Welcome to chapter 11. The following chapter is called Basics of genetic diagnosis and embryo biopsy. The author is Dr. Anick De Vos.

Transcription

1 Welcome to chapter 11. The following chapter is called Basics of genetic diagnosis and embryo biopsy. The author is Dr. Anick De Vos.

2 After finishing this chapter, the student should be familiar with the following important concepts: The two major methods currently used for diagnostic purposes in preimplantation genetic diagnosis (PGD): are fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). Furthermore, the student should know the different indications for PGD. The student should be familiar with the different aspects of the biopsy procedure, including opening of the zona pellucida and cell removal. And, finally, the student should be aware of other developmental stages in which cellular material for diagnosis can be removed. 2

3 Two major methods are currently used for diagnostic purposes in PGD. FISH allows the visualization of chromosomal regions and is used in sexing (in case of X-linked diseases), aneuploidy testing and PGD for translocations. The obvious limitation of FISH is that only a restricted number of chromosomes can be tested in a single cell. PCR, which allows the exponential amplification of a short DNA fragment, is widely used for diagnosis of monogenic diseases. Today, multiplex PCR (a technique allowing simultaneous amplification of two or more DNA sequences) is used extensively in PGD, especially for more frequently occurring diseases. Allowing simultaneous testing of linked markers alongside the mutation test significantly increased the accuracy of single-cell PCR. More recent developments include comparative genomic hybridization (CGH) and whole genome amplification (WGA). 3

4 In FISH, fluorochrome-labeled DNA probes that are complementary to DNA sequences specific to individual chromosomes are used. This technique is useful for identifying chromosomal abnormalities within a single cell: both numerical and structural abnormalities can be detected. 4

5 The PCR procedure involves exponential amplification of short DNA fragments using two short, single-stranded DNA pieces (primers) that are complementary to the boundaries of the sequence to be amplified. PCR allows analysis of the genetic content of a single cell. In PGD, DNA amplification is used to detect single-gene defects, so-called monogenic diseases. PCR protocols are, however, subject to a variety of pitfalls, such as variable amplification efficiency, contamination and allele drop out (ADO). The management of these pitfalls is of extreme importance because they may result in misdiagnosis. Multiplex PCR with linked markers represents a straightforward strategy to do so. Allowing simultaneous testing of linked markers alongside the mutation test significantly increases the accuracy of single cell PCR (Lewis et al., 2001). 5

6 A promising recent development, however not yet routinely used, is the technique of comparative genomic hybridization (CGH), which allows all chromosomes to be simultaneously identified in a single cell. Initial reports showed its application in assessing aneuploidy and structural chromosomal aberrations in polar bodies and blastomeres (Malmgren et al., 2002 ; Wells et al., 2002). The use of metaphase CGH to screen all human chromosomes for aneuploidy in preimplantation embryos is hindered by the time required to perform the analysis. Microarray CGH could alleviate this time constraint (Hu et al., 2004). For further reading see a recent review article regarding this subject (Wilton, 2005). 6

7 Also, more recently developed, is the use of whole genome amplification (WGA) as a universal first step to generate a high yield, accurate and complete representation of the original single-cell template, allowing subsequent simultaneous analysis of more than one mutation by standard PCR protocols or microarrays. The inclusion of several polymorphic markers allows haplotype analysis. This WGA technique is isothermal and depends on multiple displacement amplification (MDA), using bacteriophage phi 29 DNA polymerase and random hexamer primers. Reports indicated its use at the single-cell level as successful (Handyside et al., 2004; Hellani et al., 2004). For further reading, see two recent review articles on this subject (Coskun and Alsmadi, 2007; Spits and Sermon, 2009). 7

8 The PGD procedure is a very early form of prenatal diagnosis for patients with a preexisting genetic risk. Oocytes and embryos obtained in vitro through assisted reproductive techniques, in particular ICSI (Intra Cytoplasmic Sperm Injection), undergo biopsy and the cells (polar bodies, blastomeres, or trophectoderm cells) are used for genetic diagnosis. In principle, the indications for PGD are similar to those for prenatal diagnosis after chorionic villus sampling or amniocentesis, given that PGD at the single-cell level is technically possible. The main reasons for PGD (in addition to the genetic risk) are previous termination of pregnancy and objection to pregnancy termination in case of a spontaneous affected pregnancy. The genetic risk can also be combined with subfertility or infertility. 8

9 The PGD-FISH procedure is performed for couples at risk for chromosomal disorders, which can be divided into two groups: structural and numerical abnormalities. Structural aberrations involve reciprocal translocations, robertsonian translocations, inversions, deletions and microdeletions. In the group of numerical abnormalities, patients with Klinefelter syndrome can benefit from PGD-FISH because the normal sex chromosome content of the embryos is determined before transfer. Aneuploidy testing is performed for the following indications: advanced maternal age, recurrent miscarriages, repeated IVF failures and eventually in cases of non-obstructive azoospermia. A main disadvantage of sexing for X-linked diseases is that half of the male embryos are healthy and will not be transferred, and the transfer of female carrier embryos cannot be avoided. Couples may opt for a specific DNA diagnosis, given that the mutation is known and a single-cell test is available, rather than simple sexing. 9

