Preimplantation Diagnosis by Fluorescence In Situ Hybridization Using 13-, 16-, 18-, 21-, 22-, X-, and Y-Chromosome Probes

Purpose: Our purpose was to select the proper chromosomes for preimplantation diagnosis based on aneuploidy distribution in abortuses and to carry out a feasibility study of preimplantation diagnosis for embryos using multiple-probe fluorescence in situ hybridization (FISH) on the selected chromosomes of biopsied blastomeres. Methods: After determining the frequency distribution of aneuploidy found in abortuses, seven chromosomes were selected for FISH probes. Blastomeres were obtained from 33 abnormal or excess embryos. The chromosome complements of both the biopsied blastomeres and the remaining sibling blastomeres in each embryo were determined by FISH and compared to evaluate their preimplantation diagnostic potential. Results: Chromosomes (16, 22, X, Y) and (13, 18, 21) were selected on the basis of the high aneuploid prevalence in abortuses for the former group and the presence of trisomy in the newborn for the latter. Thirty-six (72%) of 50 blastomeres gave signals to permit a diagnosis. Diagnoses made from biopsied blasotmeres were consistent with the diagnoses made from the remaining sibling blastomeres in 18 embryos. In only 2 of 20 cases did the biopsied blastomere diagnosis and the embryo diagnosis not match. Conclusions: If FISH of biopsied blastomere was successful, a preimplantation diagnosis could be made with 10% error. When a combination of chromosome-13, -16, -18, -21, -22, -X, and -Y probes was used, up to 65% of the embryos destined to be aborted could be detected.     

Simultaneous Detection of Chromosomes X, Y, 13, 18, and 21 by Fluorescence In Situ Hybridization in Blastomeres Obtained from Preimplantation Embryos

Purpose. Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. Methods: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. Results: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. Conclusions: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.     

Detection of chromosomal abnormalities in human preimplantation embryos using FISH

Purpose: Multicolour FISH has been used for the preimplantation diagnosis of sex for X-linked disorders and to examine the chromosome constitution of early human embryos. Materials and Methods: Single blastomeres and whole embryos were spread using HCl and Tween 20. Multicolour FISH was performed using directly-labelled human DNA probes for chromosomes X, Y, and I in a two hour FISH procedure. Results: Four groups of chromosome arrangements have been found in human preimplantation embryos (i) normal, all nuclei uniformly diploid, (ii) diploid mosaics, majority of the nuclei diploid, with a small number of nuclei aneuploid (iii) chromosomally abnormal, all nuclei uniformly chromosomally abnormal, e.g. XO, XXY, XXX and (iv) chaotic, all nuclei showing different chromosome complements. Conclusions: For the preimplantation diagnosis of sex, an XX nucleus has always been representative of a female embryo. However, for the diagnosis of dominant disorders or chromosome abnormalities, two cells should be analysed to reduce the chance of misdiagnosis which may arise from chromosomal mosaicism. Implantation and further embryo development may be possible from mosaic or chromosomally abnormal embryos, but those showing chaotic chromosome arrangements would be unlikely to implant.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization

Purpose Our purpose was to assess the clinical application of dual fluorescent in situhybridization (FISH) for the diagnosis of sex in the human preimplantation embryo. Results Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in vitro fertilization (IVF) treatment cycles led to nine pregnancies; 7 reached the stage of clinical recognition, of which 2 spontaneously aborted. There were five live births, three singleton and two twin: none in disagreement with the diagnosed sex. The diagnosis was corroborated in 51 of the 74 nontransferred embryos. The efficiency of the procedure improved throughout the four treatment cycles. This was reflected in the increased proportion of double embryo transfers (from 50% in series 1 and 2 to 100% in series 3 and 4), with a consequent improvement in pregnancy rate (from 28 to 71% per embryo transfer). The excess of male embryos (male∶female, 60∶40 overall) and the high proportion of biopsied embryos with abnormal numbers of X and Y chromosome signals (14.5%) effectively reduced the number of normal female embryos available for transfer. Conclusion Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.     

