La importancia del asesoramiento genético para el diagnóstico genético preimplantacional de aneuploidías (PGT-A)

The development of new genomic technologies has strengthened the influence of genetics in all medical specialties; reproductive medicine is no exception. The introduction of new genetic tests to daily clinical practice, together with the complexity of genetic information and its potential psychological burden, make specialized genetic counseling essential. Preimplantation genetic testing for aneuploidies (PGT-A) has become an almost routine procedure in assisted reproduction treatments. Nevertheless, in Peru, patients usually receive none or inadequate corresponding genetic counseling, which hinders informed decision-making.

El desarrollo de las nuevas tecnologías genómicas ha potenciado la influencia de la genética en todos los campos de la medicina, en donde la medicina reproductiva no es la excepción. La frecuente introducción de nuevas pruebas genéticas en la práctica clínica diaria, junto con la complejidad de la información genética y su potencial carga psicológica, hacen indispensable el asesoramiento genético especializado. En este contexto, el diagnóstico genético preimplantacional para aneuploidías (PGT-A) se ha convertido en un procedimiento casi de rutina en los tratamientos de reproducción asistida. Sin embargo, en el Perú, en la gran mayoría de casos los pacientes no reciben el asesoramiento genético correspondiente o este no es el adecuado, que no permite una toma de decisiones informada.

El uso de la tecnología Time-Lapse y algoritmo predictivo KIDScore 5 ayuda a mejorar la selección de embriones euploides para la transferencia

Objectives: To determine if the use of the KIDScore 5 algorithm (known implantation data) can help select between euploid embryos in order to improve pregnancy and implantation rates in patients undergoing assisted reproductive procedures. Methods: Retrospective cohort study in a fertility clinic, from October 2016 to December 2018, of 1 049 embryos from 328 patients. All the embryos were cultured in the Time-Lapse, Embryoscope® incubator (Vitrolife®, Canada) for 5-6 days. Of these, 896 embryos (85.4%) were biopsied and analyzed by NGS, and assessed with the predictive KIDScore 5 algorithm (Vitrolife®, Canada). The 153 remaining embryos (14.6%) were assessed with the predictive KIDScore 5 algorithm only. 256 single euploid embryos were transferred in couples undergoing IVF treatments at the Inmater clinics laboratory of assisted reproduction in Lima - Peru. Results: The implantation rate was significantly higher (p = 0.004) in euploid embryos transferred when selected by the KIDScore 5 algorithm (Group 2) versus those selected using only genetic study by NGS technology (Group 1) (71.2% vs. 48.8%). The rate of implantation of the euploid embryos transferred with KIDScore value = 6 versus those transferred with KIDScore = 1 was statistically different (73.5% vs. 50.8%; p = 0.030). When assessing the relationship between the rate of euploid embryos versus the result of the KIDScore 5 value, we found highly significant differences in the rates of euploid embryos with values 6 and 5 versus those with KIDScore 0 and 1 (60.5% vs. 45.7%; p = 0.0004). Conclusions: The embryo selection with the KIDScore 5 algorithm offers advantage on implantation and pregnancy rates only when euploid embryos are transferred. Its use as an additional criterion to embryo selection should be considered when accompanied by genetic study of the embryos to be transferred. Euploid embryos with a higher value in the KIDScore 5 algorithm scale have better rates of implantation and euploidy than embryos with the minimum value of this algorithm.

