Causes and estimated incidences of sex-chromosome misdiagnosis in preimplantation genetic diagnosis of aneuploidy.

Preimplantation genetic diagnosis of aneuploidy (PGD-A) with comprehensive chromosome analysis has been known to improve pregnancy outcomes. Accuracy in detecting sex chromosomes becomes important when selecting against embryos at risk for sex-linked disorders. A total of 21,356 PGD-A cycles consisting of day-3 (cleavage) or day-5 (blastocyst) biopsies were received at the same laboratory for PGD-A via fluorescence in situ hybridization (FISH) or array comparative genome hybridization (aCGH) from multiple fertility centres. The misdiagnosis rates were 0.12% (Wilson 95% CI 0.05 to 0.25%) in day-3 FISH cycles, 0.48% (Wilson 95% CI 0.19 to 1.22%) in day-3 aCGH cycles and 0.0% (Wilson 95% CI 0 to 0.26) in day-5 aCGH cycles. Although rare, the likely causative biological event for true misdiagnosis is embryonic XX/XY mosaicism. Reanalysis of 1219 abnormal cleavage-stage research embryos revealed a 73% incidence of minor and major mosaicism. Only four (0.3%) embryos were found to be diploid and contained XX and XY cells that could potentially account for the misdiagnosis of sex. Our investigation identified errors leading to misdiagnosis and their attribution to specific events during PGD-A testing. The reported misdiagnosis rates suggest that PGD-A for sex determination is highly accurate, particularly when using aCGH applied to blastocyst biopsies.

Validation of array comparative genome hybridization for diagnosis of translocations in preimplantation human embryos.

Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were ∼6 Mbp and ∼5 Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy.

Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos.

OBJECTIVE: To validate and determine the best array-comparative genomic hybridization (aCGH; array-CGH) protocols for preimplantation genetic screening (PGS). DESIGN: Embryos had one cell removed as a biopsy specimen and analyzed by one of two array-CGH protocols. Abnormal embryos were reanalyzed by fluorescence in situ hybridization (FISH). SETTING: Reference laboratory. PATIENT(S): Patients donating embryos or undergoing PGS. INTERVENTION(S): Embryo biopsy, array-CGH, FISH reanalysis. MAIN OUTCOME MEASURE(S): Diagnosis, no result rate and error rate. RESULT(S): Method one produced 11.2% of embryos with no results and a 9.1% error rate compared with 3% and 1.9% for method two, respectively. Thereafter, only method two was used clinically. The aneuploidy rate for cleavage-stage embryos was 63.2%, significantly increasing with maternal age. The chromosomes most involved in aneuploidy were 16, 22, 21, and 15. We report the first live births after array-CGH combined with single blastomere biopsy. CONCLUSION(S): Array-CGH is proved to be highly robust (2.9% no results) and specific (1.9% error rate) when applied to rapid (24-hour) analysis of single cells biopsied from cleavage-stage embryos. This comprehensive chromosome analysis technique is the first to be validated by reanalyzing the same embryos with another technique (e.g., FISH). Unlike some alternative techniques for comprehensive chromosome screening, array-CGH does not require prior testing of parental DNA and thus advance planning and careful scheduling are unnecessary.

Preimplantation genetic diagnosis (PGD) improves pregnancy outcome for translocation carriers with a history of recurrent losses.

OBJECTIVE: To determine if preimplantation genetic diagnosis (PGD) for translocation carriers with three or more pregnancy losses reduces loss rates. DESIGN: Retrospective review of data. SETTING: Preimplantation genetic diagnosis laboratory servicing IVF groups. PATIENT(S): Patients (n = 192) undergoing PGD for either a reciprocal translocation or Robertsonian translocation who had three or more previous pregnancy losses. INTERVENTION(S): Preimplantation genetic diagnosis for translocations. MAIN OUTCOME MEASURE(S): Pregnancy loss rate, pregnancy success rate defined as delivery of at least one child or an ongoing pregnancy in the third trimester, and length of time to success. RESULT(S): Pregnancy loss rate was significantly reduced to 13% post-PGD compared with 88.5% in previous non-PGD pregnancies and to 35% to 64% from naturally conceived pregnancies as reported in the literature. Pregnancy success rate was 87%. Conception occurred after an average of 1.4 cycles or <4 months. CONCLUSION(S): Individuals with translocations who have experienced three or more losses benefit from PGD by realizing a significant reduction in loss rate and improvement in rate of success of pregnancy. Length of time to conceive is also dramatically reduced compared with data in the literature for similar populations not undergoing PGD.

