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1.  The expression of DBC1/CCAR2 is associated with poor prognosis of ovarian carcinoma 
Background
Recent reports have shown that deleted in breast cancer 1 (DBC1/CCAR2) is an indicator of poor prognosis of various human cancers. However, its expression in ovarian carcinoma has not been reported.
Methods
We investigated the immunohistochemical expression of DBC1 and BRCA1 and their prognostic significance in 104 ovarian carcinomas. Survival analyses were performed according to the Kaplan-Meier method, as well as univariate and multivariate Cox proportional hazard regression analysis.
Results
Positive expression of DBC1 and BRCA1 were seen in 63% (66/104) and 44% (46/104) of overall ovarian carcinomas, respectively. DBC1 expression was significantly associated with advanced clinicopathological factors such as high tumor stage, latent distant metastasis, platinum-resistance, elevated serum levels of CA125, high histologic grade, and BRCA1 expression. In the histological subtypes of ovarian carcinomas, DBC1 expression was more common in serous carcinoma (72%, 54/75) than mucinous carcinoma (15%, 3/20). BRCA1 expression was significantly associated with latent distant metastasis, platinum-resistance, and higher histologic grade. In addition, DBC1 expression was significantly associated with shorter overall survival (OS) and relapse-free survival (RFS) in 104 ovarian carcinomas (OS; P < 0.001, RFS; P < 0.001) and 63 high-grade serous carcinomas (OS; P = 0.008, RFS; P = 0.023) by univariate analysis. BRCA1 expression was significantly associated with OS and RFS in 104 ovarian carcinomas (OS; P = 0.005, RFS; P = 0.002) and 75 serous carcinomas (OS; P = 0.047, RFS; P = 0.038) by univariate analysis. Moreover, DBC1 expression was an independent prognostic indicator for OS in both 104 ovarian carcinomas (P = 0.021) and 63 high-grade serous carcinomas (P = 0.011) by multivariate analysis.
Conclusions
These results indicate that the expression of DBC1 and BRCA1 are closely related with in the progression of ovarian carcinomas and may have clinical utility in the prediction of prognosis of ovarian carcinomas. Especially, DBC1 expression could be employed as a significant prognostic indicator for ovarian carcinomas especially in high-grade serous carcinomas.
doi:10.1186/s13048-015-0129-3
PMCID: PMC4335761
Ovarian neoplasms; Serous carcinoma; Deleted in breast cancer 1; BRCA1; Prognosis
2.  Sequential combination therapy with flavopiridol and autocatalytic caspase-3 driven by amplified hTERT promoter synergistically suppresses human ovarian carcinoma growth in vitro and in mice 
Background
Induction of cell apoptosis and regulation of cell cycle are very attractive for treatments of tumors including ovarian carcinoma. Flavopiridol is a potent small molecular cyclin-dependent kinase(cdk) inhibitor, but its antitumor efficacy is not satisfied yet. Caspase-3 play a major role in the transduction of apoptotic signals and the execution of apoptosis in mammalian cells. We have successfully constructed the recombinant adenovirues AdHTVP2G5-rev-casp3 containing autocatalytic caspase-3 (rev-caspase-3) driven by amplified hTERT promoter system (TSTA-hTERTp). In this study, we applied it with flavopiridol to investigate their antitumor effect on ovarian cancer in vitro and in vivo.
Methods
Cell viabilities were determined using Cell Counting Kit 8 and flow cytometry. RT-PCR and immunoblotting assays were used to detect cellular apoptotic activities. Tumor growth and survival of mice bearing tumors were studied.
Results
Flavopiridol or AdHTVP2G5-rev-casp3 at low dosage alone was mildly cytotoxic in vitro with a viability rate of 86.5 ± 4.7% for 300 nM flavopiridol and 88.9 ± 5.4% for AdHTVP2G5-rev-casp3 (MOI 20). By contrast, significant synergism of their sequential combination was observed, and the treatment of AdHTVP2G5-rev-casp3 (MOI 20) infection for 72 h, followed by flavopiridol (300 nM) for 48 h, can result in the most synergistic cell death, with cell survival rate and apoptotic rate of 11.6% and 69.7%, respectively. The sequential combination showed synergistic tumor suppression rate of 77.8%, which was significantly higher than that of AdHTVP2G5-rev-casp3 (33.6%) or flavopiridol (40.1%) alone. The mean survival of mice treated with the combination was 286 ± 8 d, which was synergistically longer than that of mice treated with AdHTVP2G5-rev-casp3 (141 ± 14d), flavopiridol (134 ± 10 d) or controls (106 ± 11 d) (P < 0.01).
