Copy‐number analysis of Y‐linked loci in young men with non‐obstructive azoospermia: Implications for the rarity of early onset mosaic loss of chromosome Y

Abstract Purpose Mosaic loss of chromosome Y (mLOY) is a common feature in elderly men. If mLOY can also occur in young men, it may lead to spermatogenic failure due to loss of spermatogenic genes. Indeed, previous studies detected the 45,X/46,XY karyotype in a few young men with spermatogenic failure. The present study aimed to clarify the frequency of cryptic mLOY in reproductive‐aged men with spermatogenic failure. Methods We studied 198 men at ages 24‐55 years who presented with etiology‐unknown non‐obstructive azoospermia. Prior this study, these patients underwent G‐banding analysis for 20 leukocytes and were found to have 46,XY karyotype. We analyzed copy numbers of chromosome Y in blood cells by using semi‐quantitative multiplex PCR for AMELY/AMELX, array‐based comparative genomic hybridization (CGH) for the AMELY locus, and droplet digital PCR for SRY, USP9Y, and UTY. Results Multiplex PCR showed borderline low AMELY/AMELX ratios in three patients. However, for the three patients, CGH excluded deletion of the AMELY locus, and droplet digital PCR suggested preserved copy numbers of all tested loci. Conclusion This study highlights the rarity of leukocyte mLOY in reproductive‐aged men with spermatogenic failure. In addition, our data imply that standard karyotyping is sufficient to screen early onset mLOY.

Human chromosome Y encodes several spermatogenic genes. 6 Hemizygous interstitial deletions of chromosome Y, particularly those in the azoospermia factor (AZF) region, are known as the risk factor of non-obstructive azoospermia. 7 Thus, mLOY, if it occurs in testis of young men before or during reproductive ages, may cause spermatogenic failure. In 2016, Shin et al 8  previous studies. The present study aimed to clarify the frequency of cryptic mLOY in young men with spermatogenic failure. To this end, we screened 198 samples using a semi-quantitative multiplex PCR method, whose sensitivity has been confirmed in previous studies. [10][11][12] 2 | MATERIAL S AND ME THODS

| Patients
The study group consisted of 198 Japanese patients with non-obstructive azoospermia at ages 24-55 years (median 34 years). The patients were numbered in order of their age. These patients visited our clinic because of infertility and were diagnosed with nonobstructive azoospermia of unknown etiology. Prior to the present study, all patients underwent conventional G-banding analysis for 20 peripheral leukocytes and were found to have a normal 46,XY karyotype. In addition, the patients were subjected to copy-number analysis of the AZF regions, 7,13 which showed the lack of AZF deletion in 132 of 198 patients. The common gr/gr, AZFb, AZFc, and AZFb+c deletions were detected in 49, 1, 15, and 1 patient(s), respectively. The frequencies of these AZF deletions were comparable to previous data in Japan. 7

| Screening of mLOY by semi-quantitative multiplex PCR
We performed semi-quantitative multiplex PCR for all samples to screen mLOY. Genomic DNA samples were obtained from peripheral blood of each patient. We utilized a previously established method with slight modifications. 10 In brief, copy numbers of chromosome Y relative to chromosome X were assessed by comparing the area under the curve of PCR products for AMELY at Yp11.2 to that for AMELX at Xp22.2. The PCR products were analyzed using the 3130 Genetic Analyzer with LIZ500 (Applied Biosystems). The area under the curve was calculated using the GeneMapper 3.7 software (Applied Biosystems). Samples which showed AMELY/AMELX ratios of ≤0.89 in two independent assays were subjected to the second analysis by droplet digital PCR. This cutoff value, 0.89, was determined based on the previous report 11 and our own data on 42 fertile men aged between 31 and 39 years (range, 0.89-1.04; mean, 0.97).

| Detection of copy-number alterations involving AMELY
Low AMELY/AMELX ratios are indicative of mLOY; however, they can also result from interstitial deletions involving the AMELY locus.
To exclude such Y-linked interstitial deletions, we performed arraybased comparative genomic hybridization (CGH) for samples with a low AMELY/AMELX ratio. We used a human catalog array (4 × 180 k format; Agilent Technologies, Palo Alto, CA), which contains three probes for the AMELY gene. Copy number of the AMELY locus was

| Detection of mLOY by droplet digital PCR
To examine the copy number of chromosome Y in the samples with possible mLOY, we performed droplet digital PCR. 14 Genomic DNA samples were analyzed using the QX200 system (Bio-Rad Laboratories). We examined copy numbers of three loci, that is, SRY (Bio-Rad Laboratories, Assay ID: dHsaCP2500472), USP9Y (Assay ID: dHsaCP2506328), and UTY (Assay ID: dHsaCNS782024066).
RPP30 at 10q23.31 (Assay ID: dHsaCP2500350) was utilized as the internal reference. For each locus, two independent assays were performed.

| Detection of copy-number alterations involving AMELY
Array-based CGH for patients 7, 147, and 164 detected no copynumber alterations at the AMELY locus ( Figure S1). Thus, the low AMELY/AMELX ratios in these patients were not ascribable to an interstitial deletion of chromosome Y.

| Detection of mLOY by droplet digital PCR
Droplet digital PCR for patients 7, 147, and 164 showed apparently normal copy numbers for the three tested loci ( Figure 1C). Thus, these patients were unlikely to have mLOY.

| D ISCUSS I ON
None of the 198 men with non-obstructive azoospermia and normal karyotype had recognizable mLOY. Whereas semi-quantitative multiplex PCR showed low AMELY/AMELX ratios in three patients, droplet digital PCR for these individuals suggested normal copy numbers of three loci on chromosome Y. The semi-quantitative multiplex PCR method have been used in several previous studies [10][11][12] showing that its detection limit of mLOY is as low as 4.6%. 10 Thus, it is unlikely that this method failed to detect leukocyte mLOY in our subjects. In this regard, the false-positive results of patients 7, 147, and 164 were not ascribable to interstitial deletion of the AMELY locus, because array-CGH demonstrated a normal copy number for this locus. The low AMELY/AMELX ratios of these patients may reflect the relatively low specificity of this screening method. Actually, the ratios in patients 7, 147, and 164 were only slightly lower than the cutoff value.
We cannot completely exclude the possibility that some of our 198 subjects carried mLOY exclusively in the testis. Indeed, previous studies detected mLOY not only in blood cells, but also in buccal samples. [15][16][17] Considering that strong expression of the sex-determining gene SRY was observed in the fetal testis but not in the postnatal testis, 18 postnatal mLOY of young men may impair spermatogenesis but likely permits normal male-type sexual development. However, the probability of testis-specific occurrence of mLOY is low, because mLOY is predicted to occur predominantly in high-turnover cells such as peripheral leukocytes. 1,2 The results of this study, in conjunction with the previous reports by Shin et al 8  Furthermore, there are no data whether mLOY in young men is associated with early death and the risk of various disorders, as is the case for elderly people. 1,4,5 In this context, Thompson et al 3 raised questions about the pathogenicity of mLOY in blood cells.
It is possible that leukocyte mLOY itself has no deleterious effects on men's health, but reflects genomic instability in other tissues.
Large-scale studies are needed to clarify the clinical significance of leukocyte mLOY in young men.
In summary, the results of this study demonstrated the rarity of mLOY in reproductive-aged men with spermatogenic failure. Thus, early onset mLOY is unlikely to play a major role in the development of spermatogenic failure. In addition, our data imply that standard karyotyping is sufficient to screen early onset mLOY.