Preimplantation diagnosis: Preimplantation diagnosis or chorionic villus biopsy? Women's attitudes and preferences

The objective of this work was to assess women's attitudes andpreferences to two methods of prenatal diagnosis for geneticdisease: embryo and chorionic villus biopsy (CVS). The designinvolved a structured postal questionnaire sent to women inthe Grampian region with different reproductive experiences.The population sample included 46 women who had had geneticcounselling because of a family history of a single gene disorder,18 women who had had CVS for a single gene disorder, 158 womenwho had had CVS for other reasons, 200 women who had recentlydelivered a normal baby and 50 women who had experience of in-vitrofertilization (IVF). The main outcome measures were attitudesto family limitation, prenatal diagnosis, termination for fetalabnormality, embryo biopsy and CVS. Of the women surveyed, 38%preferred embryo diagnosis, whereas 42% favoured CVS and termination.Women with previous experience of CVS preferred this optionwhereas those with experience of IVF as infertility treatmentwere more likely to favour embryo diagnosis, as were women whohad had genetic counselling for a single gene disorder. It wasconcluded that a substantial number of women find embryo diagnosismore acceptable than CVS when the pregnancy is at high risk.This is especially true amongst those with experience of IVFor who are at risk themselves. A demand for embryo diagnosishas been demonstrated.     

Preimplantation genetic diagnosis for achondroplasia: genetics and gynaecological limits and difficulties

BACKGROUND: We report the first attempts at preimplantation genetic diagnosis (PGD) and IVF and their accompanying difficulties for achondroplasia (ACH) patients. METHODS: A PGD test was developed using fluorescent single cell PCR on lymphoblasts from patients and controls and from blastomeres from surplus IVF embryos. A specific digestion control based on the use of two fluorochromes was elaborated. Ovarian stimulation and oocyte retrieval were carried out using conventional protocols. RESULTS: We performed 88 single cell tests for which amplification was obtained in 86 (97.7%) single lymphoblasts. Allele drop out (ADO) was observed in two out of 53 (3.7%) heterozygous lymphoblasts. If we combine the results from the blastomere testing from surplus embryos with those from PGD cycles and re-analysis after PGD, we obtained a PCR signal in 84% of cases of which 91% were correctly diagnosed at the G380 locus. A total of six cycles were performed resulting in three embryo transfers. We observed difficulties in ovarian stimulation and oocyte retrieval with affected female patients. No pregnancy was obtained. CONCLUSION: A PGD test for ACH is now available at our centre but our initial practice raises questions on the feasibility of such a test, specially with affected female patients.     

Preimplantation diagnosis: Blood analysis of mice born following single-cell embryo biopsy

Single-cell embryo biopsy is an important technique in preimplantationdiagnosis. The development of the mouse embryo and fetus, andthe results of some analyses after birth following embryo biopsy,have been demonstrated to be normal. Histopathological analysisof mice born following single-cell embryo biopsy with a physicalmethod (zona puncture) also showed normal organ and cell structure,thus demonstrating the safety of embryo biopsy. This experimentanalysed the parameters of blood cells and the blood chemistryof 28 mice born following single-cell embryo biopsy. White cellcount, red cell count, haemoglobin level and platelet countof the blood, and plasma sodium, potassium, chloride, bicarbonate,calcium, glucose, albumin, protein, creatinine, urea, bilirubin,aspartate transaminase, creatine kinase and lactate dehydrogenaseof the blood were not significantly different between the micefrom the biopsied and the control embryo groups, which againdemonstrated the safety of single-cell embryo biopsy. The remainingtotipotent cells in the biopsied embryos would thus be expectedto develop to the correct cell counts and normal organic functionsaccording to the intact hereditary messages. Further studieson the safety of embryo biopsy (including long-term observationafter birth) and the improvement of the different biopsy techniquesand skills for preimplantation diagnosis are necessary.     

