Rapid comparative genomic hybridization protocol for prenatal diagnosis and its application to aneuploidy screening of human polar bodies

GENETICS Rapid comparative genomic hybridization protocol for prenatal diagnosis and its application to aneuploidy screening of human polar bodies Christina Landwehr, M.Sc.,a Markus Montag, Ph.D.,b Katrin van der Ven, M.D.,b and Ruthild G. Weber, M.D.a a

Department of Human Genetics, and b Department of Gynecological Endocrinology and Reproductive Medicine, Rheinische Friedrich-Wilhelms-University, Bonn, Germany

Objective: To establish, validate, and apply a rapid protocol for comparative genomic hybridization (CGH), a technique that detects aneuploidies of all chromosomes in a single experiment. Design: Experimental study. Setting: University human genetics and IVF unit. Patient(s): Sixteen patients 33 to 44 years of age with advanced maternal age or repeated implantation failure. Intervention(s): Each step of the conventional CGH protocol was evaluated and shortened (rapid CGH). Rapid CGH was validated by analysis of DNA with known aberrations and applied to aneuploidy screening of 32 first polar bodies. Main Outcome Measure(s): Duration of CGH protocol, results of validation experiments, and aneuploidy type and rate in polar bodies from patients with advanced maternal age or repeated implantation failure. Result(s): The protocol was shortened from 76 to 12 hours (16 h for single-cell analysis). Gains of chromosomes 18 and 21 could be detected by using rapid CGH on single cells with trisomy 18 and 21. In the polar bodies that were analyzed by rapid CGH, an average of 1.8 chromosomal aberrations (range, 0–5) was found, involving almost all chromosomes at least once. Conclusion(s): The rapid CGH protocol allows a fast screen for aneuploidies and can analyze single cells in 16 hours. Rapid CGH revealed aneuploidies in 75% of polar bodies from patients with advanced maternal age or repeated implantation failure. (Fertil Steril 2008;90:488–96. 2008 by American Society for Reproductive Medicine.) Key Words: Comparative genomic hybridization, prenatal diagnosis, polar body, rapid analysis, aneuploidy

Comparative genomic hybridization (CGH) is a molecular cytogenetic method that allows the detection of all unbalanced chromosomal aberrations of R10 Mb in size, that is, aneuploidies of all chromosomes, in one experiment (1–3). In this technique, fluorescence-labeled test DNA is hybridized to normal metaphase chromosomes, together with control DNA from a normal proband labeled with a different fluorophore. The intensity of the two fluorescent dyes along each chromosome is detected by using a fluorescence microscope. By using software analysis, fluorescence ratios are calculated, indicating chromosomes or chromosomal regions showing a relative gain or loss. Since CGH was established 15 years ago, the technique has been used very successfully in tumor genetics (for review, see James [4]). Comparative genomic hybridization also would be an ideal technique to use in globally screening for Received May 10, 2007; revised and accepted July 17, 2007. Reprint requests: Ruthild G. Weber, M.D., Department of Human Genetics, Rheinische Friedrich-Wilhelms-University, Wilhelmstrasse 31, D-53111 Bonn, Germany (FAX: 49-228-287-22380; E-mail: ruthild. [email protected]).

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aneuploidies in preimplantation genetic diagnosis (PGD) to improve pregnancy and baby take-home rates by selecting oocytes or embryos without numerical chromosome aberrations for implantation. However, for an application in PGD, it was a prerequisite that DNA from a single cell would be sufficient to allow the analysis. Protocols were developed for universal amplification of target DNA by a single degenerate primer (5), as well as for global amplification of DNA from single cells including blastomeres and amniocytes and subsequent CGH analysis (6, 7). These techniques then were applied in PGD to screen for aneuploidies in biopsied blastomeres from 5- to 12-cell embryos from women with repeated implantation failure (RIF) (8–10). These analyses took 4 to 5 days and could be performed because the embryos were cryopreserved during this time. The first clinical application of aneuploidy screening by CGH in 10 polar bodies from a patient with advanced maternal age (AMA) required an analysis time of around 2 days (11). To improve the usefulness of CGH in a clinical setting of prenatal and preimplantation diagnosis, it would be desirable if the analysis took little more than overnight. Thus, it was the

Fertility and Sterility Vol. 90, No. 3, September 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.

