Next Generation DNA Sequencing in Patients With Idiopathic Male Infertility

Infertility involves about 15% of couples who attempt to conceive and the male factor accounts for about half of the cases. Idiopathic, azoospermia, or oligozoospermia, which may be genetically based, constitute a significant number of male infertility cases.In the recent years, there is increasing recognition of the genetic abnormalities as a cause of male infertility. Genetic factors contribute up to 15 - 30% of male infertility cases. These abnormalities may be numerical chromosomal abnormality; the most common is Klinefelter syndrome, or structural e.g. Y-chomosome microdeletion, DNA mutations or mitochondrial DNA mutations. They account for 5%-10% cases of oligozoospermia, to 15%-25% cases with non-obstructive azoospermia. In azoospermia, sex chromosome abnormalities are predominant. However, autosomal structural chromosomal abnormalities are the chief genetic cause of oligozoospermia and may be balanced or unbalanced. In a substantial proportion of cases (32% of infertile men, or 1.5% of the male population), the underlying causes are unknown (idiopathic infertility). While cytogenetic studies have been successful in revealing some causes most non-obstructive azoospermic men are still idiopathic. They are believed to carry unknown autosomal mutations based on studies of familial male infertility that often involves two or more siblings.Regulation of the complex process of spermatogenesis depends on the cooperation of many genes which are expressed at various stages of differentiation, where mitotic, meiotic cell divisions, and postmeiotic development finally lead to the spermatozoa. Identification of genes playing a crucial role in spermatogenesis is mainly based on the observations of rodents, particularly the mouse model. In humans, such identification remains largely unavailable because of the difficulty in gaining access to tissue samples from a male gonad and the limited possibilities of functional verification in humans. Hence the genetic causes of male infertility have not yet been well recognized. Next-generation sequencing (NGS) is an effective tool for Mendelian gene identification. So far, more than 30 Mendelian disease genes have been identified using NGS, including recessive and dominant diseases. NGS data can also be used to discover linkage and homozygosity intervals, as well as determine copy number number of specific intervals (Becker et al. 2011, Krawitz et al. 2010). 15, 16 The two major branches of next-generation DNA sequencing are whole-exome (WE) and whole genome (WG) sequencing. While WGS is more comprehensive and useful for population genetics or for diseases in which complex non-exonic inheritance is suspected, WES remains a more powerful, cost-effective tool for detecting rare mutations within a patient population. Our use of WGS or WES will depend on the nature of the patients recruited for the study. In cases where depth of coverage requirements are high, WES would be better-suited as it allows for 4-6 exomes to be covered at such depth for the same cost of 1 genome at 30X. This would aid in sampling rarer alleles within the population and discovering novel mutations in patients that are not present in their unaffected family members. This is especially useful in cases of diseases where wide allelic heterogeneity may exist, as patients who test negative for the most common mutations may have a novel mutation that would otherwise have been undetectable by traditional methods. Multiple members of the same family may be affected by infertility with similar or different presentations of testicular failure with different histopathologies. In the presence of normal karyotyping and Y-chromosome microdeletion, we are usually left with unknown genetic abnormality. Genetic causes of male factor infertility can be broadly assumed to be a result of either de novo mutations inherited by the patient from one of his parents or from polymorphisms circulating in the population. In both cases, a search for potential genetic causes for infertility must rely on comparison of the DNA sequence of the patient with a controlled fertile member of his family.