Human single follicle growth in vitro from cryopreserved ovarian tissue after slow freezing or vitrification.

STUDY QUESTION: What is the effect of human ovarian tissue cryopreservation on single follicular development in vitro? SUMMARY ANSWER: Vitrification had a greater negative effect on growth and gene expression of human ovarian follicles when compared with fresh follicles. WHAT IS KNOWN ALREADY: For human ovarian cortex cryopreservation, the conventional option is slow freezing while more recently vitrification has been demonstrated to maintain good quality and function of ovarian tissues. STUDY DESIGN, SIZE, DURATION: Ovarian tissues were collected from 11 patients. For every patient, the ovarian cortex was divided into three samples: Fresh, slow-rate freezing (Slow) and vitrification (Vit). Tissue histology was performed and follicles were isolated for single-cell mRNA analysis and in vitro culture (IVC) in 1% alginate for 8 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicle morphology was assessed with hematoxylin-eosin analysis. Follicles were individually embedded in alginate (1% w/v) and cultured in vitro for 8 days. Follicle survival and growth were assessed by microscopy. Follicle viability was observed after Calcein-AM and ethidium homodimer-I (Ca-AM/EthD-I) staining. Expression of genes, including GDF9 (growth differentiation factor 9), BMP15 (bone morphogenetic protein 15) and ZP3 (zona pellucida glycoprotein 3) in oocytes and AMH (anti-Mullerian hormone), FSHR (FSH receptor), CYP11A (cholesterol side-chain cleavage cytochrome P450) and STAR (steroidogenic acute regulatory protein) in GCs, was evaluated by single-cell mRNA analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 129 follicles were separated from ovarian cortex (Fresh n = 44; Slow n = 40; Vit n = 45). The percentage of damaged oocytes and granulosa cells was significantly higher in both the Slow and Vit groups, as compared with Fresh control (P< 0.05). The growth of follicles in vitro was significantly delayed in the Vit group compared with the Fresh group (P< 0.05). Both slow freezing (P< 0.05) and vitrification (P< 0.05) down-regulated the mRNA levels of ZP3 and CYP11A compared with Fresh group, while there was no significant difference between the Slow and Vit groups (P> 0.05). Vitrification also down-regulates AMH mRNA levels compared with Fresh group (P< 0.05). LIMITATIONS, REASONS FOR CAUTION: Only short-term IVC studies (8 days) are reported. Further study should be performed to examine and improve follicular development in a long-term culture system after cryopreservation. WIDER IMPLICATIONS OF THE FINDINGS: This is the first comparison of gene expression and growth of single human ovarian follicles in vitro after either slow freezing or vitrification. With the decreased gene expression and growth during IVC, damage by cryopreservation still exists and needs to be minimized during the long-term IVC of follicles in the future for eventual clinical application. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (31230047, 81571386, 81471508, 31429004 and 81501247), National Natural Science Foundation of Beijing (7142166) and Mega-projects of Science Research for the 12th five-year plan (2012ba132b05). There are no conflicts of interest to declare.

Basic fibroblast growth factor promotes the development of human ovarian early follicles during growth in vitro.

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.

[Expression of PPARγ mRNA in granulosa cells and its correlation with clinical characteristics of polycystic ovary syndrome].

OBJECTIVE: To study the expression of PPARγ mRNA in granulosa cells of patients with polycystic ovary syndrome (PCOS) and the impact of testosterone, insulin and PPARγ agonist rosiglitazone on granulosa cells (GCs). METHODS: The expression of PPARγ mRNA in GCs of patients with PCOS and normal controls were analyzed by Real-time PCR. We assessed the level of PPARγ mRNA in GCs from normal controls after treatment with testosterone, insulin, and rosiglitazone. RESULTS: The expression of PPARγ mRNA was lower in the GCs of PCOS than that of the controls (P<0.05). When testosterone concentration was 1 nmol/L, the expression of PPARγ mRNA was lower in the GCs as compared with the blank control (P<0.05). When testosterone concentration was 10 nmol/L, PPARγ mRNA increased in the GCs as compared with the blank control, which was of no significance (P>0.05). When insulin concentration was 10 nmol/L, the expression of PPARγ mRNA was higher in the GCs as compared with the blank control (P<0.05). When insulin concentration was 100 nmol/L, the expression of PPARγ mRNA increased, but the difference was not statistically significant (P>0.05). When rosiglitazone concentration was 1 nmol/L, the expression of PPARγ mRNA in ovarian GCs significantly increased, as compared with the blank control (P<0.05). When rosiglitazone concentration was at 10 nmol/L, the PPARγ mRNA expression significantly increased, as compared with the concentration at 1 nmol/L (P<0.05). CONCLUSION: PPARγ mRNA expression is down-regulated by testosterone, and up-regulated by insulin and rosiglitazone with different dosages. Decreased PPARγ mRNA in the GCs of PCOS is related to the clinical characteristics of PCOS.

