Preimplantation Genetic Diagnosis (PGD): Screening for Aneuploidy in Human Oocytes and Polar Bodies

What Makes PGD for Aneuploidy Screening (AS) by Polar Body (PB) Analysis Interesting and Possible?

Since it has been reported that in humans 90% of embryos are aneuploid as a result of malsegregation mechanisms in maternal meiosis I,¹ the detection of abnormal oocytes in IVF treatments has become of considerable importance.

Taking into account the results obtained from the study of recognized pregnancies,² and gametes,^3,4 the conclusion is that the segregation of chromosomes during meiosis is extremely abnormal in humans, and especially during the first meiotic division of the oocytes. This is in agreement with the fact that the first female meiotic division starts during the prenatal stages (at about 11th week of pregnancy) and is not completed until time of ovulation during the fertile life of the woman, between, approximately, 11 and 40 years later² (fig. 1). Advances in the procedures of assisted human reproduction have facilitated the development of methods of diagnosis which are more precocious than prenatal diagnosis, and corresponds to what is known as Preimplantational Genetic Diagnosis (PGD).⁵ This has given rise to an availability of biological material (gametes and human embryos), that has provided information for a better estimation of the frequency of aneuploidy in the first stages of embryo development.

The results of the application of PGD to aneuploidy screening (AS) are periodically compiled by the European Society of Human Reproduction. The last collection⁶ includes a total of 2,854 PGD AS cycles performed until december 2001 (71 through the study of the 1PB and 1,783 through the study of blastomeres). Of a total of 24,374 COCs obtained, 20,930 were inseminated and 15,002 were fertilized. A diagnosis was obtained in 9,045 of the 10,760 cells successfully biopsied. There were 3,625 transferable embryos, of which 2,932 were transferred, in 1,342 IVF cycles. A total of 431 biochemical pregnancies were obtained, with 336 pregnancies detected by means of the heartbeat. The success of PGD AS, related to the total number of oocytes, is 18%.

The development of PGD by means of the analysis of the 1PB (PGD-1PB) arose subsequent to PGD on blastomeres.⁷ The challenge was to detect genetic anomalies in the gametes generated in individuals at risk even before fertilization. This is only feasible if the parent at the risk is the female. The differential characteristics that exist between male and female gametogenesis allow this analysis. While spermatozoa are haploid cells which result from the two meiotic divisions, the oocytes, at the time of ovulation, have not completed meiosis II and are arrested in metaphase of the second meiotic division (fig. 2). The female gamete is made up of two cells, the first of which is called the oocyte in metaphase II (MII oocyte), and the other which is the first polar body (1PB). Both cells contain complementary chromosomal sets. Only the MII oocyte receives the totality of the cytoplasm and has the reproducing function that will be developed upon being fertilized by a sperm. The 1PB does not have any reproductive function and will degenerate. Thus, the 1PB can be biopsied and can be used to carry out an indirect analysis of the genetic contents of the MII oocyte.

Methodologies of Analysis of Aneuploidies in Human Oocytes: Cytogenetic Analysis of 1PB and MII of the Oocyte

To carry out a study of aneuploidies of female origin, in a research project, both chromosomal sets should be studied. The result of the 1PB analysis constitutes an internal control of the segregation of the chromosomes, which is essential.

Although one can try to fix both cells at the same time, it is recommended to proceed to the individual fixation of each of them. The cells should be well separated after the total elimination of the zona pellucida with Tyrode's acid or with trypsin solution, or by perforating the zona pellucida, applying diverse alternative methods which allow for obtaining the 1PB, without damaging the MII oocyte.

There are diverse biopsy methods of 1PB available, all of which are applied to the mature oocyte, free of cumulus cells, which is placed in an inverted microscope equipped with a micromanipulator, and held with a holding pipette (fig. 3). One of the most common methods is a mechanical system. The oocyte is subjected with its 1PB placed at 12 o'clock, and using a sharp needle pipette in the micromanipulator's arm, a simple or a double slit is made in the zona pellucida.^8-10 Then, the needle pipette is removed, and using a biopsy pipette with a diameter of 20-30 μm, the 1PB is gently removed from the oocyte and transferred to a clean, just-filtered drop of medium. The faster system is the laser system that has been used in the mouse.¹¹ The hole is made with just one or two laser shots. The third method, which is the Tyrode's acid system, is not recommended for PGD-1PB because it affects negatively the fertilization capacity of the MII oocyte.^12,13

For the analysis of aneuploidies in oocytes, chromosome spreads of the 1PB and its corresponding MII oocyte fixed on a slide have been mainly used. To obtain adequate chromatin material spread two methods of fixation have been used: the methods of Tarkowsky¹⁴ and of Mikamo and Kamiguchi.¹⁵

The method of Tarkowsky,¹⁵ which is widely used, consists of dropping carnoy fixative solution (methanol: acetic acid (3:1 v/v)) on a previously hypotonized single cell placed on a slide.

