Blastomere fixation techniques and risk of misdiagnosis for preimplantation genetic diagnosis of aneuploidy

RBMOnline - Vol 4. No 3. 210–217 Reproductive BioMedicine Online; www.rbmonline.com/Article/467 on web 25 February 2002

Articles Blastomere fixation techniques and risk of misdiagnosis for preimplantation genetic diagnosis of aneuploidy Esther Velilla began her studies in biology in 1990 at Universitat Autonoma de Barcelona (Spain) and was awarded her Bachelor degree in 1995. In 1998 she obtained her MSc degree in Cellular Biology in the Science Faculty at the same University. She then moved to the Veterinary Faculty to develop her PhD thesis on in-vitro maturation and fertilization studies on prepubertal and adult goat oocytes. During the same period she began her work in human reproduction at the Clinic Instituto de Reproduccion CEFER (Barcelona). In 2001 she began working at The Institute for Reproductive Medicine and Science of Saint Barnabas (Livingston, NJ, USA) in the field of preimplantation genetic diagnosis under the direction of Santiago Munné. Esther Velilla Esther Velilla, Tomas Escudero, Santiago Munné1 Institute for Reproductive Medicine and Science of Saint Barnabas Medical Centre, 101 Old Short Hills Road, Suite 501, West Orange, NJ-07052, USA 1Correspondence: Tel: +1-973–3226236, Fax: +1-973–3226235, e-mail: [email protected]

Abstract One of the most critical steps in preimplantation genetic diagnosis (PGD) studies is the fixation required to obtain good fluorescence in-situ hybridization (FISH) nuclear quality without losing any of the cells analysed. Different fixation techniques have been described. The aim of this study was to compare three fixation methods (1, acetic acid/methanol; 2, Tween 20; 3, Tween 20 and acetic acid/methanol) based on number of cells lost after fixation, average rate of informative cells, rate of signal overlaps and FISH errors. A total of 100, 106 and 114 blastomeres were fixed using techniques 1, 2 and 3 respectively. Technique 2 gave the poorest nuclear quality with higher cytoplasm, number of overlaps and FISH errors. Although technique 1 showed better nuclear quality in terms of greater nuclear diameter, fewer overlaps and FISH errors, it is difficult to perform correctly. However, technique 3 shows reasonably good nuclear quality and is both easier to learn and use for PGD studies than the others. Keywords: cell fixation, FISH, fluorescence in-situ hybridization, preimplantation genetic diagnosis, signal overlap

Introduction Preimplantation genetic diagnosis (PGD) for gender selection (Griffin et al., 1992; Munné et al., 1993a), aneuploidy (Munné et al., 1993b, 1995, 1999; Verlinsky et al., 1995; Gianaroli et al., 1999), and structural abnormalities (Munné et al., 1996, 1998; Verlinsky and Evsikov, 1999) involves the biopsy of one or both polar bodies or the biopsy of one or two blastomeres, fixation to glass slides, followed by fluorescence in-situ hybridization (FISH) analysis. One of the most critical steps in this process is cell fixation, because ideally not a single cell should be lost, and each cell should be informative in order to have the best possible results for each embryo.

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The traditional fixation method, first developed by Tarkowski et al. (1966) and later modified in different ways by many authors, involves the use of acetic acid/methanol (Carnoy) solution applied to a small drop of hypotonic solution containing the blastomere to be fixed. This process requires

considerable practice and skill, and for this reason at least, two other cell fixation methods have been developed, one using Tween 20 solution (Coonen et al., 1994) and the other a combination of Tween 20 and Carnoy solution (Dozortsev and McGinnis, 2001). These two last methods require less skill and seem to be less prone to cell loss during fixation (Xu et al., 1998; Dozortsev and McGinnis, 2001). However, because the two newer methods rely on total or partial drying of the cell, a large nuclear diameter is hard to achieve (Hliscs et al., 1997); large diameters are desirable because they have been inversely correlated to signal overlaps and FISH errors (Munné et al., 1996). To date, no study has yet analysed the FISH error rate of the two last methods; the traditional Carnoy’s method produces about 10–15% errors (Munné and Weier, 1996; Munné et al., 1998, 1999). The purpose of this study was to compare the three fixation methods based on number of cells lost after fixation, average rate of informative cells, rate of signal overlaps and FISH errors.

Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.

excess of the ones transferred, were disaggregated and analysed, because cryopreservation of biopsied embryos is considered inefficient (Magli et al., 1999).

Materials and methods Source of embryos Embryos were obtained from patients undergoing IVF at The Institute for Reproductive Medicine and Science at Saint Barnabas Medical Centre (Livingston, NJ, USA) in accordance with guidelines approved by the internal review board. Written patient consent for donated embryo research and for PGD was provided for all embryos. There were two sources of embryos. The first source was supernumerary embryos with compromised morphology and/or development not used in embryo replacement or cryopreservation. Severely compromised development included embryos with fewer than four cells on day 3, or with a normal number of cells but with >35% fragmentation or multinucleation on day 2 of development (Alikani et al., 1999, 2000). The other source was embryos that after PGD were found to be chromosomally abnormal. Only those embryos with three or more cells with observed nuclei after fixation were included in this study.

Blastomere fixation and FISH The PGD embryos had one cell biopsied on day 3 of development (Grifo, 1992). Those embryos considered to be chromosomally, morphologically and developmentally normal were replaced. Many of the embryos classified as normal by PGD were transferred to the patient on the same day as the analysis. The non-transferred embryos, either chromosomally normal or abnormal, had their zonae pellucida removed by exposure to Pronase solution (3 mg/ml, Sigma), were transferred to Ca/Mg-free media for 10 min, and were then fixed each cell individually as described below. Infrequently, some chromosomally and morphologically normal embryos, in

Only one person performed the fixation for all three methods. This person had no previous experience in fixation, and started the study once she felt proficient in all three methods. Embryos were randomly assigned to one of three fixation methods: (1) acetic acid/methanol (Tarkowski, 1966, modified by Munné et al., 1998); (2) Tween 20-HCl (Coonen et al., 1994); or (3) Tween 20-HCl and acetic acid/methanol (Dozortev and McGinnis, 2001).

Method 1 This method was described by Tarkowski (1966) and modified for single blastomere fixation by Munné et al. (1998). The whole process was performed using a stereoscope (Leica MZ9.5, Leica Wild MZ8) with a base having a mirror that could move from vertical to horizontal position; for this process, the mirror was at a 30° angle. The blastomere was exposed to hypotonic solution [0.075 mol/l KCl supplemented with 0.6% BSA (w/v)] for 2 min. Then the blastomere was placed onto the microscope slide in a small hypotonic drop (1–2 µl) using a 0.16 mm inner-diameter microneedle. After that, 1 drop of methanol:acetic acid (3:1) fixative was added over the blastomere. Often, the blastomere moved after the first fixative drop, but was easily detectable if the slide was clean of dust because the position of the stereoscope mirror produced a three-dimensional effect that allowed localization of objects protruding from the glass slide (Figure 1). Blastomeres before cytoplasm breakdown appear refringent and with smooth circular edges. After the blastomere settled, but before the cytoplasm burst, a second drop of hypotonic was added. While the second drop was drying, humidity was added by breathing over the blastomere to facilitate cytoplasm

Table 1. Analysable cells depending on different fixation methods and studies. Method

Fixed

Cells Lost (%)

No. nuclei

Nucleated Analysable (%)

Found

Xu et al. (1998) 1

121

26 (21.5)a

95

ND

ND

76 (62.8)c

2 Dozortsev and McGinnis (2001)

131

8 (6.1)b

123

ND

ND

60 (45.8)d

1

16

2 (13.0)

ND

ND

ND

13 (81.0)

2

16

1 (16.0)

ND

ND

ND

14 (87.0)

3

18

0 (0.0)

ND

ND

ND

18 (100.0)

1

110

4 (3.6)

106

15

91

89 (84.0)e

2

106

3 (2.8)

103

22

81

71 (68.9)f

3

114

3 (2.6)

111

10

101

92 (82.9)

Present study

a versus b, c versus d: P < 0.001; e versus f: P < 0.025. ND = not determined.

