High Frequency of 45,X/46,XY mosaicism carring a structurally abnormal Y chromosome in patients with Y chromosome microdeletions: a 8-year period retrospective study

Background: Structural abnormalities of Y chromosome are commonly described in associated with the occurrence of a 45,X/46,XY chromosomal mosaicism. In recent years, evidences of an association between Y chromosome microdeletions and 45,X/46,XY mosaicism had been investigated, and 45,X/46,XY mosaicism was found to be particularly common in patients with Y chromosome microdeletions. The aim of our study was to investigate the presence of 45,X/46,XY mosaicism carrying a structurally abnormal Y chromosome in patients with Y chromosome microdeletions. Results: A 8-year retrospective study was conducted on 6545 infertile men with nonobstructive azoospermia or oligozoospermia. A total of 19 patients with 45,X/46,XY mosaicism or its variants were found, of which 78.95% (15/19) had a structural Y chromosome abnormalities in 46,XY cell line. Thirteen of 19 (68.42%, 13/19) patients with 45,X/46,XY mosaicism had Y chromosome microdeletions, and 12/13 (92.31%) of them exhibited a structurally abnormal Y chromosome. Conclusions: Our results were consistent with previous studies that a high frequency of 45,X/46,XY mosaicism or its variants were detected in patients with Y chromosome microdeletions. Different from previous studies, we found that 45,X/46,XY mosaicism in patients with Y chromosome microdeletions almost all exhibited a structurally abnormal Y chromosome. We conducted a retrospective study of infertile men with nonobstructive azoospermia or oligozoospermia from male outpatients of the Women's Hospital of Zhejiang University between January 1st 2011 and December 31st 2019. A total of 6,545 cases of infertility men were recruited in the study. The patients’ ages ranged from 17 to 63 (mean±SD, 31.45±5.29) years, and infertility lasted from 2 to 21 years old. Karyotype analysis and Y chromosome microdeletions were performed in all patients. This study was approved by the Scientic Research Ethics Committee of the Women's Hospital of Zhejiang University. This was a retrospective study of the clinical database with no intervention and no informed consent was required. All the methods used in the study followed the current approved guidelines.


Background
Chromosomal abnormalities and Y chromosome microdeletions are considered to be the two more common genetic causes of spermatogenic failure. The mosaic 45,X/46,XY karyotype is a common sex chromosomal abnormality associated with male infertility. Structural abnormalities of Y chromosome are estimated to affect 10-20% of men with non-obstructive azoospermic infertility [1], which are commonly described in associated with the occurrence of a 45,X/46,XY chromosomal mosaicism due to a probable mitotic instability of the abnormal chromosome [2]. A previous report showed that more than half of the patients with Y chromosome structural abnormalities might present with 45,X mosaic cell lines [3]. Y chromosome microdeletions are associated with severe spermatogenic failure and represent the most frequent molecular genetic cause of azoospermia and severe oligozoospermia [4]. Azoospermia Factor (AZF) region is responsible for the Y chromosome microdeletions. This region located on the long arm of Y (Yq11.23) and was mapped to three non-overlapping regions described as AZFa, AZFb, and AZFc [5]. The frequency of Y chromosome microdeletions was reported to be in the range of 3-55% according to previous studies with an average frequency of 7% [6].
In recent years, evidences of an association between Y chromosome microdeletions and the formation of 45,X/46,XY mosaicism had been investigated, and found that 45,X/46,XY mosaicism to be particularly common in patients with Y chromosome microdeletions. The aim of our study was to investigate the presence of 45,X/46,XY mosaicism carryiny a structurally abnormal Y chromosome in patients with Y chromosome microdeletions. Here, we retrospectively reviewed a single center experience over a 8-year period (2012-2019) at a university teaching hospital in southern China. Our study included 19 clinically well-characterized mosaic patients whose karyotype consisted of a 45,X cell line and a second cell line containing a normal or an abnormal Y chromosome.