Conclusions
The sequential combination of rev-caspase-3 and flavopiridol result in significant synergistic cell killing effects, significant tumor growth suppression and extended survival of mice bearing OVCAR3 cells. The combination should be further explored as a potential clinically useful regimen against ovarian cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0121-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0121-3
PMCID: PMC4302516  PMID: 25528169
Amplified hTERT promoter; Autocatalysis; Caspase-3; Flavopiridol; Human ovarian carcinoma
3.  The role of KIF14 in patient-derived primary cultures of high-grade serous ovarian cancer cells 
Objective
Previously, it has been shown that KIF14 mRNA is overexpressed in ovarian cancer (OvCa), regardless of histological subtype. KIF14 levels are independently predictive of poor outcome and increased rates of recurrence in serous OvCa patients. Furthermore, it has been shown that KIF14 also controls the in vivo tumorigenicity of OvCa cell lines. In this study, we evaluate the potential of KIF14 as a therapeutic target through selective inhibition of KIF14 in primary high-grade serous patient-derived OvCa cells.
Methods
To assess the dependence of primary serous OvCa cultures on KIF14, protein levels in 11 prospective high grade serous ovarian cancer samples were increased (KIF14 overexpression by transfection) or decreased (anti-KIF14 shRNA) in vitro, and proliferative capacity, anchorage independence and xenograft growth were assessed.
Results
Seven of eleven samples demonstrated increased/decreased in vitro proliferation in response to KIF14 overexpression/knockdown, respectively. When examining in vitro tumorigenicity (colony formation) and in vivo growth (subcutaneous xenografts) in response to KIF14 manipulation, none of the samples demonstrated growth in soft agar (11 samples), or xenograft growth (4 samples).
Conclusions
Although primary high-grade serous OvCa cells may depend on KIF14 for in vitro proliferation we were unable to demonstrate a role for KIF14 on tumorigenicity or develop an in vivo model for assessment. We have, however developed an effective in vitro method to evaluate the effect of target gene manipulation on the proliferative capacity of primary OvCa cultures.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0123-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0123-1
PMCID: PMC4302703  PMID: 25528264
KIF14; Ovarian tumor tissue; Primary culture; shRNA; Proliferation; Colony formation
4.  Evaluating influence of the genotypes in the follicle-stimulating hormone receptor (FSHR) Ser680Asn (rs6166) polymorphism on poor and hyper-responders to ovarian stimulation: a meta-analysis 
Background/aims
Reported associations of controlled ovarian hyperstimulation response (COH) with genotypes of the Ser680Asn (N680S) polymorphism in the follicle stimulating hormone receptor (FSHR) gene have conflicting results.
Methods
PubMed and Embase databases were searched for studies that investigated the N680S polymorphism in the FSHR gene in COH. Parameters used to examine ovarian response were poor and hyper-responses to COH. Using the meta-analytic approach, we estimated ovarian response risk (odds ratio [OR] with 95% confidence intervals) according to genotype.
Results
Our findings showed that SS genotype carriers were most likely to be poor responders (OR 1.61, p = 0.08) compared to the NN and NS genotypes which showed no associations (OR 0.93-0.95, p = 0.75-0.78). Heterogeneity of these pooled ORs warranted examining its sources. We detected outlying studies in each of the three N680S genotypes. Omitting these outliers erased the heterogeneity of the recalculated pooled outcomes. It also materially altered the SS effects where carriers became slightly unlikely to be poor responders (OR 0.90, p = 0.52). The S allele carrier effect was modulated for poor responders (OR 1.24, p = 0.39) in the Non-Hispanic Caucasian (NHC) subgroup. The likelihood of the S allele carriers (OR 1.47, p = 0.02) and the unlikelihood of the N allele carriers (OR 0.64, p = 0.007) were significant in our hyper-response findings. Confined to NHC retained significance of the S allele effects (OR 1.57, p = 0.01) but not among the N allele carriers (OR 0.68, p = 0.18).