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Figure 1. An expanded blastomere before cytoplasm burst, showing a three-dimensional effect.

Figure 2. Blastomeres were analysed by FISH with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold). Blastomeres (nucleus diameter = 62 µm) were fixed using method 1.

Figure 3. Blastomere (nucleus diameter = 17 µm) shows overlaps between chromosomes 16 and 13, 16 and 21, 21 and 22, and 22 and 18. Blastomeres were analysed by FISH with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold) and fixed using method 2.

Figure 4. A binucleated blastomere (nucleus diameter = 20 and 10 µm) shows overlaps in the top nucleus between chromosomes 13 and 18, and 21 and 22. In addition excess cytoplasm can be seen. Blastomeres were analysed by FISH with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold) and fixed using method 2.

Table 2. Number of signal overlaps and FISH errors in the present study. Method

Analysablea

Diameter ± SD (µm)

Nuclei with errors (%)

No. total FISH (%) overlaps

1

89

58.5 ± 20.7

12 (13.5)a

16d

9 (10.1)g

2

71

30.8 ± 12.9

41 (57.7)b

88e

21 (29.6)h

46.0 ± 18.7

(39.1)c

45f

16 (17.4)

3

212

92

36

a versus b, a versus c, d versus e, d versus f: P < 0.001; g versus h: P < 0.005. aSee differences between techniques in Table 1 for ‘Analysable’.

Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.

Figure 5. Nucleus (diameter = 21 µm) with overlaps between chromosomes 16, 18 and 21 and between 16 and 18, with considerable cytoplasm. Blastomeres were analysed by FISH with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold) and fixed using method 2.

Figure 6. Another binucleated blastomere (nucleus diameter = 24 and 27 µm) with overlaps between chromosomes 13, 16 and 18 and between 13 and 16 in the top nucleus, and overlaps between chromosomes 13, 16 and 21 and between 13 and 16 in the bottom nucleus. Blastomeres were analysed by FISH with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold) and fixed using method 2.

Method 3 This method was described by Dozortsev and McGinnis (2001). The blastomere was placed (5–10 s) in hypotonic solution (1% sodium citrate in 0.2 mg/ml BSA) and then washed in Tween 20 solution (1% of Tween 20 in 0.01 N HCl, 1% in 0.01 N HCl) for 40 s. After that, it was placed onto a glass slide with 3–4 µl of Tween 20 under a stereoscope microscope and allowed to dry completely. In order to remove the remaining cytoplasm, several drops of methanol: acetic (3:1) fixative were added.

Figure 7. A nucleus with split signals for chromosomes 21 and 22. Blastomeres (nucleus diameter = 85 µm) were fixed using method 1 and analysed by FISH with probes for chromosomes 13 (red), 16 (pale blue), 18 (dark blue), 21 (green), 22 (gold).

For all three methods, the temperature and humidity conditions were the same (22ºC and 30–45% respectively), based on previous observations that nuclear diameters are a function of temperature and humidity (Spurbeck et al., 1996).

membrane breakdown. Once cytoplasm breakdown occurred, the nucleus could be seen under the stereoscope for a few seconds; but not once the fixative dried completely.

FISH analysis was performed using probes for chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22 following the previously published protocol (Munné et al., 1998), except that instead of a locus-specific probe for chromosome 14, a centromeric one was used for chromosome 17, also labelled in Spectrum Orange (Vysis).

Method 2

Data evaluation and scoring criteria

This method was first described by Coonen et al. (1994). The blastomere was placed (5–10 s) into hypotonic solution (1% sodium citrate in 0.2 mg/ml BSA) and transferred onto a Petri dish containing Tween 20 solution for 2 min. After that, it was transferred onto a glass slide within approximately 3 µl of Tween 20 solution. Tween 20 solution was continuously added to the drop until cell membrane breakdown under stereoscope observation. After membrane breakdown, the slide was allowed to dry completely and the slide was treated with 1% pepsin to digest the remaining cytoplasm.

