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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.


Huang X.,Peking University | Huang X.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Bai Q.,Peking University | Yan L.,Peking University | And 5 more authors.
Chinese Medical Journal | Year: 2012

Background It is still controversial whether the serum inhibin B level is a superior predictor of the presence of sperm in testicular sperm extraction (TESE) in azoospermic men compared with serum follicle-stimulating hormone (FSH). In this study, we evaluated the diagnostic accuracy of serum inhibin B levels as a predictor of the outcome of TESE in Chinese non-obstructive azoospermic men and compared it with the traditional marker serum FSH and testicular volumes. Methods Basal values of serum hormone levels, testicular volumes and histological evaluation of 305 Chinese non-obstructive azoospermic men were analyzed. The level of inhibin B was measured using a three-step enzyme-linked immunoassay before sperm extraction, and the diagnostic accuracy of prediction of the outcome of TESE was compared for different markers by the receiver operating characteristics (ROC) curve analysis. Results Testicular sperm was successfully retrieved in 137 of 305 patients (44.9%). The serum level of inhibin B, the FSH and the testicular volume were significantly different between the successful TESE group and the unsuccessful group. According to the ROC curve analysis, for inhibin B, the cut-off value for discriminating between successful and failed TESE was 28.39 pg/ml (sensitivity 83.5%, specificity 79.1%). For FSH, the best cut-off value for discriminating was 11.05 pg/ml (sensitivity 83.5%, specificity 74.5%). The area under the ROC curve of serum inhibin B was similar to that of FSH. Combining the serum inhibin B with FSH levels did not improve the predictive value for successful TESE. Conclusions Serum inhibin B and FSH levels are correlated with spermatogenesis. However, inhibin B is not superior to FSH in predicting the presence of sperm in TESE. And the combination of them does not improve the diagnostic accuracy on TESE outcome.


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.


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.


Li M.,Peking University | Li M.,Key Laboratory of Assisted Reproduction | Li M.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Lin S.,Peking University | And 14 more authors.
Fertility and Sterility | Year: 2015

Objective To determine the value of transferring embryos formed from nonpronuclear (0PN) zygotes. Design A case-control study. Setting Not applicable. Patient(s) The current study was a retrospective analysis of embryo transfers of just 0PN embryos using fresh cleavage-stage embryos (0PN cleavage fresh), frozen-thawed cleavage-stage 0PN embryos (0PN cleavage frozen), and frozen 0PN blastocyst-stage embryos (0PN blast frozen). Intervention(s) To study the effect of 0PN transfer, comparison groups were used: fresh cycles of 2PN (2PN cleavage fresh-C) and frozen-thawed cycles cleavage-stage (2PN cleavage frozen-C) and blastocyst-stage (2PN blast frozen-C). Comparison groups were matched for cycle and patient characteristics to the 0PN group. Main Outcome Measure(s) Implantation rate (IR), pregnancy rate, and transferable embryo rate. Result(s) For fresh cycles, the IR in the 0PN cleavage fresh was lower than that in the 2PN cleavage fresh-C (8.04% vs. 19.50%, respectively). For frozen-thawed cycles, the IR in the 0PN cleavage frozen was lower than that in the 2PN cleavage frozen-C (15.38% vs. 28.24%, respectively), but the IR in 0PN blast frozen was comparable to that of 2PN blast frozen-C (39.56% vs. 48.18%, respectively). Conclusion(s) Transfer of 0PN embryos from fresh or frozen-thawed cycles results in pregnancies and live births. Nonpronuclear embryos have a lower IR than 2PN embryos, but if the embryos are cultured to the blastocyst stage and then are frozen, their IRs approach that of 2PN embryos in subsequent frozen-thawed cycles. The culture of 0PN embryos to the blastocyst stage may select for embryos with a near-normal IR. © 2015 American Society for Reproductive Medicine.


