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  • Research article
  • Open Access

Is sperm FISH analysis still useful for Robertsonian translocations? Meiotic analysis for 23 patients and review of the literature

  • 1, 3,
  • 2, 3, 4,
  • 2,
  • 3, 4,
  • 2, 3, 4,
  • 2, 3, 4,
  • 2,
  • 2,
  • 5,
  • 6,
  • 1,
  • 1, 3 and
  • 1, 3, 4Email author
Contributed equally
Basic and Clinical AndrologyJournal officiel de la Société d'andrologie de langue française201828:5

https://doi.org/10.1186/s12610-018-0069-z

  • Received: 6 December 2017
  • Accepted: 8 March 2018
  • Published:

Abstract

Background

Robertsonian translocations (RobT) are common structural chromosome rearrangements where carriers display a majority of chromosomally balanced spermatozoa from alternate segregation mode. According to some monotony observed in the rates of balanced segregation, is sperm FISH analysis obsolete for RobT carriers?

Methods

Retrospective cohort research study on 23 patients analyzed in our center from 2003 to 2017 and compared to the data of 187 patients in literature from 1983 to 2017.

Robertsonian translocation carriers were divided in six groups according to the chromosomes involved in the translocation: 9 patients from our center and 107 from literature carrying 45,XY,der(13;14) karyotype, 3 and 35 patients respectively with 45,XY,der(14;21), 5 and 11 patients respectively with 45,XY,der(13;15), 4 and 7 patients respectively with 45,XY,der(14;15), 1 and 4 patients respectively with 45,XY,der(13;22),and 1 and 10 patients respectively with 45,XY,der(14;22).

Results

Alternate segregation mode is predominant in our group of Robertsonian translocation carriers with 73.45% ±8.05 of balanced spermatozoa (min 50.92%; max 89.99%). These results are compliant with the data from literature for all translocations types (p > 0.05) and are consistent among the different types of Robertsonian translocations (p > 0.05) except for der(13;15) that exhibit lower balanced spermatozoa rates (p < 0.05 versus der(13;14), der(14;21), (13;21) and der(15;22)). Normozoospermic patients also display a significantly (p < 0.01) higher rate of balanced sperm cells than patients with abnormal seminograms whatever the defect implied.

Conclusions

According to the discrepancies observed between der(13;15) and all the other Rob T carriers, the differences observed among patients presenting normal and abnormal sperm parameters and the input in genetical counselling, sperm FISH does not seem obsolete for these patients. Moreover, it seems important to collect more data for rare RobT.

Résumé

Contexte

Le mode de ségrégation chromosomique le plus fréquemment observé chez les patients porteurs de translocation robertsonienne est. un mode équilibré. Les données semblent varier peu selon la translocation analysée. La relative constance des résultats dans le cas de ces translocations robertsoniennes rend elle inutile ces analyses chromosomiques pour ces patients?

Patients et méthodes

Nous avons analysé de façon rétrospective les données spermatiques et de ségrégation méiotique de 23 patients porteurs de translocation robertsonienne, de 2003 à 2017 et comparé les résultats observés à ceux décrits dans la littérature pour 187 patients.

Résultats

Le mode de ségrégation alterne est. prépondérant dans notre série de patients avec 73.45% ±8.05 de spermatozoïdes équilibrés (min 50.92%; max 89.99%). Ces résultats sont en accord avec les données de la littérature, toutes translocations confondues et selon le type de translocation (p > 0.05) sauf pour la translocation der(13;15) où ces taux sont significativement plus faibles (p < 0.05 vs der(13;14), der(14;21), (13;21) et der(15;22)). Nous observons également des taux de spermatozoïdes équilibrés significativement plus élevés chez les patients à spermogramme normal (p < 0.01).

Conclusions

Les différences observées dans les taux d’aneuploïdies entre les translocations der(13;15) et les autres translocations robertsoniennes et entre les porteurs de translocation à spermogramme normal ou altéré, et l’utilité de ces données dans le conseil génétique conduisent à poursuivre l’analyse systématique de la ségrégation méiotique pour les patients porteurs de translocations robertsoniennes et ceci particulièrement pour les translocations rares.

Keywords

  • Robertsonian translocation
  • Sperm FISH
  • Meiotic segregation
  • Spermatozoa
  • Preimplantation genetic diagnosis

Mots-clés

  • Translocation robertsonienne
  • hybridation in situ
  • ségrégation méiotique
  • spermatozoïde
  • diagnostic génétique préimplantatoire

Background

Robertsonian translocation (RobT) is a frequent structural chromosomal aberration with an incidence of 1.23 per thousand births [1]. Carriers present a karyotype with 45 chromosomes resulting from centromeric fusion of two acrocentric chromosomes (13; 14; 15; 21 or 22). Most common Robertsonian translocations are der (13;14) and der(14;21) with a frequency of 73% and 10% respectively [2]. Unbalanced segregation of these chromosomes through meiosis can result in recurrent pregnancy loss if the unbalanced chromosomal content is not viable, or birth of a child with severe malformations and mental retardation in case of viability. The prevalence of RobT carriers in recurrent pregnancy loss and infertile male population are at least ten times higher (respectively 1.1% and 3% versus 0,1%) than in general population [25]. Knowing the rates of balanced and unbalanced segregation including the different types of unbalanced modes is thus of great importance in genetic counselling for these couples. Moreover, male carriers can present oligoasthenoteratozoospermia leading to procreation issues.