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aim of this study to establish a rapid CGH protocol that would allow aneuploidy screening within a time frame desirable in prenatal diagnostics and PGD and that would be applicable for single-cell analysis. This protocol was validated by successfully analyzing small DNA amounts and single cells with known unbalanced chromosomal aberrations. Rapid CGH was then applied to aneuploidy screening of 32 first polar bodies from 16 patients, 33 to 44 years of age, who had been treated mainly for AMA or RIF.

were used (CustomTip 02/240, Eppendorf, Germany) (14). Further processing of polar bodies, transfer to glass slides, and fixation were performed as described elsewhere (15). The polar bodies were released for research after aneuploidy testing by multicolor FISH had been performed. The corresponding oocytes could not be examined because most were fertilized and transferred to the patient. The few oocytes with aneuploidies in the corresponding polar bodies according to multicolor FISH were discarded before this study was designed.

MATERIALS AND METHODS Materials Used to Establish and Validate the Rapid CGH Protocol To establish the rapid CGH protocol, DNA was used from the ovarian adenocarcinoma cell line COLO 704 (German Collection of Microorganisms and Cell Culture [DSMZ], Braunschweig, Germany), because this cell line carries imbalances of entire chromosomes of different sizes, as well as a gain of a chromosome arm. Comparative genomic hybridization analysis according to standard protocols (12) had determined the following karyotype: rev ish enh(1) (q),enh(7),enh(8),enh(19),enh(20).

Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) with the commercial probes LSI 13 and LSI 21 (Vysis, Germany) to a lymphocyte interphase nucleus with trisomy 21 was performed according to standard protocols. Fluorescence in situ hybridization with clones RP11-120L14, located at 13q31.1, and RP11-430M15, located at 13q33.1, to chromosome preparations of a patient with the karyotype 46,XX,add(13)(p12) was performed according to standard protocols. For polar body FISH before rapid CGH analysis, a six-probe FISH set (for chromosomes 13, 16, 18, 21, 22, X) was used as described elsewhere (16). In short, co-denaturation was performed by using a hybridization oven (Hybrite; Abbott Laboratories, Abbott Park, IL), programmed at a melting temperature of 69 C for 8 minutes, followed by hybridization at 37 C overnight. Coverslips were removed, and preparations were washed in 0.7  SSC, 0.3% NP-40 at 73 C for 7 minutes, followed by 1 minute in 2  SSC, 0.1% NP-40 at room temperature. Slides were mounted with anti-fade solution (Vectashield, Alexis, Germany). Fluorescent signals were evaluated with an inverted microscope that was equipped with a 100W fluorescent bulb (DMIRB; Leica, Wetzlar, Germany) and appropriate filter sets for the relevant fluorophores (Abbott).

To investigate whether structural aberrations can be identified by rapid CGH, DNA was used from peripheral blood lymphocytes of a patient carrying a derivative chromosome 13 containing additional material of unknown origin after informed consent of the patient’s parents. The karyotype of the patient had been determined to be 46,XX,add(13)(p12). To validate the rapid CGH protocol for the analysis of single cells, cells were analyzed that had known chromosomal aberrations and that were obtained from the Cytogenetics Laboratory of the Department of Human Genetics, Rheinische Friedrich-Wilhelms-University Bonn (Bonn, Germany). These included single lymphocytes from a cell suspension that was generated from the peripheral blood of a patient with trisomy 21 and a single fibroblast from a cultivated tendon biopsy of an aborted fetus with trisomy 18. These cells had been cultivated and analyzed by GTG banding for diagnostic purposes. Both chromosome aberrations were present in all cells investigated. Patients and Polar Body Biopsies Thirty-two first polar bodies from 16 patients were analyzed by using rapid CGH. The age range of the patients was between 33 and 44 years, with an average age of 38 years. The patients were undergoing infertility treatment at the Department of Gynecological Endocrinology and Reproductive Medicine, Rheinische Friedrich-Wilhelms-University Bonn (Bonn, Germany). They were being treated for the following indications: RIF (n ¼ 7), AMA (n ¼ 5), RIF and/or AMA (n ¼ 3), and reciprocal translocation (n ¼ 1). Polar body biopsies were performed for diagnostic purposes after patients provided informed consent as described elsewhere (13), except that for zona drilling, the Octax laser was used (MTG, Altdorf, Germany), and for aspiration, custom-made capillaries Fertility and Sterility

