Preimplantation genetic diagnosis for couples with a Robertsonian translocation: practical information for genetic counseling

Purpose  

To evaluate the proportions of abnormal and normal embryos detected by preimplantation genetic diagnosis (PGD) of infertile couples of whom one was a Robertsonian translocation (RT) carrier, and to provide practical information, including details of reproductive outcomes, to aid in genetic counseling of such couples.     

Preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) is an exciting new approach for the prevention of transmission of genetic disorders between generations. The use of genetically screened, healthy embryos to establish a pregnancy avoids the need for termination of an affected pregnancy, a procedure which can be traumatic physically and emotionally for potential patients, and is sometimes not available when needed. PGD usually follows the processes of ovarian hyperstimulation and in vitro fertilization (IVF). After carrying out appropriate genetic tests, only normal embryos are transferred to the patient's uterus to achieve an unaffected pregnancy and the birth of a healthy infant.     

Preimplantation genetic diagnosis: an update on current technologies and ethical considerations

The aim of reproductive medicine is to support the birth of healthy children. Advances in assisted reproductive technologies and genetic analysis have led to the introduction of preimplantation genetic diagnosis (PGD) for embryos. Indications for PGD have been a major topic in the fields of ethics and law. Concerns vary by nation, religion, population, and segment, and the continued rapid development of new technologies. In contrast to the ethical augment, technology has been developing at an excessively rapid speed. The most significant recent technological development provides the ability to perform whole genome amplification and sequencing of single embryonic cells by microarray or next‐generation sequencing methods. As new affordable technologies are introduced, patients are presented with a growing variety of PGD options. Simultaneously, the ethical guidelines for the indications for testing and handling of genetic information must also rapidly correspond to the changes.     

植入前遗传学诊断四例临床分析

目的 探讨对遗传病高危夫妇采用单细胞荧光原位杂交（FISH）进行胚胎植入前遗传学诊断（PGD）的临床价值。方法 对曾生育过遗传病患儿的4对夫妇通过超排卵获得卵子,体外受精,体外培养至6-8细胞胚胎,每个胚胎取1-2个细胞,采用FISH进行遗传学分析。筛选无遗传病发病风险的胚胎移植入子宫。结果 4例患者共进行4个治疗周期,获得可供活检的胚胎12个,活检细胞20个,固定后有核细胞17个,FISH后除2个细胞无杂交信号外,其余杂交信号清楚,结果明确,活检后的12个胚胎继续发育,结合遗传学诊断,8个胚胎可供移植,其中1例妊娠,于2001年9月14日足月剖宫产分娩一女婴,发育正常,体重4270g,出生后染色体检查为正常女性核型。结论 对遗传病高危夫妇采用FISH技术进行PGD具有临床应用价值。     

Preimplantation genetic diagnosis and the welfare of the child

Preimplantation genetic diagnosis is a form of very early prenatal diagnosis. The technique combines assisted reproductive technology with molecular genetics and cytogenetics to allow the identification of abnormalities in embryos prior to implantation. Since its introduction in 1990 this approach has been applied to an increasing number of single gene disorders, chromosomal rearrangements, and more recent indications such as aneuploidy screening and HLA matching. Since its inception the technology has attracted much attention: geneticists have expressed concerns about the robustness and validity of diagnosis based on single cell analysis, perinatologists were anxious about the effect of embryo biopsy on normal fetal development; and philosophers and ethicists have argued the cases for and against embryo selection. This article attempts to highlight the difficult choices and ethical challenges confronting patients and clinicians in an effort to balance the recognition of parental autonomy with the obligation of clinics to consider the welfare of any child born as a result of this treatment.     

染色体易位的植入前遗传学诊断

目的 观察染色体平衡易位和罗伯逊(罗氏)易位基因携带者夫妇进行植入前遗传学诊断(PGD)后的胚胎染色体遗传特征和胚胎着床、妊娠情况,探讨PGD在染色体易位基因携带者夫妇实现正常生育中的意义.方法 用荧光原位杂交(FISH)技术对36对夫妇的胚胎进行PGD,其中14例为染色体平衡易位(平衡易位组),22例为染色体罗氏易位(罗氏易位组),并对诊断结果和胚胎着床、妊娠情况进行分析.结果 36例患者共活检胚胎253个,成功诊断胚胎225个,成功率为88.9%(225/253),获得可供移植的正常或平衡的胚胎共58个.平衡易位组和罗氏易位组PGD后胚胎着床率分别为36%(5/14)和14%(6/44),临床妊娠率分别为4/9和26%(5/19).结论 PGD可有效诊断胚胎染色体平衡易位和罗氏易位,避免反复流产和不必要的非意愿性终止妊娠,并获得理想的胚胎着床率和临床妊娠率.     