A fast and efficient method for simultaneous X and Y in situ hybridization of human blastomeres

Purpose To use a 6-hr fluorescence in situ hybridization (FISH) procedure involving fluorochrome-labeled probes to determine the gender of blastomeres from arrested biopsied human embryos.Results Simultaneous detection of X and Y chromosomes was performed on 68 blastomeres with this technique. The FISH efficiency for gender determination was 95.5% (65/68). In addition, rehybridization with chromosome 8-specific probes was performed to determine the ploidy of blastomeres with more than two sex chromosomes.Conclusion This technique offers an alternative to polymerase chain reaction for the preimplantation diagnosis of X-linked diseases and can also be used for ploidy assessment.     

Discordance among blastomeres renders preimplantation genetic diagnosis for aneuploidy ineffective

Purpose: To investigate the contribution of discordance among blastomeres from the same embryo in the interpretation of blastomeres biopsied from day 3 embryos. Methods: 228 IVF embryos had two blastomeres removed and fluorescent in situ hybridization (FISH) was used to detect aneuploidy of chromosomes 13, 15, 16, 18, 21, 22, X and Y. Of the 228 embryos, 102 had complete FISH results for both blastomeres. Results: When the 2 blastomeres of 102 embryos with successful FISH results were compared, 26 (25.5%) were concordant for all 8 chromosomes and 76 (74.5%) were discordant for one or more chromosomes. Among the 102 embryos, 12 (12%) were disomy in both blastomeres and 37 (36%) were disomic in all 8 chromosomes in one of the two blastomeres. Conclusion: Discordance among blastomeres from the same embryo appears to present a significant problem in interpreting results of embryos biopsied on day 3 and analyzed by FISH especially when most PGD’s are done on single blastomeres.     

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.     

A normal birth following preimplantation genetic diagnosis by FISH determination in the carriers of der(15)t(Y;15)(Yq12;15p11) translocations: two case reports

Purpose To investigate the clinical application of fluorescence in situ hybridization (FISH) for assessing chromosome disorders of embryos in preimplantation diagnosis of carriers with der(15)t(Y;15)(q12;p11) translocations. Methods Multicolor FISH was performed using directly-labelled DNA probes, chromosome X with one (DXZ1, Xp11.1-q11.1), but Y with two (DYZ3, Yp11.1-q11.1 and DYZ1, Yq12). Normal embryos were transferred on day 6 at blastocyst stage. Results Couple A: Three of 6 biopsied embryos were normal. Two normal blastocysts were transferred, but no pregnancy was achieved. Couple B: Three of 6 biopsied embryos were normal. Two normal blastocysts were transferred. A normal male infant weighing 3,230 g was born by cesarean section on the 39th week of gestation. All of the remaining nonreplaced embryos showed mosaic or der(15). Conclusion Embryos from carries of der(15)t(Y;15)(q12;p11) translocation showed a high frequency of chromosome abnormalities. PGD is a valuable screen tool for those couples to treat their infertility and break the transmission of der(15) chromosome for their offspring.     

Different probe combinations for assessment of postzygotic chromosomal imbalances in human embryos

Purpose: We compared three different probe combinations for detection of postzygotic mosaic imbalances in human preimplantation embryos. Methods: Two hundred and two spare cleavage stage embryos were hybridized with fluorescently labelled DNA probe mixtures specific to chromosomes X, Y, 18 (N = 67), chromosomes 2, 7, 18 (N = 71), or chromosomes 13, 16, 18, 21, 22 (N = 64). Results: An overall higher incidence of abnormalities was detected using probe mixture for five (69%) or three (72%) autosomes compared to one autosome and chromosomes X and Y (54%). The rate of aneuploidy detected increased with the number of autosomes hybridized from 4% (X, Y, 18) to 11% (2, 7, 18) to 19% (13, 16, 18, 21, 22). Postzygotic mosaicism comprised the most frequent abnormality detected by all probe combinations, and the percentage detected by each was similar, 48% (X, Y, 18), 56% (2, 7, 18(, and 50% (13, 16, 18, 21, 22). Conclusions: A probe combination of five autosomes, particularly those of clinical relevance, may be more beneficial for screening embryos from patients at risk of maternal-age-related aneuploidy. However, all three probe combinations are as efficient at identifying postzygotic mosaicism, and may be used for identifying embryos with less potential of developing to term.     