Classification of chromosomal abnormalities in cleavage-stage embryos, usually with 2–12 cells, requires scoring criteria based on the analysis of as many cells as possible to differentiate mosaicism (30% of cleavage-stage embryos; Munné et al., 1995) from FISH errors (10% of single cells analysed; Munné et al., 1998). In this study, the previously described criteria distinguishing mosaics from FISH errors were used without modification (Munné et al., 1994; Munné and Cohen, 1998). Previous criteria were also used to differentiate close signals from split signals when analysing

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Table 3. Example of 20 embryos with FISH errors. Embryo No. cells 13

16

18

21

22

XY

15

17

Embryo diagnosis

1

2 2 2 1a 2 2 4 2 3 2a nr 2 2 3a 2 2 nr 2 2 2 2 2 nr 2 2 2 3a nr 2 3a 2 1a 2 nr 3 3 3 2 2 1a 4 2 4 2 nr nr nr 2 2 1 2

2 2 2 2 2 2 4 2 3 3 2 2 3 4 2 2 2 2 2 2 2 2 2 nr 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 2 2 2 2 2 2 1 2

2 2 2 2 2 2 4 nr 3 3 2 2 2 2 2 2 2 3 3 2 2 2 2 3 2 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 2 2 nr 2 2 2 2a 2

2 2 2 2 2 2 4 1a 3 3 2 2 2 2 2 2 2 2 2 2a 3 3 2a 2 2 2 2 2 2 2 2 1 1 2 2 2 2 1 1 1 4 2 4 2 nr 2 2 2 2 1 nr

XY XY XY XY XY XY XXYY XY XXX XXX XY XY XX XX XX XX XX XY XY XX XX XX XX XX XX XX XX XY XY XY XY XY XY XY XY XY XY XY XY XY XXYY XY XXYY XX XX XX XX XY XY XX 4X

1a 2 2 2 2 2 4 2 3 3 2 2 nr nr 2 2 1a 0a 2 2 2 2 2 1 1 1 2a 1a 2 2 2 2 2 2 1a 2 2 3 2a 3 4 2 4 2 2 1a 2 2 1a 2 4

2 2 1a 2 2 2 4 2 3 3 3a 2 1 3 2 2 2 1a 2 2 2 2 2 2 2 1a 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 2 2 2 2 2 2 2 4

Normal

2 3

4 5 6

7 8 9 10 11

12 13 14 15

16

17

18

19

20

214

1 2 1 1 7 6 1 1 2 3 1 3 3 1 1 3 1 1 2 1 3 3 1 1 4 1 1 1 5 1 3 1 3 2 4 5 1 3 1 1 1 7 1 2 1 1 1 6 1 1 3

2 2 2 1a 2 2 3a 2 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1a 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 3a 2 2 2 3a 2 2 1 2

aConsidered errors. bAneuploid mosaic for the chromosomes in brackets. nr = no result

Normal Mosaic 2N/4N

Triploid Normal Trisomy 18, and Aneuploid mosaic (18,17)b Normal Trisomy 21 Trisomy 22 Trisomy 22 Monosomy 15, and Aneuploid mosaic (21)b

Normal Normal Monosomy 22 Trisomy 16

Monosomy 22

Mosaic 2N/4N

Normal

Normal

Complex mosaic

Articles - Fixation techniques for PGD of aneuploidy - E Velilla et al.

single cells in PGD cases (Munné and Weier, 1996). Embryos were classified as normal, aneuploid, polyploid, haploid and/or mosaic according to guidelines described elsewhere (Munné et al., 1995; Munné and Cohen, 1998).

Parameters to be evaluated Cells lost during fixation These were cells that in method 1 were lost after adding the fixative, and where the refringent protrusion was not observed. For methods 2 and 3, the blastomere seldom moved but the cell could burst, and these cells were also counted as lost.

