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Purpose The aim of this study was to determine the relationship between morphological parameters and the incidence of de novo chromosomal abnormalities. Methods This was a retrospective cohort study of 652 patients who underwent 921 cycles with 3238 blastocysts biopsied. The embryo grades were evaluated according to Gardner and Schoolcraft’s system. The incidence of euploidy, whole chromosomal aneuploidy (W-aneuploidy), segmental chromosomal aneuploidy (S-aneuploidy), and mosaicism in trophectoderm (TE) cell biopsies was analyzed. Results The euploidy decreased significantly with maternal age and was positively correlated biopsy day and morphological parameters. The W-aneuploidy increased significantly with maternal age and was negatively correlated biopsy day and morphological parameters. Parental age, TE biopsy day, and morphological parameters were not associated with S-aneuploidy and mosaicism, except that TE grade C blastocysts had significantly higher mosaicism than TE grade A blastocysts. Subanalysis in different female age groups showed that euploidy and W-aneuploidy had a significant correlation with TE biopsy day among women aged ≤ 30 y and 31–35 y, with expansion degree among women aged ≥ 36 y, with ICM grade among women aged ≥ 31 y, and with TE grade among all female age ranges. Conclusion Female age, embryo developmental speed and blastocyst morphological parameters are associated with euploidy and whole chromosomal aneuploidy. The predictive value of these factors varies across female age groups. Parental age, embryo developmental speed, expansion degree, and ICM grade are not associated with the incidence of segmental aneuploidy or mosaicism, but TE grade seemingly has a weak correlation with segmental aneuploidy and mosaicism in embryos.

What are the correlations between male age, traditional semen parameters, sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) in a sufficiently large sample size?Retrospective cohort study of 18,441 semen samples, with data divided into seven age groups according to male age: ≤25, 26-30, 31-35, 36-40, 41-45, 46-50 and ≥51 years.Age was negatively correlated with semen volume, total sperm count, motility and HDS, and positively correlated with sperm concentration and DFI (P 0.001). After 35 years of age, semen volume and total sperm count began to decline. After 30 years of age, motility and HDS decreased consistently. Sperm concentration and DFI increased from 26-30 years of age. DFI was negatively correlated with sperm concentration, total sperm count, motility and normal morphology (P 0.001) and positively correlated with semen volume and HDS (P 0.001). HDS was negatively correlated with all parameters (P 0.001) except semen volume (r = -0.013, P = 0.074) and DFI (r = 0.124, P 0.001). Patients aged ≥40 years had higher DFI than those aged40 years in the entire cohort, in the abnormal semen parameters cohort, and in the normal semen parameters cohort (OR 2.145, 2.042, 1.948, respectively, P 0.001). The ≥40 years age group had a lower HDS than the40 years age group in the entire cohort and abnormal semen parameters cohort (OR 0.719, 0.677, respectively, P 0.001).Ageing is a negative effector of sperm quantity and quality, and routine sperm parameters have weak but significant correlations with sperm DNA/chromatin integrity.

Background: Many studies have assessed the association between sperm DNA fragmentation and outcomes of ART. But the published papers have not offered enough evidence about whether sperm DNA fragmentation tests could make suggestions to predict intrauterine insemination (IUI) outcomes. The aim of this study was to assess whether the sperm chromatin structure assay (SCSA) parameters, sperm DNA fragmentation index (DFI) and high DNA stainability (HDS), could be used as predictors for treatment outcomes in IUI program.Methods: A retrospective cohort study was conducted at a large reproductive medicine center. 1139 IUI cycles from 1139 couples were studied. The association of SCSA parameters with the clinical pregnancy and early pregnancy loss after IUI were analyzed. Results: Clinical pregnancy rate per cycle in DFI15% groups were 9.8% and 7.5%, respectively, with no significant difference (P=0.468), and the pregnancy loss rate were 26.2% and 0, respectively, and also no statistical difference (P=0.191). Multivariate logistic regression analysis showed a higher rate of clinical pregnancy in couples with a younger female (OR=0.90, 95% CI: 0.83-0.97, P=0.007), and in couples with a male who had higher sperm concentration after washing (OR=1.02, 95% CI: 1.00-1.04, P=0.035). A higher risk of pregnancy loss was observed with increased female age (OR= 1.43, 95% CI:1.09-1.89, P=0.010) and lower sperm concentration after washing (OR= 0.92, 95% CI: 0.84-0.99, P= 0.029). Conclusions: Sperm DFI and HDS were not significantly correlated with clinical pregnancy and pregnancy loss in cycles of IUI. Female age and motile sperm concentration had statistically significant effects on both clinical pregnancy and pregnancy loss after IUI treatment.