Objetivos. Evaluar si el uso del algoritmo KIDScore 5 (known implantation data) puede ayudar a seleccionar entre los embriones euploides, para mejorar las tasas de embarazo e implantación en pacientes sometidas a procedimientos de reproducción asistida. Métodos. Estudio de cohorte retrospectivo en una clínica de fertilidad, desde octubre 2016 a diciembre 2018. Se estudió 1 049 embriones provenientes de 328 pacientes. Todos los embriones fueron cultivados en la incubadora Time-Lapse, Embryoscope® (Vitrolife®, Canadá) durante 5 a 6 días. De estos, 896 embriones (85,4%) fueron biopsiados y analizados mediante NGS y recibieron una valoración otorgada por el algoritmo predictivo KIDScore 5 (Vitrolife®, Canadá). Los 153 embriones restantes (14,6%) únicamente recibieron la valoración mediante el algoritmo predictivo KIDScore 5. Se realizó 256 transferencias únicas de embriones euploides en parejas sometidas a tratamientos de FIV en el laboratorio de reproducción asistida de la Clínica Inmater, Lima - Perú. Resultados. La tasa de implantación de los embriones euploides transferidos con valores de KIDScore = 6 versus los transferidos con valores de KIDScore = 1 tuvo diferencia estadísticamente significativa (73,5% vs. 50,8%; p=0,030). Al evaluar la relación entre la tasa de euploidia embrionaria versus el resultado del valor de KIDScore 5, se obtuvo diferencias altamente significativas en las tasas de euploidia en los embriones con resultados de KIDScore 6 y 5 versus los de KIDScore 0 y 1 (60,5% vs. 45,7%; p=0,0004). Conclusiones. La selección embrionaria con ayuda del algoritmo KIDScore 5 ofrece ventaja en las tasas de implantación y embarazo únicamente cuando se transfieren embriones euploides. Su uso como criterio adicional a la selección embrionaria debiera ser considerado siempre que se acompañe estudio genético a los embriones a transferir. Los embriones euploides con valor más alto en la escala del algoritmo KIDScore 5, tienen mejores tasas de implantación y euploidía que los embriones con el valor mínimo de dicho algoritmo.

Application of NGS-based SNP haplotyping for preimplantation genetic diagnosis for beta-thalassemia and HLA matching / 中华医学遗传学杂志

Objective@#To assess the value of next-generation sequencing (NGS)-based single nucleotide polymorphism (SNP) haplotyping for preimplantation genetic diagnosis (PGD) for beta-thalassemia coupled with human leukocyte antigen (HLA) matching.@*Methods@#Three couples were recruited. Couple 1 both carried a βIVS-2-654 variation and had previously given birth to a son with β thalassemia major. Couple 2 respectively carried βcd41-42 and βIVS-2-654 but had no history of pregnancy. Couple 3 respectively carried βCD17and βIVS-2-654, and had a daughter carrying βCD17.@*Results@#For couple 1, NGS-SNP typing identified two embryos not only unaffected with thalassemia but also with matched HLA. One blastocyst was transferred and resulted in successful pregnancy. A healthy baby was born at 39th week of gestation. Its umbilical blood was used to treat the sick brother through hemopoietic stem cell transplantation. For couple 2, seven blastocysts were obtained. Second transplantation has resulted in successful pregnancy. Prenatal diagnosis was consistent with PGD. For couple 3, two blastocysts not only unaffected with thalassemia but also with no pathogenic copy number variations were obtained. Transfer of one blastocyte resulted in successful pregnancy, and prenatal diagnosis was consistent with PGD.@*Conclusion@#NGS-based SNP typing is an useful tool for selecting embryos unaffected with beta-thalassemia and matched HLA through PGD.

Clinical application of SNP haplotype analysis in the preimplantation genetic diagnosis (PGD) of monogenic diseases / 临床检验杂志

Objective@#To investigate the clinical application value of single nucleotide polymorphism (SNP) haplotype analysis in the preimplantation genetic diagnosis (PGD) of monogenic diseases. @*Methods@#The whole genome amplification products of biopsied trophectoderm cells were analyzed by SNP haplotype analysis and verified by Sanger sequencing. @*Results@#A total of 205 embryos were performed SNP haplotype analysis and Sanger sequencing. Among them, Sanger sequencing failed in 14.63% (30/205) of embryos, and SNP haplotype analysis failed in 0.98% (2/205) of embryos. The failure rate of the latter was significantly lower than that of the former (P＜0.05). There were consistent results in 155 (75.61%) embryos, and inconsistent results in 18 (8.78%) embryos. Forty-five embryos in 41 cycles were performed embryo transplantation. The clinical pregnancy rate was 70.73% (29/41) and the implantation rate was 71.11% (32/45). The results of prenatal diagnosis of amniotic fluid during the second trimester of pregnancy were completely consistent with those of SNP haplotype analysis. @*Conclusion@#SNP haplotype analysis is accurate, and its failure rate is lower than that of Sanger sequencing. It can be effectively used in the PGD of clinical monogenic diseases.