In the method of Mikamo and Kamiguchi,¹⁵ a gradual fixation of the previously hypotonized oocytes is performed as follows: first, fixative I (methanol: acetic acid: distilled water (5:1:4 v/v, 5 min)); second, fixative II (methanol: acetic acid (3:1 v/v, 10 min)); and third, fixative III (methanol: acetic acid: distilled water (3:3:1 v/v, 1 min)). The quality of the chromosomes is good enough to allow R-banding.^16,17

For 1PBs, two fixation methods have been applied: the method of Tarkowsky,¹⁴ which is applied in a similar fashion as for the MII oocyte, and the method of Durban.¹⁸ This method was developed especially for the fixation of 1PBs and should be performed under stereomicroscopic control. Each previously hypotonized 1PB is fixed on a slide previously treated with trimethoxysylane. Once the 1PB is left on the slide, a soft flow of fixative solution (ethanol: acetic acid; 1:1 v/v) is created which is directed toward the 1PB until it reaches it, dissolving its plasma membrane (fig. 4). This method, applied to 1PBs of fresh oocytes, achieves in 88% of cases, spreads of the chromosomal complement of the 1PB with good chromosomal quality (fig. 5) and has provided the best coincidence between the chromosome complements of the 1PB and the MII oocyte after in situ hybridization techniques (FISH) (fig. 4). In aged oocytes good chromosomal morphology and concordant results are observed with much lower frequency.¹³

In general, while the fixed chromosomes of the MII oocyte appear with the two chromatids separated and zig-zagging and with the chromatin quite compact, the chromosomes of the 1PB have their two chromatids also separated, but the chromatin appears more fuzzy and disorganized. Although banding techniques^16,17 can be applied to MII chromosomes, it is more recommendable to use FISH techniques with whole-chromosome painting probes, which are noninformative in interphase nuclei, as well as specific probes: locus-specific, centromeric and telomeric among others. FISH has been used for the study of a restricted and predetermined number of chromosomes in one, two or three rounds of FISH. Initially, chromosomes 18 and X¹⁹ were analyzed, and subsequently other chromosomes implicated in viable and nonviable aneuploidies in humans were gradually included, eventually reaching, at present, up to nine chromosomes (1, 13, 15, 16, 17, 18, 21, 22 and X).²⁰ These chromosomes have been identified as responsible for aneuploidies in spontaneously abortions and also in blastomeres of embryos of women of advanced age^21-23 which in turn would reduce the rate of implantation.^19,20,24-26 Treatment of the slide with microwaves for one hour at 75w immediately before hybridization allows reducing the time of FISH and intensifies the fluorescent signals.²⁷

FISH has also been applied to the analysis of the whole chromosomes set^4,28,29 by Spectral karyotyping (SKY). The system employs 24 chromosome-specific painting probes. Each probe is labeled with a different proportion of five separate fluorochromes, which on being observed by spectral imaging and with the adequate software, show distinct colors for each chromosome, which permits to correctly identify each chromosome pair in the karyotype. At the same time, a variant of FISH, cenM-FISH, which is a new multicolor-FISH approach applying centromere-specific multicolor-FISH probes directly or indirectly labeled with one, two, three or four distinct fluorochromes,³⁰ has been already applied to the study of meiotic cells.³¹ This system, when applied to good chromosomal spreads of metaphase II oocytes (fig. 6, unpublished data), appears as an alternative of great interest for the AS analysis in oocytes, because in one single round of FISH all centromeres of the chromosomal complement can be analyzed.

