Robertsonian translocations—reproductive risks and indications for preimplantation genetic diagnosis

P.N. Scriven1,4, F.A. Flinter2, P. R. Braude3 and C. Mackie Ogilvie1

1 Guy's & St Thomas' Centre for PGD, Cytogenetics Department 5th Floor, Guy's Tower, St Thomas Street, London SE1 9RT, 2 Clinical Genetics, 7th Floor, New Guy's House, St Thomas Street, London SE1 9RT and 3 Assisted Conception Unit, 10th Floor North Wing, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK4


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Robertsonian translocations carry reproductive risks that are dependent on the chromosomes involved and the sex of the carrier. We describe five couples that presented for preimplantation genetic diagnosis (PGD). METHODS: PGD was carried out using cleavage-stage (day 3) embryo biopsy, fluorescence in-situ hybridization (FISH) with locus-specific probes, and day 4 embryo transfer. RESULTS: Couple A (45,XX,der(14;21)(q10;q10)) had two previous pregnancies, one with translocation trisomy 21. A successful singleton pregnancy followed two cycles of PGD. Couple B (45,XX,der(13;14)(q10;q10)) had four miscarriages, two with translocation trisomy 14. One cycle of PGD resulted in triplets. Couple C (45,XX,der(13;14)(q10;q10)) had four years of infertility; two cycles were unsuccessful. Couple D (45,XY,der(13;14)(q10;q10)) presented with oligozoospermia. A singleton pregnancy followed two cycles of PGD. Couple E (45,XY,der(13;14)(q10;q10)) had a sperm count within the normal range and low levels of aneuploid spermatozoa. PGD was therefore not recommended. No evidence for a high incidence of embryos with chaotic or mosaic chromosome complements was found. CONCLUSIONS: For fertile couples, careful risk assessment and genetic counselling should precede consideration for PGD. Where translocation couples need assisted conception for subfertility, PGD is a valuable screen for imbalance, even when the risk of viable chromosome abnormality is low.

Key words: PGD/reproductive risks/Robertsonian translocation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Robertsonian translocations (centric fusion of two acrocentric chromosomes) occur with a prevalence of ~1 in 1000 in the general population (Gardner and Sutherland, 1996Go). By far the most common are the nonhomologous forms, i.e. those involving two different acrocentric chromosomes—either two different D group chromosomes (chromosomes 13, 14 and 15), two different G group chromosomes (21 and 22), or a D group and a G group chromosome. At meiosis, these rearrangements form trivalents, segregation of which may result in gametes nullisomic or disomic for one of the chromosomes involved in the rearrangement and consequently to a zygote with trisomy or monosomy for one of the chromosomes involved. Zygotes with monosomy are not compatible with life and most translocation trisomy conceptuses are expected to result in first trimester loss or earlier; however, some survive beyond the second trimester and to term.

The most common Robertsonian translocation is between chromosomes 13 and 14. This D/D translocation makes up ~75% of all Robertsonians (Gardner and Sutherland, 1996Go). The potential liveborn chromosomally unbalanced outcome of this is translocation trisomy 13 (Patau syndrome); there is an empirical risk of occurrence at second trimester prenatal diagnosis of <0.4% (Boué and Gallano, 1984Go; Gardner and Sutherland, 1996Go). There is also potential for uniparental disomy (UPD) for chromosome 14 following trisomy rescue, with an estimated risk of ~0.1–0.5% (Gardner and Sutherland, 1996Go). Translocation trisomy 14 is expected to result in first trimester loss. For der(13;14) carriers the overall risk of miscarriage is not expected to be significantly different from the background risk of 15% (Harris et al., 1979Go) (up to two miscarriages); however, some individuals with a der(13;14) present with infertility or recurrent spontaneous abortions. Other D/D Robertsonians are much less frequent and specific risks have not been derived; however, der(13;15) and der(14;15) might be expected to have similar risks to the der(13;14) (Gardner and Sutherland, 1996Go).

