Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22).

In this study we describe the pre-clinical development and clinical application of preimplantation genetic diagnosis (PGD) by fluorescence in-situ hybridization (FISH) for two non-related carriers (one male and one female) of the most common balanced reciprocal translocation: t(11;22)(q25;q12). For the couple with the female carrier, enumeration of the sex chromosomes in the embryos was also indicated (husband: 47,XXY karyotype). Four-colour FISH analysis was performed on six blastomeres from three embryos. No embryo transfer was possible because all the embryos were unbalanced. Three PGD cycles, with two-colour FISH, were carried out for the couple with the male translocation carrier. A total of 35 embryos were biopsied and diagnosed by FISH; nine out of the 35 embryos (25. 7%) were normal and seven of them were transferred (two embryos from the first and four from the third cycle), six out of 35 embryos (17%) were unbalanced, three out of 35 embryos (5.7%) were triploid or polyploid, 10 out of 35 embryos (28.6%) were mosaic and seven out of 35 embryos (20%) were chaotic. Diagnosis failed in 2.9% of the embryos. The spermatozoa of the male carrier were also analysed using three-colour FISH. Only 29.1% of the sperm cells seemed to be balanced or normal. By choosing probes lying on both sides of the breakpoints and by using a combination of sub-telomeric or locus-specific probes and centromeric probes, the use of three-colour FISH enabled detection of all the imbalances in sperm and/or cleavage-stage embryos in the patients. This may improve risk assessment and genetic counselling in the future for translocation carriers.     

PGD for a complex chromosomal rearrangement by array comparative genomic hybridization

Patients carrying a chromosomal rearrangement (CR) have an increased risk for chromosomally unbalanced conceptions. Preimplantation genetic diagnosis (PGD) may avoid the transfer of embryos carrying unbalanced rearrangements, therefore increasing the chance of pregnancy. Only 7-12 loci can be screened by fluorescence in situ hybridization whereas microarray technology can detect genome-wide imbalances at the single cell level. We performed PGD for a CR carrier with karyotype 46,XY,ins(3;2)(p23;q23q14.2),t(6;14)(p12.2;q13) using array comparative genomic hybridization. Selection of embryos for transfer was only based on copy number status of the chromosomes involved in both rearrangements. In two ICSI-PGD cycles, nine and seven embryos were analysed by array, leaving three and one embryo(s) suitable for transfer, respectively. The sensitivity and specificity of single cell arrays was 100 and 88.8%, respectively. In both cycles a single embryo was transferred, resulting in pregnancy following the second cycle. The embryo giving rise to the pregnancy was normal/balanced for the insertion and translocation but carried a trisomy 8 and nullisomy 9 in one of the two biopsied blastomeres. After 7 weeks of pregnancy the couple miscarried. Genetic analysis following hystero-embryoscopy showed a diploid (90%)/tetraploid (10%) mosaic chorion, while the gestational sac was empty. No chromosome 8 aneuploidy was detected in the chorion, while 8% of the cells carried a monosomy for chromosome 9. In summary, we demonstrate the feasibility and determine the accuracy of single cell array technology to test against transmission of the unbalanced meiotic products that can derive from CRs. Our findings also demonstrate that the genomic constitution of extra-embryonic tissue cannot necessarily be predicted from the copy number status of a single blastomere.     

一例罗伯逊易位携带者的胚胎植入前遗传学诊断

目的　探讨植入前遗传学诊断 (preimplantation genetic diagnosis,PGD)用于筛选罗伯逊易位携带者无遗传缺陷后代的可行性及风险。方法　 1对因男方携带易位 (13;14 )染色体并伴少、弱精的原发不孕夫妇 ,经激素超促排卵和单精子卵胞浆内注射 (intracytoplasmic sperm injection,ICSI)进行体外受精(in vitro fertilization,IVF) ,当胚胎发育到 6～ 8细胞阶段 (受精后第 3天 )时 ,用酸化法活检 ,从每个胚胎中取出单个分裂球 ,用 L SI 13q和 Tel 14 q探针进行荧光原位杂交 (fluorescence in situ hybridization,FISH)检测 ,继续培养活检后的胚胎到第 2天 ,并选择正常胚胎移植 ,获临床妊娠后 ,于妊娠中期行羊水细胞染色体检查。结果　活检 10个胚胎 ,获得 8个 FISH诊断结果 :5 0 % (4/8)正常或平衡的胚胎 ,37.5 % (3/8)不平衡的胚胎 ,12 .5 % (1/8)不确定。将诊断正常或平衡的胚胎 3枚于活检第 2天移植入母体宫腔 ,获临床单胎妊娠 ,产前诊断证实胎儿核型为 4 6 ,XY,完全正常 ,现分娩一正常男婴。结论　需行辅助生殖技术治疗的患者 ,当携带有罗伯逊易位时 ,PGD用于筛除异常胚胎 ,解决患者的生育障碍、预防严重遗传病胎儿的产生具有重要价值。     