Subjects
We conducted a retrospective study of infertile men with nonobstructive azoospermia or oligozoospermia from male outpatients of the Women's Hospital of Zhejiang University between January 1st 2011 and December 31st 2019. A total of 6,545 cases of infertility men were recruited in the study. The patients' ages ranged from 17 to 63 (mean±SD, 31.45±5.29) years, and infertility lasted from 2 to 21 years old. Karyotype analysis and Y chromosome microdeletions were performed in all patients. This study was approved by the Scienti c Research Ethics Committee of the Women's Hospital of Zhejiang University. This was a retrospective study of the clinical database with no intervention and no informed consent was required. All the methods used in the study followed the current approved guidelines.

Karyotype Analysis
Metaphase chromosomes with targeted 400-band level were performed as previously described [7]. Brie y, about 1 mL peripheral blood was inocubated aseptically into culture bottle containing of 5 mL lymphocyte culture solution in 5% CO 2 incubator at 37°C for 72 h. Then 20 μg ml -1 colchicine was added to the culture system half an hour before the termination of cell culture to arrest the chromosomes at metaphase. G-banding of metaphase chromosomes were obtained by hypotension, xation, trypsinization, Giemsa staining, and so forth. At least 30 metaphases were counted and 5 metaphases were analyzed for each sample by two certi ed physicians according to the International System for Human Cytogenetic Nomenclature guidelines (ISCN, 2016, 5th edition) [8].
Y Chromosome Microdeletions Y chromosome microdeletions were performed by multiplex PCR ampli cation as previously described [7]. Six speci c sequence-tagged sites (STS) that recommended by the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMGQ) were used for the detection of Y chromosome microdeletions, including sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255 for AZFc.
The Human Y Chromosome Microdeletion Gene Detection Kit was used for the detection of Y chromosome microdeletion. Multiplex PCR ampli cation was performed on the LightCycler 480 II thermocycler. PCR was performed with the following thermal cycling conditions: 95ºC for 3 min, then 10 cycles at 95ºC for 15 s, 63ºC for 20 s, 72ºC for 20 s, followed by 30 cycles at 95ºC for 15 s, 63ºC for 32 s, 72ºC for 20 s, and a nal extension at 72ºC for 10 min. An STS marker was considered to be deleted only after at least two failed PCR ampli cation attempts with single primer pairs. of 45,X/46,XY mosaicism in patients with Y chromosome microdeletions exhibited a structural Y chromosome abnormalities in 46,XY cell line; six with Yqh-, ve with del(Y) (q12), and one with Y ≤ 21. All patients (100.00%, 11/11) with AZFb + c microdeletions and one of two patients (50%, 1/2) with AZFc microdeletions exhibited a structurally abnormal Y chromosome. Moreover, a totla of 315 patients with AZF microdeletion were identi ed in this study. The pattern/number and characteristic of the patients showing these deletions were shown in Table 2. Only the deletions of AZFc region and AZFb + c region invovled 45,X/46,XY mosaicism, including eleven of 60 (18.33%, 11/60) men with the AZFb + c deletion and two of 208 (0.96%, 2/208) men with the AZFc deletion. Table 1 The results of AZF microdeletions in men with 45,X/46,XY mosaicism or its variants reported in the literature.

Discussion
The prevalence of 45,X/46,XY mosaicisms or its variants in this male infertility study was 0.29% (19/6545) (approximately 29/10,000), which was consistent with previous studies (0.27%) [9]. However, this is much higher than its incidence (1.5/10,000) in newborns [10], indicating that 45,X/46,XY mosaicisms are common chromosomal aberrations associated with male infertility. In this study, we analysed 6545 infertile men and found that 68.42% (13/19)  University. This was a retrospective study of the clinical database with no intervention and no informed consent was required.

Consent for publication
Not applicable Availability of data and materials The data supporting the conclusions of this article is included within the article.

Competing interests
The author declares that there are no competing interests.

Funding
None declared.