Five patients, four of whom were diagnosed with azoospermia and one with cryptospermia, had identical changes in the AZF region STS markers: sY1192, sY153 and sY1191 marker deletions. The results obtained when the sY1191, sY153 and sY1192 markers were not amplified indicate partial Y chromosome microdeletions of the AZF region: sY1192 and sY153 markers are specific to the AZFb region of the Y chromosome (Yq.11.223) and sY1191 for the AZFc (gr/gr) region (Yq11.223) (Devyser AZF v2 Art. No.: 8-A019.2, Devyser AZF Extension Art. No.: 8-A020).
The deletion combination of STS markers identified during the study is not described in the literature, so the results are not interpreted in detail. However, complete microdeletions of the AZFb region can lead to meiosis errors and changes in sperm maturation, and the significance of partial microdeletions of this region for spermatogenesis has not yet been investigated (Punjani et al. 2020; Colaco and Modi 2018). Microdeletions occurring in the AZFc region are known to account for 80% of YCM and consequences include azoospermia, SCO syndrome, and oligospermia (Witherspoon et al. 2021). Partial microdeletions of AZFc (gr/gr) are known to remove about half of the AZFc region. However, gr/gr microdeletions and association with infertility is debatable. Part of the scientific research associate the gr/gr microdeletions with the changes in motility and number of spermatozoa, and results in reduced sperm production and gene expression in the AZFc region. In the other part – no negative changes in spermatogenesis were found (Colaco and Modi 2018). Due to the lack of research on the significance of partial AZFb and AZFc (gr/gr) microdeletions in male infertility, more detailed conclusions cannot be given and further investigation is mandatory.
Regarding the analysis of NGS data in this study, one patient had a pathogenic variant that explained the clinical phenotype associated with male infertility - globozoospermia. This variant is in the DPY19L2 gene, while changes in this gene are associated with globozoospermia – a rare phenotype of primary male infertility, which is characterized by the formation of a round head without an acrosome in most spermatozoa (https://www.omim.org/). The identified change is described in Table 3.
Table 3
Pathogenic variant of the DPY19L2 gene and suspected deletion of the DPY19L2 gene.
No. | Gene | Type of inheritance | Change location | Zygosity | Variant | Functional consequence | dbSNP database | ClinVar database | InterVar database | Varsome database |
1. | DPY19L2 | AR | Exon (22 of 22) | Likely compound heterozygous | NM_173812.5(DPY19L2):c.2242C > T(;)(2242C > T) | Termination codon | – | – | VUS | VUS |
2. | Whole gene (exons 1–22) | Whole gene deletion | There is no DPY19L2 gene product | – | – | – | – |
AR - autosomal recessive inheritance, VUS – variant of unknown significance.
It is worth noting that the deletion of the whole DPY19L2 gene (exons 1–22) was also suspected after CNV analysis. Variants of this gene have been shown to account for 50–70% of all cases of globozoospermia, and deletion of the whole gene is one of the most commonly identified causes (Celse et al. 2021).
Likely pathogenic variants were identified for two patients that explain the phenotype associated with male infertility. Variants were detected in the MTHFR and DCC genes. A variant in the MTHFR gene was found in a patient with oligoasthenospermia. DCC gene variant was identified in patient with oligospermia. Publications mention that MTHFR gene variants are associated with non-obstructive azoospermia (Li et al. 2015). Also, scientific studies describe the association of MTHFR gene variants with azoospermia and oligoasthenoteratospermia – a clinically significant NM_005957.5(MTHFR):c.[677C > T];[677=] variant was identified in this gene (Han et al 2020). Variants in the DCC gene are found in cases of Kallman syndrome, which is also associated with male infertility (Bouilly et al. 2027). Also, the highest expression of the DCC gene is observed in testicular tissue ( https://varsome.com/), and phenotypic features associated with this gene include reduced fertility, changes in testicular size, and penile hypoplasia (https://monarchinitiative.org). Identified variants in the discussed genes are described in Table 4.
Table 4
MTHFR and DCC gene likely pathogenic variants explaining male infertility.