Advances in preimplantation genetic diagnosis / 中华内分泌代谢杂志

In the past 20 years,molecular genetic technology has developed rapidly.The leap forward development of single-cell genetic diagnosis technologies represented by whole genome amplification combined with microarray technology or next-generation sequencing not only increased the accuracy of preimplantation genetic diagnosis (PGD) but also greatly expanded the variety and scope of detectable diseases.This paper systematically reviews the clinical application of PGD as well as recent progress of related technologies.

Birth of a healthy baby after preimplantation genetic diagnosis in a carrier of mucopolysaccharidosis type II: The first case in Korea / 대한생식의학회지

Mucopolysaccharidosis type II (MPS II) is a rare X-linked recessive lysosomal storage disease caused by mutation of the iduronate-2-sulfatase gene. The mutation results in iduronate-2-sulfatase deficiency, which causes the progressive accumulation of heparan sulfate and dermatan sulfate in cellular lysosomes. The phenotype, age of onset, and symptoms of MPS II vary; accordingly, the disease can be classified into either the early-onset type or the late-onset type, depending on the age of onset and the severity of the symptoms. In patients with severe MPS II, symptoms typically first appear between 2 and 5 years of age. Patients with severe MPS II usually die in the second decade of life although some patients with less severe disease have survived into their fifth or sixth decade. Here, we report the establishment of a preimplantation genetic diagnosis (PGD) strategy using multiplex nested polymerase chain reaction, direct sequencing, and linkage analysis. Unaffected embryos were selected via the diagnosis of a single blastomere, and a healthy boy was delivered by a female carrier of MPS II. This is the first successful application of PGD in a patient with MPS II in Korea

Ethical problems with the preimplantation genetic diagnosis of human embryos / Problemas éticos con el diagnóstico genético preimplantacional de embriones humanos / Problemas éticos com diagnóstico pré-implantacional de embriões humanos

Abstract: 14. The purpose of preimplantation genetic diagnosis by embryonary biopsy is to identify genetic alterations prior to the implantation of embryos produced by in vitro fertilization. The most important aim is the selection of genetically healthy embryos due to their genetic indemnity, but it can also be used to select the sex or, eventually, other detectable traits accrding to the wishes of the parents. This procedure has been the subject of scientific debates, in relation to the harm that it can cause to healthy embryos that are going to be implanted, and in relation to the interpretation of the genetic tests made. Ethical debates have also focused on the production of and respect for the life and the integrity of developing human beings. In this work, it is argued that most of the uses of PGD are morally reprehensible, because they are done with disregard to the dignity that should be granted to embryos as human persons.

Resumen: 18. El diagnóstico genético preimplantacional (DGP) mediante biopsia embrionaria tiene como objeto la detección de alteraciones genéticas previamente a la implantación de embriones producidos por fertilización in vitro (FIV). Su finalidad más significativa es la selección de embriones por su indemnidad genética. También se puede emplear para seleccionar el sexo o eventualmente otras características detectadas según el deseo de los padres. Este procedimiento ha sido objeto de debates en el ámbito científico, por el eventual daño que puede ocasionar la técnica en embriones sanos que serán implantados y por las interpretaciones de los exámenes genéticos realizados. También ha sido objeto de debates en el ámbito ético-antropológico, en cuanto a la producción y al respeto a la vida e integridad de los seres humanos en desarrollo. En este trabajo se argumenta que los usos que se hacen del DGP son, en su gran mayoría, moralmente reprochables, por hacerse con desprecio de la dignidad que debe darse al embrión como persona humana.

Resumo: 22. O Diagnóstico genético pré-implantacional (PGD) por meio de biópsia embrionária visa a identificação de alterações genéticas prévias à implantação de embriões produzidos por fertilização in vitro (FIV). Seu propósito mais significativo é a seleção de embriões por sua característica genética. Ele também pode ser usado para selecionar o sexo ou, eventualmente, outras características identificadas de acordo com os desejos dos pais. Este procedimento tem sido tema de debate em âmbito científico, por eventual dano que pode ocaciosionar a técnica em embriões saudáveis que serão implantados e pela interpretações dos exames genéricos realizados. Ele também tem sido objeto de debate na área ético-antropológica, no que concerne a produção e o respeito à vida e integridade do ser humano em desenvolvimento. Este artigo argumenta que os usos que são feitos do PGD são, em sua grande maioria, moralmente condenáveis, por ser instrumentalizado com desrespeito pela dignidade que deve ser dada ao embrião como uma pessoa humana.