The most common Robertsonian after the der(13;14) is the der(14;21). The potential liveborn unbalanced outcome of this D/G Robertsonian is translocation trisomy 21 resulting in Down's syndrome; for female carriers, the empirical risk of occurrence at second trimester prenatal diagnosis is 15%, with a 10% risk of liveborn trisomy 21 plus a small risk of UPD 14, as before. For male carriers, the second trimester risk of translocation trisomy 21 is <0.5% (Boué and Gallano, 1984Go; Gardner and Sutherland, 1996Go), possibly due to the selective disadvantage for spermatozoa carrying an extra homologue of chromosome 21.

Other D/G Robertsonians which involve chromosome 21 may be expected to have similar reproductive risks to the der(14;21); those involving chromosome 22 have a lower risk since trisomy 22 has very limited potential to be viable.

Prenatal diagnosis has been available to carriers of Robertsonian translocations for many years. However, termination of pregnancy in the event of translocation trisomy is not an acceptable option for some couples and, for carriers of these translocations, there is growing interest in preimplantation genetic diagnosis (PGD) in conjunction with assisted conception using IVF or intracytoplasmic sperm injection (ICSI).

PGD is now offered by around 40 centres worldwide, including five in the UK. Fluorescence in-situ hybridization (FISH) is used for sex determination for X-linked conditions (Handyside and Delhanty, 1997Go; Kuo et al., 1998Go; Staessen et al., 1999Go; Pettigrew et al., 2000Go) and to investigate the status of embryos from couples where one partner carries a chromosome rearrangement. PGD for chromosome rearrangements began with the work-up of specific probes for each reciprocal or Robertsonian translocation (Munné et al., 1998aGo), which allowed discrimination between normal embryos and those carrying the balanced form of the translocation. A more general approach to reciprocal translocations became possible (Handyside et al., 1998Go; Scriven et al., 1998Go) with the development of subtelomeric probes specific for each chromosome (National Institutes of Health and Institute of Molecular Medicine Collaboration, 1996Go). This approach has led to successful pregnancies for reciprocal translocation carriers (Van Assche et al., 1999Go; Munné et al., 2000Go; Scriven et al., 2000Go) but does not allow the discrimination between ‘normal’ and ‘balanced’ embryos. A pregnancy rate of 19% per embryo transfer has been reported for chromosome rearrangement PGD (ESHRE PGD Consortium Steering Committee, 2000Go).

PGD for Robertsonian translocations has been undertaken successfully by several centres worldwide, both by polar body biopsy (Munné et al., 1998aGo,bGo), and by blastomere biopsy, where one or two blastomeres are removed from the embryo at the 6–10 cell stage (day 3 post-fertilization) (Escudero et al., 2000Go; Munné et al., 2000Go). However, some centres have reported high levels of mosaicism and chaos in embryos from Robertsonian translocation carriers (Conn et al., 1998Go, 1999Go) resulting in reduced pregnancy success rates. These observations have led to the suggestion that Robertsonian translocations may in some way predispose to malsegregation and/or lack of normal embryo development.

At our own centre we have assessed a number of Robertsonian couples and have undertaken to date seven cycles for four couples resulting in three pregnancies and four babies born. This paper presents our experience of Robertsonian couples exploring the possibility of PGD and the details of cycles carried out, including data indicating that previous reports of high levels of abnormal embryos in these translocation carriers may be at least in part artefactual.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Karyotyping and cytogenetic work-up for PGD
Karyotyping by G-banded metaphase chromosome analysis of cultured peripheral blood lymphocytes was performed using standard techniques. FISH studies on metaphase and interphase nuclei used the probe combinations and method described below. Both partners were assessed to ensure that the probes hybridized as expected; 10 metaphase spreads were examined from each partner and 200 interphase nuclei scored. Probe signal patterns in interphase nuclei were scored using conventional scoring criteria (Munné et al., 1998bGo). The probe combinations were assessed qualitatively with respect to signal discreteness and brightness, and the quantitative data were used to estimate the efficiency of each probe independently and the specificity of each assay. As is required in the UK, a licence was obtained from the Human Fertilisation and Embryology Authority (HFEA) to undertake PGD for each different probe combination.

