Progress of research on chromosomal mosaicism embryos / 中华医学遗传学杂志

Chromosomal mosaicism (CM) is a common phenomenon in preimplantation genetic testing (PGT). In embryos with CM, genetic contents of trophoblastic ectodermal (TE) cells may be different from that of the inner cell mass (ICM) which will develop into the fetus. Embryos with low mosaic proportion could give rise to healthy live births after transplantation, but are accompanied with high pregnancy risks such as high abortion rate. In order to provide a more comprehensive understanding for CM embryos, this article has systematically summarized the recent progress of research on the definition, mechanism, classification, PGT techniques, self-correction mechanism, transplantation outcome and treatment principles for CM embryos.

Preimplantation genetic testing for monogenic/single gene disorders in a family with Molybdenum co-factor deficiency / 中华医学遗传学杂志

OBJECTIVE@#To carry out preimplantation genetic testing for monogenic/single gene disorders (PGT-M) for a Chinese family affected with Molybdenum co-factor deficiency due to pathogenic variant of MOCS2 gene.@*METHODS@#A family with molybdenum co-factor deficiency who attended to the Maternal and Child Health Care Hospital of Guangxi Zhuang Autonomous Region in April 2020 was selected as the research subject. Trophoblast cells were biopsied from blastocysts fertilized by intracytoplasmic sperm injection. Embryos carrying the MOCS2 gene variant and chromosome copy number variation (CNV) of more than 4 Mb were detected by single-cell whole genome amplification, high-throughput sequencing and single nucleotide polymorphism typing. Embryos without or carrying the heterozygous variant and without abnormal chromosome CNV were transplanted. During mid-pregnancy, amniotic fluid sample was collected for prenatal diagnosis to verify the results of PGT-M.@*RESULTS@#Eleven oocytes were obtained, among which three blastocysts were formed through culturing. Results of genetic testing suggested that one embryo was heterozygous for the maternally derived MOCS2 gene variant and without chromosomal CNV. Following embryo transfer, intrauterine singleton pregnancy was attained. Prenatal diagnosis by amniocentesis at 18 weeks of gestation revealed that the MOCS2 gene variant and chromosomal analysis results were both consistent with that of PGT-M, and a healthy male infant was born at 37+5 weeks of gestation.@*CONCLUSION@#PGT-M has helped the couple carrying the MOCS2 gene variant to have a healthy offspring, and may become an important method for couples carrying other pathogenic genetic variants.

Born to donate: proposals for "savior sibling" regulation in Latin America / Niños creados para servir de donantes de órganos: propuestas para una regulación en América Latina

Abstract A Savior Sibling is a child who is born to provide an organ, bone marrow or cell transplant, to a sibling that is affected with a fatal disease. There are created with the in vitro fertilization and pre-implantation genetic diagnosis and, in the process, the ethical standards for organ donation of children become less demanding. Therefore, we propose that the authorization of the technique considers, unavoidably, the opinion of an impartial third party that can guarantee the welfare of the child. We develop a critical analysis of the laws that regulate the creation of babies to serve as organ donors. We evaluate under what circumstances the organizations that play a part in the decisions, fulfill the ethical standards to allow the organ donation of children.

Resumen Los llamados Savior Sibling son bebés creados con la técnica de la fertilización in vitro y el diagnóstico preimplantacional genético, con el fin de servir como donantes a un hermano afectado por una enfermedad fatal. Se crean con el diagnóstico genético de fertilización in vitro y preimplantación y, en el proceso, las normas éticas para la donación de órganos a niños son menos exigentes. Por esta razón, proponemos que la autorización para llevar a cabo esta técnica considere, como obligatorio, la opinión de un tercero que sea imparcial y que pueda garantizar el bienestar del niño. Se hizo un análisis crítico de las leyes que regulan la creación de estos bebés que sirven como donantes de órganos. Evaluamos bajo qué circunstancias, las organizaciones que participan en estas decisiones cumplen con los estándares éticos para permitir la donación de órganos a niños.