Higher degree of chromosome mosaicism in preimplantation embryos from carriers of robertsonian translocation t(13;14) in comparison with embryos from karyotypically normal IVF patients

Purpose : To compare the frequency and the degree of mosaicism in human embryos from Robertsonian translocation (RT) t(13;14) carriers, with embryos from karyotypically normal IVF patients. Methods : FISH analysis of embryos from PGD cycles for RT t(13;14), with probes for chromosomes 13, 14, and 18 (Group I) and of embryos from karyotypically normal IVF patients with probes for chromosomes 13, 18, 21, X, and Y (Group II). Results : The incidence of abnormal mosaic embryos was significantly higher in group I (38/51) as compared with group II (6/45) (²: P < 0.01). Furthermore, in group I the percentage of diploid cells per embryo was lower for chromosome 13 and 14 in comparison with 18, while in group II no differences were observed between the five chromosomes analyzed. Conclusions : RT induces a high frequency of mosaicism specifically for the chromosomes implicated in the translocation; the analysis by FISH of two blastomeres is strongly recommended for these patients.     

In vitro fertilization plus preimplantation genetic diagnosis in patients with recurrent miscarriage: an analysis of chromosome abnormalities in human preimplantation embryos

Objective: To analyze the incidence of numeric chromosomal abnormalities in preimplantation embryos from women with unexplained recurrent miscarriage (RM) so as to seek an etiology and to determine whether the use of IVF may be indicated to treat these cases.

Design: Prospective controlled study.

Setting: University laboratory of reproductive genetics and a tertiary referral center for infertility.

Patient(s): Nine women with a mean (±SD) of 3.9 ± 0.6 RMs who were undergoing IVF and preimplantation genetic diagnosis, and a control group of young (n = 10) and older (n = 6) patients who were undergoing preimplantation genetic diagnosis because of sex-linked diseases.

Intervention(s): In vitro fertilization, embryo culture for 72 hours, blastomere biopsy, and analysis of chromosomes 13, 16, 18, 21, 22, X, and Y with the use of fluorescent in situ hybridization. Transfer of chromosomally normal embryos into the uterus.

Main Outcome Measure(s): Numeric chromosomal abnormalities in human embryos.

Result(s): Sixty-six embryos from patients with RM were compared with 62 embryos from young patients and 41 embryos from older patients. There was a significant increase in the rate of abnormal embryos in the patients with RM and the older patients compared with the controls. Abnormalities in most of the chromosomes studied were higher in the RM group than in the control group, especially those affecting chromosome 13.

Conclusion(s): There was an increase in numeric chromosomal abnormalities in preimplantation embryos from women with RM that could be the cause of infertility in many couples with unexplained RM. The use of IVF in such circumstances may be indicated if successful preimplantation genetic diagnosis is added to the procedure.     

Chromosomal Mosaicism in Cleavage-Stage Human Embryos and the Accuracy of Single-Cell Genetic Analysis

Purpose: Our purpose was to assess the effect of chromosomal mosaicism in cleavage-stage human embryos on the accuracy of single-cell analysis for preimplantation genetic diagnosis. Methods: Multicolor fluorescence in situ hybridization with X, Y, and 7 or X, Y, 7, and 18 chromosome-specific probes was used to detect aneuploidy in cleavage-stage human embryos. Results: Most nuclei were diploid for the chromosomes tested but there was extensive mosaicism including monosomic, double-monosomic, nullisomic, chaotic, and haploid nuclei. Conclusions: Identification of sex by analysis of a single cleavage-stage nucleus is accurate but 7% of females are not identified. One or both parental chromosomes 7 were absent in at least 6.5% of the nuclei. With autosomal recessive conditions such as cystic fibrosis, carriers would be misdiagnosed as normal or affected. With autosomal dominant conditions, failure to analyze the affected parents allele (1.6–2.5%) would cause a serious misdiagnosis and analysis of at least two nuclei is necessary to reduce errors.     