Conclusions
In summary, this is a meta-analytical confirmation of the FSHR SS genotype role in COH response. Hyper-responder analysis strengths lie on the non-heterogeneity and robustness of its results. Non-robustness and heterogeneity of the poor-responder results compose its limitations. Thus, poor response findings probably require caution as to the interpretation as a susceptibility marker for ovarian response.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0122-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0122-2
PMCID: PMC4279698  PMID: 25526787
Follicle-stimulating hormone receptor; FSHR; Polymorphisms; Ovarian response; Meta-analysis
5.  ESR1 and ESR2 gene polymorphisms are associated with human reproduction outcomes in Brazilian women 
Background
Important candidate genes involved in the ovarian response to exogenous FSH are the estrogen receptor genes (ESRs), since the effects of estrogens on follicle growth, maturation and oocyte release. It is known that some markers of ovarian stimulation can help to personalize the treatment, adjusting the dose of exogenous rFSH, thus preventing excessive wear of the patient. Inspired on this information we aimed to analyze four different polymorphisms in the estrogen receptor genes ESR1: rs2234693/T-397C (PvuII) and rs9340799/A-351G (Xbal) and ESR2: rs4986938/G1082A (RsaI) and rs1256049/A + 1730G (AluI), and their association with assisted reproduction outcomes in Brazilian women that underwent in vitro fertilization (IVF).
Methods
A cross-sectional study was performed involving 136 infertile women less than 39 years of age with normal ovarian reserve. Patients were divided according to the same COH protocol for statistical analysis. The Taqman assay was used for PvuII and XbaI of ESR1, and RsaI and AluI of ESR2 genotyping. Serum estradiol and FSH were measured by Elisa assay.
Results
The PvuII (ESR1) TT and RsaI (ESR2) GG genotypes were associated with a longer induction period and higher doses of medication (p < 0.03). The XbaI (ESR1) AA genotype was associated with better COH results, including a larger number of follicles, mature oocytes, embryos, and good quality embryos (p < 0.05). The AluI GG genotype showed an association with the Ovarian Hyperstimulation Syndrome (OHSS) (p = 0.03). According to the haplotype analysis of ER1 (PvuII/XbaI), we demonstrated that the CA combination increases by 0.68 the number of good quality embryos while the TG decreases it by 0.71 (p = 0.04).
Conclusion
ER polymorphisms have an association with the assisted reproduction outcomes in Brazilian women.
doi:10.1186/s13048-014-0114-2
PMCID: PMC4302593  PMID: 25526766
Estrogen receptor gene; Polymorphism; In vitro fertilization; Controlled ovarian hyperstimulation
6.  Anti Müllerian Hormone (AMH) level and expression in mural and cumulus cells in relation to age 
Background
Serum AMH is declining with age and is highly associated with ovarian follicular reserve and disordered folliculogenesis. However, the precise role of AMH in the process of human follicular aging has still to be determined.
Aim
This study investigates AMH level in the follicular fluid (FF) and mRNA expression pattern in cumulus and mural granulosa cells of human ovarian follicles in relation to age.
Methods
We conducted a prospective study. Sixty-eight women undergoing In vitro fertilization (IVF) treatment were enrolled in the study. We obtained FF, mural and cumulus granulosa cells from large preovulatory follicles (17-20 mm) of 21–35 years old women (n = 40) and 40–45 years old women (n = 28) during oocyte pickup.
Results
Higher level of AMH mRNA expression in cumulus cells was observed in the older age group compared to the younger (P <0.01). In accordance with AMH mRNA expression results, FF AMH protein levels were significantly higher in the older group than in the younger group (4.7 ± 1.1 ng\ml and 2.3 ± 0.2 ng\ml respectively, p < 0.002).
