Molecular Human Reproduction, Vol. 5, No. 7, 682-690,
July 1999
© 1999 European Society of Human Reproduction and Embryology
Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22)
1 Centre for Medical Genetics and 2 Centre for Reproductive Medicine, University Hospital, Dutch-speaking Free University of Brussels (Vrije Universiteit Brussel) Belgium, and 3 First Department of Obstetrics and Gynaecology, University of Milan, Milan, Italy
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In this study we describe the pre-clinical development and clinical application of preimplantation genetic diagnosis (PGD) by fluorescence in-situ hybridization (FISH) for two non-related carriers (one male and one female) of the most common balanced reciprocal translocation: t(11;22)(q25;q12). For the couple with the female carrier, enumeration of the sex chromosomes in the embryos was also indicated (husband: 47,XXY karyotype). Four-colour FISH analysis was performed on six blastomeres from three embryos. No embryo transfer was possible because all the embryos were unbalanced. Three PGD cycles, with two-colour FISH, were carried out for the couple with the male translocation carrier. A total of 35 embryos were biopsied and diagnosed by FISH; nine out of the 35 embryos (25.7%) were normal and seven of them were transferred (two embryos from the first and four from the third cycle), six out of 35 embryos (17%) were unbalanced, three out of 35 embryos (5.7%) were triploid or polyploid, 10 out of 35 embryos (28.6%) were mosaic and seven out of 35 embryos (20%) were chaotic. Diagnosis failed in 2.9% of the embryos. The spermatozoa of the male carrier were also analysed using three-colour FISH. Only 29.1% of the sperm cells seemed to be balanced or normal. By choosing probes lying on both sides of the breakpoints and by using a combination of sub-telomeric or locus-specific probes and centromeric probes, the use of three-colour FISH enabled detection of all the imbalances in sperm and/or cleavage-stage embryos in the patients. This may improve risk assessment and genetic counselling in the future for translocation carriers.
FISH/PGD/reciprocal translocation/sperm analysis
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Introduction |
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Balanced reciprocal translocations are among the most frequent structural chromosomal aberrations in man. An estimate of the birth prevalence is reported (Nielsen and Wohlert, 1991
), 50 balanced reciprocal translocations were found among 34 910 unselected live births (one out of 712; 0.14%). Except for one author (Fryns et al., 1986), who found an excess of mental retardation and/or congenital malformation in apparently inherited reciprocal translocations, carriers of balanced translocations are in general considered to be phenotypically normal (Cans et al., 1993). However, they have an increased risk of producing chromosomally unbalanced offspring (Cans et al., 1993). The risk of miscarriages, stillbirths or chromosomally unbalanced live births with multiple congenital anomalies depends on the probability of different types of unbalanced gametes being produced and, after fertilization of such gametes, on the probability of different types of unbalanced embryos being able to survive.
The 11q;22q translocation seems to be the most frequently observed reciprocal translocation in humans; approximately 110 families from different ethnic and racial groups have been described (Fraccaro et al., 1980, Iselius et al., 1983). Since, among viable offspring, the tertiary trisomy for the derivative chromosome 22 [der(22)] due to 3:1 non-disjunction was the only imbalance observed (Iselius, 1983), the minimal risk of an unbalanced karyotype at birth resulting from this translocation was estimated to be 3.75 ± 1.22% (nine out of 241) for female carriers of the translocation and <0.65% (none out of 77) for male carriers (Stengel-Rutkowski et al., 1988). According to the same authors, the early abortion rate was 26.87 ± 2.86% (65 out of 241) for female carriers and 22.08 ± 4.73% for male carriers (17 out of 77).
The birth of chromosomally unbalanced offspring to translocation carriers can be prevented through prenatal diagnosis followed by termination of pregnancy. This approach is often a stressful and traumatic experience (Lloyd and Laurence, 1985; Black, 1989). Since translocation carriers are often faced with repetitive miscarriages or infertility (Cans et al., 1993) preimplantation genetic diagnosis (PGD), a relatively new procedure, provides an alternative reproductive option. Using assisted reproductive technology (ART), preimplantation embryos are obtained and on day 3 post-insemination one or two blastomeres can be biopsied and genetically investigated. Only chromosomally balanced embryos are transferred to the uterus to lead to pregnancy and the birth of an unaffected child. Several healthy normal children have been born after PGD of X-linked genetic diseases, single gene defects and aneuploidy (Harper et al., 1996; Verlinsky et al., 1996; Liebaers et al., 1998).
