A rare Robertsonian translocation rob(14;22) carrier with azoospermia, meiotic defects, and testicular sperm aneuploidy

Abstract

Male infertility is a serious problem in an increasing number of couples. We report an infertile man with non-obstructive azoospermia and karyotype 45,XY,rob(14;22). The immunofluorescence analysis of his testicular tissue using antibodies to SYCP1, SYCP3, HORMAD2, MLH1, and centromeres showed delayed synapsis of the chromosomes involved in the translocation, a varying extent of trivalent asynapsis and its association with sex chromosomes. The mean frequency of meiotic recombination per cell was within the range of normal values. Fluorescence in situ hybridization (FISH) with probes for chromosomes 14 and 22 revealed 5.83% of chromosomally abnormal testicular spermatozoa. FISH with probes for chromosomes X, Y, and 21 showed frequencies of disomic and diploid testicular spermatozoa increased when compared to ejaculated sperm of healthy donors, but comparable with published results for azoospermic patients. PGD by FISH for the translocation and aneuploidy of chromosomes X, Y, 13, 18, and 21 showed a normal chromosomal complement in one out of three analyzed embryos. A healthy carrier girl was born after the embryo transfer. This study shows the benefits of preimplantation genetic diagnosis in a case of a rare Robertsonian translocation carrier with azoospermia and a relatively low frequency of chromosomally unbalanced testicular spermatozoa.

• Aneuploidy
• azoospermia
• FISH
• meiosis
• PGD
• Robertsonian translocation
• testicular sperm

Introduction

Azoospermia is a serious cause of male infertility. Among genetic disorders contributing to azoospermia, deletions in the azoospermia factor (AZF) genes and cystic fibrosis transmembrane regulator (CFTR) mutations, as well as chromosomal abnormalities are frequently described [Shah et al. 2003]. In vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) of spermatozoa obtained by testicular sperm extraction (TESE) is a technique, which can help such couples achieve pregnancy. However, Robertsonian translocation carriers often show an increased frequency of spermatozoa aneuploid for the chromosomes involved in the translocation and for other chromosomes (interchromosomal effect) [Roux et al. 2005; Vozdova et al. 2013]. This represents a serious risk factor for a successful assisted reproduction treatment in such infertile couples. The frequency of chromosomally abnormal spermatozoa can be assessed by fluorescence in situ hybridization (FISH) and results of this sperm analysis have a predictive value for aneuploid embryo formation risk assessment [Escudero et al. 2003]. Preimplantation genetic diagnosis (PGD) for aneuploidy is recommended to increase the chance of delivering a healthy child after IVF using either ejaculated or surgically retrieved testicular spermatozoa from translocation carriers [Munné 2005]. The cause of the spermatogenetic impairment and sperm aneuploidy formation can be elucidated by immunofluorescent analysis of the testicular tissue using antibodies to proteins involved in bivalent formation (synaptonemal complex proteins 1 and 3, SYCP1, SYCP3), meiotic recombination (Mut-L homolog 1, MLH1) and inactivation of unsynapsed chromosomes (e.g., BRCA1, HORMAD2, γH2AX) [Barlow and Hultén 1998; Kogo et al. 2012]. In this study, we report the results of the immunofluorescent analysis of the first meiotic prophase, FISH analysis of the testicular sperm aneuploidy, and IVF-PGD for translocation and common aneuploidy in a couple where a man is an azoospermic carrier of a rare Robertsonian translocation rob(14;22).

Results

Meiotic synapsis

The ratio of leptotene, zygotene, and pachytene cells was 4.0:10.8:85.2%, based on scoring of 324 meiotic cells after immunofluorescent detection of SYCP1 and SYCP3 proteins. As much as 45% of the pachytene cells showed intensive SYCP1 and SYCP3 staining and aberrantly condensed, curly structure of the synaptonemal complexes (Figure 1).

Figure 1. Immunofluorescent analysis of spermatocytes. (A) Closed trivalent showing heterologous synapsis between the p-arms of the acrocentric chromosomes 14 and 22. (B) Open trivalent showing unsynapsed p-arms of acrocentric chromosomes 14 and 22 in the cis position. (C) An association between the trivalent and the sex chromosomes. (D) HORMAD2 staining of p-arms of acrocentric chromosomes 14 and 22 and on sex chromosomes. (E) Closed trivalent showing MLH1 foci. (F) A pachytene spermatocyte showing aberrant synaptonemal complex morphology compared with normal bivalents. SCP1: synaptonemal complex protein 1; SCP3: synaptonemal complex protein 3; cen: centromeres.