During meiosis, pairing and segregation is possible through formation of a trivalent during prophase I (Fig. 1). Alternate segregation results in two balanced gametes containing either normal chromosomes A and B or the derivate der(A;B). FISH analysis does not allow differentiating these, neither in sperm, nor in the embryos. The karyotype of the conceptus is then either normal or presents the same translocation as the parent, possibly leading to abnormality in the child’s offspring at adulthood. The adjacent segregation modes lead either to sperm nullosomy or sperm disomy. In case of nullosomy, the conceptus presents a monosomy which is not viable, while in case of a sperm disomy, the conceptus presents a trisomy, which can be viable (from several hours to several years or more in trisomy 21). The 3:0 mode of segregation leads to sperm double nullosomy or disomy, leading to unviable monosomic or trisomic conceptus. Detailed analysis of the sperm chromosomal content can thus help genetical counselling through a quantification of (i) the chances of a viable pregnancy (balanced content of sperm and conceptus) and the risk of (ii) recurrent pregnancy loss (unviable monosomy or trisomy) or (iii) possibly viable trisomy. The risk of unbalanced conceptus highlights the importance of chromosomal meiotic segregation analysis. Preimplantation genetic diagnosis (PGD) for RobT carriers reduces the risk of pregnancy loss or multiple congenital anomalies and intellectual disability (MCA-ID) through selection and transfer of normal/balanced embryos.
Fig. 1
Fig. 1

Formation of trivalent and its segregation in meiosis

The first analyses of meiotic segregation variants were done by heterospecific oocyte fertilization followed by sperm karyotyping. This technique was long and fastidious. It only allowed the analysis of a few number of sperm, moreover restricted to the fertile ones. One advantage of this technique was to distinguish between normal and balanced sperm. Development of fluorescence in-situ hybridization (FISH) technique has simplified the analysis of sperm chromosomal content and has enabled to collect numerous data on meiotic segregation and balanced and unbalanced rearrangements. This technique combined to automated slides scanning allows the analysis of a large number of sperm cells and is for several years used in routine practice.

The primary objective of this study was to assess the variability of meiotic segregation in sperm of RobT carriers. We thus, analyzed 23 new carriers and literature data of 187 patients. We also looked for factors influencing meiotic segregation rates.

Methods

Patients

Twenty three male patients aged 26 to 40 years, carrying a RobT, were included in this retrospective cohort study. They consulted for fertility issues in the genetic and procreation department of university hospital of Grenoble between january 2003 and april 2017, except for three of them who were referred by three other french centers (service de génétique, CHU de Reims; service de génétique, CH de Chambéry; centre d’AMP, HFME, CHU de Lyon, France).

Karyotype performed on blood cells was 45,XY,der(13;14)(q10;q10) in 9 patients, 45,XY,der(13;15)(q10;q10) in 5 patients, 45,XY,der(14;15)(q10;q10) in 4 patients, 45,XY,der(14;21)(q10;q10) in 3 patients, 45,XY,der(13;22)(q10;q10) in one patient and 45,XY,der(14;22)(q10;q10) in one patient.

Sperm FISH analyses performed between 2004 and 2006, as a research project, were submitted to a signed inform consent of all the patients with approval of the study by the ethic committee of the University Hospital of Grenoble. Since 2006, the analysis was achieved as a routine test, ruled by a signed informed genetic consent for all patients. The sperm preparation and sperm FISH techniques remained identical over the entire period of study.

Sperm preparation

Semen samples were collected in a sterile container after masturbation. Liquefaction was obtained after 30 min at 37 °C. Sperm concentration, motility and morphology were determined according to WHO criteria (World Health Organization, 1999 for the analyses done until 2009 and WHO, 2010 for the analyses performed later on) [6].

Sperm FISH technique

Samples were washed twice with 5 ml of phosphate-buffered saline (PBS) 1X and fixed in a methanol/acetic acid (3:1, v/v) solution. Cells were spread on Superfrost© (Kindler, Freidburg Germany) slides and air dried at room temperature. Sperm head decondensation was performed in NaOH 1 M solution, followed by two washes in 2X standard saline citrate (SSC) and dehydration in a 70, 90% and pure ethanol solution. Samples were then hybridized overnight with probes of interest for dual-color FISH, depending on the chromosomes involved (Table 1). The scoring of the fluorescent signals was performed by two independent investigators, using an epifluorescence microscope (Nikon Eclipse 80i or Leica DM 5000B) with adapted filter DAPI, FITC, Orange or triple-band. Manual spot count was performed following strict criteria [7]. Automated FISH results were obtained with Metafer Slide Scanning System and MetaCyte software (Metasystems®, Germany), as reported previously [8], with over one thousand cells analyzed when preparation allowed it.
Table 1

Probes used in FISH analysis

Patient

Translocation

Probes

P1 to P9

der(13;14)(q10;q10)

LSI® 13q14 SG (Vysis®, ABBOTT) & TelVysion 14q SO (Vysis®, ABBOTT)

P10 to P14

der(13;15)(q10;q10)

LSI® 13q14 SG (Vysis®, ABBOTT) & TelVysion 15q SO (Vysis®, ABBOTT)

aP10 and P11

der(13;15)(q10;q10)

13q32.1 orange (BlueGnome) & CEP 15 SA (Vysis®, ABBOTT)

P15

der(13;22)(q10;q10)

13q32.1 orange (BlueGnome) & LSI® 22 (BCR) SG (Vysis®, ABBOTT)

P16 and P17

der(14;15)(q10;q10)

TelVysion 14q SO (Vysis®, ABBOTT) & CEP 15 SA (Vysis®, ABBOTT)

aP16

der(14;15)(q10;q10)

Subtelomere 14q green (Cytocell Aquarius) & CEP 15 SA (Vysis®, ABBOTT)

P18 and P19

der(14;15)(q10;q10)

TelVysion 14q SO (Vysis®, ABBOTT) & CEP 15 SA (Vysis®, ABBOTT)

P20 to P22

der(14;21)(q10;q10)

TelVysion 14q SO (Vysis®, ABBOTT) & Subtelomere 21q green (Cytocell Aquarius)

P23

der(14;22)(q10;q10)