Conclusions
AMH is highly expressed and secreted from cumulus GCs of advanced age patients. This remarkable correlation between AMH mRNA levels in cumulus cells in respect to age suggests that AMH may be involved in follicular aging process.
doi:10.1186/s13048-014-0113-3
PMCID: PMC4269874  PMID: 25500128
7.  Does bariatric surgery improve ovarian stimulation characteristics, oocyte yield, or embryo quality? 
Background
Obesity is a major global health concern associated with multiple co-morbidities. Bariatric surgery has been considered a good treatment option in cases of morbid obesity. This preliminary study aims to investigate the effect of bariatric surgery on ovarian stimulation characteristics and IVF treatment cycle outcome.
Methods
A retrospective study that was performed in a tertiary, university-affiliated medical center and included all patients who underwent IVF treatment both before and after bariatric surgery. Data on ovarian stimulation variables of IVF treatment cycle prior and following the bariatric surgery were reviewed and compared.
Results
From January 2005 to June 2014, seven women fulfilled the inclusion criteria. After the operation, BMI was significantly reduced (mean ± SD) (43.1 ± 3.3 vs. 29.6 ± 7.33, p = 0.018), as was the number of gonadotropin ampoules required during stimulation (69.3 ± 10.5 vs. 44.5 ± 17, p = 0.043). No between-cycle differences were observed in peak estradiol level, the number of oocytes retrieved, and percentage of mature oocytes.
Conclusions
To the best of our knowledge, this preliminary case series is the first comparison of IVF cycle characteristics prior to and following bariatric surgery. The operation seems to reduce treatment costs without affecting oocyte or embryo quality. Further large studies are required to establish the surgery’s effect on IVF outcome among infertile women.
doi:10.1186/s13048-014-0116-0
PMCID: PMC4265478  PMID: 25491501
Bariatric surgery; Morbid obesity; IVF; Female fertility
8.  Protein kinase C isoforms α, δ and ε are differentially expressed in mouse ovaries at different stages of postnatal development 
Background
The protein kinase C (PKC) is a family of serine/threonine kinases that consists of 12 different isoforms. Since PKC isoform expressions are known to be specific for different cell types and postnatal developmental stages, we aimed to determine immunolocalizations and protein expression levels of different PKC isoforms in pre-pubertal, pubertal and adult mouse ovaries.
Methods
Ovaries were obtained from postnatal day 1 (PND1) and PND7 of pre-pubertal, PND21 of pubertal and PND60 of adult mice. Immunolocalizations of PKCα, PKCδ and PKCε isoforms were determined and immunostainings in different cellular components of all follicular stages were evaluated by H-Score. PKCα, PKCδ and PKCε protein expression levels were determined by Western blot. The bands were quantified via ImageJ software. The data obtained from H-Score and ImageJ evaluations were analyzed by ANOVA statistical test.
Results
PKCα immunostainings were more intense in oocytes when compared to granulosa and theca cells at different follicular stages of all groups. The Western blot analysis revealed that PKCα expression was significantly higher in PND60 adult ovaries. Conversely, PKCδ immunostainings were more intense in granulosa cells. According to the Western blot analysis, PKCδ protein expression was also higher in PND60 and significantly lower in PND1 ovaries. PKCε immunostaining was more apparent in oocytes. PKCε protein expression was significantly higher in adult PND60 and pubertal PND21 ovaries when compared to pre-pubertal PND7 and PND1 ovaries. Interestingly, PKCε immunostaining was significantly higher in primordial follicles, though PKCα and PKCδ immunostainings were more apparent in larger follicles. PKCα immunostainings of corpora lutea (CL) were significantly higher when compared to follicles in PND60 ovaries.
Conclusions
This study demonstrates that PKCα, PKCδ and PKCε isoforms are differentially expressed in particular cellular components of pre-pubertal, pubertal and adult mouse ovarian follicles. Therefore, we suggest that each PKC isoform has unique functions that are controlled by gonadotropin dependent mechanisms during follicular growth, oocyte maturation, ovulation and luteinization.
doi:10.1186/s13048-014-0117-z
PMCID: PMC4271327  PMID: 25491605
Protein kinase C; Ovary; Postnatal development
9.  Wilms’ tumor suppressor gene (WT1) suppresses apoptosis by transcriptionally downregulating BAX expression in immature rat granulosa cells 
Background
The important role of WT1 in early folliculogenesis was evident from its restricted expression pattern in immature follicles and from its involvement in transcriptional control of inhibin-α and FSH receptor. There is also considerable evidence that WT1 is a potent inhibitor of apoptotic cell death in the developing kidney and male germ cells, suggesting that it could play a role in the regulation of follicle survival. Therefore, we evaluated if WT1 involves in cell survival of granulosa cells (GCs) during the FSH-independent stage.