Polycystic ovary syndrome (PCOS) is a common reproductive disorder that has many characteristic features including hyperandrogenemia, insulin resistance and obesity, which may have significant implications for pregnancy outcomes and long-term health of women. Daughters born to PCOS mothers constitute a high-risk group for metabolic and reproductive derangements, but no report has described potential growth and metabolic risk factors for such female offspring. Hence, we used a mouse model of dehydroepiandrosterone (DHEA)-induced PCOS to study the mechanisms underlying the pathology of PCOS by investigating the growth, developmental characteristics, metabolic indexes and expression profiles of key genes of offspring born to the models. We found that the average litter size was significantly smaller in the DHEA group, and female offspring had sustained higher body weight, increased body fat and triglyceride content in serum and liver; they also exhibited decreased energy expenditure, oxygen consumption and impaired glucose tolerance. Genes related to glucolipid metabolism such as Pparγ, Acot1/2, Fgf21, Pdk4 and Inhbb were upregulated in the liver of the offspring in DHEA group compared with those in controls, whereas Cyp17a1 expression was significantly decreased. However, the expression of these genes was not detected in male offspring. Our results show that female offspring in DHEA group exhibit perturbed growth and glucolipid metabolism that were not observed in male offspring.

The influence of 24-epibrassinolide (EBR) on the regulation of antioxidant and pesticide detoxification systems in grape seedlings (Vitis vinifera L.) was investigated. The grapevine leaves were pretreated with different concentrations of EBR (0.10, 0.21 and 0.42 μM) before spraying with chlorothalonil (CHT) (600 times diluent). Compared with the control, CHT treatment showed an adverse effect on osmoregulation substances (soluble protein and free proline) and increased the content of malondialdehyde (MDA), activities of antioxidant enzymes and detoxification enzymes as well as the content of some non-enzymatic antioxidants. Compared to the CHT treatment alone, application of three concentrations of EBR resulted in an overall enhancement of these enzyme activities, soluble protein and free proline contents and reduction in MDA content. In addition, the expression of some key genes involved in CHT degradation including glutathione synthetase (GSH), glutathione S-transferase (GST), glutathione reductase (GR), P450 monooxygenase (P450) and MRP-type transporter (MRP) was analyzed. The transcriptional level of these genes increased in response to CHT treatment with the exception of GSH. EBR treatment of 0.10 μM could effectively promote expression of most genes and accelerate CHT degradation by 29 % as compared to only CHT treatment in grapevine leaves.

Summary The potential of various concentrations of 24-epibrassinolide (EBR) treatments on the control of grey mould in artificial Botrytis cinerea inoculation test and on the maintenance of postharvest quality of table grapes without inoculation in cold storage were researched. In the cold storage, clusters of grapes with exogenous EBR application (EBR1, 0.1 mg L−1 EBR; EBR2, 0.4 mg L−1 EBR; EBR3, 0.8 mg L−1 EBR) were stored at 0 ± 1 °C. Fruits’ quality attributes including berry firmness, weight loss, berry drop, decay berry percentages, total sugar and titratable acidity were measured at 15, 30 and 60 days. The results showed that EBR treatments exhibited a higher berry firmness and a lower percentage of weight loss, berry drop and decay berry. In the artificial inoculation trial, single berries were immersed in EBR1, EBR2 and EBR3, then inoculated by spraying with a B. cinerea spore suspension and stored at 25 ± 2 °C and 95% relative humidity. Disease severity (DS), lipid peroxidation and defence-related enzymes of these berries were analysed every 2 days. EBR-treated berries resulted in an increase in activities of defence-related enzymes including superoxide dismutase, peroxidase, catalase and phenylalanine ammonia-lyase compared with control berries inoculated by B. cinerea. Simultaneously, significant decreases in DS and production of superoxide anion radical and malondialdehyde were observed. These results suggested that exogenous application of EBR could play a protective role in controlling the grey mould of postharvest grapes and maintaining the postharvest quality during cold storage.

