Clinical, cytogenomic, and molecular characterization of isodicentric Y-chromosome and prediction of testicular sperm retrieval outcomes in azoospermic and severe oligozoospermic infertile men

Abstract

Purpose

Sex chromosome abnormalities are associated with male infertility. The aim of this study was to characterize the clinical, cytogenetic, and molecular findings of 12 infertile men with isodicentric Y-chromosome [idic(Y)] abnormalities diagnosed over a period of 13 years.

Materials and methods

Chromosomal analyses of peripheral blood samples were done using standard procedures. Fluorescence in situ hybridization (FISH) analysis was performed on metaphase spreads of the patients. Multiplex polymerase chain reaction (PCR) using several sequence-tagged site (STS) primer sets within the long arm of Y-chromosome was used to detect AZF deletions.The breakpoints and copy number variations (CNV) were identified by array comparative genomic hybridization analysis (aCGH) analysis.The short-stature homeobox (SHOX) gene deletions were verified using multiplex ligation-dependent probe amplification (MLPA) analysis.

Results

Twelve infertile men were diagnosed cytogenetically with idic(Y). The karyotypes of two of the patients were non-mosaic, and the remaining karyotypes showed various degrees of mosaicism. SHOX gene deletion was found in two of the four patients with short stature, and the remaining two patients had shown a 45,X dominant cell line (33.3%). The most common breakpoints for idic(Yq) and idic(Yp) were found to be in Yq11.222 and Yp11.32, respectively. Semen analysis of ten patients (83.3%) demonstrated azoospermia, and the remaining two patients (16.7%) showed severe oligoasthenoteratozoospermia (OAT). In total, 33% (4/12) of idic(Y) patients with or without microsurgical testicular sperm extraction (microTESE) had sperm retrieval.

Conclusions

Twelve patients with idic(Y) and different breakpoints of Y-chromosome were characterized using multiple detection strategies. Sperm retrieval outcomes of patients either with idic(Yp) or idic(Yq) showed the possibility to find sperm by microTESE.

Introduction

Isodicentric Y-chromosomes [idic(Y)] are the most common structural rearrangements of Y-chromosome [1, 2]. Structural chromosomal rearrangements are relatively common involving one or more sex and/or autosomal chromosomes, which result from the breakage of the chromosome arm and then the exchange of strands, followed by the reformation in abnormal recombination [3]. The location of breakage and fusion of the short arm of the Y-chromosome (Yp) and the long arm of the Y-chromosome (Yq) is variable, generating either a dicentricYp or Yq with deletions or duplications [4]. Most of the affected patients are reported to bear a mosaic karyotype, especially with a 45,X cell line [5]. The phenotype of individuals with mosaic idic(Y) chromosome varies greatly from infertile males and females with or without Turner syndrome to individuals with ambiguous genitalia and mixed gonadal dysgenesis depending on the percentage of mosaic cells [6].

In the present study, 12 infertile male patients diagnosed with idic(Y) using different screening approaches including cytogenetic analyses such as karyotype analysis and fluorescence in situ hybridization (FISH), molecular techniques such as multiplex polymerase chain reaction (PCR), and array comparative genomic hybridization (aCGH) verified with multiplex ligation-dependent probe amplification (MLPA) were presented. Therefore, we aimed a better understanding of phenotype-genotype associations in phenotypically normal infertile men with isodicentric Y-chromosomes that are diagnosed with various screening techniques.

Materials and methods

Patients

One-thousand three-hundred infertile men with severe male factor consulted on standard cytogenetic and Y-chromosomal micro deletion analysis, between 2004 and 2017 from the Urology Department of  the Medical Faculty, Ondokuz Mayis University, were retrospectively reviewed, and 12 infertile men with idic(Y) were selected, and then, a particular genetic characterization of these patients was performed. All patients underwent a detailed physical examination, evaluation of hormone profiles, and at least two consecutive semen analyses. The medical and family history of all patients was evaluated. Testicular volumes of these patients were obtained by testicular ultrasound and calculated using the formula [length (L) × width (W) × height (H) × 0.52 with the dimensions]. Serum follicular-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and total testosterone (TT) levels were routinely measured in all patients. The clinical data of these patients with idic(Y) were listed in Table 1. Men with non‐obstructive azoospermia (NOA) underwent testicular sperm extraction (microTESE) under general anesthesia.

Table 1.