Subtelomere 14q green (Cytocell Aquarius) & Tel22q SO (Amplitech)

apatients for whom a second analysis was performed because of insufficient initial count

Literature analysis

Literature analysis was mainly performed on PUBMED. Searching was performed using the following MESH terms: Robertsonian translocation/sperm FISH/meiotic segregation. A total of 171 publications were found. Among them, 44 publications [952] about meiotic segregation of sperm from Robertsonian translocation carriers were found between 1983 and 2017 (Table 2).
Table 2

Robertsonian translocation carriers with meiotic segregation analysis in literature

 

13;14

14;21

13;15

14;15

14;22

13;21

13;22

21;22

15;22

15;21

Wang et al. 2017

6

3

 

1

  

1

1

  

Song et al. 2016

   

1

      

Godo et al. 2015

10

     

1

   

Sobotka et al. 2015

    

1

     

Xu et al. 2014

   

1

      

Perrin et al. 2013

1

1

1

       

Pylyp et al. 2013

5

3

   

1

    

Rouen et al. 2013

1

1

        

Rouen et al. 2013

7

1

1

       

Vozdova et al. 2013

11

1

      

1

 

Bernicot et al 2012

     

1

  

1

 

Cassuto et al. 2011

1

1

        

Ferfouri et al. 2011

16

8

1

 

3

    

1

Mahjoub et al. 2011

5

         

Anton et al. 2010

 

3

    

1

   

Brugnon et al. 2010

1

1

1

 

2

     

Perrin et al. 2010

3

         

Perrin et al. 2009

3

1

        

Nishikawa et al. 2008

1

2

   

1

    

Chen et al. 2007

4

   

1

1

    

Kekesi et al 2007

1

         

Brugnon et al. 2006

3

 

1

       

Hatakeyama et al 2006

     

1

    

Moradkhani et al 2006

  

2

2

      

Moradkhani et al 2006

    

3

     

Ogur et al 2006

7

2

2

2

   

1

  

Tang et al 2006

 

1

        

Anahory 2005

      

1

   

Rives et al 2005

  

1

       

Roux et al 2005

3

         

Anton et al 2004

7

         

Frydman et al 2001

3

3

        

Morel et al 2001

3

         

Escudero et al 2000

2

         

Honda et al 2000

 

1

        

a Ogawa et al. 2000

1

         

Mennicke et al. 1997

       

1

  

Rousseaux et al 1995

 

1

        

a Martin et al 1992

        

1

 

a Syme et al 1992

       

1

  

a Pellestor et al. 1990

  

1

       

a Martin et al. 1988

1

         

a Pellestor et al. 1987

1

         

a Balkan et al. 1983

 

1

        

Literature

107

35

11

7

10

5

4

4

3

1

Our study

9

3

5

4

1

0

1

0

0

0

Total

116

38

16

11

11

5

5

4

3

1

% total

55,24

18,10

7,62

5,24

5,24

2,38

2,38

1,90

1,43

0,48

a Studies using sperm karyotyping after heterospecific fertilization

Data analyzed

Variables analyzed were: sperm concentration (106/ml), motility (%), morphology (%) and meiotic segregation rates of different variants (%).

Statistical analysis

Data were treated with R software (version number 2.14.1). A probability value of less than 0.05 was considered to be statistically significant.

Results

Semen parameters

As summarized in Tables 3, 4 patients were normozoospermic, 6 were oligoasthenozoospermic, 6 were oligoasthenoteratozoospermic (OAT), 3 were oligoteratozoospermic, 2 were asthenozoospermic, and 2 were oligozoospermic.
Table 3

Robertsonian translocation carriers age, karyotype and semen parameters

Patient

Age

Karyotype

Semen parameters

Seminogram

Concentration (×10^6/ml)

Motility (%)

(%) Normal morphology

P1

36

45,XY,der(13;14)(q10;q10)

4,5

20

4

Oligoasthenozoospermia

P2

26

45,XY,der(13;14)(q10;q10)

0,6

36

3

Oligoasthenoteratozoospermia

P3

34

45,XY,der(13;14)(q10;q10)

0,48

17

3

Oligoasthenoteratozoospermia

P4

38

45,XY,der(13;14)(q10;q10)

28

40

9

Normozoospermia

P5

29

45,XY,der(13;14)(q10;q10)

22,3

50

16

Normozoospermia

P6

33

45,XY,der(13;14)(q10;q10)

2,2

20

2

Oligoasthenoteratozoospermia

P7

38

45,XY,der(13;14)(q10;q10)

25

5

2

Oligoasthenoteratozoospermia

P8

36

45,XY,der(13;14)(q10;q10)

2

30

4

Oligoasthenozoospermia

P9

32

45,XY,der(13;14)(q10;q10)

0.007

17

0

Oligoasthenoteratozoospermia

P10

32

45,XY,der(13;15)(q10;q10)

13

34

8

Oligoasthenozoospermia

P11

27

45,XY,der(13;15)(q10;q10)

23

60

4

Normozoospermia

P12

28

45,XY,der(13;15)(q10;q10)

0,9

48

13

Oligozoospermia

P13

35

45,XY,der(13;15)(q10;q10)

0,02

13

2

Oligoasthenoteratozoospermia

P14

27

45,XY,der(13;15)(q10;q10)

5

45

0

Oligoteratozoospermia

P15

35

45,XY,der(13;22)(q10;q10)

7,3

45

3

Oligoteratozoospermia

P16

38

45,XY,der(14;15)(q10;q10)

27

30

39

Asthenozoospermia

P17

33

45,XY,der(14;15)(q10;q10)

35

30

21

Asthenozoospermia

P18

31

45,XY,der(14;15)(q10;q10)

2,2

30

59

Oligoasthenozoospermia

P19

30

45,XY,der(14;15)(q10;q10)

5

35

15

Oligoasthenozoospermia

P20

40

45,XY,der(14;21)(q10;q10)

