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Guo F.,Peking University | Yan L.,Peking University | Yan L.,Key Laboratory of Assisted Reproduction | Guo H.,Peking University | And 45 more authors.
Cell | Year: 2015

Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from themigrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes. © 2015 Elsevier Inc.

Yu Y.,Peking University | Yu Y.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Yu Y.,Key Laboratory of Assisted Reproduction | Yan J.,Peking University | And 15 more authors.
Human Reproduction | Year: 2012

Background Human cloned blastocysts generated from oocytes following in vitro maturation (IVM) are a potential resource for embryonic stem cells (ESC) with homologous immune systems. The purpose of this study was to evaluate the effects of multiple growth factors [epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1)] on human oocyte maturation, early embryo development, blastocyst formation and ESC line generation. Methods Patients (n 344) undergoing IVF owing to male factor infertility were enrolled in this study. Metaphase II oocytes were separated into four grades based on their morphology. Spindle assembly from IVM oocytes with or without growth factor treatment was assessed by immunostaining. Piezo-assisted micromanipulation technology was used to produce fertilized (ICSI) and cloned [(somatic cell nuclear transfer (SCNT)] embryos. Embryos received four different growth factor treatments; embryo development rates from pronuclear to blastocyst stage and embryo grading (for quality) at the 8-cell stage were analyzed. The presence of receptors on human cumulus cells and IVM oocytes was assessed by immunofluorescence. The blastocysts generated from fertilized and cloned embryos were used for ESC derivation. Results The combination of EGF, BDNF and IGF-1 can effectively increase oocyte maturation rate in vitro, and significantly improve the oocyte quality in terms of morphology and normal spindle levels (P< 0.05). Also, the developmental competence of fertilized oocytes to 8-cell and blastocyst stages was improved by the addition of growth factors (P< 0.05). However, there were no significant differences among the four groups in 8-cell grading. Blastocyst formation in cloned embryos cultured with the three growth factors was higher than the control group (23.1 versus 4.3, P< 0.05). Receptors for the three growth factors were present in cumulus cells and IVM oocytes, and four human ESC lines were derived from fertilized blastocysts but none from cloned blastocysts. Conclusion This study demonstrated that EGF, BDNF and IGF-1 can improve oocyte maturation rate and quality in vitro, and consequently increase early embryo development and blastocyst formation, which is very beneficial in improving the reprogramming efficiency of SCNT. The present study has identified a valuable culture system for IVM and cloned human embryos, potentially using these embryos to derive human therapeutic ESC. © The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Huang J.,Peking University | Huang J.,Key Laboratory of Assisted Reproduction | Huang J.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Zhang L.,Translational Medicine Center | And 10 more authors.
Molecular Reproduction and Development | Year: 2015

The molecular pathogenesis of Klinefelter Syndrome (KS) is not fully understood. The aim of this study was to determine differences in gene expression patterns between KS patients and control individuals to help identify disease-related genes and biological pathways. Gene expression profiles of five KS patients and five healthy men were determined by microarray; 21 differentially expressed genes with a fold-change >1.5 and q-value <0.05 were identified between the groups. Genes associated with metabolism regulation and encoding liver fatty acid-binding protein (FABP1), aldehyde dehydrogenase 1 family member L1 (ALDH1L1), and vitronectin (VTN) were the most-significantly down-regulated in KS, as confirmed by quantitative reverse transcription PCR. Notably, none of these differentially expressed genes are normally found on the X chromosome. Thus, our results indicate that aberrant metabolism is involved in the pathogenesis of KS. Further elucidation of the how aberrant expression of metabolism-related genes affect the pathogenesis of KS may lead to the development of novel preventative and therapeutic strategies. © 2014 Wiley Periodicals, Inc.

Wang T.-R.,Peking University | Wang T.-R.,Shenyang University | Yan L.-Y.,Peking University | Yan L.-Y.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | And 21 more authors.
Human Reproduction | Year: 2014

