Yanwen Xu

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

ABSTRACTGenome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

Relationship between antithyroid antibody and pregnancy outcome following in vitro fertilization and embryo transfer.

Objective: To investigate the impact of antithyroid antibody on pregnancy outcome following the in vitro fertilization and embryo transfer (IVF-ET). Methods: A total of 90 patients (156 cycles) positive for antithyroid antibody (ATA+ group) and 676 infertile women (1062 cycles) negative for antithyroid antibody (ATA- group) undergoing IVF/ICSI from August 2009 to August 2010 were retrospectively analyzed. Results: There was no significant difference in the days of ovarian stimulation, total gonadotropin dose, serum E2 level of HCG day and number of oocytes retrieved between the two groups. The fertilization rate, implantation rate and pregnancy rate following IVF-ET were significantly lower in women with antithyroid antibody than in control group (64.3% vs 74.6%, 17.8% vs 27.1% and 33.3% vs 46.7%, respectively), but the abortion rate was significantly higher in patients with antithyroid antibody (26.9% vs 11.8%). Conclusion: Patients with antithyroid antibody showed significantly lower fertilization rate, implantation rate and pregnancy rate and higher risk for abortion following IVF-ET when compared with those without antithyroid antibody. Thus, the presence of antithyroid antibody is detrimental for the pregnancy outcome following IVF-ET.

High follicle-stimulating hormone increases aneuploidy in human oocytes matured in vitro

OBJECTIVE To study the effect of FSH on the aneuploidy risk of human oocytes matured in vitro. DESIGN Prospective study. SETTING Hospital-based IVF center. PATIENT(S) Patients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S) Immature oocytes were put into five groups according to the FSH concentration (0, 5.5, 22, 100, and 2,000 ng/mL) in in vitro maturation (IVM) medium. Spindles were observed under a polarized microscope before polar body biopsy. Fixed polar bodies and corresponding oocytes were examined on chromosomes 13, 16, 18, 21, and 22 by fluorescence in situ hybridization. Oocytes matured in 5.5 and 2,000 ng/mL FSH were immunostained for tubulin and chromatin. MAIN OUTCOME MEASURE(S) Aneuploidy rate, spindle visualization rate, and spindle morphology. RESULT(S) The frequency rates of aneuploidy were 26.7%, 23.3%, 36.75%, 46.67%, and 63.3% in the five FSH groups, respectively. There was a significantly higher aneuploidy rate in oocytes matured in the 2,000 ng/mL FSH group. The spindle visualization rates assessed under PolScope were not significantly different between aneuploid and normal oocytes. There was no difference in spindle morphology between the 2,000 and 5.5 ng/mL FSH groups. CONCLUSION(S) High-concentration FSH in IVM medium significantly increased the first meiotic division error, resulting in more aneuploid oocytes during IVM.

Correction of β-thalassemia mutant by base editor in human embryos

Abstractβ-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G) mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Endometrial thickness as a predictor of pregnancy outcomes in 10787 fresh IVF-ICSI cycles.

This retrospective study assessed the predictive value of endometrial thickness (EMT) on HCG administration day for the clinical outcome of fresh IVF and intracytoplasmic sperm injection (ICSI) cycles. A total of 8690 consecutive women undergoing 10,787 cycles over a 5-year period were included. The 5th, 50th and 95th centiles for EMT were determined as 8, 11 and 15 mm, respectively. Group analysis according to these centiles (Group 1: < 8 mm; Group 2: ≥ 8 and ≤11 mm; Group 3: > 11 and ≤15 mm; Group 4: > 15 mm) demonstrated significant differences (P < 0.001) in clinical pregnancy rates (23.0%, 37.2%, 46.2% and 53.3%, respectively), live birth rates per clinical pregnancy (63.3%, 72.0%, 78.1% and 80.3%, respectively), spontaneous abortion rates (26.7%, 23.8%, 19.9% and 17.5%, respectively), and ectopic pregnancy rates (10.0%, 4.3%, 2.1% and 2.2%, respectively). Logistic regression analyses showed EMT as one of the independent variables predictive of clinical pregnancy (OR = 1.097; P < 0.001), live birth (OR = 1.078; P < 0.001), spontaneous abortion (OR = 0.948; P < 0.001), and ectopic pregnancy (OR = 0.851; P < 0.001). Future research should aim to understand the underlying mechanisms relating EMT to conception, ectopic implantation and spontaneous abortion.