10 The PGD-PCR procedure is used for couples at risk for monogenic disorders. Three disease groups can be distinguished according to the mode of inheritance: X-linked disorders, autosomal recessive disorders and autosomal dominant disorders. Several laboratories offer patients custom-made PGD tests for monogenic disease, making it difficult to establish the exact number of indications for which PGD is offered. More recently, combined PGD and human leukocyte antigen (HLA) matching has been reported (Van de Velde et al., 2004 ; Fiorentino et al., 2004), representing an innovative way to preselect an HLA identical healthy embryo for an affected sibling, requiring hematopoietic stem cell (HSC) transplantation (in cases of Fanconi anemia, leukemia, Wiskott-Aldrich syndrome, and beta-thalassemia). At birth, the cord blood which contains HSCs of the newborn can be used to cure the affected sibling. 10

11 Cleavage-stage embryo biopsy is performed in the morning of day three, hours post-microinjection (or insemination), when the embryos are preferably in the eight-cell stage. The removal of cells requires an opening in the zona pellucida, which may be created in a mechanical or chemical way, or otherwise by laser energy. One or two blastomeres may be removed primarily by aspiration. Incubating the embryos in calcium magnesium free decompaction medium may facilitate the biopsy procedure. The cellular material removed should be suitable for genetic analysis, either by FISH or by PCR. This means that the cell should be intact and it should contain only one, clearly visible nucleus. Further development of the embryo may not be impaired as a result of the biopsy procedure (Liebaers et al., 2010). 11

12 Three different biopsy procedures according to the developmental stage are wellestablished: polar body removal from oocytes and zygotes, cleavage-stage embryo biopsy, and biopsy at the blastocyst stage. The European Society of Human Reproduction and Embryology (ESHRE), PGD Consortium data collection (Goossens et al., 2009) show that the most widely used biopsy procedure is cleavage-stage biopsy. One or two blastomeres are removed from the embryo on day 3. The embryos are preferably in the third mitotic division at that time, presenting at least five cells. More ideal for biopsy, of course, is a further advanced division of all blastomeres resulting in synchronous eight-cell embryos at about hours after microinjection. 12

13 In order to avoid contamination with naked sperm DNA, embryos for PGD are ideally obtained by microinjection of a single sperm cell. Also, care should be taken at the moment of oocyte denudation, that all remaining cumulus and corona cells, which could also be a source of DNA contamination, are removed. 13

14 Cleavage-stage biopsy of human preimplantation embryos always involves two steps: opening of the zona pellucida and subsequent removal of cellular material by aspiration. Laser-assisted zona drilling results in an opening in the zona pellucida with an average size of 20 to 30µm. A biopsy pipette is introduced through the hole and a first blastomere is aspirated. Aspiration of a second blastomere, if needed, can be done in a similar way. Preferably, blastomeres with only one clearly visible nucleus are aspirated. 14

15 The zona pellucida can be opened in three different ways: either mechanically (direct puncture, partial zona dissection, or zona slitting), chemically (using acid Tyrode solution), or by the recently introduced laser technology. Chemical zona drilling remains a widely used approach, probably because it is a much cheaper option compared to laser technology. Local acidification may cause cell lysis or subtle damage to cells and should be considered when evaluating the safety of this approach. Laser technology is certainly less detrimental, extremely accurate, and simple in use (Joris et al., 2003). The number of PGD cycles where laser is used to open the zona pellucida starts to exceed the number of PGD cycles where acidic Tyrode is still used (Goossens et al. 2009). Human zona pellucida opening procedures date from the late 1980s and early 1990s, when applied in order to assist human oocyte fertilization and in order to help the hatching process of the embryo. It was a small step toward cell removal for the purpose of PGD. 15

16 The removal of human blastomeres for clinical PGD is done primarily by aspiration (Goossens et al., 2009). Other methods used include extrusion (Levinson et al., 1992) and flow displacement (Pierce et al., 1997). Apart from the reduction in cellular mass, the development of human embryos to the blastocyst stage in vitro is unaffected by biopsy and removal of one or two cells at the eight-cell stage (Hardy et al., 1990), which means that up to one-quarter can be removed without impairment of its further in vitro development. However, more recently, evidence has accumulated that two-cell biopsy seems more detrimental than one-cell biopsy: indirect evidence from cryopreservation analogy and direct evidence through randomized studies (Goossens et al., 2007; De Vos et al., 2009). 16