全染色体涂抹探针在女性罗伯逊易位携带者植入前遗传学诊断中的临床应用

目的应用全染色体涂抹探针（whole chromosome painting probe，WCP）对女性罗伯逊易位携带者进行卵母细胞第一极体的植入前遗传学诊断（preimplantation genetic diagnosis，PGD）。方法应用全染色体涂抹探针进行第一极体荧光原位杂交，对4例女方罗伯逊易位携带者进行了4个周期的PGD。患者染色体核型均为45，XX，der（13；14），（q10；q10）。所有周期取卵后6h内通过活检取出第一极体，采用WCP探针进行荧光原位杂交，受精后第3天选择染色体组成正常或平衡的胚胎进行宫腔内移植。结果4个周期共获卵61个，其中54个成熟可进行活检，活检成功率92．6％（50／54），固定成功率90．O％（45／50）。40个获得明确诊断，总体诊断率为74．1％（40／54）。卵胞浆内单精子注射后受精率64．8％（35／54），优质胚胎率为65．7％（23／35）。获得2例临床妊娠。其中1例于孕9周胚胎停止发育，绒毛染色体分析核型为45，X；另1例产前诊断证实核型为46，XX。2006年6月足月分娩一正常活女婴。结论全染色体涂抹探针可准确区分正常、平衡以及异常卵子，从而可有效应用于女性染色体易位携带者的PGD。     

应用全基因组扩增技术对β-地中海贫血进行胚胎植入前遗传学诊断

目的 探讨对β-地中海贫血进行胚胎植入前遗传学诊断的方法。方法 夫妇双方分别为β41-42(-TCTT)及IVS-I 654(C→T)突变杂合子，在本中心进行体外受精-胚胎移植和胚胎植入前遗传学诊断。结果 13个胚胎中共有11个胚胎经PCR分析后获得明确诊断，正常胚胎2个(18．1％)；杂合子胚胎6个(54．5％)；双重杂合子胚胎3个(27．3％)。共移植3个胚胎，其中2个正常胚胎、1个杂合子胚胎。在胚胎移植后5周B超示三胎妊娠，孕8周自然减一胎，并于孕20周时经产前诊断，证实均为健康胎儿。现已分娩双胎分别为正常和杂合子。结论 成功应用全基因组扩增技术对β-地中海贫血进行胚胎植入前遗传学诊断，并分娩健康双胎。     

Preimplantation genetic diagnosis for an insertional translocation carrier

BACKGROUND: While preimplantation genetic diagnosis (PGD) is well established for carriers of reciprocal terminal translocations, reports on PGD for insertional translocation carriers are lacking. Here, we report on the PGD of an insertional translocation carrier with karyotype 46,XX,ins(14;2)(q21;q31q35). Due to the possibility of crossovers within the inserted region, rather than a single probe, four probes are required for proper embryo selection. METHODS: Probes were generated for PGD using fluorescence in situ hybridization and two PGD cycles. RESULTS: Analysis of 10 embryos revealed four embryos to be normal diploid. Two embryos were consistent with 3:1 segregation of the theoretical quadrivalent and one was consistent with 2:2 or 1:1 segregation. Furthermore, one embryo was mosaic abnormal and one remained without diagnosis. CONCLUSIONS: With increased acceptance of PGD, it is likely that more carriers of complex translocations will enter PGD programmes. The present results suggest that a careful genetic work-up of complex translocations is essential for proper embryo selection. While theoretical modelling may predict that quadrivalents will form during the meiosis of insertional translocations, experimental proof for the occurrence of quadrivalents is still lacking and more research on the meiotic process of both female and male insertional translocation carriers is warranted.     