Fluorescence in-situ hybridization (FISH) is the method of choice for the enumeration of specific chromosomes in interphase cells from preimplantation embryos. Due to the complex organization of DNA in interphase nuclei, specific breakpoint-spanning yeast artificial chromosome (YAC) DNA probes must be developed for each reciprocal translocation to detect the imbalances (Tkachuk et al., 1991). Since the development of translocation-specific probes requires labour-intensive techniques, PGD for translocations has not been applied routinely. However, PGD cycles for Robertsonian translocations t(13;14), t(13;21) and t(14;15) on cleavage-stage embryos (Conn et al., 1998; Munné et al., 1998) have already been performed. These authors used YAC–DNA probes to detect unbalanced embryos. PGD for reciprocal translocations [t(5;8)(p13;p23), (Pierce et al., 1998); t(12;20)(p13.1;q13.3X); t(3;4)(p24;p15);t(6;11)(p22.1;p15.3); (Munné et al., 1998a)] have also been reported. These authors used a combination of YAC–DNA probes and/or 
-satellite and/or locus-specific probes. In the first study only imbalances could be detected with their probe mixture, which were compatible with live births and resulted from 2:2 meiotic segregations (Pierce et al., 1998). Tertiary aneuploidies and interchange aneuploidies causing miscarriages were not detectable. The latter study was able to detect all possible imbalances with their probe mixtures (Munné et al., 1998).
In this paper we describe the development and outcome of two clinically applied PGDs for two unrelated carriers of the balanced reciprocal translocation involving the long arms of chromosomes 11 and 22 [t(11;22)(q25;q12)]. The first couple was referred to our centre because of male infertility due to azoospermia. GTG-banded karyotyping of peripheral blood lymphocytes from the couple, showed the 36 year old husband as having a 47,XXY chromosome complement in 20 analysed metaphases and his 36 year old wife was a carrier of the t(11;22)(q25;q12) translocation. The second couple suffered from repeated spontaneous miscarriages (G5P0A5). In this case the 29 year old husband was a carrier of the translocation (11;22)(q25;q12) and his 29 year old wife had a 46,XX normal karyotype. The objective of the PGD for both couples was to obtain an ongoing pregnancy involving a fetus with a normal or balanced chromosome complement in the first place and to avoid any miscarriage due to imbalance or pregnancy involving a fetus with the viable chromosome der(22) imbalance. Since analysis of spermatozoa of Klinefelter patients shows a significant increase in 24,XY-bearing sperm cells (Cozzi et al., 1994; Chevret et al., 1995; Martini et al., 1996), evaluation of the number of sex chromosomes in embryos of the first couple was also indicated. Spermatozoa from the male translocation carrier were analysed by FISH in order to collect data on the different modes of meiotic segregation.
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Preparation of metaphase chromosome spreads from patient peripheral lymphocytes
Blood (10 ml) was drawn into tubes containing heparin to prevent clotting. Metaphase spreads were made from phytohaemagglutinin-stimulated peripheral lymphocytes using standard cytogenetic techniques (Rooney and Czepulkowski, 1992
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Preparation of the semen sample from the male carrier of the translocation
The semen sample was washed in phosphate-buffered saline (PBS), pH 7.4 and centrifuged at 280 g for 10 min. The pellet was fixed in acetic acid/methanol (1:3) and stored at –20°C until slide preparation. Spermatozoa were spread on Superfrost Plus glass slides (Kindler GmbH, Freiburg, Germany) and were kept at room temperature for 1–3 days. Then the slides were washed in 2x sodium chloride/sodium citrate (SSC). Decondensation of the spread spermatozoa was performed by incubation for 5 min in 1 M Tris buffer, pH 9.5, containing 25 mM dithiothreitol (DTT) (Martini et al., 1995). After this decondensation, the slides were washed in 2x SSC and in 1x PBS, dehydrated through an ethanol series and finally air-dried.