Fertility preservation is an important type of frontier scientific research in the field of reproductive health. The culture of ovarian cortices to i) initiate primordial follicle growth and ii) procure developing follicles for later oocyte maturation is a promising fertility preservation strategy, especially for older women or cancer patients. At present, this goal remains largely unsubstantiated in primates because of the difficulty in attaining relatively large follicles via ovarian cortex culture. To overcome this hurdle, we cultured macaque monkey ovarian cortices with FSH, kit ligand (KL), basic fibroblast growth factor (bFGF), and/or epidermal growth factor (EGF). The various factors and factor combinations promoted primordial follicle development to different extents. Notably, both bFF (bFGF, 100 ng/ml and FSH, 50 ng/ml) and KF (KL, 100 ng/ml and FSH, 50 ng/ml) contributed to the activation of primordial follicles at day 12 (D12) of culture, whereas at D18, the proportions of developing follicles were significantly higher in the bFF and KF groups relative to the other treatment groups, particularly in the bFF group. Estradiol and progesterone production were also highest in the bFF group, and primary follicle diameters were the largest. Up until D24, the bFF group still exhibited the highest proportion of developing follicles. In conclusion, the bFGF–FSH combination promotes nonhuman primate primordial follicle developmentin vitro, with the optimal experimental window within 18 days. These results provide evidence for the future success of human ovarian cortex culture and the eventual acquisition of mature human follicles or oocytes for fertility restoration.

STUDY QUESTION What 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 ANSWER The addition of 200 ng bFGF/ml improves human early follicle growth, survival and viability during growth in vitro. WHAT IS KNOWN ALREADY It 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, DURATION The 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, METHODS Individual 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 CHANCE After 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 CAUTION The 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 FINDINGS The 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. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Basic Research Program of China (2011|CB944504, 2011CB944503) and the National Natural Science Foundation of China (81200470, 81000275, 31230047, 8110197). There are no conflicts of interest to declare.

Objective One of the major obstacles to ovarian tissue preservation is delayed angiogenesis that leads follicles lost after transplantation. The aim of the present study was to investigate the effects of bFGF and VEGF on heterotopic transplanted ovarian tissue using a mouse model. Methods Female mice underwent bilateral ovariectomy. Ovarian tissues encapsulated by fibrin hydrogels were transplanted subcutaneously into recipient mice, in which ovarian hormonal cyclicity was absent. The fibrinogen solution was mixed with bFGF, VEGF, or a mixture of bFGF and VEGF. The grafts were recovered 21 days after transplantation. Follicle morphology and follicle numbers were observed by H&E staining. Blood vessels were observed in transplanted intra-ovarian tissue by CD31 antibody IHC staining. Daily vaginal cytology was performed to determine estrous cycle and functional restoration of transplanted ovarian tissue. Blood was collected weekly and serum FSH levels were measured with a radioimmunoassay kit. Apoptosis analysis was performed by anti-AC-3 staining and survivin mRNA expression. Results The number of primordial follicles and secondary follicles in the bFGF+VEGF group was significantly higher than in the control group. The vascular density in the bFGF+VEGF groups were significantly higher than in the bFGF and the VEGF groups; there was no significant difference between the bFGF and VEGF groups. Estrous cycle was earlier in the bFGF+VEGF group compared with the control group; all mice in this group restored ovarian function. Serum FSH levels in the bFGF+VEGF group were significantly lower than in the control group by day 14 post-transplantation. The AC-3-positive in control group was significantly higher compared with bFGF group and VEGF group, and in bFGF+VEGF group was significantly lower than bFGF group and VEGF group. Survivin mRNA expression in bFGF+VEGF group was significantly higher than control group. Conclusion The combination of bFGF and VEGF has beneficial effects on follicle survival, angiogenesis, and resumption of estrous cycles.

