To explore the content and clinical value of genetic testing for spermatogenic dysfunction. Methods We combined our own clinical practice and reviewed the relevant domestic and international literature in the past decade. The genetic tests related to patients with spermatogenic disorders mainly included karyotype analysis, spermatogenic gene test, androgen receptor gene test and cystic fibrosis gene test. It is concluded that the necessary genetic testing components should be given high priority when clinically treating patients with spermatogenic disorders, especially when performing assisted human reproduction techniques for pregnancy.
Infertility is a worldwide problem. According to WHO statistics, its prevalence accounts for 10-15% of couples of reproductive age. Among the male causes, sperm production disorders are important, accounting for 30% to 65% of male infertility causes. The performance is azoospermia, severe spermatozoa, weak sperm, deformed sperm, and dead sperm.
With the promotion of assisted human reproduction technology, the number of patients treated with artificial insemination and intracytoplasmic single sperm injection for male infertility is increasing year by year. At the same time, the genetic risk for these patients is increasing. Therefore, it is particularly important to perform genetic analysis and fertility risk assessment before the implementation of assisted human reproductive technologies, as well as prenatal screening and prenatal diagnosis after the female partner becomes pregnant.
Spermatogenic disorders can be caused by various factors, such as age, severe systemic diseases, severe malnutrition, neuroendocrine-immune dysfunction, genetic defects, environmental pollution (radiation, chemical toxicity, etc.), and testicular trauma. The genetic causes include chromosome number and structure abnormalities, Y chromosome microdeletion, androgen receptor gene mutation and cystic fibrosis disease.
I. Chromosome number and structure abnormalities
Spermatogenesis is controlled by many genes that are expressed in an orderly manner. Abnormalities in chromosome number and structure can affect the function of these genes, which in turn affects spermatogenesis.
The incidence of chromosome number and structure abnormalities in patients with azoospermia ranges from 13% to 39%; the incidence of chromosome number and structure abnormalities in patients with severe oligospermia ranges from 4.9% to 13.2%, while the incidence of chromosome number and structure abnormalities in the general population is only 0.5%.
Patients with azoospermia due to chromosomal number and structure abnormalities have small testes and poor secondary male sexual characteristics on physical examination. Most patients with oligospermia due to chromosome number and structure abnormalities have normal testes and external genitalia, and acceptable male secondary sexual characteristics on physical examination. The testes of individual patients are small.
1. Creutzfeldt-Jakob sign.
The most common karyotype leading to azoospermia is 47,XXY, also known as Klinefeler’s disease (Klinefeler syndrome), accounting for 10% to 50% of chromosomal abnormalities in azoospermia. As a result of the X chromosome abnormality, the patient has vitreous hyaline degeneration and fibrosis of the testicular seminiferous tubules, death of spermatogenic epithelial cells, and no sperm production. Patients clinically present with tall stature, abundant subcutaneous fat, and small testes. In a very small number of patients, spermatozoa are found in the seminiferous tubules, and there are reports of pregnancy in the wife through ICSI technique.
2.Robertsonian translocation.
It can be azoospermia or oligospermia. Since the majority of chromosomal breakpoints are located in the non-transcribed region of DNA, the phenotype is normal because the number of functional genes is generally kept in balance and can perform normal functions despite the change in chromosome structure. However, during the development of germ cells, the breakage and rejoining of genes cause a small number of bases to be free and result in a relatively unbalanced translocation, which affects normal meiosis and leads to abnormal spermatogenesis.
3.Autosomal structure abnormalities
Autosomal translocations and inversions mainly lead to miscarriage and delivery of deformed children, but they have also been reported to cause oligospermia. The main autosomes involved are #1, #3, #5, #6, #7, #8, #9, #10, #12, #13, #14, #15, #17, #21, and #22.
The inter-arm inversion of chromosome 9, previously considered a polymorphic phenomenon, has been reported in a number of articles with genetic effects. in Misic’s analysis of 820 cases of azoospermia, there were 23 cases of inter-arm inversion of chromosome 9 (9 cases of azoospermia and 14 cases of oligospermia). It may be related to the hindering effect of inversion on the formation of bivalent bodies during the first meiosis.