Medium-based Noninvasive Preimplantation Genetic Diagnosis for Human α-thalassemias-sea

AbstractTo develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for &agr;-thalassemias-SEA.The embryos of &agr;-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of &agr;-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium.The diagnosis efficiency of medium-based &agr;-thalassemias–SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based &agr;-thalassemias–SEA detection is Day 5 (D5) following IVF.Medium-based &agr;-thalassemias–SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification

OBJECTIVE To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). DESIGN MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. SETTING Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. PATIENT(S) Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. INTERVENTION(S) The MDA of single cells and fluorescent PCR assays for PGD. MAIN OUTCOME MEASURE(S) The ability to analyze single blastomeres for DMD using MDA. RESULT(S) The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. CONCLUSION(S) We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9

The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5′-NGG-3′) recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5′-NNGRRT-3′) preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models.

Solid-surface vitrification is an appropriate and convenient method for cryopreservation of isolated rat follicles

BackgroundCryopreservation of isolated follicles may be a potential option to restore fertility in young women with cancer, because it can prevent the risks of cancer transmission. Several freezing protocols are available, including slow-rate freezing, open-pulled straws vitrification (OPS) and solid-surface vitrification (SSV, a new freezing technique). The purpose of our study was to investigate the effects of these freezing procedures on viability, ultrastructure and developmental capacity of isolated rat follicles.MethodsIsolated follicles from female Sprague-Dawley rats were randomly assigned to SSV, OPS and slow-rate freezing groups for cryopreservation. Follicle viability assessment and ultrastructural examination were performed after thawing. In order to study the developmental capacity of thawed follicles, we performed in vitro culture with a three-dimensional (3D) system by alginate hydrogels.ResultsOur results showed that the totally viable rate of follicles vitrified by SSV (64.76%) was slightly higher than that of the OPS group (62.38%) and significantly higher than that of the slow-rate freezing group (52.65%; P < 0.05). The ultrastructural examination revealed that morphological alterations were relatively low in the SSV group compared to the OPS and slow-rate freezing groups. After in vitro culture within a 3D system using alginate hydrogels, we found the highest increase (28.90 ± 2.21 μm) in follicle diameter in follicles from the SSV group. The estradiol level in the SSV group was significantly higher than those in the OPS and slow-rate freezing groups at the end of a 72-hr culture period (P < 0.05).ConclusionsOur results suggest that the SSV method is an appropriate and convenient method for cryopreservation of isolated rat follicles compared with the conventional slow-rate freezing method and the OPS method.

Evidence for Decreased Expression of ADAMTS-1 Associated With Impaired Oocyte Quality in PCOS Patients

CONTEXT Polycystic ovary syndrome (PCOS) is the most common cause of dysfunctional ovulation-affecting female fertility. A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS-1) is required for normal ovulation and subsequent fertilization, and the expression of ADAMTS-1 may be altered in PCOS granulosa cell (GC)-reflecting abnormalities in ovulatory signaling. OBJECTIVE The purpose of this paper is to analyze the differential expression of ADAMTS-1 in PCOS patients associated with impaired oocyte quality. DESIGN AND SETTING A prospective comparative experimental study was performed at a clinical reproductive medicine center. PATIENTS Women with PCOS (n = 40) and normovulatory controls (n = 40) undergoing controlled ovarian hyperstimulation and in vitro fertilization were recruited in our study. MAIN OUTCOME MEASURES Differential expression of ADAMTS-1 in GCs was analyzed with immunocytochemistry in PCOS patients and normal controls, and ADAMTS-1 mRNA expression was quantified by RT-PCR. Furthermore, the correlation between ADAMTS-1 mRNA and oocyte quality was analyzed. RESULTS The expression of ADAMTS-1 was decreased in PCOS patients compared with normally ovulating women and was closely related to lower oocyte recovery, oocyte maturity, and fertilization rate. CONCLUSION Our study provides evidence that the dysregulated expression of ADAMTS-1 in PCOS may influence oocyte quality-via GCs-oocyte paracrine and endocrine mechanism.