Another technique called the primer in situ (PRINS) has been successfully tested on chromosomal spreads of fixed 1PBs. It is a fast inexpensive method of labeling chromosomes 13, 16, 21 or X, which are frequently involved in aneuploidies.³² Chromosomes are labeled by in situ annealing with chromosome-specific oligonucleotide primers, followed by primer extension with labeled nucleotides using the Taq DNA polymerase. Each 1PB chromosome spread undergoes three successive double-labeling reactions, and it in less than 40 min intense signals are obtained, which is of great interest in the application of PGDs for AS.

But all of the methodologies indicated have a common limitation. They are applied on chromosomal spreads of cells fixed on slides and it is never possible to discard that the hypohaploidies detected are not artifactual losses that occur during fixation.

The Comparative Genomic Hybridization (CGH) cytogenetic method applied to isolated cells^33,34 is a very favorable alternative for the study of aneuploidies in human oocytes. This method permits one to study all chromosomes of the chromosomal complement of the 1PBs and of the corresponding MII oocyte. The possibility of artifactual losses of chromosomes does not exist, because the cells are introduced, lysed and processed separately inside PCR tubes (fig. 7). This system, given the limitation in the amount of DNA present in an isolated cell (approximately 6pg), requires a preamplification of the whole cell genome using DOP-PCR.³⁵ Next, the preamplified material can be used in CGH. CGH is a competitive DNA hybridization on chromosomal spreads in metaphases of control lymphocytes, with a mixture in equimolecular amounts of the two previously prepared DNAs: DNA of the isolated test cell, labeled with a red fluorochrome (Spectrum red), and a reference DNA, labeled with a green fluorochrome (Spectrum green) coming from an individual with a normal karyotype. The chromosomal losses of the problem cell appear in green, chromosomal gains appear in red and the chromosomal regions that show identical proportions appear in yellow. The limitation of this methodology is that it is not informative for heterochromatic or telomeric regions, and chromosomes 17, 19 and 22 usually show profiles that are difficult to interpret. These limitations are also seen when analyzing biological samples in which there is no limitation in DNA amount.³⁶ Occasionally the results in the 1PB and its corresponding MII oocyte show profiles with losses or gains which do not allow assurance as to whether they affects a whole chromosome or a chromatid.^37,38

Study of Aneuploidies in Human Oocytes: 1PB and MII Oocytes

A relevant byproduct of in vitro fertilization techniques has been the availability of discarded oocytes, a fact that has promoted a considerable progress in the knowledge of oocyte and embryo chromosome anomalies and their mechanisms of production. In the best of cases, though sporadically, the oocytes are fresh and have not been inseminated. However, in most cases they are oocytes which are nonfertilized after being inseminated in vitro. In other case, the oocytes are immature. These are aged oocytes that do not represent the oocytes of a general population.

In a recent review,³⁹ the mean frequency of chromosomal alterations seen in the studies that have used the fixation technique of Tarkowsky is 35.98%, which includes 26.5% of aneuploidies (15.5% hypohaploidies, 7.3% hyperhaploidies and 3.6% complex aneuploidies) and 6.02% structural aberrations (deletions, acentric fragments chromosome fragmentation).

No significant differences were observed when cytogenetic results obtained from inseminated oocytes discarded from IVF cycles were compared with those non inseminated or with obtained without hormonal induction.⁴⁰ No differences observed either when comparing them to those fertilized by ICSI or IVF.^38,41

In a recent review,³⁹ a great variability in the incidence of aneuploidies, varying between 2.0% and 59.6%, as well as contradictory results in the effects of maternal age were observed.^16,42 In this review the divergences were attributed to the reduced size of the sample of oocytes analyzed and to the difficulty in identifying the chromosomes in MII oocytes, because most studies used only uniform-staining techniques, and in a limited number of studies,^43-46 low quality banding techniques.

But the uniform-staining techniques on spreads of chromosomal sets of 1PBs and MII oocytes allowed the identification of a new alteration that affected the chromatids of the chromosomes that appeared as a premature separation of sister chromatids (PSSC), which occurred during the first meiotic division.^47,48

A balanced PSSC is characterized by balanced chromosomal sets with separation of sister chromatids. This anomaly is not considered as responsible for aneuploidies although it may indicate a predisposition to nondisjunction.^16,28,49 An unbalanced PSSC, characterized by chromosomal sets with gains or losses of chromatids, is considered as a numerical cytogenetic anomaly, resulting from the early separation and malsegregation of the chromatids of one of the chromosomes of the dyad (fig. 8).

