Molecular Human Reproduction, Vol. 5, No. 4, 382-389,
April 1999
© 1999 European Society of Human Reproduction and Embryology
Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis
1 Centre for Reproductive Medicine and 2 Centre for Genetics, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090 Brussels, Belgium
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In our centre we started using fluorescent in-situ hybridization (FISH) technique for sexing in couples with sex-linked diseases in May 1995. Probes specific for chromosomes X, Y and 18 were applied, allowing us to detect simultaneously both gender and ploidy status. The efficiency of the FISH procedure is 90.4% per biopsied blastomere or 95.2% per biopsied blastomere with a distinct nucleus visible at spreading. Up to December 1997, we treated 15 couples (20 treatment cycles) at risk for X-linked recessive disease and two couples with Yq deletion (two treatment cycles) with the aim of transferring only female embryos. In one cycle, no embryos suitable for biopsy were obtained and in five cycles no normal female embryos were available at diagnosis. In the remaining 16 cycles, transfer was possible and six pregnancies ensued: one miscarriage has occurred and six children have been born from the other five pregnancies. The implantation rate (fetal sacs) per transferred embryo was 20.8%. In 98 (61%) of the 161 diagnosed embryos, a diploid status was observed in one or in both biopsied blastomeres. In 10 out of the 161 (6.2%) embryos a heterogeneity among the two biopsied blastomeres was found: a diploid nucleus in one blastomere and a non-diploid pattern or binuclear status in the other. In the remaining 53 (32.9%) out of 161 diagnosed embryos, the biopsied blastomeres were abnormal. The embryos that were not transferred or frozen were further analysed. When two sex chromosomes and two autosomes were present in the biopsied blastomere, the sex determination of the biopsied blastomere was never in conflict with the sex determination in the rest of the embryo. Furthermore, if the biopsied cell was diagnosed as abnormal (triploid, aneuploid, chaotic) the embryo was indeed completely abnormal or at least mosaic. A FISH error could not be excluded in two embryos (1.2%); however, a wrong gender determination did not result from this.
FISH/PGD/X-linked disease
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The successful development of artificial reproductive technology, micromanipulation procedures on human embryos allowing blastomere biopsy and molecular genetic techniques on single cells has led to the clinical application of preimplantation genetic diagnosis (PGD). The first successful clinical application of PGD was reported by Handyside (1990). Using polymerase chain reaction (PCR) procedure, the presence of a Y-chromosome-specific sequence was determined in single blastomeres of couples carrying X-linked diseases. After the occurrence of a misdiagnosis due to amplification failure (Kontagianni et al., 1991), PCR-based methods now employ both X- and Y-specific sequences (Liu et al., 1994
) for sexing the embryo.
Meanwhile, fluorescent in-situ hybridization (FISH) was adapted for application to single cells and became the preferred method for gender determination. The efficiency of the technique is high (Harper et al., 1994) and the sex of an embryo can be unequivocally determined when two X-chromosome signals in the absence of a Y-chromosome signal or one X-chromosome and one Y-chromosome signal are detected (Griffin et al., 1992, 1994; Grifo et al., 1994; Veiga et al., 1994). Furthermore, multicolour FISH with probes for chromosomes X, Y and one or more autosomes has the additional advantage that it can detect simultaneously the sex, ploidy status and numerical abnormalities of the investigated chromosomes. The application of FISH to human embryos has led to the discovery that chromosomal mosaicism is common at the cleavage stage of arrested and/or abnormally developing embryos as well as in normally developing embryos (Coonen et al., 1994a; Munné et al., 1994a, 1995; Harper et al., 1995; Delhanty et al., 1997).
So far PGD has been performed in a limited number of centres worldwide. Because of the novelty of the technique, it is worthwhile reporting in detail on the first clinical experiences from the different centres in order to evaluate the accuracy, efficiency and reproducibility of the technique.
In May 1995 we started using sex determination by FISH for PGD of sex-linked diseases, resulting in a total of 22 treatment cycles by December 1997. The aim of the present study is to report our clinical experience with these PGD cycles. Embryos unsuitable for transfer or cryopreservation were donated for research which made it possible to evaluate the diagnostic accuracy of PGD on the biopsied blastomeres.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Patient characteristics and assisted reproductive techniques (ART)
A total of 17 couples at risk for sex-linked diseases were treated; 15 of the couples were at risk for an X-linked disease as mentioned in Table I
. Two couples had a Y deletion of the AZF region resulting in severe oligoasthenoteratozoospermia (OAT) requiring intracytoplasmic sperm injection (ICSI) as fertility treatment in any case. The couples wished to combine the treatment with PGD so as to have only female embryos replaced in order to avoid transmission of the deletion. The patients' ages, the husbands' semen characteristics and the prior history of the couples are summarized in Table I. The mean age (±SD) of the females was 30.8 ± 4.9 years and of the males 33.9 ± 5.7 years. Almost all couples with X-linked disease revealed normal sperm characteristics, with the exception of one couple (S16) with severe OAT requiring ICSI as fertility treatment. Previously 12 out of the 17 couples had conceived spontaneously. The outcome of the spontaneous pregnancies is summarized in Table I for each couple separately. In total, of the 32 spontaneous pregnancies obtained, two ended in the delivery of a genetically unaffected child, five ended in the delivery of a genetically affected child, 20 were terminated in the first trimester after prenatal diagnosis (PND), and five underwent spontaneous abortion.
|
Ovarian stimulation was performed by a desensitizing protocol using gonadotrophin-releasing hormone (GnRH) agonist in association with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) (Ubaldi et al., 1995
). Oocyte retrieval was carried out by ultrasound-guided puncture 36 h after HCG administration.
