Preimplantation genetic diagnosis of β-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells (∼5 cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38 weeks’ gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.     

Distinguishing between carrier and noncarrier embryos with the use of long-read sequencing in preimplantation genetic testing for reciprocal translocations

Balanced reciprocal translocation carriers are usually phenotypically normal but are at an increased risk of infertility, recurrent miscarriage or having affected children. Preimplantation genetic testing on chromosomal structural rearrangement (PGT-SR) offers a way to screen against unbalanced embryos. Here, we demonstrated a new method to distinguish carrier from noncarrier embryos. Translocation breakpoints were first delineated by nanopore sequencing followed by polymerase chain reaction (PCR) across breakpoints. High-resolution breakpoint mapping was successful in all (9/9) balanced reciprocal translocation carriers. Retrospective analysis of their embryo biopsies with breakpoint PCR showed 100% concordant results with PGT-SR on trophectoderm biopsies (40/40) and 53% concordance on blastomere biopsies (8/15). The low concordant rate in blastomeres was due to failure in the amplification of derivative chromosomes involving large deletions. Breakpoint PCR also showed 100% concordant results with prenatal/postnatal outcomes on 5 pregnancies, indicating that our new method can accurately distinguish carrier from noncarrier embryos.     

Preimplantation genetic diagnosis in Saudi Arabia

Preimplantation genetic diagnosis (PGD) testing is the practice of obtaining a cellular biopsy sample from a developing human oocyte or embryo, acquired via a cycle of in vitro fertilization (IVF); evaluating the genetic composition of this sample; and using this information to determine which embryos will be optimal for subsequent uterine transfer. PGD has become an increasingly useful adjunct to IVF procedures. The ability to provide couples who are known carriers of genetic abnormalities the opportunity to deliver healthy babies has opened a new frontier in reproductive medicine. The purpose of the PGD is enables us to choose which embryos will be implanted into the mother. In the present study 137 families who had undergone IVF at Habib Medical Centre, were enrolled for the PGD analysis. The couple visited the clinic for the sex selection, recurrent fetal loss and with the recurrent IVF failure. 802 embryos were tested by the biopsy method and 512 are found to be normal and 290 were abnormal embryos. In this study only 24% of the embryos were transferred and the remaining was not transferred because of the abnormalities or undesired sex of the embryos. The structural and numerical abnormalities were found to be 16.8%.     

Preimplantation genetic diagnosis for spinal and bulbar muscular atrophy (SBMA)

X-linked spinal and bulbar muscular atrophy is characterized by adult onset motor neuron disease and results from a defect in the androgen receptor. The disease is caused by a dynamic mutation in the first exon of the androgen receptor gene, involving a CAG trinucleotide repeat. We have developed a single-cell polymerase chain reaction assay for the androgen receptor gene and describe the application of this assay for preimlantation genetic diagnosis (PGD) in a couple at risk, where the female partner is a carrier of 47 repeats. Diagnosis was based on the detection of both normal and expanded alleles. Allele dropout of the expanded allele was observed in only 1 of 25 lymphoblasts of the carrier and of a non-expanded allele in 1 of 20 research blastomeres tested before the actual PGD. One contraction of four repeats was also found in the carrier's lymphoblasts. Neither expansions nor contractions were observed in the blastomeres biopsied from 11 embryos. Two embryos were unaffected, eight were female carriers and one was an affected male embryo.     

PID in der Reproduktionsmedizin

Preimplantation genetic diagnosis in assisted reproduction Preimplantation genetic diagnosis (PGD) describes the biopsy of one or more cells of an in vitro fertilized embryo and its genetical analysis before the transfer of the embryo into the uterus. Since the modification of the Embryonenschutzgesetz a PGD can be lawfully performed in Germany. Several diagnostic methods can be performed: FISH for the detection of maternal translocations on polar bodies, at the moment the most applied the array‐CGH for the screening of aneuploidies and the detection of translocations and also in the case of monogenic diseases PCR followed by sequencing. In the near future next generation sequencing will possibly replace all mentioned methods.     