Additionally, in the studies of aneuploidies in oocytes other chromosomal alterations, such as chromosomal loss or gain, produced by non disjunction phenomena (ND) have been described.^16,50 While some studies^48,51 have observed almost exclusively PSSC, in other cytogenetic studies of oocytes, using FISH with a predetermined number of chromosomes^{16,20,24,26,28,49} or analyzing all chromosomes simultaneously by CGH,^37,38 the presence of both PSSC and ND of chromosomes has been described.

While some works that analyze nonfertilized oocytes and oocytes matured in vitro, suggest that PSSC and ND do not significantly increase over culture time,^37,49,52 other studies suggest the opposite.^16,53

A study of 1,397 oocytes, fixed by means of the gradual method, indicated that PSSC was significantly increased with respect to ND.⁵⁴ Similar results have been obtained with different methods of analysis.^28,37 At the same time, in fresh, noninseminated oocytes and fresh, unfertilized oocytes from women 35-year old and over, the phenomenon of PSSC is more frequent than ND.²⁸ The authors observed a higher frequency of balanced, rather than unbalanced PSSC in oocytes of women 35 years of age or older, with respect to those of women 24 to 34 years old. This study made it quite clear that balanced PSSC affected more frequently chromosomes of a smaller size.

PSSC may result from premature degradation, before Anaphase I, of the cohesins that maintain sister chromatids joined.⁵⁵ Some authors have related the presence of PSSC, to advanced maternal age.^17,28 Women of advanced age would have an accumulation of mutations in the mitochondrial DNA, not only in the oocytes but also in the follicular cells.⁵⁶ This fact would reduce mitochondrial function.⁵⁷ Since the activity of the cohesins depends on ATP,⁵⁸ a deficiency in oxidative energy metabolism could result in an increase of PSSC in women of advanced age. Also, some authors^59,60 have suggested that the presence of free chromatids during the first division could affect the segregation of other chromosomes.

The frequency of aneuploidy varies enormously according to different studies: between 4.7% and 47.5%, with an average of 27.7%, including the studies carried out using R-banding,^16,17 FISH techniques with up to 9 chromosomes,^19,20,24-26 the SKY system,^4,28 or multicolor fluorescent FISH (M-FISH).⁶⁰ While some authors^17,50,61 observe that approximately 16% of the oocytes are aneuploid, the rate of aneuploidy observed in oocytes analyzed by the SKY system^4,28 or by the M-FISH system²⁹ varies between 16.7% to 39%, with an average value of 31.3%. At the same time the values obtained by FISH for a limited and predetermined number of chromosomes show even more variation. So, while some groups find frequencies of 3.2%, 9.5% and 4.3% analyzing 2, 6 and 7 chromosomes, respectively (chromosomes 18 and X; 9, 13, 16, 18, 21 and 22 and 1, 9, 13, 16, 18, 21 and X respectively),^26,62,63 other groups find higher rates of aneuploidy. Recently, by analyzing 5, 6 or 9 chromosomes (13, 16, 18, 21 and 22; 1, 7, 13, 18, 21 and X, or 1, 13, 15, 16, 17, 18, 21, 22 and X), frequencies of aneuploidies of 41.7%, 44% and 47.5%, have been, respectively, found.^20,64,65

The rate of aneuploidy depends on maternal age; thus aneuploidy frequencies of 4%, 9%, 11.5% and 29.8% in the oocytes of females belonging to the maternal age groups of 25-34, 35-39 and 40-45 years old, respectively, had been already estimated.¹⁶

In a series of 3,042 nonfertilized oocytes from 792 patients between the ages of 19 and 46 years included in an IVF program, chromosomal alterations which originated by nondisjunction of the chromosomes of the dyad were also correlated to advanced maternal age.¹⁷

