Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.

Digitization of clinical and epidemiological data from the Kyoto Collection of Human Embryos: maternal risk factors and embryonic malformations.

Understanding the causes of congenital anomalies is of prime importance to develop management and/or prevention strategies. It is widely accepted that the occurrence of congenital malformations in fetuses and neonates is heavily correlated with maternal genetic makeup and lifestyle. However, very few epidemiologic analyses have been conducted on the embryonic developmental period because of the rarity of data available. Instigated in 1961, the Kyoto Collection of Human Embryos comprises approximately 45,000 specimens of embryos and fetuses. The collection's most unique feature is that most specimens were added to the collection along with epidemiologic information on the respective mothers. This is the first report on the digitization of data from the collection. A total of 22,262 embryonic specimens were selected on the basis of data integrity. Data related to the embryos were then classified according to the following criteria: developmental stage, sampling period, geographical area, maternal determinant, and external malformation. Results indicate that 7.8% of the embryos exhibit external anomalies and 92.2% are without anomalies. The three most common anomalies were nuchal bleb, holoprosencephaly and spina bifida. A special emphasis was placed on the potential association between maternal determinants and embryonic external anomalies, allowing for statistical analyses. The present study provides further evidence that this collection represents a unique source of information to conduct epidemiological analyses, not only to further the understanding of congenital anomalies but also to help establish preventive health guidelines for pregnant women.