Analysable cells A cell was considered analysable when it had at least five informative chromosomes out of the eight analysed.

Nuclear diameter This was measured in microns under phase contrast observation before FISH analysis.

Nuclei with overlaps Overlaps between the two-homologue chromosomes cannot be precisely quantified in a single cell and are one source of misdiagnosis (see below, FISH errors). However, any other overlap between non-homologue chromosomes can be easily measured if these chromosomes were labelled in different colours, as is the case here. Thus the parameter ‘nuclei with overlaps’ indicated the presence of overlaps between nonhomologous chromosomes in a specific nucleus.

Total number of overlaps This is the measure of the total number of overlaps between non-homologous chromosomes in a specific nucleus.

FISH errors To differentiate between FISH errors and mosaicism in this study, previously described criteria were used (Munné et al., 1994). A chi-square test using the algorithm GENSTAT (1988 version) was used to evaluate statistical differences between proportions. The significance chosen for the test was P < 0.025.

Table 4. Nuclear diameter in relation to overlaps and FISH errors. Diameter Analysable Average No. nuclei Total no. No. FISH (µm) diameter with overlaps errors (µm) overlaps (%) (%)

<30 30–60 >60

55

21.63

28 (50.9)a 62

14 (25.5)c

139

42.97

45 (32.4) 71

29 (20.9)

78.72

(27.6)b

3 (5.2)d

58

16

a versus b: P < 0.025; c versus d: P < 0.005.

16

Results The three fixation techniques were evaluated according to the following parameters: cells lost during fixation, nucleated cells with no result, analysable cells, nuclear diameter, nuclei with overlaps, and total number of overlaps, and FISH errors, as defined in the materials and methods (Tables 1 and 2). These parameters were also compared with other previously published studies (Table 1), although FISH errors and overlaps had not been previously evaluated in relation to blastomere fixation methods. The present results indicate that similar rates of lost cells were observed for the three methods evaluated (Table 1), ranging from 2.8% to 3.6%. This contrasts markedly with data published by others reporting lost cells ranging from 0% to 21.5% (Table 1). The fraction of nucleated cells that was analysable after FISH was significantly higher for method 1 than for method 2, in this study as well as in that of Xu et al. (1998) (Table 1). As far as is known, no other study has yet evaluated the number of signal overlaps and FISH errors according to fixation technique. The present results indicate a significant differences in nuclear diameter after fixation, with an average of 58 µm for method 1, 31 for method 2, and 46 for method 3 (P < 0.001) (Table 2), which translates to higher rates of nuclei with signal overlaps (from 14% for method 1 to up to 58% for method 2, P < 0.001), total number of overlaps, and FISH errors (from 10% for method 1 to 30% for method 2, P < 0.005) (Table 2). Examples of embryos with FISH errors are shown in Table 3 and examples of overlaps in Figures 3–7. The correlation between overlaps and FISH errors and small nuclear diameter was also observed irrespective of the type of fixation. For instance, taking all embryos together and grouping them by diameters, those cells of <30 µm in diameter had more overlaps and FISH errors than those cells of >60 µm in diameter (P < 0.005) (Table 4).

Discussion The two most important aspects of cell fixation are to ensure that each single cell is fixed and that the fixed nucleus is informative. One of the steps in method 1 involves the mixture of fixative with the drop of hypotonic solution containing the blastomere. This act produces turbulence, during which the cell may be lost, and the risk is about 3% in expert hands; but could be higher for technicians using method 1 only occasionally (Xu et al., 1998; Dozortsev and McGinnis, 2001). In contrast, methods 2 and 3 overcome the turbulence step and are easier to learn, but they have other problems. For instance, the presence of cytoplasm interferes with probe binding to the nucleus especially with locus specific probes. These probes are longer than the repetitive ones and easily attach to the cytoplasm debris, increasing the background signal and limiting the attachment of the probes to their target. Moreover, cytoplasm is refringent by itself, masking the signals. In short, cytoplasm can increase misdiagnosis or render the nucleus non-informative (Figures 4 and 6). This is a considerable problem in method 2, although modifications