Methods
GCs were obtained from small preantral follicles of immature rat ovary. Bax and bcl-2 mRNA and protein levels in GCs transfected with WT1 (−KTS) or WT1 (+KTS) were analyzed by Real-time RT-PCR and immune-blotting analysis. Cell viability was measured with MTT assays and apoptosis was analyzed with caspase 3/7 activity and TUNEL assay. The mechanism by which WT1 regulates Bax expression was investigated using Bax promoter-luciferase reporter assay and ChIP assays from GCs.
Results
Here, we showed that WT1 (−KTS) suppressed endogenous Bax transcript and protein expression, and this inhibition resulted from direct binding of WT1 in the Bax promoter region in vivo. In addition, anti-apoptotic effects of WT1 (−KTS) were demonstrated based on MTT assays, a sensitive bioluminescence caspase 3/7 assay and TUNEL assays. On the other hand, WT1 has no role on bcl-2 expression in GCs.
Conclusion
These findings suggest that activation of WT1 is necessary for maintenance of GC survival during early stage of follicles and WT1 can play a role in protecting apoptosis through the regulation of upstream activator (Bax), as well as through regulation of downstream effecter (caspases 3 and 7).
doi:10.1186/s13048-014-0118-y
PMCID: PMC4279686  PMID: 25491731
WT1; Apoptosis; Bax; bcl-2; Caspase; Granulosa cell; Rat
10.  Succinate dehydrogenase subunit B inhibits the AMPK-HIF-1α pathway in human ovarian cancer in vitro 
Background
Ovarian carcinoma is one of the most common gynecological cancers with high mortality rates. Numerous evidences demonstrate that cancer cells undergo metabolic abnormality during tumorigenesis in tumor microenvironment and further facilitate tumor progression. Succinate dehydrogenase (SDH or Complex II) is one of the important enzymes in the tricarboxylic acid (TCA) cycle. Succinate dehydrogenase subunit B (SDHB) gene, which encodes one of the four subunits of SDH, has been recognized as a tumor suppressor. However the role of SDHB in ovarian cancer is still unclear.
Methods
Using the SDHB specific siRNA and overexpression plasmid, the expression of SDHB was silenced and conversely induced in ovarian cancer cell lines SKOV3 and A2780, respectively. The possible role of SDHB in ovarian cancer was investigated in vitro, using proliferation, migration and invasion assays. To explore the mechanism, proliferation and migration related proteins such as Bcl-2, cleaved caspase 3, p-ERK, MMP-2, and p-FAK were examined by western blot. P-P38, p-AMPKα, and HIF-1α were also examined by western blot. CoCl2 was used to induce HIF-1α expression in SKOV3 and A2780 cells.
Results
SDHB silencing promoted cell proliferation, invasion, and migration, but inhibited apoptosis of SKOV3 and A2780 cells. In contrast, overexpression of SDHB inhibited cell proliferation, invasion, migration, and promoted apoptosis in SKOV3 cells. It was observed that up-regulation of Bcl-2 and MMP-2, activation of p-P38, p-ERK, and p-FAK, inhibition of cleaved caspase 3 in SDHB-silenced cells. Meanwhile, decreased Bcl-2 and MMP-2, inhibition of p-P38, p-ERK, and p-FAK, activation of cleaved caspase 3 were shown in SDHB-overexpressed SKOV3 cells. HIF-1α, an essential factor in tumor progression, was up-regulated in SDHB-silenced cells with the activation of p-AMPKα and down-regulated in SDHB-overexpressed cancer cells with the decreased p-AMPKα. And SDHB was proved to be decreased due to upregulation of HIF-1α expression in CoCl2-treated cancer cells.