Isolation of Single Cells and Polar Bodies for Rapid CGH Single lymphocytes with trisomy 21 were isolated in one of two ways. First, a drop of a strongly diluted cell suspension of lymphocytes with trisomy 21 was placed on a glass slide, and a single lymphocyte was isolated by using a glass needle under an inverted microscope. Second, a single lymphocyte nucleus with trisomy 21 that was fixed on a glass slide after standard chromosome preparation was microdissected by using a glass needle under an inverted microscope. The fibroblast with trisomy 18 was isolated by using a glass needle under an inverted microscope from a drop of a strongly diluted cell suspension, which was obtained after trypsinizing the cell culture of a tendon biopsy from an aborted fetus. Polar bodies fixed on glass slides were microdissected with a glass needle by using an inverted microscope, after aneuploidy testing had been performed by using multicolor FISH. Each cell or polar body was transferred into a 0.2-mL tube containing 5 mL of PCR buffer. Extraction of DNA High molecular weight DNA from the tumor cell line COLO 704, from peripheral blood of the patient with a derivative 489

chromosome 13, and from peripheral blood of normal male probands as control DNA for CGH analysis, was extracted according to standard procedures. Lysis of isolated single cells and polar bodies to extract DNA took place by adding 5 mL of proteinase K (125 mg/ mL) to the 5-mL PCR buffer containing the cell or polar body and incubating at 37 C for 30 minutes, followed by an incubation at 95 C for 10 minutes to inactivate the enzyme. Universal DNA Amplification Two protocols for universal DNA amplification were tested to establish the rapid CGH protocol. These were used to universally amplify small DNA amounts that were approximately equivalent to the DNA content of a single diploid cell (5 pg), DNA from single cells and polar bodies, and male control DNA used in the CGH experiments. The protocols applied were a modified degenerate oligonucleotide–primed PCR protocol published elsewhere (17), based on the method of Telenius et al. (5), and the single-cell PCR protocol according to Klein et al. (6). Negative (water) and positive (normal genomic DNA) controls were included in each PCR reaction. To avoid contamination, pipetting of PCR reactions was performed in a laminar flow hood. Amplifications were performed in a thermocycler (MJ Research, Waltham, MA). Comparative Genomic Hybridization As a target for CGH analysis, metaphase spreads were prepared according to standard procedures from stimulated peripheral blood lymphocytes that were obtained from a healthy male proband (46,XY). The standard chromosomal CGH protocol used was described in detail elsewhere (12). Various modifications were made to establish the rapid CGH protocol (see Results). Image capture and processing of CGH data was performed with the Leica CW4000 system. The diagnostic threshold values used to score losses and gains were 0.8 (lower threshold) and 1.2 (upper threshold). Because CGH is not a precise quantitative method, no distinction was made between chromatid and chromosome aberrations. Test and control samples were sex mismatched, so that the ratio profiles obtained for the gonosomes could serve as internal controls for hybridization quality. Statistical Analyses Comparisons between groups were performed by using Spearman’s rank order correlation or Mann-Whitney rank sum test. A P value of < .05 was considered statistically significant. RESULTS Establishing a Rapid CGH Protocol It takes 76 hours to perform the standard CGH protocol used in our laboratory (12). To establish a rapid CGH protocol that is usable in prenatal diagnostics and PGD, we tried to reduce 490