Preimplantation genetic diagnosis: present and future

Purpose: Preimplantation genetic diagnosis (PGD) was developed more than a decade ago and aims to identify embryos free of genetic disease attributed either to gene mutations or chromosome errors. The purpose of this article is to provide an update on the current status and future prospects of PGD. Methods: Review of studies employing different strategies for the detection of single gene defects, and chromosome abnormalities, both structural and numerical in the context of PGD. Results: Amplification of several DNA fragments is feasible via multiplex PCR for the PGD of single gene disorders, whilst current FISH protocols employ up to 10 probes to identify embryos with a normal chromosome complement. New methods are being developed which will enable the assessment of the entire chromosome complement of embryonic blastomeres. Conclusions: PGD has come a long way since its first application, and has become very accurate and reliable. Technical advances in the field of preimplantation genetics mean that PGD holds great promise for the future.     

Preimplantation genetic diagnosis (PGD) for SHOX-related haploinsufficiency in conjunction with trisomy 21 detection by molecular analysis

Purpose  

Development of a molecular PGD protocol for a male with an X-linked deletion in the SHOX gene region, located in the pseudoautosomal region of the X/Y chromosomes. Due to excessive recombination in this region, the deletion can be found in male offspring.     

胚胎染色体非整倍体筛查用于平衡易位携带者植入前的遗传学诊断

目的 交信号和1个Y染色体杂交信号者,则诊断为整倍体胚胎;异常杂交信号的胚胎则诊断为非整倍体胚胎.结果 (1)11个平衡易位的PGD周期中,选出杂交信号完整的130个细胞核进行分析,FISH共分析了937个荧光杂交信号,其中整倍体细胞核38个,共有304个杂交信号;其余92个为非整倍体细胞核.(2)在92个非整倍体细胞核中,Ⅰ、Ⅱ及Ⅲ级胚胎的比例分别为20个(22%)、36个(39%)及36个(39%);38个整倍体细胞核中,Ⅰ、Ⅱ及Ⅲ级胚胎的比例分别为13个(34%)、17个(45%)及8个(21%),两者的Ⅰ、Ⅱ及Ⅲ级胚胎数分别比较,差异均无统计学意义(P＞0.05).虽然染色体整倍体率在不同级别胚胎中的分布比较,差异均无统计学意义(P＞0.05),但优质胚胎(Ⅰ级+Ⅱ级)中非整倍体率仍为60%(56/92).(3)平衡胚胎来源的卵裂球细胞核整倍体率(71.4%,30/42)明显高于非平衡胚胎来源的卵裂球细胞核整倍体率(9.1%,8/88),两者比较,差异有统计学意义(P＜0.05).平衡胚胎来源的卵裂球细胞核非整倍体率(包括三体、单体、复杂非整倍体、单倍体、多倍体)明显低于非平衡胚胎来源的卵裂球细胞核非整倍体率(P＜0.05).结论 平衡易位携带者的胚胎中有较高的非整倍体率,因此,胚胎非整倍体筛查在平衡易位携带者的PGD中有重要价值和临床意义.

Abstract：

Objective To determine the importance of aneuploidy screening in preimplantation genetic diagnosis for the couples of chromosome translocation carriers. Methods To perform 11 prenatal genetic disgnosis (PGD) cycles for 7 couples of chromosome translocation carriers from January 2006 to March 2009 in the Reproductive Medical Center, First Affiliated Hospital of Zhengzhou University. To re-analyze the well-fixed, non-multinuclear and non-debris nuclei using the probes of LSI 13, 18, 21,CEPX, CEPY to detect the aneuploidy rate which come from the PGD cycles of the couples of chromosome translocation carriers. The euploid embryo was defined as two fluorescence in situ hybridization (FISH)signals of LSI 13, 18, 21 respectively and two signals of CEPX, or one signal of CEPX and one signal of CEPY. The other abnormal signals were defined as aneuploid embryo. Results (1) A tolal of 130 nuclei from 11 PGD cycles got the integrated re-FISH signals. Nine hundred and thirty-seven FISH signals were analysized, including 304 signals from 38 euploid nuclei and the others from 92 aneuploid nuclei. (2) The number of the aneuploid nuclei from grade Ⅰ , Ⅱ and Ⅲ embryo was 20 (22%), 36(39%), and 36(39%). The number of the euploid nuclei from grade Ⅰ , Ⅱ and Ⅲ embryo was 13(34%), 17(45%),and 8(21%). There was no significant difference of aneupioidy rate between the embryos form different grades (P＞0.05). However, the rate of aneuploid nucleus from good quality embryos (grade Ⅰ + grade Ⅱ) was 60% (59/92). (3) The euploidy rate was 71.4% (30/42) from balanced embryos, while 9.1%(8/88)from unbalanced embryos. There was significant difference between them (x2=53.4, P＜0.05).The rate of aneuploidy from balanced embryos was lower than those from unbalanced embryos (P＜0.05).Conclusions Since higher rate of aneuploidy was detected in embryos of the couples of chromosome translocation carriers. It is advisable to recommend the FISH re-analysis for aneuploidy screening to preimplantation genetic diagnosis for the couples of chromosome translocation carriers.     