Research progress on new techniques for preimplantation genetic diagnosis / 医学研究生学报

Preimplantation genetic diagnosis (PGD) was developed to play a supporting role in assisted reproductive technology. With this kind of detection method, embryos with copy number variations, chromosome translocations or single mutations were excluded and the normal embryos were chosen and implanted. Theoretically, the application of these procedures could improve the implantation and pregnancy rate and help to delivery healthy offspring. PGD was considered to be more precise, higher specific and non-invasive with the appearance of microarray hybridization technology, the next generation sequencing and time-lapse monitoring technology. This paper presented a review of new Methods used in PGD, including fluorescence in situ hybridization, array comparative genomic hybridization, SNP array, next generation sequencing, MicroSeq-PGD, MaReCs, time-lapse monitoring and cfDNA-based method, and their advantages and disadvantages as well as efficacy in the procedures in which they are used.

Preimplantation genetic diagnosis for infertile males with autosomal dominant polycystic kidney disease / 中华男科学杂志

<p><b>Objective</b>Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic renal diseases, which may cause oligoasthenospermia and azoospermia and result in male infertility. This study aimed to analyze the outcomes of preimplantation genetic diagnosis (PGD) in male patients with ADPKD-induced infertility.</p><p><b>METHODS</b>We retrospectively analyzed the clinical data on 7 male patients with ADPKD-induced infertility undergoing PGD from April 2015 to February 2017, including 6 cases of oligoasthenospermia and 1 case of obstructive azoospermia, all with the PKD1 gene heterozygous mutations. Following intracytoplasmic sperm injection (ICSI), we performed blastomere biopsy after 5 or 6 days of embryo culture and subjected the blastomeres to Sureplex whole-genome amplification, followed by haplotype linkage analysis, Sanger sequencing, array-based comparative genomic hybridization to assess the chromosomal ploidy of the unaffected embryos, and identification of the unaffected euploid embryos for transfer.</p><p><b>RESULTS</b>One PGD cycle was completed for each of the 7 patients. Totally, 26 blastocysts were developed, of which 12 were unaffected and diploid. Clinical pregnancies were achieved in 6 cases following 7 cycles of frozen embryo transplantation, which included 5 live births and 1 spontaneous abortion.</p><p><b>CONCLUSIONS</b>For males with ADPKD-induced infertility, PGD may contribute to high rates of clinical pregnancy and live birth and prevent ADPKD in the offspring as well. This finding is also meaningful for the ADPKD patients with normal fertility.</p>

Importance of genetic and preimplantation genetic diagnosis in the prevention and treatment of inherited pediatric oculopathies / 中华实验眼科杂志

Inherited pediatric oculopathies are a group of pediatric eye chiseases caused by genetic defects,and they are the primary causes of irreversible pediatric blindness.Common inherited pediatric oculopathies include inherited retinopathies,congenital cataract and retinoblastoma.Inherited pediatric oculopathies present distinct genetic and clinical heterogeneity.Lots of disease causative genes are involved in pathogenesis inherited of pediatric oculopathies,which brings many difficulties for genetic diagnosis.Concealed and complicated phenotypes and poor cooperation make it even more difficult for precise clinical diagnosis.Therefore,exploring molecular causes of inherited pediatric oculopathies,providing professional genetic concealing and guiding eugenic measures are important bases for better clinical diagnosis,disease intervention and gene therapy.It is also one of the current hotspots for ophthalmic research.