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made by Xu et al. (1998) to this method reduced the number of nuclei with cytoplasm to <5%. Removal of cytoplasm by pepsin may also be detrimental because overexposure to pepsin may degrade the DNA (Xu et al., 1998; Dozortsev and McGinnis, 2001).

chromosomes 13, 21, and 22) are different. The three fixation methods were evaluated for their use in PGD of aneuploidy and method 2 may still be used for gender determination based on FISH errors or translocation PGD cases, but it is certainly not recommended for PGD of aneuploidy.

Another source of background interference found in this study was poly-L-lysine, used in method 2 to avoid nuclear loss in attaching the cell to the slide. This substance produces a mask along the slide, which interferes with the analysis of the signals. In methods 1 and 3, the cells are fixed to the slide by methanol:acetic acid instead of poly-L-lysine. In consequence, these fixation methods show clearer nuclei for FISH analysis (Figure 7).

The FISH error rate observed for method 1, 10%, was comparable to that found in previous studies using the same fixation method (Munné et al., 1996). Although in this study PGD was not performed using the three techniques, if the error rates observed here for methods 2 and 3 are maintained when performing PGD, as for method 1, a 30% error rate for method 3 would render it useless for PGD of aneuploidy.

Another factor in PGD analysis is the nuclei diameter. Small nuclei preserve their three-dimensional structure, and as a consequence the signals lie on different focus planes, making the analysis more prone to misdiagnosis (Figures 3 and 6). In addition, two signals close to each other or overlapping could be misdiagnosed as a single signal (Munné et al., 1996). Methods 1 and 3 provide flat nuclei with all the signals in the same focus plane, thereby reducing the frequency of overlaps. It was observed in this study that the ideal diameter is 60 µm and above (Figure 2). However, nuclei >80 µm show excessively decondensed chromatin, as the signals are spread more widely and are weaker than regular sized nuclei. Sometimes they are almost imperceptible, which leads to misdiagnosis of false nullisomies, monosomies or disomies. It is important to mention that nuclear diameter is not only dependent on the fixation technique, but also on temperature and humidity (Spurbeck et al., 1996; Hliscs et al., 1997). Previous observations that nuclear diameter is linked to FISH errors (Munné et al., 1996) were confirmed. In this respect, method 1, which produced the largest nuclei, produced the fewest number of signal overlaps and the fewest errors after FISH (Figure 2). In contrast, method 2 (Coonen et al., 1994) produced the highest number of overlaps and three times more FISH errors than method 1 (Figures 2–5).

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Method 2 has been previously used in PGD for gender determination (Harper et al., 1994, 1995; Coonen et al., 1996; Soussis et al., 1996) and PGD diagnosis on translocation carriers (Van Asche et al., 1999; Coonen et al., 2000; Iwarsson et al., 2000; Scriven et al., 2001) using probes for one or two chromosome pairs. The probes used for gender determination bind to repetitive sequences in the satellite regions (centromere X and 18, or most of the q arm of chromosome Y), producing a large signal, usually even under the worst fixation conditions. In addition, misdiagnosis of gender using FISH can be improved because the mere presence of different colours (irrespective of the number of signals) can identify the sex, even though signal overlaps could still misdiagnose the ploidy. For translocation carriers, only the chromosomes involved in the translocation are diagnosed, usually involving only a centromeric probe and a pair of distal probes instead of the five probes simultaneously used for the first aneuploidy hybridization. In addition, for PGD of translocations, either telomeric or home-made locus-specific (LSI) probes are used, while the LSI probes used for PGD of aneuploidy (for

In conclusion, method 1 gives the best nuclear quality for PGD analysis, even though it is the hardest to accomplish. Method 3 gives reasonably good nuclear quality, and is easy to learn. Method 2 is also easy to learn, but under the conditions of this study, the quality of the results was not sufficiently good for PGD of aneuploidy.

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