Preimplantation genetic diagnosis for a known cryptic translocation: follow-up clinical report and implication of segregation products

This report describes preimplantation genetic diagnosis (PGD) of a couple with a known paternally-derived balanced cryptic translocation 46,XY.ish t(2q;17q)(210E14-,B37c1+;B37c1-,210E14+) in embryos from a couple who previously had a child with severe mental retardation and was previously described in this journal [Bacino et al., 2000]. This child inherited the unbalanced product of translocation from her father: 46,XX.ish der(2)t(2q;17q)pat(210E14-,B37c1+). The couple desired a normal offspring and sought PGD to avoid clinical pregnancy termination. They were treated three times with in vitro fertilization followed by PGD. Two sequential FISH hybridizations were performed. In the first hybridization, telomeric probes to 2q and 17q and a chromosome 17 centromere probe were employed. The second hybridization screened for maternal age-related aneuploidy (X,Y,13,18,21). Of the 18 informative embryos, only 4 (22%) were normal. The remaining 12 (67%) were abnormal; most with unbalanced products (10/12) from the paternally-derived rearrangement. The most frequent mode of segregation observed for this cryptic translocation was adjacent-1 (7/18, 39%). This suggests cryptic translocations are amenable to PGD and, as are traditional translocations, demonstrate higher frequencies of unbalanced segregants than the empiric risk of 10-15% observed at amniocentesis or chorionic villus sampling. Thus, cryptic translocations presumably behave like overt translocations, in that PGD must be performed on a relatively large number of embryos to assure even 2-3 transferable embryos.     

Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations

We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.     

Cryptic familial t(11;18)(q25;q23) incidentally detected by interphase FISH

During a prospective prenatal study of numerical abnormalities of chromosomes 13, 18, 21, X and Y using locus-specific probes, we incidentally found a case with only one signal for chromosome 18 per cell in a chorionic villus sampling (CVS) associated with an otherwise apparently normal G-banded karyotype. This led us to discover a cryptic t(11;18) segregating in a four-generation family. The CVS was performed because of mental retardation in the brother to the father of the fetus. A subtelomeric chromosome 18 probe revealed one signal on 18qter and one on 11qter of the father. Thus the father had a balanced reciprocal t(11;18) in spite of the apparently normal G-banded karyotype. Using the same probes, we found an unbalanced translocation 46,XX,-18,+der (18)t(11;18)-(q25;q23)pat in the fetus. Further investigation of the family showed the translocation in balanced and unbalanced form in four generations in mentally normal and retarded individuals, respectively. The study emphasizes the need for a follow-up with molecular cytogenetic techniques in dysmorphic and retarded children.     

Analysis of a familial three way translocation involving chromosomes 3q, 6q, and 15q by high resolution banding and fluorescent in situ hybridisation (FISH) shows two different unbalanced karyotypes in sibs.

We report on a familial three way translocation involving chromosomes 3, 6, and 15 identified by prometaphase banding and fluorescence in situ hybridisation (FISH). Two mentally retarded sibs with different phenotypic abnormalities, their phenotypically normal sister and mother, and two fetuses of the phenotypically normal sister were analysed. The terminal regions of chromosomes 3q, 6q, and 15q were involved in a reciprocal translocation, in addition to a paracentric inversion of the derivative chromosome 15. Conventional cytogenetic studies with high resolution GTG banding did not resolve this rearrangement. FISH using whole chromosome paints (WCPs) identified the chromosomal regions involved, except the aberrant region of 3q, which was undetectable with these probes. Investigation of this region with the subtelomeric FISH probe D3S1445/D3S1446 showed a balanced karyotype, 46,XX,t(3;15;6) (q29;q26.1;q26), inv der(15) (q15.1q26.1) in two adult females and one fetus. It was unbalanced in two sibs, showing two different types of unbalanced translocation resulting in partial trisomy 3q in combination with partial monosomy 6q in one patient and partial trisomy 15q with partial monosomy 6q in the other patient and one fetus. These represent apparently new chromosomal phenotypes.     

Aneuploidy 12 in a Robertsonian (13;14) carrier: Case report

In translocation carriers, the presence of aneuploidy for the chromosomes unrelated to the rearrangement may lead to an additional risk of abnormal pregnancy or implantation failure. Consequently, it may be important to analyse not only the chromosomes involved in the rearrangement but also the rest of chromosomes. We combined spectral karyotyping (SKY) and comparative genomic hybridization (CGH) to karyotype one unfertilized oocyte and its first polar body (1PB) from a Robertsonian translocation carrier t(13;14) aged 29 years who was undergoing IVF and preimplantation genetic diagnosis (PGD) for translocations and aneuploidy screening. Two out of four embryos were aneuploid, as a result of an adjacent segregation. The unfertilized oocyte had a normal/ balanced constitution of the chromosomes involved in the reorganization. However, this 1PB-metaphase II doublet was aneuploid for chromosome 12, the oocyte being hyperhaploid (24, X, +12) and its 1PB hypohaploid (22, X, -12). The application of CGH for the study of Robertsonian translocations of maternal origin will be useful to study imbalances of the chromosomes involved in the rearrangement, as well as alterations in the copy number of any other chromosome. The combination of PGD for translocations with aneuploidy screening could help to reduce the replacement of chromosomally abnormal embryos.     