The oocytes were fertilized in one cycle (S2) by conventional in-vitro fertilization (IVF) (Staessen et al., 1995) and in the other cycles by ICSI (Van Steirteghem et al., 1995). Fertilization was assessed 16–18 h after injection or insemination. Embryo evaluation was performed on days 2 and 3 (Staessen et al., 1995). Grade A embryos are defined as embryos without anucleate fragments. Grade B embryos have blastomeres of equal or unequal size, with a maximum of 20% of the volume of the embryo filled with anucleate fragments. In a third category of grade C embryos, anucleate fragments are present in 20–50% of the volume of the embryo.
Pregnancy
Implantation was confirmed when two HCG concentrations at least 10 days following embryo transfer revealed a gradual increase. A clinical pregnancy was defined by an intrauterine gestational sac seen by vaginal ultrasound at least 5 weeks post-embryo transfer. An ongoing pregnancy was defined as a clinical pregnancy with a fetal heart beat >12 weeks. When a pregnancy was achieved we recommended the couple undertake a prenatal diagnosis, either chorionic villus sampling (CVS) or amniocentesis, in order to confirm the PGD diagnosis.
Embryo transfer and cryopreservation
In agreement with the normal transfer policy in our centre, two or three embryos were transferred. Spare female embryos of sufficient morphological quality were cryopreserved (Van den Abbeel et al., 1997).
Procedure of preimplantation diagnosis
Embryo biopsy
Embryos of grade A, B or C with at least four blastomeres were biopsied (Sermon et al., 1997) in the morning of day 3 after insemination or microinjection. From the 7-cell stage onward, two blastomeres per embryo were removed.
Blastomere spreading
The individually biopsied blastomeres were spread onto a Superfrost Plus glass slide (Kindler GmbH, Freiburg, Germany) using a 0.01 N HCl/0.1% Tween 20 solution (Coonen et al., 1994b; Staessen et al., 1996).
FISH diagnosis
Two different FISH procedures were used. In the first eight cycles, the FISH procedure of Staessen et al. (1997) was used. In the other cycles, we used a protocol modified as follows: (i) fixation after spreading in methanol:acetic acid (3:1 v/v) instead of in a 1% paraformaldehyde solution; and (ii) post-hybridization steps according recommendations of the probe providing company. Although the efficiency was not different between both procedures, with the second procedure the signals were of higher intensity and defined more clearly.
Briefly, the nuclei were digested with pepsin (from porcine stomach mucosa: 100 µg/ml; Sigma) in 0.01 N HCl for 15 min at 37°C. The slides were rinsed in Milli Q water and 1x phosphate-buffered saline (PBS) and fixed for 10 min in methanol:acetic acid (3:1 v/v) solution at 4°C. After fixation, the slides were first rinsed in 1x PBS and then in Milli Q water and dehydrated by means of an ethanol series. The triple-target FISH technique was performed using directly-labelled DNA probes specific for chromosomes X (Vysis, Alpha Satellite DNA, Spectrum Green), Y (Vysis, Alpha Satellite III DNA (DYZI locus), Spectrum Orange) and 18 (Vysis, Alpha Satellite DNA, 1:1 mixture Green/Orange spectrum). The nuclear and probe DNA were denatured simultaneously for 1 min at 75°C. The slides were then incubated in a humidified chamber at 42°C for 1–2 h to allow hybridization of the DNA probes. After hybridization, the slides were washed for 2 min with 0.4x SSC at 73°C followed by several dips in a 0.1% NP-40/2x SSC solution at room temperature. The slides were mounted in the antifade medium Vectashield (Vector) containing 1.25 ng/ml 4',6-diamino-2-phenyl indole (DAPI) to counterstain the nuclei. The nuclei were examined using a Zeiss Axioskop fluorescence microscope with the adequate filterset. All the slides were observed and the signals were interpreted by two independent observers using the scoring criteria as described by Hopman et al. (1988). The efficiency of the FISH procedure was tested for each PGD cycle on interphase nuclei from methanol:acetic acid fixed suspension of male human lymphocytes. Before the treatment cycle, the probe for the Y chromosome (Vysis; Alpha Satellite III DNA, Spectrum Orange) was tested on interphase nuclei of lymphocytes from the male partner to ensure the presence of a large probe signal. In one case, the DYZ1 locus gave a very small signal and the Alpha Satellite DNA DYZ3 was used instead.
Confirmation of diagnosis in embryos which were not transferred
All the patients involved signed the consent form for further research on these embryos. The remaining cells of all embryos classified as male or genetically heterogeneous were reanalysed to confirm the initial diagnosis. On day 4, the blastomeres of the rejected embryos were spread separately (Staessen et al., 1997) and FISH was performed using probes for chromosomes X, Y, 18.