Preimplantation diagnosis: Histopathological analysis of mice born following single cell embryo biopsy

Single cell embryo biopsy is a useful but invasive techniquefor preimplantation diagnosis. Biopsy may be performed by physical(direct zona puncture) or chemical methods (zona drilling withacid solution). This study has analysed the safety of a physicalmethod of embryo biopsy in the mouse. Six adult mice (male andfemale), three from biopsied embryos and three from a controlgroup (non-biopsied) were subjected to histopathological analyses.Macroscopically, the anatomy and morphology of the internalorgans in both groups were normal. Microscopic analyses of 15major organs, which included the brain, heart, lung, liver,kidney, stomach, intestine, voluntary muscle, spleen, pancreas,adrenal, thymus, skin, testis (male) and ovary (female), inboth groups were all normal. These results showed that carefulsingle cell embryo biopsy by direct zona puncture performedat the 8-cell embryo stage had no adverse influence on the macroscopicand microscopic structure of the organs. The remaining pluripotentialcells of biopsied embryos developed normal microstructure accordingto the hereditary messages. Ideally, the safety of embryo biopsyrequires observation of three stages after embryo biopsy, namelyembryonic and fetal development before birth, neonatal assessmentand long-term monitoring after birth.     

Abnormal Development at Early Postimplantation Stage in Mouse Embryos After Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD) is an established procedure for the genetic analysis of embryos. To assess the effect of the procedure on early embryonic development, we generated a murine experimental system, including mice implanted with biopsied in vitro cultured embryos, control mice implanted with in vitro cultured embryos without biopsy, and mice with naturally conceived embryos. Embryos at the 7.5‐dpc stage were isolated from all three groups and the embryo implantation rate, the survival rate of implanted embryos, and the developmental stage of surviving embryos were carefully assessed and compared among all three groups. We found the implantation rate was similar between biopsied and control group embryos (67.92% vs. 66.67%). However, the survival rate of implanted embryos in the biopsied group (49.31%) was significantly lower than that of the control (60.91%) and normal groups (96.24%) at 7.5 dpc. In addition, the survival rate of control group embryos was significant lower than that of normal group embryos. Classification of the precise developmental stages of randomly selected live implanted embryos at 7.5 dpc revealed no differences among the three groups. Our results indicate that blastomere biopsy does not adversely affect embryo implantation. The PGD procedure, in particular blastomere biopsy, increases the rate of embryo death at 4.5–7.5 dpc, but does not affect the development of surviving 7.5 dpc embryos. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Preimplantation diagnosis of aneuploidy using fluorescent in-situ hybridization: evaluation using a chromosome 18-specific probe

Fluorescent detection of in-situ hybridization (FISH) with achromosome 18-specific probe (P5041 B.5 D18Z1) has been usedto assess the use of this method for preimplantation diagnosisof aneuploidy. Interphase nuclei (n = 802) have been analysedfrom 59 normally fertilized embryos developing in vitro at thenormal rate between days 2 and 7 post-insemination. The efficiencyof hybridization in control cells, as assessed by the proportionwith two signals in normal female lymphocytes was 88.9% (n =353) and with three signals in a trisomic (48, XXX+18) fibroblastcell line 74.0% (n = 290). Fifty-four of the human embryos wereconsidered to be diploid on the basis that the majority of nucleihad two signals. Some nuclei in these embryos had one or nosignal, especially on day 2, and tetraploid nuclei were alsowidespread. Among the remaining five embryos, one 5-cell embryoon day 2 had three hybridization signals in 4/5 nuclei and wastrisomic for chromosome 18, one 4-cell embryo on day 2 had onlyone signal in 4/4 nuclei and was monosomic, and the three otherembryos were aneuploid mosaics and/or had multi-nucleated blastomeres.Analysis of the incidence of interphase nuclei with more orless than the diploid number of hybridization signals indicatesthat more than a single nucleus will be necessary for accuratepreimplantation diagnosis of aneuploidy.     

