Cohort characteristics and the landscape of genomic variants
A total of 101 unrelated male patients presenting with NOA (n=63) or severe oligozoospermia (n=38) (Table 1) without a positive finding from routine standard-of-care tests was included in this study. The clinical workup included detailed hormone analysis (follicle stimulating hormone [FSH] and testosterone), two semen analyses, and testicular ultrasound imaging. Overall, low-pass mate-pair GS identified clinically significant SVs, and multiple regions with AOH in 18 patients (17.8%, Tables 2 and 3), all findings were confirmed by orthogonal methods. Follow-up studies were conducted for each of these patients for the outcomes of sperm retrieval and pregnancy with in vitro fertilization technology.
Clinically significant variants in patients with NOA
Among the 63 NOA patients, mate-pair GS identified clinically relevant SVs (n=9), and multiple regions with copy-number neutral AOHs (n=3) in an overall of 10 patients (15.9%, Table 2). Patient 20C1113 was found to have two SVs, while patient 20C0911 had an SV in chromosome X, and two regions with AOH in chromosome 6.
Case 20C1113 was a 33 year-old patient presenting with small testes and NOA. Mate-pair GS identified a forward tandem duplication of Xp21.2 of 535 kb, seq[GRCh37] dup(X)(p21.3) chrX:g.29957001_30492292dup, involving NR0B1 and MAGEB gene clusters (Figure 1A and B). NR0B1 and MAGEB gene clusters have been curated as triplo-sensitive regions by ClinGen (TS=3; Dosage #ISCA-46302). Duplications of similar size were reported in cases with isolated 46,XY gonadal dysgenesis. In addition, a 4 Mb inversion, inv(13)(q31.1q32.1), which was missed by former G-banded chromosome analysis (Figure 1C), was detected without gene disruption at the breakpoint junctions. Follow-up of the patient indicated that no natural conception was achieved for more than one year since receiving former negative karyotyping results.
Case 21C0764 was diagnosed as NOA (testicular failure). Ultrasound examination revealed small testes (5 cc) and a small epididymal cyst. A hemizygous 1.14 Mb deletion in Xq28 was detected (seq[GRCh37] del(X)(q28) chrX:g.150838316_151978891del) by mate-pair GS involving MAGEA and CSAG gene clusters as well as FATE1 gene. The MAGEA gene cluster is crucial in maintaining normal testicular size in mice(Hou et al. 2016), knockout of which suggested that specific defects occurred during the first wave of spermatogenesis. ScRNA-seq data from fetal and postnatal testicular development indicated that MAGEA gene cluster expressed in primordial germ cells (Figure 2A). The protein of FATE1 is known to control the early testicular differentiation and cell proliferation, and high expression of gene FATE1 was only observed in Sertoli cells (Figure 2A) in early testicular development. In comparison, depletion of CSAG1 disrupts centrosomes and leads to multipolar spindles during mitosis(Sapkota et al. 2020), but no expression of gene CSAG1 was identified from fetuses (Figure 2A). Both FATE1 and CSAG1 are highly expressed in adult germ cells (Figure 2B and C). Therefore, the lack of the expressions of these genes by the hemizygous deletion in this case likely led to small testes (MAGEA gene cluster and FATE1) and absence of sperms (CSAG1). Follow-up study indicated that the patient pursued an IVF pregnancy using donor sperm.
Case 21C2529 was a 38 year-old NOA male with bilateral small testes. A heterozygous intragenic forward tandem duplication of 16.3 kb (seq[GRCh37] dup(8)(q21.11) chr8:g.74500678_74517010dup) was identified involving exons 10 and 11 (NM_001164380) of STAU2, likely resulting in gene truncation (loss-of-function, Figure 3A). STAU2 mRNA is expressed in spermatocytes found in seminiferous tubules at stages VI-XII of the spermatogenic cycle and plays a role in mammalian gametogenesis(Saunders et al. 2000), and scRNA-seq data indicated STAU2 likely expressed in most of the cell types in fetal testicular development as well as in adult germ cells (Supplementary Figure S3). In addition, STAU2 is likely haploinsufficient (pLI=0.97 in gnomAD). Stau2 knockdown disrupted spindle formation and chromosome alignment during meiotic progression in mouse oocytes(Cao et al. 2016), while depletion or interference with STAU2 function during early zebrafish development results in aberrant migration and subsequent extinction of primordial germ cells (Ramasamy et al. 2006). Furthermore, down regulation of Stau2 in vivo resulted in a small tissue size(Cockburn et al. 2012). Follow-up reported the patient did not proceed to TESE.