Clinical and laboratory findings of infertile with idic(Y)

Clinical findings	Idic(Yp) cases					Idic(Yq) cases
	Case 1	Case 2	Case 3	Case 4	Case 5	Case 6	Case 7	Case 8	Case 9	Case 10	Case 11	Case 12
Diagnosis age (yrs)	29	27	31	28	27	26	36	29	46	30	40	36
Height (cm)/SD	160/ − 2.7	184/1.7	168/ − 1.2	158/ − 3.1	190/2.8	175/0.0	168/ − 1.2	160/ − 2.7	176/0.2	178/0.6	151/ − 4.4	166/ − 1.6
Body weight (kg)	69	115	76	80	120	85	65	50	87	72	52	110
BMI (kg/m2)	26.9	33.9	26.9	32	33.2	27.8	26.0	19.5	28	22.7	NA	NA
Gynecomastia	No	Yes	No	No	No	No	Yes	No	No	No	No	No
Onset of puberty (yrs)	14	14	14	15	13	14	16	13	13	15	14	13
Testicular volume (mL) (R/L)	10/8	10/10	16/14	12/non-palpable	22/22	18/18	6/6	15/12	24/24	25/23	10/6	10/10
FSH (mU/mL) (1.5–12.4)	9.29	15.77	16.61	17.8	2.53	13.19	26.86	25.77	16.3	3.84	49.4	16.0
LH (mIU/mL) (1.7–8.6)	10.99	13.57	7.37	7.82	3.19	8.36	14.12	9.75	9.8	3.38	17.2	18.0
Prolactin (ng/mL) (2.0–18.0)	7.87	18.9	14.27	16.52	4.49	12.83	14.1	10.96	24.3	7.96	24.45
TT (ng/mL) (2.8–8.0)	4.41	2.01	2.22	2.74	5.63	2.86	2.09	3.93	3.87	5.98	6.7	1.4
E2 (pg/mL) (7.6–43.0)	42.62	20.4	22.19	29.82	21	22.67	27.79	NA	21.6	18.46	33.6	37.6

BMI, body mass index; E2, estradiol; FSH, follicle-stimulating hormone; L, left; LH, luteinizing hormone; NA, not available; R, right; SD, standard deviation; SDS, standard deviation score, TT, total testosterone; yrs, years

Ethics statement

The study protocol and the informed consent were approved by the institutional review board of Ondokuz Mayis University (Reg. No. 58–2007), and an informed written consent form was signed by all volunteer patients.

Semen analysis

Semen samples of the patients were collected by masturbation after 3–5 days of sexual abstinence into sterile containers. All semen samples were evaluated for sperm concentration, motility, morphology, pH, and volume according to the World Health Organization (WHO) 2010 guideline [7]. Two semen analyses were done for each patient; centrifugation and pellet analysis were performed according to best practice statements and previous guidelines. Azoospermia was diagnosed based on the absence of spermatozoa after at least two semen samples received more than 2 weeks after the first one [8]. The diagnosis of NOA was based on the finding of no spermatozoa in semen samples with small testes volumes and/or elevated serum FSH and previous testicular histopathology (if available).

Lymphocytes culture and karyotyping

Cytogenetic evaluations were done as part of the routine evaluation in patients with a sperm count less than 5 × 10⁶/mL. Peripheral blood lymphocytes of all patients were cultured and harvested using the methotrexate and thymidine synchronization method based on the routine protocols [9, 10] and karyotyped using CytoVision software (version 3.93; Applied Imaging), and at least 50 metaphases were assessed with a 550–700 band resolution following a standard procedure. The karyotypes were reported according to the International System for Human Cytogenetic Nomenclature 2016 (ISCN2016).

FISH analysis

FISH analysis was performed on metaphase chromosomes and in situ cell of patients with chromosome abnormalities using centromeric (CEPX, CEPY) and locus-specific (SRY) probes to assess the mosaicism statuses of patients according to the protocol recommended by the manufacturer (Cytocell, Cambridge, UK or Vysis, IL, USA). Cell line 46,X,idic(Yp/q) (single signal for CEPX, double signal for CEPY or SRY), cell line 46,XY (single signal for CEPX and single signal for CEPY or SRY), or cell line 45,X (single signal for CEPX and no signal for CEPY or SRY) were identified in patients. In addition, a dominant cell line was defined in mosaic patients. At least 100 cells were evaluated per case of suspected mosaicism to rule out 3% mosaicism with a 95% confidence level.

DNA extraction

A salting-out method [11] or automated QIAcube systems (QIAGEN, Hilden, Germany) [12] were used for genomic DNA isolation from peripheral venous blood samples of patients.