[Clinical features of the metabolic syndrome in patients with polycystic ovary syndrome].

OBJECTIVE: To study characteristics of the metabolic syndrome (MS) in patients with polycystic ovary syndrome (PCOS) according to 2004 Diagnostic Criteria by the Chinese Diabetes Society (CDS). METHODS: A 75 g oral glucose tolerance test (OGTT) with plasma glucose and serum insulin (INS) levels was performed in 232 PCOS patients. Also, serum triglycerides (TG), total cholesterol (TC), high density lipoprotein (HDL) cholesterol and low density lipoprotein (LDL) cholesterol (HDL-C and LDL-C) were measured. RESULTS: The rates of MS prevalence was 31.9% (74/232) in patients with PCOS. The rate of hyperglycaemia, high blood pressure, obesity and lipid metabolic disturbance were 30.6% (71/232), 5.6% (13/232), 48.3% (112/232) and 48.3% (112/232), respectively. The rate of MS prevalence and all metabolic parameters were higher in obese than in non-obese patients with PCOS. Age, BMI and all metabolic parameters were higher in PCOS patients with MS than in those without MS. CONCLUSION: According to the CDS criteria, the rate of MS prevalence was lower than that of the West in our study, and similar to that of some Asian countries. MS prevalence increased with age and BMI in patients with PCOS.

[Characteristics of abnormal glucose tolerance in patients with polycystic ovary syndrome].

OBJECTIVE: To analyze the characteristics of abnormal glucose tolerance in patients with polycystic ovary syndrome (PCOS). METHODS: A 75 g oral glucose tolerance test (OGTT) with plasma glucose and serum insulin (INS) levels was performed in 257 PCOS patients. The states of glucose tolerance were classified by fasting plasma glucose (FPG) and plasma glucose at 120 minutes according to the Word Health Organization (WHO) criterion. RESULTS: According to the WHO criteria, 69.3% were classified as normal glucose tolerance (NGT) and 30.7% as with abnormal glucose metabolism, with 26.1% of them presenting impaired glucose tolerance (IGT) and 4.7% diabetes mellitus (DM). Age, BMI, fasting glucose or 2 h glucose concentration, fasting insulin or 2 h glucose concentration and homeostasis model assessment-insulin resistance (HOMA-IR) increased gradually among NGT, IGT and 2DM. QUICKI and the ratio of fasting glucose/ fasting insulin decreased gradually. The prevalence of abnormal glucose tolerance was significantly higher after 30 years old. There was significant correlation between abnormal glucose tolerance and age or BMI. A positive correlation was found between BMI and fasting glucose or 2-h glucose concentration. CONCLUSION: Abnormal glucose tolerance was correlated with age and BMI in PCOS patients. Insulin resistance is the mechanistic basis of abnormal glucose tolerance. Thus PCOS patients should be screened by glucose tolerance at an early time and a regular interval.

[Study on endometrium receptivity in patients with polycystic ovary syndrome during implantation window time].

OBJECTIVE: To explore the gene which might influence endometrium receptivity during the implantation window time in normal women and patients with polycystic ovary syndrome (PCOS). METHODS: Transvaginal ultrasound were performed and serum estrogen and progestogen levels were measured in all women to monitor the exact time of ovulation. Endometrium biopsy was done in normal women and PCOS patients during implantation window time. Sixteen women were enrolled in this study, in which seven were normal women, and nine were PCOS patients. cDNA extraction was performed, matrix metalloproteinase 26 (MMP-26) primers were synthesized and real time fluorescent quantitative PCR was conducted using beta-actin gene as endogenous control. RESULTS: The ratios of MMP-26 were 0.31, 0.11 and 0.05 in 3 patients with PCOS by real time fluorescent quantitative PCR, obviously decreased during implantation time compared with the normal women. CONCLUSIONS: Our data suggest a lower expression of MMP-26 in implantation window time in patients with PCOS than in normal patients. This might indicate a declined capability of endometrium receptivity in implantation window time in patients with PCOS.

