Yukiko Katagiri

Effects of assisted reproduction technology on placental imprinted gene expression.

We used placental tissue to compare the imprinted gene expression of IGF2, H19, KCNQ1OT1, and CDKN1C of singletons conceived via assisted reproduction technology (ART) with that of spontaneously conceived (SC) singletons. Of 989 singletons examined (ART n = 65; SC n = 924), neonatal weight was significantly lower (P < .001) in the ART group than in the SC group, but placental weight showed no significant difference. Gene expression analyzed by real-time PCR was similar for both groups with appropriate-for-date (AFD) birth weight. H19 expression was suppressed in fetal growth retardation (FGR) cases in the ART and SC groups compared with AFD cases (P < .02 and P < .05, resp.). In contrast, CDKN1C expression was suppressed in FGR cases in the ART group (P < .01), while KCNQ1OT1 expression was hyperexpressed in FGR cases in the SC group (P < .05). As imprinted gene expression patterns differed between the ART and SC groups, we speculate that ART modifies epigenetic status even though the possibilities always exist.

Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

Characterization of S100A11, a suppressive factor of fertilization, in the mouse female reproductive tract

We recently found that Xenopus dicalcin, present in the extracellular egg‐coating envelope, suppresses the efficiency of fertilization in vitro through binding to envelope‐constituent glycoproteins. In the present study, we explored the mouse counterpart of Xenopus dicalcin, specifically its localization in the female reproductive tract and its action on mouse fertilization. Our homology and phylogenetic analyses using known S100 proteins showed that S100A11 is most closely related to Xenopus dicalcin. S100A11 was localized in the cytosol of luteal cells, but not in the follicle, in the mouse ovary, and also in the cytosol of the oviductal epithelial cells. In addition, our quantitative analyses revealed preferential expression of S100A11 in the ampullary region of the oviduct and at the estrus stage during the mouse estrous cycle. In the cumulus cell–oocyte complex dissected from the oviduct following ovulation, S100A11 was present in the plasma membrane of cumulus cells, but not in the zona pellucida, which is comparable with Ca2+‐dependent binding of exogenously applied S100A11 to the plasma membrane of cumulus cells. Pretreatment of the cumulus cell–oocyte complex with recombinant S100A11 substantially reduced the efficiency of in vitro fertilization, but S100A10, the next closest S100 protein to Xenopus dicalcin, had no effect. These results suggested that S100A11 is the mouse counterpart of Xenopus dicalcin, suppresses the fertilization process through its action on cumulus cells, and thereby plays a key role in fertilization success in the mouse. Mol. Reprod. Dev. 78:91–103, 2011.

Influence of mosaicism on sexing of human preembryos detected by the polymerase chain reaction

AbstractPurpose: Preimplantation sex determination using a single cell by the polymerase chain reaction (PCR) was investigated to elucidate the influence of mosaicism. Methods: The SRY and ZFX genes were coamplified as target sequences for the Y and X chromosomes, respectively. The sensitivity of the single and nested PCR method was examined initially followed by amplification of single amniocytes by the nested PCR. Then the sex of single blastomeres at the three- and nine-cell stages was determined by the nested PCR. Results: The nested PCR was 104-fold more sensitive than the single PCR. Sex determination was possible in 97.5% (117/120) of the blastomeres tested. However, the correspondence rate for all blastomeres within a single embryo was only 60% (12/20 embryos). Among the remaining embryos for which sexing of all blastomeres was not consistent, only one blastomere showed findings indicating the presence of mosaicism (or pseudomosaicism). Conclusions: At least two blastomeres need to be assessed when determining the sex of an embryo in order to avoid misdiagnosis due to mosaicism (or pseudomosaicism).