Sperm FISH studies were carried out on mature sperm heads decondensed with 10 mmol/l dithiothreitol (DTT) using the probe combinations and method described below.

Ovarian stimulation, embryo culture and biopsy
These are as previously described (Scriven et al., 2000Go). Briefly, luteal phase down-regulation with intranasal gonadotrophin-releasing hormone agonist buserelin acetate (Suprefact; Hoechst) was followed by ovarian stimulation with 225 i.u. daily of recombinant FSH (Gonal-F, Serono). Human chorionic gonadotrophin (HCG; Profasi, Serono) was administered when at least three follicles were >18mm in diameter. Oocyte retrieval was carried out by ultrasound guided puncture 36 h after HCG administration. Oocytes and embryos were cultured in IVF sequential media (Science Scandinavia, Gothenberg, Sweden) under oil in 5% CO2 in air. For oocyte collection and overnight fertilization IVF medium was used. Normally fertilized embryos were transferred into G1.2 micro-drops on day 1, and G2.2 micro-drops at midday on day 2 for overnight culture. On day 3 the embryo biopsy procedure was performed after decompaction in Ca/Mg-free Scandinavian Embryo Biopsy Medium (Science Scandinavia) and acidified Tyrode's solution for zona drilling. Blastomeres were assessed for the presence of nuclei prior to biopsy, and one blastomere with a distinct nucleus was identified for removal from each embryo. The embryos were then washed and replaced into G2.2 micro-drops until embryo transfer the following day.

Spreading of blastomere interphase nuclei
Each single blastomere was transferred to a 1–2 µl drop of 0.2% Tween 20 in 0.01N HCl solution on a silanized slide. The blastomere was observed under a stereomicroscope during spreading to ensure a nucleus was present. The slides were left to air dry for ~20 min, washed in PBS for 5 min and dehydrated through an ethanol series.

FISH
Biopsied blastomeres were hybridized with probes as follows: Couple A: locus-specific indicator (LSI) 21 (SpectrumOrange; Vysis, Inc., USA) and a biotinylated 14q subtelomere probe (non-commercial); Couples B, C and D: QuintEssential 13 (digoxygenin-labelled, Appligene Oncor Lifescreen) and TelVysion 14q (SpectrumOrange; Vysis Inc., USA). Target material and probe were co-denatured at 75°C for 5 min, then hybridized for a minimum of 14 h at 37°C. Stringent washing to remove unbound probe was in 2x standard saline citrate solution (SSC) at 70–72°C for 5 min. Biotinylated probe was detected with fluorescein isothiocyanate (FITC)-Avidin (Vector Labs, Burlingame, CA); digoxygenin-labelled probe was detected with FITC-anti-digoxygenin (Boehringer Mannheim, UK). Preparations were counterstained with 4,6-diamino-2-phenyl-indole (DAPI)/ Vectashield (Vector Labs) and visualized using an Olympus fluorescence microscope, fitted with a 83 000 Pinkel filter set. Images were produced using Quips imaging software (Vysis, UK).

Non-transferred embryos were disaggregated and spread on day 4 or 5 and the nuclei obtained were hybridized with the same probe mixes as above.

Classification of FISH results
FISH error rates were calculated as described above. Biopsied cells were assigned a ‘normal/balanced’ status if FISH clearly indicated two signals for each chromosome tested, as defined by published scoring criteria (Munné et al., 1998bGo). Whole embryos were assigned ‘normal/balanced’ status if follow-up FISH showed a uniform ‘normal/balanced’ signal pattern within the limitations of the FISH assay (95% confidence interval (CI) of the specificity based on the lymphocyte work-up). For instance, where the FISH assay had a specificity of 88%, a follow-up embryo would be assigned ‘normal/balanced’ status if up to 3 out of 12 nuclei had deviant signal patterns, assuming no plausible mechanism for the abnormal signal patterns (for instance, a clear indication of a second cell line) could be invoked.

