Enhancing the scope of in vitro maturation for fertility preservation: transvaginal retrieval of immature oocytes during endoscopic gynaecological procedures.

STUDY QUESTION: Could in vitro maturation (IVM) following transvaginal oocyte retrieval during gynaecological surgery (IVM-surgery) be an effective and safe strategy for fertility preservation? SUMMARY ANSWER: IVM-surgery on unstimulated ovaries is a novel option that can be considered for fertility preservation for women requiring gynaecological surgery, but more research is needed to identify appropriate patients who may benefit and to determine the cost-effectiveness of such an approach. WHAT IS KNOWN ALREADY: IVM followed by oocyte/embryo cryopreservation has been useful as a safe reproductive strategy for some infertile women. STUDY DESIGN, SIZE, DURATION: This prospective cohort study comprised 158 consecutive women with polycystic ovary syndrome (PCOS) who underwent laparoscopy or hysteroscopy for other reasons and had concomitant transvaginal oocyte retrieval followed by IVM between 2014 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 158 women with anovulatory PCOS who underwent IVM-surgery in our infertility centre were recruited for this study. Matured IVM oocytes obtained from these women were either freshly fertilized and subsequently frozen at the blastocyst stage (fresh oocyte group, n = 46) or the oocytes were frozen (frozen oocyte group, n = 112) for fertility preservation followed by later thawing for insemination and cleavage embryo transfer (ET) (n = 33). The following outcomes were then evaluated: embryological data, clinical pregnancy rate, live birth rate (LBR), neonatal outcomes, post-operative complications and post-operative ovarian function. MAIN RESULTS AND THE ROLE OF CHANCE: Among all the women who underwent IVM-surgery, the clinical pregnancy rate and LBR per initiated IVM cycle were 9.5% (15/158) and 6.9% (11/158), respectively. Women (40.6%, 20/33) who underwent the procedure with frozen-thawed oocytes (oocyte survival rate, 83.0%) obtained a high quality of cleaved embryos. In the fresh oocyte group, the clinical pregnancy rate and LBR per ET cycle were 69.2 and 53.8%, respectively. In the frozen oocyte group, the clinical pregnancy rate and LBR per ET cycle were 28.6 and 19.1%, respectively. No adverse neonatal outcomes were recorded. IVM-surgery was not associated with post-operative complications, a longer hospital stay, or impaired ovarian function. LIMITATIONS, REASONS FOR CAUTION: Because of the small sample size and the low utilization rate and cost-effectiveness per retrieval, the present findings should be interpreted with caution, and further studies are needed for the long-term follow-up of live births. WIDER IMPLICATIONS OF THE FINDINGS: This strategy can also help patients with normal ovulation to obtain available oocytes and embryos for cryopreservation and subsequent use. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the Joint Research Fund for Overseas Natural Science of China (No. 31429004), the National Key Research and Development Program of China (No. 2017YFC1002000, 2017YFC1001504, 2016YFC1000302), the Ministry of Science and Technology of China Grants (No. 2014CB943203), the Chinese Society of Reproductive Medicine Fund (No. 16020400656) and the National Natural Science Foundation of China (No. 81300456). All the authors have nothing to disclose in terms of conflicts of interest. TRIAL REGISTRATION NUMBER: chictr-ONC-17011861.

Effect of exogenous gonadotropin on the transcriptome of human granulosa cells and follicular fluid hormone profiles.