Y-chromosome microdeletion analysis

All patients were screened for the AZF region microdeletions according to the guideline of the European Academy of Andrology (EAA) and the European Molecular Genetics Quality Network (EMQN) using a multiplex PCR. sY84 and sY86 (AZFa), sY127 and sY134 (AZFb), sY254 and sY255 (AZFc), and SRY and AMXY (internal controls) markers were analyzed based on EAA/EMQN guidelines. Fertile female and male DNA samples were used as controls, and negative controls were also included in each PCR run [10, 13].

aCGH and MLPA

To clarify the break points and copy number variations (CNV) on Y-chromosome, aCGH analysis was performed for 7 patients with either non-mosaic (case 10) or dominant cell line idic(Y) patients (case 7, case 1, case 3, case 9, case 4, and case 12) using a 60 K oligonucleotide microarray (Agilent Technologies) as recommended protocol. Human male genomic DNA with 46,XY karyotype was used as a reference for the aCGH analysis. Cy3 and Cy5 labelling, washing, and scanning steps were done following a standard protocol. Data extraction and data analysis were carried out using Agilent Feature Extraction Software and Agilent CytoGenomics software (v.2.0.6.0; Agilent Technologies, USA), respectively. At least three consecutive probes set in a region and an abnormal log2 ratio were used to determine the imbalances. Gene annotations and the probe sequences were based on GRCh37/hg19 assembly [9].

MLPA analysis was done to verify the presence and copy number of the short-stature homeobox (SHOX) gene in 7 patients with either non-mosaic (case 10) or idic(Y) dominant (case 7, case 1, case 3, case 9, case 4, and case 12) cell line. Commercially available Salsa MLPA kit PO18-E1 SHOX (MRC Holland, Amsterdam, Netherlands) was used, and the results were analyzed by GeneMapper software version 4.0 (Applied Biosystems) and MRC Coffalyser MLPA-DAT software (MRC Holland, Amsterdam, Netherlands) following standard procedure and reference values. A reduction (one copy number) or increase (three copies numbers) of 40% or more in a peak area compared to normal/positive controls (two copy numbers for male 46,XY) was evaluated as an aberrant amplification [9].

aCGH and MLPA analyses did not apply to DNA samples of five patients since the DNA sample of one of a patient with non-mosaic idic(Yq) (case 5) was inadequate. The remaining 4 patients three of whom were having karyotype with 45,X (case 6, case 8, and case 11) and one with 46,XY (case 2) as a dominant cell line. Therefore, aCGH and MLPA assays have not been performed due to the limited ability of aCGH and MLPA in detecting and evaluating low-level mosaicisms.

Results

Detailed clinical, laboratory, and genetic findings of all patients were given in Table 1.

Clinical findings

Twelve infertile men were diagnosed with idic(Y) among 1300 infertile male patients with severe oligozoospermia and azoospermia. The mean age ± standard deviation (SD) was 32.0 ± 6.1 years, and the mean height ± SDS was 169.5 ± 11.4 cm. The mean maternal age ± SD was 29 ± 11.16 (17–49). Height standard deviation scores were calculated by the formula (height observed − mean height reference) ÷ (SD reference). Short stature was defined as a height of ≤  − 2 SDs compared to the average height of the corresponding gender. Therefore, four patients with a height of less than 164.1 have short stature (Table 1). The average height for Turkish men is 174.3 ± 4.9 cm [14].