Biopsied cells and whole embryos were assigned an ‘unbalanced’ status if the nuclei showed a clear and consistent deviation from the ‘normal/balanced’ signal pattern.

Biopsied cells were assigned an ‘inconclusive’ status if the signal pattern was not a clear-cut normal result, usually because two signals were lying close together, which could either be scored as two signals, or as one ‘split’ signal. Whole embryos were assigned an ‘inconclusive’ status if the quality of the nuclei obtained resulted in poor hybridization which meant that a conclusive diagnosis was not possible.

Whole embryos were assigned a ‘mosaic’ status if there was evidence of two cell lines, based on >=2 nuclei for each cell line.

Whole embryos were assigned a ‘chaotic’ status if there was an insufficient proportion (i.e. outside the 95% CI, as above) of nuclei with uniform scorable signal patterns to assign the embryo to one of the other categories.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Case A:
The 39-year-old female partner had the karyotype 45,XX,der(14;21)(q10;q10). This couple had one phenotypically normal child (karyotype not known) and a previous pregnancy had been found to have translocation trisomy 21 (Down's syndrome). FISH work-up for this couple showed that the efficiencies of the LSI 21 probe and the 14q probe were 97.4 and 90.7% respectively. The assay specificity was 88.3%. Two cycles of PGD were carried out.

In the first cycle, 11 oocytes were collected, of which seven fertilized normally, and five embryos were biopsied on day 3. Of these, four gave a normal/balanced signal pattern in the biopsied cell, and the three showing the best morphology on day 4 were transferred. No pregnancy resulted. The fifth embryo showed a +14, +21 signal pattern. The non-transferred embryos and two abnormally fertilized embryos were spread on day 4. The follow-up FISH confirmed the diagnosis on the non-transferred, normal/balanced embryo, while the fifth embryo showed a chaotic, triploid complement. The results are detailed in Table IGo.


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Table I. Results from the seven PGD cycles carried out for couples A–D.
 
In the second cycle, 15 oocytes were collected, 10 fertilized normally and nine embryos were biopsied on day 3. Of these, three showed a normal/balanced signal pattern; all three were transferred, and a singleton pregnancy resulted. The couple opted for chorionic villus sampling at 12 weeks gestation and the fetus was shown to have the balanced form of the translocation: 45,XX,der(14;21)(q10;q10). A phenotypically normal girl was born at 38 weeks gestation. Of the non-transferred embryos, the biopsy diagnosis indicated one each of +21, –21, +14 and –14, and two which were inconclusive. Follow-up FISH after embryo spreading confirmed the trisomy 21 and monosomy 21 diagnoses, but the remaining nuclei from the embryos diagnosed as aneuploid for chromosome 14 were consistent with a normal/balanced chromosome complement. In addition, one of the embryos with an inconclusive biopsy diagnosis was also consistent with normal/balanced, while the other had degenerated and only one nucleus was found after spreading and no diagnosis was obtained. An abnormally fertilized embryo was shown to be mosaic triploid.

Therefore for this couple, where it was possible to reach a diagnosis and excluding abnormally fertilized embryos, the overall percentage of embryos consistent with alternate segregation (normal/balanced) was 77%, with 15% consistent with adjacent segregation (translocation trisomy or monosomy). Two of the normal/balanced embryos were diagnosed as unbalanced on biopsy, probably as a result of error in the FISH technique. These results are detailed in Table IGo.

Case B
The 34-year-old female partner had the karyotype 45,XX,der(13;14)(q10;q10) and presented with a history of four miscarriages, two of which had been karyotyped and found to be trisomy 14. FISH work-up showed that the efficiencies of the QuintEssential 13 probe and the TelVysion 14q probe were 97.2 and 91.0% respectively. The assay specificity was 88.5%. One cycle of PGD was carried out.

