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Preimplantation genetic diagnosis



In medicine and (clinical) genetics preimplantation genetic diagnosis (PGD) (or also known as Embryo Screening) refers to procedures that are performed on embryos prior to implantation, sometimes even on oocytes prior to fertilization. PGD is considered an alternative to prenatal diagnosis. Its main advantage is that it avoids selective pregnancy termination as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology, and requires in vitro fertilization (IVF) to obtain oocytes or embryos for evaluation. The term preimplantation genetic screening (PGS) is used to denote procedures that do not look for a specific disease but use PGD techniques to identify embryos at risk. PGD is a poorly chosen phrase because, in medicine, to "diagnose" means to identify an illness or determine its cause. An oocyte or early-stage embryo has no symptoms of disease. They are not ill. Rather, they may have a genetic condition that could lead to disease. To "screen" means to test for anatomical, physiological, or genetic conditions in the absence of symptoms of disease. So, both PGD and PGS should, be referred to as types of embryo screening.

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Contents

History

In 1967, Robert Edwards and David Gardner reported the successful sexing of rabbit blastocysts, setting the first steps towards PGD (Edwards and Gardner, 1967). It was not until the 1980s that human IVF was fully developed, which coincided with the breakthrough of the highly sensitive polymerase chain reaction (PCR) technology. Handyside and collaborators' first successful attempts at testing were in October 1989 with the first births in 1990 (Handyside et al., 1992) though the preliminary experiments had been published some years earlier (Coutelle et al., 1989; Holding and Monk, 1989; Handyside et al., 1990). In these first cases, PCR was used for sex determination for patients carrying X-linked diseases.

Indications

Currently, there are mainly two groups of patients for which PGD is indicated.

  • The first group consists of couples with a high risk of transmitting an inherited condition. This can be a monogenic disorder, meaning the condition is due to a single gene only, (autosomal recessive, autosomal dominant or X-linked disorders) or a chromosomal structural aberration (such as a balanced translocation). PGD helps these couples identify embryos carrying a genetic disease or a chromosome abnormality, thus avoiding the difficult choice of abortion. In addition, there are infertile couples who carry an inherited condition and who opt for PGD as it can be easily combined with their IVF treatment.
  • The second group consists of couples who undergo IVF treatment and whose embryos are screened for chromosome aneuploidies. The technique is not used to obtaining a specific prenatal diagnosis but rather for screening, properly referred to as preimplantation genetic screening (PGS), to increase the chances of an ongoing pregnancy. The main indications for PGS are an advanced maternal age, a history of recurrent miscarriages or repeated unsuccessful implantation. It has also been proposed for patients with obstructive and non-obstructive azoospermia.

PGD is available for a large number of monogenic disorders. The most frequently diagnosed autosomal recessive disorders are cystic fibrosis, Beta-thalassemia, sickle cell disease and spinal muscular atrophy type 1. The most common dominant diseases are myotonic dystrophy, Huntington's disease and Charcot-Marie-Tooth disease; and in the case of the X-linked diseases, most of the cycles are performed for fragile X syndrome, haemophilia A and Duchenne muscular dystrophy. Though it is quite infrequent, some centers report PGD for mitochondrial disorders or two indications simultaneously.

In the case of chromosomal abnormalities, PGD is mainly carried out for reciprocal and Robertsonian translocations, and few cases for other abnormalities such as chromosomal inversions or deletions.

Aneuploidy screening is probably the most frequent indication for PGD, mainly suggested to couples undergoing IVF with an advanced maternal age and for patients with repetitive IVF failure. The principle behind it is that, since it is known that numerical chromosomal abnormalities explain most of the cases of pregnancy loss, and a large proportion of the human embryos are aneuploid, the selective replacement of euploid embryos should increase the chances of a successful IVF treatment. Different studies provide indications that PGS increases the implantation rate (Gianaroli at al., 1999; Munne et al., 1999; Munne et al., 2002; Rubio et al., 2003) and lowers the spontaneous abortion rate (Munne et al., 2005), though other studies indicate that there are no significant differences for patients with an advanced maternal age (Kahraman et al., 2000; Staessen et al., 2004), with a poor implantation rate (Kahraman et al., 2000) or with recurrent idiopathic miscarriages (Platteau et al., 2005). It is thus clear that large randomised-controlled studies are still necessary to measure the real impact of this technique for the different indications. A recent systematic review on PGS can be found in the Cochrane database (Twisk et al., 2006).