The rearrangements were not correlated with advanced maternal age. Short stature is defined as a height of ≤  − 2 SDs compared to the average height at the corresponding gender. Therefore, four patients with a height of less than 164.1 have short stature (Table 1). All cases demonstrated a normal male phenotype with normal secondary sex characteristics, external genitalia, scrotum, libido, and erection. Five of the twelve patients (41.7%) had small testis. Four patients (case 1, case 8, case 4, and case 11) (33.3%) had short stature compared to mean Turkish men’s height (174.3 ± 4.9 cm) [14]. SHOX gene deletion was found in two (case 1 and case 4) of the four patients (50%) with short stature, and the remaining two patients (cases 8 and case 11) had shown a 45,X dominant cell line. Two of twelve patients (16.7%) showed gynecomastia. The onset of puberty was within normal ranges in 11 of the patients; however, one of the patients revealed the late onset of adulthood. Mean FSH and LH levels of the patients were high (17.7 ± 12.3 mU/mL and 10.2 ± 4.7 mIU/mL, respectively). Furthermore, the mean TT and estradiol levels of patients were normal (3.6 ± 1.7 ng/mL and 27.0 ± 7.9 pg/mL, respectively). Semen analysis of ten patients (83.3%) demonstrated azoospermia, and the remaining two patients (case 5 and case 10) (16.7%) showed oligoasthenoteratozoospermia (OAT) with sperm count (200,000/mL and 2 million/mL). Histopathological findings of testicular biopsies taken from patients showed a range of histologic changes including incomplete spermatocytic arrest, Sertoli cell-only (SCO), incomplete spermatocytic arrest, and partial atrophy and germ cell aplasia. SCOS was observed in two patients with idic(Yq) with a quite different cell line compositions and a similarity in regard to AZFb and AZFc deletions and also in one patient with dominant mosaic idic(Yp) cell line and without AZF deletions. These data show that the genetic etiology shows variation in patients with idic(Y). FSH and LH serum levels showed variation among patients and were higher in patients with a higher percentage of 45,X cell line. Total testosterone and estradiol levels of all patients were normal. Prolactin level was high only in one patient with mosaic cell lines and AZF deletion.

Karyotype and FISH analysis

The cytogenetic analysis of cultured blood lymphocytes showed non-mosaic idic(Y) or various degrees of mosaicism in all patients (Table 2). The karyotype of two patients (16.7%) was non-mosaic, and the remaining patients’ karyotypes (83.3%) bearded two or three mosaic cell lines. The dominant cell lines were idic(Y) (n = 6; 60.0%), 45,X (n = 3; 30%), and 46,XY (n = 1; 10%) in patients presenting mosaicism. FISH analysis demonstrated double SRY signals, SRY loci, on the same chromosome in the ten of 12 patients with idic(Y). The karyotype and FISH images of the case 12 with 46,X,idic(Y)(q11.2) karyotype have been given in Fig. 1. Figure 2 shows FISH images of patients idic(Yq) and idic(Yp). The remaining two patients with idic(Yp) demonstrated a condensed single SRY hybridization signal (likely close double signal), SRY locus, and a double CEPY signal (Table 2). Structural rearrangements of Y-chromosomes in the present study are de novo. None of our patients has a family history of infertility. Paternal karyotype of one of the patients was found to be normal. The karyotypes of other parents could not be obtained.

Table 2.

Genetic findings of patients with Idic(Y)