Pre-implantation genetic diagnosis and pre-implantation genetic screening: two years experience at a single center

OBJECTIVE: Indications for preimplantation genetic diagnosis (PGD)/preimplantation genetic screening (PGS) cycles and clinical outcomes were evaluated at CHA Gangnam Medical Center. METHODS: This is retrospective cohort study. All patients (n=336) who went through in vitro fertilization (IVF)-PGD/PGS cycles (n=486) between January 2014 and December 2015 were included in Fertility Center of CHA Gangnam Medical Center. Patients underwent IVF-PGD/PGS with 24-chromosome screening. Patients with euploid embryos had transfer of one or 2 embryos in a fresh cycle with any subsequent frozen embryo transfer (ET) cycle. Compared implantation, clinical pregnancy, ongoing pregnancy, and early abortion rates were the main outcome measures. RESULTS: The most common indication for PGD/PGS was recurrent spontaneous abortion (n=160). The chromosome rearrangement cases (n=116) included 24 Robertsonian translocations, 60 reciprocal translocations, 3 inversions, 2 deletions, 4 additions, and 23 mosaicisms. PGS cases rather than the PGD cases showed higher implantation rates (26.4% vs. 20.3%), ongoing pregnancy rates (19.5% vs. 16.4%), and clinical pregnancy rates (28.6% vs. 23.3%). Implantation rates (30.3% vs. 23.7%), clinical pregnancy rates (39.2% vs. 25.2%), and ongoing pregnancy rates (25.7% vs. 17.5%) were significant higher in the blastocyst evaluation group than cleavage stage evaluation group. CONCLUSION: This was the largest study of PGD/PGS for 2 years at a single center in Korea. The pregnancy outcomes of PGD cases are slightly lower than PGS cases. It was confirmed again that success rate of PGD/PGS is higher if biopsy was done at blastocyst than cleavage stage.

Outcomes of Preimplantation Genetic Diagnosis Cycles by Fluorescent Hybridization of Infertile Males with Nonmosaic 47,XYY Syndrome / 中华医学杂志(英文版)

Background@#The 47,XYY syndrome could result in fertility problems. However, seldom studies reported comprehensive researches on the embryonic development and pregnancy outcomes of these patients. This study aimed to evaluate the clinical outcomes of nonmosaic 47,XYY patients performed with fluorescent in situ hybridization (FISH) and preimplantation genetic diagnosis (PGD) treatment.@*Methods@#This was a retrospective study. Between January 2012 and May 2017, 51 infertile males with nonmosaic 47,XYY syndrome underwent FISH-PGD were included in the study. According to sex chromosomal FISH results, embryos were classified as normal signal, no nuclei fixed, no signal in fixed nuclei, suspensive signal, and abnormal signal groups, respectively. The incidence of each group, the fixation rate, and hybridization rate were calculated. Embryonic development and pregnancy outcomes were also analyzed. The measurement data were analyzed with Student's t-test. The comparison of categorical data was analyzed with the Chi-square test and Fisher's exact test when expected cell count was 0.05), and were significantly lower than the normal signal group (66.4%, P < 0.001). The clinical pregnancy rates of fresh and frozen embryos transferred cycles were 70.6% and 85.7%, respectively.@*Conclusions@#Among embryos with a clear diagnosis of sex chromosome, about one-fifth showed abnormal signals. Embryos with two sex chromosomal signals are more likely to develop into good-quality ones. The application of the PGD by FISH may help to improve the clinical outcome s.

Reproductive intervention for inborn errors of metabolism / 中华实用儿科临床杂志

Inborn errors of metabolism(IEM)are a large class of genetic diseases caused by congenital metabolic disturbance.IEM results in complex clinical phenotype and it is an important cause of disability,death,and stupidity for the children.The technique of reproductive intervention including prenatal diagnosis(PND)or preimplantation genetic diagnosis(PGD)is an important approach to avoid the birth of defect.According to the two basic characteristics of IEM,metabolic disturbance and gene mutation,the clinical application of PND/PGD technology in IEM are reviewed,and put forward a new idea for reproductive intervention based on carrier screening.