A fourth group of indications includes all of the ethically difficult cases. These include situations such as human leukocyte antigen (HLA) typing of the embryo, so that the child can be a cord-blood stem cell donor for a sick sibling (Pattinson 2003). Verlinsky and collaborators described the first case in 2001. HLA typing has meanwhile become an important PGD indication in those countries where the law permits it (Verlinsky et al., 2001). The HLA matching can be combined with the diagnosis for monogenic diseases such as Fanconi anaemia or b-thalassemia in those cases where the ailing sibling is affected with this disease, or it may be exceptionally performed on its own for cases such as children with leukaemia. The main ethical argument against is the possible exploitation of the child, although some authors maintain that the Kantian imperative is not breached since the future donor child will not only be a donor but also a loved individual within the family.

Another problematic case is the non-disclosure PGD for Huntington's disease. It is applied when patients do not wish to know their carrier status but want to ensure that they have offspring free of the disease. This procedure can place practitioners in questionable ethical situations, e.g. when no healthy, unaffected embryos are available for transfer and a mock transfer has to be carried out so that the patient does not suspect that he/she is a carrier. The ESHRE ethics task force currently recommends using exclusion testing instead. Exclusion testing is based on a linkage analysis with polymorphic markers, in which the parental and grandparental origin of the chromosomes can be established. This way, only embryos are replaced that do not contain the chromosome derived from the affected grandparent, avoiding the need to detect the mutation itself.

A more recent application of PGD is to diagnose late-onset diseases and (cancer) predisposition syndromes. It can be argued that PGD for late-onset diseases is unethical since the individuals remain healthy until the onset of the disease, usually in the fourth decade of life. On the other hand, the high probability or certainty of developing some disorders, and their incurable nature, can lead to a stressful life, waiting for the first symptoms to occur and to an early death. In the case of predisposition syndromes, such as BRCA1 mutations predisposing to breast cancer, it can be argued that there is no certainty of getting the disease and that the disease can usually be treated. It is a fact that although PGD is often regarded as an early form of prenatal diagnosis, the nature of the requests for PGD often differs from those of prenatal diagnosis requests made when the mother is already pregnant. Some of the widely accepted indications for PGD would not be acceptable for prenatal diagnosis.

Increasingly, PGD is also used for sex selection for non-medical reasons. A 2006 survey[1] found that 9 per cent of clinics in the US provide this service. Half of these perform it only for “family balancing”, which is where a couple with two or more children of one sex desire a child of the other, but half do not restrict sex selection to family balancing. In India, this practice has been used to select only male embryos although this practice is illegal[citation needed]. Opinions on whether sex selection for non-medical reasons is ethically acceptable differ widely, as exemplified by the fact that the ESHRE Task Force could not formulate a uniform recommendation.

Finally, the 2006 survey reveals that PGD has occasionally been used to select against disabilities such as deafness,[2] though it does not give any details.

Technical aspects of preimplantation genetic diagnosis

PGD is a form of genetic diagnosis performed prior to implantation. This implies that the patient’s oocytes should be fertilized in vitro and the embryos kept in culture until the diagnosis is established. It is also necessary to perform a biopsy on these embryos in order to obtain material on which to perform the diagnosis. The diagnosis itself can be carried out using several techniques, depending on the nature of the studied condition. Generally, PCR-based methods are used for monogenic disorders and FISH for chromosomal abnormalities and for sexing those cases in which no PCR protocol is available for an X-linked disease. These techniques need to be adapted to be performed on blastomeres and need to be thoroughly tested on single-cell models prior to clinical use. Finally, after embryo replacement, surplus good quality unaffected embryos can be cryopreserved, to be thawed and transferred back in a next cycle.