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the duration of each step required for the analysis. For these experiments, we used DNA from the tumor cell line COLO 704 containing gains of entire large and small chromosomes as test DNA to verify whether these imbalances were detectable under the different conditions applied. First, we wanted to reduce the hybridization time, which is the most time-consuming step in standard protocols (64 h in the protocol used routinely in our laboratory). The duration of hybridization was continually decreased until the shortest time span was identified that still brought reliable results. This was a hybridization time of 6 hours. We found that the quality of CGH profiles was best when this short hybridization took place in a thermomixer for glass slides that rotated at 300 rpm (Eppendorf), rather than in a shaking water bath or an incubator. Second, we wanted to test whether reliable results could be obtained when reducing the hybridization time to 6 hours after incorporating nucleotides into the DNA that were directly coupled to fluorochromes. Using fluorochrome-conjugated nucleotides has the advantage that no detection steps are necessary after hybridization, thus reducing the time span required for the protocol. However, no reliable CGH profiles could be generated this way. Therefore, nucleotides labeled with a reporter (e.g., biotin or digoxigenin) were incorporated into the DNA instead, leading to reproducible CGH results. This required a nick-translation step and a detection step with molecules (e.g., avidin and anti-digoxigenin antibodies) that were coupled to fluorochromes. However, the time span required for these two steps could be reduced. We found that after 1 hour instead of 2 hours, DNAwas sufficiently labeled by nick translation. Also, detection with one round of fluorochrome-conjugated molecules without a signal-enhancing step was sufficient to generate CGH profiles of good quality, which reduced stringent washes and detection steps from 4 to 2 hours. When the reaction tubes were placed into a thermomixer (Eppendorf) at 1,300 rpm, the time span used for resuspension of DNA precipitated after nick translation could be reduced from 1 hour to 30 minutes. The minimal time necessary for preannealing of repetitive sequences after DNA denaturation was found to be 30 minutes and could thus be reduced from 2 hours. Digital image acquisition and analysis of 15 metaphases could be performed in 1 hour by an experienced technician or scientist. Taken together, the CGH protocol could be shortened from 76 to 12 hours for cases in which sufficient test DNA was available. For the analysis of single cells, two additional steps were required. First, a 40-minute digest with proteinase K was necessary to extract the DNA from the cell. Second, a universal DNA amplification step was required to generate sufficient DNA for the CGH experiment. We tested different DNA amplification protocols and evaluated their suitability. In the single-cell PCR protocol according to Klein et al. (6), DNA from one cell is amplified and labeled with fluorochromeconjugated nucleotides by PCR. However, using a shortened version of this protocol, we could not obtain reliable results.

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TABLE 1 Comparison of duration of individual steps in the standard and rapid CGH protocols. Parameter Protocol step 1. Proteinase K digest to extract DNA 2. Universal DNA amplification by degenerate oligonucleotide–primed PCR 3. Photometer measurement 4. DNA labeling by nick translation 5. DNA precipitation 6. DNA dissolving, pretreatment of chromosome preparations 7. Denaturation and preannealing 8. Hybridization 9. Stringent washes and antibody detection 10. Image acquisition and analysis Total duration for regular application (excluding steps 1 and 2) Total duration for single-cell application

Standard CGH protocol

Rapid CGH protocol

40 min 6h

40 min 3h

5 min 2h 1h 1h

5 min 1h 1h 30 min

2 h, 5 min 64 h 4h 2h 76 h

35 min 6h 2h 1h 12 h

83 h

16 h

Landwehr. A rapid CGH protocol for prenatal diagnosis. Fertil Steril 2008.

In the degenerate oligonucleotide–primed PCR protocol according to Telenius et al. (5), the DNA is labeled by nick translation after DNA amplification. Using this protocol and a hybridization time of 6 hours, reliable CGH profiles of good quality could be generated. To decrease the time span necessary for the DNA amplification step, the number of cycles was reduced until the shortest degenerate oligonucleotide–primed PCR protocol was identified that still brought reliable results. The short degenerate oligonucleotide–primed PCR protocol takes 3 hours and consists of the following steps: 96 C for 5 minutes; 94 C (1 min), 30 C (1 min, 30 s), ramp-up to 72 C within 3 minutes, and 72 C (3 min) for 10 cycles; and 94 C (1 min), 62 C (1 min), and 72 C (2 min, 30 s) for 20 cycles; followed by a final step at 72 C for 7 minutes. The protocol established for fast single-cell analysis was termed rapid CGH protocol, requires a time span of 16 hours, and is compared with the conventional CGH protocol in Table 1. Validating the Rapid CGH Protocol The DNA amount approximately equivalent to the DNA content of a single cell (5 pg) of COLO 704 DNA was successfully analyzed by rapid CGH. All gains known from the analysis of COLO 704 DNA by conventional CGH were found to be diagnostic, so that there was full agreement between the results obtained by conventional and rapid CGH (Fig. 1A). Rapid CGH also allowed the detection of a gain of chromosome 18 in a single fibroblast with known trisomy 18, and a gain of chromosome 21 in a single lymphocyte with known trisomy 21 (Fig. 1B). A gain of chromosome 21 could also be detected by rapid CGH when a lymphocyte nucleus with known trisomy 21 was investigated that had been fixed on a glass slide and was then microdissected (Fig. 1C, top). In a polar body with three chromatids of chromosome 22 by Fertility and Sterility