[Association of monocyte chemoattractant protein-1 and the clinical characteristics of polycystic ovary syndrome: analysis of 65 cases].

OBJECTIVE: To investigate the association of monocyte chemoattractant protein-1 (MCP-1) and the clinical characteristics of polycystic ovary syndrome: (PCOS). METHODS: Fasting peripheral venous blood samples were collected on the 2nd or 3rd day of the menstrual cycle or when there was no dominant follicle shown by ultrasonography after amenorrhea from 65 POCS patients, aged 30 +/- 3, 27 being attributed to the obese group according the body mass index (BMI) and 38 to the non-obese group, and 40 patients with infertility, aged 31 +/- 3, as controls, subdivided into obese and non-obese subgroups (both n = 20), and then the samples of serum. Were obtained. The level of MCP-1 was examined by ELISA. The levels of prolactin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol, and testosterone (T) were assayed with chemoluminescence methods, and the level of androstenedione was detected by radioimmunoassay. The level of serum insulin was detected by chemoluminescence method and the serum glucose (SG) was detected by oxidase enzymic method. Insulin sensitivity index (ISI) and homeostasis model assessment insulin resistance (HOMA-IR) were calculated. RESULTS: (1) The levels of serum LH, T, and LH/FSH of the obese and non-obese POCS subgroups were all significantly higher than the corresponding obese and non-obese control subgroups (all P < 0.05). (2) The level of MCP-1 of the non-obese PCOS subgroup was (98 +/- 67) ng/ml, significantly higher than that of the non-obese controls [(58 +/- 41 mg/L, P < 0.05)]. (3) Pearson correlation showed that serum PCP-1 was significantly positively correlated with BMI (r = 0.339, P = 0.000). LH (r = 0.224, P = 0.024)), and HOMA-IR (r = 0.239, P = 0.016), and significantly negatively correlated with ISI (r = -0.250, P = 0.0006). (3) Multiple regression analysis showed that BMI and LH were the principal factors influencing the level of MCP-1 in the POCS patients. CONCLUSION: The serum level of MCP-1 is associated with the LH level in POCS patients. POCS may a chronic inflammatory disease. MCP-1 is likely to participate in obesity, hyperinsulinemia, and insulin resistance in POCS.

[Monocyte chemoattractant protein-1 and its correlation with lipoprotein in polycystic ovary syndrome].

OBJECTIVE: To measure serum monocyte chemoattractant protein-1 (MCP-1) levels and study its associations with lipoproteins in patients with polycystic ovary syndrome (PCOS). METHODS: Sixty-five PCOS women and 20 ovulating normal women with body mass index (BMI) < 25 kg/m2 as controls were recruited. PCOS women were divided to two groups: 27 BMI >or = 25 kg/m2 patients as obese group; 38 BMI < 25 kg/m2 as non-obese group. Serum MCP-1 was assayed by enzyme-linked immunosorbent assays (ELISA). Serum prolactin (PRL), follicle stimulating hormone (FSH), luteinizing (LH), estradiol (E2) and testosterone (T) were assayed by chemoluminescence method. Serum androstenedione (A) was assayed by radioimmunity method in patients. And triglycerides (TG), total cholesterol (TC), apoprotein A (ApoA), apoprotein B (ApoB) , lipoprotein (a) [LP(a)], high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol (HDL-C and LDL-C) were measured. RESULTS: MCP-1 (P = 0.001) and ApoB (P = 0.018) levels were found to be significantly increased in PCOS groups compared with that of controls, but the ratio of ApoA/ ApoB was significantly decreased in groups PCOS (P = 0.015). PCOS obese group had markedly higher MCP-1 serum levels than non-obese group (P = 0.012), and MCP-1 serum levels in PCOS non-obese group higher than controls (P = 0.03). Univariate analysis revealed that serum MCP-1 levels were significantly and positively correlated with BMI (r = 0.350, P = 0.001), LH(r = 0.262, P = 0.016), TG (r = 0.480, P = 0.000) and ApoB (r = 0.289, P =0.008); but significantly and negatively correlated with the ratio of ApoA/ ApoB (r = -0.282, P = 0.009). Partial correlation showed that serum MCP-1 levels were correlation with LH (r = 0.2577, P = 0.020) and TG (r = 0.4611,P = 0.000). Multiple regression analysis showed that MCP-1 levels was influenced by BMI and TG. Furthermore, TG showed more effect on MCP-1 levels. CONCLUSION: PCOS obese and non-obese patients had higher serum MCP-1 levels than controls. MCP-1 was correlated with BMI, LH ,TG, ApoB and the ratio of ApoA/ ApoB. BMI and TG were two major determining factors of MCP-1 in patients with PCOS. Furthermore,TG had more effect on MCP-1 levels. Based on the above findings, we presume that MCP-1 is likely to participate in the pathophysiology and long-term complication of PCOS.