Obtaining embryos for preimplantation genetic diagnosis

Currently, all PGD embryos are obtained by assisted reproductive technology, although the use of natural cycles and in vivo fertilization followed by uterine lavage was attempted in the past and is now largely abandoned. In order to obtain a large cohort of oocytes, the patients undergo controlled ovarian stimulation (COH). COH is carried out either in an agonist protocol, using gonadotrophin-releasing hormone (GnRH) analogues for pituitary desensitisation, combined with human menopausal gonadotrophins (hMG) or recombinant follicle stimulating hormone (FSH), or an antagonist protocol using recombinant FSH combined with a GnRH antagonist according to clinical assessment of the patient’s profile (age, body mass index (BMI), endocrine parameters). hCG is administered when at least three follicles of more than 17 mm mean diameter are seen at transvaginal ultrasound scan. Transvaginal ultrasound-guided oocyte retrieval is scheduled 36 hours after hCG administration. Luteal phase supplementation consists of daily intravaginal administration of 600 µg of natural micronized progesterone.

Oocytes are carefully denudated from the cumulus cells, as these cells can be a source of contamination during the PGD if PCR-based technology is used. In the majority of the reported cycles, intracytoplasmic sperm injection (ICSI) is used instead of IVF. The main reasons are to prevent contamination with residual sperm adhered to the zona pellucida and to avoid unexpected fertilization failure. The ICSI procedure is carried out on mature metaphase-II oocytes and fertilization is assessed 16-18 h after. The embryo development is further evaluated every day prior to biopsy and until transfer to the woman’s uterus. During the cleavage stage, embryo evaluation is performed daily on the basis of the number, size, cell-shape and fragmentation rate of the blastomeres. On day 4, embryos were scored in function of their degree of compaction and blastocysts were evaluated according to the quality of the throphectoderm and inner cell mass, and their degree of expansion.

Biopsy procedures

As PGD can be performed on cells from different developmental stages, the biopsy procedures vary accordingly. Theoretically, the biopsy can be performed at all preimplantation stages, but only three have been suggested: on unfertilised and fertilised oocytes (for polar bodies, PBs), on day three cleavage-stage embryos (for blastomeres) and on blastocysts (for trophectoderm cells).

The biopsy procedure always involves two steps: the opening of the zona pellucida and the removal of the cell(s). There are different approaches to both steps, including mechanical, chemical (Tyrode’s acidic solution) and laser technology for the breaching of the zona pellucida, extrusion or aspiration for the removal of PBs and blastomeres, and herniation of the trophectoderm cells.

Polar body biopsy

The first and second polar body of the oocyte are extruded at the time of the conclusion of the meiotic division, normally the first polar body is noted after ovulation, and the second polar body after fertilization. PB biopsy is used mainly by two PGD groups in the USA (Verlinsky et al., 1990; Munne et al., 1995) and by groups in countries where cleavage-stage embryo selection is banned (Montag et al., 2004). They have been used for diagnosing translocations and monogenic disorders of maternal origin, as well as for PGS.

The first PB is removed from the unfertilised oocyte, and the second PB from the zygote, shortly after fertilization. The main advantage of the use of PBs in PGD is that they are not necessary for successful fertilisation or normal embryonic development, thus ensuring no deleterious effect for the embryo. One of the disadvantages of PB biopsy is that it only provides information about the maternal contribution to the embryo, which is why cases of autosomal dominant and X-linked disorders that are maternally transmitted can be diagnosed, and autosomal recessive disorders can only partially be diagnosed. Another drawback is the increased risk of diagnostic error, for instance due to the degradation of the genetic material or events of recombination that lead to heterozygous first PBs. It is generally agreed that it is best to analyse both PBs in order to minimize the risk of misdiagnosis. This can be achieved by sequential biopsy, necessary if monogenic diseases are diagnosed, to be able to differentiate the first from the second PB, or simultaneous biopsy if FISH is to be performed. In Germany, where the legislation bans the selection of preimplantation embryos, PB analysis is the only possible method to perform PGD. The biopsy and analysis of the first and second PBs can be completed before syngamy, which is the moment from which the zygote is considered an embryo and becomes protected by the law.

