Lingbo Cai

Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing

Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture. We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis, we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7 out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos.

Oocyte vitrification technology has made egg-sharing donation easier in China

When infertile women undergoing IVF or intracytoplasmic sperm injection (ICSI) have more than 20 mature oocytes retrieved, at least 15 oocytes are inseminated by their husbands spermatozoa. The extra oocytes are cryopreserved by vitrification. If the patients became pregnant and have healthy live births, the patients are encouraged to donate their remaining cryopreserved oocytes. Forty-seven egg-sharing donors were recruited after having normal deliveries and they donated their remaining oocytes, totalling 395 cryopreserved oocytes, to 75 recipients. The survival rate of vitrified-warmed oocytes was 83.0%. Following insemination by ICSI, the fertilization and cleavage rates were 83.8% and 89.8%, respectively. Out of 75 recipients, 71 recipients completed the treatment cycles and 30 of them became pregnant with clinical pregnancy and implantation rates of 42.3% and 25.5%, respectively. The birthweight of the new-born infants (22 from singleton and two from one set of twins) were 3344.5 ± 669.1g and 2425.0 ± 742.5 g, respectively. No birth defects were observed for the live births. These results indicate that oocyte vitrification is an effective methodology for an egg-sharing donation programme, with acceptable pregnancy and implantation rates.

Downregulation of both gene expression and activity of Hsp27 improved maturation of mouse oocyte in vitro

BackgroundHeat shock protein 27 (Hsp27), a member of the small heat shock protein family, is an apoptosis regulator. Our previous proteomic study showed that Hsp27 mainly expressed in human oocyte, and that Hsp27 expression was downregulated in the ovaries derived from women with the polycystic ovary syndrome (PCOS), a well known endocrinal disorder with abnormal apoptotic activity and folliculogenesis. However, the exact effects of Hsp27 downregulation on oocyte development have not yet been clarified.MethodsThe expression of Hsp27 gene was downregulated in the mouse oocytes cultured in vitro using siRNA adenovirus infection, while the activity of Hsp27 was decreased by microinjection of polyclonal Hsp27 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocyte maturation rate was evaluated by morphological observation. Early stage of apoptosis was determined using Annexin-V staining analysis and some critical apoptotic factors and cytokines were also monitored at both mRNA level by real time RT-PCR and protein expression level by immunofluorescence and western blot.ResultsHsp27 expressed at high level in maturing oocytes. Infection with AdshHsp27, and microinjection of Hsp27 antibody into GV oocytes, resulted in the improved oocyte development and maturation. Germinal vesicle breakdown (GVBD) rates were significantly increased in two AdshHsp27-treated groups (88.7%, 86.0%) and Hsp27 antibody-injected group (77.0%) when compared with control (76.2% in AdGFP, 64.4% in IgG-injected), respectively. In addition, the rates of metaphase II (MII) development in two AdshHsp27-treated groups (73.8%, 76.4%) and Hsp27 antibody-injected group (67.3%) were higher than that in the controls (59.6% in AdGFP, 55.1% in IgG-injected). We also found that the rates of early stage of apoptosis in Hsp27 downregulated groups (46.5% and 45.6%) were higher than that in control group (34.1%) after 8 h of IVM. Similarly, downregulation of Hsp27 caused a significantly enhanced the expression of apoptotic factors (caspase 8, caspase 3) and cytokines (bmp 15 and gdf 9).ConclusionsDownregulation of Hsp27 improved the maturation of mouse oocytes, while increased early stage of apoptosis in oocytes by inducing the activation of extrinsic, caspase 8-mediated pathway.

The clinical significance of sperm DNA damage detection combined with routine semen testing in assisted reproduction.