Preimplantation Genetic Diagnosis of X-linked Severe Combined Immunodeficiency / 中山大学学报(医学科学版)

[Objective]To establish a reliable and accurate preimplantation genetic diagnosis (PGD)method using multiple dis?placement amplification (MDA), which can be applied to the diagnosis of X-linked severe combined immunodeficiency disease (X-SCID).[Methods]Haplotype analysis for the X-SCID family was performedusing five short tandem repeats (STR) markers flanking the both sides of the interleukin-2 (IL-2) receptor gamma chain (IL2RG) gene. MDA technique was used for single-cell whole genomic amplification. The products were used as template in polymerase chain reaction (PCR) of informative STR markers found by linkage analysis for haplotype analysis as well as sequencing of the IL2RG gene exon 5.The amelogenin (AMEL) locus was used to do sex diag?nosis.[Results]Linked analysis revealed 3 STR markers were informative. The method was evaluated with 10 single lymphocytes and 10 single blastomeres. MDA was successful in all single cell. The detection efficiency of gene sequencing of pathogenic IL 2RG exon5 was 100%. The PCR efficiency of 3 STR informative markers and AMEL was 96.3%(77/80)and the average allele drop-out (ADO) rate was 11.5%(7/61). A cycle of PGD was performed on the family, and seven embryos were diagnosed, two of which were normal embry?os. Twin pregnancy occurred after transplantation which were given a healthy baby boy and a healthy baby girl.[Conclusion]In this study, multiple displacement amplification combined with specific amplification/sequencing of pathogenic gene and haplotype analysis in the single cell level of X-linked severe combined immunodeficiency disease were performed. The protocol can avoid misdiagnosis caused by contamination and ADO, and improve the diagnostic efficiency of PGD.

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.

Mutation analysis of a Chinese family with Alport syndrome and genetic diagnosis before embryo implantation / 中华实用儿科临床杂志

Objective To study the gene mutation of a Chinese family with Alport syndrome and to perform preimplantation genetic diagnosis before embryo implantation.Methods Next generation sequence analysis was done for checking COL4A3,COL4A4 and COL4A5 genes in the Alport syndrome family members.Array comparative genomic hybridization(CGH) was used to detect the embryos.Results A mutation c.2605G ＞ A was found and identified in COL4A5 gene of all of the Alport syndrome patients in the family,but COL4A3 and COL4A3 genes were normal in all of the detected people.After searching for the mutation database,the mutation c.2605G ＞ A of COL4A5 gene was related to the X-linked dominant Alport syndrome.Three embryos were detected by using the preimplantation genetic diagnosis.Among these embryos,there were two male and one female.One of the male embryos was chromosomal aneuploidy,which was 45,XY,-16 and the other was normal.This normal embryo was implanted,and after 20 weeks the prenatal amniocentesis diagnosis approved that the fetus was normal.Conclusions The mutation of COL4A5 gene (c.2605 G ＞ A) is the cause of Alport syndrome in this family,which indicates that next generation sequence analysis proves to be an accurate and rapid method to detect Alport syndrome disease.Meanwhile array CGH can be used to reduce birth rates as a useful preimplantation genetic diagnosis method.

Diagnóstico genético pré-implantacional e sua aplicação na reprodução humana assistida / Pre-implantation genetic diagnosis and application in human reproduction

INTRODUÇÃO: A infertilidade causada por fatores genéticos acomete um grande número de casais que buscam tratamento de reprodução humana assistida. O diagnóstico genético pré-implantacional (PGD) é uma técnica usada durante a reprodução humana assistida que investiga alterações cromossômicas e genéticas em embriões in vitro durante diferentes estágios de seu desenvolvimento e seleciona os embriões livres de alterações genéticas para implantação uterina. Três tipos de biópsia podem ser feitos no PGD: dos glóbulos polares, dos blastômeros e do blastocisto, em ordem cronológica de acordo com o desenvolvimento embrionário. OBJETIVO: Analisar a aplicabilidade e a relevância da técnica PGD na rotina laboratorial da reprodução humana assistida e revisar sua metodologia. MÉTODO: Revisão de literatura sobre a técnica e os questionamentos éticos envolvidos com o PGD. Resultados: Os dados existentes atualmente sugerem que não há efeitos detrimentais sobre os embriões que sofreram biópsia. No Brasil o PGD é visto como uma ferramenta no auxílio da reprodução humana assistida e apresenta questões éticas ainda em discussão. CONCLUSÕES: O PGD respeita a visão e os valores da sociedade que considera a saúde como um de seus maiores bens.