DNA probe pooling for rapid delineation of chromosomal breakpoints.

Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival, as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multiclone and multicolor mapping experiments do not generate additional information. Our pooling protocol, described here with examples from thyroid cancer research and PGD, accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as 3 to 4 days.

Rapid mapping of chromosomal breakpoints: from blood to BAC in 20 days.

Structural chromosome aberrations and associated segmental or chromosomal aneusomies are major causes of reproductive failure in humans. Despite the fact that carriers of reciprocal balanced translocation often have no other clinical symptoms or disease, impaired chromosome homologue pairing in meiosis and karyokinesis errors lead to over-representation of translocations carriers in the infertile population and in recurrent pregnancy loss patients. At present, clinicians have no means to select healthy germ cells or balanced zygotes in vivo, but in vitro fertilization (IVF) followed by preimplantation genetic diagnosis (PGD) offers translocation carriers a chance to select balanced or normal embryos for transfer. Although a combination of telomeric and centromeric probes can differentiate embryos that are unbalanced from normal or unbalanced ones, a seemingly random position of breakpoints in these IVF-patients poses a serious obstacle to differentiating between normal and balanced embryos, which for most translocation couples, is desirable. Using a carrier with reciprocal translocation t(4;13) as an example, we describe our state-of-the-art approach to the preparation of patient-specific DNA probes that span or 'extent' the breakpoints. With the techniques and resources described here, most breakpoints can be accurately mapped in a matter of days using carrier lymphocytes, and a few extra days are allowed for PGD-probe optimization. The optimized probes will then be suitable for interphase cell analysis, a prerequisite for PGD since blastomeres are biopsied from normally growing day 3--embryos regardless of their position in the mitotic cell cycle. Furthermore, routine application of these rapid methods should make PGD even more affordable for translocation carriers enrolled in IVF programs.

Increased efficiency of preimplantation genetic diagnosis for infertility using "no result rescue".

OBJECTIVE: To improve preimplantation genetic diagnosis (PGD) accuracy by using "no result rescue" (NRR) consisting of the reanalysis of dubious results with additional probes binding to a locus different from the one previously analyzed. DESIGN: Prospective study of PGD cycles with and without reanalysis of inconclusive results. SETTING: PGD laboratory. PATIENT(S): Patients undergoing PGD for infertility or Robertsonian translocations. INTERVENTION(S): Nuclei from day 3 biopsied embryos were analyzed with fluorescence in situ hybridization for chromosomes X,Y, 13, 15, 16, 17, 18, 21, and 22. When inconclusive results were obtained, NRR was performed. In addition, 100 PGD cycles using NRR were matched to controls according to maternal age, previous failed cycles, number of zygotes, number of eggs, and date of retrieval. MAIN OUTCOME MEASURE(S): Determination of frequency of inconclusive results and error rate after use of additional probes. Comparison of frequency of inconclusive results with prior PGD results when NRR was not used. Assisted reproductive technology outcome was compared between PGD using NRR and controls not using PGD. RESULT(S): After analysis of 34,831 blastomeres from 34,225 embryos, 2,609 blastomeres (7.5%) showed inconclusive results. After NRR on those 2,609 blastomeres, the number of cells with inconclusive results was reduced to 3.1% (P<.001). After the introduction of NRR, fluorescence in situ hybridization errors, measured as discrepancies between the PGD diagnosis and the analysis of the nonreplaced embryo, decreased from 13.6% to 4.7% (P<.001). PGD with NRR significantly improved implantation rates, from 20% to 31%, and reduced spontaneous abortions from 27% to 6%. CONCLUSION(S): The use of NRR has been proven to be a powerful tool to reduce the error rate and the frequency of inconclusive results in PGD, both parameters of high importance to assess quality of PGD laboratories. Indeed, these parameters are two of the few measurable criteria to measure PGD laboratories. In a parallel controlled study, PGD with NRR significantly improved implantation rates and reduced spontaneous abortions, showing that PGD is more efficient in selecting embryos that will reach term.

Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies.