4.Y chromosome abnormalities
The second most common chromosomal abnormality in azoospermia patients is Yq deletion. inversion, translocation, intermediate deletion, and ring chromosome of Yq11 fragment can cause serious spermatogenic disorders. Depending on the size of the lost fragment, different manifestations can be seen pathologically.
The genetic effects of large Y (≥chromosome 18) are still controversial. Some believe that it does not affect fertility; however, clinically, embryonic arrest, miscarriage, stillbirth, fetal malformation, and the discovery of mental retardation after birth in the wives of patients with large Y azoospermia or oligospermia are indeed seen. Some scholars believe that Big Y is an excessive duplication of DNA in heterochromatin, which can cause mitotic errors or affect gene regulation and cell differentiation, and finally lead to reproductive abnormalities. It is believed that the genetic effects of large Y will be clearly understood at the genetic level in the future.
The presence or absence of clinical genetic effects of small Y chromosomes (≤ chromosome 21) is also controversial. It is generally considered to be a chromosomal polymorphism and is seen in normal individuals. However, there are reports in the literature of patients with small Y karyotype who are azoospermic or severely oligospermic and have a history of adverse maternal outcomes in their wives. The authors of the literature believe that small Y, like large Y, cannot be considered as a normal polymorphism. There is insufficient evidence whether the spermatogenic disorder in patients with small Y is due to some association between Y chromosome heterozygosity and spermatogenesis or due to the presence of AZF microdeletions that are morphologically difficult to determine.
5. Sexual inversion
46, XX sexual inversions are clinically seen in patients with azoospermia. The currently accepted explanation is that homologous recombination and exchange of X-Y chromosomes occurs during meiosis, as the sex-determining region (SRY) of Y chromosome is very close to the proposed chromosomal region (PAR), allowing the transfer of SRY gene into X chromosome. Since the spermatogenic gene is located on Yq not transferred to X chromosome, thus 46, XX males have only testicular development but cannot produce sperm.
6.Y-chromosome abnormal chimerism
The cause of azoospermia or severe oligospermia in patients with abnormal Y chromosome chimerism may be the partial loss or deletion of the Y chromosome and the partial loss or deletion of testicular determinants (TDF) and AZF, which causes incomplete or no expression of the genetic functions associated with them and finally leads to oligospermia or azoospermia.
In clinical practice, it may sometimes be encountered that the same patient gets inconsistent results of karyotype analysis reports from different hospitals: one report has normal karyotype, while another report has chimerism with a high proportion of normal karyotype and a low proportion of abnormal karyotype. In this case, it cannot be easily assumed which report is incorrect. It should be analyzed in the context of the patient’s clinical symptoms and signs and other aspects of the examination as well as the female partner’s pregnancy.
Y chromosome microdeletion
The Y chromosome microdeletion exists in 10%-20% of azoospermia of unknown cause and 5%-15% of severe oligospermia. 1976 Tiepolo et al. found a break and deletion at Yq11 in patients with primary azoospermia and proposed that a gene related to spermatogenesis exists on Yq and named it azoospermia factor (AZF). Further studies showed that the AZF family on the Yq11.22 to Yq11.23 region could have deletion mutations at multiple loci, and microdeletions at any of these loci could lead to spermatogenesis disorders.
In the past, the detection of Y chromosome microdeletions was mostly limited to patients with idiopathic male infertility, but in recent years, it has been found that Y chromosome microdeletions may also exist in patients with cryptorchidism, varicocele, bilateral vas deferens, sex hormone abnormalities, and syringomyelia. Therefore, Y chromosome microdeletion testing should also be performed for azoospermia and severe oligospermia in these patients. Try to avoid blindly performing spermatozoal vein high ligation to improve semen analysis parameters.
AZF is generally divided into 3 regions, namely AZFa, AZFb, and AZFc. In 1999, Kent-First et al. suggested the presence of AZFd between AZFb and AZFc, and since then a series of candidate genes for infertility have been successively identified. Among them, the RBM1 (RNA bingding motif 1 ) gene cluster with RNA structural sequences and DAZ (Deleted in azoospermia) are thought to play an important role in spermatogenesis.
Zhou Zuomin et al. found that the deletion rate of DAZ was 18.2% and 31.6% in patients with azoospermia and severe oligospermia, respectively. DAZ encodes an RNA-binding protein essential for spermatogenesis, and defects in DAZ can affect spermatogenesis.
