Preimplantation Genetic Diagnosis For Translocations Research Articles

Preimplantation genetic diagnosis for translocations and interchromosomal effect assessed by array CGH

Preimplantation genetic diagnosis for translocations and interchromosomal effect assessed by array CGH

The origin and significance of additional aneuploidy events in couples undergoing preimplantation genetic diagnosis for translocations by array comparative genomic hybridization

Diagnostic application of array comparative genomic hybridization (aCGH) in preimplantation genetic diagnosis for reciprocal and Robertsonian translocations has revealed 55–65% embryos with additional aneuploidies with or without translocation-related imbalances. The occurrence of these extra abnormalities with the balanced form of the translocation reduces the number of embryos suitable for transfer. Eighty-three embryos were followed up on days 5–7 of development from 23 infertile or sub-fertile carriers for whole chromosome and segmental aneuploidies present in addition to the balanced or unbalanced translocations detected on aCGH diagnosis. Embryos were analysed by fluorescence in-situ hybridization (n = 63) and aCGH (n = 20). Meiotic aneuploidy affected 35% of embryos and 47% had mitotic events; 15% had both types. Meiotic and mitotic events were almost equal (60 versus 64), 97 affected whole chromosomes (58 meiotic, 39 mitotic) and 27 were segmental (two meiotic, 25 mitotic). In 85.5% of embryos with whole chromosome additional aneuploidies, the aneuploidy was present throughout or in more than 50% of cells. All embryos diagnosed as abnormal (translocation balanced or unbalanced) after aCGH diagnosis at cleavage stage would have remained unsuitable for transfer if tested at later stages of development. Additional aneuploidies merit full consideration when considering the choice of embryos to transfer.

Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?

To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities. Longitudinal cohort study. In vitro fertilization unit. Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30). BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs. Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies. DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs. BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing.

PGD for translocations – increased risk for multiple chromosomal abnormalities

To evaluate the frequency of chromosomal abnormalities in embryos of couples with chromosomal translocations. Retrospective cohort study with four couples were submitted to five cycles for carrying out preimplantation genetic diagnosis (PGD). Four couples were submitted to five cycles for carrying out preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridization (FISH) probes for chromosomal translocations. The median maternal age was 35,7 years. In three cases the male was the carrier of chromosomal translocation including the following abnormalities: rob(13,22); t(4 21); t(7,12), and in one case the women was the carrier of t(15,16). On the 3rd day morphologically normal embryos were biopsied for PGD using specific probes for each translocation and a FISH for 5 chromosomes (13, 18, 21, X e Y). Euploid embryos were transferred on day 5. From 37 biopsied embryos, only six were euploid and they were transferred in 4 cycles of three couples. There were two clinical pregnancies and one normal baby was born. No euploid embryos were detected for the couple in which the woman was carrying a chromosomal translocation. Eleven of 37 embryos were balanced but 5/11 presented one or more chromosomal aneuploidies. Among unbalanced embryos, 19/26 also presented one or more aneuploidies. Our results are in accordance with previous descriptions of an increased risk for multiple chromosomal aneuploidies in embryos of couples with chromosomal translocations. A complete chromosomal evaluation with comparative genomic hybridization (CGH) is a better approach for PGD in such cases. Genetic counseling should be offered prior to PGD for couples with chromosomal translocations and the possibility of a small number of euploid embryos for transferring should be previously discussed.

Comparison between microarray comparative genomic hybridization (aCGH) and fluorescence in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) in translocations carriers

FISH has been used for many years in PGD for translocations, but recently other techniques have been introduced. The aim of this study was to compare PGD outcomes of patients who underwent FISH or aCGH for detection of reciprocal or Robertsonian translocations.

O-15 Application of aCGH for pre-implantation genetic diagnosis of translocations and additional unrelated chromosome aneuploidy and identification of factors influencing outcome

O-15 Application of aCGH for pre-implantation genetic diagnosis of translocations and additional unrelated chromosome aneuploidy and identification of factors influencing outcome

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization

To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. Clinical research study. A PGD laboratory and two IVF clinics. Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Clinical analysis of 100 preimplantation genetic diagnosis cycles

To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD) techniques through clinical analysis on PGD cycles. Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. Among 354 embryos biopsied by PGD for translocations, 321 (90.7%) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38.3% (64/167), which was significantly higher than 20.8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82.5% (443/537). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34.4% (77/224), which was significantly higher than 19.6% (19/97) of biopsed embryos with <7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% (57/96), which was significantly higher than 34.2% (77/225) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43.1% (25/58). PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.

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

To determine if preimplantation genetic diagnosis (PGD) for translocation carriers with three or more pregnancy losses reduces loss rates. Retrospective review of data. Preimplantation genetic diagnosis laboratory servicing IVF groups. Patients (n = 192) undergoing PGD for either a reciprocal translocation or Robertsonian translocation who had three or more previous pregnancy losses. Preimplantation genetic diagnosis for translocations. 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. 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. 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.