42

45

17

Normozoospermia

P21

36

45,XY,der(14;21)(q10;q10)

2,7

40

11

Oligozoospermia

P22

27

45,XY,der(14;21)(q10;q10)

0,4

16

5

Oligoasthenozoospermia

P23

39

45,XY,der(14;22)(q10;q10)

8

45

2

Oligoteratozoospermia

Table 4

Meiotic segregation of Robertsonian translocation carriers

Patient

% Alt

% Adjacent

% 3:0

% unbalanced

der(13;14)

 

balanced

disomy 13

nullisomy 13

disomy 14

nullisomy 14

3:0

unbalanced

 P1

72.76

6.34

8.21

2.99

6.72

2.99

27.24

 P2

71.69

4.57

6.39

7.31

10.05

0.00

28.31

 P3

64.94

4.60

15.52

8.62

5.75

0.57

35.06

 P4

84.86

2.83

4.66

3.16

4.49

0.00

15.14

 P5

85.95

2.42

5.82

2.75

3.07

0.00

14.05

 P6

75.39

6.84

6.29

5.74

5.52

0.22

24.61

 P7

78.52

7.21

6.38

3.52

4.36

0.00

21.48

 P8

66.08

4.59

3.81

9.97

12.12

3.42

33.92

 P9

60.69

13.08

3.15

7.75

7.27

8.06

39.31

Mean

73.43

5.83

6.69

5.76

6.59

1.70

26.57

der(13;15)

 

balanced

disomy 13

nullisomy 13

disomy 15

nullisomy 15

3:0

unbalanced

 P10

66.80

6.40

7.60

8.80

10.40

0.00

33.20

 P11

68.94

4.61

7.01

4.81

14.63

0.00

31.06

 P12

81.32

1.10

6.59

3.30

6.59

1.10

18.68

 P13

50.92

9.17

12.84

10.09

16.97

0.00

49.08

 P14

81.57

3.66

2.66

3.97

3.76

4.39

18.43

Mean ± SD

69.91

4.99

7.34

6.19

10.47

1.10

30.09

der(13;22)

 

balanced

disomy 13

nullisomy 13

disomy 22

nullisomy 22

3:0

unbalanced

 P15

72.78

4.63

9.51

4.39

8.05

0.73

27.32

der(14;15)

 

balanced

disomy 14

nullisomy 14

disomy 15

nullisomy 15

3:0

unbalanced

 P16

71.80

7.00

10.60

3.60

7.00

0.00

28.20

 P17

83.90

0.85

4.24

0.21

10.81

0.00

16.10

 P18

83.26

2.48

4.75

1.24

8.26

0.00

16.74

 P19

68.26

5.49

4.51

7.33

12.56

1.85

31.74

Mean ± SD

76.80

3.95

6.03

3.10

9.66

0.46

23.20

der(14;21)

 

balanced

disomy 14

nullisomy 14

disomy 21

nullisomy 21

3:0

unbalanced

 P20

89.99

3.24

0.99

1.97

3.81

0.00

10.01

 P21

53.34

11.35

28.46

3.11

0.00

3.73

46.66

 P22

79.71

4.92

7.63

3.73

3.22

0.79

20.29

Mean ± SD

74.35

6.51

12.36

2.94

2.34

1.51

25.65

der(14;22)

 

balanced

disomy 14

nullisomy 14

disomy 22

nullisomy 22

3:0

unbalanced

 P23

76.09

5.07

5.92

4.21

6.78

1.93

23.91

Sperm FISH analysis

The number of analyzed sperm ranged from 91 to 1950 for each patient, with a total of 18,261 spermatozoa. Segregation results are illustrated in Fig. 2 and detailed in Table 4 with insight in each mode: alternate, adjacent and 3:0. Our results confirmed a majority of balanced spermatozoa for all patients with a mean ± SE of 73.45 ± 8.05% for all RobT (min 50.92; max 89.99). The rate of unbalanced spermatozoa resulting from adjacent mode of segregation represented 25.25 ± 7.63% (min 10.01%; max 49.08%). The 3:0 segregation mode represented 1.29 ± 1.50% (min 0%; max 8.06%). Mean disomy rates vary from 2.94% to 6.51% (min = 0.21%, max = 13.85%) when comparing all the translocations, while mean nullosomy rates vary from 2.34% to 12.36% (min = 0.00%; max = 16.97%). For each chromosome, mean disomy rate is always lower than mean nullosomy rate.
Fig. 2
Fig. 2

Rates of different variants in meiotic segregation

Analysis of the segregation data available in the literature

Bibliographic references about sperm FISH analysis of Robertsonian translocation carriers are presented in Table 2. Forty four articles have been published from 1983 to 2017 dealing with meiotic segregation in sperm with thirty nine for the same RobT as in our study. It overall summarized the FISH analysis of 210 patients.

Our segregation rates were compliant with the data from literature for all translocation types (our study versus literature): der(13;14) 73.43 ± 7% versus 83.29 ± 8.72%, der(13;15) 69.91 ± 12.62% versus 79.73 ± 6.73%, der(14;15) 76.80 ± 7.96% versus 84.51 ± 5.58%, der(14;21) 74.35 ± 18.9% versus 83.45 ± 8.3% (p > 0.05, t-test). Statistics were not available for der(13;22) and (14;22) as we only added one patient.