Does basic fibroblast growth factor (bFGF) in combination with fibrin hydrogel improve follicle development and revascularization of heterotopically transplanted mouse ovarian tissues?Treatment of transplanted ovarian tissues with higher concentrations (75, 100 and 150 μg/ml), but not lower concentrations (25 and 50 μg/ml), of bFGF significantly improved primordial follicle survival and angiogenesis, while apoptosis of follicles and stromal cells was significantly decreased.Use of transplanted ovarian tissues in female fertility preservation is limited by the massive loss of follicles and ischemia-reperfusion injury due to the expected delay in revascularization.Ovarian tissues from 18-day-old ICR mice were encapsulated in fibrin hydrogel mixed with different concentrations of bFGF, then transplanted under the skin of adult female mice for 1 week. The ovarian tissues treated without fibrin hydrogels and bFGF were designated as Control group I, and the ovarian tissues treated with fibrin hydrogels but without bFGF were designated as Control group II. The ovarian tissues treated with 25 and 50 μg/ml bFGF were designated as the lower concentration group, and the ovarian tissues treated with 75, 100 and 150 μg/ml bFGF were designated as the higher concentration group.Assessment of follicular quantity and follicle classification was carried out by histologic analysis. Follicle proliferation was evidenced by immunostaining with proliferating cell nuclear antigen and apoptosis was verified by anti-active caspase-3 staining. Epithelial cells of new blood vessels were stained using CD31 antibody to evaluate neoangiogenesis, and the blood vessel density was analyzed by immunohistochemistry.The ovarian tissues were recovered 1 week post-transplantation. Compared with the control group, the survival and proliferation of the follicles was significantly increased, the apoptosis of follicles and stromal cells was significantly decreased, and angiogenesis was significantly enhanced when the transplanted ovarian tissues were treated with a higher concentration of bFGF. Treatment with a lower concentration of bFGF did not improve follicle survival and blood revascularization.The results obtained may not be fully extrapolated to humans because of the physiologic differences between mice and humans.For the first time, the present study investigated the role of bFGF in transplanted ovarian tissues and demonstrated that bFGF might significantly improve the quality of transplanted ovarian tissues by increasing follicle quantity and promoting neoangiogenesis. This study sets the stage for further study and application of ovarian tissue transplantation in clinics, and may eventually benefit females for fertility preservation.This work was partially supported by the Ministry of Science and Technology of China Grants (973 Program; 2011CB944503 to Q.J.), the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (30825038 to Q.J.), and the National Natural Science Funds for Young Scholar (81200470 to Y.J. and 81000275 to Y.L.Y.). None of the authors have any conflicts of interest.

The present study evaluated the influence of hyperandrogenism on oocyte quality using a murine PCOS model induced by dehydroepiandrosterone (DHEA) and further explored the effect of metformin treatment. Female BALB/c mice were treated with a vehicle control or DHEA (6 mg /100 g body weight) or DHEA plus metformin (50 mg /100 g body weight) for 20 consecutive days. DHEA-induced mice resembled some characters of human PCOS, such as irregular sexual cycles and polycystic ovaries. After the model validation was completed, metaphase II (MII) oocytes were retrieved and subsequent analyses of oocyte quality were performed. DHEA-treated mice yielded fewer MII oocytes, which displayed decreased mtDNA copy number, ATP content, inner mitochondrial membrane potential, excessive oxidative stress and impaired embryo development competence compared with those in control mice. Metformin treatment partially attenuated those damages, as evidenced by the increased fertilization and blastocyst rate, ATP content, GSH concentration and GSH/GSSG ratio, and decreased reactive oxygen species levels. No significant difference in normal spindle assembly was observed among the three groups. During in vitro maturation (IVM), the periods of germinal vesicle breakdown (GVBD) and the first polar body (PB1) extrusion were extended and the maturation rate of GVBD oocytes was decreased in DHEA mice compared with controls. Metformin treatment decreased the time elapsed of GVBD while had no effect on PB1 extrusion. These results indicated that excessive androgen is detrimental to oocyte quality while metformin treatment is, directly or indirectly, beneficial for oocyte quality improvement.

Human parthenogenetic embryonic stem cells (hpESCs) are generated from artificially activated oocytes, however, the issue of whether hpESCs have equivalent differentiation ability to human fertilized embryonic stem cells remains controversial. hpESCs were injected into male severe combined immunodeficiency (SCID) mice and the efficiency of teratoma formation was calculated. Then the gene expression and methylation modification were detected by real time-PCR and bisulfate methods. Comparison of five hpESCs with different differentiation abilities revealed that levels of paternal genes in the Dlk1-Dio3 region on chromosome 14 in the hpESCs with high differentiation potential are enhanced, but strictly methylated and silenced in the hpESCs with lower differentiation potential. Treatment with ascorbic acid, rescued their ability to support teratoma formation and altered the expression profiles of paternally expressed genes in hpESCs that could not form teratoma easily. No differences in the expression of other imprinting genes were evident between hpESCs with higher and lower differentiation potential, except for those in the Dlk1-Dio3 region. The Dlk1-Dio3 imprinting gene cluster distinguishes the differentiation ability of hpESCs. Moreover, modification by ascorbic acid may facilitate application of hpESCs to clinical settings in the future by enhancing their pluripotency.