FISH, which was then microdissected from the glass slide and analyzed by rapid CGH, a gain of chromosome 22 could be detected (Fig. 1C, bottom). Finally, 5 pg of DNA from the peripheral blood of a patient with a structural aberration, that is, a chromosome 13 containing additional material (Fig. 1D, left), was successfully analyzed by rapid CGH. A partial gain was detected in the CGH profile of chromosome 13, identifying the additional material as originating from distal chromosome 13 (Fig. 1D, center). The rapid CGH result was verified by FISH analysis using two locus-specific probes from the trisomic chromosomal region (Fig. 1D, right).

Applying the Rapid CGH Protocol to Aneuploidy Screening in Polar Bodies In the 32 polar bodies from 16 patients, aged 33 to 44 years with an average age of 38 years, an average of 1.8 chromosomal aberrations (range, 0–5) was found (Table 2). Recurrent gains were found of chromosomes 17 and 22 (4/32 cases), 19 (3/32 cases), 21, and X (2/32 cases). Chromosomes showing recurrent losses were 6 (5/32 cases); 3, 7, 10, 15, and 16 (3/32 cases); 4, 8, 11, 12, 18, and 20 (2/32 cases). The frequency of gains and losses and the chromosomes involved are given in Figure 2A. The CGH results were in agreement with those obtained by six-color FISH analysis (Table 2). Profiles obtained after rapid CGH analysis of polar bodies are shown for two selected cases (Fig. 2B). In the polar body of a 39-year-old patient treated for AMA, five chromosomal aberrations were found (losses of chromosomes 6, 7, 8, 11, and 19; Fig. 2B, top). No aneuploidy was found in a polar body from a 41-year-old patient treated for AMA (Fig. 2B, bottom). The respective oocyte was fertilized, the embryo was transferred, and a pregnancy resulted. 491

FIGURE 1 Establishing and validating the rapid CGH protocol. In all CGH profiles shown, the centromeres of all chromosomes, the heterochromatin block on chromosome 1, as well as the short arms of the acrocentric chromosomes (13, 21, 22) were shaded to indicate regions of tandem repetitive DNA sequences, which were excluded from the CGH evaluation. (A) Chromosomal CGH profiles obtained by using the standard CGH protocol (top row) and the rapid CGH protocol (bottom row) when analyzing DNA from our test system, the ovarian adenocarcinoma cell line COLO 704, carrying gains of entire large and small chromosomes as well as a gain of a chromosome arm. Gains of chromosomes and chromosomal arms 1q, 7, 8, 19, and 20 that were detected by the standard CGH protocol (top) are also diagnostic when using rapid CGH (bottom). (B) Top, trisomy 18 is detected in the karyotype of a fibroblast from a tendon biopsy of an aborted fetus by GTG banding (top left), and a gain of chromosome 18 is found when analyzing a single fibroblast from the same tendon biopsy culture by using rapid CGH (top right). Bottom, trisomy 21 is detected in the karyotype of a lymphocyte from a patient with Down’s syndrome by GTG banding (bottom left), and a gain of chromosome 21 is found when analyzing a single lymphocyte from the same blood culture by using rapid CGH (bottom right). (C) Top, analysis of lymphocyte nuclei with trisomy 21 from a patient with Down’s syndrome by using interphase FISH and rapid CGH. Top left, FISH analysis of a lymphocyte nucleus showing three signals for chromosome 21 (red, with filled arrowheads) and two regular signals for chromosome 13 (green, with open arrowheads). Top right, another lymphocyte nucleus from the same glass slide was then microdissected, and rapid CGH was performed and showed a shift of the chromosome 21 profile to the right, equivalent to a chromosome 21 gain, whereas the profile for chromosome 13 was balanced. Bottom, consecutive analysis of the same first polar body with an extra chromatid of chromosome 22 by using interphase FISH and rapid CGH. Bottom left, polar body analyzed by FISH showing three signals for chromosome 22 (green, with open arrowheads), equivalent to an extra chromatid, and two regular signals for chromosome 18 (red, with filled arrowheads). Bottom right, the polar body was then microdissected from the glass slide, and rapid CGH was performed and showed a gain of chromosome 22, whereas the profile for chromosome 18 was balanced. (D) Identification of extra chromosomal material on a derivative chromosome 13 from a patient with mental retardation by using rapid CGH, with verification by FISH. Left, GTG banding showing a normal chromosome 13 and a derivative chromosome 13 containing extra material in the short arm. Center, rapid CGH profile of chromosome 13 demonstrating a partial gain of distal 13q. The additional material attached to the short arm of the derivative chromosome 13 is thus identified as material originating from the distal long arm of chromosome 13. Right, the rapid CGH result is verified by FISH analysis with clones RP11-120L14 (red, located at 13q31.1) and RP11-430M15 (green, located at 13q33.1), showing signals on the short arm of the derivative chromosome 13, in addition to signals at the expected bands of the long arm of chromosome 13.