A total of 101, 107, and 50 well spread pachytene spermatocytes were analyzed in the SYCP1/SYCP3, MLH1/SYCP3, and HORMAD2/SYCP1 study, respectively. The normal acrocentric chromosomes 14 and 22 formed a trivalent with the fused chromosome in pachynema. Examples of trivalent configurations are shown in Figure 1. Full synapsis of the acrocentric chromosomes 14 and 22 with the fused chromosome and a single centromeric signal was observed in 29.8% of the trivalents and was more frequent in late pachytene. Heterosynapsis between the p-arms of acrocentric chromosomes 14 and 22 characterized by SYCP1 labelling was completed in 58.6% of such closed trivalents. Most of the trivalents (70.2%) showed asynapsis of various extent which involved mainly the chromosome 22. The asynaptic parts of the trivalents did not show any SYCP1 labelling and were decorated by the anti-HORMAD2 antibody (Figure 1). Breaks in the pericentromeric region of the derivative chromosome were found in 5.8% (12/208) of the pachytene cells.

The association of the trivalent with the sex chromosomes detected as intertwining of their unsynapsed chromosomal axes was observed in 30.1% (64/208) of the pachytene spermatocytes, mainly in late pachytene (Figure 1). Chromosome 22 was more often found associated (29.3%, 61/208 of the spermatocytes) than chromosome 14 (10.6%, 22/208). No heterosynapsis or associations involving other autosomes were observed. No univalent autosomes were observed and the partial asynapsis was rare (4%). The sex chromosomes did not achieve synapsis in 4.3% (9/208) of the pachytene spermatocytes.

Meiotic recombination

A total of 107 well spread pachytene spermatocytes were analyzed in the MLH1/SYCP3 study. The mean number of MLH1 foci was 46.3 per cell (SD ± 4.0, range 39–57). An autosomal bivalent lacking an MLH1 focus was observed in 4.7% (5/107) of the spermatocytes. An MLH1 focus in the pseudoautosomal region of the sex chromosomes was observed in 55.6% of the spermatocytes.

The two arms of the trivalents were recognizable by their lengths (Figure 1E). The mean number of MLH1 foci on the q-arm of chromosomes 14 and 22 was 1.4 and 1.0, respectively. Most of the trivalents showed two MLH1 foci on the part of the trivalent corresponding to chromosome 14 (64.8% of the trivalents) and a single MLH1 focus on the part corresponding to chromosome 22 (89.2%).

Testicular sperm aneuploidy

We scored 1,030 testicular spermatozoa after FISH with probes for chromosomes 14 and 22. The frequency of chromosomally abnormal spermatozoa was 5.83%. A total of 1,016 spermatozoa were relocated after a second round of FISH with probes for chromosomes X, Y, and 21. At least 5,000 spermatozoa were scored for each probe combination in each of the control donors. A significantly higher frequency of XY and YY disomy (p < 0.001) and of the diploidy due to the error in the second meiotic division (p < 0.001) was observed in testicular sperm of our patient than in the ejaculated sperm of control donors. However, there was no difference when compared with our previously published results in testicular sperm of non-obstructive azoospermic patients [Vozdova et al. 2012]. The results are summarized in Table 1.

Table 1. Sperm FISH results.

	Patient	Control donors (%)						Azoospermicmen*
Abnormality	(%)	C1	C2	C3	C4	C5	Mean ± SD	Mean ± SD (%)
Meiotic segregation study
14,14,22	1.55^a	0.04	0.08	0.10	0.06	0.08	0.07 ± 0.02
-,22	0.39^a	0.02	0.04	0.04	0.04	0.04	0.04 ± 0.01
14,22,22	2.14^a	0.04	0.06	0.10	0.04	0.08	0.06 ± 0.03
14,-	0.97^a	0.10	0.02	0.00	0.04	0.04	0.04 ± 0.04
Diploidy	0.78
Total Abnormal	5.83
Interchromosomal effect study
21,21	0.30	0.09	0.26	0.09	0.24	0.13	0.16 ± 0.08	0.32 ± 0.35
XX	0.20	0.03	0.06	0.01	0.03	0.08	0.04 ± 0.03	0.10 ± 0.16
YY	0.59^a	0.08	0.08	0.02	0.03	0.14	0.07 ± 0.05	0.57 ± 0.32
XY	0.49^a	0.03	0.07	0.14	0.21	0.05	0.10 ± 0.07	0.29 ± 0.31
MI diploidy	0.20	0.12	0.11	0.25	0.15	0.21	0.17 ± 0.06	0.33 ± 0.24
MII diploidy	0.59^a	0.09	0.12	0.15	0.11	0.06	0.10 ± 0.03	0.47 ± 0.54
Total diploidy	0.79^b	0.21	0.23	0.39	0.26	0.27	0.27 ± 0.07	0.80 ± 0.67