Preimplantation genetic diagnosis associated to Duchenne muscular dystrophy / Diagnóstico genético pré-implantacional associado à distrofia muscular de Duchenne

ABSTRACT Duchenne muscular dystrophy is the most common muscle disease found in male children. Currently, there is no effective therapy available for Duchenne muscular dystrophy patients. Therefore, it is essential to make a prenatal diagnosis and provide genetic counseling to reduce the birth of such boys. We report a case of preimplantation genetic diagnosis associated with Duchenne muscular dystrophy. The couple E.P.R., 38-year-old, symptomatic patient heterozygous for a 2 to 47 exon deletion mutation in DMD gene and G.T.S., 39-year-old, sought genetic counseling about preimplantation genetic diagnosis process. They have had a 6-year-old son who died due to Duchenne muscular dystrophy complications. The couple underwent four cycles of intracytoplasmic sperm injection (ICSI) and eight embryos biopsies were analyzed by polymerase chain reaction (PCR) for specific mutation analysis, followed by microarray-based comparative genomic hybridisation (array CGH) for aneuploidy analysis. Preimplantation genetic diagnosis revealed that two embryos had inherited the maternal DMD gene mutation, one embryo had a chromosomal alteration and five embryos were normal. One blastocyst was transferred and resulted in successful pregnancy. The other embryos remain vitrified. We concluded that embryo analysis using associated techniques of PCR and array CGH seems to be safe for embryo selection in cases of X-linked disorders, such as Duchenne muscular dystrophy.

RESUMO A distrofia muscular de Duchenne é a doença muscular mais comum observadas em crianças do sexo masculino. Atualmente, não há terapia eficaz disponível para distrofia muscular de Duchenne, portanto, é essencial o diagnóstico pré-natal e o aconselhamento genético para reduzir o nascimento desses meninos. Relatamos um caso de diagnóstico genético pré-implantação associado à distrofia muscular de Duchenne. O casal E.P.R., 38 anos, heterozigota, sintomática para uma mutação de deleção dos éxons 2 a 47 no gene DMD e G.T.S., 39 anos, buscaram aconselhamento genético sobre o processo de diagnóstico genético pré-implantação. O casal relatou que tiveram um filho de 6 anos que morreu devido a complicações da distrofia muscular de Duchenne. Os pacientes realizaram quatro ciclos de injeção intracitoplásmica de espermatozoides (ICSI) e oito biópsias de embriões foram analisadas por reação em cadeia da polimerase (PCR) para análise de mutação específica, seguida hibridação genômica comparativa baseada em microarranjos ( array CGH) para a pesquisa de aneuploidias. O diagnóstico genético pré-implantação revelou que dois embriões haviam herdado a mutação materna no gene DMD , um embrião tinha uma alteração cromossômica e cinco embriões eram normais. Um blastocisto foi transferido e resultou em gravidez bem sucedida. Os outros embriões permanecem vitrificados. Concluímos que a análise de embriões utilizando técnicas associadas de PCR e CGH array mostrou-se segura para a seleção de embriões em casos de doenças ligadas ao X, como a distrofia muscular de Duchenne.

Preimplantation genetic diagnosis for cystic fibrosis: a case report / Diagnóstico genético pré-implantacional na fibrose cística: relato de caso

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This disorder produces a variable phenotype including lung disease, pancreatic insufficiency, and meconium ileus plus bilateral agenesis of the vas deferens causing obstructive azoospermia and male infertility. Preimplantation genetic diagnosis is an alternative that allows identification of embryos affected by this or other genetic diseases. We report a case of couple with cystic fibrosis; the woman had the I148 T mutation and the man had the Delta F508 gene mutation. The couple underwent in vitro fertilization, associated with preimplantation genetic diagnosis, and with subsequent selection of healthy embryos for uterine transfer. The result was an uneventful pregnancy and delivery of a healthy male baby.

A fibrose cística é uma doença autossômica recessiva causada por mutações no gene regulador de condutância transmembrana na fibrose cística. Produz fenótipo variável, incluindo doença pulmonar, insuficiência pancreática, íleo meconial, além de agenesia bilateral dos ductos deferentes, causando azoospermia obstrutiva e infertilidade masculina. O diagnóstico genético pré-implantacional é uma alternativa diagnóstica, que permite identificar embriões portadores de fibrose cística e outras doenças genéticas. Relatamos o caso de um casal portador de fibrose cística, sendo a mulher portadora da mutação I148 T e o homem da mutação gênica Delta F508. O casal foi submetido a técnicas de fertilização in vitro associadas ao diagnóstico genético pré-implantacional, com consequente seleção de embriões saudáveis, que foram transferidos para o útero, resultando em gravidez sem intercorrências e com feto saudável, do sexo masculino.