Clinical findings	Idic(Yp) cases				Idic(Yq) cases
	Case 1	Case 2	Case 3	Case 4	Case 5		Case 6	Case 7	Case 8	Case 9	Case 10	Case 11	Case 12
Karyotype	45,X (4%) 46,X,idic(Yp11.3) (96%) dn	45,X (10%) 46,X,idic(Yp11.3) (18%) 46,XY (72%) dn	45,X (4%) 46,X,idic(Yp11.3) (96%)	45,X (6%) 46,X(Yp11.3) (94%) dn	46,X,idic(Yq12) (100%)	45,X (52%) 46,X,idic(Yq11.2) (48%)		45,X (45%) 46,X,idic(Yq11.2) (55%)	45,X (70%) 46,XY (20%) 46,X,idic(Yq11.2)(10%)	45,X (20%) 46,XY (17%) 46,X,idic(Yq11.2) (63%)	46,X,idic(Yq11.2) (100%)	45,X (46%) 46,XY (27%) 46,X,idic(Yq11.2) (27%)	45,X (6%) 46,XY (3%) 46,X,idic(Yq11.2) (91%)
FISH	SRY*0/CEPX*1 [7%], CEPY*2/SRY*1/CEPX*1[93%]	SRY*0/CEPX*1 [9%] SRY*2/CEPX*1[12%], SRY*1/CEPX*1 [79%]	SRY*0/CEPX*1[4%], SRY*2/CEPX*1[96%]	SRY*0/CEPX*1[18%], CEPY*2/SRY*1/CEPX*1[82%]	SRY*2/CEPX*1 [100%]	SRY*0/CEPX*1[55%], SRY*2/CEPX*1 [45%]		SRY*0/CEPX*1[45%], SRY*2/CEPX*1 [55%]	SRY*0/CEPX*1 [70%], SRY*2/CEPX*1 [10%], SRY*1/CEPX*1 [20%]	SRY*0/CEPX*1 [18%], SRY*1/CEPX*1 [18%], SRY*2/CEPX*1 [64%]	SRY*2/CEPX*1[100%]	SRY*2/CEPX*1 [18%], SRY*1/CEPX*1 [13%], SRY*0/CEPX*1 [79%]	SRY*0/CEPX*1 [15%], SRY*1/CEPX*1 [15%], SRY*2/CEPX*1 [70%]
Dominant clone	Idic(Yp) dominant	46,XY dominant	Idic(Yp) dominant	Idic(Yp) dominant	Non-mosaic Idic (Yq)	45,X dominant		Idic(Yq) dominant	45,X dominant	Idic(Yq) dominant	Non-mosaic Idic (Yq)	45,X dominant	Idic (Yq) dominant
AZF regions	No deletion	No deletion	No deletion	No deletion	No deletion	AZFb and AZFc		AZFb and AZFc	AZFb and AZFc	Incomplete AZFb and complet AZFc	No deletion	Only AZFc	AZFb and AZFc
SHOX MLPA	SHOX 1 copy (deletion)	-	SHOX 3 copy (duplication)	SHOX 1 copy (deletion)	NA	-		SHOX 3 copy (duplication)	-	SHOX 3 copy (duplication)	SHOX 3 copy (duplication)	-	SHOX 3 copy (duplication)
Array-CGH (breakpoint)	724 Kb deletion Ypter-Yp11.32 (0–724,548 bp) –––––––- 58.5 Mb Duplication Yp11.32-Yqter (767,992–59,326,886 bp)	-	235 Kb deletion Ypter-Yp11.32 (0–246,520 bp) –––––––- 59.1 Mb Duplication Yp11.32-Yqter (246,520–59,326,886 bp)	2.5 Mb deletion Ypter-Yp11.32 (0–2,544,954 bp) ––––– 56.6 Mb Duplication Yp11.32-Yqter (2,635,220–59,280,569 bp)	N/A	-		19.5 Mb duplication Ypter-Yq11.222 (0–19,538,915 bp) ––––––––- 38.5 Mb Deletion Yq11.2-Yqter (20,826,207) 59,335,913 bp)	-	19.5 Mb duplication Ypter-Yq11.222 (0–19,538,915 bp) ––––––––- 38.5 Mb Deletion Yq11.222-Yqter (20,826,207 −) 59,311,250 bp)	24.5 Mb duplication Ypter-Yq11.23 (0–24,515,036 bp) ––––––- 30.8 Mb Deletion Yq11.23-Yqter (28,579,260–59,311,250 bp)	-	19.5 Mb duplication Ypter-Yq11.222 (0–19,538,915 bp) ––––––––- 38.5 Mb Deletion Yq11.222-Yqter (20,826,207 −)59,335,913 bp)
Final breakpoint (region)	Yp11.32 (PAR1)	Yp11.3	Yp11.32 (PAR1)	Yp11.32 (PAR1)	Yq11.23-q12	Yq11.2		Yq11.222 (P4-P5)	Yq11.2	Yq11.222 (P4-P5)	Yq11.23 (P1)	Yq11.223	Yq11.222 (P4-P5)
Semen volume (mL)	1.6	1.6	3.4	2.5	2.4	6		2.3	2.6	3.1	3	3.6	4.2
Sperm count	Azoospermia	Azoospermia	Azoospermia	Azoospermia	OAT	Azoospermia		Azoospermia	Azoospermia	Azoospermia	OAT	Azoospermia	Azoospermia
microTESE	Yes	Yes	Yes	NA	Yes	NA		Yes	Yes	Yes	NA	Yes	NA
Sperm retrieval	Positive	Negative	Negative	NA	Positive	NA		Negative	Negative	Positive	Positive	Negative	NA
pathological findings	Incomplete spermatocytic arrest + partial atrophy	NA	SCOS	NA	Incomplete spermatocytic arrest	NA		NA	SCOS	SCOS	NA	NA	NA

AZF, azoospermia factor; CGH, comparative genomic hybridization; NA, not available; PAR, pseudoautosomal region; SCOS, Sertoli cell-only syndrome; Yq, Y-chromosome long arm; Yp, chromosome short arm

Fig. 1.

a Karyotype of the case 12 showing 46,X,idic(Y)(q11.2). b FISH using probes for SRY locus (red) and X centromere (DXZ1; green) show two red signals on the idic(Y) and one green signal on the chromosome X

Fig. 2.