Altogether, balanced segregation rates were consistent among the different types of RobT (p > 0.05, t-test) except for der(13;15) that exhibited lower balanced spermatozoa rates (Fig. 3). Der(13;15) segregation rates were statistically different (p < 0.05, t-test) from those from the two most common Robertsonian translocation der(13;14) and der(14;21), and two less common der(13;21) and der(15;22).
Fig. 3
Fig. 3

Comparative analysis of the rates of balanced spermatozoa between each RobT. Legend: n = number of patients, *p-value < 0.05 versus der(13;15). Statistical analysis not possible for der(15;21) due to the number of patients

Correlation between segregation data and semen analysis

From the 187 selected carriers of literature, sperm analysis data were available for 159, and added to our 23 patients. Among all, 33 were normozoospermic (18.13%) and 149 exhibited abnormal seminogram (81.87%). Oligozoospermia was found in 133 patients (73.08%), asthenozoospermia in 109 (59.89%) and teratozoospermia in 119 (65.38%). Twenty three patients had a single anomaly (12.64%), 46 two anomalies (25.27) and 83 displayed OAT (45.60%).

As shown in Fig. 4, normozoospermic patients display a significantly (p < 0.01, t-test) higher rate of balanced sperm cells (85%) than patients with seminogram anomalies (81.3%), whatever the number or the type of anomalies involved (p > 0.05, t-test).
Fig. 4
Fig. 4

Balanced spermatozoa rates among normozoospermic patients and patients with abnormal seminogram. Legend: n = number of patients

Discussion

Thanks to the twenty-three new patients of this study, literature reaches more than two hundred descriptions of meiotic segregation of RobT carriers. Compiling of these data is especially important for rare RobT, like der(13;15) for which we add 5 carriers to the 11 already known (+ 45%), der(14;15) with 4 new patients to the 7 previously published (+ 57%) and der(13;22) with one addition to the 4 patients already presented (+ 25%).

Our rates of balanced segregation (73.45 ± 8.05%) are compliant with the previous studies (p > 0.05) for each translocation versus data from publications listed in Table 4, showing the predominance of alternate segregation for all carriers. Similar to literature (41.7%), most of our patients (39.1%) exhibit balanced segregation rates between 75 and 85%. Patients with rates under 65% or over 90% only represent a small proportion of global population both in our study (17.4%) and in literature (16.6%).

It is commonly assumed that rearranged chromosomes of RobT carriers have similar meiotic behavior, regardless of the chromosomes involved ([34], data from 41 carriers). This hypothesis is strongly supported by the similarity of balanced gamete rates among the different RobT carriers. We demonstrate that all RobT segregation rates are similar to each other (p > 0.05), except for der(13;15) whose rates are significantly lower (p < 0.05) than der(13;14) and der(14;21), the two most frequent translocations, and der(13;21) and der(15;22). The limited size of the cohorts of the other translocations probably explains the lack of significance in segregation rates (p = 0.13 vs der(14;15) n = 11; p = 0.17 vs der(14;22) n = 11; p = 0.46 vs der(13;22) n = 5). No clue has been found so far to explain the difference between der(13;15) segregation rate and the other RobT. It could be due either to the structure of the chromosomes involved in the translocation, or to the spermatogenesis itself. We also clearly show that mean disomy rates are lower than mean nullosomy rates, whatever the chromosome analyzed and that the discrepancies observed among the rates for patients carrying the same translocation are important. When giving genetical counselling to the patients and thinking about preimplantation diagnosis, oocyte fertilization by a nullosomic sperm leads to miscarriage, while oocyte fertilization by a disomic sperm can lead to the birth of a child with MCA-ID. The choice of preimplantation diagnosis may thus be all the more considered as the risk of MCA-ID child is high.

What about spermatogenesis for these patients and the possible links between germ cell production and meiotic segregation? Abnormal semen parameters were found in 82.6% (19 for 23) of our RobT carriers which support the fact that semen parameters of RobT carriers are significantly lower than those of men with normal karyotype [53]. Altered semen parameters have previously been correlated with aneuploidy in RobT carriers [18] and suggested implication in malsegregation rates [21]. Here we confirm that normozoospermic men have higher rates of balanced spermatozoa than men with semen anomalies, whatever the anomaly implied. The proportion of RobT carriers with abnormal seminogram was not different among the translocations analyzed in our cohort (unpublished data) and particularly not between der(13;14) and der(13;15) (p = 0.58, Fischer’s exact test). The discrepancies between der(13;15) and the other translocations cannot be explained this way.

It seems also interesting to question if the sperm preparation methods used in assisted reproductive techniques can improve the rates of balanced sperm used in these techniques. Several procedures have been developed to improve the detection or exclusion of sperm with quantitative or qualitative nuclear anomalies (translocation or DNA fragmentation) with partial results [54]. Among them, no morphological discrimination was sufficiently accurate to identify chromosomal imbalances or DNA defects [55, 56], but the use of a simple discontinuous gradient centrifugation could lead to a 30% decrease of unbalanced sperm in chromosomal structural rearrangement carriers [16]. Recent work by Rouen et al [57] suggests that the hypo-osmotic swelling test (HOST) could allow a more efficient selection of balanced sperm in translocation carriers. HOST has already shown some efficacy in normal sperm selection in patients with testicular biopsy and very low sperm count and/or little or no motility, but the efficiency of this procedure has yet to be confirmed under ICSI standard conditions.

Beyond basic cytogenetic research, these data are useful to bring better reproductive and genetic counseling when couples are engaged in PGD. Studies involving PGD for RobT carriers confirmed alternate segregation predominance [5861]. We consider that sperm FISH is a useful tool to help the management of PGD attempts.

Conclusion

According to the discrepancies observed between der(13;15) and all the other Rob T carriers, the differences observed among patients presenting normal and abnormal sperm parameters and the input in genetical counselling, sperm FISH does not seem obsolete for these patients. Moreover, it seems important to collect more data for rare RobT.

Abbreviations

FISH: 

Fluorescence In-Situ Hybridization

HOST: 

Hypo-Osmotic Swelling Test

MCA-ID: 

Multiple Congenital Anomalies and Intellectual Disability

OAT: 

OligoAsthenoTeratozoospermia

PGD: 

Preimplantation Genetic Diagnosis

RobT: 

Robertsonian translocation

SSC: 

Saline-Sodium-Citrate buffer

WHO: 

World Health Organization

Declarations

Acknowledgements

Not applicable

Funding

This work started with a grant from the Délégation de la Recherche Clinique CHU Grenoble, 2004.

