Hongzi Du

CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

Similar biological characteristics of human embryonic stem cell lines with normal and abnormal karyotypes

BACKGROUND Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar. METHODS Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines. RESULTS All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines. CONCLUSIONS The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research.

Derivation and characterization of human embryonic stem cell lines from poor quality embryos.

Poor quality embryos discarded from in vitro fertilization (IVF) laboratories are good sources for deriving human embryonic stem cell (hESC) lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses (ICMs) could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods (P>0.05). As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.

MiR-133b Regulates the Expression of the Actin Protein TAGLN2 during Oocyte Growth and Maturation: A Potential Target for Infertility Therapy

Infertility is an area of increasing in life science research. Although follicular maturation disorders and anovulation are the primary causations of infertility, its molecular mechanism is not well understood. Recent research has shown that microRNAs (miRNAs) might play an important role in the regulation of ovarian follicle development and maturation. In this study, the expression of miRNAs in metaphase I (MI) oocytes treated with or without insulin-like growth factor 1 (IGF-1) was observed by microRNA microarray analysis. Results show that 145 miRNAs were up-regulated and 200 miRNAs were down-regulated in MI oocytes after IGF-1 treatment. MiR-133b, which was up-regulated more than 30-fold, was chosen for further research. As a potential target of miR133b, transgelin 2 (TAGLN2) gene was down-regulated, at both transcription and translation levels, in miR-133b- over-expressed 293T cells, but TAGLN2 was up-regulated when the expression of miR-133b was inhibited. Furthermore, the expression level of TAGLN2 in the ovaries of 8-week- old mice was higher than that observed in 4-week-old mice. Immunofluorescence experiments showed that TAGLN2 was located in the cytoplasm. In general, our results indicate that miR-133b may play important roles in the growth and maturation of oocytes by regulating its potential target, TAGLN2, at both transcription and translation levels. Therefore, our research provides a potential new target for infertility therapy.

The effects of cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression in mice.

Cigarette smoking can harm fertility, but the existing research has targeted primarily on ovarian follicles, embryos or sex hormone. In this study, we tested cigarette smoke extract on ovulation, oocyte morphology and ovarian gene expression associated with inhibition of oxidative stress using C57BL/6 mice. Mice in the experimental group were administered a cigarette smoke extract (CSE) solution (2 mg/ml) orally daily, while the blank control group was given dimethylsulfoxide (DMSO). A positive control group (menadione) was used that received an intraperitoneal injection of 15 mg/kg menadione in oil solution daily. We found that the CSE group manifested a reduced diameter of zona pellucida-free oocyte (ZP-free OD) and a morphologically misshapen first polar body (PB). Our results suggest that CSE exposure is associated with a shrink size and poor quality of oocytes. Quitting smoking is a wise choice to ensure good fertility.

Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium.

Abstract Background: Cell-free nuclear DNA has been isolated from spent embryo culture medium. Whether this small amount of DNA can be amplified at the whole genome level and the concordance rate of karyotypes and specific alleles between biopsied cells and media has not been evaluated. Methods: Seven couples were recruited, 88 donated embryos and their corresponding media were collected for whole genome amplification (WGA). The efficiency of WGA, the concordance of chromosome status, and the HBB gene IVSII654 allele between biopsied cells and media were investigated. Results: After WGA, the DNA detection rate was 90.90% with a mean concentration of 26.15 ng/μl. The full chromosome concordance rate between biopsied cells and medium was 64.52%, and it increased to 90.00% for diploid blastocyst samples. Analysis of the mutated IVSII654 locus and SNP linkage verified that the DNA present in the medium originated from embryonic cells. Conclusion: We confirmed that nuclear DNA is present in spent culture medium and that the majority of this DNA can be amplified for subsequent analysis. Our results showed that non-invasive embryo genetic testing at the chromosomal-level using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications. KEY MESSAGES The aggressive biopsy step during PGD/PGS procedure would have a negative effect on the future development of the embryo. Cell-free nuclear DNA has been observed in spent embryo culture medium, which holds promise for the development of non-invasive PGD/PGS approaches. The presence of DNA in medium, its efficiency for WGA, and the concordance between chromosome status and the HBB gene IVSII654 allele as diagnosed from biopsied cells or medium were investigated. Non-invasive embryo genetic testing at the chromosomal-level and allele site using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications.

Defining Differentially Methylated Regions Specific for the Acquisition of Pluripotency and Maintenance in Human Pluripotent Stem Cells via Microarray

Background Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study. Methodology Here, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illuminas Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines. Conclusions This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.