Conclusions
Our results firstly revealed that SDHB played a key role in cell proliferation, invasion, migration, and apoptosis of human ovarian carcinoma via AMPK-HIF-1α pathway. SDHB-overexpression might be a new approach to inhibit tumor progression in human ovarian carcinoma.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0115-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0115-1
PMCID: PMC4279696  PMID: 25491408
Succinate dehydrogenase subunit B; HIF-1α; AMPK; Gene silence; Gene overexpression; Ovarian carcinoma
11.  A prospective cohort study of dietary indices and incidence of epithelial ovarian cancer 
Background
Several dietary indices have been developed to measure overall diet quality, including the Healthy Eating Index-2005 (HEI-2005), which measures adherence to the 2005 Dietary Guidelines from the USDA; the Alternative Healthy Eating Index-2010 (AHEI-2010), which is based on foods and nutrients predictive of chronic disease risk; and the Alternate Mediterranean Diet Score (aMDS), which is an index that characterizes traditional food patterns of Mediterranean countries. Few studies have evaluated diet quality and ovarian cancer risk.
Methods
We assessed the associations of the HEI-2005, AHEI-2010, and aMDS with risk of epithelial ovarian cancer prospectively among women in the Nurses’ Health Study. We used Cox proportional hazards models, adjusting for known ovarian cancer risk factors.
Results
During 24 years of follow-up, we documented 696 incident epithelial ovarian cancer cases among 82,948 women with diet information. The multivariate adjusted hazard ratios (95% confidence interval; Ptrend) of epithelial ovarian cancer comparing the highest with the lowest quintile were 1.03 (0.80-1.34; 0.77) for the AHEI-2010, 0.85 (0.65-1.12; 0.57) for the HEI-2005, and 0.91 (0.71-1.18; 0.44) for the aMDS.
Conclusions
We did not observe any clear association of three diet quality scores with ovarian cancer risk. Further work should other metrics of evaluating diet quality that may be more relevant cancer risk.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0112-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0112-4
PMCID: PMC4263215  PMID: 25477244
Alternative healthy eating index; Healthy eating index; Mediterranean diet score; Dietary pattern; Ovarian cancer; Prospective cohort; Epidemiology
12.  Kallikrein family proteases KLK6 and KLK7 are potential early detection and diagnostic biomarkers for serous and papillary serous ovarian cancer subtypes 
Background
Early detection of ovarian cancer remains a challenge due to widespread metastases and a lack of biomarkers for early-stage disease. This study was conducted to identify relevant biomarkers for both laparoscopic and serum diagnostics in ovarian cancer.
Methods
Bioinformatics analysis and expression screening in ovarian cancer cell lines were employed. Selected biomarkers were further validated in bio-specimens of diverse cancer types and ovarian cancer subtypes. For non-invasive detection, biomarker proteins were evaluated in serum samples from ovarian cancer patients.
Results
Two kallikrein (KLK) serine protease family members (KLK6 and KLK7) were found to be significantly overexpressed relative to normal controls in most of the ovarian cancer cell lines examined. Overexpression of KLK6 and KLK7 mRNA was specific to ovarian cancer, in particular to serous and papillary serous subtypes. In situ hybridization and histopathology further confirmed significantly elevated levels of KLK6 and KLK7 mRNA and proteins in tissue epithelium and a lack of expression in neighboring stroma. Lastly, KLK6 and KLK7 protein levels were significantly elevated in serum samples from serous and papillary serous subtypes in the early stages of ovarian cancer, and therefore could potentially decrease the high “false negative” rates found in the same patients with the common ovarian cancer biomarkers human epididymis protein 4 (HE4) and cancer antigen 125 (CA-125).