Positive outcome after preimplantation diagnosis of aneuploidy in human embryos.

usromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization (IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation; (ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.     

Identification of an unbalanced cryptic translocation t(9;17)(q34.3;p13.3) in a child with dysmorphic features

We report a case of an unbalanced cryptic telomeric translocation 46,XY,der(17),t(9;17)(q34.3;p13.3) in a boy with dysmorphic features and developmental delay. The proband had intrauterine growth retardation, postnatal short stature, and mild microcephaly. Magnetic resonance imaging showed incomplete myelination, but no evidence of lissencephaly. Cytogenetic analysis of the proband's peripheral blood showed an abnormal 17p. Fluorescence in situ hybridisation (FISH) with a Miller-Dieker cosmid probe did not detect a deletion for that area. Further analysis with a 17p telomere specific probe identified an unbalanced telomeric translocation. The same probe was used to determine the presence of an apparent balanced translocation t(9;17)(q34.3;p13.3) in the mother of the proband. The balanced translocation was confirmed with two cosmids that map distally on 9q34.3. Two phenotypically normal half sibs, a maternal aunt, a maternal uncle, and the maternal grandmother were found to be balanced translocation carriers as well. A subtle translocation carriers as well. A subtle translocation is one mechanism that can produce an abnormal phenotype in a patient who had a normal karyotype at lower band resolution levels.     

Robertsonian translocations--reproductive risks and indications for preimplantation genetic diagnosis.

BACKGROUND: Robertsonian translocations carry reproductive risks that are dependent on the chromosomes involved and the sex of the carrier. We describe five couples that presented for preimplantation genetic diagnosis (PGD). METHODS: PGD was carried out using cleavage-stage (day 3) embryo biopsy, fluorescence in-situ hybridization (FISH) with locus-specific probes, and day 4 embryo transfer. RESULTS: Couple A (45,XX,der(14;21)(q10;q10)) had two previous pregnancies, one with translocation trisomy 21. A successful singleton pregnancy followed two cycles of PGD. Couple B (45,XX,der(13;14)(q10;q10)) had four miscarriages, two with translocation trisomy 14. One cycle of PGD resulted in triplets. Couple C (45,XX,der(13;14)(q10;q10)) had four years of infertility; two cycles were unsuccessful. Couple D (45,XY,der(13;14)(q10;q10)) presented with oligozoospermia. A singleton pregnancy followed two cycles of PGD. Couple E (45,XY,der(13;14)(q10;q10)) had a sperm count within the normal range and low levels of aneuploid spermatozoa. PGD was therefore not recommended. No evidence for a high incidence of embryos with chaotic or mosaic chromosome complements was found. CONCLUSIONS: For fertile couples, careful risk assessment and genetic counselling should precede consideration for PGD. Where translocation couples need assisted conception for subfertility, PGD is a valuable screen for imbalance, even when the risk of viable chromosome abnormality is low.     

Preimplantation genetic diagnosis for complex chromosome rearrangements

Complex chromosome rearrangements (CCR) are structural rearrangements that involve more than two breakpoints. The most common ones involve three chromosomes and three breakpoints, but double translocations can also occur. Theoretically, the potential for chromosome imbalance in the gametes of CCR carriers is higher than for simple translocations, and thus they are at an even higher risk of recurrent miscarriage. Preimplantation genetic diagnosis (PGD) has been shown to significantly reduce the risk of repeated pregnancy loss in translocation carriers, and thus it is well indicated for CCR. Here we describe different approaches for PGD of CCRs. After PGD of five couples carriers of CCR, only 6.4% of embryos were found normal or balanced, but the transfer of these six embryos into four couples, resulted in two pregnancies, one with normal non-identical twins, and the other with a balanced karyotype. Thus the implantation rate was 50%, and the pregnancy per retrieval was 40%, with 0% spontaneous abortions and 0% unbalanced offspring. This compares favorably with a prior history of spontaneous abortions, unbalanced offspring, and low fertility.     

Down syndrome: characterisation of a case with partial trisomy of chromosome 21 owing to a paternal balanced translocation (15;21) (q26;q22.1) by FISH.