Cell engineering and genetic approaches to development of human embryonic stem cell models for genetic disorders

We introduced a novel approach for the establishment of genetically modified hESC lines, and have shown that mutant hESC may be derived from affected embryos after preimplantation genetic diagnosis (PGD) screening for a particular single gene disorder. Here we describe the procedure of embryo and cell manipulation, their diagnostic layout, and the analysis of the efficiency of embryo development and hESC establishment, as well as the developments for hESC derivation in animal-product-free conditions. Our study shows that a high efficiency of hESC derivation (50%) is especially crucial when working with rare and unique resources such as genetically screened embryos necessary for the derivation of hESC lines that represent specific genetic diseases.     

Refining the ethics of preimplantation genetic diagnosis: A plea for contextualized proportionality

Many European countries uphold a ‘high risk of a serious condition’ requirement for limiting the scope of preimplantation genetic diagnosis (PGD). This ‘front door’ rule should be loosened to account for forms of PGD with a divergent proportionality. This applies to both ‘added PGD’ (aPGD), as an add‐on to in vitro fertilization (IVF), and ‘combination PGD’ (cPGD), for a secondary disorder in addition to the one for which the applicants have an accepted PGD indication. Thus loosening up at the front has implications at the back of PGD treatment, where a further PGD rule says that ‘affected embryos’ (in the sense of embryos with the targeted mutation or abnormality) should not be transferred to the womb. This ‘back door’ rule should be loosened to allow for transferring ‘last chance’ affected embryos in aPGD and cPGD cases, provided this does not entail a high risk that the child will have a seriously diminished quality of life.     

Advances in preimplantation genetic diagnosis/screening

Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS.     

Eugenic Selection Benefits Embryos

The primary question to be addressed here is whether pre‐implantation genetic diagnosis (PGD), used for both negative and positive trait selection, benefits potential supernumerary embryos. The phrase ‘potential supernumerary embryos’ is used to indicate that PGD is typically performed on a set of embryos, only some of which will be implanted. Prior to any testing, each embryo in the set is potentially supernumerary in the sense that it may not be selected for implantation. Those embryos that are not selected, and hence destroyed or frozen, are ‘actually supernumerary’. The argument to be advanced is hypothetical: If embryos may be said to benefit or be harmed by our actions, then PGD used to select for an embryo or embryos with the highest expected Wellbeing benefits potential supernumerary embryos. The argument shows that the ‘non‐identity’ problem is not sufficient to show that eugenic selection does not benefit supernumerary embryos.     

Preimplantation genetic diagnosis: State of the ART 2011

For the last 20 years, preimplantation genetic diagnosis (PGD) has been mostly performed on cleavage stage embryos after the biopsy of 1–2 cells and PCR and FISH have been used for the diagnosis. The main indications have been single gene disorders and inherited chromosome abnormalities. Preimplantation genetic screening (PGS) for aneuploidy is a technique that has used PGD technology to examine chromosomes in embryos from couples undergoing IVF with the aim of helping select the chromosomally ‘best’ embryo for transfer. It has been applied to patients of advanced maternal age, repeated implantation failure, repeated miscarriages and severe male factor infertility. Recent randomised controlled trials (RCTs) have shown that PGS performed on cleavage stage embryos for a variety of indications does not improve delivery rates. At the cleavage stage, the cells biopsied from the embryo are often not representative of the rest of the embryo due to chromosomal mosaicism. There has therefore been a move towards blastocyst and polar body biopsy, depending on the indication and regulations in specific countries (in some countries, biopsy of embryos is not allowed). Blastocyst biopsy has an added advantage as vitrification of blastocysts, even post biopsy, has been shown to be a very successful method of cryopreserving embryos. However, mosaicism is also observed in blastocysts. There have been dramatic changes in the method of diagnosing small numbers of cells for PGD. Both array-comparative genomic hybridisation and single nucleotide polymorphism arrays have been introduced clinically for PGD and PGS. For PGD, the use of SNP arrays brings with it ethical concerns as a large amount of genetic information will be available from each embryo. For PGS, RCTs need to be conducted using both array-CGH and SNP arrays to determine if either will result in an increase in delivery rates.     