Preimplantation genetic diagnosis for myotonic dystrophy type 1: upon request to child

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for patients at risk of transmitting an inherited disease such as myotonic dystrophy type 1(DM1) to their offspring. In this paper, the clinical application of preimplantation diagnosis for DM1 upon request to children born is described in a large cohort of risk couples. PGD could be offered to all 78 couples opting for PGD regardless of the triplet repeat size. The incidence of major complications was minimalised following a careful assessment in affected DM1 females anticipating possible cardiological, obstetrical and anaesthetical problems. A live-birth delivery rate per cycle with oocyte retrieval of 20% was the outcome. Forty-eight of the 49 children born are in good health and have normal psychomotor development.     

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 in an HIV-serodiscordant couple carrier for sickle cell disease: lessons from a case report

Since 1999, the Erasme Hospital Fertility Clinic has carried a special programme for patients with HIV seropositivity. The philosophy of the programme is to give access to these patients in a secure environment to the same technological facilities available to any other patients. Many of these patients being native from sub-Saharan countries, they are often sickle cell disease (SCD) carriers, a common autosomal recessive disorder in these regions, and a severe affection in homozygotes. We hereby report, for the first time, the birth of a healthy sickle haemoglobin (HbS) heterozygous baby after preimplantation genetic diagnosis (PGD) for SCD in an HIV-serodiscordant couple of HbS mutation carriers with longstanding infertility. The prospective mother was 35 years old and HIV positive with an undetectable viral load under highly active antiretroviral therapy. One carrier embryo was transferred and resulted in the birth of a healthy HbS carrier baby girl. Despite stimulation difficulties, sometimes described in HIV patients, PGD represents an interesting additional technology, especially in populations where the coexistence of both diseases is frequent. PGD could even be preferred to prenatal diagnosis for couples of HbS carriers if the woman is HIV positive, as invasive prenatal samplings carry a risk of materno-foetal viral transmission.     

植入前遗传学诊断的新进展

植入前遗传学诊断作为产前诊断的一种形式，可在胎种植前进行诊断，从而防止遗传病的发生。其技术主要包括胚胎活检、聚合酶链反应及荧光原位杂交。现就这些技术在植入前遗传学诊断中的应用、存在的问题、解决及改进的方法进行综述。     

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease type 1A

Charcot-Marie-Tooth (CMT) disease is the 'common' name for a range of hereditary peripheral neuropathies. CMT1 is the most common form and is transmitted in an autosomal dominant manner. CMT1A maps to chromosome 17p11.2 and is caused, in the majority of cases, by a 1.5 Mb DNA duplication, that includes the peripheral myelin protein 22 (PMP) gene. This paper reports on preimplantation genetic diagnosis (PGD) for CMT1A in five couples. The CMT1A duplication was detected by fluorescent PCR analysis using polymorphic (CA)n markers localized within the duplication. Single-cell PCR on blastomeres allowed genetic analysis of embryos obtained after ICSI. Only healthy unaffected embryos were transferred to the uterus. PCR experiments with single EBV-transformed lymphoblasts or with research blastomeres allowed the evaluation of amplification efficiencies, as well as contamination and allele drop-out (ADO) rates for each PCR protocol. Three simplex PCR protocols (using one primer pair) and two duplex PCR protocols (using two primer pairs) were developed for CMT1A. Additionally, a protocol using all three primer pairs in triplex was also established. Thirteen clinical ICSI-PGD cycles were performed for five couples (12 simplex PCR cycles and one duplex PCR cycle), resulting in seven embryo transfers. Three singleton pregnancies ensued in two couples and three healthy babies were delivered. This report describes different fluorescent PCR-based tests which allow efficient and accurate single-cell level detection of the CMT1A duplication. On the basis of the presence of the healthy allele of the affected parent-to-be (and/or absence of the affected one), healthy embryos can be selected for transfer. The assays are suitable for PGD for other couples who present with the same CMT1A duplication [depending on their informativity for the (CA)n markers available] as described here.     