STUDY QUESTIONWhat is the effect of basic fibroblast growth factor (bFGF) on the growth of individual early human follicles in a three-dimensional (3D) culture system in vitro?SUMMARY ANSWERThe addition of 200 ng bFGF/ml improves human early follicle growth, survival and viability during growth in vitro.WHAT IS KNOWN ALREADYIt has been demonstrated that bFGF enhances primordial follicle development in human ovarian tissue culture. However, the growth and survival of individual early follicles in encapsulated 3D culture have not been reported.STUDY DESIGN, SIZE, DURATIONThe maturation in vitro of human ovarian follicles was investigated. Ovarian tissue (n= 11) was obtained from 11 women during laparoscopic surgery for gynecological disease, after obtaining written informed consent. One hundred and fifty-four early follicles were isolated by enzymic digestion and mechanical disruption. They were individually encapsulated into alginate (1% w/v) and randomly assigned to be cultured with 0, 100, 200 or 300 ng bFGF/ml for 8 days.PARTICIPANTS/MATERIALS, SETTING, METHODSIndividual follicles were cultured in minimum essential medium α (αMEM) supplemented with bFGF. Follicle survival and growth were assessed by microscopy. Follicle viability was evaluated under confocal laser scanning microscope following Calcein-AM and Ethidium homodimer-I (Ca-AM/EthD-I) staining.MAIN RESULTS AND THE ROLE OF CHANCEAfter 8 days in culture, all 154 follicles had increased in size. The diameter and survival rate of the follicles and the percentage with good viability were significantly higher in the group cultured with 200 ng bFGF/ml than in the group without bFGF (P < 0.05). The percentage of follicles in the pre-antral stage was significantly higher in the 200 ng bFGF/ml group than in the group without bFGF (P < 0.05), while the percentages of primordial and primary follicles were significantly lower (P < 0.05).LIMITATIONS, REASONS FOR CAUTIONThe study focuses on the effect of bFGF on the development of individual human early follicles in 3D culture in vitro and has limited ability to reveal the specific effect of bFGF at each different stage. The findings highlight the need to improve the acquisition and isolation of human ovarian follicles.WIDER IMPLICATIONS OF THE FINDINGSThe in vitro 3D culture of human follicles with appropriate dosage of bFGF offers an effective method to investigate their development. Moreover, it allows early follicles to be cultured to an advanced stage and therefore has the potential to become an important source of mature oocytes for assisted reproductive technology; particularly as an option for fertility preservation in women, including patients with cancer. © 2014 The Author.

Zhu J.,Peking University | Zhu J.,Key Laboratory of Assisted Reproduction | Zhu J.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Zhuang X.,Peking University | And 11 more authors.
Human Reproduction | Year: 2015

STUDY QUESTION Does embryo culture medium influence the percentage of males at birth? SUMMARY ANSWER The percentage of males delivered after ICSI cycles using G5™ medium was statistically significantly higher than after cycles where Global, G5™ PLUS, and Quinn's Advantage Media were used. WHAT IS KNOWN ALREADY Male and female embryos have different physiologies during preimplantation development. Manipulating the energy substrate and adding growth factors have a differential impact on the development of male and female embryos. STUDY DESIGN, SIZE AND DURATION This was a retrospective analysis of the percentage of males at birth, and included 4411 singletons born from fresh embryo transfer cycles between January 2011 and August 2013 at the Center for Reproductive Medicine of Third Hospital Peking University. PARTICIPANTS/MATERIALS, SETTING, AND METHODS Only singleton gestations were included. Participants were excluded if preimplantation genetic diagnosis, donor oocytes and donor sperm were used. The database between January 2011 and August 2013 was searched with unique medical record number, all patients were present in the database with only one cycle. Demographics, cycle characteristics and the percentage of male babies in the four culture media groups were compared with analysis of variance or χ2 tests. Multivariable logistic regression was done to determine the association between the sex at birth and culture media after adjusting for other confounding factors, including parental age, parental BMI, type of infertility, parity, number of embryos transferred, number of early gestational sacs, cycles with testicular sperm aspiration (TESA)/percutaneous epididymal sperm aspiration (PESA)/testicular sperm extraction (TESE), number of oocytes retrieved, cycles with blastocyst transfers, and gestational age within ICSI group. MAIN RESULTS AND THE ROLE OF CHANCE Within the IVF group, the percentage of males at birth for G5™, Global, Quinn's and G5™ PLUS media were comparable (P > 0.05); however, within the ICSI group, the percentage of male babies in cycles using G5™(56.1%) was statistically significantly higher than in cycles that used Global (47.2%; P = 0.003), G5™ PLUS (47.7%; P = 0.005) or Quinn's media (45.0%; P = 0.009). There were no statistically significant differences in the percentage of males at birth between cycles that used Global, G5™ PLUS and Quinn's media (P > 0.05). Multivariable logistic regression indicated that culture media (G5™ versus Global, G5™ PLUS, and Quinn's) were significantly associated with the sex at birth (P = 0.008) after adjusting for parental age, parental BMI, type of infertility, parity, number of embryos transferred, number of early gestational sacs, cycles with TESA/PESA/TESE, number of oocytes retrieved, cycles with blastocyst transfers, and gestational age. LIMITATIONS AND REASONS FOR CAUTION This study was not a randomized controlled trial and allocation of treatment cycles over the four media was not completely at random. Cigarette smoking was not included in the current study because this confounding factor was not registered in our database. Moreover, intra-variability of sperm selection between the five embryologists may directly affect the percentage of males. WIDER IMPLICATIONS OF THESE FINDINGS Our study suggests that human embryogenesis responds differently to G5™, Global, G5™ PLUS and Quinn's Advantage Medium. This finding can be generalized to other commercial culture media. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

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