Human preimplantation embryos
Spare embryos unsuitable for transfer or cryopreservation were used for pre-clinical experiments according to the research protocol, which was approved by the ethical committee of our Medical Campus. PGD was carried out as we have described previously (Staessen et al., 1998). Briefly, preimplantation embryos were generated using intracytoplasmic sperm injection (ICSI) (Van Steirteghem et al., 1995; De Vos et al., 1997; Joris et al., 1998) and cultured for 3 days up to the 5–10-cell stage. Depending on the developmental stage of the embryo, either one or two blastomeres with a distinct nucleus were removed (Sermon et al., 1997). After biopsy the embryos were returned immediately to normal culture conditions. Whole embryos used for the pre-clinical work as well as biopsied blastomeres used for PGD were spread on Superfrost Plus glass slides (Kindler GmbH, Freiburg, Germany) using the HCl/Tween method described in detail previously (Coonen et al., 1994). After spreading, the slides were left for 30 min to air dry, washed in 1x PBS for 5 min and dehydrated through an ethanol series.
Fluorescence in-situ hybridization
Slides with spread blastomeres were fixed and pre-treated as described previously (Staessen et al., 1998). Slides with spread and decondensed spermatozoa were only pre-treated as described below. First the nuclei were digested with pepsin (from porcine stomach mucosa: 100 µg/ml; Sigma) in 0.01 N HCl at 37°C for 15 min at 37°C. After rinses in Milli Q water and 1x PBS, the nuclei were fixed in Carnoy's fixation solution (acetic acid / methanol 1:3 vol/vol) at 4°C for 10 min. After fixation, the slides were rinsed in 1x PBS buffer and then in Milli Q water and finally dehydrated through an ethanol series.
The probe mixture for the blastomere analysis of the first couple and for the sperm analysis of the translocation carrier, was prepared as follows: 0.5 µl of Green and 0.5 µl of Orange fluorochrome-labelled probes for chromosome 11 -satellite (CEP 11, Vysis, Downers Grove, IL USA); 2 µl of the two colour probe mixture containing the Spectrum Orange TUPLE 1 probe (mapping at region 22q11.2) and the Spectrum Green Arylsulphatase A gene probe (mapping at region 22q13) (LSI DiGeorge/VCFS Region probe, Vysis, Downers Grove, IL, USA); 1 µl of Aqua fluorochrome-labelled probe for chromosome X -satellite and 1 µl of Aqua fluorochrome-labelled probe for chromosome Y -satellite III were mixed with 14 µl LSI hybridization buffer (Vysis, Downers Grove, IL USA) and 1 µl of Milli Q water to reach a final volume of 20 µl.