Multiple displacement amplification for preimplantation genetic diagnosis of fragile X syndrome

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples that have genetic risks. Despite the many advantages provided by PGD, there are several problems, including amplification failure, allele drop-out and amplification inefficiency. We evaluated multiple displacement amplification (MDA) for PGD of the fragile X syndrome. Whole genome amplification was performed using MDA. MDA products were subjected to fluorescent PCR of fragile X mental retardation-1 (FMR1) CGG repeats, amelogenin and two polymorphic markers. In the pre-clinical tests, the amplification rates of the FMR1 CGG repeat, DXS1215 and FRAXAC1 were 84.2, 87.5 and 75.0%, respectively, while the allele dropout rates were 31.3, 57.1 and 50.0%, respectively. In two PGD treatment cycles, 20 embryos among 30 embryos were successfully diagnosed as 10 normal embryos, four mutated embryos and six heterozygous carriers. Three healthy embryos were transferred to the uterus; however, no clinical pregnancy was achieved. Our data indicate that MDA and fluorescent PCR with four loci can be successfully applied to PGD for fragile X syndrome. Advanced methods for amplification of minuscule amounts of DNA could improve the sensitivity and reliability of PGD for complicated single gene disorders.     

Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program

Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300–1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7–10 hours and 63% for 1–2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the results obtained using ultrasonography in all pregnancies assessed (11/11, 100%); untreated biopsy samples were correctly diagnosed in 36 of 41 assessed pregnancies (87.8%), although the difference between treated and untreated biopsy samples was not significant. Our results demonstrated that biopsied embryos can remain in holding medium before being transferred, until gender diagnosis by PGD is complete (7–10 hours), without affecting pregnancy rates. This simplifies the management of an embryo transfer program willing to incorporate PGD for gender selection, by transferring only embryos of the desired sex. Embryo biopsy can be performed in a clinical setting on embryos of different sizes, without affecting their viability. Additionally, we showed that pretreating biopsy samples with a short incubation at 95 °C improved the overall efficiency of embryo sex determination.     

Genetic testing and PGD for unexplained recurrent fetal malformations with MAGEL2 gene mutation

Birth defects are caused by multiple factors, such as chromosome abnormality, environmental factors, and maternal factors. In this study, we focused on exploring the genetic causes of a non-consanguineous couple who suffered from four times of unsuccessful pregnancy due to unexplained recurrent fetal malformations with similar symptoms and normal chromosome copy number variations. Using trio-whole exome sequencing(trio-WES) for this couple and one of the affected fetuses, we found a mutation, c.1996 delC on the maternal imprinted gene MAGEL2 that was carried by the affected fetus and husband, leading to Schaaf-Yang syndrome. To screen this mutation, we further performed preimplantation genetic diagnosis(PGD) strategy followed by a gene pedigree validation and pathogenicity analysis. After the transfer of a PGD-screened embryo, a normal newborn without previous abnormal symptoms was born(February 15, 2019). We present the first data that identified a pathogenic gene(MAGEL2 c.1996 delC) in a fetus with Schaaf-Yang syndrome in the EAS(East Asian) database and overcame this genetic defect by using processed PGD for this couple based on the WES results.     