In spite of the fact that it has been possible to identify that only 25% of the hypohaploidies would correspond to artifacts of the fixation technique or to FISH failures,²⁰ it is still difficult, when only one of the cells is analyzed (1PB or MII oocyte), to know whether the absence of a chromosome should be considered artifactual or not. The rate of aneuploidies is conservatively calculated, in general, as twice the rate of hyperhaploidies.^25,26 The rate of aneuploidies observed when applying a combination of methods, i.e., CGH for the 1PBs and FISH for 9 chromosomes for the corresponding MII oocyte, has been estimated at 57.1%.³⁸ This high frequency of aneuploidies could be related to the fact that a greater number of chromosomes are analyzed, and to the fact that the maternal age of these patients was close to 35 years. A very similar frequency, 57.2%, has also been calculated for the 23 chromosomes of the complement from the results obtained from the analysis of 9 chromosomes by FISH.²⁰ Considering the results obtained analyzing 1PBs by CGH^37,38 the corresponding MII by FISH in 67 1PB-MII doublets the mean frequency of aneuploidies was 53.7%, with an incidence of 30.5% being in women under 35 years of age and of 69.5% in women 35 or over. The reliability of the study of aneuploidies by FISH variants would be limited by the quality of the extension of the chromosomes and the existence of chromosome overlapping.³⁸ For this reason the use of CGH and FISH for the study of aneuploidies in 1PBs and MII oocytes is a very adequate method for the correct calculation of the rate of aneuploidies.

It has been observed that in MII oocytes there is a predominance of hyperhaploidies over hypohaploidies^20,64,66 and also a greater proportion of unbalanced PSSC than of chromosome alterations (fig. 9).^20,64,66 These results are opposed to those obtained in studies of aneuploidies carried out in wide series of embryos, in which a predominance of monosomies was observed. The difference could be due to losses of chromosomes that could take place in the first mitotic divisions.⁶⁷ It is not difficult to suppose that the mechanism of chromosomal losses could easily affect free chromatids because they would be weakly joined to one of the poles of the spindle of the second meiotic division (fig. 10). If this were so, the chromatids would tend to be lost in the oocyte cytoplasm. Then, since oocytes with a single-chromatid gain would tend to result in euploid embryos and the oocytes with single-chromatid losses would tend to result in monosomic embryos, the data obtained in MII oocytes and in embryos would not be contradictory (fig. 9).

Chromosomes belonging to all-chromosome groups have been involved in aneuploidy events. Practically all chromosomes of the human karyotype have been found to be involved in aneuploidies.^37,38 The most frequently involved have been chromosomes 1 and 4, followed by chromosomes 22 and X, then by chromosomes 15, 16 and 21, then by chromosomes 2, 3, 6, 7, 8, 9, 13 and 19, and finally by chromosomes 10, 14, 17, 18 and 20. Even though aneuploidies for chromosomes 5, 11 and 12 are not seen in the series, it must be said that on performing a PGD AS analyzing 1PBs by CGH, an aneuploidy affecting chromosome 5 was observed.⁶⁸ In the same way, in a study combining CGH and SKY, an aneuploidy involving chromosome 12 in an oocyte of a woman carrier of a Robertsonian translocation was found.⁶⁹

The application of CGH to the study of aneuploidies has allowed for the identification of up to 38.1% more of the aneuploidies that would be identified with FISH for chromosomes 1, 13, 15, 16, 17, 18, 21, 22 and X. Aneuploidies for chromosomes 3, 4, 6, 7, 8, 9, 10, 14 and 19 would have remained unidentified. Despite the fact that oocytes involving multiple aneuploidies would be identified as aneuploid, by FISH, if they were aneuploid for just one of the 9 analyzed chromosomes, 28% of the aneuploid 1PBs, identified by CGH, would have been missed using FISH for the 9 chromosomes.³⁸ These results agree with the 25% of aneuploid blastomeres identified applying CGH that had been erroneously diagnosed with standard FISH.⁷⁰

Chromosomes 16 and 22 have been frequently involved in aneuploidies in 1PB and in MII oocytes as well as in embryos.^{20,28,38,64,67} Chromosomes 1, 4 and 17 also show a considerable aneuploidy rate.³⁷ Aneuploidies for chromosome, which are not involved in trisomic offspring or in spontaneous abortions,⁷¹ are probably common at the time of conception and would prevent implantation or would induce the death of the implanted embryo.