A patient with a typical Down syndrome (DS) phenotype and a normal karyotype was studied by FISH. Using painting probes, we found that the patient had partial trisomy of chromosome 21 owing to an unbalanced translocation t(15;21) (q26; q22.1) of paternal origin. To correlate genotype with phenotype as accurately as possible, we localised the breakpoint using a contig of YACs from the long arm of chromosome 21 as probes and performed FISH. We ended up with two YACs, the most telomeric giving signal on the der (15) in addition to signal on the normal chromosome 21 and the most centromeric giving signal only on both normal chromosomes 21. From these results we could conclude that the breakpoint must be located within the region encompassing YACs 280B1 and 814C1, most likely near one end of either YAC or between them, since neither YAC814C1 nor 280B1 crossed the breakpoint (most likely between marker D21S304 and marker D21S302) onband 21q22.1. The same study was performed on the chromosomes of the father and of a sister and a brother of the patient; all three carried a balanced translocation between chromosomes 15 and 21 and had a normal phenotype. We also performed a prenatal study using FISH for the sister. The fetus was also a carrier of the balanced translocation.     

Partial trisomy 3q syndrome inherited from familial t(3;9)(q26.1; p23)

A five-year-old girl was referred to prometaphase chromosome analysis because of mental retardation, facial dysmorphic features suggestive of Cornelia de Lange syndrome, cleft palate and additional minor congenital malformations of the cardiac system and fingers and toes. A familial balanced translocation (3;9)(q26.1; p23) was found. The karyotype of the proposita was 46,XX,der(9),t(3;9)(q26.1;p23). Thus the patient was trisomic for 3q26.1-qter and monosomic for 9p23-pter. The unbalanced chromosome constitution was not detected by standard Q-banding analysis shortly after birth. The karyotype was misdiagnosed as 46,XX,9(p+) in the proposita and her mother, and thought to be a normal variant of chromosome 9. The repeated cytogenetic study led to the diagnosis of the translocation and to the possibility of prenatal diagnosis in the translocation carriers. A survey of 22 published cases of dup(3q) showed that nearly 60% were secondary to familial balanced rearrangements with an excess of maternally derived abnormal chromosomes 3. Red blood cell galactose-1-phosphate-uridyltransferase (GALT) activity was normal in the patient, consistent with previous assignment of the gene locus for GALT to 9p13 (Shih et al. 1982).     

Molecular and clinical characterization of a recurrent cryptic unbalanced t(4q;18q) resulting in an 18q deletion and 4q duplication

Recurrent constitutional non-Robertsonian translocations are very rare. We present the third instance of cryptic, unbalanced translocation between 4q and 18q. This individual had an apparently normal karyotype; however, after subtelomere fluorescence in situ hybridization (FISH), he was found to have a cryptic unbalanced translocation between 4q and 18q [ish der(18)t(4;18)(q35;q23)(4qtel+,18qtel-)]. Oligonucleotide array comparative genomic hybridization (aCGH) refined the breakpoints in this child and in the previously reported child and indicated that the breakpoints were within 20 kb of each other, suggesting that this translocation is, indeed, recurrent. A comparison of the clinical presentation of these individuals identified features that are characteristic of both 18q- and 4q+ as well as features that are not associated with either condition, such as a prominent metopic ridge, bitemporal narrowing, prominent, and thick eyebrows. Individuals with features suggestive of this 4q;18q translocation but a normal karyotype warrant aCGH or subtelomere studies.     

运用全染色体探针FISH技术进行罗伯逊易位携带者胚胎植入前诊断

目的运用全染色体探针荧光原位杂交技术(fluorescent in situ hybridization ,FISH)对14/21罗伯逊易位携带者的胚胎进行植入前遗传学诊断,达到优生目的.方法应用FITC、Texas标记的21/14全染色体探针对14/21罗伯逊易位携带者的胚胎活检后的单个卵裂球进行遗传学检测,选择正常信号或携带者信号核型胚胎进行移植.结果常规单精子显微注射-胚胎移植(intracytoplasmic sperm injection,ICSI)后获取胚胎12个,活检优质胚胎11个,杂交后有信号胚胎7个,其中1个正常胚胎,1个携带者胚胎;1个14单体胚胎,1个21单体胚胎,1个21三体胚胎,另2个胚胎活检后均为21号二体,无14号信号.结论运用14/21全染色体探针荧光原位杂交技术对14/21罗伯逊易位携带者的胚胎进行植入前遗传学诊断是能够将正常核型胚胎挑选出来进行移植,从而达到优生目的.