Cleavage-stage biopsy (Blastomere biopsy)

Cleavage-stage biopsy is generally performed the morning of day three post-fertilization, when normally developing embryos reach the eight-cell stage. The biopsy is usually performed on embryos with less than 50% of anucleated fragments and at an 8-cell or later stage of development. A hole is made in the zona pellucida and one or two blastomeres containing a nucleus are gently aspirated or extruded through the opening. The main advantage of cleavage-stage biopsy over PB analysis is that the genetic input of both parents can be studied. On the other hand, cleavage-stage embryos are found to have a high rate of chromosomal mosaicism, putting into question whether the results obtained on one or two blastomeres will be representative for the rest of the embryo. It is for this reason that some programs utilize a combination of PB biopsy and blastomere biopsy. Furthermore, cleavage-stage biopsy, as in the case of PB biopsy, yields a very limited amount of tissue for diagnosis, necessitating the development of single-cell PCR and FISH techniques. Although theoretically PB biopsy and blastocyst biopsy are less harmful than cleavage-stage biopsy, this is still the prevalent method. It is used in approximately 94% of the PGD cycles reported to the ESHRE PGD Consortium. The main reasons are that it allows for a safer and more complete diagnosis than PB biopsy and still leaves enough time to finish the diagnosis before the embryos must be replaced in the patient’s uterus, unlike blastocyst biopsy. Of all cleavage-stages, it is generally agreed that the optimal moment for biopsy is at the eight-cell stage. It is diagnostically safer than the PB biopsy and, unlike blastocyst biopsy, it allows for the diagnosis of the embryos before day 5. In this stage, the cells are still totipotent and the embryos are not yet compacting. Although it has been shown that up to a quarter of a human embryo can be removed without disrupting its development, it still remains to be studied whether the biopsy of one or two cells correlates with the ability of the embryo to further develop, implant and grow into a full term pregnancy.

Blastocyst biopsy

In an attempt to overcome the difficulties related to single-cell techniques, it has been suggested to biopsy embryos at the blastocyst stage, providing a larger amount of starting material for diagnosis. It has been shown that if more than two cells are present in the same sample tube, the main technical problems of single-cell PCR or FISH would virtually disappear. On the other hand, as in the case of cleavage-stage biopsy, the chromosomal differences between the inner cell mass and the TE can reduce the accuracy of diagnosis, although this mosaicism has been reported to be lower than in cleavage-stage embryos.

TE biopsy has been shown to be successful in animal models such as rabbits (Gardner and Edwards, 1968), mice (Carson et al., 1993) and primates (Summers et al., 1988). These studies show that the removal of some TE cells is not detrimental to the further in vivo development of the embryo.

Human blastocyst-stage biopsy for PGD is performed by making a hole in the ZP on day three of in vitro culture. This allows the developing TE to protrude after blastulation, facilitating the biopsy. On day five post-fertilization, approximately five cells are excised from the TE using a glass needle or laser energy, leaving the embryo largely intact and without loss of inner cell mass. After diagnosis, the embryos can be replaced during the same cycle, or cryopreserved and transferred in a subsequent cycle.

There are two drawbacks to this approach, due to the stage at which it is performed. First, only approximately half of the preimplantation embryos reach the blastocyst stage. This can restrict the number of blastocysts available for biopsy, limiting in some cases the success of the PGD. Mc Arthur and coworkers (2005) report that 21% of the started PGD cycles had no embryo suitable for TE biopsy. This figure is approximately four times higher than the average presented by the ESHRE PGD consortium data, where PB and cleavage-stage biopsy are the predominant reported methods. On the other hand, delaying the biopsy to this late stage of development limits the time to perform the genetic diagnosis, making it difficult to redo a second round of PCR or to rehybridize FISH probes before the embryos should be transferred back to the patient.

Genetic analysis techniques

Fluorescent in situ hybridization (FISH) and Polymerase chain reaction (PCR) are the two most commonly used technologies in PGD, although other approaches have been proposed or are currently in development (such as whole genome amplification and comparative genomic hybridization) . PCR is generally used to diagnose monogenic disorders and FISH is used for the detection of chromosomal abnormalities (for instance, aneuploidy screening or chromosomal translocations).

FISH

FISH is the most commonly applied method to determine the chromosomal constitution of an embryo. In contrast to karyotyping, it can be used on interphase chromosomes, so that it can be used on PBs, blastomeres and TE samples. The cells are fixated on glass microscope slides and hybridised with DNA probes. Each of these probes are specific for part of a chromosome, and are labelled with a fluorochrome. Currently, a large panel of probes are available for different segments of all chromosomes, but the limited number of different fluorochromes confines the number of signals that can be analysed simultaneously.

