X-chromosome inactivation pattern of amniocytes predicts the risk of dystrophinopathy in fetal carriers of DMD mutations.

OBJECTIVE: To predict the risk of dystrophinopathy in fetal carriers of dystrophin gene (DMD) mutations. METHODS: Twenty-three pregnant women, with a total of 25 female fetuses carrying DMD mutations, were recruited. Among them, 13 pregnant women who participated in this study were only used to analyse the incidence of induced abortion after fetuses were diagnosed as dystrophinopathy carriers. Eleven fetal carriers from 10 pregnant women were tested to analyse X-chromosome inactivation (XCI) using amniocytes to assess the risk of dystrophinopathy. Follow-ups were conducted on all cases. RESULTS: Approximately one-third of fetuses were aborted before assessing the risk of dystrophinopathy. XCI analysis of amniocytes showed that 10 fetuses had random XCI patterns, and one fetus exhibited a highly skewed XCI pattern (100:0) with primary expression of the maternal X chromosome that carried the mutant allele. These 11 fetal carriers were born, and follow-up showed that the girl who showed the skewed XCI pattern as a fetus was diagnosed with Duchenne muscular dystrophy (DMD) at the age of four. The others did not present with dystrophinopathy-associated symptoms. CONCLUSIONS: XCI was significantly implicated in symptomatic female carriers of dystrophinopathies, and XCI pattern analysis of amniocytes may be useful in predicting the risk of dystrophinopathy in fetal carriers.

Identification of biparental and diploid blastocysts from monopronuclear zygotes with the use of a single-nucleotide polymorphism array.

OBJECTIVE: To select normal fertilized diploid blastocysts in patients who had only monopronucleated (1PN) embryos for transfer. DESIGN: Experimental study. SETTING: University-affiliated center. PATIENT(S): Couples who were undergoing intracytoplasmic sperm injection treatment and had 1PN blastocysts. INTERVENTION(S): In a preliminary test, limited cells of parthenogenetic human embryonic stem cells (phESCs) and normal fertilized blastocysts were analyzed with the use of a low-density single-nucleotide polymorphism (SNP) array to identify the distribution pattern and rate of heterozygosity. In the clinical application, 1PN blastocysts were analyzed with the use of the SNP array. Only diagnosed normal blastocysts were transferred. The diagnosed uniparental blastocysts were validated by imprinted gene expression. MAIN OUTCOME MEASURE(S): Distribution pattern and rate of heterozygosity between parthenogenesis and normal fertilization. RESULT(S): In the pretest, phESCs exhibited distinct distribution pattern and lower rate of heterozygosity, compared with normal fertilized blastocysts after SNP analysis. In particular, homozygous hESCs showed a panhomozygosity distribution pattern, hybrid phESCs showed a partial homozygosity distribution pattern, and normal fertilized blastocysts exhibited a panheterozygosity distribution pattern with an average of 20.21% heterozygosity rate; 13.6% was found to be the minimum cutoff to predict normal fertilized samples. In the clinical application, 24 1PN blastocysts were analyzed; 10/24 showed chromosomal abnormalities, 3/24 showed panhomozygosity with 0.45%-0.8% heterozygosity, and 1/24 showed partial homozygosity with 6.54% heterozygosity. The remaining 10 blastocysts, with a panheterozygosity distribution pattern and higher genomic heterozygosity rate, were diagnosed as normal-fertilization diploid embryos; three were transferred and resulted in two healthy newborns. CONCLUSION(S): The low-density SNP array might serve as a cost-effective method to identify biparental origin and diploid 1PN blastocysts for transfer.

Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation.

PURPOSE: We aimed to determine the developmental potential of human reconstructed oocytes after polar body genome transfer (PBT) and to report the case of a woman with multiple cycles of severe embryo fragmentation. METHODS: Fresh and cryopreserved first polar bodies (PB1s) were transferred to enucleated metaphase II oocytes (PB1T), while fresh PB2s were removed from fertilized oocytes and used instead of the female pronucleus in donor zygotes. Reconstructed oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured to blastocyst. Biopsied trophectoderm cells of PBT-derived blastocysts were screened for chromosomes by next-generation sequencing (NGS). Then, cryopreserved PB1T was carried out in one woman with a history of several cycles of extensive embryo fragmentation, and the blastocysts derived from PB1T were screened for aneuploidy but not transferred to the patient. RESULTS: There were no significant differences in the rates of normal fertilization and blastocyst formation between fresh and cryopreserved PB1T and control oocytes. Of the three fresh and three cryopreserved PB1T-derived blastocysts, two and one blastocysts exhibited normal diploidy respectively. In contrast, 17 PB2 transfers yielded 16 two pronuclei (2PN) zygotes with one normal and one small-sized pronucleus each and no blastocyst formation. In the female patient, 18 oocytes were inseminated by ICSI in the fourth cycle and the PB1s were biopsied. Although the embryos developed from the patient's own oocytes showed severe fragmentation, the oocytes reconstructed after PB1T produced three chromosomally normal blastocysts. CONCLUSIONS: Normal blastocysts can develop from human reconstructed oocytes after PB1T. The application of the first PB transfers may be beneficial to patients with a history of poor embryo development and excessive fragmentation.

Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.

How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.