FISH: fluorescence in situ hybridization; *Vozdova et al. 2012; ^a,bsignificant difference vs. control donors (^ap < 0.001; ^bp < 0.01)

PGD

FISH with probes for chromosomes 14 and 22 showed two red and two green signals indicating a normal/balanced karyotype in embryo No.1. Embryo No. 2 showed ambiguous results due to extensive fragmentation and embryo No. 3 was unbalanced (monosomy of the chromosome 14). Further analysis of the blastocysts from embryo No. 1 with the MultiVysion PGT probe did not reveal any other chromosomal abnormality. This embryo was transferred on developmental day 5 at the stage of expanded blastocyst. Prenatal diagnosis at the 12th week of gestational age showed the balanced karyotype 45,XX,rob(14;22)(q10;q10). A healthy girl (3,280 g, 50 cm) was born in the 39th week of gestation.

Discussion

Infertility in Robertsonian translocation carriers is usually caused by meiotic errors leading to low sperm counts and increased sperm aneuploidy. A prerequisite for the completion of meiotic division and for the equal segregation of chromosomes to gametes is pairing and synapsis of homologous chromosomes and meiotic recombination between the non-sister chromatids during the first meiotic prophase. All meiotic stages were observed in the testicular tissue of the azoospermic translocation carrier, as well as spermatids and a limited number of spermatozoa. Synapsis of the trivalent chromosomes proceeded from the most distal parts of the chromosomes towards the centromeres and was delayed compared to normal bivalents. Chromosome 14 achieved full synapsis including the centromere more frequently than chromosome 22, which also showed more frequent association with the sex chromosomes. Such associations between the unsynapsed parts of the trivalent and the sex chromosomes were observed previously in Robertsonian translocation carriers in an even higher frequency [Navarro et al. 1991; Sciurano et al. 2012]. The frequency of cells with partially asynapsed bivalents (splits) of other chromosomes (4%) was comparable with the frequency found in normal men by Sun et al. [2007] and lower than published for men with severe oligospermia (17.6%) [Egozcue et al. 2005]. The unsynapsed parts of the trivalents displayed HORMAD2 staining similar to the staining of the sex chromosomes in our patient. HORMAD2 is a protein involved in the surveillance of unsynapsed regions by the recruitment of the ATR-kinase activity and formation of the phosphorylated histone γH2AX during meiotic sex chromosome inactivation (MSCI; [Kogo et al. 2012]). Our results show that HORMAD2 is involved in general meiotic silencing of unsynapsed chromatin (MSUC) in Robertsonian translocation carriers. The analysis of the ratio of the first meiotic prophase substages in our patient showed 85.2% of pachytene cells which is within the range published for normal men [Sun et al. 2007]. The pachytene cells exhibiting aberrant synaptonemal complex morphology could be technical artifacts, but they can also represent arrested pachytene spermatocytes heading to apoptosis. Manterola et al. [2009] reported in Robertsonian heterozygous mice that cells with trivalent synapsis defects and correct MSUC response are not eliminated by apoptosis in pachytene, but are recognized later, at metaphase I. Meiotic arrest and cell death mediated by both pachytene and spindle assembly checkpoints due to the chromosome rearrangement could contribute to the patient’s azoospermia.

The mean number of MLH1 foci per cell (46.3) as well as the percentage of spermatocytes with an autosomal bivalent lacking an MLH1 focus (4.7%) were comparable with the data published for normal men, but the mean number of MLH1 foci on the q-arm of chromosome 14 was lower [Sun et al. 2005; Sun et al. 2006a; 2006b; Sun et al. 2007], probably due to synaptic defects. In accordance with results in oligoasthenoteratospermic men [Codina-Pascual et al. 2005], the frequency of pachytene spermatocytes showing MLH1 focus in the pseudoautosomal region of the sex chromosomes was reduced in our azoospermic patient.