Availability of data and materials

All data generated or analysed during this study are included in this published article (and its supplementary information files).

Authors’ contributions

AL, GM, FD and SH analyzed the data and wrote the manuscript. AL, GM and FD performed sperm FISH experiments. AL, GM and JPH performed the literature analysis and statistical analysis. VS, CC, FA, RH, JB, JL, MB and SB included patients and/or provided clinical samples and data. SH designed the study, had full access to all of the data in the study and takes responsibility for the integrity of the data and its accuracy. All authors contributed to the report. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All patients signed inform consent and study was approved by the ethic committee of the University Hospital of Grenoble.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
CHU de Grenoble, UF de Biologie de la procréation, F-38000 Grenoble, France
(2)
CHU de Grenoble, UF de Génétique Chromosomique, F-38000 Grenoble, France
(3)
Université Grenoble Alpes, F-38000 Grenoble, France
(4)
Team ‘Genetics Epigenetics and Therapies of Infertility’, Institute for Advanced Biosciences INSERM U1209, CNRS UMR5309, F-38000 Grenoble, France
(5)
Service de génétique CH de Chambéry, Chambery, F-38000, France
(6)
Centre d’AMP, HFME, CHU de Lyon, Lyon, F-69000, France

References

  1. Nielsen J, Wohlert M. Chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Hum Genet. 1991;87:81–3.View ArticlePubMedGoogle Scholar
  2. Therman E, Susman B, Denniston C. The nonrandom participation of human acrocentric chromosomes in Robertsonian translocations. Ann Hum Genet. 1989;53:49–65.View ArticlePubMedGoogle Scholar
  3. Fryns JP, van Buggenhout G. Structural chromosome rearrangements in couples with recurrent fetal wastage. Eur J Obstet Gynecol Reprod Biol. 1998;81:171–6.View ArticlePubMedGoogle Scholar
  4. Escudero T, Abdelhadi I, Sandalinas M, Munne S. Predictive value of sperm fluorescence in situ hybridization analysis on the outcome of preimplantation genetic diagnosis for translocations. Fertil Steril. 2003;79(Suppl3):1528–34.View ArticlePubMedGoogle Scholar
  5. Mau-Holzmann UA. Somatic chromosomal abnormalities in infertile men and women. Cytogenet Genome Res. 2005;111:317–36.View ArticlePubMedGoogle Scholar
  6. World Health Organization. WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press; 1999.Google Scholar
  7. Molina O, Sarrate Z, Vidal F, Blanco J. FISH on sperm: spot-counting to stop counting? Not yet. Fertil Steril. 2009;92:1474–80.View ArticlePubMedGoogle Scholar
  8. Martinez G, Le Mitouard M, Borye R, Esquerré C, Satre V, Bujan L, et al. FISH and tips: a large scale analysis of automated versus manual scoring for sperm aneuploidy detection. Basic And Clinical Andrology. 2013;23:13.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Wang B, Nie B, Tang D, Li R, Liu X, Song J, et al. Analysis of meiotic segregation patterns and interchromosomal effects in sperm from 13 robertsonian translocations. BJMG. 2017;20:43–50.PubMedPubMed CentralGoogle Scholar
  10. Song J, Li X, Sun L, Xu S, Liu N, Yao Y, et al. A family with Robertsonian translocation: a potential mechanism of speciation in humans. Mol Cytogenet. 2016;9(48)Google Scholar
  11. Godo A, Blanco J, Vidal F, Sandalinas M, Garcia-Guixe E, Anton E. Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers. Reprod BioMed Online. 2015;31:79–88.View ArticlePubMedGoogle Scholar
  12. Sobotka V, Vozdova M, Heracek J, Rubes J. A rare Robertsonian translocation rob(14;22) carrier with azoospermia, meiotic defects, and testicular sperm aneuploidy. Syst Biol Reprod Med. 2015;61:245–50.View ArticlePubMedGoogle Scholar
  13. Xu S, Tang D, Fang K, Xia Y, Song J, Wang W, et al. Analysis of meiotic segregation patterns and interchromosomal effects in sperm from a Robertsonian translocation family. Biomed Res. 2014;25:233–9.Google Scholar
  14. Perrin A, Nguyen MH, Bujan L, Vialard F, Amice V, Gueganic N, et al. DNA fragmentation is higher in spermatozoa with chromosomally unbalanced content in men with a structural chromosomal rearrangement. Andrology. 2013;1:632–8.View ArticlePubMedGoogle Scholar
  15. Pylyp LY, Zukin VD, Bilko NM. Chromosomal segregation in sperm of Robertsonian translocation carriers. J Assist Reprod Genet. 2013;30:1141–5.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Rouen A, Balet R, Dorna M, Hyon C, Pollet-Villard X, Chantot-Bastaraud S, et al. Discontinuous gradient centrifugation (DGC) decreases the proportion of chromosomally unbalanced spermatozoa in chromosomal rearrangement carriers. Hum Reprod. 2013;28:2003–9.View ArticlePubMedGoogle Scholar