Técnicas emergentes en medicina reproductiva: diagnóstico cromosómico del primer corpúsculo polar del ovocito / Emerging techniques in reproductive medicine: chromosomal diagnosis of the oocyte first polar body

El Diagnóstico Genético Preimplantacional (PGD) se ha convertido en una herramienta de rutina para la detección de anormalidades cromosómicas o genéticas, en muchos países del mundo. Se han reportado más de 20.000 ciclosde PGD, desde su desarrollo hace más de 20 años, habiendo nacido más de 4.000 niños hasta el año 2007. En Chile, esta técnica es realizada por la Unidad de Medicina Reproductiva de Clínica Las Condes, y se realiza sólo en la variante previa a la fecundación, en donde se biopsia el primer corpúsculo polar y sólo se insemina a los ovocitos encontrados cromosómicamente sanos. Las indicaciones más comunes para este tratamiento son: 1) evitar el aborto en pacientes con aborto recurrente sin explicación anatómica ni clínica; 2) mejorar las tasas de implantación en mujeres mayores de 37 años con antecedentesde procedimientos anteriores en los que se transfirieron embriones de buena calidad; 3) evitar el nacimiento deniños con enfermedades de origen cromosómico en mujeres mayores de 39 años.

Pre-implantational Genetic Diagnosis has become a common tool in most countries of the world. In almost 20 years since its development, it has been reported more than 20,000 cycles of PGD and till 2007, more than 4,000 children have been born. In Chile, this technique is done by the Unit of Reproductive Medicine of Clínica Las Condes. It is done only in the mode previous to fertilization. In where we study polar bodies and only chromosomically healthy oocytes are inseminated. The most common indications for this treatment are: 1) to avoid abortions in patients with recurrent abortion without anatomical nor clinical explanation; 2) to improve implantation rates in women older than 37 years of age, with previous procedures in which good quality embryos were transferred; 3) to avoid birth of children with diseases of chromosomal origin in women over 39 year of age.

DGP: técnica e indicaciones / PGD: technique and indications

El DGP (diagnóstico genético preimplantatorio), conocido también con las siglas del inglés PGD (preimplantation genetic diagnosis) es una herramienta más dentro del arsenal diagnóstico y terapéutico con que cuenta hoy la Medicina Asistencial. El diagnóstico genético efectuado con esta técnica puede involucrar diferentes alteraciones, tanto a nivel cromosómico como génico. El procedimiento se efectúa en los zigotos clivados obtenidos mediante las técnicas de Reproducción Asistida de Alta Complejidad, antes de la transferencia de los mismos al útero materno. Constituye una alternativa válida dentro de la metodología del diagnóstico prenatal, tendiente a identificar anomalías genéticas antes de la implantación del embrión. Pueden ser estudiadas alteraciones numéricas de los cromosomas sexuales, anomalías estructurales, enfermedades con herencia ligada al cromosoma X y enfermedades monogénicas. La aplicación de esta técnica también es especialmente importante en el área de la medicina reproductiva, debido a que es frecuente la asociación entre esterilidad y factores genéticos. Así, sus principales indicaciones serían la edad materna avanzada, las parejas con aborto a repetición o con fallo recurrente de implantación y el factor masculino severo. Para efectuar el DGP requiere previamente de la realización técnica de los procedimientos de Fecundación asistida, ya sea fecundacion in vitro (FIV) o por inyección intracitoplásmatica espermática (ICSI, según su sigla en inglés por Intracitoplasmatic Sperm Injection). Luego de la obtención de los embriones a través de dichas técnicas, se procede a la extracción del material que será objeto del estudio citogenético (cuerpos polares o una a dos blastómeras) en forma mecánica, química o física. El material a analizar podrá ser la biopsia del cuerpo polar (CP), la biopsia en estado de cigoto hasta mórula avanzada o la biopsia en estado de blastocito...

Diagnóstico genético pré-implantacional: uma ferramenta importante para a rotina de fertilização in vitro? / Preimplantation genetic diagnosis: an important tool for the in vitro fertilization routine?