Ten oocytes were collected, of which eight fertilized normally using IVF. Eight embryos were biopsied on day 3, of which five showed a normal/balanced signal pattern (Table IGo). Three of these were transferred, resulting in a triplet pregnancy and the subsequent birth of two boys and a girl, all phenotypically normal and carriers of the translocation. Of the remaining embryos, two were diagnosed at biopsy as +13 and one as +14. The trisomy 14 and one of the trisomy 13 diagnoses were confirmed on follow-up; the third abnormal embryo was found to have a chaotic chromosome complement. Two abnormally fertilized embryos were also spread; one was a triploid mosaic and the other was haploid.

Of the embryos, 63% were therefore consistent with alternate segregation and 25% were consistent with adjacent segregation. These results are detailed in Table IGo.

Case C
The 37-year-old female partner had a 45,XX,der(13;14)(q10;q10) karyotype. No pregnancy had been achieved after four years without contraception. Two PGD cycles were carried out.

In the first cycle, six oocytes were collected of which two were fertilized using IVF. One embryo was suitable for biopsy and was diagnosed as normal/balanced and transferred. No pregnancy resulted. An abnormally fertilized embryo was spread and found to be haploid.

In the second cycle, five oocytes were collected of which two were suitable for injection using ICSI. Both resulted in embryos suitable for biopsy, were diagnosed as normal/balanced and transferred. No pregnancy resulted (see Table IGo).

Case D
The 35-year-old male partner (female partner was 34 years) had the karyotype 45,XY,der(13;14)(q10;q10) and presented with oligozoospermia (0.2–2x106/ml). The couple had not previously achieved a pregnancy. Sperm FISH indicated that 14% of gametes were aneuploid for either chromosome 13 or chromosome 14. Two cycles of PGD were carried out.

In the first cycle, five oocytes were collected and four fertilized normally after ICSI. Four embryos were biopsied on day 3; three of these were diagnosed as normal/balanced and two were transferred, but no pregnancy resulted. The third normal/balanced embryo was spread and found to be normal/balanced on follow-up. The fourth embryo was inconclusive on biopsy and found to have monosomy 14 on follow-up.

In the second cycle, five oocytes were collected and all fertilized normally after ICSI. Five embryos were biopsied on day 3, of which three were diagnosed as normal/balanced and transferred on day 5. Of the two non-transferred embryos, one was diagnosed as +13 and one as –14. However, in both cases the follow-up was inconclusive. Of the embryos, 67% were therefore consistent with a normal/balanced complement for the translocation chromosomes. A singleton pregnancy resulted and the couple declined prenatal diagnosis. A detailed fetal anomaly scan at 20 weeks gestation showed significant fetal abnormalities including agenesis of the corpus callosum, a neural tube defect and a ventral septum heart defect. The fetal karyotype was found to be primary trisomy 18 following amniocentesis. This abnormality was probably unrelated to the translocation, but an interchromosomal effect cannot be ruled out (Blanco et al., 2000Go).

Case E:
The 41-year-old male partner (female partner was 37 years) (karyotype 45,XY,der(13;14)(q10;q10)) had a sperm count within the normal range. The translocation had been an incidental finding and the fertility of the couple was not established. Sperm studies showed that 1.5% of spermatozoa were disomy 13 or 14; PGD was not recommended because the couple have a high chance of achieving a viable, chromosomally normal pregnancy without PGD.

These results are summarized in Tables I and IIGoGo. Figure 1Go shows two different embryo biopsies and follow-up results.


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Table II. Summary of the factors taken into consideration when counselling for PGD and the outcome for the five couples described in this paper
 