INTRODUCTION: Infertility caused by genetic factors affect a large number of couples seeking treatment assisted human reproduction. The preimplantation genetic diagnosis (PGD) is a technique used during assisted human reproduction that investigates chromosomal and genetic abnormalities in vitro embryos during different stages of development and select genetic healf embryos to the uterine implantation. Three types of biopsy may be performedin PGD: biopsy of polar cells, biopsy of blastomere and biopsy of the blastocyst in chronological order according to embryonic development. OBJECTIVE: to analyze the applicability and relevance ofthe PGD technique on human assisted reproduction laboratory routine and review its methodology. METHOD: This article provides an overview on the technical and ethical issues involved with PGD. RESULTS: Many studies suggest that there is currently no detrimental effects on embryos that underwent biopsy. PGD in Brazil is seen as a tool to aid in the assisted human reproduction and presents ethical issues still under discussion. CONCLUSIONS: The PGD respects the vision and values of the society that considers health asone of its greatest assets.

Origen de las aneuploidías en blastocistos humanos, analizados por la técnica de polimorfismo de nucleótido único con parental support

Introducción: El diagnóstico genético preimplantacional (PGD) por medio de las técnicas de aCGH y polimorfismo de nucleótido único (SNPs - single nucleotide polymorphism) se ha convertido en una herramienta útil en los ciclos de reproducción asistida, superando al PGD por hibridación fluorescente in situ (FISH). Estas técnicas nos permiten conocer las aneuploidías en cada uno de los cromosomas. Además, la SNPs con Parental Support (Natera, Inc) posibilita conocer el origen de la aneuploidía en los blastocistos, si por el espermatozoide o por el ovo-cito. Objetivos: Determinar la tasa de aneuplodías únicas en embriones humanos obtenidos por reproducción asistida, utilizando la técnica de polimorfismo de nucleótido único. Diseño: Estudio retrospectivo. Institución: Grupo PRANOR, Sede Monterrico, y Genomics Perú, Lima, Perú. Material biológico: Embriones humanos. Intervenciones: Análisis de los registros de 429 embriones estudiados con PGD por SNPs, embriones obtenidos de 105 ciclos de reproducción asistida, entre 2011 y 2013. Principales medidas de resultados: Aneuploidía de embriones, relación con edad materna y origen paterno o materno. Resultados: El 48,8% de embriones resultó normal, con tasa de aneuploidía de 51,2%. La proporción de embriones sanos varió de acuerdo a la edad de la madre, disminuyendo cuando la edad aumentaba. En todos los grupos etarios estudiados más de 66% de las aneuploidías fue de origen materno, incluyendo el grupo de ovodonación (OD). Conclusiones: El diagnóstico preimplantacional mediante SNPs tendría gran valor pronóstico y sería una herramienta útil para conocer el origen de las aneuploidías en los embriones de las pacientes que se someten a procedimientos de reproducción asistida.

Background: Preimplantation genetic screening (PGS) using comparative genomics hybridization (aCGH) and single nucleotide polymorphism (SNP) technology has become a useful tool in assisted reproduction by improving clinical outcomes. Both techniques identify aneuploidies and chromosomal rearrangement. However, SNPs with Parental Support (Natera Inc.) give extra information of the aneuploidy origin either from the sperm or the oocyte. Objectives: To determine single aneuplodies rate in human embryos obtained by assisted reproduction using single nucleotide polymorphism microarray. Design: Retrospective study. Setting: Grupo PRANOR, Sede Monterrico, and Genomics Peru, Lima, Peru. Biologic material: Human embryos. Interventions: From 2011 to 2013, 105 patients underwent IVF/ ICSI with SNPs and Parental Support. In total, 429 embryos were analyzed and records were reviewed. Main outcome measures: Embryos aneuploidy, relation with maternal age and paternal or maternal origin. Results: From the 429 embryos 208 (48.8%) were chromosomally normal. The proportion of normal embryos decreased with increasing maternal age. In all age groups more than 66% of aneuploidies had maternal origin, including the ovodonation group (OD). Conclusions: PGS by SNPs with Parental Support resulted in good prognostic value and would be useful in determining the origin of aneuploidy in embryos of patients who underwent assisted reproduction technology.