A 3.2 Mb inversion, inv(16)(q22.2q23.1), was identified in case 20C0011 with NOA. Both breakpoint junctions were located in intergenic regions (i.e., no gene was disrupted, Figure 3B). However, by the analysis of the topologically associated domain (TAD), RFWD3, mutations of which is known to cause Fanconi anemia with variable expressivity of azoospermia (GeneReviews# NBK1401), was located in the TAD of the distal breakpoint junction of the inversion (Supplemental Figure S4). This likely resulted in a position effect. Follow-up reported that MESA was performed with limited sperm obtained, and an ongoing pregnancy achieved by IVF with ICSI (Intracytoplasmic Sperm Injection).
Mate-pair GS also detected multiple regions with AOH in three NOA patients (Table 2). For case 20C0911, a complex insertion was identified resulting in disruption of gene ANOS1 (ClinGen dosage sensitivity map: HI=3, Table 2), which is known to cause Kallmann syndrome (Thakker et al. 2020). In addition, two interstitial regions with AOH were identified with an overall size of 18.7Mb. Uniparental disomy in chromosome 6 (UPD6) was suspected; however, no parental samples were available for confirmation. High read-depth GS excluded the presence of causative SNVs/Indels in genes related to male infertility. In comparison, the overall sizes of regions with AOH identified in cases 22C0195 and 21C3049 were 68 Mb and 218.6 Mb, respectively (Table 2), suggesting parental consanguinity. High read-depth GS in patient 22C0195 revealed a pathogenic homozygous SNV NC_000011.9:g.5248155C>G in gene HBB (Figure 3Ci) mapped in the region with AOH in chromosome 11 (ClinVar ID: #15447). It is a well-established cause for beta thalassemia. As beta-thalassemia is classically considered to result in iron deposition in the endocrine glands leading to male infertility(De Sanctis et al. 2013), this finding potentially explains the infertility in this patient. In comparison, high read-depth GS in patient 21C3049 revealed a homozygous AC deletion NC_000017.10:g.78064149_78064150delAC in gene CCDC40 (NM_001243342.2:c.3044_3045del, Figure 3Cii). Loss-of-function mutations in CCDC40 are known to cause Ciliary dyskinesia, primary, 15 (OMIM # 613808), and clinically significant SNVs in this gene are identified in male infertility(Precone et al. 2020). Further study was warranted to investigate whether it potentially caused RNA decay. Follow-up indicated that the patient suffered Sertoli-Cell-Only (SCO) syndrome revealed by MESA.
Clinically significant variants in patients with severe oligozoospermia
Among the 38 patients with severe oligozoospermia, low-pass mate-pair GS identified SVs (n=8) in an overall of eight patients (21.1%, Table 2). They included three recurrent CNV syndromes (Supplementary Figure S5), one intragenic heterozygous deletion, one intragenic forward tandem duplication and three complex insertions.
Patient 21C1102 was diagnosed with severe oligozoospermia and dry eye syndrome. A 2.6 Mb duplication (seq[GRCh37] dup(22)(q11.21) chr22:g.18862504_21453290dup at 22q11.2) was detected. This is consistent with 22q11.2 microduplication syndrome (OMIM# 608363). 22q11.21 duplication syndrome exhibits variable expressivity and incomplete penetrance(Wentzel et al. 2008). However, affected individuals may have intellectual or learning disability and/or developmental delay as well as infertility(Abdullah et al. 2021). Follow-up revealed that the couple had undergone IVF but failed to achieve a pregnancy.