No. | Gene | Type of inheritance | Change location | Zygosity | Change | Functional consequence | dbSNP database | ClinVar database | InterVar database | Varsome database |
1. | MTHFR | AD | Exon (6 of 12) | Heterozygous | NM_005957.5(MTHFR):c.[1004C > T];[1004=] | Nonsynonymous | rs543016186 | Complicated assessment of pathogenicity | Likely pathogenic | Likely pathogenic |
3. | DCC | AD | Splicing site (19 of 28 introns) | Heterozygous | NM_005215.4(DCC):c.[2935 + 1G > A];[2935 + 1G=] | – | – | – | – | Likely pathogenic |
AD - autosomal dominant inheritance |
Also, in one patient diagnosed with azoospermia, a likely pathogenic variant of the genome in the already discussed DPY19L2 gene was identified. After evaluating the CNV data, the individual has a suspected deletion of the whole DPY19L2 gene (exons 1–22). This is a novel find, because this gene is known to be associated with globozoospermia, but not with azoospermia (Celse et al. 2021). Therefore, the latter genomic variant does not directly explain the clinical phenotype. In this case, a repeated spermogram and a more detailed analysis of the DPY19L2 gene are required to find functional links between the gene variant and the azoospermia phenotype.
Variants of uncertain clinical significance in genes associated with male infertility were identified in five patients. These variants were detected in BRCA1, BRCA2, TP63, PKD1, CSMD1, SBF1 and DNAH8 genes. Pathogenic (pathogenic in oncology) variants were identified in two patients, the clinical significance of which is not clear in relation to the infertility of the men studied. Variants have been identified in the BRCA1 and BRCA2 genes are usually associated with oncogenetics (breast and ovarian cancer) (https://civicdb.org). A variant in the BRCA1 gene was found in a patient diagnosed with azoospermia, and BRCA2 in a patient with severe oligoasthenospermia. According to scientific publications that BRCA1 gene variants can be associated with male infertility, and BRCA2 gene variants are also found in cases of idiopathic male infertility, including azoospermia, oligospermia, asthenospermia and teratospermia (Hershlag et al. 2019; https://www.auanet.org). It is also worth noting that BRCA1 and BRCA2 gene expression was observed in testicular tissue, which confirms that these genes may also be important in spermatogenesis processes (https://varsome.com/). Variants of the BRCA1 gene are also associated with prostate cancer, which may affect the quality of prostate secretion, while BRCA2 is associated with the absence of testes (https://string-db.org). A variant of TP63 oncogene of uncertain clinical significance was identified in an individual with azoospermia clinic. Variants of the TP63 gene have been found to be associated with male infertility, including azoospermia, oligospermia, teratospermia, or asthenospermia (Hershlag et al. 2019).
This analysis show that well-known oncogenes may also have an important role in men infertility and further functional analysis is needed.
Also, for a patient with the already discussed BRCA2 gene variant (a case of severe oligoasthenospermia), a variant of uncertain clinical significance was identified in the PKD1 gene, which may be associated with male infertility. Variants of this gene are not only associated with polycystic kidney disease, but also with asthenospermia and azoospermia (He et al. 2018). Furthermore, this gene is also associated with prostate cancer (https://varsome.com/). The scientific publication also describes a case where two de novo PKD1 gene variants were identified by NGS, which lead to autosomal dominant polycystic kidney disease and Klinefelter's syndrome, one of the symptoms of which is hypogonadism and azoospermia characteristic of male infertility (Peces et al. 2022).
CSMD1 and SBF1 gene variants were identified in one patient with azoospermia. Variants of the CSMD1 gene are associated with infertility in both women and men, with non-obstructive azoospermia in nonobstructive azoospermia (Lee et al. 2019). Also, variants of the CSMD1 gene are found in cases of prostate carcinoma (https://monarchinitiative.org). It is known that the prostate is the main male reproductive gland, and it is important for male fertility due to the secreted prostate secretion (Verze et al. 2016). Variants of the SBF1 gene also are associated with testicular failure (Kuzmin et al. 2009) and shows that it is necessary to include this gene in the analysis of infertile men
A variant of the DNAH8 gene of unclear clinical significance was identified in a patient diagnosed with cryptospermia. Variants of the DNAH8 gene are characteristic of spermatogenic failure (https://www.omim.org/). Changes in this gene are also associated with sperm flagella abnormalities (short, twisted flagella), complete absence of flagella and reduced sperm motility (Liu et al. 2022). And this leads to changes in the motility of male gametes associated with cryptospermia. This is also confirmed by publications on infertile individuals due to multiple morphological abnormalities of sperm flagella and reduced sperm motility which may lead to male infertility (Verze et al. 2016; Yang et al. 2020).
