Azoospermia: Suatu Tinjauan Genomik

Azoospermia or the absence of sperm in semen is one of the sperm disorders that results in male infertility. There are two types of azoospermia, the first one isazoospermia caused by obstruction of the vas deferens (obstructive azoospermia) and the second one is azoospermia due to the damage of testes (nonobstructive azoospermia). The etiology of azoospermia could be genetic or non-genetic. Genetic factors may occur in genomics starting from chromosome until gene level or single nucleotide polymorphism (SNPs). At the chromosome level, there is Klinefelter’s syndrome (47, XXY) to the Y chromosome microdeletion, whereas at the gene level there is mutation of jsd, Bmp8b and other genes. At the level of SNPs, Genome Wide SNP Association Study (GWAS) had uncovered 20 SNPs which were related significantly to azoospermia. Extensive knowledge of genomics review on male infertility, is expected to promote the development of investigation and management of azoospermia.

Best practice policies for male infertility

Azoospermia may occur because of reproductive tract obstruction (obstructive azoospermia) or inadequate production of spermatozoa, such that spermatozoa do not appear in the ejaculate (non-obstructive azoospermia). Azoospermia is diagnosed based on the absence of spermatozoa after centrifugation of complete semen specimens using microscopic analysis. History and physical examination and hormonal analysis (FSH, testosterone) are undertaken to define the cause of azoospermia. Together, these factors provide a >90% prediction of the type of azoospermia (obstructive v. non-obstructive). Full definition of the type of azoospermia is provided based on diagnostic testicular biopsy. Obstructive azoospermia may be congenital (congenital absence of the vas deferens, idiopathic epididymal obstruction) or acquired (from infections, vasectomy, or other iatrogenic injuries to the male reproductive tract). Couples in whom the man has congenital reproductive tract obstruction should have cystic fibrosis (CF) gene mutation analysis for the female partner because of the high risk of the male being a CF carrier. Patients with acquired obstruction of the male reproductive tract may be treated using microsurgical reconstruction or transurethral resection of the ejaculatory ducts, depending on the level of obstruction. Alternatively, sperm retrieval with assisted reproduction may be used to effect pregnancies, with success rates of 25-65% reported by different centres. Non-obstructive azoospermia may be treated by defining the cause of low sperm production and initiating treatment. Genetic evaluation with Y-chromosome microdeletion analysis and karyotype testing provides prognostic information in these men. For men who have had any factors potentially affecting sperm production treated and remain azoospermic, sperm retrieval from the testis may be effective in 30-70% of cases. Once sperm are found, pregnancy rates of 20-50% may be obtained at different centres with in vitro fertilisation and intracytoplasmic sperm injection.

In infertile couples, a male contribution to infertility is found in 45-50%. The cause of male factor infertility remains largely unexplained, but varicocele and genetic disorder are recognized as major causes leading to spermatogenesis disability. Genetic disorder leads to male infertility include chromosomal abnormalities and Y chromosome microdeletions. Chromosomal abnormalities (numerical or structural abnormalities) can be detected routine karyotype analysis. In non-obstructed azoospemia (NOA) patients, the incidence of chromosomal abnormalities is about 15%, and Europe Association of Urology (EAU) recommend the karyotype analysis in men with azoospermia or oligozoospermia (sperm concentration <10 million/mL). The most common chromosomal aberration causing male infertility is 47, XXY, Klinefelter’s syndrome (KFS). In genetic disorder, dose the extra X chromosome play a most important role? In NOA patients, it’s true. But, in patients with severe oligozoospermia, Y chromosome microdeletions are the most major causes but they cannot be detected by routine karyotyping. Conventional diagnostic testing for the Y chromosome microdeletions is performed by PCR amplification of selected regions of the Y chromosome. Sequence tag site (STS) markers, which are specific for the loci, are amplified and the presence of the PCR products is detected by electrophoresis. Europe Andrology Association showed the guidelines for the diagnostic testing, and recommended six screening markers. In the past, we had used several STS markers including this recommended markers. But, the sensitivity of the recommended markers was insufficient for the Japanese population. To improve the sensitivity and specificity of the testing, we developed a new kit for the detection of molecular Y-chromosome deletions by re-selecting STS markers and carrying out multiplex target detection on the Luminex suspension array platform.In this session, I will introduce this kit in detail.