The DAZ gene was isolated in 1995, is unique to humans and orangutans, and is highly exclusively expressed in the testis. It is a specific gene in a non-specific gene cluster on the Y chromosome, with several copies and homology to autosomes. In addition, DAZ is a member of a polygenetic family in which the SPGY gene or DAZL on autosomes is located on chromosome 3.
AZFa deletions are uncommon and can lead to spermatozoal arrest at puberty, manifesting as a pathological “support cell only syndrome” and microtesis;
AZFb deletion is more common and presents with normal pre-meiotic epithelial cells and a lack of post-meiotic cells, suggesting disruption of spermatogenesis before or during puberty; AZFc deletion is more common and accounts for approximately 60% of Y chromosome microdeletions.
The pathological histological changes and clinical manifestations of patients are diverse and can be either azoospermia, oligospermia or normal sperm concentration but abnormal morphology. Testicular pathology reveals a number of empty varicocele surrounded by a number of varicocele with spermatogonia and spermatocytes and reduced spermatogenesis.
Although the Y chromosome is largely intact in most patients with primary azoospermia and severe oligospermia, and individuals with AZF deletion are not always sterile, the Y chromosome AZF microdeletion test has developed into an important technical tool and routine test for the diagnosis of genetic male infertility.
In China, with the increasing rate of male infertility visits in recent years and the widespread development of assisted human reproduction techniques, it is particularly important to perform testing for AZF deletion. Before ICSI treatment of patients with AZF deletion, it is important to inform them of the risk of producing male offspring with the same deletion to avoid doctor-patient disputes.
Partial deletions in the AZFc region can be passed vertically to the offspring through assisted human reproduction techniques, and the type, length, and number of DNA copies of partial deletions in the AZFc region of the next generation of males are identical to those of the father.
The chromosomal abnormality is accompanied by a Y chromosome microdeletion. Both are important genetic factors contributing to azoospermia and oligospermia. The simultaneous testing of the two indicators can provide a more comprehensive picture of the presence of genetic defects in male infertility spermatogenic disorders.
Case 1: A patient with azoospermia, who had been seen in a fertility clinic. His wife underwent ovulation treatment, and on the day of egg retrieval, he underwent percutaneous testicular puncture to retrieve sperm, but none of the sperm was retrieved.
After bilateral testicular biopsy, no mature sperm was found and the treatment cycle had to be cancelled. Later, after Y-chromosome microdeletion testing, the entire AZFb was found to be missing. If the Y-chromosome microdeletion test had been done beforehand, his wife would not have experienced unnecessary pain and huge expenses.
Case 2: A fertility center performed ICSI for three patients with azoospermia and severe oligospermia, resulting in four boys. Because of the lack of prior knowledge of the importance of Y chromosome microdeletion testing, this test was not performed. Later, in order to collect data for the study, a Y chromosome microdeletion test was performed on both the father and son, and it was found that all four boys and their father had AZFc deletion. This suggested that the AZFc deletion in the father could be passed on to the male offspring.
The above case illustrates that ICSI is not safe without prior Y-chromosome microdeletion testing.
Third, androgen receptor gene abnormalities
Androgen receptor (AR) gene defect causes testicular feminization syndrome, also known as androgen insensitivity syndrome. Patients with complete testicular feminization present with female ectopic reproduction, gynecomastia, absence of blind vagina, uterus and ovaries, testes in the abdominal cavity or inguinal cavity, and sterility, but with a karyotype of 46, XY.
The AR gene is located in Xq11-Xq13 and encodes a protein with 3 functional regions. The N-terminal region with regulatory function is encoded by exon 1; the DNA-binding region is encoded by exons 2,3. The androgen-binding region is encoded by five exons.
More than 300 different mutations have been identified, mainly in the DNA-binding and androgen-binding regions. Notably, in the N-terminal region, a CAG repeat sequence is present within exon 1. Patients with >40 times of this sequence exhibit spinal demyelination with azoospermia and testicular atrophy; the number of CAG repeats correlates with spermatogenic function in Chinese and Americans.
IV. Cystic fibrosis disease (CF)
CF is common in populations of European origin. The CF gene is located at 7q31. 70% of the more than 800 CF mutations identified to date are SF508 deletions.