[Relations of serum adipocyte fatty acid binding protein to sex hormone and lipoproteins in patients with polycystic ovary syndrome].

OBJECTIVE: To investigate the relationship of serum adipocyte fatty acid binding protein (FABP4) level with sex hormone and lipoprotein in patients with polycystic ovary syndrome (PCOS). METHODS: Peripheral blood samples were collected from 29 out-patients of PCOS patients, aged 31 +/- 3 with a body mass index (BMI) of (26 +/- 4) kg/m2, and 14 out-patients of infertility, aged (31 +/- 3), with a BMI of (21 +/- 2) kg/m2 as normal controls. ELISA was used to detect the serum FABP4. Serum prolactin, follicle stimulating hormone, luteinizing hormone, estradiol, and testosterone were assayed by chemoluminescence method. Serum androstenedione was assayed by radioimmunity method. And triglycerides, total cholesterol, high density lipoprotein (HDL-C), and low density lipoprotein cholesterol (LDL-C) were measured by automatic biochemistry analyzer. The relations of FABP4 with sex hormone and lipoprotein in patients with PCOS were analyzed. RESULTS: The FABP4 level of the PCOS patients was (24 +/- 15) ng/ml, significantly higher than that of the controls (9 ng/ml +/- 8 ng/ml, P = 0.000). There were no significant differences in serum prolactin, follicle stimulating hormone, luteinizing hormone, estradiol, and testosterone between the 2 groups. Univariate analysis revealed that serum FABP4 level was significantly positively correlated with BMI (r = 0.621, P = 0.000) and testosterone (r = 0.658, P = 0.016), and significantly negatively correlated with HDL-C (r = -0.331, P = 0.030). Partial correlation analysis showed that serum FABP4 level was significantly positively correlated with testosterone (r = 0.507, P = 0.008). CONCLUSION: The increase of serum FABP4 in the PCOS patients may be correlated with the high androgen level. FABP4 is likely to participate in the lipid metabolic.

A study on human urine in a high-selenium area of China by 1H-NMR spectroscopy.

In this study, high-resolution 600-MHz 1H-NMR (nuclear magnetic resonance) spectroscopies were used to compare the urinary metabolic profiles of healthy humans and humans in a high-selenium area of China. NMR biomarkers for renal and liver lesions were observed by comparing the urine 1H-NMR spectra. In urinary excretion, the concentrations in human urine samples of formate, lactate, acetate, hippurate, and alanine in overexposure to selenium were increased, whereas citrate, creatine, and TMAO excretion were decreased compared with that of the healthy human--some of them even disappeared. An interesting result was the appearance of formate in urine, which has previously been shown to lead to acidosis and chronic renal failure and interfere with the lumen and proximal tubular cells. The level of creatine was associated with the seminal activity. The changes of acetate and citrate may explain the disorder of the cellular energy metabolism caused by selenium, and the changes of other amino acids were a result of the reuptake of these compounds that had been blocked in the glomerulus and proximal tubule. The results elucidate the renal/liver lesion in humans in high-selenium area by 1H-NMR spectroscopy and offer the molecular basic of selenium toxicity.