The type and number of probes that are used on a sample depends on the indication. For sex determination (used for instance when a PCR protocol for a given X-linked disorder is not available), probes for the X and Y chromosomes are applied along with probes for one or more of the autosomes as an internal FISH control. More probes can be added to check for aneuploidies, particularly those that could give raise to a viable pregnancy (such as a trisomy 21). The use of probes for chromosomes X, Y, 13, 14, 15, 16, 18, 21 and 22 has the potential of detecting 70% of the aneuploidies found in spontaneous abortions.

In order to be able to analyse more chromosomes on the same sample, up to three consecutive rounds of FISH can be carried out. In the case of chromosome rearrangements, specific combinations of probes have to be chosen that flank the region of interest. The FISH technique is considered to have an error rate between 5 and 10%.

The main problem of the use of FISH to study the chromosomal constitution of embryos is the elevated mosaicism rate observed at the human preimplantation stage. Sandalinas and collaborators found that up to 70% of the embryos they studied by FISH were mosaic for some kind of chromosomal abnormality (Sandalinas et al., 2001). Li and co-workers (2005) found that 40% of the embryos diagnosed as aneuploid on day 3 turned out to have a euploid inner cell mass at day 6. Staessen and collaborators found that 17.5% of the embryos diagnosed as abnormal during PGS, and subjected to post-PGD reanalysis, were found to also contain normal cells, and 8.4% were found grossly normal (Staessen et al., 2004). As a consequence, it has been questioned whether the one or two cells studied from an embryo are actually representative of the complete embryo, and whether viable embryos are not being discarded due to the limitations of the technique.

PCR

Kary Mullis conceived PCR in 1985 as an in vitro simplified reproduction of the in vivo process of DNA replication. Taking advantage from the chemical properties of DNA and the availability of thermostable DNA polymerases, PCR allows for the enrichment of a DNA sample for a certain sequence. PCR provides the possibility to obtain a large quantity of copies of a particular stretch of the genome, making further analysis possible. It is a highly sensitive and specific technology, which makes it suitable for all kinds of genetic diagnosis, including PGD. Currently, many different variations exist on the PCR itself, as well as on the different methods for the posterior analysis of the PCR products.

When using PCR in PGD, one is faced with a problem that is inexistent in routine genetic analysis: the minute amounts of available genomic DNA. As PGD is performed on single cells, PCR has to be adapted and pushed to its physical limits, and use the minimum amount of template possible: one strand. This implies a long process of fine-tuning of the PCR conditions and a susceptibility to all the problems of conventional PCR, but several degrees intensified. The high number of needed PCR cycles and the limited amount of template makes single-cell PCR very sensitive to contamination. Another problem specific to single-cell PCR is the allele drop out (ADO) phenomenon. It consists of the random non-amplification of one of the alleles present in a heterozygous sample. ADO seriously compromises the reliability of PGD as a heterozygous embryo could be diagnosed as affected or unaffected depending on which allele would fail to amplify. This is particularly concerning in PGD for autosomal dominant disorders, where ADO of the affected allele could lead to the transfer of an affected embryo.

Establishing a diagnosis

The establishment of a diagnosis in PGD is not always straightforward. The criteria used for choosing the embryos to be replaced after FISH or PCR results are not equal in all centres. In the case of FISH, in some centres only embryos are replaced that are found to be chromosomally normal (that is, showing two signals for the gonosomes and the analysed autosomes) after the analysis of one or two blastomeres, and when two blastomeres are analysed, the results should be concordant. Other centres argue that embryos diagnosed as monosomic could be transferred, because the false monosomy (i.e. loss of one FISH signal in a normal dipoloid cell) is the most frequently occurring misdiagnosis. In these cases, there is no risk for an aneuploid pregnancy, and normal diploid embryos are not lost for transfer because of a FISH error. Moreover, it has been shown that embryos diagnosed as monosomic on day 3 (except for chromosomes X and 21), never develop to blastocyst, which correlates with the fact that these monosomies are never observed in ongoing pregnancies.