BACKGROUND: Superovulation treatment had some adverse effects on maturity and development of oocytes. Can superovulation dose of gonadotropins (Gns) affect the transcriptome of granulosa cells and follicular fluid (FF) hormone levels? METHODS: One leading pre-ovulatory follicle per subject was used from three natural-cycle and four Gn-stimulated patients. Granulosa cells and FF samples were collected from the same leading follicle of each patient. RNA was extracted from granulosa cells and subjected to deep sequencing and analysis. Follicle-stimulating hormone (FSH), estradiol (E2), androstenedione (AND), testosterone (T), luteinizing hormone (LH), and progesterone (P4) levels in FF were measured by immunoassays. Student's t test was used for statistical analysis. RESULTS: A total of 715 genes were up-regulated, and 287 genes were down-regulated, in the Gn-stimulated group relative to the control group. Gene Ontology analysis revealed that the down-regulated genes were enriched in cell cycle and meiosis pathways, primarily those associated with follicle or oocyte maturation and quality. On the other hand, the up-regulated genes were enriched in functions related to immunity and cytokine-cytokine receptor interactions. Compared to the follicles of natural cycle, the E2 and LH concentrations were significantly reduced (P < 0.001), the P4 concentration was significantly increased (P = 0.003), and the concentrations of FSH, T and AND had no difference in the follicles of Gn-stimulated cycle. CONCLUSIONS: Cell cycle- and meiosis-associated genes were down-regulated by Gns stimulation, whereas immune- and cytokine-associated genes were up-regulated. Hormone levels were also affected by Gns stimulation. Compared with natural-cycle follicles,putative markers associated with oocyte quality and follicle maturation were significantly different from those in Gn-stimulated follicles. Hormone levels in follicles were compatible with the steroidogenic patterns of granulosa cell, which reflects the follicle maturation and oocyte quality.

Downregulation of miR-218 by nicotine promotes cell proliferation through targeting CDK6 in non-small cell lung cancer.

BACKGROUND: Nicotine, an important component of tobacco, is a major risk factor of lung cancer, but the mechanism through which nicotine promotes lung cancer development remains unclear. METHODS: Eighty patients with lung cancer were enrolled in this study, 34 of whom did not smoke and the others did. The expression of miR-218 and CDK6 messenger RNA (mRNA) was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). A luciferase reporter system was used to identify the direct target of miR-218. The protein expression of CDK6 was analyzed by using Western blotting. Cell proliferation was analyzed using an approach of calculation of cell number under a microscope. RESULTS: Nicotine decreased miR-218 expression in non-small cell lung cancer (NSCLC) cells and promoted proliferation of NSCLC cells. Smoking patients with NSCLC had lower expression of miR-218 in tumor compared with NSCLC patients who did not smoke. We found that miR-218 directly targeted the CDK6 mRNA 3'untranslated region and inhibited its expression in NSCLC cells and also observed a negative correlation between the expression of miR-218 and CDK6 mRNA in lung cancer tissues. Furthermore, miR-218- or nicotine-induced proliferative effects of NSCLC cells were rescued by the recovery of the expression level of CDK6. CONCLUSION: Nicotine promotes proliferation of NSCLC cells through regulating the miR-218/CDK6 axis, which may be a potential therapeutic target for lung cancer.

Transcriptome Landscape of Human Folliculogenesis Reveals Oocyte and Granulosa Cell Interactions.

The dynamic transcriptional regulation and interactions of human germlines and surrounding somatic cells during folliculogenesis remain unknown. Using RNA sequencing (RNA-seq) analysis of human oocytes and corresponding granulosa cells (GCs) spanning five follicular stages, we revealed unique features in transcriptional machinery, transcription factor networks, and reciprocal interactions in human oocytes and GCs that displayed developmental-stage-specific expression patterns. Notably, we identified specific gene signatures of two cell types in particular developmental stage that may reflect developmental competency and ovarian reserve. Additionally, we uncovered key pathways that may concert germline-somatic interactions and drive the transition of primordial-to-primary follicle, which represents follicle activation. Thus, our work provides key insights into the crucial features of the transcriptional regulation in the stepwise folliculogenesis and offers important clues for improving follicle recruitment in vivo and restoring fully competent oocytes in vitro.

Transcriptome analysis of PCOS arrested 2-cell embryos.

In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

Activation of hedgehog signaling and its association with cisplatin resistance in ovarian epithelial tumors.