  17. Rouen A, Pyram K, Pollet-Villard X, Hyon C, Dorna M, Marques S, et al. Simultaneous cell by cell study of both DNA fragmentation and chromosomal segregation in spermatozoa from chromosomal rearrangement carriers. J Assist Reprod Genet. 2013;30:383–90.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Vozdova M, Oracova E, Kasikova K, Prinosilova P, Rybar R, Horinova V, et al. Balanced chromosomal translocations in men: relationships among semen parameters, chromatin integrity, sperm meiotic segregation and aneuploidy. J Assist Reprod Genet. 2013;30:391–405.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Bernicot I, Schneider A, Mace A, Hamamah S, Hedon B, Pellestor F, et al. Analysis using fish of sperm and embryos from two carriers of rare rob(13;21) and rob(15;22) robertsonian translocation undergoing PGD. Eur J Med Genet. 2012;55:245–51.View ArticlePubMedGoogle Scholar
  20. Cassuto NG, Le Foll N, Chantot-Bastaraud S, Balet R, Bouret D, Rouen A, et al. Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation: relationship between segregation modes and high-magnification sperm morphology examination. Fertil Steril. 2011;96:826–32.View ArticlePubMedGoogle Scholar
  21. Ferfouri F, Selva J, Boitrelle F, Gomes DM, Torre A, Albert M, et al. The chromosomal risk in sperm from heterozygous Robertsonian translocation carriers is related to the sperm count and the translocation type. Fertil Steril. 2011;96:1337–43.View ArticlePubMedGoogle Scholar
  22. Mahjoub M, Mehdi M, Brahem S, Elghezal H, Ibala S, Saad A. Chromosomal segregation in spermatozoa of five Robertsonian translocation carriers t(13;14). J Assist Reprod Genet. 2011;28:607–13.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Anton E, Blanco J, Vidal F. Meiotic behavior of three D;G Robertsonian translocations: segregation and interchromosomal effect. J Hum Genet. 2010;55:541–5.View ArticlePubMedGoogle Scholar
  24. Brugnon F, Janny L, Communal Y, Darcha C, Szczepaniak C, Pellestor F, et al. Apoptosis and meiotic segregation in ejaculated sperm from Robertsonian translocation carrier patients. Hum Reprod. 2010;25:1631–42.View ArticlePubMedGoogle Scholar
  25. Perrin A, Morel F, Douet-Guilbert N, Le Bris MJ, Amice J, Amice V, et al. A study of meiotic segregation of chromosomes in spermatozoa of translocation carriers using fluorescent in situ hybridisation. Andrologia. 2010;42:27–34.View ArticlePubMedGoogle Scholar
  26. Perrin A, Caer E, Oliver-Bonet M, Navarro J, Benet J, Amice V, et al. DNA fragmentation and meiotic segregation in sperm of carriers of a chromosomal structural abnormality. Fertil Steril. 2009;92(2)Google Scholar
  27. Nishikawa N, Sato T, Suzumori N, Sonta S, Suzumori K. Meiotic segregation analysis in male translocation carriers by using fluorescent in situ hybridization. Int J Androl. 2008;31:60–6.PubMedGoogle Scholar
  28. Chen Y, Huang J, Liu P, Qiao J. Analysis of meiotic segregation patterns and interchromosomal effects in sperm from six males with Robertsonian translocations. J Assist Reprod Genet. 2007;24:406–11.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Kekesi A, Erdei E, Torok M, Dravucz S, Toth A. Segregation of chromosomes in spermatozoa of four Hungarian translocation carriers. Fertil Steril. 2007;88:212.e5–11.View ArticleGoogle Scholar
  30. Brugnon F, Van Assche E, Verheyen G, Sion B, Boucher D, Pouly JL, et al. Study of two markers of apoptosis and meiotic segregation in ejaculated sperm of chromosomal translocation carrier patients. Hum Reprod. 2006;21:685–93.View ArticlePubMedGoogle Scholar
  31. Hatakeyama C, Gao H, Harmer K, Ma S. Meiotic segregation patterns and ICSI pregnancy outcome of a rare (13;21) Robertsonian translocation carrier: a case report. Hum Reprod. 2006;21:976–9.View ArticlePubMedGoogle Scholar
  32. Moradkhani K, Puechberty J, Bhatt S, Lespinasse J, Vago P, Lefort G, et al. Rare Robertsonian translocations and meiotic behaviour: sperm FISH analysis of t(13;15) and t(14;15) translocations: a case report. Hum Reprod. 2006;21:3193–8.View ArticlePubMedGoogle Scholar
  33. Moradkhani K, Puechberty J, Bhatt S, Vago P, Janny L, Lefort G, et al. Meiotic segregation of rare Robertsonian translocations: sperm analysis of three t(14q;22q) cases. Hum Reprod. 2006;21:1166–71.View ArticlePubMedGoogle Scholar
  34. Ogur G, Van Assche E, Vegetti W, Verheyen G, Tournaye H, Bonduelle M, et al. Chromosomal segregation in spermatozoa of 14 Robertsonian translocation carriers. Mol Hum Reprod. 2006;12:209–15.View ArticlePubMedGoogle Scholar
  35. Tang YP, Liu XS, Liu Y, Yang ZR, Chen Y, Xiong CL. Somatic cell and sperm cell cytogenetics in a patient with t(14; 21). Yi Chuan Xue Bao. 2006;33:488–94.PubMedGoogle Scholar
  36. Anahory T, Hamamah S, Andreo B, Hedon B, Claustres M, Sarda P, et al. Sperm segregation analysis of a (13;22) Robertsonian translocation carrier by FISH: a comparison of locus-specific probe and whole chromosome painting. Hum Reprod. 2005;20:1850–4.View ArticlePubMedGoogle Scholar
  37. Rives N, Ravel C, Duchesne V, Siffroi JP, Mousset-Simeon N, Mace B. Molecular cytogenetics analysis with whole chromosome paint probes of sperm nuclei from a (13;15) Robertsonian translocation carrier. J Hum Genet. 2005;50:360–4.View ArticlePubMedGoogle Scholar