In this study, at least two chromosome pairs were analysed and most or all the cells of each embryo were assessed. A blastomere was considered to be diploid when two signals for each chromosome pair were present. A blastomere was considered to be triploid (tetraploid) when three (four) signals for each chromosome pair were present. Similarly, when a single chromosome of each kind was detected the blastomere was classified as haploid. A blastomere was considered to be aneuploid when an extra (trisomic) or missing (monosomic) signal for one kind of chromosome was observed, in the presence of two signals for the remaining chromosomes analysed. A blastomere was considered chaotic when it was neither diploid nor haploid, triploid, tetraploid or aneuploid. An embryo was classified as mosaic when two or more groups of cells from that embryo had different chromosome complements. The combinations could be normal/aneuploid, normal/polyploid or normal/haploid or multiple combinations. An embryo was classified as chaotic when all the blastomeres contained a different genetic constitution.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Outcome of PGD cycles
We have treated 17 couples (22 treatment cycles) with a risk of sex-linked disease with the aim of transferring only female embryos. A total of 303 cumulus–oocyte complexes were retrieved, providing a mean of 13.8 ± 6.7 (range 2–28) cumulus–oocyte complexes per cycle. In all, 206 oocytes (68%) showed two distinct pronuclei after insemination or injection. On day 3 of development, of the 196 (a mean of 8.9 ± 5.4 embryos per patient) embryos obtained, 169 (86%) reached at least the 4-cell stage and were of good enough morphological quality for biopsy. A mean of 7.6 ± 4.9 embryos per patient were available for biopsy; in one couple no embryos suitable for biopsy were obtained. A diagnosis was obtained in 161 (95.3%) embryos. In five cycles, on the basis of the chromosome probes used, no normal female embryos were available after diagnosis; in three out of these five cycles only one or two embryos were available for diagnosis. In the remaining 16 cycles, a total of 53 female embryos considered for transfer were obtained. Of these female embryos, 37 were transferred (a mean of 2.3 ± 0.8 embryos per transfer). Six pregnancies ensued, giving a pregnancy rate of 37.5% per transfer or 27.2% per started cycle. All the pregnancies occurred after the transfer of embryos which cleaved again in the time interval between biopsy and transfer. One clinical abortion and five evolutive pregnancies (three singletons, two twin implantations, one of whom resulted in a triplet gestation by monozygotic twinning) were obtained, resulting in an implantation rate of 22.9% per transferred embryo. The pregnancies were confirmed by amniocentesis or at birth. In one cycle a pregnancy ensued and by ultrasound a singleton pregnancy was detected but a miscarriage occurred at 7 weeks without providing cellular tissue to confirm the diagnosis.
FISH analysis: efficiency of the technique
From the 169 embryos biopsied, two blastomeres were removed from 93 (55%) embryos and only one blastomere from 76 (45%) embryos, resulting in a total of 262 blastomeres available for diagnosis. No results were obtained from 25 blastomeres due to: (i) the biopsy of an anucleate blastomere in eight cases; (ii) the presence of fragmented nuclear material in four cases; (iii) a successfully spread single nucleus lost during the FISH procedure in one case and (iv) the absence of signals in 12 cases. Interpretable FISH signals were obtained in the remaining 237 blastomeres. The efficiency of the FISH procedure is, therefore, 90.4% per biopsied blastomere or 95.2% if expressed as the proportion of blastomeres with clearly distinct nuclear material. Moreover, in 161 (95.3%) out of the 169 embryos, a diagnosis was possible: 76 embryos with a diagnosis based on two blastomeres and 85 embryos based on one blastomere. From the eight embryos without diagnosis only one blastomere was retrieved.
FISH analysis of biopsied blastomeres and confirmation in embryos unsuitable for transfer or freezing
From a total of 161 embryos a diagnosis was made. According to the FISH results of the biopsied blastomeres, we defined three different groups: (i) 98 (60.9%) embryos in which a diploid pattern was observed in one or in both blastomeres biopsied; (ii) 10 (6.2%) in which one of the two blastomeres analysed showed a diploid pattern and the other an abnormal pattern; and (iii) 53 (32.9%) embryos in which one or both biopsied blastomeres showed an abnormal pattern. For these three different categories of embryos, the FISH results of the biopsied blastomeres and of the further analysis on day 4 of the non-transferred or non-frozen embryos are summarized in Tables II, III and IV.
|
|
|
Of the first group of embryos or the 98 embryos found to be normal diploid on the basis of the applied probes, 50 were diagnosed as female and 48 as male, as shown in Table II
. Of the female embryos, 40 were transferred or frozen. From the male embryos and the female embryos which were neither transferred nor frozen, 49 were analysed further. We observed uniformly diploid cells (2N) in 21 embryos. Furthermore, we found a combination of diploid cells with: (i) tetraploid cells in seven embryos (2N/4N); (ii) binuclear blastomeres with both nuclei diploid in four embryos (2N/BNB both 2N); (iii) tetraploid cells and binuclear blastomeres with both nuclei diploid in two embryos (2N/4N/BNB both 2N); (iv) haploid cells in three embryos (2N/N); (v) tetraploid and haploid cells in two embryos (2N/4N/N), (vi) aneuploid blastomeres in four embryos (2N/aneuploid); (vii) chaotic cells in four embryos (2N/chaotic); and (viii) tetraploid and chaotic cells in two embryos (2N/4N/chaotic). The haploid cells observed were all X1821. The aneuploid cells involved the loss of chromosome 18 in one embryo, and reciprocal non-disjunction of chromosome 18 or Y in the three other embryos.