Few studies have evaluated Hedgehog (Hh) signaling pathway activation in different types of ovarian tumors including benign, borderline and malignant ovarian tumors. The present study investigated the expression of Hh signaling pathway components (SHH, SMO, PTCH, and GLI1) in 193 ovarian epithelial tumor specimens (including 147 malignant epithelial ovarian cancers, 30 borderline ovarian tumors, 16 benign ovarian epithelial tumors) and 11 normal ovarian epithelial tissues by immunohistochemistry. The results demonstrated widespread expression of Hh pathway molecules in ovarian tumors. However, there was no significant difference in the expression intensity of SHH among the four groups (P>0.05). Statistically significant differences were identified in the expression intensity of the SMO, PICH and GLI1 among groups (P<0.001). In addition, significant differences were also revealed in the expression levels of SMO (P=0.013) and GLI1 (P=0.0005) between the platinum drug-sensitive and drug-resistant groups. The overexpression of SMO and GLI1 was further confirmed in the cisplatin-resistant ovarian cancer cell line A2780/DDP by immunofluorescence, flow cytometry and western blotting. The results revealed that the Hh pathway components SMO, PICH and GLI1 are activated in ovarian epithelial tumors. Novel potential associations between cisplatin resistance and the overexpression of SMO and Gli1 in malignant epithelial ovarian cancer were also observed, which may provide an innovative approach to the treatment of drug resistant ovarian epithelial cancer.

Pretreatment with coenzyme Q10 improves ovarian response and embryo quality in low-prognosis young women with decreased ovarian reserve: a randomized controlled trial.

BACKGROUND: Management of women with reduced ovarian reserve or poor ovarian response (POR) to stimulation is one of the major challenges in reproductive medicine. The primary causes of POR remain elusive and oxidative stress was proposed as one of the important contributors. It has been suggested that focus on the specific subpopulations within heterogeneous group of poor responders could assist in evaluating optimal management strategies for these patients. This study investigated the effect of anti-oxidant treatment with coenzyme Q10 (CoQ10) on ovarian response and embryo quality in young low-prognosis patients with POR. METHODS: This prospective, randomized controlled study included 186 consecutive patients with POR stratified according to the POSEIDON classification group 3 (age < 35, poor ovarian reserve parameters). The participants were randomized to the CoQ10 pre-treatment for 60 days preceding IVF-ICSI cycle or no pre-treatment. The number of high quality embryos was a primary outcome measure. RESULTS: A total of 169 participants were evaluated (76 treated with CoQ10 and 93 controls); 17 women were excluded due to low compliance with CoQ10 administration. The baseline demographic and clinical characteristics were comparable between the groups. CoQ10 pretreatment resulted in significantly lower gonadotrophin requirements and higher peak E2 levels. Women in CoQ10 group had increased number of retrieved oocytes (4, IQR 2-5), higher fertilization rate (67.49%) and more high-quality embryos (1, IQR 0-2); p < 0.05. Significantly less women treated with CoQ10 had cancelled embryo transfer because of poor embryo development than controls (8.33% vs. 22.89%, p = 0.04) and more women from treatment group had available cryopreserved embryos (18.42% vs. 4.3%, p = 0.012). The clinical pregnancy and live birth rates per embryo transfer and per one complete stimulation cycle tended to be higher in CoQ10 group but did not achieve statistical significance. CONCLUSION: Pretreatment with CoQ10 improves ovarian response to stimulation and embryological parameters in young women with poor ovarian reserve in IVF-ICSI cycles. Further work is required to determine whether there is an effect on clinical treatment endpoints.

Current perspectives on in vitro maturation and its effects on oocyte genetic and epigenetic profiles.