Preimplantation genetic diagnosis (PGD) for translocations has been shown to significantly reduce the risk of recurrent miscarriage, but because the majority of embryos produced are unbalanced, pregnancy rate is relatively low since 20% or more cycles have no normal or balanced embryos to transfer. The purpose of this study was to evaluate whether PGD could improve pregnancy outcome in translocation carriers with a history of two or more consecutive miscarriages and no live births. PGD for translocations was offered to translocation carriers with two or more previous miscarriages (average 3.5) and no live births (0/117 pregnancies) using a combination of distal and proximal probes to the breakpoints. After PGD, only 18.3% of embryos were normal or balanced. Only 5.3% of pregnancies were lost after PGD compared with 100% before PGD (P < 0.001). The cumulative pregnancy rate was 57.6% and the cumulative ongoing pregnancy rate was 54.5% in the short period of time of 1.24 IVF cycles, or 46.3% and 43.9% respectively per cycle. In conclusion, PGD significantly reduced losses and increased the number of viable pregnancies (P < 0.001). IVF plus PGD are a faster method of conceiving a live child than natural conception, at least for translocation carriers with recurrent miscarriages and no previous live births.

Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages.

OBJECTIVE: To determine whether preimplantation genetic diagnosis (PGD) and transfer of euploid embryos would decrease spontaneous abortion rates in recurrent miscarriage (RM) patients. DESIGN: Controlled clinical study. SETTING: In vitro fertilization centers and PGD reference laboratory. PATIENT(S): Recurrent-miscarriage patients with three or more prior lost pregnancies with no known etiology. INTERVENTION(S): Biopsy of a single blastomere from each day 3 embryo, followed by fluorescence in situ hybridization analysis. MAIN OUTCOME MEASURE(S): The rate of spontaneous abortions in RM subjects undergoing PGD were compared with [1] their own a priori expectations and [2] a comparison group of women undergoing PGD for advanced maternal age (> or =35 years). RESULT(S): Before PGD, RM patients had lost 87% (262/301) of their pregnancies, with an expected loss rate of 36.5%. After, they only lost 16.7% pregnancies. This difference was mostly due to reduction in pregnancy loss in the > or =35-years age subgroup, to 12% from an expected 44.5%. CONCLUSION(S): Preimplantation genetic diagnosis aneuploidy screening has a beneficial effect on pregnancy outcome in RM couples, especially those in which the woman is aged > or =35 years. Our data indicate that PGD reduces the risk of miscarriage in RM patients to baseline levels.

Patterns of ovarian response to gonadotropin stimulation in female carriers of balanced translocation.

OBJECTIVE: To determine whether maternal balanced translocation is a risk factor for poor ovarian response to controlled ovarian hyperstimulation (COH). DESIGN: A retrospective analysis. SETTING: Private IVF center. PATIENT(S): All couples presenting to a single center for preimplantation genetic diagnosis (PGD) for autosomal balanced translocation in either partner from 1995 through 2001. INTERVENTION(S): Cycle parameters and embryology outcomes were examined and compared between two groups: 61 cycles in 46 women with balanced translocations compared with 42 cycles in 32 women whose male partner had a balanced translocation. MAIN OUTCOME MEASURE(S): Response to ovarian stimulation. RESULT(S): In couples undergoing IVF/PGD to avoid transmission of an unbalanced karyotype, a significantly higher proportion of women carrying balanced translocations (female carrier) responded very poorly (E2 on the day of hCG <1,000 pg/mL) to ovarian stimulation compared to women whose partner had a balanced translocation (male carrier) (23.0% vs. 7.1%). CONCLUSION(S): In couples undergoing IVF/PGD for balanced translocation, the risk for poor response to ovarian stimulation may be increased when the female partner carries the balanced translocation compared to when the male partner carries the translocation. Given significant embryo attrition due to chromosomal imbalance, aggressive stimulation should be considered if the patient does not have risk factors for ovarian hyperstimulation syndrome (OHSS).

Negligible interchromosomal effect in embryos of Robertsonian translocation carriers.

It has been suggested that translocations, and perhaps other chromosome rearrangements, disturb meiotic disjunction of chromosome pairs not involved in the translocation, resulting in non-disjunction in those chromosomes (interchromosomal effect) and predisposition to trisomy offspring. Other reports have suggested an increased risk of mosaicism and chaotic embryos in translocation carriers. This study was designed to determine if such interchromosomal effects are producing significantly more chromosome abnormalities than those expected from unbalanced gametes. For that purpose, two groups of PGD patients were compared, Robertsonian translocation carriers (RBT) and carriers of X-linked diseases (XLI), of similar age. Both groups were analysed by FISH with similar DNA probes. The results indicate that overall, the higher rate of chromosome abnormalities in the RBT group was solely due to unbalanced gametes and not to an interchromosomal effect or higher incidence of mosaicism. If unbalanced and normal were combined, this proportion was 53% in XLI and 59% in RBT. However, when specific RBT translocations were studied, only a slight increase in embryos with aneuploidy for chromosome 22 was found for the t(13;14) translocation carriers, while a higher rate of post-zygotic abnormalities was observed in the more rare RBT. In conclusion, the overall rate of non-translocation related abnormalities was not increased in the RBT group compared with the control group, but a slight interchromosomal effect may exist, as some Robertsonian translocations may be more prone to produce mosaic and chaotic embryos.