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TABLE 2 Summary of clinical data and FISH and rapid CGH results for 32 first polar bodies from 16 patients. FISH results Case Patient Indication no. no. PB no. Age(y) for IVF Losses Gains 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 2 2 3 3 4 4 4 4 4 4 5 5 6 7 8 9 9 9 9 9 9 10

PB1A PB2A PB2B PB3A PB3B PB4A PB4B PB4C PB4D PB4E PB4F PB5A PB5B PB6A PB7A PB8A PB9A PB9B PB9C PB9D PB9E PB9F PB10A

34 44 44 33 33 33 33 33 33 33 33 44 44 40 37 39 39 39 39 39 39 39 ND

24

11

PB11A

ND

25

12

PB12A

ND

26 27 28 29 30 31 32

13 13 14 15 15 16 16

PB13A PB13B PB14A PB15A PB15B PB16A PB16B

39 39 37 41 41 34 34

RIF AMA AMA RIF RIF RIF RIF RIF RIF RIF RIF AMA AMA AMA RIF AMA RIF RIF RIF RIF RIF RIF RIF and/or AMA RIF and/or AMA RIF and/or AMA RIF RIF t AMA AMA RIF RIF

Rapid CGH results Losses

Gains

— — — — 6 — — 4, X — 22 6, 7, 10, 12, 18 — 20 — 4, 8 — 2, 3, 10, 12 7 6, 7, 20 — 16 — 15 10, 18, 21, X 15 14 18 — 15 17 6, 7, 8, 11, 19 — 4, 5 17, 22 — 17 — — — 16, 19 — 19, 22 — 21 3, 16 —

Aberrations per polar body

— — — ND ND ND — ND — ND 16 ND — ND ND — ND ND — — — — ND

— — — ND ND ND — ND — ND — ND — ND ND — ND ND — 16 22 21 ND

0 0 1 2 1 5 1 2 5 3 1 5 2 1 2 5 4 1 0 2 2 1 2

ND

ND

10, 16

—

2

ND

ND

—

—

0

— — — — — ND —

— — — — — ND —

3, 6, 11 — — — — — —

17, 19 — 8 — — 22 —

5 0 1 0 0 1 0

Note: PB ¼ polar body; ND ¼ no data; t ¼ translocation. Landwehr. A rapid CGH protocol for prenatal diagnosis. Fertil Steril 2008.