Blastomere removal from cleavage-stage mouse embryos alters steroid metabolism during pregnancy

Preimplantation genetic diagnosis (PGD) is a genetic screening of embryos conceived with assisted reproduction technologies (ART). A single blastomere from an early-stage embryo is removed and molecular analyses follow to identify embryos carrying genetic defects. PGD is considered highly successful for detecting genetic anomalies, but the effects of blastomere biopsy on fetal development are understudied. We aimed to determine whether single blastomere removal affects steroid homeostasis in the maternal-placental-fetal unit during mouse pregnancy. Embryos generated by in vitro fertilization (IVF) were biopsied at the four-cell stage, cultured to morula/early blastocyst, and transplanted into the oviducts of surrogate mothers. Nonbiopsied embryos from the same IVF cohorts served as controls. Cesarean section was performed at term, and maternal and fetal tissues were collected. Embryo biopsy affected the levels of steroids (estradiol, estrone, and progesterone) in fetal and placental compartments but not in maternal tissues. Steroidogenic enzyme activities (3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase, and cytochrome P450 19) were unaffected but decreased activities of steroid clearance enzymes (uridine diphosphate-glucuronosyltransferase and sulfotransferase) were observed in placentas and fetal livers. Although maternal body, ovarian, and placental weights did not differ, the weights of fetuses derived from biopsied embryos were lower than those of their nonbiopsied counterparts. The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.     

The establishment and application of preimplantation genetic haplotyping in embryo diagnosis for reciprocal and Robertsonian translocation carriers

Background

Preimplantation genetic diagnosis (PGD) is now widely used to select embryos free of chromosomal copy number variations (CNV) from chromosome balanced translocation carriers. However, it remains a difficulty to distinguish in embryos between balanced and structurally normal chromosomes efficiently.

Methods

For this purpose, genome wide preimplantation genetic haplotyping (PGH) analysis was utilized based on single nucleotide polymorphism (SNP) microarray. SNPs that are heterozygous in the carrier and, homozygous in the carrier’s partner and carrier’s family member are defined as informative SNPs. The haplotypes including the breakpoint regions, the whole chromosomes involved in the translocation and the corresponding homologous chromosomes are established with these informative SNPs in the couple, reference and embryos. In order to perform this analysis, a reference either a translocation carrier’s family member or one unbalanced embryo is required. The positions of translocation breakpoints are identified by molecular karyotypes of unbalanced embryos. The recombination of breakpoint regions in embryos could be identified.

Results

Eleven translocation families were enrolled. 68 blastocysts were analyzed, in which 42 were unbalanced or aneuploid and the other 26 were balanced or normal chromosomes. Thirteen embryos were transferred back to patients. Prenatal cytogenetic analysis of amniotic fluid cells was performed. The results predicted by PGH and karyotypes were totally consistent.

Conclusions

With the successful clinical application, we demonstrate that PGH was a simple, efficient, and popularized method to distinguish between balanced and structurally normal chromosome embryos.

     

Chromosome Segregation Analysis in Human Embryos Obtained from Couples Involving Male Carriers of Reciprocal or Robertsonian Translocation

The objective of this study was to investigate the frequency and type of chromosome segregation patterns in cleavage stage embryos obtained from male carriers of Robertsonian (ROB) and reciprocal (REC) translocations undergoing preimplantation genetic diagnosis (PGD) at our reproductive center. We used FISH to analyze chromosome segregation in 308 day 3 cleavage stage embryos obtained from 26 patients. The percentage of embryos consistent with normal or balanced segregation (55.1% vs. 27.1%) and clinical pregnancy (62.5% vs. 19.2%) rates were higher in ROB than the REC translocation carriers. Involvement of non-acrocentric chromosome(s) or terminal breakpoint(s) in reciprocal translocations was associated with an increase in the percent of embryos consistent with adjacent 1 but with a decrease in 3∶1 segregation. Similar results were obtained in the analysis of nontransferred embryos donated for research. 3∶1 segregation was the most frequent segregation type in both day 3 (31%) and spare (35%) embryos obtained from carriers of t(11;22)(q23;q11), the only non-random REC with the same breakpoint reported in a large number of unrelated families mainly identified by the birth of a child with derivative chromosome 22. These results suggest that chromosome segregation patterns in day 3 and nontransferred embryos obtained from male translocation carriers vary with the type of translocation and involvement of acrocentric chromosome(s) or terminal breakpoint(s). These results should be helpful in estimating reproductive success in translocation carriers undergoing PGD.     