In vitro maturation (IVM), the maturation in culture of immature oocytes, has been used in clinic for more than 20 years. Although IVM has the specific advantages of low cost and minor side effects over controlled ovarian stimulation, the prevalence of IVM is less than 1% of routine in vitro fertilization and embryo transfer techniques in many reproductive centers. In this review, we searched the MEDLINE database for all full texts and/or abstract articles published in English with content related to oocyte IVM mainly between 2000 and 2016. Many different aspects of the IVM method may influence oocyte potential, including priming, gonadotrophin, growth factors, and culture times. The culture conditions of IVM result in alterations in the oocyte or cumulus cell transcriptome that are not observed under in vivo culture conditions. Additionally, epigenetic modifications, such as DNA methylation or acetylation, are also different between in vitro and in vivo cultured oocytes. In sum, current IVM technique is still not popular and requires more systematic and intensive research to improve its effects and applications. This review will help point our problems, supply evidence or clues for future improving IVM technique, thus assist patients for fertility treatment or preservation as an additional option.

Increased incidence of ectopic pregnancy after in vitro fertilization in women with decreased ovarian reserve.

The incidence of ectopic pregnancy after assisted reproductive technology is increased approximately 2.5-5-fold compared with natural conceptions.Strategies were used to decrease the incidence of ectopic pregnancy, but ectopic pregnancy still occurs. In the present study, women were selected with decreased ovarian reserve (defined as FSH > 10 IU/L) aged 20 to 38 years who underwent IVF-ET between 2009 and 2014. These 2,061 women were age-matched with an equal number of women with normal ovarian reserve (defined as FSH ≤ 10 IU/L). During cycles following fresh embryo transfer, 93 patients were diagnosed with ectopic pregnancy. The incidence of ectopic pregnancy in clinical pregnancies was significantly higher in the decreased ovarian reserve than in the normal ovarian reserve group (5.51% vs. 2.99%). After adjusting for confounding factors, the incidence of ectopic pregnancy was significantly associated with decreased ovarian reserve. Our results showed that decreased ovarian reserve is an independent risk factor for ectopic pregnancy after in vitro fertilization-embryo transfer.

The 'normal' range of FMR1 triple CGG repeats may be associated with primary ovarian insufficiency in China.

The aim of this study was to investigate the relationship between normal Fragile X mental retardation gene 1 (FMR1) CGG repeat numbers and primary ovarian insufficiency (POI) occurrence or subsequent resumption of ovarian function. A total of 122 women with POI and 105 controls were followed up and analysed in our centre. The prevalence of premutation and intermediate range of FMR1 CGG repeats in Han Chinese women with POI was only 0.81% (1/122) and 1.64% (2/122), respectively. The risk of POI occurrence for less than 26 CGG repeats and 29 or more CGG repeats in allele1 (smaller allele) was significantly higher than that for 26-28 CGG repeats (odds ratio 13.50, 95% confidence interval: 3.21 to 56.77 and 6.32, 95% confidence interval: 2.49 to 16.09 respectively; both P < 0.001). No significant difference was found in the CGG repeat distribution (<26, 26-28, or ≥29) in FMR1 allele1 between POI cases whose ovarian function resumed and those whose ovarian function did not. It is suggested that the CGG repeat number in allele1, but not that in allele2 (longer allele), was significantly associated with POI occurrence (P < 0.001). Fewer than 26 or more than 28 CGG repeats in FMR1 allele1 were both risk factors of POI occurrence.

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.

Increased Incidence of Mitochondrial Cytochrome C Oxidase 1 Gene Mutations in Patients with Primary Ovarian Insufficiency.

Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), is defined as more than six months of cessation of menses before the age of 40 years, with two serum follicle stimulating hormone (FSH) levels (at least 1 month apart) falling in the menopause range. The cause of POI remains undetermined in the majority of cases, although some studies have reported increased levels of reactive oxygen species (ROS) in idiopathic POF. The role of mitochondrial DNA in the pathogenesis of POI has not been studied extensively. This aim of this study was to uncover underlying mitochondrial genetic defects in patients with POI. The entire region of the mitochondrial genome was amplified in subjects with idiopathic POI (n=63) and age-matched healthy female controls (n=63) using nine pair sets of primers, followed by screening of the mitochondrial genome using an Illumina MiSeq. We identified a total of 96 non-synonymous mitochondrial variations in POI patients and 93 non-synonymous variations in control subjects. Of these, 21 (9 in POI and 12 in control) non-synonymous variations had not been reported previously. Eight mitochondrial cytochrome coxidase 1 (MT-CO1) missense variants were identified in POI patients, whereas only four missense mutations were observed in controls. A high incidence of MT-CO1 missense variants were identified in POI patients compared with controls, and the difference between the groups was statistically significant (13/63 vs. 5/63, p=0.042). Our results show that patients with primary ovarian insufficiency exhibit an increased incidence of mitochondrial cytochrome c oxidase 1 gene mutations, suggesting that MT-CO1 gene mutation may be causal in POI.

Gonadotropin-mediated dynamic alterations during bovine oocyte maturation in vitro.

Gonadotropins have been widely used in human-assisted reproduction and animal science for the past four decades. However, the effects of gonadotropins on oocyte maturation at the molecular and biochemical levels are poorly understood. To determine the effects of gonadotropins (recombinant follicle stimulating hormone and urinary human menopausal gonadotropin) on oocyte maturation, we used the bovine oocyte in vitro maturation model. First, we studied the effects of increasing gonadotropin concentrations on nuclear maturation and mitochondrial function in oocytes. Gonadotropins at concentrations of 0.075 and 0.75 IU/ml improved nuclear maturation and increased inner mitochondrial membrane potential and ATP levels; however, there were no beneficial effects at concentrations of 7.5 and 75 IU/ml. Second, we studied the effects of increasing gonadotropin concentrations on the status of methylation in matured (MII) oocytes. Aberrant methylation and demethylation of H19, SNRPN, and PEG3 genes were observed in MII oocytes at all concentrations except 0.075 IU/ml. The expression of genes that function in spindle formation, cell cycle control, and methylation was also downregulated by high gonadotropin concentrations. In conclusion, we established the optimal gonadotropin concentration (i.e., 0.075 IU/ml) to be used for bovine oocyte in vitro maturation studies. These results may provide a guide for clinical stimulation protocols and help to reduce the risks associated with gonadotropin administration during in vitro fertilization treatment.

Advances in preimplantation genetic diagnosis/screening.

Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS.

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.

ART do not increase the risk of Y-chromosome microdeletion in 19 candidate genes at AZF regions.

Y-chromosome microdeletions (YCMs) have been found at a much higher rate in infertile men than fertile controls. A specific deletion in the azoospermia factor locus (AZF) at Yq11 is significantly associated with male infertility. Whether assisted reproductive technology (ART) increases the risk of YCM in ART-derived offspring remains unclear. In this study the occurrence of YCM in 199 fathers and their 228 sons (Chinese, Han ethnicity), including 85 offspring conceived by IVF, 73 by intra-cytoplasmic sperm injection (ICSI) and 70 by natural conception, was investigated. Nineteen candidate genes related to YCM were analysed by multiplex ligation-dependent probe amplification. We identified one de novo YCM from 70 naturally-conceived offspring and none from 158 ART-conceived offspring and found no statistical significance between these two groups. There was no statistically-significant difference in the detection rate of the father's Y-chromosome microdeletion group: IVF 10.7% (8/75), ICSI 3.2% (2/63), natural conception 8.2% (5/61). These results suggest that ART does not increase the risk of YCM in male offspring.

A bronchofiberoscopy-associated outbreak of multidrug-resistant Acinetobacter baumannii in an intensive care unit in Beijing, China.