About 15% of couples have infertility problems, half of which are related to male factors. Cytogenetic and genetic disorders account for about 10% of the male infertility problems. The aim of this study was to determine the frequency and types of both cytogenetic abnormalities and AZF microdeletions of Y chromosome in idiopathic azoospermic and oligospermic infertile men in west of Iran. In this case-control study, a total of 108 infertile men including 62 azoospermic and 46 oligospermic men were studied for the cytogenetic and AZF microdeletions. Moreover, 90 fertile men served as a control group. Detailed clinical and laboratory examination was done for all participants. Karyotyping was done on peripheral blood lymphocytes to detect the cytogenetic abnormalities; likewise, multiplex-PCR method was performed to identify the presence of microdeletion in AZFa, AZFb or AZFc regions. Chromosomal abnormalities were detected in 6.5% (7/108) of cases, including two oligospermic men with balanced autosomal rearrangements, one oligospermic and four azoospermic men with Klinefelter syndrome. Y chromosome microdeletions were detected in 4.6% (5/108) of infertile men (AZFc: 3.7%, AZFbc: 0.9%). No AZFa deletion was detected in any of the patients. No chromosomal abnormality and Y chromosome microdeletion was detected in control group. The prevalence of chromosomal abnormalities and Y chromosome microdeletions shows the importance of genetic factors in male infertility. The analysis of karyotype and Y microdeletions in infertile men provide a proper understanding about the causes of infertility, the choice of the appropriate assisted reproduction technique and reducing the risk of transmission of these genetic defects to the future generation.

To investigate the incidence of abnormal karyotypes and Y chromosome microdeletion in Chinese men with azoospermia, and the relationship with reproductive hormones. Four hundred and eighty nine cases of azoospermic patients and 20 fertile men were studied. Karyotypes and Y chromosome microdeletion were analyzed by G-banding and mutiplex polymerase chain reaction, respectively. Chemiluminescene immunoassay technique was applied to measure the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), and prolactine (PRL). Chromosome abnormalities were found in 102 out of 489 azoospermic patients (20.86%), among them 86 (84.31%) cases had sex chromosome abnormalities, with 73 cases being Klinefelter syndrome. Y chromosome microdeletions were detected in 58 (11.86%) cases out of the 489 patients, and deletion of the AZFc region was the leading group (63.8% of all deletions), followed by AZFbc (19.0%), AZFabc (10.3%), AZFb or AZFa (3.4%). FSH, LH levels were significantly increased and T level was decreased in azoospermic patients compared with the fertile men group (P<0.01). Furthermore, in the azoospermic patients with Klinefelter syndrome or AZFabc microdeletions, FSH and LH levels were increased more significantly, and were statistically different from azoospermic patients with normal karotype or without Y chromosome microdeletion (P<0.05). In the Chinese men with azoospermia, the incidence of abnormal karyotype and Y chromosome microdeletion were similar to those described previously in other populations. In azoospermia with Klinefelter syndrome or AZFabc microdeletions, FSH and LH levels increased markedly indicating the protracted stimulation of gonadotrophs due to lack of androgen feedback.

Report on evaluation of the azoospermic male

Introduction Azoospermia, absence of sperm in the ejaculate is classified as obstructive (OA) and non-obstructive azoospermia (NOA). In OA, sperm are produced, but due to physical obstruction in the male reproductive tract, they are not released in the ejaculate. NOA, on the other hand, is defined as the absence of sperm in the ejaculate due to testicular dysfunction. In NOA, spermatogenesis is frequently preserved in specific sites, and proteomics studies have been employed in order to identify men with preserved spermatogenesis. Areas covered Differential protein expression in patients with male infertility is an indicator of impaired spermatogenesis. Here, we reviewed proteins with a potential role as biomarkers of spermatogenesis that could help in the management of non-obstructive and obstructive azoospermia. The following keywords were used for bibliographic research: seminal plasma, proteomics, male infertility, nonobstructive, obstructive, azoospermia, oligospermia. Expert opinion Biopsy is an invasive and potentially harmful technique for detecting spermatogenesis in men with OA and NOA. Seminal plasma proteins are highly promising as biomarkers for spermatogenesis. Current literature presents a number of potential candidate biomarkers for determining preserved spermatogenesis.