Traditional routine semen analysis and biochemical assays are insufficient for the determination of fertility status in individual men. In recent years, clinical evidence has shown that damaged human sperm DNA may adversely affect assisted reproductive outcomes and that the spermatozoa of infertile men possess substantially more DNA damage than the spermatozoa of fertile men. In this study, we combined these two methods to test their clinical significance in assisted reproductive techniques (ART). A total of 302 in vitro fertilization (IVF) and 67 intracytoplasmic sperm injection (ICSI) couples were included in the study. The acridine orange technique (AOT) was used to detect the DNA integrity of the sperm. Correlation analysis showed that the DNA fragmentation index (DFI) was negatively related to the no. of good quality embryos and the clinical pregnancy rate in IVF patients. No significant correlations were found between DFI and ICSI patients. We then combined the results of the routine semen testing of IVF patients with their DFI values. Statistical analysis revealed notable differences in the no. of good quality embryos (P=0.015) and the clinical pregnancy rate (P=0.015) between subgroups divided according to DFI value in a group with normal semen test results (Group 1). In a group with abnormal semen test results (Group 2), the fertilization rate (P=0.034) and the pregnancy rate (P=0.018) showed remarkable variation between DFI subgroups. In conclusion, the detection of damaged DNA in spermatozoa needs to be conducted along with standard semen analysis. This might prove to be a promising predictor of ART outcome, particularly in IVF.

Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa.

Purpose: In vitro maturation (IVM) of immature human oocytes is an attractive option for the treatment of infertility. Similarly, intracytoplasmic sperm injection (ICSI) followed by testicular fine needle aspiration (TEFNA) is an important treatment for primarily male-factor infertility. This report highlights the combination of these two advanced assisted reproduction techniques, namely IVM and fertilization with TEFNA-retrieved spermatozoa by ICSI to overcome both of male and female infertility problems.Methods: Before immature oocyte retrieval (IOR), gonadotropin stimulation was given for 3 or 5 days. Following IVM, and mature oocytes were inseminated by ICSI followed by TEFNA.Results: Four couples with five completed treatment cycles were performed, and total of 36 immature oocytes were retrieved. Following 36 to 48 h of culture, 32 (88.89%, 32/36) oocytes became mature. The mature oocytes were inseminated with TEFNA-retrieved sperm, and 18 (56.25%, 18/32) oocytes were fertilized normally following ICSI. Eleven embryos were transferred in five cycles and two pregnancies and two singleton births were achieved in two patients.Conclusions: This result demonstrates that the successful pregnancies and live births can be established from embryos produced from {in vitro} matured oocytes that fertilized with testicular sperm.

Successful PGD for late infantile neuronal ceroid lipofuscinosis achieved by combined chromosome and TPP1 gene analysis

Late infantile neuronal ceroid lipofuscinosis (NCL-2) is a severe debilitating autosomal recessive disease caused by mutations in TPP1. There are no effective treatments, resulting in early childhood death. A couple with two affected children presented for reproductive genetic counselling and chose to undertake IVF and preimplantation genetic diagnosis (PGD) to avoid the possibility of another affected child. However, DNA testing revealed only one mutation in the proband inherited from mother. Linkage analysis identified five informative linked short tandem repeat markers to aid the genetic diagnosis. Following IVF, five cleavage-stage embryos were biopsied and blastomeres were first subjected to whole-genome amplification, then a series of down-stream molecular genetic analyses to diagnose TPP1 genotype and finally array comparative genomic hybridization (CGH) to assess the chromosomal ploidy of each embryo. Two unaffected euploid embryos were identified for transfer. One was transferred on day 5 resulting in an ongoing pregnancy. Confirmatory prenatal diagnosis by amniocentesis showed concordance of the embryo and fetal diagnosis. As far as is known, this is the first successful report of PGD for NCL-2 using double-factor PGD with simultaneous single-gene testing and array CGH to identify an unaffected and chromosomally normal embryo for transfer.

Effects of Upregulation of Hsp27 Expression on Oocyte Development and Maturation Derived from Polycystic Ovary Syndrome

Heat shock protein 27 (Hsp27) is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated Hsp27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS) however, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS is unclear. The effect of Hsp27 over-expression was studied in vitro using oocytes derived from PCOS patients. An artificial GFP-plasmid was injected into human oocyte to increase Hsp27 protein level. Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our results revealed that Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile, fertilization rate and high quality embryo rate were similar between the Hsp27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Expression analysis revealed that the oocyte-secreted factors, BMP15 and GDF9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in Hsp27 overexpressing oocytes. In conclusion, upregulation of Hsp27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.

Downregulated expression of peroxiredoxin 4 in granulosa cells from polycystic ovary syndrome.