There was no significant relationship between the number of aberrations per polar body and patient age (P¼.3). There also was no statistically significant difference between the number of aberrations per polar body in the two patient groups treated for AMA and RIF failure (P¼.5). DISCUSSION Because using CGH allows the detection of aneuploidies of all chromosomes in one experiment, it would be the ideal tool to use in screening for numerical and also unbalanced structural chromosome aberrations in prenatal diagnostics. Fertility and Sterility

However, its application has been hampered by the long duration of normal CGH protocols, which require >3 days’ analysis time. Therefore, it was the first aim of this study to develop a fast but reliable CGH protocol. To allow aneuploidy screening of polar bodies or blastomeres in PGD, such a protocol must be applicable to single cells. Therefore, our second aim was to adapt the fast CGH protocol to the analysis of single cells (rapid CGH). The third aim was to apply the established rapid CGH protocol to aneuploidy screening of polar bodies from patients undergoing routine infertility treatment, mainly for AMA or RIF. 493

FIGURE 2 Applying the rapid CGH protocol to aneuploidy screening of first polar bodies. In all CGH profiles shown, the centromeres of chromosomes were shaded to indicate regions of tandem repetitive DNA sequences, which were excluded from the CGH evaluation. (A) Frequency of chromosomal imbalances detected in 32 first polar bodies from 16 patients aged 33 to 44 years (average age, 38 y) who were undergoing infertility treatment mainly for AMA or RIF. Gains (black upward columns) and/or losses (light-gray downward columns) were detected in 75% of polar bodies, involving almost all chromosomes at least once. Among the detected aberrations in polar bodies, 40% were gains resulting in monosomies in the fertilized oocytes. (B) Comparative genomic hybridization profiles obtained after rapid CGH analysis of polar bodies from two selected cases. Top row, CGH analysis of polar body PB8A from a 39-year-old patient treated for AMA showing multiple aneuploidies (losses of chromosomes 6, 7, 8, 11, and 19). Bottom row, CGH analysis of polar body PB15B from a 41-year-old patient treated for AMA. No imbalances of any chromosome were detected. The profiles from the same chromosomes as in the top row are shown for comparison. A pregnancy resulted after implantation of the corresponding fertilized oocyte.

Landwehr. A rapid CGH protocol for prenatal diagnosis. Fertil Steril 2008.

To establish a fast CGH protocol, we evaluated each step required for the analysis and modified it if possible to reduce its duration. We then analyzed a tumor cell line with known aneuploidies of big and small chromosomes and could show that the rapid CGH protocol reliably detects these imbalances within 12 hours. This means a reduction in analysis time of around 64 hours, as compared with standard protocols (12). For single-cell application, two additional steps had to be added to the protocol for DNA extraction and universal DNA amplification. This increased the duration of the rapid protocol to 16 hours. We could then show that numerical and unbalanced structural chromosomal aberrations could be detected reliably within this short time period in small DNA amounts that were equivalent to the DNA content of a single cell and single lymphocytes and fibroblasts. Thus, by using the rapid CGH protocol, it is now possible to perform an aneuploidy screen overnight, even if only one cell or very small DNA amounts are available. This is the time span required for the rapid FISH assay that is used routinely in prenatal diagnostics to screen for aneuploidies of chromosomes 13, 18, 21, X, and Y in uncultivated amniocytes (18). However, CGH analysis has the advantage of allowing the numerical analysis of 19 additional chromosomes.

Thus, we used the rapid CGH protocol to screen for aneuploidies in 32 first polar bodies that were biopsied from human oocytes. In these first polar bodies from 16 patients who were 33 to 44 years of age (average age, 38 y) and were undergoing infertility treatment mainly because of AMA or RIF, the aneuploidy rate was 75%. This implies an aneuploidy rate in the corresponding oocytes that is at least as high, because meiosis II errors that can occur in oocytes are not detected when first polar bodies are analyzed. When screening first polar body–oocyte complexes from 46 patients by using a standard CGH protocol, Fragouli et al. (19) found an aneuploidy rate of 22%. This much lower number of numerical aberrations is most likely due to the different treatment indications of the patients whose polar bodies and oocytes were analyzed. Only 2 of the 46 patients reported by Fragouli et al. (19) were treated for RIF or AMA. However, these two indication groups are most representative for our IVF center and others, especially with respect to PGD, so we focused our analysis on polar bodies from these patients.

We then wanted to know whether polar bodies fixed on glass slides could be analyzed. By performing control exper-

The aneuploidy rate of 75% in the polar bodies studied here is in better agreement with the study of Wells et al.

