic diagnosis was identified in 48 (76%) 46, XY DSD patients. Forty eight mutation sites in eight genes were identified and 30 mutants were novel. AR gene mutations were found in 16 (16/63,25%) patients, SRD5A2 gene mutations in 10 (10/63,19%) patients and NR5A1 gene mutations in 13 (13/63,21%) patients. Mutated HSD17B3 and SRY genes were found in 3 patients, respectively. Another 3 patients were identified with MAP3K1, POR and CYP17A1 gene mutation respectively.3. Patients caused by AR, SRD5A2 and NR5A1 gene mutations accounted for 39 out of 63 (62%) 46, XY DSD patients. Testosterone level were similar between patients with mutated AR gene and SRD5A2 gene, but significantly higher than that of patients with mutated NR5A1 gene (P< 0.001, P = 0.022). Palpable mass in labia or inguinal area or testes in scrotum without uterus could be found in all patients with AR and SRD5A2 gene mutations, while half of patients (7/13) with NR5A1 gene mutations were found a mass and 38%(5/13) had a uterus. All of the 6 patients with AR gene mutations who undergone puberty had breast development,1 of them experienced spontaneous virilization, their LH was (10.8 ± 3.2)IU/L, FSH(7.0 ± 6.7)IU/L. Four patients with SRD5A2 gene mutations undergone puberty without breast development and half of them experienced spontaneous virilization, their LH was (12.6 ± 4.1)IU/L, FSH(15.1 ± 8.9)IU/L. A total of 8 patients with mutated NR5A1gene undergone puberty,3 patients had breast development and 3 had spontaneous virilization, their LH was (50.4 ± 45.6)IU/L, FSH(38.9 ± 27.1)IU/L.Conclusion1. For 46, XY DSD patients who were raised as females, patients with palpable mass in labia or inguinal area or ambiguous genitalia were main complaints for clinical evaluation before puberty. During or after puberty, patients always visited doctors for spontaneous virilization or primary amenorrhea. For those who were raised as males, most of them complained of ambiguous genitalia or gynaecomastia.2. Forty eight mutants in 8 different genes were identified in 76% patients, indicating that targeted gene capture with second-generation is a reliable and effective method to screen the pathogenic genes of 46, XY DSD.3. Most 46, XY DSD patients (62%) were caused by AR, SRD5A2 and NR5A1 gene mutations.4. Patients with AR and SRD5A2 gene mutations had testes or testis-like tissue, no uterus, and higher testosterone level than that of patients with NR5A1 gene mutations. Patients with AR gene mutations will experience breast development during puberty while a high portion of patients with SRD5A2 gene mutations experience spontaneous virilization. Patients with NR5A1 gene mutations have high LH and FSH level.Part 2 Efficacy and outcome predictors of gonadotropin-induced spermatogenesis for male patients with congenital hypogonadotropic hypogonadism: a retrospective study of 223 patientsIntroductionCongenital hypogonadotropic hypogonadism (CHH), caused by deficiency or dysfunction of GnRH, is a disorder characterized by delayed puberty development and infertility. CHH is comprised of Kallmann syndrome and normosmic CHH. Kallmann syndrome is a subgroup of CHH associated with a defective sense of smell (anosmia or hyposmia). The incidence of CHH is 1:8,000-10,000 in live births. When fertility is required, combined human chorionic gonadotropin (HCG) and human menopausal gonadotropin (HMG) therapy may promote testis development, androgen synthesis and spermatogenesis. However, data on spermatogenesis of CHH patients is still very limited due to the low incidence of this disease. There are different results referring to the efficacy and outcome predictors of gonadotropin-induced spermatogenesis for CHH patients. In this study, a total of 223 male CHH patients were included to investigate the efficacy of gonadotropin treatment and analyze putative predictors for successful spermatogenesis, aiming to provide more evidence for gonadotropin-induced spermatogenesis treatment.Objective1. To investigate the efficacy of gonadotropin treatment in a large cohort of male CHH patients and analyze putative predictors for successful spermatogenesis.2. To investigate the efficacy of gonadotropin treatment in CHH patients with cryptorchidism.Subjects and Methods1. Subjects:A total of 223 male CHH patients referred to Peking Union Medical College Hospital between 2005.1 and 2014.12 were included. Forty patients were reported to have histories of cryptorchidism.2. Methods:Retrospective study.3. Treatment and follow-up:All patients were intramuscularly injected with combined gonadotropins (HCG 2000-5000 U and HMG 75-150 U, twice a week) and were followed up for more than 6 months. Regular follow-ups were conducted at an interval of 3-6 months. Testicular size, plasma gonadotropins, plasma testosterone and sperm count were measured on each visit. Self-reported pregnancy was noted.Results1. All CHH patients (n=223) were followed up at a mean time of 23 ± 13 months. After gonadotropin therapy, serum total testosterone was elevated from 0.9 ± 0.5 nmol/1 to 15.1 ± 8.2 nmol/1 (P< 0.001) and testicular size was enlarged from 2.1 ± 1.6 ml to 8.1 ± 4.6 ml (P< 0.001). Spermatogenesis (> 0/ml) occurred at a median period of 15 months (95% confidence interval (CI) 13.5-16.5). Larger basal testicular volume (P= 0.012) and non-cryptorchidism history (P = 0.028) are independent predictors for earlier sperm appearance. Sixty four percent (143/223) of patients succeeded in producing sperms and the average time for initial sperm detection was 14 ± 8 months. Of the 34 patients who desired for fathering children,19 patients impregnanted their partners during the treatment.2. For CHH patients with cryptochidism histories (n = 40) who were followed up 24 (15, 33) months, sperm (> 0/ml) initially appeared in semen at a median of estimated 24 months (95% CI 17.8-30.2). Twenty (20/40,50%) patients succeeded in producing sperms and five pregnancies were achieved in 9 (5/9,56%) couples who were eager for children.3. Compared with CHH patients without cryptorchidism, cryptorchid patients had longer median time for first sperm detection in semen (24 months vs.15 months, P< 0.001), lower success rate of spermatogenesis (50% vs.67%, P = 0.032) and lower mean sperm concentration (1.9 (0.5,8.6) million/ml vs.11.1(1.0,25.0) million/ml, P = 0.006) at the last visit.Conclusion1. Gonadotropin therapy can successfully induce spermatogenesis in male CHH patients. A larger basal testicular size and non-cryptorchidism history are favorable indicators for earlier spermatogenesis.2. Gonadotropin therapy can induce spermatogenesis in half male CHH patients with cryptorchidism. Their partners still had chance to conceive babies regardless long treatment time for spermatogenesis and low sperm concentration in cryptorchid CHH patients.