It is important to mention that the highest expression of the discussed genes (CSMD1, SBF1, DNAH8) is observed in testicular tissue, which confirms that these genes may play an important role in sperm development processes (https://varsome.com/), and the identified variants in these genes may be the cause of infertility in the individuals. The identified variants in the discussed genes are described in Table 5.
Table 5
SNV of uncertain clinical significance.
No. | Gene | Type of inheritance | Change location | Zygosity | Change | Functional consequence | dbSNP database | ClinVar database | InterVar database | Varsome database |
1. | BRCA1 | – | Exon (4 of 23) | Heterozygous | NM_007294.4(BRCA1):c.[181T > G];[181T=] | Nonsynonymous | rs28897672 | Pathogenic | Pathogenic | Pathogenic |
2. | BRCA2 | – | Exon (11 of 27) | Heterozygous | NM_000059.4(BRCA2):c.[3682_3685del];[3682_3685=] | A deletion that causes a reading frameshift | rs80359396 | Pathogenic | – | Pathogenic |
3. | PKD1 | AD | Exon (29 of 46) | Heterozygous | NM_001009944.3(PKD1):c.[9829G > A];[9829=] | Nonsynonymous | rs148812376 | Ambiguous assessment of pathogenicity | VUS | VUS |
4. | CSMD1 | – | Exon (14 of 70) | Heterozygous | NM_033225.6(CSMD1):c.[1954G > T];[1954=] | Nonsynonymous | rs773431047 | – | VUS | VUS |
5. | SBF1 | – | Exon (2 of 40) | Heterozygous | NM_002972.4(SBF1):c.[68G > A];[68=] | Nonsynonymous | – | – | VUS | VUS |
6. | DNAH8 | AR | Exon (45 of 93) | Heterozygous | NM_001206927.2(DNAH8):c.[6328T > C];[6328=] | Nonsynonymous | rs201462063 | VUS | VUS | VUS |
7. | TP63 | AD | Exon (13 of 14) | Heterozygous | NM_003722.5(TP63):c.[1697C > T];[1697C=] | Nonsynonymous | rs745687224 | VUS | Likely pathogenic | VUS |
AD - autosomal dominant inheritance, AR - autosomal recessive inheritance, VUS – variant of unknown significance |
Nine of the 18 (50%) individuals had no variants in candidate genes associated with changes in sperm count and/or morphology. Of the nine individuals mentioned, eight had no YCM, the following phenotypes were diagnosed in these patients by spermogram tests: oligoasthenospermia, severe oligospermia, oligospermia, oligoasthenospermia/azoospermia and azoospermia (for four cases). This shows the need for further investigation, analysing whole exome data. However, one individual of nine (clinical phenotype – azoospermia) had Y chromosome changes – sY153, sY1192 and sY1191 markers of AZF region deletion. After analyzing the genomic variants of candidate genes four variants (two in the DPY19L2, one in the DCC and one in the MTHFR genes), confirming the infertility phenotype, were detected for three individuals.
The obtained data were the first to examine a sensitive group of infertile men at the gene panel level. With the increasing problem of infertility in the world, it is important to identify and understand its causes, therefore, NGS facilitates the research we do. Usually, the diagnosis of male infertility in Lithuania is limited to individual molecular tests, such as the determination of complete YCM and the sequencing of particular single genes, while the determination of partial YCM and the application of NGS allowed to deepen knowledge in the etiopathology of male infertility and to raise new questions. We also compiled a list of genes that could be used for further diagnosis of infertile patients. This will speed up the explanation of the cause. For patients with an undetermined cause, whole-exome or genome-wide testing can be used. Despite the identification of genomic variants that explain the clinical features associated with male infertility, there may be an opportunity for assisted reproduction techniques in some cases.
Although the identified VUS variants have not been confirmed by other research methods, they pave the way for future studies using functional research methods to confirm pathogenicity. Particularly interesting findings with the BRCA1, BRCA2 and TP63 genes, which are usually associated with cancer and are rarely emphasized and associated in the field of infertility.