Preimplantation genetic diagnosis: the ethics of intermediate cases

According to the current guiding principle regarding preimplantation genetic diagnosis (PGD), the technique should focus on the diagnosis of genetic defects which (may) affect the health of this particular potential child--the so-called 'medical model'. I argue in favour of a more permissive view, also allowing PGD of characteristics which may be relevant for the health of 'third parties'. Two cases are analysed: PGD/HLA typing in order to save a sib, and PGD/sex selection in order to prevent the birth of healthy female carriers of X-linked recessive disorders, who are at high risk of conceiving affected sons. While these cases are at odds with the medical model stricto sensu, they do have a link with health problems. In the first case, the health benefit hoped for is intrafamilial, in the second case the health benefit is transgenerational. These cases illustrate that the traditional dichotomy between the medical model on the one hand and the 'designer' or autonomy model on the other hand is simplistic--they represent an intermediate category.     

Genetics: Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients

In some 47,XXY Klinefelter patients without evidence of mosaicism,testicular spermatozoa can be successfully recovered and usedfor intracytoplasmlc sperm Injection (ICSI). To ensure the replacementof embryos with a normal X and Y chromosome pattern, preimplantationdiagnosis can be performed. This paper reports on three 47,XXYKlinefelter patients from whom it was possible to retrieve testicularspermatozoa in order to perform ICSI. From their healthy wivesa total of 27 oocyte-cumulus complexes were retrieved from which22 metaphase-II oocytes were obtained and injected; 19 of thesewere intact after injection. Two distinct pronuclel were observedin eight oocytes (42.1%) 18 h after injection. On day 3 of development,five embryos (62.5%) had reached at least the 6-cell stage andwere of sufficient quality to undergo biopsy and subsequentpreimplantation diagnosis for sex chromosome analysis by fluorescentin-situ hybridization. The four embryos diagnosed as normalwere transferred to three respective patients, resulting Inone biochemical pregnancy. The remaining cells of the fifthembryo were analysed afterwards, revealing a normal X and Ychromosome constitution. So far, in the five embryos diagnosed,a normal sex chromosome pattern has been observed.     

Preimplantation diagnosis: Primer extension preamplification for detection of multiple genetic loci from single human blastomeres

A new technology called primer extension preamplification (PEP),which has been applied to single spermatozoa, increases theamount of polymerase chain reaction (PCR) templates by amplifyingDNA of the whole genome. The current investigation was aimedat applying PEP to single human blastomeres. Two blastomereswith nuclei from arrested embryos were selected for this study.Using three different PEP protocols (experiments I, II and III),DNA from single blastomeres was amplified using 15-base oligonucleotiderandom primers. The efficiency of the procedure was determinedby further amplifications of aliquots of the PEP products withtwo specific sequences. Three aliquots from each PEP productwere used as PCR templates for the human X chromosome (X) orthe exon 10 of the cystic fibrosis gene (CF). PCR amplifiedproducts were analysed by gel electrophoresis. In experimentI, when X primers were used, positive signals were detectedin all 10 embryos (100%), 90.0% (18/20) of the blastomeres,and in 80.0% (96/120) of the replicates. When CF primers wereamplified, all embryos (100%, 10/10), 90.9% (18/20) of the blastomeresand 78.3% (47/60) of the replicates were positive. In experimentII, efficiency was significantly reduced when total time forthe procedure was minimized from 8 h to 5 h and 45 min. Althoughthe time was further reduced to 4 h and 40 min in experimentIII, the efficiency remained the same as in experiment I whenthe volume of PEP was reduced from 60 µl (experimentsI and II) to 40 µl. One out of 132 control replicates(0.8%) was contaminated. The present study indicates that PEPcan be successfully applied to single blastomeres allowing forsimultaneous detection of approximately 20 DNA sequences.     

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.     

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。     

Precise sex selected births of mice following single cell embryo biopsy and Y-linked testis-specific gene analysis

We report on the birth of 39 mice following single cell embryobiopsy and precise sex determination following in-vitro fertilization.Polymerase chain reaction was used to amplify fragments of themouse testis-specific gene sequence (pYMT2/B) on the Y chromosomeand the ovary-specific gene (ZP₃) sequence on chromosome 5 fromthe single biopsied cell. Embryo biopsy was not associated withany deleterious effects.     