Liu N.,Peking University | Liu N.,Key Laboratory of Assisted Reproduction | Liu N.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Ma Y.,Peking University | And 14 more authors.
Reproductive Biology and Endocrinology | Year: 2012

Background: High circulating luteinizing hormone (LH) level is a typical biochemical feature of polycystic ovary syndrome (PCOS) whose pathophysiology is still unclear. Certain mutations of LH and LH receptor (LHR) may lead to changes in bioactivity of these hormones. The aim of this study was determine the role of the LH and LHR polymorphisms in the pathogenesis of PCOS using a genetic approach.Methods: 315 PCOS women and 212 controls were screened for the gene variants of LH G1052A and LHR rs61996318 polymorphisms by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP).Results: PCOS patients had significantly more A allele frequency of LH G1052A mutations than controls (p=0.001). Within PCOS group, carriers of LH 1052A allele had lower LH (p=0.05) and higher fasting glucose levels (p=0.04). No subjects were identified with LHR rs61996318 polymorphisms. A new LHR single nucleotide polymorphism (SNP) was found without clear association with PCOS.Conclusions: Results suggested LH G1052A mutation might influence PCOS susceptibility and phenotypes. © 2012 Liu et al.; licensee BioMed Central Ltd.


Zhu J.,Peking University | Zhu J.,Key Laboratory of Assisted Reproduction | Zhu J.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Lin S.,Peking University | And 17 more authors.
Human Reproduction | Year: 2014

STUDY QUESTIONDoes prolonged in vitro culture influence newborn birthweight?SUMMARY ANSWERThe absolute mean birthweight and gestational age-and gender-adjusted birthweight (Z scores) of singletons born from blastocyst transfer are higher than singletons born from Day 3 transfer.WHAT IS KNOWN ALREADYAn increased proportion of large-for-gestational age (LGA) newborns occurs after blastocyst transfer compared with Day 2 transfer, and Z scores for newborns after blastocyst transfer are higher than newborns after transfer on Day 2 or Day 3.STUDY DESIGN, SIZE AND DURATIONThis study was a retrospective analysis of newborn birthweight, including 2929 singletons at the Reproductive Medical Center of Peking University Third Hospital between January 2009 and June 2012. The number of singletons after Day 3 transfer was 2833 and the number of singletons after blastocyst transfer (Day 5-6) was 96.PARTICIPANTS/MATERIALS, SETTING, AND METHODSOnly cycles with fresh embryo transfer were included. Patients ≤40 years of age with a BMI < 30 kg/m2 were analyzed. Only data from singleton newborns born alive after the 20th week of gestation were included in the data analysis. Patients with more than one fetal sac diagnosed by ultrasound but who delivered singletons were excluded. Patients who received PGD and cycles with donor oocytes were excluded. Multiple linear regression analysis was performed to determine the significance of individual factors on absolute birthweight of singleton newborns. The absolute birthweight and Z scores of singletons were compared.MAIN RESULTS AND THE ROLE OF CHANCEMultiple linear regression analysis indicated that maternal age, maternal BMI, paternal BMI, type of infertility, gestational age, infant gender and culture period were significantly associated with birthweight. The absolute birthweight for singletons resulting from blastocyst transfer was significantly greater than singletons resulting from Day 3 transfer (3465.31 ± 51.36 versus 3319.82 ± 10.04 g respectively, P = 0.009). The Z scores for singletons after blastocyst transfer were significantly higher than singletons after Day 3 transfer (0.347 versus 0.029 respectively, P = 0.016).LIMITATIONS AND REASONS FOR CAUTIONIn our clinic, blastocyst culture is mainly offered to patients with unsuccessful IVF cycles but also to patients with uterine malformations, and therefore this protocol introduced a potential selection bias in our study. Moreover, as certain culture media are associated with fetal overgrowth, the media used may be also a confounding factor, even though the absolute birthweights of singletons were comparable.WIDER IMPLICATIONS OF THESE FINDINGSOur study suggests that a prolonged (5-6 days) in vitro culture period has a significant effect on the mean absolute birthweight and Z scores of singleton newborns. The effect of prolonged in vitro culture on epigenetic changes in the embryo needs further study. © 2013 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.


Huang J.,Peking University | Huang J.,Key Laboratory of Assisted Reproduction | Huang J.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction | Zhang L.,Guangdong Women and Childrens Hospital | 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.


PubMed | Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, CAS Technical Institute of Physics and Chemistry and Peking University
Type: | Journal: Scientific reports | Year: 2016

Recent studies have shown that L-proline is a natural osmoprotectant and an antioxidant to protect cells from injuries such as that caused by freezing and thawing in many species including plant, ram sperm and human endothelial cells. Nevertheless, this nontoxic cryoprotectant has not yet been applied to mammalian oocyte vitrification. In this study we evaluated the efficiency and safety of the new cryoprotectant in oocyte vitrification. The results indicated that L-proline improves the survival rate of vitrified oocytes, protects mitochondrial functions and could be applied as a new cryoprotectant in mouse oocyte vitrification.

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