Ten-year experience with preimplantation genetic diagnosis (PGD) at the New York University School of Medicine Fertility Center

We describe our experience of over 300 cycles of preimplantation genetic diagnosis (PGD) and report clinical pregnancy rates (35%-67%) that support using this technology to screen for genetic disorders and chromosomal abnormalities. In clinical practice for over ten years, PGD offers couples the earliest form of genetic screening and may help improve ongoing pregnancy rates in poor-prognosis patients.

Clinical Application of Preimplantation Genetic Diagnosis for Translocations in Couples with Recurrent Miscarriages

Balanced carriers of chromosome structural abnormalities, although phenotypically normal, may present with reduced fertility, recurrent miscarriages, or offspring with an abnormal phenotype. While balanced translocations are found in 0.35% of neonatal population, they occur in 1.4% of IVF patients with implantation failure and 2% of patients with recurrent miscarriages. At meiosis I, translocated chromosomes are paired with their corresponding normal chromosomes and form a quadrivalent (in the case of reciprocal translocation) or a trivalent (in the case of Robertsonian translocation). The unbalanced gametes are produced through malsegregation of these unique complexes. The occurrence risk of the unbalanced gametes depends on the number and the breakpoints of the chromosomes involved in translocation, and the extent of the genetic imbalance resulting from abnormal segregation could determine the viability of the gamete or the conception. Recently, PGD (preimplantation genetic diagnosis) has been performed worldwide for translocation carriers in order to select embryos with no genetic imbalance and increase their pregnancy and delivery rates. Although there is a possibility of successful natural outcome in many translocation carriers, the introduction of PGD into ART is expected to improve reproductive performance of these couples.

Reply to: Preimplantation genetic diagnosis for translocations

Reply to: Preimplantation genetic diagnosis for translocations

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.

Results of PGD for Translocations in 539 Cycles

To evaluate the results of Preimplantation Genetic Diagnosis (PGD) for translocations. 52 USA-based IVF centers referring PGD samples to a single reference PGD laboratory. Cycles of PGD for translocations from 1995 to February 2005 were included in the study. Samples from 52 IVF centers were received in our PGD laboratories the same or next day after biopsy. All results were provided within 30 hours after sample arrival. FISH methods evolved during this period, and included polar body biopsy using painting probes, breakpoint-spanning probes in interphase blastomeres, and the last and preferred method, the use of telomeric and proximal probes in interphase blastomeres. A total of 539 cycles were performed, with an average maternal age of 34.0 years (compared to 37.5 for PGD of aneuploidy), involving the analysis of 4,597 embryos, of which only 19.0% were normal, and the rest unbalanced. Reciprocal translocations produced 15% normal embryos while Robertsonian translocations produced 28% normal embryos. Of the 539 cycles, 422 received a replacement (78%), with an average of 1.3 normal embryos replaced. 117 cycles had no replacement as all embryos were found to be abnormal. Information regarding pregnancy results was obtained from 462 pick ups, of which 138 became pregnant with the presence of a fetal heart, producing a pregnancy rate of 30% per pick- up, and 40% per replacement. The pregnancy rate, however, varied depending on the IVF center and the etiology of patients visiting each clinic, from a low 0% to 52% per oocyte retrieval. Also it varied depending on the type of translocation, with 25% and 40% pregnancy rate per oocyte retrieval in Reciprocal and Robertsonian translocations, respectively. In total, there were 176 fetal heartbeats. Of those, 1 was ectopic, 15 spontaneously aborted (9%), 7 were elective reduced due to multiple pregnancy, 40 are still ongoing, and 113 delivered . These patients had aborted previously 88% of their pregnancies compared to 9% after PGD (p<0.001). There have not been any misdiagnoses in this group to date. PGD has been shown in the past to reduce spontaneous abortions in patients with translocations. We have confirmed this observation with the largest dataset so far on PGD of translocations. The pregnancy rate obtained is dependent on the type of translocation but also it is proportionally linked to the overall pregnancy rate of each clinic and their experience in performing embryo biopsy, indicating the presence of a learning curve in the procedure. Some European PGD laboratories emphasize biopsy of two cells rather than one to further reduce the error rate, but clearly a very acceptable error rate can be attained with only 1 cell biopsied with the advantage of a reduction personnel time and stress upon the growing embryo.

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.

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. Setting 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. Result(s) 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 × 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.