The 10 (6.2%) embryos in which one of the two analysed blastomeres showed a diploid pattern at biopsy and the other an abnormal pattern are summarized in Table III together with the results of the further analysis of the embryo. In three embryos (XX1818 n = 1; XY1818 n = 2) the diploid blastomere was combined with a tetraploid blastomere. The embryo diagnosed as female was transferred for the reasons discussed below. The other two embryos were analysed further; one showed only diploid blastomeres while the other had a combination of diploid and tetraploid blastomeres. In three embryos, the diploid blastomere was combined with a binuclear blastomere with both nuclei diploid. Further analysis of these embryos revealed only normal diploid blastomeres. In one embryo, we observed a combination of a diploid with a haploid nucleus at diagnosis. In this embryo, further analysis here showed a mosaic embryo with diploid, haploid and tetraploid blastomeres. In one embryo, we observed a diploid blastomere in combination with a binuclear blastomere, both of these nuclei were haploid X18. The further analysis revealed the presence of solely diploid XY1818 blastomeres. In one embryo we observed an XY1818 pattern in one blastomere combined with an aneuploid XYY1818 blastomere. However, the same trisomy or reciprocal monosomy was not found in the rest of the embryo, while some cells revealed the presence of monosomies for chromosomes 18 or 21. Re-evaluation of the slide suggested that a split spot may have been involved in the XYY1818 case. In one embryo the diploid blastomere was combined with a XXXXY blastomere. Further analysis did not confirm the diagnosis and only diploid cells were found. Re-evaluation of the slide suggested that cross-hybridization combined with failed signals from chromosome 18 had occurred. Therefore, both these observations (XYY1818 and XXXXY) may be considered to be FISH errors.
In Table IV, the remaining 53 (32.9%) embryos with one or both blastomeres showing an abnormal pattern are summarized in combination with the results of the analysis of the rest of the embryo.
In nine embryos, the single biopsied blastomere was tetraploid (XXXX18181818 n = 5; XXYY18181818 n = 4). Seven embryos were analysed further and we found only tetraploid cells in two embryos; diploid cells in combination with tetraploid cells in two embryos; tetraploid cells in combination with haploid cells in one embryo; and two embryos were chaotic. From four embryos the single biopsied blastomere was binuclear with both nuclei diploid; two were transferred for the reasons discussed below. Two embryos were analysed further and we found diploid cells in combination with tetraploid cells in one case and diploid cells in combination with binuclear blastomeres with both nuclei diploid in the other embryo.
In seven out of the eight embryos with a X01818 pattern, further analysis was carried out. In two embryos all the remaining cells were monosomic X, possibly in combination with other anomalies, indicating that a plausible explanation is an error before fertilization. In two embryos we found the monosomy in combination with a trisomic form (in one embryo XXX1818 and in one embryo XYY1818) indicative of a reciprocal mitotic non-disjunction. In the other two, we found the combination of XX- and X0-bearing cells without the presence of a trisomic complement indicative of chromosome loss during the mitotic division. In one embryo we found only cells with a normal XX pattern, although both biopsied blastomeres clearly indicated X01818. Since mostly adjacent cells were biopsied, an explanation is that the abnormal cells from the mosaic embryo were removed. Three out of the four embryos analysed further showed a monosomy for chromosome 18, further analysis revealed that one embryo was completely monosomic for chromosome 18, one was completely disomic for chromosome 18 and one showed the disomic, monosomic and trisomic for chromosome 18 mosaicism.
Of the three triploid embryos found, the results were confirmed; one was completely triploid while the other two showed triploid cells together with chaotic blastomeres. Tetraploid/chaotic mosaicism was observed in the single embryo with the tetraploid cell combined with the abnormal XXXX1818 pattern. The haploid embryo was confirmed by further analysis. The embryo with the Y1818 and X1818 pattern in the biopsied blastomeres, further analysis revealed an abnormal embryo with the particularity that XY1818 blastomeres were observed in combination with X18, XX1818 and XXXX18181818 blastomeres.
Furthermore, 10 blastomeres from which five were multinuclear showed an abnormal pattern containing a signal for the Y chromosome. Only four embryos could be analysed further; in two we found male diploid cells in combination with chaotic patterns and two were completely chaotic with Y signals in some cells. Of the 12 biopsied blastomeres showing an abnormal pattern without a Y signal, six were multinuclear. Of the seven embryos analysed; three embryos were found to be chaotic without Y signals; in two embryos female diploid cells were found in combination with chaotic cells and in two embryos male diploid cells were found in combination with chaotic cells.
Finally, a Y signal was observed in 82 (50.9%) of the 161 embryos diagnosed, indicating a 50/50 ratio between male and female sex distribution.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We started the FISH technique for gender determination in sex-linked genetic disease in May 1995, and accumulated a total of 22 treatment cycles in 17 couples by December 1997. On day 3 of development, embryos suitable for biopsy were available in 21 out of 22 treatment cycles. After diagnosis, diploid female embryos were available for transfer in 16 cycles. For five patients no transfer took place; the main reason being the limited number of embryos available for diagnosis. In such patients, stimulation should therefore be optimized to obtain as many oocytes as possible (Vandervorst et al., 1998
).
