Study Of The Relationship Between Genetic Phenotype, Serum FSH And FSH Receptor And Testicular Spermatogenesis Of Idiopathic Azoospermic Patients

Infertility exists in 15% of all couples in the world and about half of the infertility problem was caused by men. On the whole, factors resulting in male infertility could be classified into three types: pre-testicular factor, testicular factor (including the genetic factor) and post-testicular factor. It is known well that Y chromosome is very importanct in testicular differentiation and that Y chromosome abnormality, in either number or structure, will result in the disorder of testicular spermatogenesis, such as oligozoospermia or severe oligozoospermia. A genetic factor has been suggested to be responsible for some cases of idiopathic severe oligozoospermia and azoospermia. The existence of a gene, or a gene complex, associated with normal spermatogenesis first was postulated on the basis of a cytogenetic analysis that showed microscopic deletions of the distal part of the Y chromosome (Yq11) in men with azoospermia, defined as the azoospermia factor (AZF). Recently, it has been suggested that AZFs are present in four subregions, AZFa, AZFb, AZFc and AZFd, which were all associated with male infertility.Although many causes can lead to male infertility, such as infections, cryptorchidism, systemic diseases or endocrine disorders, and so on. The etiology of male infertility was still not very clear. Nearly 10% of those infertile men areazoospermic which was no spermatozoa in semen. In half the cases testicular biopsies identify the men as belonging to the category of idiopathic spermatogenic defects and show a total range of spermatogenic failure from complete absence of germ cells: SCOS, through MA up to Hypo. Follicle stimulating hormone (FSH) is a pituitary glycoprotein hormone, which is necessary for normal reproduction in both male and female mammals. In men, FSH determines the number and the quantity of Sertoli cell and quality of spermatogenesis. Therefore FSH was thought to be the most important hormone in spermatogenesis and spermatozoa maturation. FSH level in the serum was regarded as an important marker to estimate male spermatogenic arrest or seminiferous obstruction. It is known that Sertoli cells can express FSH receptor (FSHR) in their plasma membrane. The biochemical actions of FSH are exerted by binding to membrane receptor (FSHR) and initiating signaling events via the G-protein and/or other receptor coupling mechanisms. FSHR signaling is required to stimulate proliferation of Sertoli cells and to maintain normal sperm production.Although many investigators have found that male infertility was correlated with the microdeletion of Y chromosome, the relationship between the microdeletion of Y chromosome and serum FSH level and testicular histological phenotype of idiopathic azoospermia have not yet been clear. It was found that both FSH and FSHR signaling are required to stimulate proliferation of Sertoli cells and to maintain normal sperm production and that the decrease of testicular FSH receptor binding ability existed in infertile men. But it was not still clear that whether abnormality of the FSHR expression existed in testicular tissue of the patients with spermatogenic defects, whether the FSHR expression level was related to the degree of spermatogenesis. In addition, a definitive relationship between serum FSH level and spermatogenic defects of the patients with idiopathic azoospermia had not yet been clear. These questions still need to be clarified.In our present study, we detected serum FSH level, microdeletions in Y chromosome, the degree of testicular spermatogenesis and FSHR expression level in different testicular histological phenotype of the patients with idiopathic azoospermia. On the basis of this, we explore the relationship between microdeletions in Ychromosome and serum FSH level and the degree of testicular spermatogenesis, and investigate the correlation between the expression level of FSHR in the testis and the degree of spermatogenesis, and evaluate the predictive power of serum FSH level in relation to successful sperm retrieval from testis in idiopathic azoospermia.Part OneThe detection of Y chromosome microdeletions in patients with idiopathic azoospermiaObjective To clarify the relationship between gene microdeletion of Y chromosome and the phenotype of testicular histology and serum FSH level of idiopathic azoospermia.Materials and Methods Among adult men referred to our infertility center from Mar. 2002 to Dec. 2005, 126 azoospermic subjects with normal (n=90) or high (n=36) serum FSH levels were recruited. A complete medical history was obtained from each patient, and a physical examination was performed for each patient. Azoospermia was confirmed by two different tests, each performed after a 7-day period of sexual abstinence and separated by a 3-week interval, according to World Health Organization guidelines. The diagnosis of azoospermia was established by pellet analysis after centrifugation of semen sample. Subjects showing azoospermia on both occasions were recruited for this study. Among the patients with azoospermia, those with defective spermatogenesis secondary to infection, obstruction of the seminal