FISH with probes for chromosomes 14 and 22 revealed 5.83% of chromosomally abnormal testicular spermatozoa. This is within the range observed in ejaculated sperm of Robertsonian translocation carriers (3.4%–40%, reviewed by [Roux et al. 2005]), but significantly less than published by Moradkhani et al. [2006] in ejaculates of three rob(14;22) carriers (17.59–20.94%). The relatively low frequency of aneuploidy of chromosomes 14 and 22 in our patient was probably caused by a checkpoint-controlled meiotic arrest. In agreement with our meiotic study, which showed a higher frequency of chromosome 22 asynapsis compared to chromosome 14, we observed more spermatozoa to be aneuploid for chromosome 22 than for chromosome 14.

In translocation carriers, a controversial phenomenon of interchromosomal effect, i.e., increased levels of sperm aneuploidy of chromosomes not involved in the rearrangement, is often discussed [Anton et al. 2011; Vozdova et al. 2013]. The presence of meiotic associations between the trivalent and sex chromosomes and low recombination in the XY pair indicated an increased risk of formation of gametes with sex-chromosome aneuploidy. The use of the sperm-FISH analysis for chromosomes X, Y, and 21 revealed that the frequency of XY and YY disomy and diploidy originating in the second meiotic division were significantly increased in testicular spermatozoa of our patient compared to ejaculated sperm from control donors, but comparable with testicular spermatozoa of azoospermic men. A similar increase in testicular sperm aneuploidy rates was reported previously in non-obstructive azoospermic and oligoasthenoteratospermic men with normal karyotypes [Gianaroli et al. 2005; Vozdova et al. 2012]. Thus, the increased frequency of disomic and diploid spermatozoa in some translocation carriers is probably associated with general spermatogenetic defects and cannot be attributed to the interchromosomal effect. Accordingly, in our patient, increased rates of YY disomic and diploid spermatozoa originating in the second meiotic division indicate a complex disruption of the meiotic process. This can be explained by a combination of factors, including a compromised testicular environment due to varicocele and hormonal imbalance after surgical correction of congenital cryptorchidism. It is known that some patients with corrected cryptorchidism develop azoospermia even if orchidopexy is performed in early childhood [Hadziselimovic 2006; Hadziselimovic et al. 2007]. The presence of the Robertsonian translocation in our patient, who was born with cryptorchidism, is most probably coincidental, but meiotic abnormalities associated with the translocation can contribute to potentiation of the spermatogenic impairment leading to azoospermia.

High rates of chromosome abnormalities related to the translocation as well as common aneuploidies were reported in embryos from Robertsonian translocation carriers [Munne 2005; Gianaroli et al. 2002]). On one hand, a relatively low frequency of gametes unbalanced for the chromosomes involved in the translocation observed in our patient (5.1%) suggested a good prognosis for the infertility treatment. On the other hand, the use of testicular sperm with an increased frequency of aneuploidy for ICSI in a patient with azoospermia brings an additional risk of formation of chromosomally abnormal embryos [Platteau et al. 2004]. Assuming that sperm FISH is a prognostic tool [Sanchez-Castro et al. 2009], PGD by FISH was performed in our couple. Despite the low frequency of aneuploidy of chromosomes 14 and 22 in sperm, just one out of the three embryos analyzed showed normal/balanced FISH results for these chromosomes.

It is known, that fertilization by an unbalanced gamete giving rise to a monosomic or trisomic zygote can be followed by a post-zygotic correction of the aneuploidy by either loss (in trisomy) or duplication (in monosomy) of the chromosome involved in aneuploidy. Thus, carriers of Robertsonian translocations are at increased risk of formation of uniparental disomy (UPD) in the offspring [Ruggeri et al. 2004; Shaffer 2006; Yip 2014]. In the case of Robertsonian translocations involving chromosomes 14 and 15, uniparental disomy assessment can be recommended because UPD results in an abnormal phenotype due to the differential expression of paternal and maternal imprinted genes [Yip 2014]. However, the estimated 0.6–0.8% risk of UPD formation [Shaffer 2006] is relatively low and prenatal UPD testing is not routinely performed in our country, neither was it performed in this case. A healthy carrier girl was born showing normal prenatal and postnatal development.