For the PGD of the second couple, the X and Y probes were omitted from the previous probe cocktail and only 7 µl of the LSI hybridization buffer were added to reach a final volume of 10 µl. Two µl of the appropriate probe mixtures were applied to the dehydrated slides and covered with a 12 mm round coverslip. After simultaneous denaturation of the probes and the target-DNA at 75°C for 1 min the coverslips were sealed with rubber cement and hybridization took place for 4–5 h in a humidified chamber at 37°C. The post-hybridization washing steps were performed according to the recommendations of the manufacturer. Briefly, the slides were washed for 2 min in 0.4x SSC at 73°C followed by several dips in 2x SSC/0.1% NP-40 at room temperature. Finally, the slides were mounted in Vectashield antifade medium (Vector Laboratories) containing 125 ng/ml 4',6-diamidino-2-phenyl-indole (DAPI) counterstain. The nuclei were examined using a Zeiss-Axioskop fluorescence microscope with the appropriate filter sets (filter 10 for the Fluorochrome Green, filter 02 for the fluorochrome DAPI, filter Omega for the Orange fluorochrome and the double bandpass filter Aqua/Orange from Vysis). As a result, the centromeric region of chromosome 11 was visualized as an orange signal, the 22q11.1 region as a red signal, the 22q13 as a green FISH signal and the X centromeric region and the Yq12 region as a blue FISH signal if the probes for the sex chromosomes were added (see Figure 1a). The balanced form (presence of two normal chromosomes 11 and 22 and the two translocation chromosomes 11 and 22) or the absence of the translocation could easily be visualized at the microscope as the presence of two orange, two green and two red FISH signals in case of diploid cells (blastomeres, lymphocytes) (see Figure 1a) or one FISH signal of each colour in case of haploid cells (spermatozoa). So the unbalanced forms of the translocation could also easily be recognized (see Figure 1d–g). Embryos formed after fertilization of a normal gamete with a gamete with the adjacent 1 segregation of the translocation will possess either two normal chromosomes 11 and one normal and one derivative 22 (two orange signals + two red signals + one green signal) or one normal and one derivative chromosome 11 and two normal chromosomes 22 (two orange signals + two red signals + three green signals: Figure1d). The presence of three orange, one red and two green signals (two normal and one derivative chromosome 11 plus one normal 22) or one orange, three red and two green signals (one normal chromosome 11 and two normal and one derivative 22: Figure 1e) in an embryo will point to adjacent 2 segregation. Embryos with a tertiary trisomy for the translocation will show three orange, two red and three green signals (Figure 1f) (two normal and a derivative 11 and two normal chromosomes 22) or two orange, three red and two green signals (two normal chromosomes 11 and two normal and a derivative 22). Finally, embryos with an interchange trisomy will show three orange, two red and two green signals (Figure 1g, two normal plus a derivative chromosome 11 and one normal plus a derivative 22) or two orange signals, three red and three green signals (normal plus a derivative chromosome 11 and two normal plus a derivative chromosome 22).
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Two independent observers using an established scoring criteria (Hopman et al., 1988
) interpreted the signals in the blastomeres and in the spermatozoa. |
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Pre-clinical FISH analysis
The different modes of meiotic segregations were considered after determination of the translocation breakpoint position of high-level G-banded metaphases (between 550 and 850 bands visible) as well as after FISH with painting probes specific for chromosomes 11 and 22 identifying the translocation as a `single segment imbalance'-type (Stengel-Rutkowski et al., 1988
) (see Figure 1
). As a consequence of the translocation breakpoint located at band 22q12, the LSI DiGeorge probe mixture mapping at the regions 22q11.2 and 22q13 and detected as a red and a green signal respectively on the normal chromosome 22 was considered to be suitable for PGD purposes. In our opinion, triple-colour FISH was considered sufficient to detect all possible imbalances for couple 2, i.e. the -satellite chromosome 11 probe ratio labelled is used for enumeration of the normal chromosome 11 and the LSI DiGeorge probe mixture is used for enumeration of the normal chromosome 22.
Since enumeration but not identification of the sex chromosomes was important for couple 1, 4-colour FISH was necessary for PGD, i.e. the -satellite chromosome X and the classical satellite Yq12 probe were added to the mixture described previously (see Figure 1c). Although the probes for the sex chromosomes were labelled with the same fluorochrome, identification was possible. The FISH signal of the Yq12 probe was much larger, due to the presence of a large heterochromatic Yq12 region, than the one for the X centromeric region.
The hybridization specificity and efficiency of probes were tested on lymphocytes of both carriers of the translocation and on a control individual with a normal constitutional karyotype. The two probe mixtures (with and without the sex chromosomes) showed good specificity and signal intensity with minimal background fluorescence on spreads of control lymphocytes.
The expected number of hybridization signals for three- and four-colour FISH were detected in 89% of lymphocyte nuclei of a control individual and in 90 and 89% of lymphocyte nuclei of the male and female carrier respectively.