Preimplantation genetic diagnosis

Pre-implantation genetic diagnosis (PGD) is generally defined as the testing of pre-implantation stage embryos or oocytes for genetic defects. It has been developed for couples whose potential offspring are at risk of severe Mendelian disorders, structural chromosome abnormalities or mitochondrial disorders. Pre-implantation embryo diagnosis requires in vitro fertilization, embryo biopsy and either using fluorescent in situ hybridization or polymerase chain reaction at the single cell level. Therefore, it is a complex procedure which requires much experience. Aneuploidy screening to improve medically assisted reproduction ( in vitro fertilization/intracytoplasmic sperm injection) is a variant type of PGD. The past, present and future of this development are strongly related to the natural occurrence of chromosomal mosaicism in the pre-implantation embryo. PGD should be included in each reproductive health care programme. It is recognized as an important alternative to pre-natal diagnosis. However, diagnosis from a single cell remains a technically challenging procedure, and the risk of misdiagnosis cannot be eliminated. An ethical discussion of the question of whether PGD is acceptable at all–the 'desirability question'–is a rearguard action. Discussion must primarily focus on the conditions of exercising due caution in and the dynamics of PGD.     

Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis

BACKGROUND: Preimplantation genetic diagnosis or screening (PGD, PGS) involves embryo biopsy on Day 3. Opting for one- or two-cell biopsy is a balance between the lowest risk for misdiagnosis on the one hand and the highest chance for a pregnancy on the other hand. METHODS: A prospective controlled trial was designed and 592 ICSI cycles were randomly assigned to the one-cell (group I) or the two-cell group (group II). Primary outcomes were diagnostic efficiency and embryonic development to delivery with live birth (analysed by cycle). The false-positive rate for the PCR cycles is presented as a secondary outcome (analysed by embryo). RESULTS: A strong significant correlation was observed between embryonic developmental stage on Day 3 and post-biopsy in vitro development on Day 5 (P < 0.0001). The influence of the intervention on Day 3 was less significant (P = 0.007): the biopsy of one cell is less invasive than the biopsy of two cells. PCR diagnostic efficiency was 88.6% in group I and 96.4% in group II (P = 0.008). For the fluorescence in situ hybridization (FISH) PGD cycles no significant difference in efficiency was obtained (98.2 and 97.5% in group I and II, respectively). Similar delivery rates with live birth per started cycle were obtained [58/287 or 20.2% in group I versus 52/303 or 17.2% in group II, P = 0.358; the absolute risk reduction = 3.05%; 95% confidence interval (CI): -3.24, 9.34]. Post-PGD PCR reanalysis showed six false positives in 97 embryos (6.2%) in group II and none in group I (91 embryos reanalysed). No false negatives were found. CONCLUSIONS: While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, Day 3 in vitro developmental stage is a stronger predictor for Day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. Delivery rates with live birth per started cycle were not significantly different.     

Molecular genetics of Krabbe disease (globoid cell leukodystrophy): Diagnostic and clinical implications

Galactocerebrosidase (GALC) is a lysosomal β-galactosidase responsible for the hydrolysis of the galactosyl moiety from several galactolipids, including galactosylceramide and psychosine. The deficiency of this enzyme results in the autosomal recessive disorder called Krabbe disease. It is also called globoid cell leukodystrophy (GLD), because of the characteristic storage cells found around cerebral blood vessels in the white matter of affected human patients and animal models. Although most patients present with clinical symptoms before 6 months of age, older patients, including adults, have been diagnosed by their severe deficiency of GALC activity. More than 40 mutations have been identified in patients with all clinical types of GLD. While some mutations clearly result in the infantile type if found homozygous or with another severe mutation, it is difficult to predict the phenotype of novel mutations or when mutations are found in the heterozygous state. A high incidence of polymorphic changes on apparent disease-causing alleles also complicates the interpretation of the effects of mutations. The detection of mutations has greatly improved carrier identification among family members and will permit preimplantation diagnosis for some families. The molecular characterization of the naturally occurring mouse, dog, and monkey models will permit their use in trials to evaluate different modes of therapy. Hum Mutat 10:268–279, 1997. © 1997 Wiley-Liss, Inc.