Diagnóstico genético preimplantacional: análisis de aneuploidías únicas / Preimplantational genetic diagnosis: single aneuploidies analysis

Introducción: De las causas más conocidas en cuanto a la falta del éxito en el embarazo con tratamientos de reproducción asistida son aquellas relacionadas a las aneuploidías cromosómicas presentes en los embriones. El diagnóstico genético preimplantacional (PGD) es una técnica empleada en reproducción asistida para detectar estas anomalías, seleccionando aquellos que sean cromosómicamente normales, para luego transferirlos al útero de la paciente. Los embriones con aneuploidías únicas podrían tener la capacidad de sobrevivir y lograr la implantación, y por lo tanto, sin diagnóstico previo, estas podrían pasar desapercibidas. Objetivos: Determinar la incidencia de aneuploidías únicas en embriones de buena calidad embrionaria en el día 3 de desarrollo hasta blastocisto. Diseño: Estadístico y experimental. Instituciones: Reprogenetics Latinoamérica y Centro de Reproducción asistida, de la Clínica Concebir. Material Biológico: Muestras de biopsia embrionaria. Metodología: Análisis comparativo de resultados a partir de la evaluación de cada muestra obtenida por biopsia en el día tercero y día quinto de desarrollo embrionario, realizando el PGD por hibridación in situ (FISH) y genómica comparada (aCGH), respectivamente. Resultados: El 62,9% de embriones que presentaron monosomías únicas al tercer día de desarrollo embrionario resultaron ser de 8 células. Pero cuando se evaluó por aCGH en día cinco, 42,3% resultó anormal, y de estos 37,5% perteneció al estadio de 8 células. El índice de monosomías únicas en blastocisto resultó ser 57,9% de un total de 84,2% de aneuploidías únicas. Conclusiones: Los embriones de 8 células en el tercer día de desarrollo embrionario son los más probables de llegar al estadio de blastocisto, así como presentar aneuploidías únicas.

Background: Known causes of unsuccessful pregnancy in couples undergoing assisted reproduction treatment include embryo aneuploidies. Preimplantation genetic diagnosis (PGD) is a technique used in assisted reproduction in order to detect these abnormalities, select embryos chromosomally normal and subsequently transfer to the patients’ uterus. Embryos with single aneuploidies may have the ability to survive and achieve unnoticed implantation. Objectives: To determine incidence of single aneuploidies in good quality embryos in third day of development to blastocyst. Design: Statistical and experimental study. Setting: Reprogenetics Latin-America and Assisted Reproduction Center - Concebir. Biologic material: Samples of embryo biopsies. Methods: Comparative analysis of results from evaluation of each sample obtained by embryo biopsy on the third and fifth days of embryonic development, performing PGD by respectively in situ hybridization (FISH) and comparative genomics (aCGH). Results: On third day of embryonic development 62.9% of embryos with single monosomy had 8-cell morphology. Though when evaluated by aCGH in the blastocyst stage 42.3% were abnormal and 37.5% of these belonged to the 8-cell stage. Single monosomies index in the blastocyst stage was 57.9% in 84.2% of single aneuploidies. Conclusions: Eight-cell embryos on the third day of embryonic development are most likely to reach blastocyst stage and have single aneuploidies.

Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p.N540K) / 대한생식의학회지

Hypochondroplasia (HCH) is an autosomal dominant inherited skeletal dysplasia, usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3). A 27-year-old HCH woman with a history of two consecutive abortions of HCH-affected fetuses visited our clinic for preimplantation genetic diagnosis (PGD). We confirmed the mutation in the proband (FGFR3:c.1620C>A, p.N540K), and established a nested allele-specific PCR and sequence analysis for PGD using single lymphocyte cells. We performed this molecular genetic analysis to detect the presence of mutation among 20 blastomeres from 18 different embryos, and selected 9 embryos with the wild-type sequence (FGFR3:c.1620C). A successful pregnancy was achieved through a frozen-thawed cycle and resulted in the full-term birth of a normal neonate. To the best of our knowledge, this is the first report of a successful pregnancy and birth using single-cell allele-specific PCR and sequencing for PGD in an HCH patient.