Meiotic studies and sperm FISH provide useful information for genetic counselling and personalized risk assessment in infertile chromosome translocation carriers undergoing assisted reproduction. Our study showed that the frequency of aneuploidy of chromosomes 14 and 22 was lower in testicular sperm of our azoospermic patient than what was previously reported for ejaculated sperm of rob(14;22) carriers. However, it was shown that PGD is useful even to Robertsonian translocation carriers with an apparently low frequency of chromosomally abnormal spermatozoa.

Materials and Methods

Patient and control donors

A 28-year-old man and his 26-year-old wife were trying to conceive for 3 years. Cytogenetic analysis revealed a karyotype of 45,XY,rob(14;22)(q10;q10) in the patient and a normal karyotype 46,XX in his wife. Neither an AZF deletion, nor any of the thirty common CFTR mutations were found. The patient was evaluated by compiling a comprehensive history, physical examination, testicular size measurement, two semen analyses and hormonal parameters analysis. The patient reported surgical correction of bilateral cryptorchidism at the age of 4.5 y. Physical examination showed small testicles (3.8 cm³ on the left and 9.2 cm³ on the right) and Grade III varicocele on the left side. The hormone evaluation revealed a luteinizing hormone level of 7.9 IU/L (normal range 0.8–7.6), follicle stimulating hormone 15.1 IU/L (normal range 0.7–11.1), prolactin 10.5 ng/mL (normal range 2.5–17.0), and total testosterone 310 ng/dL (normal range 262.0–1,593.0). The semen analysis repeatedly showed volume 6 mL, pH 8.0, and azoospermia. A written informed consent, approved by the Ethical Committee of the Third Faculty of Medicine, Charles University in Prague was obtained from the patient. A control group consisted of 5 normospermic semen donors (age 26 – 29 years) with normal karyotype.

Testicular sample

The testicular tissue containing spermatozoa was retrieved by microdissection TESE using optical x20–25 magnification on a Zeiss OPMI Pico/S100 microscope. M-TESE involves ‘bivalve’ opening of the testicle by means of an equatorial or longitudinal incision under general anaesthesia and removal of single tubules observed to have the largest diameter and testicular pulp, using an operating microscope. The biopsy sample was used as a source of spermatozoa for IVF. A part of the testicular material was used for an immunofluorescent analysis and sperm FISH.

Immunofluorescent analysis

A small piece of the testicular tissue was homogenized with scissors in a tube with phosphate buffered saline (PBS). After sedimentation of larger pieces, the cell suspension was transferred to a clean tube and centrifuged for 5 min at 600 g. The supernatant was discarded and the pellet was resuspended in PBS. A 10 µl drop of the cell suspension was mixed on a microscope slide with 10 µl of a spreading solution containing 0.05% Triton (Fluka Chemie, Buchs, Germany), smeared, and left to dry slowly in a humid chamber. Then the smears were covered with 80 µl of 0.015% Igepal (Sigma-Aldrich, St. Louis, MO, USA) in H₂O for 5 min and fixed with 120 µl of 1% paraformaldehyde/0.016% Triton/PBS for 10 min in a humid chamber. Finally, the slides were rinsed with distilled water, and placed in a jar with Tris-HCl/NaCl/0.1% Tween (TNT). The fixed samples were immunostained with three combinations of antibodies. The first set of slides was immunostained with primary antibodies specific to SYCP3 (rabbit, Abcam, Cambridge, UK), SYCP1 (goat, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and centromeres (human, Antibodies Inc. Davis, CA, USA) detected by anti-rabbit-FITC (donkey, Santa Cruz Biotechnology), anti-goat-Texas Red (chicken, Santa Cruz Biotechnology), and anti-human-AMCA (donkey, Jackson Immunoresearch, USA), respectively. Another set of slides was subjected to immunostaining with a primary antibody to MLH1 (rabbit, Santa Cruz Biotechnology) detected by anti-rabbit-FITC secondary antibody (donkey, Santa Cruz Biotechnology), and the subsequent second round of immunostaining with primary antibodies specific to SYCP3 (rabbit, Abcam) and centromeres (human, Antibodies Inc.) detected by anti-rabbit-Texas Red (donkey, Santa Cruz Biotechnology) and anti-human-AMCA (donkey, Jackson Immunoresearch). Finally, the third set of slides was immunostained with primary antibodies specific to HORMAD2 (rabbit, Santa Cruz Biotechnology), SYCP1 (goat, Santa Cruz Biotechnology), and centromeres (human, Antibodies Inc.) detected by anti-rabbit-Texas Red (donkey, Santa Cruz Biotechnology), anti-goat-FITC (bovine, Santa Cruz Biotechnology), and anti-human-AMCA (donkey, Jackson Immunoresearch). The primary and secondary antibodies were diluted 1:50 and 1:100, respectively, in PBS/0.55% BSA/0.1% Tween. The slides were incubated in a humid chamber at 37 °C overnight (primary antibodies) or for 1 h (secondary antibodies). After a wash in TNT (two times, 3 min each), the slides were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and evaluated using an Olympus BX60 microscope equipped with a motorized stage and all necessary fluorescence filters. Images of well spread pachytene spermatocytes were captured using a CCD camera, and analyzed using Isis3 software (MetaSystems, Altlussheim, Germany).