Chromosome groups E and G are involved in a higher number of aneuploidies due to nondisjunction of the chromosomes of the dyad,^48,61,72,73 and also epidemiological results in spontaneous abortions and livebirths show a prevalence of aneuploidies of female origin for the chromosomes of these groups.^1,74 Considering these chromosomes of groups A, B and C, including the X chromosome as long chromosomes, and those of groups D, E, F and G as short chromosomes it is seen that short chromosomes are more involved in aneuploidies.^{26,28,37,38,54} In 1996, errors identified as produced during the second meiotic division were interpreted as derived from anomalies in the first meiotic division, as a result of an increase in the rate of recombination, which would lead to a failure in the separation of the bivalents.⁷⁵ Currently, it is also believed that a reduction in recombination foci, in the small chromosomes, such as has been shown in human oocytes⁷⁶ and in human spermatocytes,⁷⁷ would results in an increased risk of inadequate disjunction of the chromosomes of the dyad and, thus in the production of aneuploid oocytes.^65,78,79

The simultaneous study by FISH of both cells, the 1PB and the MII oocyte, in women with a 46XX karyotype indicates the existence of a third mechanism producing aneuploid gametes that originate from trisomic oogonia.^20,25 This type of absence of complementarity in studies combining CGH and FISH occurs in approximately 10% of the oocytes studies.³⁷ This fact suggests the existence of gonadal mosaicism in some women who undergo IVF.^20,25,26,38 During cell proliferation in the first weeks of embryonic development, some oogonia (fig. 1) would have undergone an anomalous mitotic segregation originating two new distinct cell lines with chromosomal losses and gains, respectively, coexisting with cytogenetically normal lines. This behavior, is individually-dependent and unpredictable, and makes a prenatal diagnosis quite recommendable.

Results of PGD-1PB in AS

In 1990 a PGD variant was proposed, which was initially called Preconception Genetic Diagnosis, and was based on the indirect analysis of oocytes through the genetic study of the 1PB.⁷

The study of 1PBs is of great interest since, as has been indicated, at least 90% of the chromosomal errors originate in female meiosis. Additionally, this analysis is not affected by the existence of mosaicism, which is very frequent during the first embryonic divisions, and thus makes PGD difficult.^80,81 Another of the advantages of this analysis is that in 1PBs, which are arrested in metaphase II, the interpretation of FISH signals is easier than in proliferating cells which can be in any of the stages of the cell cycle.⁸² This fact can result in errors of interpretation during PGD.⁸¹ But the PGD-1PBs has a serious limitation, compared to the study of embryos: they do not provide information on the paternal contribution. This can be solved through the study of one or two blastomeres of the embryo at the 6-8 cell stage.^27,83 Neither the biopsy of the 1PBs (and 2PBs) nor that of blastomeres has adverse effects on the development of the embryos.^66,83

The Future of DGP-1PBs

The application of CGH is a very powerful tool in the diagnosis of aneuploidies that can be applied to the study of 1PBs but which is not applicable to the study of embryo blastomeres at present unless normal embryos are crypreserved to be transferred in the next cycle. The technique requires a very intense dedication of the specialist, or the availability of a good team of molecular cytogeneticists that is well coordinated with a team of embryologists. Recently, methodological advances have been described that can help to make the timing of CGH to be shorter. The use of variants of DOP-PCR-CGH protocols has been proposed applying different Taq polymerases and labeling methods that, more than shortening the procedure allow for the availability of a product of DOP-PCR that is three times greater.¹⁰¹ Additionally, the emerging development of molecular cytogenetic procedures for isolated cells,^102,103 along with the implementation of the amplification of multiple displacements (MDA), would permit the detection of aneuploidies using a CGH array.¹⁰⁴ A type of CGH array has already permitted the detection of trisomies 13 and 15 in DNA of single cells preamplified with DOP-PCR that should be optimized for the analysis of all chromosomes.¹⁰⁵ The Peptide Nucleic Acids (PNAs) can also help to develop DNA chips applicable to the diagnosis of the human chromosomal complement.¹⁰⁶ All together, it will soon be possible to carry out PGD in blastomeres without embryos cryopreservation.70 But, in women at risk this should be done simultaneously with the analysis of all of the chromosomal complements of the PBs since that will contribute to a greater understanding of the meiotic chromosomal segregation in women involved in IVF cycles. Moreover, when by law, PGD through blastomere analysis is not possible, PGD through the study of 1PBs, becomes vital since it is the only one in which prefertilization aneuploidy screening is available that avoids embryo biopsy.¹⁰⁷

Acknowledgements

This study was supported by Ministerio de Sanidad (FIS PI-020168) and CIRIT (2001 SGR-00201).

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