Preimplantation genetic diagnosis for the Kell genotype

OBJECTIVE: To use preimplantation genetic diagnosis (PGD) to achieve a Kell 1 (K1) allele-free pregnancy in couples at risk for producing a child with hemolytic disease of the newborn (HDN) caused by maternofetal incompatibility in sensitized mothers. DESIGN: DNA analysis of biopsied blastomeres from cleavage-stage embryos in IVF-ET with the goal of identifying and transferring back to patients the K1 allele-free embryos. SETTING: IVF program at the Reproductive Genetics Institute, Chicago, Illinois, and IVF Michigan, Rochester Hills, Michigan. PATIENT(S): Two at-risk couples with a history of neonatal death caused by HDN due to K1/K2 genotype in a male partner. INTERVENTION(S): Biopsy of single blastomeres and testing for paternal K1 allele in each embryo after standard IVF. MAIN OUTCOME MEASURE(S): DNA analysis of blastomeres indicating whether corresponding embryos were K1 allele-free for the purpose of transferring only embryos without the K1 allele. RESULT(S): Of 36 embryos tested in five cycles from two couples, 18 were predicted to be K1 allele-free. Of these, 9 were transferred, resulting in a K1 allele-free twin pregnancy and the birth of two healthy children. CONCLUSION(S): PGD of the K1 genotype resulted in the birth of healthy twins confirmed to be free of the K1 allele. PGD in couples with a heterozygous K1/K2 male partner provides an option for avoiding HDN in sensitized mothers.     

Substandard application of preimplantation genetic screening may interfere with its clinical success

The intent of this study was to evaluate a recent randomized clinical trial evaluating the effect of preimplantation genetic screening (PGS) that reports a negative effect on pregnancy outcome. This article reviews appropriate PGS techniques and how they differ from the trial in question. A closer look at the clinical trial in question reveals significant lack of expertise in biopsy, cell fixation, genetic analysis, and patient selection. At most, this trial demonstrates that in inexperienced hands, PGS can be detrimental. No other conclusions concerning the effect of PGS on pregnancy results can be drawn from the trial.     

Predictive value of preimplantation genetic diagnosis for aneuploidy screening in repeated IVF-ET cycles among women with recurrent implantation failure

Objective To determine the predictive value of euploid embryos in women with recurrent implantation failure undergoing repeated IVF-ET cycles with PGD (PGD). Design Cohort of IVF-PGD cycles in a tertiary care ART facility. Materials and method(s) Fifty-five consecutive patients with repeated implantation failure (more than three failed IVF-ET cycles) underwent two or more PGD cycles for aneuploidy testing. Mean maternal age was 37.6 ± 5.3 years. Biopsies were performed on day 3. One blastomere was removed from each pre-embryo, fixed and analyzed by multicolor and multi-probe FISH for chromosomes X and Y, 13, 15, 16, 17, 18, 21, and 22. Result(s) Forty-three of 55 patients (78%) undergoing PGD had at least one euploid embryo for transfer. Of these 31 patients (72%) also had at least one euploid embryo available for transfer with the second cycle. Of the 12 (28%) patients with no euploid embryos available for transfer with the second IVF/PGD cycle, five had a third cycle of PGD and two of these had euploid embryos available for transfer. Seventeen of the 31 patients (55%) who had euploid embryos on the second PGD cycle conceived. The ongoing pregnancy and implantation rates in patients with at least one euploid embryo were 40% and 18%, respectively. Twelve of the 55 patients (22%) had no euploid embryos available for transfer on the first PGD cycle, but on the second PGD cycle, six (50%) of these had euploid embryos for transfer. Only two pregnancies were achieved among this group of women, yielding a pregnancy rate of 17%, but both conceptions resulted in miscarriage. Of the six patients with no euploid embryos available after the second PGD cycle, four patients had a third IVF/PGD cycle, but none had euploid embryos available for transfer. Also, among women with euploid embryos available only in either the first or second PGD cycle, but not both, no ongoing pregnancy was achieved. No woman who had a PGD cycle productive of no euploid embryos had an ongoing pregnancy. Significant differences were found in terms of ongoing pregnancy (40%, P < 0.05) and implantation rates (18%, P < 0.05) in women with euploid embryos available for transfer with the first and second IVF/PGD cycles, compared to women with no euploid embryos available for transfer with either the first or second cycle. The positive predictive value of the first euploid cycle predicting a second euploid cycle was 72%, 95% CI 0.66–0.78. The negative predictive value of an aneuploid cycle was 50%, 95% CI 0.27–0.72. The sensitivity and specificity of the first PGD cycle predicting the second was 84%, 95% CI 0.77–0.91 and 33%, 95% CI 0.18–0.48, respectively. Conclusion(s) Even with a history of recurrent implantation failure, the availability of euploid embryos, especially on two, consecutive PGD cycles is associated with high ongoing pregnancy and implantation rates. Conversely, the absence of euploid embryos for transfer predicts poor reproductive outcome, even if subsequent cycles do yield euploid embryos.     