Patient 20C2873 was diagnosed as severe oligozoospermia. A 1.5 Mb microdeletion, (seq[GRCh37] del(16)(p13.11) chr16:g.14991714_16507667del) was identified in 16p13.11 (involving MYH11), which is the well-established 16p13.11 microdeletion syndrome with variable congenital anomalies, incomplete penetrance and variable expressivity reported. Although paternally inherited 16p13.11 deletions have been reported, immunohistochemical analysis clearly showed that specific contractile markers including MYH11 were reduced or lost in peritubular cells of seminiferous tubules of men with mixed atrophy in testicular biopsies(Welter et al. 2013). ScRNA-seq analysis indicated that MYH11 is mainly expressed in myocytes during fetal testicular development and in early/late spermatids in adult germ cells (Supplementary Figure S6). Follow-up revealed a pregnancy achieved by IVF resulted in a live-birth.
Patient 19C1235 was diagnosed with severe oligozoospermia with normal testicular size. Bilateral varicocele was observed by ultrasound screening. A 7.3 Mb deletion (seq[GRCh37] del(5)(p15.33p15.31) chr5:g.10379_7361801del in 5p15.33p15.31) was identified, consistent with 5p- syndrome. One case with heterozygous deletion with similar size was reported with azoospermia(Rossi et al. 2005). Follow-up indicated an IVF pregnancy was achieved.
Patient 20C2993 was diagnosed with severe oligozoospermia. Mate-pair GS revealed a 22.5 kb heterozygous deletion (seq[GRCh37] del(9)(p21.2) chr9:g.26972539_26995048del) involving exons 3 to 8 (NM_001099223) of IFT74 in 9p21.2 (Supplementary Figure S7). Mutations in IFT74 are known to cause spermatogenic failure 58 (OMIM# 619585) in an AR manner. High read-depth GS did not reveal any clinically significant SNV/Indels in IFT74 nor in other genes related to male infertility. However, IFT74 is highly expressed in spermatocytes and early spermatids in adult germ cells. Knockdown of Ift74 in spermatocyte-derived GC-2 cells resulted in a significant reduction of protein levels of cell-adhesion molecules such as E-cadherin protein that is required for the initial cell division of spermatogonial stem cells(Yamashita et al. 2003). The ITF74 intragenic deletion could be causative of oligozoospermia in this patient.
Case 21C0925 had with severe oligozoospermia. A 53 kb copy-number gain (seq[GRCh37] dup(2)(q31.1) chr2:g.171170285_171223517dup) involving the 8th exon (NM_001083615) of MYO3B on 2q31.1 was inserted into the 4th intron (NM_021951) of DMRT1. Flanking the insertion was a duplication (seq[GRCh37] dup(9)(p24.3) chr9:g.910281_931988dup) involving the 4th exon of DMRT1 (Figure 4A). DMRT1 was likely disrupted by the insertion. DMRT1 is highly expressed in Sertoli cells and primordial germ cells during fetal and postnatal testicular development (Figure 4B). High expression of DMRT1 was also specifically observed in spermatogonia and early spermatids (Figure 4C) indicating an important role of DMRT1 in testicular development and spermatogenesis. In addition, heterozygous deletion of DMRT1 is known to be causative of nonsyndromic 46,XY disorders of sexual development (DSD, GeneReviews# NBK1547)(Ledig et al. 2012). The presentation in case 21C0925 differed from reported studies, and a possible explanation would be the affected exon(s) was different [e.g., the insertion resulted in RNA decay in the last exon (5th)] and the report showed deletion involving exons 2-4 in 46,XY DSD(Ledig et al. 2012).
Comparison of the detection yields by mate-pair GS and follow-up
Overall, mate-pair GS identified clinically significant variants in 10 patients with NOA (15.9%) and 8 patients with severe oligozoospermia (21.1%). There was no significant difference of the detection yields between two groups (Chi-square test: P>0.05).
In the present study, follow-up study was conducted for all 18 cases. Among the patients with NOA, follow-up information was obtained for seven (70%) individuals. Four opted out of testicular biopsy (one pursued IVF pregnancy with donor sperm) and three opted in (one with limited sperm retrieved and two without sperm retrieved, Table 2). In comparison, among these eight cases, five had follow-up and four of them pursued IVF pregnancy with ICSI with one unsuccessful (Table 3).