FISH on testicular samples

The slides with smears of testicular tissue containing spermatozoa were washed in TNT for 2 min, dehydrated in an ethanol series (70%, 85%, 96% ethanol) and the chromatin was decondensed and denatured by 2-min incubation in 1 M NaOH. The slides were washed in 2xSSC twice for 2 min and dehydrated in an increasing ethanol series. Two rounds of FISH were performed on the slides. The first FISH using probes for the 14q (red, Kreatech, Amsterdam, The Netherlands) and 22q (green, Q-Biogene, Illkirch Cedex, France) subtelomeric regions was performed according to the manufacturer’s protocol. After the evaluation using an Olympus BX60 microscope equipped with a motorized stage and all necessary fluorescence filters, the slides were subjected to the second round of FISH using CEP X (α-satellite, Spectrum Green), CEP Y (satellite III, Spectrum Aqua), and LSI 21 (locus specific, Spectrum Orange) probes (Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer’s protocol. The spermatozoa evaluated in the first round of FISH were relocated and evaluated for the additional probes. The ejaculated semen of the control donors was smeared on slides and decondensed as described above. Hybridization with probes for chromosomes 14 and 22, and X, Y, and 21was performed on two separate slides from each donor. Strict scoring criteria were used [Rubes et al. 2005].

The frequencies of chromosomally abnormal spermatozoa found in our patient were statistically compared with results obtained in the control group and with data published previously for a group of 17 non-obstructive azoospermic patients [Vozdova et al. 2012] by the Chi-square test using the SPSS software package, version 18 for Windows (SPSS, Inc., Chicago, IL, USA). The results were considered statistically significant when p < 0.05.

IVF and PGD on blastomeres

A total of 11 oocytes were fertilized by ICSI. Three out of the six developing embryos were biopsied on developmental day 3 and two cells (embryos No. 1 and 2) or one cell (embryo No. 3) were fixed on a microscope slide using the Tween/HCl method [Coonen et al. 1994]. The PGD test was performed by FISH using Tel 14q (red, Q-Biogene) and LSI 22 (Spectrum Green, Abbott Molecular) probes, followed by a second round of FISH with MultiVysion PGT probe for chromosomes X, Y, 13, 18, and 21 (Abbott Molecular) on embryo No. 1.

Abbreviations
SYCP1	=	synaptonemal complex protein 1
SYCP3	=	synaptonemal complex protein 3
MLH1	=	MutL homolog 1
FISH	=	fluorescence in situ hybridization
AZF	=	azoospermia factor
CFTR	=	cystic fibrosis transmembrane conductance regulator
IVF	=	in vitro fertilization
PBS	=	phosphate buffered saline
PGD	=	preimplantation genetic diagnosis
TESE	=	testicular sperm extraction
TNT	=	tris-HCl/NaCl/0.1% tween
UPD	=	uniparental disomy

Declaration of interest

Supported by CEITEC – Central European Institute of Technology (ED1.05/1.1.00/02.0068) from the European Regional Development Fund. The authors report no declarations of interest.

Author contributions

Designed and coordinated the study including the genetic, hormone, semen, and embryo analysis: VS; Performed the meiotic and sperm-FISH analysis: MV; Performed the TESE: JH; Performed the statistical analysis: JR. All authors participated in preparation of the manuscript.