Differences in chromosome susceptibility to aneuploidy and survival to first trimester.

The purpose of this study was to find specific rates of aneuploidy in cleavage-stage embryos compared with first trimester data and to evaluate post-zygotic selection against aneuploidy. A total of 2058 embryos were analysed by flurorescence in-situ hybridization (FISH), and specific aneuploidy rates were obtained for 14 chromosomes. Data from morphologically abnormal embryos could be pooled with data from preimplantation genetic diagnosis (PGD) cycles because it was observed that they had similar rates of aneuploidy; thus, for the purpose of studying aneuploidy they could be, and were, pooled. Specific chromosome aneuploidy rates were not related to morphology or development of the embryos. The average maternal age of patients with aneuploid embryos was significantly higher than the overall analysed population. Monosomy appeared more commonly than trisomy. The chromosomes most frequently involved in aneuploidy were (in order) 22, 16, 21 and 15. When compared with first trimester pregnancy data, aneuploidies detected at cleavage stage seem to die in excess of 90% before reaching first trimester, with the exception of chromosome 16 and gonosomes (76% and 14% respectively). Differences in chromosome-specific aneuploidy rates at first trimester conceptions are probably produced by different chromosome-specific aneuploidy rates at cleavage stage and different survival rates to first trimester.

Improved implantation after preimplantation genetic diagnosis of aneuploidy.

The objective of this study was to assess the improvement in implantation rates after preimplantation genetic diagnosis (PGD) of numerical abnormalities for the sole indication of advanced maternal age when compared with a control group. Each PGD patient was matched to a control patient according to several parameters prior to obtaining pregnancy results. The diagnosis was based on the analysis of chromosomes X, Y, 13, 15, 16, 18, 21 and 22 plus a ninth probe (1, 7, 14 or 17) on a single cell per embryo. The results were also analysed in relation to the previous number of IVF cycles and the number of dipronucleated zygotes obtained, when replacing presumptively chromosomally normal embryos on day 4 of development. It was found that women of advanced reproductive age (average age 40 years) had a higher implantation rate (18%) than their matched controls treated with standard IVF (11%) (P < 0.05). This increase was not observed in patients with two or more previous IVF cycles or patients with fewer than eight zygotes. Patients with eight or more 2PN zygotes and one or no previous cycles showed the greatest improvement in implantation rate, from 8.8% in controls to 19.2% in the PGD group (average age 40 years) (P < 0.025).

Predictive value of sperm fluorescence in situ hybridization analysis on the outcome of preimplantation genetic diagnosis for translocations.

OBJECTIVE: To determine whether the proportion of abnormal sperm is predictive of the proportion of abnormal embryos from couples in which the males are translocation carriers. DESIGN: Controlled clinical study. SETTINGS: Private in vitro fertilization (IVF) center. PATIENT(S): Eleven cases of reciprocal translocation male carriers. INTERVENTION(S): Blood sample and sperm sample collection from each male partner. Embryo biopsy of the embryos produced in each cycle. MAIN OUTCOME MEASURE(S): Fluorescence in situ hybridization on lymphocyte slides to characterize each translocation case, then fluorescent in situ hybridization (FISH) with specific probes for each of the sperm samples. Preimplantation genetic diagnosis of the translocations in the 11 cases. RESULTS: A correlation was found between the percentage of abnormal gametes and the percentage of abnormal embryos, and a predictive equation is proposed for this relationship: A = -55 + (1.9 x B), where A is the percentage of abnormal embryos and B the percentage of abnormal sperm. CONCLUSION(S): The predictive value of the sperm analysis was established. Patients with 65% or less chromosomally abnormal sperm have a good chance at conceiving; patients with higher rates would need to produce 10 or more good quality embryos to have reasonable chances of conceiving.

Preimplantation genetic diagnosis of numerical abnormalities for 13 chromosomes.