Erros cromossômicos numéricos são comuns durante as primeiras etapas do desenvolvimento embrionário humano, contribuem significativamente com processos de falta de implantação e são causadores da perda gestacional recorrente em pelo menos 50 por cento dos abortos ocorridos no primeiro trimestre. Tradicionalmente, a prevenção das anomalias genéticas cromossômicas em pacientes de alto risco é realizada por exames pré-natais, como a biópsia do vilo coriônico, aminiocentese e a cordocentese. Uma vez diagnosticada a anomalia, não existe tratamento eficaz para portadores de aberrações genéticas e a interrupção da gestação nestes casos ainda é ética e legalmente questionável. O diagnóstico genético pré-implantacional (DGPI) representa uma ferramenta valiosa aos casais de alto risco, por permitir a seleção de embriões saudáveis obtidos através de programas de fertilização in vitro antes de estes serem transferidos para um útero materno. Os primeiros relatos da utilização do DGPI datam da década de 90, quando eram utilizadas metodologias da reação em cadeia da polimerase para determinação do sexo do embrião, permitindo desta maneira transferir embriões que não fossem portadores de doenças ligadas ao cromossomo X. O DGPI é uma técnica extremamente eficaz, que analisa uma única célula do embrião que é biopsiado a partir do terceiro dia de desenvolvimento. Esta metodologia tem como finalidade identificar embriões gerados por processos de reprodução assistida, os quais sejam portadores de aberrações cromossômicas numéricas que envolvam os cromossomos X, Y, 13, 16, 18, 21 e 22. A metodologia mais utilizada para a realização do DGPI é a técnica de hibridização in situ, utilizando-se sondas fluorescentes para os cromossomos citados. Este é um método eficiente e que deve ser discutido com casais cuja idade da mulher seja acima dos 39 anos, casais com cariótipo alterado ou ainda casais com histórico familiar de presença de portadores de cromossomopatias. A eficiência...

The potential transmission of genetic disorders to the offspring has been a major problem for many couples when contemplating pregnancy. Numerical chromosomes errors are common during the first stages of the human embryonic development and contribute significantly with processes of implantation failure and it is also related to recurrent miscarriage, in at least 50 percent of the abortions occurred in the first trimester. Traditionally, the prevention of genetic chromosomal abnormalities in high risk patients is achieved by pre-natal examinations such as biopsy of the chorionic villi, aminiocentese of cordocentesis. Once diagnosed the genetic error, there is no effective treatment for patients with genetic aberrations and the interruption of pregnancy in these cases are ethically and legally questionable. The risk has been greatly decreases by the evaluation of the family history of the age of the mother, and the implementation of prenatal diagnosis in those couples in which the risk was increased compared to the general population. An alternative approach that is available with assisted reproductive technologies is the preimplantation genetic diagnosis (PGD), which it is possible to observe X, Y, 13, 16, 18, 21 and 22 chromosomes or to perform gene screen by PCR for knowing genetic diseases before the corresponding embryo is transferred to the uterus of the mother. PGD was first clinically applied in the early nineties, and was initially used in sexing cases for couples who were at risk of transmitting an X-linked recessive disorder. PGD involves the analysis of either polar bodies, extruded from oocytes during meiosis, or single cells (blastomeres) biopsied from embryos after fertilization. PGD tests have largely focused on two methodologies: fluorescent in situ hybridization and polymerase chain reaction. The efficiency of the method is about 98 percent and its extremely invasive technique demands high level professional.

A preliminary study on chromosome aneuploidy & mosaicism in early pre-implantation human embryo by fluorescence in situ hybridization.

BACKGROUND & OBJECTIVE: Chromosome aneuploidy plays an important role in infertility, early pregnancy wastage and perinatal mortality. Cytogenetic & fluorescent in situ hybridization (FISH) studies on developmentally arrested and morphologically poor embryo have shown high frequency of chromosomal abnormality and mosaicism. In this study, we attempted to evaluate chromosome aneuploidy and mosaicism on human embryos through the use of FISH. METHODS: Sixty one grade IV un-transferable embryos were obtained from 25 patients undergoing in vitro fertilization (IVF). Forty six embryos were studied by FISH; 15 were lost during transport and handling. FISH probes (non-commercial) for centromeres of chromosome X, Y, 1 and 18 were used for the study. Zona of embryos were dissolved in 0.01N HCl containing 0.1 per cent Tween 20 for 2-3 min. RESULTS: Interpretable FISH results were obtained in 24 embryos. Nineteen embryos (79.2%) were disomic (normal) for chromosome X/Y or 1/18 and five (20.8%) were abnormal. Among five abnormal embryos two were triploidy (from same patient), one was double mosaic aneuploidy, one was mosaic aneuploidy and one was trisomy for sex chromosome (XXY). There was eleven embryos with presence of Y chromosome i.e., male and three embryos were female. INTERPRETATION & CONCLUSION: Skewing of sex ratio (11M vs. 3F) and low chromosome aneuploidy were observed in this preliminary study, however, it will be premature to conclude as the numbers of embryos studied were limited and so were the numbers of FISH probes used.