The final proof of the viability of an embryo is its ability to implant. The data provided by this report indicate that the embryos retained their viability after biopsy. In our centre, treatment cycles without PGD result in ~20% of the transferred embryos becoming implanted (Staessen et al., 1995). After the PGD, six transfers out of the 16 led to pregnancy, i.e. a pregnancy rate of 27.3% per cycle or of 37.5% per transfer. The implantation rate per transferred embryo was 20.8%, which is comparable to that for embryos after ART without biopsy. However, direct comparison is not strictly possible, since different parameters may be creating beneficial or adverse effects. Possible beneficial effects are that the patients treated were mostly fertile women, as opposed to the generally infertile patient population treated by ART. The embryos were screened for ploidy and obvious chromosomal imbalances before transfer, with a possible beneficial effect on the pregnancy rate (Gianoroli et al., 1997). After genetic diagnosis, a limited number of embryos were available for implantation. This may have influenced the pregnancy rate negatively (Staessen et al., 1992).
This study confirmed that FISH of single cells is an efficient method for gender determination. Gender determination was possible in 95.3% of the successfully biopsied embryos. The main cause of failure of diagnosis was the absence of clear nuclear material in the biopsied blastomere, although at the time of biopsy blastomeres with a distinct nucleus were removed when possible. The obtained efficiency is in agreement with the figures described by others (Harper et al., 1994; Munné et al., 1994b; Coonen et al., 1996) using FISH.
During the PGD treatment cycles we encountered biopsied blastomeres with all previously described aspects of human embryos (Munné et al., 1994, 1995; Harper et al., 1995), such as multinucleation, mosaicism, aneuploidy and other abnormalities. A single diploid nucleus was observed in the biopsied blastomere(s) in 98 (61%) of the 161 diagnosed embryos. In 10 out of the 161 (6.2%) embryos we observed a combination of a diploid single nucleus in one blastomere with a non-diploid nucleus (n = 6) or multinuclear (n = 4) second blastomere. In the remaining 53 (32.9%) embryos one or both biopsied blastomeres were abnormal (n = 37) or multinuclear (n = 16). For this reason, the reliability of the comparison with the other cells of the embryo became a matter of great interest. The main goal was to confirm the determined sex.
A first observation is that when two sex chromosomes and two autosomes were present in the nucleus of the biopsied blastomere, the sex determination was never in conflict with the sex shown by analysis of the remainder of the embryo. Our experience agreed with others (Munné et al., 1994a,b; Harper et al., 1996) confirming that an XX cell in an otherwise completely diploid male embryo has not yet been observed. However, we found X18, XX1818, XXXX18181818 nuclei together with XY1818 and other abnormal cells in the same embryo. Wrong diagnosis of gender could have occurred when other cells where biopsied. A possible explanation for these embryos is the inclusion of genetic material from the first or second polar body. Also, one X01818 cell at diagnosis was revealed to be a male XY1818/XYY1818/X01818 mosaic embryo after analysis of the remainder of the embryo, highlighting the need to identify both sex chromosomes by FISH. From the chaotically dividing embryos, the observation of biopsied cells showing by coincidence two signals for the X chromosome but no signals for chromosome 18 involved in one case a mosaic XY1818/chaotic embryo. This shows the importance of the addition of at least one autosome probe during PGD.
Initially, we adhered to the criterion of replacing only embryos in which the analysed blastomere(s) was mononuclear with two signals for the X chromosomes and two signals for chromosome 18 in the absence of signals for the Y chromosome. However, this posed the problem of dealing with cases in which, after rejection of the male embryos, the biopsied blastomeres from the embryos without Y signals were binuclear with both nuclei female diploid or were mononuclear but tetraploid.
We observed a binuclear blastomere at diagnosis with both nuclei diploid in seven out of 237 (3%) biopsied blastomeres diagnosed. The analysis of the remainder of these embryos indicated that where both nuclei were diploid, they predicted the sex and ploidy status of the embryo, as already observed (Munné et al., 1993). Furthermore, 8% of the analysed embryos also contained binuclear blastomeres with both nuclei diploid in otherwise normal embryos. A large proportion of these blastomeres can develop further and give rise to normal daughter cells (Staessen et al., 1998). Therefore, we concluded that when the biopsied cell is binuclear with each nucleus showing two signals for the X chromosomes and two signals for chromosome 18 in the absence of signals for the Y chromosome the embryo can be considered for transfer.