FISH images of patients with isodicentric Y chromosome using probes for SRY locus (red) and X centromere (DXZ1; green) show two red signals on the idic(Y) and one green signal on the chromosome X

AZF microdeletion analysis

Six of eight patients with idic(Yq) were observed to have AZF deletion in various length, especially in both AZFb and AZFc or only AZFc. When these results were evaluated together with karyotype analysis, the data showed the chromosomal breakpoints were located in Yq11.221-q11.223 region. The remaining two patients with idic(Yq) display no deletion in the AZF regions. As expected, no AZF region deletion was detected in 4 patients with mosaic idic(Yp), and two patients with non-mosaic idic(Yq) presented OAT (Table 2).

aCGH and MLPA analysis

The findings of aCGH analyses in seven patients were presented in Table 2. The breakpoints of 3/4 (75%) patients with idic(Yq) was defined in the long arm of Y-chromosome in band Yq11.222. The distal region of the breakpoint (q11.222-qter) was deleted, while the proximal region (pter-q11.222) was duplicated. In the remaining one patient, the breakpoint was detected at Yq11.23 (Fig. 3). The breakpoint was defined at Yp11.32 in 3/3 (100%) idic(Yp) patients (Fig. 3). The distal part of this region (p11.32-pter) including SHOX gene was deleted, while the proximal (qter-p11.32) was duplicated in two patients. In the remaining one patient, the proximal region of this breakpoint (p11.32-qter) was duplicated, while the distal region (p11.32-pter) was deleted except for the SHOX gene. After the evaluation of all findings, the final breakpoints were shown in Table 2. The most common breakpoints for idic(Yq) and idic(Yp) were found to be in Yq11.222 or Yp11.32, respectively. Figure 4 shows a schematic drawing of possible mechanisms for the origin of the formation of idic(Yq) and idic(Yp). Figure 5 shows the array CGH data from the all patients analyzed with idic(Yq) and idic(Yp) with their breakpoints. MLPA SHOX analyses were carried out for three patients with idic(Yp) and four patients with idic(Yq). Two of three patients with idic(Yp) showed SHOX deletion, and the remaining one patient revealed SHOX duplication. The discrepancy may be attributed to the presence of different breakpoints on Yp of the patient with idic(Yp) (Table 2). Indeed, both patients with SHOX haploinsufficiency had short stature. Additionally, four patients with idic(Yq) had SHOX duplication. The height of five patients with idic(Y) with duplicated SHOX was either normal or higher than normal.

Fig. 3.

a Representation of 46,X,idic(Yq11.222) (case 12) breakpoint region by chromosome and array-CGH analysis. b Representation of 46,X,idic(Yp11.32) (case 10) breakpoint region by chromosome and array-CGH analysis

Fig. 4.

Schematic representation of proposed possible mechanisms of origin of the formation of idic(Yq) and idic(Yp)

Fig. 5.

Array CGH data from the patients with isodicentric Y chromosome

Fertility outcomes

Two patients with non-mosaic idic(Yq) presented OAT did not show any cell line mosaicism with 45,X or AZF deletion, and none of these two couples achieves pregnancy. All six patients with mosaic idic(Yq) were azoospermic with complete/partial deletions in AZFb and/or AZFc regions. In the mosaic idic(Yq) cases, mature spermatozoa have been obtained in 1/4 (25%) of patients who underwent microTESE. Five sperms have been collected from case 10 than two oocytes were fertilized by ICSI. Embryo transfer was not performed due to poor embryo quality. All four patients with mosaic idic(Yp) were azoospermic, and no deletion was observed in the AZFa, AZFb, or AZFc regions. In the mosaic idic(Yp) group, mature spermatozoa were obtained in 1/3 of patients (33%) who underwent microTESE. Four sperms have been obtained from case 2, three oocytes II were fertilized by ICSI, and two embryos were transferred with no pregnancy achievement. One of these two patients with a breakpoint at Yp11.32 pseudoautosomal region 1 (PAR1) had no AZF microdeletion, and the other one had incomplete AZFb and complete AZFc deletion with breakpoint Yq11.222 (P4-P5). Finally, sperm retrieval rate was found to be two out of seven (28%), and none of these two couples achieves pregnancy in patients idic(Y) who underwent microTESE.