植入前遗传学诊断的原理、方法及适应症

植入前遗传学诊断是一种非常早的产前诊断,指在胚胎着床之前即对配子或胚胎的遗传物质进行分析,检测配子或胚胎是否有遗传物质异常,选择正常胚胎进行移植。与传统的产前诊断相比,能避免选择性流产异常妊娠给妇女带来的心身痛苦。本文就该领域的发展及现状和其诊断原理、方法及适应症进行了总结和综述。 Abstract:Preimplatation genetic diagnosis (PGD) is a very early form of prenatal diagnosis.Gametes or embryos are biopsied and a genetic diagnosis is carried out on the biopsied cells to investigate if the gametes or embryos is free of genetic disease.And the normal embryos is transferred to the mother.Comparing to the traditional prenatal diagnosis,PGD is a method that can avoid aborting a abnormal pregnant and reduce pains of women.In this review,we introduce the history of development and statues in quo,principle,method and application of PGD.     

Non-Invasive Prenatal Diagnosis of Multiple Endocrine Neoplasia Type 2A Using COLD-PCR Combined with HRM Genotyping Analysis from Maternal Serum

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.     

A single strand conformation polymorphism-based carrier test for spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a newborn prevalence of 1 in 10,000, and a carrier frequency of 1 in 40-60 individuals. The SMA locus has been mapped to chromosome 5q11.2-13. The disease is caused by a deletion of the SMN gene, often encompassing other genes and microsatellite markers. The SMN gene is present in two highly homologous copies, SMN1 and SMN2, differing at five nucleotide positions. Only homozygous SMN1 mutations cause the disease. The sequence similarity between the SMN1 and SMN2 genes can make molecular diagnosis and carrier identification difficult. We developed a sensitive and reliable molecular test for SMN1 carrier identification, by setting up a nonradioactive single strand conformation polymorphism (SSCP)-based method, which allows for the quantification of the amount of the SMN1 gene product with respect to a control gene. The assay was validated in 56 obligate (ascertained) carriers and 20 (ascertained) noncarriers. The sensitivity of the test is 96.4%, and its specificity, 98%. In addition, 6 of 7 SMA patients without homozygous deletions presented with a heterozygous deletion, suggesting a concomitant undetected point mutation on the nondeleted SMN1 allele. Therefore, the present test is effective for detecting compound hemizygote patients, for testing carriers in SMA families, and for screening for SMA heterozygotes in the general population.     

Chromosome breakage in human preimplantation embryos from carriers of structural chromosomal abnormalities in relation to fragile sites, maternal age, and poor sperm factors

Chromosome breakage is a fairly widespread phenomenon in preimplantation embryos affecting at least 10% of day 3 cleavage stage embryos. It may be detected during preimplantation genetic diagnosis (PGD). For carriers of structural chromosomal abnormalities, PGD involves the removal and testing of single blastomeres from cleavage stage embryos, aiming towards an unaffected pregnancy. Twenty-two such couples were referred for PGD, and biopsied blastomeres on day 3 and untransferred embryos (day 5/6) were tested using fluorescence in situ hybridisation (FISH) with appropriate probes. This study investigated whether chromosome breakage (a) was detected more frequently in cases where the breakpoint of the aberration was in the same chromosomal band as a fragile site and (b) was influenced by maternal age, sperm parameters, reproductive history, or the sex of the carrier parent. The frequency of breakage seemed to be independent of fragile sites, maternal age, reproductive history, and sex of the carrier parent. However, chromosome breakage was very significantly higher in embryos from male carriers with poor sperm parameters versus embryos from male carriers with normal sperm parameters. Consequently, embryos from certain couples were more prone to chromosome breakage, fragment loss, and hence chromosomally unbalanced embryos, independently of meiotic segregation.