Quantificação do DNA e genotipagem de biópsias de embriões bovinos Gir e Girolando produzidos in vitro / DNA quantification and genotyping of biopsies obtained from Gyr and Girolando in vitro produced bovine embryos

A biópsia embrionária associada à genotipagem permite a obtenção de informações genômicas antes mesmo da transferência dos embriões. Neste estudo, foram avaliadas amostras biopsiadas de blastocistos bovinos transferidos para receptoras (n=47), sob a hipótese de que a raça (Gir ou Girolando), o estádio embrionário (blastocisto ou blastocisto expandido) e a competência para estabelecimento de prenhez (positiva ou negativa) afetariam a quantidade e a qualidade do DNA da amostra obtida. O DNA foi extraído, amplificado, quantificado por eletroferograma e genotipado. O parâmetro call rate (CR) foi adotado para mensurar a qualidade da genotipagem. Obteve-se concentração de DNA de 86,07±171,66ng/µL e CR 0,73±0,17. O CR não variou em função da quantidade de DNA nas amostras. As variáveis raça e estádio embrionário não influenciaram a concentração de DNA, nem o CR. Houve efeito da prenhez sobre o CR (P=0,0187), mas, como houve maior CR nas amostras provenientes do grupo prenhez negativa, não foi possível associar esse parâmetro à qualidade embrionária. Concluiu-se que a raça e a qualidade embrionária não afetam os parâmetros aqui estudados em amostras embrionárias, ou seja, embriões com maiores chances de implantação não refletem alta qualidade nas amostras de biópsia genotipadas.(AU)

Embryo biopsy associated with genotyping allows genomic information before embryo transfer. In this study, blastocyst biopsy samples from embryos transferred to recipients (n= 47) were evaluated, under the hypothesis that breed (Gyr or Girolando), embryonic stage (blastocyst or expanded blastocyst) and competence to establish pregnancy (positive or negative) would affect the quantity and DNA quality of samples. DNA was extracted, amplified, quantified by electropherogram and genotyped. The parameter call rate (CR) was used to measure the quality of genotyping. DNA concentration of 86.07±171.66ng/µl, and CR 0.73±0.17 was obtained. CR did not vary according to the amount of DNA in the samples. The variables breed and embryonic stage had no influence on DNA concentration or CR. There was pregnancy effect on the CR (P= 0.0187), but since there was a higher CR in the samples from the negative pregnancy group, it was not possible to associate this parameter with the embryonic quality. We conclude that the breed and embryo quality do not affect the evaluated parameters in embryonic samples. Embryos with higher chances of implantation do not reflect high quality in embryo biopsy genotyped samples.(AU)

Outcomes of preimplantation genetic diagnosis using either zona drilling with acidified Tyrode's solution or partial zona dissection / 대한생식의학회지

OBJECTIVE: To review the outcomes of preimplantation genetic diagnosis (PGD) using zona drilling with acid Tyrode's solution (chemical zona pellucida drilling, chemical ZD) and those of partial zona dissection (PZD). METHODS: Clinical outcomes of seventy-one couples undergoing 85 PGD cycles from January 2005 to December 2010 were included. Blastocyst formation and the hatching rate, clinical pregnancy rate, ongoing pregnancy rate, implantation rate, and fetal gender ratio of the PZD and chemical ZD groups were compared. RESULTS: Application of PZD resulted in a significantly higher rate of clinical pregnancy (40.7% vs. 15.4%, p=0.022), ongoing pregnancy (35.6% vs. 11.5%, p=0.023), and implantation (18.1% vs. 5.7%, p=0.007) compared with chemical ZD. Among non-transferred embryos, the rate of blastocyst formation on day 5 (49.1% vs. 39.5%, p=0.016) and hatching on day 6 (47.2% vs. 26.5%, p<0.001) were also significantly higher in the PZD group. CONCLUSION: The mechanical zona dissection method showed better outcomes than chemical ZD in terms of the blastocyst development and pregnancy rate. In this study, the fact that chemical ZD was conducted in different period from mechanical method should be considered in interpreting the result.

Avaliação genética pré-implantacional: visão geral / Preimplantation genetic testing: an overview

Identificar um embrião com alto potencial de implantação é um dos maiores desafios da medicina reprodutiva e um passo fundamental em direção à transferência de um único embrião. O diagnóstico genético pré-implantacional permite identificar alterações genéticas ou cromossômicas nos embriões em divisão antes de serem transferidos, enquanto o rastreamento genético pré-implantacional realiza a triagem em embriões quanto às anomalias cromossômicas frequentes. Dentre as limitações encontradas, o mosaicismo é responsável por grande parcela de erros de diagnóstico, e os altos custos das técnicas de hibridização in situ por fluorescência e reação em cadeia da polimerase ainda dificultam a implantação do diagnóstico genético pré-implantacional e do rastreamento genético pré-implantacional de forma rotineira. Este artigo pretende apresentar uma visão geral acerca das modalidades de avaliação genética préimplantacional disponíveis e seus resultados e avaliar criticamente a inserção dessas tecnologias entre os recursospropedêuticos para assistência reprodutiva.

The identification of an embryo with high implantation potential is one of the greatest challenges in reproductive medicine and a vital step towards transferring a single pre-embryo. Preimplantation genetic diagnosis allows the identification of genetic or chromosomal alterations in pre-embryos before they are transferred to the uterus, while preimplantation geneticscreening determines alleatory frequent chromosomal abnormalities. Among limitations, mosaicism is responsible for a large proportion of misdiagnosis, and the high costs of fluorescent in situ hybridization and polymerase chain reaction still hinder the implementation of preimplantation genetic diagnosis and preimplantation genetic screening as a routine. Thisarticle aims to present an overview of possible modalities of preimplantation genetic evaluation and their results and to criticallyevaluate the insertion of those technologies in the propaedeutical tools in reproductive assistance.

Preimplantation development of the cleavage stage embryos after laser assisted embryo biopsy.

This study was carried out to determine the effect of the duration of exposure to the infrared 1.48 mm diode laser, on the developmental potential of cleavage stage embryos. A total of 69 mouse embryos were included in the study. Twenty-two of which (group A) were biopsied using the laser with a longer duration of exposure (600 ms), meanwhile 47 (group B) were biopsied using the same laser with a shorter period (5 ms). The blastocyst formation rate of group B (46/47, 97.8%) was significantly higher than that of group A (12/22, 54.4%). There were no grade 1 blastocysts or hatching in group A. In contrast, 35 of 46 (76.0%) blastocysts in group B were grade 1 and the hatching rate of group B was 84.7% (39/46). In conclusion, the infrared 1.48 mm diode laser may be effective and safe with cautious application. A long duration of exposure to the laser can adversely affect the developmental potential of the biopsied embryos. The laser system with a shorter duration of exposure, therefore, is recommended for laser assisted embryo biopsy.