Discussion

The clinical manifestations of patients with idic(Y) range from a classical Turner phenotype, through mixed gonadal dysgenesis, ambiguous external genitalia to phenotypically normal infertile males [15]. The present study includes a subgroup of infertile men with idic(Yp) or idic(Yq), non-mosaic karyotype or in various ranges of mosaicism. Eight patients were found to have idic(Yq) karyotypes, and four patients were bearing idic(Yp) in their karyotypes. All patients demonstrated normal secondary sex characteristics, external genitalia, scrotum, libido, and erection. The mean estrogen and total testosterone levels of patients were normal; however, mean FSH and LH levels were significantly high than normal [16, 17]. Bettio et al. (2006) revealed that the percentage of cells with the SRY gene in the gonads specify the phenotypic sex of a subject. Therefore, this finding indicates that a cytogenetic study only be conducted on the blood cells is not informative regarding SRY gene percentage and mosaicism [18].

aCGH results showed a deletion at distal region (q11.222-qter) and a duplication at the proximal region (pter-q11.222) in various lengths in three of four patients with idic(Yq) karyotypes. In the fourth patient with idic(Yq), the breakpoint was detected at Yq11.23 and caused deletion at the distal part of Yq (Yq12) roughly in 40 Mb length [19]. The breakpoint involved in this rearrangement leads to the loss of PAR2 region which predominantly contains two repetitive satellite DNA families, named DYZ1 and DYZ2, and fundamentally consists of constitutive heterochromatin [20]. The lower number of copies of DYZ1 was found to be in infertile men, and epigenetic regulation of Y-chromosome heterochromatin is suggested as a possible cause of infertility by acting indirectly as resource-rich chromatin-modifying proteins [21]. Despite this region is known to exhibit a much lower frequency in pairing and recombination than PAR1, the loss of PAR2 sequence might lead to impaired semen parameters in these patients. These sequences are necessary for a correct pairing of Y-chromosome with the X-chromosome PAR regions during the prophase of spermatocyte meiosis. Therefore, the failure of meiosis may be a result of impaired spermatogenesis [22]. Maturation arrest is observed in one of these patients sample leads to OAT. SPRY3, SYBL1, IL9R, and CXYorf1 genes are identified to be located on PAR2. SYBL1 is a highly conserved gene coding a member of vehicle-associated membrane protein (VAMP)/synaptobrevins which is implicated in cellular exocytosis [23]. VAMP/synaptobrevins are involved in membrane fusion during the acrosome reaction and discriminators of fertility potential of human sperm [24].

aCGH data of three patients with idic(Yp) demonstrated a deletion at distal Ypter-Yp11.32 and a duplication at Yp11.32-Yqter in various lengths. Deletion of the distal part of the short arm of Y-chromosome is suggested to lead unpairing between X and idic(Y) chromosomes during meiosis [25]. Patsalis et al. (2005) reported that there is a strong association between Y-chromosome deletions and sex chromosome mosaicism or complete loss of the Y-chromosome (45,X/46,XY) (5). Most karyotypes of patients were observed with 45,X cell line mosaicism because of the mitotic instability of idic(Y) chromosome and tendency to be lost during mitosis [5, 15, 17, 26–28]. Studies showed that a higher proportion of 45,X cell line in gonad is correlated with closer clinical characters to the Turner syndrome [29]. Nonetheless, short stature was also observed in two (case 8 and case 11) of three patients with 45,X dominant cell lines besides patients (case 1 and case 4) with SHOX deletion. Except short stature, mixed gonadal dysgenesis or other signs have not been found in these three patients. In the present study, infertile patients bearing AZF deletions showed a higher percentage of 45,X cell line in their somatic cells. The cause of short stature in mosaic 45,X/46,XY is still controversial, and the most probable cause is reported to be the haploinsufficiency of SHOX [30]. Aberrations in PAR1 on the short arm of rearranged Y-chromosome may result in SHOX gene haploinsufficiency and primarily lead to the short stature. Indeed, SHOX gene deletions were found in two patients with larger deletions at Ypter-Yp11.32, and these deletions were verified by MLPA. The clinical findings of patients with SHOX gene deletions were similar except the left testicle was not palpable in one of these patients. Alterations in SHOX are known to lead Leri-Weill dyschondrosteosis (LWD) characteristics such as cubitus valgus and short fourth metacarpals [18, 23]. However, both of these patients with complete SHOX deletion did not show characteristics of LWD. SHOX duplications were detected in 5/7 patients. These duplications have been described in individuals with tall, normal, or short stature [31]. The variations in the stature of individuals with the duplications could be explained reduced or ablated nature of these duplications. 5′ or 3′ regulatory sequences could be degraded depending on breakpoints and alter the SHOX gene expression [32]. Complete/incomplete AZFb and AZFc deletions were identified in three of seven patients. Complete deletions of AZFb regions leading to azoospermia due to SCO syndrome and AZFb region partial deletions are reported to be associated with residual sperm production [33]. Indeed, residual sperm production was found in a patient with incomplete AZFb and AZFc deletion. Pathological findings of these patients reveal SCO syndrome, and only in one of these patients sperm retrieval was positive; however, no pregnancy was observed in the patient. Indeed, genetic aberrations in SCO syndrome show high variation ranging from numerical and structural chromosomal abnormalities, AZF deletions, and single gene aberrations [34]. Incomplete spermatogenic arrest was observed in a patient with non-mosaic idic(Yq) and in a patient with idic(Yp) showing mosaicism with partial atrophy. Spermatocytic arrest is highly heterogeneous and may result from impaired meiosis due to chromosomal abnormalities [35]. In addition, AZFb deletions [36] and abnormalities in genes are essential for meiosis such as RBMY [37] and SYCP3 [38]. In our study, only two of the twelve patients had non-mosaic idic(Yq) karyotypes with the duplication at the same breakpoints of the proximal short arm and the entire duplication of long arm with preservation of AZF regions. These patients oligoasthenoteratozoospermia and based on their genetic rearrangement, a phenotypic expression resembling a 47,XYY male could be expected. Sperm could be obtained in 2/7 (28%) idic(Y) patients who underwent microTESE. In total, 33% (4/12) of idic(Y) patients with or without microTESE had sperm. Our findings are consistent with those described by those who found mature spermatozoa rate 0–55% [17] in azoospermic infertile men. The studies performing sperm retrieval in patients with non-mosaic idic(Yq) are limited only to four patients and show no success in retrieving of mature spermatozoa [39–41]. Although the mature spermatozoa were found in one of our patients with non-mosaic idic(Yq) embryo transfer could not be performed due to poor quality (case 10). Kalantari et al. (2014) detected aneuploidy of sex chromosomes with 22,-X or 22,-idic(Y) or 24,Xidic(Y) chromosomes (nullisomic haploid) in haploid seminal germ cells [17]. These paternal sex chromosome aneuploidies give rise to a low fertilization rate and higher levels of numerically aneuploid embryos.