Ovarian response prediction in controlled ovarian stimulation for IVF using anti-Müllerian hormone in Chinese women: A retrospective cohort study

Abstract The predictive value of anti-Müllerian hormone (AMH) in Chinese women undergoing in vitro fertilization (IVF) treatment is data deficient. To determine the attributes of AMH in IVF, oocyte yield, cycle cancellation, and pregnancy outcomes were analyzed. All patients initiating their first IVF cycle with gonadotropin-releasing hormone agonist treatment in our center from October 2013 through December 2014 were included, except patients diagnosed with polycystic ovarian syndrome. Serum samples collected prior to IVF treatment were used to determine serum AMH levels. A total of 4017 continuous cycles were analyzed. The AMH level was positively correlated with the number of oocytes retrieved. Overall, AMH was significantly correlated with risk of cycle cancellation, poor ovarian response (POR, 3, or fewer oocytes retrieved) and high response (>15 oocytes), with an area under the curve (AUC) of 0.83, 0.89, and 0.82 respectively. An AMH cutoff of 0.6 ng/mL had a sensitivity of 54.0% and a specificity of 90.0% for the prediction of cycle cancellation, and cutoff of 0.8 ng/mL with a sensitivity of 55.0% and a specificity of 94.0% for the prediction of POR. Compared with AMH >2.0 ng/mL, patients with AMH < 0.6 ng/mL had a 53.6-fold increased risk of cancellation (P < 0.001), and AMH <0.80 ng/mL were 17.5 times more likely to experience POR (P < 0.001). However, AMH was less predictive of pregnancy and live birth, with AUCs of 0.55 and 0.53, respectively. Clinical pregnancy rate, ongoing pregnancy rate, and live birth rate per retrieval according to the AMH level (⩽0.40, 0.41–0.60, 0.61–0.80, 0.81–1.00, 1.01–1.50, 1.51–2.00, and >2.00 ng/mL) showed no significant differences. Even with AMH⩽0.4 ng/mL, 50.0% of all the patients achieved pregnancy and 34.8% of patients achieved live birth after transfer. Our results suggested that AMH is a fairly robust metric for the prediction of cycle cancellation and oocyte yield for Chinese women, but it is a relatively poor test for prediction of pregnancy outcomes. Patients with low levels of AMH still can achieve reasonable treatment outcomes and low AMH levels in isolation do not represent an appropriate marker for withholding fertility treatment.

Highly efficient ssODN-mediated homology-directed repair of DSBs generated by CRISPR/Cas9 in human 3PN zygotes

Ma et al. published data in Nature Journal using human zygotes that showed that double-strand breaks (DSBs) generated by CRISPR/Cas9 could be repaired by homology-directed repair (HDR) using the wild-type allele as a template, by inter-homologue recombination (Ma et al., 2017). This result received great attention from the scientific community, however, in a recent publication, researchers have raised some concerns about Mas interpretation of their results (Egli et al., 2017). Our previous work showed that ssODN-mediated HDR could repair 75% of DSBs generated by CRISPR/Cas9 at G6PD mutant alleles (Tang et al., 2017), while no ssODN-mediated HDR repair was reported in Mas work. Thus, we set out to repeat Mas work using clinically discarded human tripronuclear (3PN) zygotes to exclude the influences of external experimental conditions, such as injected mixture preparation, injection equipment, or injection procedure.

Elevated incidence of monozygotic twinning is associated with extended embryo culture, but not with zona pellucida manipulation or freeze-thaw procedure

OBJECTIVE To identify the incidence and risk factors associated with IVF-conceived monozygotic twinning (MZT). DESIGN Retrospective study. SETTING Academic hospital. PATIENT(S) A total of 3,463 women with clinical pregnancies between January 2014 and February 2015 were analyzed. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The measures were the incidence of MZT based on the number of embryos that were replaced, type of insemination method (conventional IVF or intracytoplasmic sperm injection [ICSI]), with or without the use of assisted hatching (AH), and day of embryo transferred in fresh and frozen cycles. RESULT(S) Ninety-three women (2.69%) with MZT were observed. No statistically significant differences were observed in the cycle parameters of fresh or frozen cycles between MZT and other non-MZT pregnancies. Specific IVF procedures or techniques, such as the number of embryo replaced, zona pellucida manipulation (ICSI and AH), and freeze-thaw procedure, did not significantly increase the rate of MZT, except for the day of embryo transferred. Compared with day 3 transferred, day 4 and 5/6 transferred showed an increased probability of MZT (odds ratio [OR], 2.73; 95% confidence interval [CI], 1.16-6.42 for day 4 transferred and OR, 3.68; 95% CI, 2.29-5.93 for day 5/6 transferred). CONCLUSION(S) Extended culture (advanced embryo stage) in fresh and frozen cycles appeared to be associated with increased rates of MZT. The effect of the number of embryos transferred, ICSI and AH, and freeze-thaw procedures on the risk for MZT was not demonstrated.