Another frequent observation was a single nuclear diploid blastomere in combination with a tetraploid blastomere. Of 237 of the biopsied blastomeres, 13 (5.5%) were tetraploid and in 10 out of 94 (10.6%) of the embryos analysed further at least one tetraploid cell in an otherwise diploid embryo was present. Benadiva et al. described tetraploid cells in 7.1% of morphologically and developmentally normal embryos. The tetraploid blastomeres always corresponded with the sex of the embryo. Some tetraploid nuclei have a symmetrical distribution of the FISH signals and can be interpreted as a nucleus in division (Staessen et al., 1998) or can indicate the mechanism of endomitosis leading to tetraploid cells. It has been shown that tetraploid cells are a common component of the trophoblast of human embryos (Benkhalifa et al., 1993), leading to speculation that the generation of tetraploid cells in a human embryo may be part of a normal process. On the other hand, complete tetraploid embryos were also observed, demonstrating the importance of having two blastomeres for diagnosis not so much to determine the gender but in order to judge the suitability of an embryo for transfer. Where there are two blastomeres, one of which is diploid and the other tetraploid, the replacement of the embryo is justified. If the only blastomere biopsied is tetraploid the embryo is not considered for transfer.
Another frequently observed mosaicism was the presence of haploid cells in combination with diploid blastomeres and is difficult to explain. In this series of embryos only X18(X1821) haploid sets were observed, even in embryos diagnosed as male, and frequently only one or two nuclei per embryo were observed, giving us the idea, although we have no direct proof, that this involved the genetic residue of the polar body.
If the biopsied cell or cells were diagnosed as abnormal, as was the case in 53 embryos, there is a high probability that the entire embryo was completely abnormal or at least extensively mosaic. A similar conclusion may be drawn if a multinuclear blastomere with a total number of signals corresponding to a non-diploid status is found for diagnosis.
Despite the visualization of two pronuclei, triploid embryos were observed. Possible explanations are that the third pronucleus was not detected, that a segregation error of the polar body formation had occurred, or that fertilization by a diploid spermatozoon had occurred, resulting in the presence of two pronuclei. The observed mosaicism in these embryos was as described previously (Staessen et al., 1997). The completely haploid embryo was confirmed by further analysis. The history of this embryo shows that a regularly dividing 8-cell embryo of grade A was observed on day 3, but without the presence of two pronuclei on day 1. This indicates that in an IVF/ICSI programme, strict evaluations of pronucleus formation should not be neglected.
In the aneuploid embryos, examples of three different mechanisms were observed: (i) involvement of an aneuploid gamete leading to an uniformly aneuploid embryo; (ii) reciprocal mitotic non-disjunction leading to mosaic embryos with monosomic, disomic and trisomic blastomeres for the chromosome involved; and (iii) chromosomal loss alone leading to a combination of monosomic and disomic blastomeres. As expected, we found examples of the three mechanisms involved in the three chromosomes under investigation. These mechanisms are also involved in in-vivo conceptions as reviewed recently (Nicolaidis and Petersen 1998).
The high incidence of mosaic embryos obtained on day 4 of development may indicate that the in-vitro culture conditions were sub-optimal and likely to induce the abnormalities observed (as demonstrated by Munné et al., 1998). Another plausible explanation (Edwards et al., 1997) is that in an embryo, from the very first divisions onwards, the cell line leading to the inner cell mass is differentiated and that in fact only the genetic constitution of these cells is of crucial importance. On the other hand, if an embryo is considered at the 64-cell stage, only 3–4 cells are destined to give rise to a fetus, while the other cells will form placenta and fetal annexes. Thus, a mitotic error occurring early in development is more likely to occur in an extrafetal cell lineage than in a fetal lineage. During CVS and pregnancy, the existence of confined placental mosaicism and generalized mosaicism within fetus and placenta have been reported by Kalousek and Vekemans (1996).
To conclude, these results indicate that, for gender determination by FISH, the diagnosis based on 1 or 2 cells is representative of the complete embryo in cases where the biopsied cell(s) show a diploid nucleus with two sex chromosomes. Where the biopsied cell was diagnosed as abnormal (triploid, aneuploid, chaotic) the embryo was indeed completely abnormal or at least mosaic. A FISH error could not be excluded in two embryos (1.2%) but a wrong gender determination did not result from this.
|
Acknowledgments |
|---|
The authors wish to thank the clinical, scientific, nursing and technical staff of the Centre for Reproductive Medicine and especially Peter Nagy, Hilde Van De Velde, Anick Devos. Furthermore, we are grateful to Sylvie Mertens for technical assistance with the FISH technique. We thank Frank Winter of the Language Education Centre for correcting the English text. Grants from the Belgian Fund for Medical Research are gratefully acknowledged.
|
Notes |
|---|
3 To whom correspondence should be addressed
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Benadiva, A., Kligman, I. and Munné, S. (1996) Aneuploidy 16 in human embryos increases significantly with maternal age. Fertil. Steril., 66, 248–255.[Web of Science][Medline]
Benkhalifa, M., Janny, L., Vye et al. (1993) Assessement of polyploidy in human morulae and blastocysts using co-culture and fluorescent in-situ hybridization. Hum. Reprod., 8, 895–902.
Coonen, E., Harper, J., Ramaekers, F.C.S. et al. (1994a) Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescent in-situ hybridization. Hum. Genet., 94, 609–615.[Web of Science][Medline]
Coonen, E., Dumoulin, J.C.M., Ramaekers, F.C.S. and Hopman, A.H.N. (1994b) Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescent in-situ hybridization. Hum. Reprod., 9, 533–537.
Coonen, E., Dumoulin, J.C.M., Dreesen, C.F.M. et al. (1996) Clinical application of FISH for sex determination of embryos in preimplantation diagnosis of X-linked diseases. J. Assist. Reprod. Genet., 10, 82–90.