17 The essence of the biopsy procedure is a balance between cell retrieval in order to allow a safe and correct diagnosis of the embryo, and the preservation of the implantation potential of the remaining embryo. The prime concern is that the diagnosis should be accurate and efficient, and misdiagnosis should be avoided at any cost. Technical improvements at the diagnostic level have allowed similar diagnostic accuracy to be obtained on one blastomere as compared to two blastomeres for certain indications or tests (Lewis et al., 2001). However, certain types of analysis where one-cell biopsy is not compatible with an accurate diagnosis (if no multiplex PCR test is available, translocations) will benefit, or will even require, having two cells available for diagnosis. In respect of safeguarding the implantation potential of the biopsied embryos, the removal of only one blastomere is recommended, provided that diagnostic safety measures are in place to ensure a correct diagnosis. 17

18 Indirect evidence for the possible detrimental effect of embryo biopsy has been sought in embryo cryopreservation data, assuming that cell loss from biopsy can be compared with cell loss after thawing of frozen cleaved embryos (Cohen et al., 2007). A disproportionate reduction in implantation potential according to the cell number lost has been described. Chromosomal aneuploidy testing on one cell however, does not interrogate chromosomal mosaicism, which is commonly seen in cleavage-stage embryos and represents a serious obstacle for proper aneuploidy testing. One cell removal gives more embryos with a normal test result; however, it has a lower predictive value. Two cell removal, results in fewer embryos for transfer but has a higher predictive value. The risk of aggravation of mosaicism is greater in cases of a two-cell than in one-cell biopsies (Los et al., 2004). 18

19 Removal of two blastomeres decreases the likelihood of blastocyst formation compared with the removal of one blastomere (Goossens et al., 2008). Delivery rates with live birth per started cycle however, were not significantly different (Goossens et al., 2008). Nevertheless, with an absolute difference of 3 % in live birth delivery rates, these data show that for every 33 cycles, there will be one more live birth delivery with one-cell than with two-cell biopsy. For this reason, one-cell biopsy is recommended as a valid alternative for two-cell biopsy, provided methods are in place for an accurate diagnosis on one cell. 19

20 In a prospective analyzed cohort of single blastocyst transfers, the clinical outcome from one-cell biopsy was significantly better than that of a two-cell biopsy and comparable to the situation where no intervention was performed on day three, on the eight-cell embryos (De Vos et al., 2009). The present data should facilitate the informed choice between biopsy of one cell, given sufficient safe guards for a correct diagnosis; or two cells, at the expense of the implantation potential. 20

21 Whereas full compaction does not occur before the 16- to 32-cell stage, relatively early assembly of tight junctions does occur between human blastomeres. These membrane adhesions may cause the biopsy procedure at the seven- or eight-cell stage to be rather difficult to perform because the blastomeres show a strong tendency to adhere to each other. Ca 2+ /Mg 2+ -free medium is used to loosen the membrane adhesions between blastomeres, resulting in an easier removal of blastomeres, less cell lysis and a shorter procedure time (Dumoulin et al., 1998). 21

22 Embryo biopsy may either be performed completely in decompaction medium or otherwise in order to limit the exposure time embryos can be preincubated for 5 to 10 minutes (normally sufficient for full decompaction) prior to the biopsy procedure. 22

23 Multinucleation is a frequently observed phenomenon in cleavage-stage embryos. The knowledge, that the chromosome constitution of multinucleated blastomeres is frequently different from that of their sibling blastomeres makes them unsuitable for PGD, because they are not representative for the entire embryo (Munné and Cohen, 1993). Of course, the proportion of multinucleated blastomeres within one embryo is an important parameter. Embryos presenting more than 50 % of their blastomeres with multinucleation are not considered for embryo biopsy or implantation. They have been associated with impaired cleavage and increased fragmentation, with a poor prognosis for blastocyst formation and ongoing implantation rate, most likely due to chromosomal abnormalities (Kligman et al., 1996). 23

24 The incidence of anucleate blastomeres is also high in normally fertilized embryos, especially in those of poor morphology. Efficient PGD requires a careful assessment of the nuclear status by light microscopy, in order to exclude both multinucleated and anucleate cells from diagnosis. Sometimes extreme granularity of the blastomeres may complicate this assessment. An incorrect interpretation of the nuclear status of a blastomere may be noticed at fixation in cases of FISH procedure. A PCR procedure lacks this intermediate step, because cells are immediately transferred to the PCR tubes and the amplification result is available only later. 24

25 Successful cleavage-stage biopsy involves two aspects. First, the cellular material should be suitable for genetic analysis either by FISH or by PCR. This means that the cell should be intact and it should contain one clearly visible nucleus. Secondly, further development may not be impaired as a result of the biopsy procedure. 25