Deletion detection for diagnosis of Duchenne muscular dystrophy in the Japanese population—Comparisom between the polymerase chain reaction and the Southern blot analysis

SummaryWe compared the efficacy of the multiplex PCR with that of the cDNA analysis for detection of deletions of the DMD gene in the Japanese patients. Thirty males with DMD from 27 Japanese families were studied by the multiplex PCR, and 24 of them were also investigated by Southern blot analysis. We used five dystrophin cDNA probes for deletion analysis. A total of 19 regions were amplified by the PCR to detect deletions, 9 regions by the method of Chamberlain et al. and another 10 regions by the method of Beggs et al. Deletions were detected in 14 (52%) out of 27 DMD families by the PCR. Southern blot analysis detected deletions in 14 (64%) out of 22 families. Thirteen (93%) of the 14 DMD families with deletions detected by Southern blotting were also confirmed by the multiplex PCR. Provided care is taken in cases where the deletion is limited to a single exon, the multiplex PCR appears to be an efficient and useful alternative to conventional Southern blot analysis for detecting deletions during the prenatal and postnatal diagnosis of DMD.

Factors affecting parental decisions to terminate pregnancy in the presence of chromosome abnormalities: A Japanese multicenter study

To investigate the rates of termination of pregnancy (TOP) for fetal chromosomal abnormalities and factors related to such parental decision in Japan.

Current status of non‐invasive prenatal testing in Japan

The purpose of this study was to report the 3‐year experience of a nationwide demonstration project to introduce non‐invasive prenatal testing (NIPT) of maternal plasma for aneuploidy, and review the current status of NIPT in Japan.

Prenatal Diagnosis of Duchenne Muscular Dystrophy by Polymerase Chain Reaction Analysis

The efficacy of the polymerase chain reaction (PCR) in the first-trimester prenatal diagnosis of Duchenne muscular dystrophy (DMD) was examined. Twenty-seven fetuses from 26 Japanese pedigrees at risk for DMD were analyzed. PCR-restriction fragment length polymorphism analysis, multiplex PCR, and dinucleotide repeat polymorphism analysis were used. Of 16 males, 11 were determined to be unaffected, 4 were affected, and the remaining 1 was undetermined. Of the 11 female fetuses, 1 was diagnosed as a noncarrier, 4 were carriers, and the carrier status of the remaining 6 was not determined at the option of the patients, although DNA polymorphisms could be detected in those patients. Prenatal diagnosis by PCR analysis was possible in 96% of the fetuses tested (26 of 27).

A survey on awareness of genetic counseling for non-invasive prenatal testing: The first year experience in Japan

The purpose of this study is to summarize the results from a survey on awareness of genetic counseling for pregnant women who wish to receive non-invasive prenatal testing (NIPT) in Japan. As a component of a clinical study by the Japan NIPT Consortium, genetic counseling was conducted for women who wished to receive NIPT, and a questionnaire concerning both NIPT and genetic counseling was given twice: once after pre-test counseling and again when test results were reported. The responses of 7292 women were analyzed. They expressed high satisfaction with the genetic counseling system of the NIPT Consortium (94%). The number of respondents who indicated that genetic counseling is necessary for NIPT increased over time. Furthermore, they highly valued genetic counseling provided by skilled clinicians, such as clinical geneticists or genetic counselors. The vast majority (90%) responded that there was sufficient opportunity to consider the test ahead of time. Meanwhile, women who received positive test results had a poor opinion and expressed a low-degree satisfaction. We confirmed that the pre-test genetic counseling that we conducted creates an opportunity for pregnant women to sufficiently consider prenatal testing, promotes its understanding and has possibilities to effectively facilitate informed decision making after adequate consideration. A more careful and thorough approach is considered to be necessary for women who received positive test results.

Epigenetics in assisted reproductive technology

It has been reported that the rates of epigenetic disorders such as Angelman syndrome (AS) and Beckwith-Wiedemann syndrome (BWS) are high in offspring conceived by assisted reproductive technology (ART). Angelman Syndrome is characterized by intellectual disability and BWS is known as large offspring syndrome (LOS). Weight abnormalities have also been reported in cloned animals. Possible factors underlying these findings include inherent gamete characteristics, influence of in vitro culture and peculiarity of ART methods. It is important to conclusively determine whether such epigenetic abnormalities are present in children conceived by ART, so as to consider the health of next generations.