Preimplantation genetic diagnosis: State of the art

Preimplantation genetic diagnosis (PGD) is used to analyze embryos genetically before their transfer into the uterus. It was developed first in England in 1990, as part of progress in reproductive medicine, genetic and molecular biology. PGD offers couples at risk the chance to have an unaffected child, without facing termination of pregnancy. Embryos are obtained by in vitro fertilization with intracytoplasmic sperm injection (ICSI), and are biopsied mostly on day 3; blastocyst biopsy is mentioned as a possible alternative. The genetic analysis is performed on one or two blastomeres, by fluorescent in situ hybridization (FISH) for cytogenetic diagnosis, or polymerase chain reaction (PCR) for molecular diagnosis. Genetic analysis of the first or second polar body can be used to study maternal genetic contribution. Only unaffected embryos are transferred into the uterus. To improve the accuracy of the diagnosis, new technologies are emerging, with comparative genomic hybridization (CGH) and microarrays.     

Preimplantation genetic diagnosis

Since its introduction in 1990, preimplantation genetic diagnosis (PGD) has allowed embryo genetic analysis prior to transfer into the uterus during an in vitro fertilisation (IVF) cycle. The commonest indications are for detection of single-gene, X-linked, structural chromosomal and mitochondrial disorders as well as for Human Leucocyte Antigen matching. Traditional cytogenetic and molecular techniques of fluorescent in situ hybridization and polymerase chain reaction are complemented by newer technologies e.g. array comparative genomic hybridization and whole genome amplification. Following a successful PGD cycle, pregnancy complications are comparable to those in standard IVF/intra-cytoplasmic sperm injection (ICSI) cycles, which are mostly related to preterm delivery due to multiple pregnancies. There are similar rates of congenital malformations, neonatal morbidity and developmental outcomes in children born after PGD compared with those born after IVF/ICSI. Clinical outcome data is collected by the Preimplantation Genetics International Society and the European Society for Human Reproduction and Embryology PGD Consortium.     

Preimplantation genetic diagnosis and 'savior siblings'.

From its emergence, preimplantation genetic diagnosis (PGD) has been opposed by religious, feminist, and disability-rights advocates. PGD has developed, however, to extend beyond genetic diagnosis of embryos to diagnose chromosomal abnormalities. Evidence shows that PGD is safe, children born after in vitro fertilization (IVF) and PGD having no higher rate of birth defects than children of normal pregnancies. Laws may accommodate PGD directly or indirectly, but some prohibit PGD totally or except to identify sex-linked genetic disorders. When children suffer severe genetic disorders and require stem-cell transplantation, compatible donors may be unavailable. Then, IVF and PGD of resulting embryos may identify some whose gestation and birth would produce unaffected newborns, and placental and cord blood from which stem-cells compatible for implantation in sick siblings can be derived. Ethical issues concern conscientious objection to direct participation, discarding of healthy but unsuitable embryos, and valuing savior siblings in themselves, not just as means to others' ends.     

应用荧光原位杂交技术进行植入前胚胎染色体诊断的价值

目的 初步探讨应用荧光原位杂交(FISH)技术进行植入前胚胎染色体诊断的价值。方法 对10对不孕夫妇进行植入前遗传学诊断(PGD)周期的超促排卵和卵母细胞浆内单精子注射,于受精后第3天进行胚胎活检及FISH分析,第4天选择染色体组成正常或平衡的胚胎进行移植。结果 10个PGD周期共获卵158个,对其中54个胚胎进行活检,51个胚胎获得明确诊断,诊断率为94％(51／54)。对染色体组成正常或平衡的24个胚胎进行官腔内移植,共4例获得妊娠,其中3例已足月分娩健康婴儿,1例为异位妊娠。结论 应用FISH技术进行植人前胚胎染色体诊断,是预防流产和染色体异常患儿出生的有效手段。