Preimplantation Genetic Diagnosis for Ornithine Transcarbamylase Deficiency by Simultaneous Analysis of Duplex-nested PCR and Fluorescence In Situ Hybridization: A Case Report

Ornithine transcarbamylase (OTC) deficiency is an X-linked co-dominant disorder. A couple, with a previous history of a neonatal death and a therapeutical termination due to OTC deficiency, was referred to our center for preimplantation genetic diagnosis (PGD). The female partner has a nonsense mutation in the exon 9 of the OTC gene (R320X). We carried out nested polymerase chain reaction (PCR) for R320X mutation and fluorescence in situ hybridization (FISH) for aneuploidy screening. Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex-nested PCR and FISH, and one blastomere per embryo from 2 embryos by only duplex-nested PCR. As a result of PCR and restriction fragment length polymorphism analysis, four embryos were diagnosed as unaffected embryos having the normal OTC gene. Among these embryos, only one embryo was confirmed as euploidy for chromosome X, Y and 18 by FISH analysis. A single normal embryo was transferred to the mother, yielding an unaffected pregnancy and birth of a healthy boy. Based on our results, PCR for mutation loci and FISH for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the PGD for single gene defects.

Uses and limitations of two molecular cytogenetic techniques for the study of arrested embryos obtained through assisted reproduction technology

We studied chromosomal abnormalities in arrested embryos produced by assisted reproductive technology with fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in order to determine the best technique for evaluating chromosomal aneusomies to be implemented in different situations. We examined individual blastomeres from arrested embryos by FISH and arrested whole embryos by CGH. All of the 10 FISH-analyzed embryos gave results, while only 7 of the 30 embryos analyzed by CGH were usable. Fifteen of the 17 embryos were chromosomally abnormal. CGH provided more accurate data for arrested embryos; however, FISH is the technique of choice for screening in preimplantation genetic diagnosis, because the results can be obtained within a day, while the embryos are still in culture.

Preimplantation genetic diagnosis of chromosomal abnormalities by multicolour fluorescence in situ hybridisation.

Preimplantation genetic diagnosis (PGD) is an early diagnosis of genetic disorders, prior to the onset of pregnancy. PGD incorporates the latest techniques in assisted reproduction and molecular genetics. Embryos or oocytes are biopsied during culture in vitro and genetic analysis is carried out on the blastomeres or polar bodies. Embryos shown to be free of the genetic disease under investigation are transferred to the uterus. Multicolour fluorescence in situ hybridisation (FISH) is used to diagnose numerical and certain structural abnormalities of chromosomes in the embryo. The common probes used are for chromosomes 13, 18, 21, X and Y. FISH can also be used for PGD of translocations, when one of the parents is a carrier. PGD was carried out recently in 4 cases using multicolour FISH. In one of the embryos, trisomy 18 was detected. Tetraploidy was seen in another embryo. Only chromosomally normal embryos were transferred back to the uterus. Care has to be taken while interpreting FISH signals as the signal may be split, diffused, superimposed or in a different focus.

Diagnostico genetico pre-implantacional / Polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH) and primed in situ (PRINS)

O diagnostico genetico pre-implantacional (PGD) e um novo procedimento que pode ser utilizado como diagnostico precoce pre-natal em casais com risco de transmissao de doencas geneticas. Utilizando "polymerase chain reaction" (PCR), "fluorescence in-situ hybridization" (FISH) e "primed in-situ" (PRINS) pode-se determinar o genotipo ou sexo genetico do embriao biopsiado obtido por...

Potential usefulness of preimplantation genetic diagnosis in the control and prevention of genetic diseases

Prenatal diagnosis of molecular mutations can be of immense value, since diagnosis followed by genetic counselling provides the most appropriate approach to genetic diseases control and prevention. However, ethical, psychosocial and religious considerations hamper adoption of prenatal diagnosis in communities where termination of a pregnancy may not be acceptable. Recently, preimplantation genetic diagnosis has attracted considerable interest. This involves in vitro fertilization, followed by genetic disorder diagnosis using polar bodies or cells extracted from a blastomere stage. The normal blastomere is implanted in the womb and pregnancy proceeds naturally. If an abnormality is diagnosed, the blastomere is not implanted, thus preventing pregnancy with the affected fetus. This paper outlines the potential usefulness of preimplantation genetic diagnosis in the control and prevention of genetic disease in our part of the world