Peroxiredoxin 4 (PRDX4), a member of Peroxiredoxin (PRDX) family, is a typical 2-Cys PRDX. PRDX4 monitors the oxidative burden within cellular compartment and reduces hydrogen peroxide and alkyl hydroperoxide related to oxidative stress and apoptosis. Antioxidant, like PRDX4, may promote follicle development and participate in the pathophysiology of PCOS. In our previous study, we found that PRDX4 was expressed in mice oocyte cumulus oophorus complex, and that PRDX4 could be associated with follicle development. In this study, we explored the expression of PRDX4 in human follicles and possible role of PRDX4 in PCOS pathophysiology. Our data showed that PRDX4 was mainly expressed in granulosa cells in human ovaries. When compared to control group, both PRDX4 mRNA level and protein level decreased in PCOS group. The lowered levels of PRDX4 may relate to oxidative stress in the pathophysiologic progress of PCOS. Furthermore, expression of PRDX4 in the granulosa cells of in vivo or in vitro matured follicles was higher than that in immatured follicles, which suggested that PRDX4 may have a close relationship with follicular development. Altogether, our findings may provide new clues of the pathophysiologic mechanism of PCOS and potential therapeutic strategy using antioxidant, like PRDX4.

Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes

A normal fertilized human zygote contains two pronuclei, but zygotes may also display one, three, or even more pronuclei resulting from irregular insemination or meiotic division. Today diploid and triploid human embryonic stem cell (hESC) lines have been derived from tripronuclear (3PN) triploid zygotes, and an in‐vitro fertilization (IVF) baby was born from a rescued diploid zygote by removing the extra male pronucleus of the 3PN zygote. However, whether hESCs can be derived from a rescued 3PN zygote is still unknown. Here, by microsurgical pronuclear removal, we restored 61 diploid zygotes from 3PN zygotes donated by 35 couples, and 11 blastocysts developed with a blastocyst rate of 18.0%, which seems higher than that of nonrescued 3PN zygotes according to previous reports. After the whole zona pellucida free embryos were plated onto feeder cells to grow and passage, 2 hESC lines (CCRM‐hESC‐22 and CCRM‐hESC‐23) were generated and both carried normal karyotype (46, XY). The hESC lines were then characterized by morphology, expansion in vitro, and expression of specific markers of alkaline phosphatase, OCT4, SSEA4, TRA‐1‐60 and TRA‐1‐81. Furthermore, the pluripotency of these 2 hESC lines was confirmed by in vitro embryoid body formation and in vivo teratoma production. Our study indicates that depronucleared 3PN zygotes can improve the blastocysts formation rate, and normal hESC lines can be derived from those corrected 2PN embryos. Based on their multi‐directional differentiation potential in vitro, the established hESC lines could be applied to the developmental risk assessment for IVF babies born from restored zygotes. J. Cell. Biochem. 114: 2016–2023, 2013.

Effect of Hsp27 on early embryonic development in the mouse

Previous studies by this study group have showed that heat shock protein 27 (Hsp27), expressed in the oocyte of growing follicles, is down-regulated in polycystic ovary syndrome ovaries and that down-regulation of Hsp27 improves the maturation of mouse oocytes and increases early apoptosis of oocytes. In this study, the effect of Hsp27 on early embryo development in the mouse was observed. Following microinjection of AdCMV-Hsp27 or AdsiRNA-Hsp27 into the cytoplasm of mouse zygotes, blastocyst morphology was observed and cell apoptosis of blastocysts was detected by TUNEL. After culture in vitro for 96h, blastocysts were analysed for Hsp27 expression by real-time PCR and immunofluorescence. The blastocyst formation rate and embryo quality were evaluated. The expression of Hsp27 was significantly increased in embryos with Hsp27 overexpression (AdCMV-Hsp27), while it was significantly suppressed by 75% in embryos with the gene silenced (AdsiRNA-Hsp27; both P<0.05). Cell apoptosis in blastocysts, blastocyst formation rate and embryo quality were unaffected by Hsp27 overexpression or gene silencing. In conclusion, overexpression or down-regulation of Hsp27 in zygotes, as a single factor, does not significantly affect the subsequent embryonic development.