Nuclear transfer for full karyotyping and preimplantation diagnosis for translocations

Preimplantation genetic diagnosis (PGD) for chromosomal disorders is currently performed by interphase fluorescence in-situ hybridization (FISH) analysis. This technique is known to have limitations for detecting some translocations and complete karyotyping, so visualization of chromosomes in single cells is required to improve the PGD accuracy for chromosomal disorders. To achieve this, single blastomeres were fused with enucleated or intact mouse zygotes, followed by fixing the resulting heterokaryons at the metaphase of the first cleavage division, or treating them with okadaic acid to induce premature chromosome condensation. This method allowed a significant improvement in the accuracy of testing both maternally- and paternally-derived translocations. In all, 437 blastomeres were tested, achieving full karyotyping in as many as 383 (88%), making it possible to pre-select only normal embryos or those with balanced chromosomal complements for transfer. Overall, PGD for translocations was applied in 94 clinical cycles, resulting in 66 transfers and 20 (30.3%) clinical pregnancies, with healthy deliveries of 15 children. Fifty-two of these cycles were performed using a nuclear transfer (conversion) technique, which resulted in 38 transfers of balanced or normal embryos, demonstrating that the technique is accurate and reliable for karyotyping single blastomeres for PGD of translocations.

Preimplantation genetic diagnosis (PGD), a collaborative activity of clinical genetic departments and IVF centres.

Preimplantation genetic diagnosis (PGD) requires the combined efforts of geneticists and workers in the field of reproductive medicine. This was studied on the basis of a questionnaire, sent to 35 members of the PGD Consortium of the European Society of Human Reproduction and Embryology (ESHRE). A reply was obtained from 20 centres. They represent the majority of activities in the field of PGD in the world. It is obvious that many of the activities (in vitro fertilisation, embryo culture and biopsy) take place in IVF units while others (counselling and diagnosis) are the responsibility of genetic diagnostic centres. The distances between both units vary considerably. In all but one centre sex determination is offered. Aneuploidy screening is offered in 13 out of 20 centres. PGD of translocations and other structural chromosome abnormalities is offered in all but one centre. The number of monogenic diseases offered varies considerably. In comparison to prenatal diagnosis PGD is more expensive. The majority of these costs are due to the IVF or ICSI procedure. The charges for PGD vary between about 600 euro and 4000 euro. In 16 out of 20 centres the parents to be must sign an informed consent form.

Preimplantation genetic diagnosis of structural abnormalities

Preimplantation genetic diagnosis (PGD) of translocations can be achieved through a variety of methods. For female carriers, a possibility is by polar body biopsy and analysis of its metaphase chromosomes using painting probes. For male carriers or female carriers with terminal breakpoints, metaphase chromosomes can also be studied by fusing blastomeres to enucleated oocytes. Otherwise, interphase analysis of the translocation can be performed using distal, subtelomeric or breakpoint spanning probes. The results obtained after PGD of translocations indicate a significant decrease in spontaneous abortions after the procedure, a good selection against unbalanced oocytes and embryos, and pregnancy rates that depend on the type of translocation involved. Balanced translocations occur in 0.2% of the neonatal population, but at a higher rate among infertile couples and patients with recurrent abortions. In a recent report, balanced translocations were found in 0.6% of infertile couples, 3.2% of couples that failed over ten IVF cycles, and 9.2% among fertile couples experiencing three or more consecutive first-trimester abortions (Hum. Reprod. 14 (1999) 2097). They were also found in 2–3.2% of males requiring ICSI (Hum. Reprod. 11 (1996) 2609; Hum. Reprod. 13 (1998) 576). PGD can be offered to carriers of balanced translocations as an alternative to prenatal diagnosis and pregnancy termination of unbalanced fetuses. In recent years, PGD for structural chromosome abnormalities has been attempted by a variety of approaches. The aims of PGD for translocations are to reduce the rate of spontaneous abortions that this population suffers and to minimize the risk of conceiving an unbalanced baby.

Effect of chromosomal translocations on the development of preimplantation human embryos in vitro

Objective: To determine the reliability of a new technique for single human blastomere karyotyping during clinical cases for preimplantation genetic diagnosis of translocations. Design: Controlled clinical study. Setting: Preimplantation genetic diagnosis and IVF program Patient(s): Nineteen preimplantation genetic diagnosis cases with 11 types of translocations (10 reciprocal and one Robertsonian) involving chromosomes 1, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 18, 20, 21, and 22. Intervention(s): Blastomere biopsy followed by blastomere nucleus conversion into metaphase chromosomes. Fluorescent in situ hybridization (whole chromosome painting) was used for the detection of chromosomally unbalanced preimplantation human embryos. Main Outcome Measure(s): Percentage of informative metaphase plates and effect of unbalanced translocations on preimplantation embryo development. Result(s): Informative metaphases were obtained for 84% of the blastomeres. Analysis of preimplantation development of the resulting embryos showed that an unbalanced chromosomal complement does not affect embryo ability to reach the blastocyst stage in vitro. Conclusion(s): For the translocations tested, there is no evident selection against chromosomally unbalanced embryos at the preimplantation stage of embryo development.