Tests were also performed on at least five nuclei of spare embryos (from one or three pronuclear eggs, or two pronuclear eggs arrested during cleavage or showing multinucleated blastomeres) obtained from unrelated in-vitro fertilization (IVF) cycles. It was impossible to determine the accuracy of chromosome detection, as it is known that such embryos are often chromosomal mosaics (Coonen et al., 1994; Munné et al., 1994a,b; Harper et al., 1995; Delhanty et al., 1997). Nevertheless, the intensity of the FISH signals themselves was even better than in lymphocyte nuclei. Previous studies have demonstrated that FISH efficiency is indeed higher in blastomeres when they are fixed individually, because larger fixed nuclear diameters are achieved, avoiding signal overlapping and allowing better probe penetration (Munné et al., 1996b).
Clinical FISH analysis in four PGD cycles
Case 1: mother 46,XX,t(11;22)(q25;q12); father 47,XXY
The PGD cycle was carried out with four-colour FISH to exclude an imbalance of chromosomes 11, 22 and an aneuploidy of the sex chromosomes. A total of 20 oocyte–cumulus complexes were retrieved and 15 were injected with spermatozoa present in the ejaculate (total count: 12 spermatozoa/ml). On day 3 of development, three embryos (60%) had reached at least the 5-cell stage and were of sufficient quality to undergo biopsy. Diagnosis was possible on six blastomeres (two per embryo). The results of the FISH diagnosis are given in Table I. No transfer could be carried out because all the nuclei were unbalanced. In two embryos the abnormal result was confirmed on day 4, the remaining embryo was no longer suitable for analysis.
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Case 2: 46,XY,t(11;22)(q25;q12)
For this couple, 3 PGD cycles were performed to exclude imbalances of chromosomes 11 and 22 using three-colour FISH. In all, 55 oocyte–cumulus complexes were retrieved and 48 were inseminated. In all, 35 embryos (97%) had reached at least the 5-cell stage and were of sufficient quality to undergo biopsy. Diagnosis was possible on 57 blastomeres. The results of the FISH diagnosis are given in Table II
. In the first and the third cycle 3 and 4 embryos respectively were observed with the normal number of FISH signals and considered as normal with respect of its content of chromosomes 11 and 22. They were, therefore, transferred but no pregnancy ensued. No transfer could be carried out in the second cycle because all the nuclei were unbalanced.
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Most of the embryos diagnosed as abnormal by PGD were re-investigated on day 4 (Table II
). The abnormal result was confirmed in 23/27 embryos. In embryo 18, which was diagnosed as abnormal by PGD, FISH post-PGD revealed a normal number of signals in five out of five blastomeres. In summary, nine out of 35 (25.7%) embryos were considered normal, seven of which were transferred, six out of 35 (17%) embryos were unbalanced, two out of 35 (5.7%) were triploid or polyploid, 10 out of 35 (28.6%) were mosaics and seven out of 35 (20%) were chaotic. Diagnosis failed in one out of 35 (2.9%) embryo.
Results of FISH analysis on spermatozoa of the 46,XY,t(11;22)(q25;q12) carrier
Four-colour FISH was used on spermatozoa of the male carrier of the translocation in order to collect data on the different modes of meiotic segregation and on sex distribution (see Table III). In all, 1012 spermatozoa were analysed: 29.1% of the spermatozoa were balanced or normal and 36.4 and 34.6% respectively, were unbalanced as a result of adjacent 1 and 2 and 3:1 meiotic segregations. The distribution of sex chromosomes was comparable, i.e. 47.8% of the cells carried on X chromosome and 52.2% on Y. The enumeration of the sex chromosomes showed no abnormalities.
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Discussion |
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It is known that carriers of reciprocal translocations have an increased risk of chromosomally unbalanced offspring at birth and for this reason prenatal diagnosis is offered, but they also have an increased risk of recurrent miscarriages. To avoid unbalanced pregnancies, PGD, which is an early form of prenatal diagnosis (Lissens and Sermon 1997
), can now be offered for these cases. Conventional karyotyping and comparative genomic hybridization (Kallioniemi et al., 1992) are used to detect structural abnormalities in metaphase cells from cultures but are not apt to produce results from a single interphase cell. The present study demonstrates that chromosomal imbalances expected in embryos of carriers of the t(11;22) translocation can be detected, in an efficient and reliable way, by the FISH technique using the probes shown in Figure 1. It has to be noted that the balanced form of the translocation cannot be distinguished from the normal complement. Unbalanced embryos are not transferred and as a result the chances of unbalanced pregnancies can be efficiently reduced. However, where pregnancy occurs, a control chorionic villi sampling or amniocentesis is still recommended to exclude misdiagnosis.