Delhanty, J.D.A., Harper, J.C., Ao, A. et al. (1997) Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum. Genet., 99, 755–760.[Web of Science][Medline]
Edwards, R.G. and Beard, H.K. (1997) Oocyte polarity and cell determination in early mammalian embryos. Mol. Hum. Reprod., 3, 863–905.
Gianaroli, L., Magli, M.C., Ferraretti, A.P. et al. (1997) Preimplantation genetic diagnosis increases the preimplantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos. Fertil. Steril., 68, 1128–1131.[Web of Science][Medline]
Griffin, D.K., Handyside, A.H., Harper, J.C. et al., (1994) Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridisation. J. Assist. Reprod. Genet., 11, 132–143.[Web of Science][Medline]
Griffin, D.K., Leeanda, J.W., Handyside, A.H. et al. (1992) Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for sexing of human preimplantation embryonic nuclei. Hum. Genet., 89, 18–22.[Web of Science][Medline]
Grifo, J.A., Tang, Y.X., Munné, S. et al. (1994) Healthy deliveries from biopsied human embryos. Hum. Reprod., 9, 912–916.
Handyside, A.H., Kontogianni, E.H., Hardy, K. et al. (1990) Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature, 344, 768–770.[Medline]
Harper, J.C., Coonen, E., Handyside, A.H. et al. (1995) Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenatal Diagn., 15, 41–49.[Web of Science][Medline]
Harper, J.C., Coonen, E., Ramaekers, F.C.S. et al. (1994) Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescence in-situ hybridization (FISH) using directly labelled probes. Hum. Reprod., 4, 721–724.
Hopman, A.H.N., Ramaekers, F.C.S., Raap, A.K. et al. (1988) In situ hybridisation as a tool to study numerical chromosome aberrations in solid bladder tumours. Histochemistry, 89, 307–316.[Web of Science][Medline]
Kalousek, D.K. and Vekemans, M. (1996) Confined placental mosaicism. J. Med. Genet., 33, 529–533.
Kontogianni, E.H., Hardy, K. and Handyside, A.H. (1991) Co-amplification of X- and Y-specific sequences for sexing of preimplantation human embryos. In Verlinsky, Y. and Strom, C. (eds), Preimplantation Genetics. Plenum Press, New York, USA, pp. 139–145.
Liu, J., Lissens, W., Devroey, P. et al. (1994) Amplification of X- and Y chromosome-specific regions from single human blastomeres by polymerase chain reaction for sexing of preimplantation embryos. Hum. Reprod., 9, 716–720.
Munné, S. and Cohen, J. (1993) Unsuitability of multinucleated human blastomeres for preimplantation genetic diagnosis. Hum. Reprod., 8, 1120–1125.
Munné, S., Weier, H.U.G., Grifo, J. and Cohen, J. (1994a) Chromosome mosaicism in human embryos. Biol. Reprod., 51, 373–379.[Abstract]
Munné, S., Grifo, J., Cohen, J. et al. (1994b) Chromosome abnormalities in human arrested preimplantation embryos: a multiple-probe FISH study. Am. J. Hum. Genet., 55, 150–159.[Web of Science][Medline]
Munné, S., Alikani, M., Tomkin, G. et al. (1995) Embryo morphology, developmental rates, and maternal age are correlated with chromosomal abnormalities. Fertil. Steril., 64, 382–391.[Web of Science][Medline]
Munné, S., Magli, C., Adler, A. et al. (1997) Treatment-related chromosome abnormalities in human embryos. Hum. Reprod., 12, 780–784.
Nicolaidis, P. and Petersen, M.B. (1998) Origin and mechanisms of non-disjunctions in human autosomal trisomies. Hum. Reprod., 13, 313–319.
Sermon, K., Lissens, W., Joris, H. et al. (1997) Clinical application of preimplantation diagnosis for Myotonic Dystrophy. Prenat. Diagn., 10, 919–925.
Staessen, C. and Van Steirteghem, A.C. (1997) The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum. Reprod., 10, 3305–3312.
Staessen, C. and Van Steirteghem, A.C. (1998) The genetic constitution of multinuclear blastomeres and their derivative daughter blastomeres. Hum. Reprod., 13, 1625–1631.
Staessen, C., Camus, M., Bollen, N. et al. (1992) The relationship between embryo quality and the occurrence of multiple pregnancies. Fertil. Steril., 57, 626–630.[Web of Science][Medline]
Staessen, C., Nagy, Z.P., Liu, J. et al. (1995) One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes. Hum. Reprod., 10, 3305–3312.
Staessen, C., Coonen, E., Van Assche, E. et al. (1996) Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients. Hum. Reprod., 11, 1650–1653.
Ubaldi, F., Smitz, J., Wisanto, A. et al. (1995) Pregnancy and birth after high serum progesterone concentrations during the follicular phase in an in-vitro fertilization cycle with gonadotrophin-releasing hormone agonist suppression. Hum. Reprod., 10, 211–213.
Van den Abbeel, E., Camus, M., Van Waesberghe, L. et al. (1997) Viability of partially damaged human embryos after cryopreservation. Hum. Reprod., 12, 2006–2010.