  38. Roux C, Tripogney C, Morel F, Joanne C, Fellmann F, Clavequin MC, Bresson JL. Segregation of chromosomes in sperm of Robertsonian translocation carriers. Cytogenet Genome Res. 2005;111:291–6.View ArticlePubMedGoogle Scholar
  39. Anton E, Blanco J, Egozcue J, Vidal F. Sperm FISH studies in seven male carriers of Robertsonian translocation t(13;14)(q10;q10). Hum Reprod. 2004;19:1345–51.View ArticlePubMedGoogle Scholar
  40. Frydman N, Romana S, Le Lorc'h M, Vekemans M, Frydman R, Tachdjian G. Assisting reproduction of infertile men carrying a Robertsonian translocation. Hum Reprod. 2001;16:2274–7.View ArticlePubMedGoogle Scholar
  41. Morel F, Fellmann F, Roux C, Bresson JL. Meiotic segregation analysis by FISH investigation of spermatozoa of a 46,Y,der(X),t(X;Y)(qter-->p22::q11-->qter) carrier. Cytogenet Cell Genet. 2001;92:63–8.View ArticlePubMedGoogle Scholar
  42. Escudero T, Lee M, Carrel D, Blanco J, Munne S. Analysis of chromosome abnormalities in sperm and embryos from two 45,XY,t(13;14)(q10;q10) carriers. Prenat Diagn. 2000;20:599–602.View ArticlePubMedGoogle Scholar
  43. Honda H, Miharu N, Samura O, He H, Ohama K. Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization. Hum Genet. 2000;106:188–93.View ArticlePubMedGoogle Scholar
  44. Ogawa S, Araki S, Araki Y, Ohno M, Sato I. Chromosome analysis of human spermatozoa from an oligoasthenozoospermic carrier for a 13;14 Robertsonian translocation by their injection into mouse oocytes. Hum Reprod. 2000;15:1136–9.View ArticlePubMedGoogle Scholar
  45. Mennicke K, Diercks P, Schlieker H, Bals-Pratsch M, Al Hasani S, Diedrich K, Schwinger E. Molecular cytogenetic diagnostics in sperm. Int J Androl. 1997;20:11–9.PubMedGoogle Scholar
  46. Rousseaux S, Chevret E, Monteil M, Cozzi J, Pelletier R, Delafontaine D, Sele B. Sperm nuclei analysis of a Robertsonian t(14q21q) carrier, by FISH, using three plasmids and two YAC probes. Hum Genet. 1995;96:655–60.View ArticlePubMedGoogle Scholar
  47. Martin RH, Ko E, Hildebrand K. Analysis of sperm chromosome complements from a man heterozygous for a robertsonian translocation 45,XY,t(15q;22q). Am J Med Genet. 1992;43:855–7.View ArticlePubMedGoogle Scholar
  48. Syme RM, Martin RH. Meiotic segregation of a 21;22 Robertsonian translocation. Hum Reprod. 1992;7:825–9.View ArticlePubMedGoogle Scholar
  49. Pellestor F. Analysis of meiotic segregation in a man heterozygous for a 13;15 Robertsonian translocation and a review of the literature. Hum Genet. 1990;85:49–54.View ArticlePubMedGoogle Scholar
  50. Martin RH. Cytogenetic analysis of sperm from a male heterozygous for a 13;14 Robertsonian translocation. Hum Genet. 1988a;80:357–61.View ArticlePubMedGoogle Scholar
  51. Pellestor F, Sele B, Jalbert H. Chromosome analysis of spermatozoa from a male heterozygous for a 13;14 Robertsonian translocation. Hum Genet. 1987;76:116–20.View ArticlePubMedGoogle Scholar
  52. Balkan W, Martin RH. Segregation of chromosomes into the spematozoa of a man heterozygous for a 14;21 Robertsonian translocation. Am J Med Genet. 1983b;16:169–72.View ArticlePubMedGoogle Scholar
  53. Pastuszek E, Kiewisz J, Kulwikowska PM, Lukaszuk M, Lukaszuk K. Sperm parameters and DNA fragmentation of balanced chromosomal rearrangements carriers. Folia Histochem Cytobiol. 2015;53:314–21.View ArticlePubMedGoogle Scholar
  54. Sakkas D. Novel technologies for selecting the best sperm for in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril. 2013;99:1023–9.View ArticlePubMedGoogle Scholar
  55. Garolla A, Fortini D, Menegazzo M, de Toni L, Nicoletti V, Moretti A, et al. High-power microscopy for selecting spermatozoa for ICSI by physiological status. Reprod BioMed Online. 2008;17:610–6.View ArticlePubMedGoogle Scholar
  56. Avendano C, Franchi A, Taylor S, Morshedi M, Bocca S, Oehninger S. Fragmentation of DNA in morphologically normal human spermatozoa. Fertil Steril. 2009;91:1077–84.View ArticlePubMedGoogle Scholar
  57. Rouen A, Carlier L, Heide S, Egloff M, Marzin P, Ader F, et al. Potential selection of genetically balanced spermatozoa based on the hypo-osmotic swelling test in chromosomal rearrangement carriers. Reprod BioMed Online. 2017;35:372–8.View ArticlePubMedGoogle Scholar
  58. Conn CM, Harper JC, Winston RM, Delhanty JD. Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions. Hum Genet. 1998;102:117–23.View ArticlePubMedGoogle Scholar
  59. Iwarsson E, Malmgren H, Inzunza J, Ahrlund-Richter L, Sjöblom P, Rosenlund B, et al. Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers. Prenat Diagn. 2000;20:1038–47.View ArticlePubMedGoogle Scholar
  60. Scriven PN, Flinter FA, Braude PR, Ogilvie CM. Robertsonian translocations--reproductive risks and indications for preimplantation genetic diagnosis. Hum Reprod. 2001;16:2267–73.View ArticlePubMedGoogle Scholar
  61. Alves C, Sousa M, Silva J, Barros A. Preimplantation genetic diagnosis using FISH for carriers of Robertsonian translocations: the Portuguese experience. Prenat Diagn. 2002;22:1153–62.View ArticlePubMedGoogle Scholar

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