Elevated incidence of chromosomally chaotic embryos among frozen-thawed preimplantation embryos

OBJECTIVE: The aim of the study was to evaluate the effect of cryopreservation on the formation of chromosomal abnormalities in human preimplantation embryos. STUDY DESIGN: The chromosomal constitutions of cleavage stage embryos (n = 61) were assessed using fluorescent in situ hybridisation (FISH) technique, applying probes for chromosomes 13, 16, 18, 21, X and Y. Study group embryos frozen at zygote or two-cell stage (n = 29) were cultured in vitro post-thawing until they reached four- to six-cell stage, after which their chromosomal constitutions were assessed. Control group embryos frozen at four- to six-cell stage (n = 32) were analysed immediately after thawing in order to exclude any post-thaw effect. The proportions of genetically normal and abnormal embryos were compared between study and control group. RESULTS: The proportions of normal, aneuploid and mosaic embryos were similar in both groups. However, significantly (P < 0.05) higher proportion of chaotic embryos in study (24.1%) compared to control group (6.3%) was observed. CONCLUSION: The elevated level of chromosomally chaotic embryos among embryos that had undergone cellular division after thawing as compared to embryos analysed immediately after thawing indicates a potential negative impact of cryopreservation on the formation of chromosomal abnormalities in preimplantation embryos.     

Preimplantation Diagnosis by FISH: The Rambam Experience

Purpose: Our purpose was to summarize our experience gained using fluorescence in situ hybridization for preimplantation diagnosis at the Rambam Medical Center. Methods: Seventy-three embryos (29 cycles) were analyzed for preimplantation diagnosis for the following indications: advanced maternal age (>39 years), X-linked diseases, poor-quality embryos, repeated failure in vitro fertilization cycles and fast-dividing embryos. An additional 38 embryos with unequal pronuclei size were examined for ploidy. Biopsy of embryonic blastomeres was performed at the six- to eight-cell stage. Five fluorescence probes, for chromosomes X, Y, 13, 18, and 21, were applied for ploidy detection. Results: Eighty-four (87%) blastomeres of the 73 embryos analyzed showed clear signals. Six of the blastomeres were lost during spreading. Two of the embryos were destroyed following biopsy. No nucleus was found in five of the blastomeres, while in nine, more than one nucleus was verified. Transfer was performed in 10 patients (32 embryos). Two pregnancies were achieved. Two healthy babies were born. Fifty-seven percent of the fast-dividing embryos demonstrated normal signals. In two groups of embryos of unequal pronuclei size, following conventional insemination and intracytoplasmic sperm injection, 50 and 11.4% demonstrated normal signals. Conclusions: The efficiency of florescence in situ hybridization for aneuploidy detection is 87 and 97% for autosomes and gonosomes, respectively. The preimplantation genetic diagnosis is suitable for selected in vitro fertilization cases including fast-dividing embryos and embryos with unequal pronuclei size following regular in vitro fertilization.     

Fluorescentin Situ hybridization analysis of chromosomally normal gametes and abnormal arrested embryos

Objective The purpose of the present study was to investigate if the arrested embryos from a couple with several previously failed IVF treatments were chromosomally normal. Probes for chromosomes X, Y and 18 were used.Results A couple had undergone 7in vitro fertilization treatments over a 2 1/2-year period without achieving a pregnancy. In each cycle, where fertilization was obtained, the development of the embryos was arrested. Fluorescentin situ hybridization probes for chromosomes X and Y (and 18) was carried out on gametes and on embryos in 2 separate cycles. Sperm and oocytes were normally haploid X0 or Y0. The nuclei of the blastomeres were fragmented and mosaic for X or Y, or monosomic X0, despite the fact that 2 pronuclei had been assessed on day 1 following intracytoplasmic sperm injection.Conclusion Chromosomally normal gametes can result in abnormal embryos manifested by arrested development and unexplained infertility.     

Increased efficiency of preimplantation genetic diagnosis for aneuploidy by testing 12 chromosomes

One of the most important factors in increasing the screening potential of preimplantation genetic diagnosis (PGD) for aneuploidy is to increase the number of chromosomes analysed. Inclusion of chromosomes 8, 14 and 20 to the standard set of chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22 allows the analysis of 12 chromosomes in three rounds of fluorescent in-situ hybridization (FISH) without decreasing the efficiency of the technique. Pregnancy rate was significantly increased when only embryos that had been diagnosed as normal for the 12 chromosomes analysed were transferred compared with transfer of embryos with any abnormality for chromosomes 8, 14 or 20 (P < 0.05). This study proves that the high efficiency and practical feasibility of FISH analysis of 12 chromosomes in PGD for aneuploidy is a superior approach than the standard nine-chromosome analysis in order to screen for abnormalities.     