tract, pituitary failure, chromosomal anomaly, and other causes of possible testicular damage shown on clinical examination were excluded. Chromosomal analysis was performed on cultured lymphocytes by the GTG banding technique. Serum FSH, LH, and testosterone levels were measured by ECLIA. Extraction of genomic DNA from peripheral blood lymphocytes was performed using a standard technique. PCR was carried out on 2ul (50 ng/ul) of genomic DNA in 50ul finalreaction volume. PCR reaction products were separated on agarose gel by electrophoresis and observed with ultraviolet transmission instrument. A total of 20 normal healthy men of proven fertility and 20 normal women were included in this study as positive and negative controls respectively. Testicular biopsies were performed with the use of conventional window technique. A small piece of testicular tissue was fixed in Bouin's solution, routinely embedded in paraffin, and cut at a section thickness of 5 urn, in order to evaluate the type of testicular pathology. Spermatozoa were detected with microscope from the rest testicular tissue obtained from testicular biopsy. Then, the testicular tissue used to detect spermatozoa was immediately snap-frozen in liquid nitrogen and stored at -80°C. All patients had a normal level of serum LH and a normal testicular volume.Results Microdeletions were observed in 6 of 90 patients and the deletion ratewas 6.7% (6/90). Three types of microdeletions were detected, which were AZFa(SY82) (n=3)x AZFa (SY82)) +AZFb (SY109+SY131) (n=l ) and AZFc( SY152+SY254+SY255 ) (n=2) respectively. Among these patients withmicrodeletions, spermatozoa were detected in 4 patients and testicular histologicdiagnosis identified these patients as slight hypospermatogenesis. Whereas the twopatients, who had no spermatozoa existing in their testes, were identified asmaturation arrest with testicular histologic diagnosis. Microdeletions were observed in9 of 36 patients and the deletion rate was 25.0% (9/36). Five types of microdeletionswere detected, which were AZFc (SY152) (n=l),AZFc (SY152+SY254) +AZFd(SY153) (n=4),AZFc (SY152+SY254+SY255) +AZFd (SY153) (n=2),AZFc(SY152+SY158+SY255) +AZFd (SY153) (n=l) and AZFb (SY130) +AZFc(SY158+SY254+SY255 ) +AZFd (SY153) (n=l) respectively. Among these patientswith microdeletions, spermatozoa were detected in 3 patients and testicular histologicdiagnosis identified these patients as slight hypospermatogenesis. But in the sixpatients, who had no spermatozoa existing in their testes, five were identified asmaturation arrest and one were identified as sertoli cell-only syndrome with testicularhistologic diagnosis. The deletion rate in idiopathic azoospermic patients with highserum FSH was significantly higher than that in normal serum FSH patients (p=0.01). The various deleted regions of the azoospermic patients with hypospermatogenesis ranged from AZFa to AZFd, and in the patients with MA, the deleted regions ranged fromAZFbtoAZFd.Conclusions 1. Microdeletion of the Y chromosome was one of the important reasons of the patients with normal and high serum FSH azoospermia. 2. There was no definite relationship existing between the type of microdeletion and the testicular histological phenotype of idiopathic azoospermia. 3. There was a correlation existing between serum FSH level and the rate of microdeletions in patients with idiopathic azoospermia.Part TwoExpression of follicle stimulating hormone receptor in the different testicular histological phenotype of the patients with idiopathic azoospermiaObjective To clarify the relationship between serum FSH level and expression level of FSHR in the testis and the degree of spermatogenesis and investigate whether different expression level of FSHR existed in the different testicular histology phenotype of the idiopathic azoospermic patients.Materials and Methods Among adult men referred to our infertility center from Mar. 2002 to Dec. 2005, we selected 57 idiopathic azoospermic subjects. A complete medical history was obtained from each patient, and a physical examination was performed for each patient. Azoospermia was confirmed by two different tests, each performed after a 7-day period of sexual abstinence and separated by a 3-week interval, according to World Health Organization guidelines. The diagnosis of azoosperraia was established by pellet analysis after centrifugation of semen sample. Subjects showing azoospermia on both occasions were recruited for this study. Among the patients with azoospermia, those with defective spermatogenesis secondary to infection, obstruction of the seminal tract, pituitary failure, chromosomal anomaly, and other causes of possible testicular damage shown on clinical examination were excluded. Chromosomal analysis was performed on cultured lymphocytes by the GTG banding technique. Serum FSH, LH, and testosterone levels were measured by ECLIA. Testicular biopsies were performed with the use of conventional window technique. One small piece of testicular tissue was fixed in Bouin's solution, routinely embedded in paraffin, and cut at a section thickness of 5 um, in order to evaluate the phenotype of testicular histology. The other small piece of testicular tissue was immediately snap-frozen in liquid nitrogen and stored at -80 V. All patients had normal levels of serum LH and testosterone, and had a normal testicular volume. Fifty-seven cases of idiopathic azoospermia were classified into three groups: the patients with hypospermatogenesis (Hypo), the patients with maturation arrest (MA) and the patients with sertoli cell-only syndrome (SCOS) according to the results of testicular biopsy. 