Diagnosis and misdiagnosis in PGD using PCR have been mathematically modelled in the work of Navidi and Arnheim and of Lewis and collaborators (Navidi and Arnheim, 1991; Lewis et al., 2001). The most important conclusion of these publications is that for the efficient and accurate diagnosis of an embryo, two genotypes are required. This can be based on a linked marker and disease genotypes from a single cell or on marker/disease genotypes of two cells. An interesting aspect explored in these papers is the detailed study of all possible combinations of alleles that may appear in the PCR results for a particular embryo. The authors indicate that some of the genotypes that can be obtained during diagnosis may not be concordant with the expected pattern of linked marker genotypes, but are still providing sufficient confidence about the unaffected genotype of the embryo. Although these models are reassuring, they are based on a theoretical model, and generally the diagnosis is established on a more conservative basis, aiming to avoid the possibility of misdiagnosis. When unexpected alleles appear during the analysis of a cell, depending on the genotype observed, it is considered that either an abnormal cell has been analysed or that contamination has occurred, and that no diagnosis can be established. A case in which the abnormality of the analysed cell can be clearly identified is when, using a multiplex PCR for linked markers, only the alleles of one of the parents are found in the sample. In this case, the cell can be considered as carrying a monosomy for the chromosome on which the markers are located, or, possibly, as haploid. The appearance of a single allele that indicates an affected genotype is considered sufficient to diagnose the embryo as affected, and embryos that have been diagnosed with a complete unaffected genotype are preferred for replacement. Although this policy may lead to a lower number of unaffected embryos suitable for transfer, it is considered preferable to the possibility of a misdiagnosis.

Preimplantation Genetic Haplotyping

Preimplantation Genetic Haplotyping (PGH) is a new clinical method of Preimplantation genetic diagnosis (PGD).

Embryo transfer and cryopreservation of surplus embryos

Embryo transfer is usually performed on day three or day five post-fertilization, the timing depending on the techniques used for PGD and the standard procedures of the IVF centre where it is performed.

With the introduction in Europe of the single-embryo transfer policy, which aims at the reduction of the incidence of multiple pregnancies after ART, usually one embryo or early blastocyst is replaced in the uterus. Serum hCG is determined at day 12. If a pregnancy is established, an ultrasound examination at 7 weeks is performed to confirm the presence of a foetal heartbeat. Couples are generally advised to undergo PND because of the, albeit low, risk of misdiagnosis.

It is not unusual that after the PGD, there are more embryos suitable for transferring back to the woman than necessary. For the couples undergoing PGD, those embryos are very valuable, as their current cycle may not lead to an ongoing pregnancy. The cryopreservation and later thawing and replacement of these embryos would give them a second chance to pregnancy without undergoing another time the cumbersome and expensive ART and PGD procedures.

Cryopreservation of mammal embryos has proven difficult, causing severe cellular damage, and, consequently, low survival rates and implantation rates. Numerous modifications have been assayed on the different cryopreservation protocols, and applied to cleavage-stage embryos and blastocysts, leading to increasing success rates. On the other hand, biopsied embryos have shown a much lower survival rate after cryopreservation than intact embryos, the biopsy hole in the zona pellucida being hold responsible for the higher susceptibility to freeze-thaw damage.

Ethical issues

PGD has raised ethical issues. The technique can be used to determine the gender of the embryo, and thus can be used to select embryos of one gender in preference of the other in the context of “family balancing”. It may be possible to make other "social selection" choices in the future. While controversial, this approach is less destructive than fetal deselection during the pregnancy.

Costs are substantial and insurance coverage may not be available. Thus PGD widens the gap between people who can afford the procedure versus a majority of patients who may benefit but cannot afford the service.

PGD has the potential to screen for genetic issues unrelated to medical necessity. The prospect of a “designer baby” is closely related to the PGD technique.

By relying on the result of one cell from the multi-cell embryo, it assumed that this cell is representative of the remainder of the embryo. This may not be the case as the incidence of mosaicism is often relatively high. On occasion, PGD may result in a false negative result leading to the acceptance of an abnormal embryo, or in a false positive result leading to the deselection of a normal embryo.

References in Popular Culture

  • PGD features prominently in the 1997 film Gattaca. The movie is set in a near-future world where PGD/IVF is the most common form of reproduction. In the movie parents routinely use PGD to select desirable traits for their children such as height, eye color and freedom from even the smallest of genetic predispositions to disease. The ethical consequences of PGD are explored through the story of the main character who faces discrimination because he was conceived without such methods.
 
This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Preimplantation_genetic_diagnosis". A list of authors is available in Wikipedia.
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