Four PGD cycles were carried out for two couples with the t(11;22) translocation. In the first couple, the fertilization rate was very low due to the fact that the male had a 47,XXY karyotype. Only three out of five embryos were diagnosed and unfortunately all were unbalanced and could not be transferred. FISH post-PGD confirmed the abnormal results in two out of three embryos. No aneuploidy of the sex chromosomes was observed in any of the three embryos. Assuming that the gametes of the 47,XXY husband carry a normal haploid number of chromosomes 11 and 22, the modes of meiotic segregation of the t(11;22) carrier can be determined (see Figure 1). As a result of this, embryos 1 and 2 are the results of 3:1 segregation and embryo 3 is the result of adjacent 1 segregation.
Three PGD cycles were carried out for couple 2 with the male carrier of the t(11;22) translocation. In all, 48 out of 55 oocytes were collected and 35 of these were suitable for biopsy and PGD. Three and four embryos were transferred in the first and third cycle respectively, but no pregnancy resulted. Combined results for the three cycles show 25.7% (nine out of 35) of embryos analysed were normal and 74.3% (26/35) were abnormal. These abnormalities were categorized as 17% unbalanced, 5.7% triploid and polyploid, 28.6% mosaics and 20% chaotic (Table II).
In the case of embryo 18 an abnormal result was detected by PGD, while FISH post-PGD showed a normal embryo in five out of five blastomeres. Two explanations for this observation are possible: (i) the FISH signals of 22q11 were overlapping (three-dimensionally) at the time of the initial PGD so that instead of two 22q11 signals, only one was observed. In the other blastomere, three 22q11 signals were counted according to established scoring criteria (Hopman et al., 1988). However, these three signals could have been the result of a split signal at one of the 22q11 loci; (ii) two abnormal and five normal blastomeres could have been present in the embryo. At the time of the PGD, the two blastomeres of embryo 18 were diagnosed as abnormal and so the embryo was not transferred, although for our further calculations the embryo was considered normal.
Analysis of the untransferred embryos showed that 28.6% of them were mosaics. Mosaics in embryos is a common finding in many studies using multicolour FISH. Levels of mosaics ranging from 24 to 53% have been found in embryos with inferior morphology or in embryos at risk of a Robertsonian translocation (Benkhalifia et al. 1993; Munné et al., 1993; Harper et al. 1995; Delhanty et al. 1997; Conn et al. 1998). A mosaic as high as 70% in arrested spare embryos has been found previously (Munné et al., 1994).
Seven out of the 35 embryos (20%) were categorized as chaotic. This phenomenon has already been observed in many studies on spare embryos (Harper and Delhanty 1996; Delhanty et al. 1997; Laverge et al. 1997). Previous studies (Munné et al., 1996; Delhanty et al., 1997) have shown that some patients do produce significantly higher levels of chaotic embryos than others. It can be concluded that the most accurate and reliable diagnosis can be made only if two blastomeres are biopsied from an embryo.
The modes of meiotic segregation from embryos from couple 2 were determined assuming that the woman was producing gametes with a normal haploid set of chromosomes 11 and 22 (Table II). Embryos 1, 28 and 33 were the result of adjacent 1 type of segregation after 2:2 disjunction and embryos 4 and 30 of adjacent 2. Embryo 17 was an example of tertiary trisomy as a result of 3:1 non-disjunction.
For most reciprocal translocations, unbalanced offspring result most frequently from 2:2 adjacent 1 segregation of chromosomes. Nevertheless, for the 11q;22q translocation it is the tertiary trisomy for the der(22) due to 3:1 non-disjunction that has always been observed among viable offspring (Iselius, 1983). This type of abnormality was observed only once among the unbalanced embryos. This preferential 3:1 segregation mode observed in live-born babies can be explained as a result of characteristics of the specific chromosomes involved in the translocation or the production of non-viable conceptions by other types of segregations (Iselius, 1983).