BACKGROUND: Bronchofiberscopy, a widely used procedure for the diagnosis of various pulmonary diseases within intensive care units, has a history of association with nosocomial infections. Between September and November 2009, an outbreak caused by multidrug-resistant Acinetobacter baumannii (MDR-Ab) was observed in the intensive care unit of a tertiary care hospital in Beijing, China. This study is aimed to describe the course and control of this outbreak and investigate the related risk factors. METHODS: Clinical and environmental sampling, genotyping with repetitive extragenic palindromic polymerase chain reaction (REP-PCR), and case-control risk factor analysis were performed in the current study. RESULTS: During the epidemic period, 12 patients were infected or colonized with MDR-Ab. Sixteen (72.7%) of twenty-two MDR-Ab isolates from the 12 patients and 22 (84.6%) of 26 MDR-Ab isolates from the bronchofiberscope and the healthcare-associated environment were clustered significantly into a major clone (outbreak MDR-Ab strain) by REP-PCR typing. Seven patients carrying the outbreak MDR-Ab strain were defined as the cases. Six of the seven cases (83%) received bronchofiberscopy versus four of the 19 controls (21%) (odds ratio, 22.5; 95% confidence interval, 2.07-244.84; P = 0.005). Several potential administrative and technical problems existed in bronchofiberscope reprocessing. CONCLUSIONS: Bronchofiberscopy was associated with this MDR-Ab outbreak. Infection control precautions including appropriate bronchofiberscope reprocessing and environmental decontamination should be strengthened.

Fragile X premutation RNA is sufficient to cause primary ovarian insufficiency in mice.

Spontaneous 46,XX primary ovarian insufficiency (POI), also known as 'premature menopause' or 'premature ovarian failure', refers to ovarian dysfunction that results in a range of abnormalities, from infertility to early menopause as the end stage. The most common known genetic cause of POI is the expansion of a CGG repeat to 55-199 copies (premutation) in the 5' untranslated region in the X-linked fragile X mental retardation 1 (FMR1) gene. POI associated with the FMR1 premutation is referred to as fragile X-associated POI (FXPOI). Here, we characterize a mouse model carrying the human FMR1 premutation allele and show that FMR1 premutation RNA can cause a reduction in the number of growing follicles in ovaries and is sufficient to impair female fertility. Alterations in selective serum hormone levels, including FSH, LH and 17ß-estradiol, are seen in this mouse model, which mimics findings in humans. In addition, we also find that LH-induced ovulation-related gene expression is specifically altered. Finally, we show that the FMR1 premutation allele can lead to reduced phosphorylation of Akt and mTOR proteins. These results together suggest that FMR1 premutation RNA could cause the POI associated with FMR1 premutation carriers, and the Akt/mTOR pathway may serve as a therapeutic target for FXPOI.

MicroRNA-146a modulates TGF-ß1-induced phenotypic differentiation in human dermal fibroblasts by targeting SMAD4.

During wound healing and tissue repair the dermal fibroblast-to-myofibroblast transdifferentiation plays an important role, transforming growth factor-ß1 (TGF-ß1) is considered to be the main stimuli factor of transdifferentiation. MicroRNAs (miRNAs) have recently emerged as key post-transcriptional regulators of gene expression. The involvement of miRNAs and their roles in TGF-ß1-induced myofibroblast transdifferentiation remains to be determined in detail. The current study found that the expression of miR-146a was upregulated in human dermal fibroblasts cells in response to TGF-ß1 stimulation in dose-dependent manner by quantitative RT-PCR. Bioinformatic analyses predict that signaling effectors mothers against decapentaplegic protein 4 (SMAD4) is a miR-146a target gene. Luciferase assay demonstrated that miR-146a mimics suppressed SMAD4 3'-UTR reporter construct activity. Furthermore, miR-146a overexpression in dermal fibroblast did not decrease target mRNA levels, but significantly reduced target protein expression. In addition, dermal fibroblasts transfected with miR-146a mimics exhibited attenuated TGF-ß1 -induced α-smooth muscle actin (α-SMA) expression compared with the control. This study demonstrated that miR-146a may function as a novel negative regulator to modulate myofibroblast transdifferentiation during TGF-ß1 induction by targeting SMAD4.