26 Although a good quality control measure for the biopsy procedure, blastomere lysis rates (expressed per aspirated blastomere) are hard to find in literature. Cell lysis may occur at both steps of the procedure: at the moment of zona opening, related to the method used; or when aspirating the blastomeres, related to excessive mechanical stress or to strong adherence of the blastomeres to each other. Cell lysis most often means the loss of the nuclear material for diagnosis. Occasionally, the nucleus can be recovered for FISH analysis. Lysed cells are not used for PCR analysis, because contamination with maternal DNA cannot be excluded. In cases of cell lysis, it is advised that the aspiration pipette is changed before continuing biopsy of other embryos as a safety measure in order to avoid crosscontamination. 26

27 Further embryo development should not be impaired as a result of the biopsy procedure. Embryo post-biopsy development can be evaluated on day four, where a doubling of cells and/or signs of compaction represents a good evolution. Or otherwise, extending the culture until day five allows the quality of the inner cell mass and trophectoderm to be evaluated. Further in vivo development post-biopsy may be reflected by the implantation rates obtained post-pgd. In this respect, large data collections are very much valued to serve as a standard reference of good laboratory and clinical practice (Goossens et al., 2009). 27

28 Theoretically, cells for genetic diagnosis can be removed at any preimplantation developmental stage. As early as from the oocyte, polar bodies can be removed. The first polar body preconceptionally before injection of the oocyte and the second polar body, from the zygote stage, after insemination and fertilization. Both polar bodies represent extra-embryonic material. The advantage of biopsy at later developmental stages, such as the blastocyst stage, is that comparatively more cells can be collected than with biopsy at earlier developmental stages. At the blastocyst stage, extra-embryonic trophectoderm cells can be removed, leaving the inner cell mass (representing the future fetus) fully intact. 28

29 Initially only the first polar body was removed prior to insemination, however both first and second polar body can now be sampled simultaneously following insemination (Verlinsky et al., 1997). Both polar bodies represent extra-embryonic material and are expected to have no biological role in the development of the future embryo (Kaplan et al., 1995). However, a major limitation of polar body analysis is that only the maternal contribution to the future embryo can be evaluated. Polar body analysis represents an indirect method in which the genotype or chromosomal constitution of the oocyte is derived from the complement of the polar bodies. The accuracy of polar body analysis depends on information from both polar bodies, allowing possible recombination to be detected. Polar body biopsy is used for monogenic diseases, aneuploidies and translocations of female origin, albeit to a much lesser extent than cleavage-stage biopsy. 29

30 Blastocyst biopsy involves removal of extra-embryonic trophectoderm cells. Zona slitting is used to open the zona pellucida with a microneedle, or otherwise laser energy is used (Veiga et al., 1997). After herniation through the opening, trophectoderm cells are removed by excision using a glass needle or laser energy. A major limitation is the uncertainty of whether the trophectoderm cells are a proper representative of the embryo. The degree of blastocyst mosaicism seems lower than in cleavage-stage embryos (Los et al., 2004). However, no single study has compared the genetic constitution of the trophectoderm and that of the inner cell mass of single human blastocysts. Additionally, there is a time limitation to obtain the diagnostic result if a fresh transfer is attempted. Also, only half or fewer of the embryos from IVF are capable of reaching the blastocyst stage, resulting in fewer embryos for genetic analysis. Only limited clinical application has been reported. 30

31 To conclude this chapter, let me reinforce that FISH and PCR are routinely used diagnostic techniques for clinical PGD, in order to analyze the presence of chromosomal abnormalities and monogenic diseases in preimplantation embryos before transfer into the uterus. Comparative genome hybridization and whole genome amplification represent more recent developments that are showing promising results. However, their routine use awaits the result of further technical improvements. 31

32 It is important to remember that cleavage-stage embryo biopsy, in contrast to PB biopsy or TE biopsy at the blastocyst stage remains the main approach for obtaining cellular material in view of PGD. Embryos for PGD are preferentially obtained by single sperm microinjection in order to avoid DNA contamination, either from maternal cumulus cells or paternal sperm cells. Cleavage-stage biopsy s main advantage is that the paternal contribution to the embryo can be analyzed and that enough time for testing is available in case of fresh transfer on day five. 32

33 In order to summarize the aspects of embryo biopsy, the procedure is performed on day three, when the embryos are preferably in the eight-cell stage. The removal of one cell is recommended when safety measures are in place to ensure a correct diagnosis. Otherwise, at the expense of the implantation potential, two cells are removed. Decompaction might facilitate the biopsy procedure. It is important that the cells removed are suitable for genetic diagnosis; this means that they are intact and contain only one visible nucleus. 33

34 Finally, post-biopsy development of the embryo should not be impaired by the biopsy procedure, as evidenced by blastocyst formation on day five, implantation rates and live birth rates per started cycle. In this respect, large data collections are very much valued to serve as a standard reference of good laboratory and clinical practice. 34

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