Chromosome 21 Detection in Human Oocyte Fluorescence In Situ Hybridization: Possible Effect of Maternal Age

Purpose: The purpose of this study was to evaluate, among 100 uncleaved oocytes, the incidence of numerical and structural chromosome 21 and X abnormalities and to analyze the influence of various factors, such as in vitro (IVF) indications, follicle stimulation protocols, and women's age. Methods: We investigated 150 uncleaved oocytes from 128 patients after an IVF attempt. After cytogenetic analysis (Giemsa) 100 oocytes (66%) were selected for fluorescence in situ hybridization (FISH). Fluorescent probes for human chromosomes X and 21 were used simultaneously according to standard procedures for their hybridization and detection. Results and Conclusions: We analyzed by the FISH protocol 100 metaphase II oocytes with 22 to 25 chromosomes. Our results demonstrate a high rate of disomy for chromosome 21 in human oocytes. Among them, eight were disomic (8%) and three were nullosomic (3%) for chromosome 21. Only one disomy of chromosome X was noted. The various indications of IVF and the different folliculogenesis stimulating protocols did not seem to influence the results but suggested a correlation between the maternal age and the aneuploidy rate of chromosome 21.     

Comparison between fluorescence in situ hybridization (FISH) and quantitative-fluorescent polymerase chain reaction (QF-PCR) for the detection of aneuploidies in single blastomeres

OBJECTIVES: The aim of our investigation was to compare the efficiencies of the fluorescence in situ hybridization (FISH) and the quantitative-fluorescent PCR (QF-PCR) methods for the detection of sexing and numerical chromosome disorders in single blastomeres collected from the same preimplantation human embryos. METHODS: FISH analysis was carried out on 145 blastomeres from the 79 surplus embryos with probes specific for chromosomes 13, 18, 21, X, and Y. QF-PCR was performed with each one or two of the primers specific for the same chromosomes on 151 blastomeres from the same embryos obtained from patients undergoing IVF treatment. RESULTS: Analyses were possible on 135 blastomeres (93%) by FISH and on 117 blastomeres (77%) by QF-PCR. Of 65 embryos, which could be analyzed by both methods, 20 embryos (31%) were diagnosed as abnormal. CONCLUSION: The present study shows that FISH tests are more accurate than QF-PCR assays for the detection of numerical chromosome disorders when performed on single blastomeres.     

The Relationship of Pronuclear Stage Morphology and Chromosome Status at Cleavage Stage

Purpose : Infertile couples undergoing in vitro fertilization-embryo transfer (IVF-ET) program were included to study the relationship of pronuclear stage morphology and chromosome status at cleavage stage. Methods : Eighteen to twenty-one hours after fertilization, zygotes were checked for pronuclear morphology with modified Scott Z-score system. After embryo transfer on day 3, arrested or non-transferred 2 PN embryos were spread for fluorescence in situ hybridization (FISH) staining of probes to chromosomes 18, X and Y. Results : Ninety-eight embryos were successfully fixed and stained. The chromosome status were recorded in each 2 PN score group: 7 (54%) of 13 embryos in Z2 group, 14 (35%) of 40 in Z3 group and 10 (36%) of 28 in Z4 group being normal diploid. Z1 group has 12 (71%) of 17 embryos being normal diploid, which is significantly more than Z3 (p=0.020) and Z4 group (p = 0.033). Conclusion : Our results demonstrated a high probability to get normal diploid embryos if good morphology at pronuclear stage was used as selection criteria, especially for Z1 score embryos.     

Reduction in signal overlap results in increased FISH efficiency: Implications for preimplantation genetic diagnosis

Background: In the absence of mosaicism, one of the problems of preimplantation genetic diagnosis with FISH is the occurrence of false-negative hybridization results. It has been hypothesized that missing signals are produced by spatial overlap of signals. Methods and Results: To investigate the relation among cell density, signal overlap, and hybridization signal detection, 371 blastomeres and 4556 lymphocytes were fixed in different cellular concentrations and analyzed by FISH using probes for chromosomes X, Y, and 18, and their nuclear diameters and FISH results scored. The results showed that the lower the diameter of fixed nuclei, the higher the number of signal overlaps and missing signals. The minimum number of missing signals was obtained when lymphocyte and blastomere nuclei had 40 or more microns in diameter after fixation and FISH. Since blastomeres were fixed individually, results with blastomeres were invariably better than with lymphocytes.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.