Van Steirteghem, A., Joris, H. and Liu et al. (1995) Protocol for intracytoplasmic sperm injection. Hum. Reprod. Update, 1, CD-ROM.
Vandervorst, M., Liebaers, I., Sermon, K. et al. (1998) Successful preimplantation genetic diagnosis is related to the number of available cumulus–oocyte complexes. Hum. Reprod., 13, 3169–3176.
Veiga, A., Santalo, J., Vidal, F. et al. (1994) Twin pregnancy after preimplantation diagnosis for sex selection. Hum. Reprod., 9, 2156–2159.
Submitted on July 9, 1998; accepted on January 29, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  W. Verpoest, P. Haentjens, M. De Rycke, C. Staessen, K. Sermon, M. Bonduelle, P. Devroey, and I. Liebaers Cumulative reproductive outcome after preimplantation genetic diagnosis: a report on 1498 couples Hum. Reprod., November 1, 2009; 24(11): 2951 - 2959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Verpoest, L. Van Landuyt, S. Desmyttere, A. Cremers, P. Devroey, and I. Liebaers The incidence of monozygotic twinning following PGD is not increased Hum. Reprod., November 1, 2009; 24(11): 2945 - 2950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Goossens, M. De Rycke, A. De Vos, C. Staessen, A. Michiels, W. Verpoest, A. Van Steirteghem, C. Bertrand, I. Liebaers, P. Devroey, et al. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis Hum. Reprod., March 1, 2008; 23(3): 481 - 492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Donoso, W. Verpoest, E.G. Papanikolaou, I. Liebaers, H.M. Fatemi, K. Sermon, C. Staessen, J. Van der Elst, and P. Devroey Single embryo transfer in preimplantation genetic diagnosis cycles for women <36 years does not reduce delivery rate Hum. Reprod., April 1, 2007; 22(4): 1021 - 1025. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Michiels, E. Van Assche, I. Liebaers, A. Van Steirteghem, and C. Staessen The analysis of one or two blastomeres for PGD using fluorescence in-situ hybridization Hum. Reprod., September 1, 2006; 21(9): 2396 - 2402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pujol, J. Benet, C. Staessen, E. Van Assche, M. Campillo, J. Egozcue, and J. Navarro The importance of aneuploidy screening in reciprocal translocation carriers. Reproduction, June 1, 2006; 131(6): 1025 - 1035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Feyereisen, A. Amar, V. Kerbrat, J. Steffann, A. Munnich, M. Vekemans, R. Frydman, and N. Frydman Myotonic dystrophy: does it affect ovarian follicular status and responsiveness to controlled ovarian stimulation? Hum. Reprod., January 1, 2006; 21(1): 175 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Amir, V. R. Sutton, and I. B. Van den Veyver Newborn Screening and Prenatal Diagnosis for Rett Syndrome: Implications for Therapy J Child Neurol, September 1, 2005; 20(9): 779 - 783. [Abstract] [PDF]
|
|
|
|
|
|
R. E. Amir, V. Reid Sutton, and I. B. Van den Veyver Newborn Screening and Prenatal Diagnosis for Rett Syndrome: Implications for Therapy J Child Neurol, August 1, 2005; 20(8): 779 - 783. [Abstract] [PDF]
|
|
|
|
|
|
A.R. Thornhill, C.E. deDie-Smulders, J.P. Geraedts, J.C. Harper, G.L. Harton, S.A. Lavery, C. Moutou, M.D. Robinson, A.G. Schmutzler, P.N. Scriven, et al. ESHRE PGD Consortium 'Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)' Hum. Reprod., January 1, 2005; 20(1): 35 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.D. Daphnis, J.D.A. Delhanty, S. Jerkovic, J. Geyer, I. Craft, and J.C. Harper Detailed FISH analysis of day 5 human embryos reveals the mechanisms leading to mosaic aneuploidy Hum. Reprod., January 1, 2005; 20(1): 129 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Girardet, S. Hamamah, H. Dechaud, T. Anahory, C. Coubes, B. Hedon, J. Demaille, and M. Claustres Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy Mol. Hum. Reprod., July 1, 2003; 9(7): 421 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Tachdjian, N. Frydman, F. Audibert, P. Ray, V. Kerbrat, P. Ernault, R. Frydman, and J.-M. Costa Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre Hum. Reprod., August 1, 2002; 17(8): 2183 - 2186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Malpani, A. Malpani, and D. Modi Preimplantation sex selection for family balancing in India Hum. Reprod., January 1, 2002; 17(1): 11 - 12. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
ESHRE PGD Consortium Steering Committee ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001) Hum. Reprod., January 1, 2002; 17(1): 233 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.N. Scriven, F.A. Flinter, P. R. Braude, and C. M. Ogilvie Robertsonian translocations--reproductive risks and indications for preimplantation genetic diagnosis Hum. Reprod., November 1, 2001; 16(11): 2267 - 2273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
ESHRE PGD Consortium Steering Committee ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: data collection II (May 2000) Hum. Reprod., December 1, 2000; 15(12): 2673 - 2683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Van Assche, C. Staessen, W. Vegetti, M. Bonduelle, M. Vandervorst, A. Van Steirteghem, and I. Liebaers Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22) Mol. Hum. Reprod., July 1, 1999; 5(7): 682 - 690. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||