Approximately 15% of couples cannot conceive a child after 1 year of regular, unprotected intercourse. Male factor infertility is contributory in another 30% to 40%. Most causes of male infertility are treatable and the goal of many treatments is to restore the ability to conceive naturally. The dramatic recent improvements in the management of male infertility are largely contributable to improved surgical techniques and assisted reproductive technology (ART). Specifically in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) allow us to overcome even the most severe defects in spermatogenesis in which only a few are available.Varicocele repair may be considered as the primary treatment option when a man with a varicocele has suboptimal semen quality and the female partner is normal. varicocele repair can reverse a pathologic condition and halt further damage to testicular function, and improve spermatogenesis. Preferred approaches of most experts are microsurgical inguinal and subinguinal operations. Studies have shown that varicocele repair can improve semen parameters, testicular function and pregnancy rates in couples with male-factor infertility associated with varicocele. Also varicocele repair can result in sperm in the ejaculate of azoospermia men when severe hypospermatogenesis or maturation arrest spermatid stage is present.Obstructive azoospermia may result from epididymal, vasal or ejaculatory duct abnormalities. Microsurgical reconstruction remains the safest and most cost-effective treatment option for these patients (vasovasostomy, vasoepididymostomy). It is controversial that the technique of sperm retrieval (open or percutaneous) or the source of sperm (testicular, epididymal, vasal or seminal vesicular) affects pregnancy rate. Sperm extraction or aspiration for IVF via ICSI is needed in cure of surgically uncorrectable azoospermia or failed microsurgical reconstruction and the majority of patients with congenital bilateral absence of the vas deferens (CBAVD). Also sperm retrieval with IVF/ICSI is preferred to surgical treatment when the advanced female partner age, female infertility requiring IVF.Nonobstructive azoospermia (NOA) is the most challenging type, but no specific treatment was available previously. With advent of ICSI in conjunction with sperm retrieval via testicular sperm extraction (TESE), many of nonobstructive azoospermic patients are able to father own babies. However, 20-50% of NOA patients are not able to have sperm retrieved for ART. Microsurgical TESE is an advanced type of TESE that applies microsurgical techniques. This microsurgical TESE is an effective sperm retrieval from men with NOA for ICSI. The advantages of this technique are minimally invasive technique, removal of minimal amount of testicular tissue and minimalizing negative impact on testicular function. Microsurgical TESE is more effective in men with NOA than conventional TESE.Treatment strategies for male infertility have changed as dramatically over the past decade. These advances are largely contributable to microsurgical varicocele repair, microsurgical reconstructive techniques, and microsurgical techniques for surgical sperm retrieval and ART specifically ICSI.

Recent investigations have pointed to a high prevalence of Y chromosome submicroscopic deletions in men with severely impaired spermatogenesis. We report on the incidence in 128 infertile men, in whom karyotype, sperm count, and hormonal parameters were evaluated. Patients with abnormal karyotype (other than an abnormal Y chromosome) or sperm concentration of more than 2 million/mL were excluded. Genomic DNA was extracted from the peripheral leukocytes of 57 men with azoospermia and 71 with severe oligospermia. Molecular analysis was performed by 3 multiplex polymerase chain reactions using a set of 9 sequence tagged sites (STSs) from 3 different regions of the Y chromosome: AZFa, AZFb, and AZFc. In 7% of the studied patients Yq microdeletions were detected, with a high prevalence in men with azoospermia (14%). No deletions were detected in the AZFa region. Deletions were present in AZFb, AZFc, or both regions. The deletion observed in 1 patient that did not overlap with the DAZ region demonstrates that genes other than DAZ may also be involved in the pathogenesis of some subsets of male infertility. Furthermore, common Yq deletions present different testicular pictures, suggesting that some unknown factors may be disturbing spermatogenesis. Because men with severe infertility suffer a high risk of Y chromosome deletion, screening for these men is recommended prior to treatment with assisted reproduction.

Objective To investigate the effect of chromosome polymorphism on the male spermatogenesis and reproductive outcomes. Methods Totally 3 203 cases of male (infertile group), who were divided into normozoospermia group, aligozoospermia group, azoospermia group according to the sperm count of semen analysis, and 4 893 cases of donor (donor group) were involved the study. Semen analysis, karyotype analysis, and Y chromosome microdeletion test were compared between the groups. The reproductive outcomes of sperm donors and infertile patients were investigated. Results The incidence of polymorphism in infertile group and donor group was 4.62% and 3.78% respectively, and the difference was not statistically significant. The incidence of Yqh- in infertile patients increased significantly with the decrease of sperm count (0.15% in normozoospermia group, 0.22% in aligozoospermia group, 0.99% in azoospermia group), and the difference was statistically significant (P=0.033, compared between normozoospermia group and azoospermia group; P=0.027, compared between aligozoospermia group and azoospermia group), the rate of Y chromosome microdeletion in group C with Yqh- was 56.25% (9/16), significantly higher than that of other groups (P 0.05). Follow-up investigation found that the spontaneous abortion rate was 6.25% (3/48) and 6.67% (2/30) in people with polymorphic of donor group and infertile group respectively, no statistical significance (P>0.05), and no child birth defects were observed. Conclusion Part with azoospermia factor (AZF) microdeletion in Yqh- may lead to spermatogenesis, and other types of polymorphism have no obvious effect on male fertility. Key words: Chromosomal polymorphism; Male fertility; Assisted reproductive techonology(ART); Birth defect; Reproductive outcome