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iments on a lymphocyte nucleus and a polar body with known numerical aberrations that was microdissected from a glass slide, we could show that rapid CGH can detect these chromosomal aneuploidies in diploid and haploid cells.

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(11) showing chromosomal imbalances in 9 of 10 polar bodies from a 40-year-old IVF patient by CGH. In the report of Voullaire et al. (9) analyzing blastomere biopsies of 20 patients with implantation failure by CGH, 60% of the embryos investigated had at least one chromosome abnormality. On the one hand, postzygotic errors should increase the aneuploidy rate detected in blastomeres compared with that in polar bodies. On the other hand, a slightly lower aneuploidy rate in embryos vs. polar bodies can be well explained by an early genetic selection process. If an oocyte carries certain numerical chromosome aberrations, the respective embryo has a higher risk of not reaching the 6- to 12-cell stage. This is in line with the fact that many embryos, especially of patients with AMA and RIF, spontaneously arrest in vitro at very early stages after IVF. In addition, the average age of the patients investigated by Voullaire et al. (9) was 34 years, vs. 38 years in our study, which is another explanation for the higher aneuploidy rate found here. Evidence that there is a genetic selection process in early embryonic development comes from the spectrum of aneuploidies found in abortion material. Certain chromosomal aberrations, such as most monosomies, or trisomies of chromosomes containing many genes (e.g., chromosomes 1, 5, 6, 11, or 19), are not or are only very rarely (approximately 1 in 1,000) found in first-trimester miscarriages (20). This must be due to the selective loss of the abnormal embryos during early gestation, because in the polar bodies of our series, almost all chromosomes were found to be involved in imbalances at least once, including chromosomes 5, 6, 11, and 19. In particular, 40% of the aneuploidies detected in the polar bodies were gains, which would double the monosomy rate in the fertilized oocytes compared with that in first-trimester losses (21). Aneuploidy screening of blastomeres by CGH also showed involvement of many chromosomes (9), indicating that early embryos with various numerical aberrations may reach the 6- to 12-cell stage. Our data suggest, however, that the likelihood of an early in vitro arrest is increased in embryos with aneuploidies. A standard procedure for aneuploidy screening in PGD is FISH with probes for five chromosomes, 13, 16, 18, 21, and 22 (22), or for six chromosomes, with the X chromosome in addition (16). Had we used the FISH five-probe set when analyzing the polar bodies, we would only have detected 23% of the aneuploidies that were found by CGH. The FISH sixprobe set would have yielded 26%. On the basis of a six-color FISH analysis, 13 of 32 oocytes (40%) would have been discarded because of one or more numerical aberrations in their first polar bodies. When CGH was used, aneuploidies were found in an additional 11 of 32 first polar bodies (35%) that would have escaped detection by FISH analysis using six probes, adding up to a total aneuploidy rate of 75% identified by CGH. It has been suggested that the majority of oocytes in women aged R40 may be aneuploid (23). Although this was true for a 44-year-old woman who had two and five aberrations, respectively, in the two polar bodies studied, there Fertility and Sterility

was another 44-year-old woman and one 41-year-old woman with at least one euploid polar body among two analyzed. However, in a 33-year-old woman who was treated for RIF, all of the six polar bodies investigated were aneuploid. Overall, in our study, there was no statistically significant difference between the number of aberrations per polar body in patients treated for AMA or RIF. This suggests that patients with RIF may benefit from PGD by CGH analysis, even if they are only in their early 30s. In summary, we established and validated a rapid CGH protocol that takes 12 hours and allows single-cell, including polar body, analysis within 16 hours. This is a time period that opens up new applications for this genome-wide screening technique in prenatal diagnostics including PGD. By applying rapid CGH to 32 first polar bodies of patients who were treated mainly for AMA and RIF, we detected an aneuploidy rate of 75% involving almost all chromosomes at least once. These data are of interest with respect to reproductive biology and meiosis I errors, as well as for the application of PGD in assisted reproduction. Acknowledgments: The authors thank Richard Nissl, M.D., for performing the statistical analysis and Christina Ergang for excellent technical assistance.

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A rapid CGH protocol for prenatal diagnosis

Vol. 90, No. 3, September 2008