Several types of FISH protocols for PGD have been used to maximize results from a limited number of fluorochomes to study as many chromosomes as possible. The major purpose of the present study was to optimize the use of three sequential hybridizations to analyse up to 15 chromosome types in single cells. A secondary purpose was to study the frequency of aneuploidy of other chromosomes not yet extensively studied in preimplantation embryos. Patients underwent PGD of aneuploidy, and the biopsied cells were analysed with three sequential hybridizations, the first for chromosomes 13, 16, 18, 21 and 22, the second for X, Y, 15 and 17 and the third for 2, 3, 4 and 11. Overall, only 27% of embryos were normal. The chromosomes most involved in aneuploidy were, in order, chromosome 16, 15, 21, 22, 13, 18, 17, 3, 2, 4, 11, and gonosomes. Of the abnormal embryos, only 3% would have been missed without the third set of probes. This protocol allows the simultaneous analysis of up to 15 chromosomes although only 13 were analysed in this study. Results so far show that the chromosomes most involved in abnormalities are those already covered with the two first sets of probes.

Chromosomal abnormalities in embryos derived from testicular sperm extraction.

OBJECTIVE: To compare the rate of chromosome abnormalities in embryos obtained from karyotypically normal patients with nonobstructive azoospermia undergoing testicular sperm extraction (TESE) to those from patients undergoing intracytoplasmic sperm injection (ICSI) with ejaculated sperm. DESIGN: Retrospective analysis. SETTING: IVF centers. PATIENT(S): Male partners had either nonobstructive zoospermia or oligospermia. INTERVENTION(S): Preimplantation genetic diagnosis. Chromosome enumeration was performed by fluorescence in situ hybridization (FISH). Embryos classified as abnormal were reanalyzed to study mosaicism. MAIN OUTCOME MEASURE(S): Chromosome abnormalities in embryos. RESULT(S): Embryos from ICSI cycles with ejaculated sperm (group 1) were 41.8% normal, 26.2% aneuploid, and 26.5% mosaic. In contrast, the embryos from ICSI cycles with TESE for nonobstructive azoospermia (group 2) were 22% normal, 17% aneuploid, and 53% mosaic. The difference in mosaicism rate between the two groups of embryos was highly significant. CONCLUSION(S): The present study results indicate a high incidence of mosaicism in embryos derived from TESE in men with a severe deficit in spermatogenesis. Sperm derived from TESE for nonobstructive azoospermia may have a higher rate of compromised or immature centrosome structures leading to mosaicism in the embryo.

First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy.

OBJECTIVE: To develop a preimplantation genetic diagnosis (PGD) protocol that allows any form of chromosome imbalance to be detected. DESIGN: Case report employing a method based on whole-genome amplification and comparative genomic hybridization (CGH). SETTING: Clinical IVF laboratory. PATIENT(S): A 40-year-old IVF patient. INTERVENTION(S): Polar body and blastomere biopsy. MAIN OUTCOME MEASURE(S): Detection of aneuploidy. RESULT(S): Chromosome imbalance was detected in 9 of 10 polar bodies. A variety of chromosomes were aneuploid, but chromosomal size was found to be an important predisposing factor. In three cases, the resulting embryos could be tested using fluorescence in situ hybridization, and in each case the CGH diagnosis was confirmed. A single embryo could be recommended for transfer on the basis of the CGH data, but no pregnancy ensued. CONCLUSION(S): Evidence suggests that preferential transfer of chromosomally normal embryos can improve IVF outcomes. However, current PGD protocols do not allow analysis of every chromosome, and therefore a proportion of abnormal embryos remains undetected. We describe a method that allows every chromosome to be assessed in polar bodies and oocytes. The technique was accurate and allowed identification of aneuploid embryos that would have been diagnosed as normal by standard PGD techniques. As well as comprehensive cytogenetic analysis, this protocol permits simultaneous testing for multiple single-gene disorders.

Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect.

The present study evaluated mosaicism in a large series of cleavage-stage human embryos analysed by fluorescence in-situ hybridization. Only embryos with at least three cells analysed were included (n = 1235), of which 556 were mosaics. The most common types of mosaicism were chaotic (48%), diploid/polyploid (26%), and those caused by mitotic non-disjunction (25%). The number of abnormal cells per embryo ranged from 44% in diploid/polyploid to 84% in chaotic mosaics. Chromosome 16 was most commonly involved in mitotic non-disjunction mosaics. While overall mosaicism did not increase with maternal age, the average maternal age of the embryos that had mosaics caused by mitotic non-disjunction was significantly higher than that for normal or other mosaic embryos (P < 0.001). During the cleavage stage, the embryonic genome is not yet fully activated and consequently the mRNA and protein pools are still similar to those found in the oocyte. We therefore propose that the malfunctioning of the meiosis apparatus, which is similar to the mitotic one, may cause either meiotic errors or mitotic non-disjunction at cleavage-stage embryo development.

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

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.