We also performed FISH on spermatozoa of the male carrier of the translocation. We observed that all possible 2:2 (alternate, adjacent 1, adjacent 2) and 3:1 segregations were present (Table III). In all, 1012 spermatozoa were analysed: 29.1% were balanced and 70.9% were not balanced including only 2.1% showing tertiary trisomy. Another study (e.g. Martin, 1984) which studied sperm chromosomes from an 11–22 carrier support this. Only 13 sperm chromosome complements were analysed and 77% appeared to be unbalanced, which is comparable with our results. In 1984, before the PGD period, this author concluded that the results indicated that adjacent-type segregations either produce non-viable conceptions or were unable to fertilize. In this study we observed unbalanced embryos as a result of adjacent 1 and 2 segregation, supporting the fact that all types of unbalanced gametes are able to fertilize but that unbalanced embryos of the adjacent-1 and -2 types are not viable. It has to be noted also that ICSI was used as insemination method and that this may also have an influence on the modes of segregation that were observed. These results offer a risk estimation that is reliable enough to be used for genetic counselling. Yet, it has to be taken into account that exchanges of interstitial chiasmata between the centromere and the translocation breakpoint render alternate and adjacent 1 segregation products which are cytogenetically indistinguishable (Armstrong and Hultén, 1998; Martini et al., 1998).
This contribution is the first report of PGD for a translocation where all the segregations can be determined using commercially available probes. Recently, a study on PGD of translocations using case-specific probes was published (Munné et al., 1998b). Using this approach, it is possible to determine not only the different segregation types but also the difference between the balanced and unbalanced form of the translocation. An advantage of this is that if sufficient embryos with a normal set of chromosomes involved in the translocation were available, they could be transferred preferentially. Presently, PGD of translocations with case-specific probes is feasible but requires a major effort in developing translocation-probes (Munné et al., 1998). Two-colour FISH with commercially available probes has been used to detect only the viable imbalances (Pierce et al., 1998), but other possible imbalances, i.e. after 3:1 segregation, might be possible. These embryos would then be transferred and can result in miscarriages. This technique, in other words, does avoid the birth of chromosomally unbalanced offspring but miscarriages will still occur. Although this novel procedure aims at the selection of embryos for transfer of only balanced offspring, prenatal diagnosis will still be offered in the case of pregnancy, so as to exclude any misdiagnosis. A possible alternative for blastomere biopsy is polar-body biopsy for cases in which the translocation is of maternal origin. The test is based on the observation that first-polar-body chromosomes are mostly in metaphase shortly after oocyte retrieval and therefore FISH analysis with chromosome-painting probes can be performed easily for PGD of translocations (Munné et al., 1998a). So far we have had no experience with this technique and, furthermore, only female carriers can be considered. In our study we chose a more general approach for the detection of imbalances which is applicable to both male and female carriers. By choosing probes lying on both sides of the breakpoints, a balanced form of the translocation cannot be distinguished from an unbalanced. In fact, using a combination of sub-telomeric or locus-specific probes and centromeric probes, all the imbalances can be detected by three-colour FISH. Few sub-telomeric probes are as yet commercially available and development in this field will allow PGD to be performed for any translocation. More information on meiotic segregations through evaluation of sperm and/or cleavage stage embryos will also become available. This may improve risk assessment and genetic counselling in the future.
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Acknowledgments |
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The authors wish to thank the clinical, scientific, nursing and technical staff of the Centre for Medical Genetics and Reproductive Medicine and especially Hubert Joris, Hilde Van De Velde and Anick De Vos. Furthermore, we are grateful to Sylvie Mertens and Dirk De Smedt for technical assistance with the FISH-technique. We thank Frank Winter of the Language Education Centre for correcting the English text. Grants from the Flemish Fund for Medical Research are gratefully acknowledged.
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4 To whom correspondence should be addressed
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References |
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Submitted on November 23, 1998; accepted on April 12, 1999.
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