Additionally, our study demonstrated that patients with a high percentage of 45,X cell line and AZF deletions did not show any sperm in microTESE. Finally, these findings show that one of the most critical predictors in the success of sperm retrieval of these patients is AZF deletion testing [42, 43].

The present study demonstrates a detailed evaluation of infertile men with idic(Y), including the clinical features, hormonal values, cytogenetic, Y-chromosome testing, and aCGH findings with verification of SHOX gene copy number with MLPA and fertility outcomes. The findings show that the pathologic etiology shows significant variation in patients with idic(Y) and the importance of Y-chromosome microdeletion test in the success of sperm retrieval. The main limitation of the study is the inclusion of only severe oligozoospermic and azoospermic infertile men with idic(Y). Therefore, these findings could not be applied to all patients with idic(Y). Detailed clinical, cytogenomic, and molecular characterization of twelve patients with idic(Y) is the strength, and sperm retrieval findings of these patients are the novelty of the study. On the other hand, the ability of aCGH and MLPA to detect and evaluate low-level mosaicism of idic(Y) is limited; therefore, it may not be meaningful to use these techniques in the detection of breakpoints, deletions, and duplications.

In conclusion, twelve patients with idic(Y) and different breakpoints were characterized using multiple detection strategies in the present study. Sperm retrieval outcomes of patients either with idic(Yp) or idic(Yq) showed the possibility to find sperm by microTESE. It is important to know that if the patient with idic(Y) is candidate for assisted reproduction techniques, the genetic composition of the patient should be interpreted accurately using two or more different techniques.

Author contribution

Conceptualization, UA and SG. Data curation, RA, ÜA, ÖSA, and EA. Formal analysis, ÜA, NH, ÖSA, and EA. Investigation, ÜA, NH, ÖSA, and EA. Methodology, ÜA. Resources, ÜA. Supervision, ÜA and SG. Validation, ÖSA and EA. RA was responsible for the diagnosis of patients. UA, SG, NH, EA, OSA, and RA contributed to manuscript revision, read, and approved the final version.

Data availability

The datasets generated during the study are available from the corresponding author on request.

Declarations

Ethics approval and consent to participate

The present study protocol was reviewed and approved by the institutional review board of the Ondokuz Mayis University Hospital (Reg. No. 58–2007). Informed consent was submitted by all subjects when they were enrolled.

Conflict of interest

The authors declare no competing interests.