13 azoospermic patients identified as complete spermatogenesis by testicular biopsy acted as a control group. Immunohistochernistry and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were performed in each case.Results Serum FSH level of patients with SCOS was significantly higher than that in patients with Hypo, MA or complete spermatogenesis (p<0.01), and serum FSH level of patients with MA was significantly higher than mat in patients withHypo or complete spermatogenesis (p<0.05). But there was no difference existing in two groups: the patients with Hypo and the patients with complete spermatogenesis (p>0.05). FSHR expression level of testicular samples with SCOS was significantly higher than that in testicular samples with Hypo, MA or complete spermatogenesis (both p<0.05), but no significant difference was observed among three groups: Hypo, MA and complete spermatogenesis testicular samples (p>0.05).Conclusions 1. Serum FSH level and differential FSHR expression level in testicular tissue of patients with idiopathic azoospermia may be associated with the degree of spermatogenesis. 2. Testicular histological phenotype of the patient with idiopathic azoospermia may be SCOS, when serum FSH level and FSHR expression of the testicular tissue are both high.Part ThreePrediction for successful sperm retrieval in idiopathic azoospermia with abnormal serum follicle stimulating hormoneObjective To evaluate the predictive power of the follicle-stimulating hormone (FSH) levels in relation to successful sperm retrieval from testis in idiopathic azoospermia with abnormal serum FSH levels, and to provide reference for testicular sperm extraction (TESE) before intracytoplasmic sperm injection (ICSI) is executed.Materials and Methods Among adult men referred to our infertility centerfrom May 2002 to Dec. 2005, we selected 65 azoospermic subjects with abnormal serum FSH. Their age distribution was from 21 to 40 years old with a medium of 29. A complete medical history was obtained from each patient along with a physical examination. Azoospermia was confirmed by two different tests, each performed after a 7-day period of sexual abstinence and separated by a 3-week interval, according to World Health Organization guidelines. The diagnosis of azoospermia was established by pellet analysis after centrifugation of semen sample. Subjects showing azoospermia on both occasions were recruited for this study. Among the patients with azoospermia, those with defective spermatogenesis secondary to infection, obstruction of the seminal tract, pituitary failure, chromosomal anomaly, and other causes of possible testicular damage shown on clinical examination were excluded. Chromosomal analysis was performed on cultured lymphocytes by the GTG banding technique. All patients had normal testicular volumes. Serum FSH, luteinizing hormone (LH), and testosterone levels were measured by ECLIA, using the El70 (Roche, Basel, Switzerland). All patients had normal levels of serum LH (2.6~12.6IU/L) and testosterone (12~37nmol/L), The normal range of serum FSH is 3.5~5.5 IU/L. Testicular biopsies were performed with the use of conventional window technique with local anesthesia. A part of biopsy material was used for histological examination, which was fixed in Bouin's solution, routinely embedded in paraffin, and cut at a section thickness of 5um. The other parts were kept in a small amount of Zarle (E2888, Sigma) culture and used for TESE. The testicular tissue was minced, and the presence of motile sperm was observed with the microscope. No quantification of the sperm was performed. 65 cases of idiopathic azoospermia with abnormal serum FSH levels were divided into two groups according to serum FSH levels: the patients with FSH levels less than 3.5IU/L and the patients with FSH levels more than 5.5IU/L.Results There were 20 patients with serum FSH levels less than 3.5IU/L and 45 patients with serum FSH levels more than 5.5IU/L. Spermatozoa were found in 19 patients with FSH levels less than 3.5IU/L and 17 patients with FSH levels more than5.5IU/L by TESE, and all of them were living. Spermatid existed in the testis of the patient with FSH level less than 3.5IU/L, who had no sperm found by TESE. For the 45 patients with serum FSH levels higher than 5.5 IU/L, those with no sperm retrieved had much higher FSH levels than that of patients with successful sperm retrieved (p<0.01). With respect to the successful sperm retrieval in TESE, the sensitivity of the FSH level >9.1IU/L was 67.9% and the specificity was 94.1%.Conclusions 1. There was a definitive relationship existing between serum FSH level and spermatogenic defects of the patients with idiopathic azoospermia. 2. Serum FSH level could be considered as a better parameter to predict successful sperm retrieval for those idiopathic azoospermic patients who had normal serum LH and testosterone levels as well as normal testicular volumes.