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Figure 1. Embryo biopsy and follow-up nuclei from two embryos. The nucleus from the biopsied cell is contained within the white box; the remaining nuclei were obtained following lysis of the whole embryo on day 4. (a) An embryo consistent with monosomy 21 from a der (14;21) cycle illustrating that unbalanced chromosome complements not expected to survive to prenatal diagnosis are found in cleavage stage embryos. The dimorphic nuclei contained within the red box suggest post-zygotic nondisjunction of chromosome 21. However, further investigation with probes for additional chromosomes showed a random tetraploid distribution of chromosomes between the two nuclei and it is highly likely that both nuclei came from a single binucleate blastomere. (b) An embryo consistent with translocation trisomy 13 from a der(13;14) cycle.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Of the normally fertilized embryos described here, 20% were the result of abnormal segregation of the translocation. This is considerably higher than the theoretical risks at prenatal diagnosis, probably because in vivo most abnormal embryos would fail to establish a pregnancy. The screening out of embryos with an unbalanced product of the Robertsonian prior to transfer would be expected to increase the chance of a successful pregnancy. Since overall ongoing pregnancy rates after PGD are of the same order as those which follow IVF/ICSI performed for infertility, PGD may be seen as a useful adjunct to assisted conception for couples with Robertsonian translocations who also have fertility problems. This is regardless of the nature of the Robertsonian or of the empirical reproductive risks associated with it.

However, counselling is necessary for couples with proven fertility. A history of recurrent pregnancy loss may not be associated with the translocation, particularly in the case of 13;14 Robertsonian translocations; this link can only be established by karyotyping the products of conception, as in case B above, where two out of four miscarriages had been shown to be trisomy 14. If PGD is requested for couples where a link has not been established, it should be considered whether it is appropriate to subject a fertile woman to IVF procedures when the role of the translocation is not known. The possibility of other contributing factors such as antiphospholipid syndrome should be investigated thoroughly and the couple counselled accordingly.

The empirical reproductive risks for male carriers of 13;14 Robertsonian translocation carriers are low. Sperm FISH using probes for the translocation chromosomes can be used to establish the level of aneuploidy, and in the case of a normal sperm count and low aneuploid levels, PGD may not be indicated, as in Case E. For some males who present with oligozoospermia and a Robertsonian translocation, such as Case D, ICSI may be necessary to overcome the infertility, in which case PGD would be a useful adjunct, as discussed above.

High levels of mosaicism and chaos in embryos from Robertsonian translocation carriers have been reported (Conn et al., 1998Go, 1999Go). These authors found only 13% of the embryos tested were normal or balanced for the translocation chromosomes. In the cycles reported here, excluding the abnormally fertilized embryos, 70% of embryos were normal or balanced for the translocation chromosomes. Only two embryos (6%) were chaotic, and true mosaicism was only seen in the abnormally fertilized embryos (Table IGo). Some embryos had tetraploid cells, indicating failure of karyokinesis and/or cytokinesis in the cell sampled and regarded as a normal observation. These embryos were therefore not classified as mosaic. Our findings differ from the published figure of 51% of embryos classified as mosaic or chaotic; these high levels may be a reflection of embryo culture conditions (Scriven et al., 2000Go). We conclude that Robertsonian translocations do not predispose to abnormal cell division in cleavage-stage embryos, although it remains possible that some couples may produce a high proportion of chromosomally abnormal embryos (Munné et al., 1996Go; Delhanty et al., 1997Go), possibly due to defects in cell cycle control mechanisms not associated with any chromosome rearrangements.

In conclusion, this paper does not support the contention that Robertsonian translocations predispose to embryos with abnormal cleavage divisions. PGD can therefore be considered, and has been shown to be an effective strategy for carriers of these chromosome rearrangements. The lack of a successful outcome for one of the couples described is likely to be due to overriding fertility problems, unconnected with the translocation. We are hopeful that a successful pregnancy may be established for this couple in a future cycle. In any case of subfertility, PGD may be viewed as a valuable screen for imbalance, even where the risk of viable chromosome abnormality is low. Counselling of couples carrying these translocations should take into account the previous obstetric history of the couple and other carriers in the family, in conjunction with the established risk figures for miscarriage and chromosome abnormality at birth; PGD may not always be indicated.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to acknowledge the contribution of the other members of the Guy's and St Thomas' Centre for PGD.


    Notes
 
4 To whom correspondence should be addressed. E-mail: paul.scriven{at}gstt.sthames.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 31, 2001; accepted on August 7, 2001.