Low levels of serum inhibin B do not exclude successful sperm recovery in men with nonmosaic Klinefelter syndrome

Sperm cells can be retrieved directly from the testis (testicular sperm extraction [TESE] procedure) and used for intracytoplasmic sperm injection (ICSI), circumventing underlying spermatogenetic defects. Thus, it is important that added information be available on the genetic defects in men undergoing TESE for the ICSI procedure and on the transmission of genetic factors associated with infertility to the offspring. We report a three-generation genetic analysis of a family with a case of male factor infertility. The proband, previously diagnosed as infertile, was physically examined and laboratory tested for gonadotrophic hormones, semen analysis, karyotype and Y-chromosome microdeletion screening in the blood and testis. The Y-chromosome microdeletion screening was performed by multiplex polymerase chain reaction with 20 Y-chromosome sequenced, tagged sites located at the Y chromosome. A microdeletion including the AZF-c region was detected in the azoospermic patient. His father, four brothers, and three offspring born after ICSI also underwent Y-chromosome microdeletion screening. The genetic analysis of the male members of the patient's family did not reveal similar microdeletions. The newborn male was found to bear a Y-chromosome microdeletion similar to that of his father. The fertilization capacity of the proband testicular microdeleted spermatozoa by the ICSI procedure is described. The transfer of the genetic defect raises the possibility that the son will have the same fertility problem as his father.

Harnessing the full potential of reproductive genetics and epigenetics for male infertility in the era of “big data”

A male factor is solely responsible in approximately 20% of cases of infertility and contributory in another 30-40%. Azoospermia is present in 15-20% of infertile males. Although the main goal of the evaluation of the infertile men is to identify the reversible conditions, to identify the irreversible causes that can or cannot be managed by assisted reproductive techniques (ART) is also important. Etiologies for azoospermia can be categorized as pre-testicular, testicular and post-testicular. Azoospermia is defined as the absence of sperm from at least two centrifuged semen samples. The initial evaluation of the azoospermia men includes a thorough history, physical examination, and hormonal tests. Physical examination should focus on testis size and presence of vas deferens and varicocele. Hormonal evaluation should include measurement of serum testosterone and follicle stimulating hormone (FSH) levels. When the vasa are palpable, testis size, semen volume and serum FSH are key factors in determining the etiology of the azoospermia. If the semen volume is reduced and this is not an artifact, the first laboratory test is post-ejaculatory urinalysis to exclude a retrograde ejaculation. After exclusion of retrograde ejaculation, transrectal ultrasonography (TRUS) should be considered to identify ejaculatory duct obstruction (EDO). Dilation of the seminal vesicles serves as a sign of EDO. Seminal vesicle aspiration to identify sperm at the time of TRUS can increase the diagnostic accuracy. In our center, TRUS-guided opacification of the seminal tracts with a mixture of contrast media and dye is performed to facilitate effective transurethral resection (TUR) of ejaculatory duct. Based on our experiences, patients with midline cysts who are treated by TUR are expected to have the best outcome. Testis size and level of serum FSH in azoospermic males with normal semen volume are critical factors in determining diagnostic strategies. Men with elevated FSH and bilateral small testis have non-obstructive azoospermia (NOA). Diagnostic testicular biopsy is not required in cases of NOA. Elevated FSH is indicative of a significant problem with spermatogenesis whereas a normal serum FSH does not guarantee intact spermatogenesis. Therefore, patients with normal testis size and FSH level should undergo a testicular biopsy to provide a definitive diagnosis. If the testicular biopsy is normal, most men have bilateral epididymal obstruction. Epididymal obstruction can be identified only by surgical exploration. Vasography is performed at the time of reconstructive surgery. Once sperm are found from the epididymal tubule, vasoepididymostomy is performed. The best results can be achieved by surgeons with training and on-going experience in microsurgery. There is some evidence that a small percentage of men with NOA may benefit from treatment of a clinical varicocele. Therefore, it is reasonable to offer men with NOA and clinical varicoceles a varicocelectomy. However, the most men will still need to use intracytoplasmic sperm injection (ICSI) to conceive. Testicular sperm extraction (TESE) should be offered to all men with NOA. Microsurgical TESE increases retrieval rates, and should be preferred in severe cases of NOA.