Conclusion
KLK6 and KLK7 mRNA and protein overexpression is directly associated with early-stage ovarian tumors and can be measured in patient tissue and serum samples. Assays based on KLK6 and KLK7 expression may provide specific and sensitive information for early detection of ovarian cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0109-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0109-z
PMCID: PMC4271347  PMID: 25477184
Biomarker; Ovarian cancer; Diagnostic; Early detection; Bioinformatics
13.  The transcriptome of corona radiata cells from individual MІІ oocytes that after ICSI developed to embryos selected for transfer: PCOS women compared to healthy women 
Background
Corona radiata cells (CRCs) refer to the fraction of cumulus cells just adjacent to the oocyte. The CRCs are closely connected to the oocyte throughout maturation and their gene expression profiles might reflect oocyte quality. Polycystic ovary syndrome (PCOS) is a common cause of infertility. It is controversial whether PCOS associate with diminished oocyte quality. The purpose of this study was to compare individual human CRC samples between PCOS patients and controls.
Methods
All patients were stimulated by the long gonadotropin-releasing hormone (GnRH) agonist protocol. The CRC samples originated from individual oocytes developing into embryos selected for transfer. CRCs were isolated in a two-step denudation procedure, separating outer cumulus cells from the inner CRCs. Extracted RNA was amplified and transcriptome profiling was performed with Human Agilent® arrays.
Results
The transcriptomes of CRCs showed no individual genes with significant differential expression between PCOS and controls, but gene set enrichment analysis identified several cell cycle- and DNA replication pathways overexpressed in PCOS CRCs (FDR < 0.05). Five of the genes contributing to the up-regulated cell cycle pathways in the PCOS CRCs were selected for qRT-PCR validation in ten PCOS and ten control CRC samples. qRT-PCR confirmed significant up-regulation in PCOS CRCs of cell cycle progression genes HIST1H4C (FC = 2.7), UBE2C (FC = 2.6) and cell cycle related transcription factor E2F4 (FC = 2.5).
Conclusion
The overexpression of cell cycle-related genes and cell cycle pathways in PCOS CRCs could indicate a disturbed or delayed final maturation and differentiation of the CRCs in response to the human chorionic gonadotropin (hCG) surge. However, this had no effect on the in vitro development of the corresponding embryos. Future studies are needed to clarify whether the up-regulated cell cycle pathways in PCOS CRCs have any clinical implications.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0110-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0110-6
PMCID: PMC4302704  PMID: 25432544
Corona radiata cells; Transcriptome; Gene expression; PCOS; Oocyte quality
14.  The level of RECQL1 expression is a prognostic factor for epithelial ovarian cancer 
Background
The human RECQ DNA helicase family is involved in genomic stability. Gene mutations of RECQL2, RECQL3, and RECQL4 are associated with genetic disorders and induce early aging and carcinogenesis. Although previous studies have reported that the level of RECQL1 expression is correlated with the prognosis of some of malignancies, the function of RECQL1 is not yet clarified. The present study aimed to examine the relationship between prognosis and the level of RECQL1 expression in epithelial ovarian cancer (EOC), and to identify the role of RECQL1 in EOC cells.
Methods
The level of RECQL1 expression was determined immunohistochemically in 111 patients with EOC who received initial treatment at Hirosaki University hospital between 2006 and 2011. Effects of RECQL1 on cell growth or apoptosis were examined in vitro using wild-type and OVCAR-3 cells (RECQL1(+) cells) and similar cells transfected with RECQL1 siRNA transfected (RECQL1(−) cells).
Results
The level of RECQL1 expression was not related to histological type, clinical stage, or retroperitoneal lymph node metastasis, but the expression level was significantly higher (P = 0.002) in patients with recurrence than those without recurrence, and progression-free survival and complete response rate to chemotherapy were also improved in patients with RECQL1-low expression (n = 39) stage III/IV EOC (P = 0.02 and P <0.05 vs RECQL1-high expression patients (n = ), respectively). A cell proliferation and colony formation assays revealed significantly less growth of RECQL1(−) cells compared to RECQL1(+) cells. A flow cytometry using annexin V -FITC and propidium iodide (PI) staining revealed a significant increase in apoptotic RECQL1(−) cells. Cell cycle analysis showed a significantly greater distribution in subG1 phase indicating apoptotic cells in RECQL1(−) cells than in RECQL1(+) cells.
Conclusions
These results suggest that RECQL1 is a prognostic factor for EOC and that RECQL1 contributes to potential malignancy by inhibiting apoptosis.
doi:10.1186/s13048-014-0107-1
PMCID: PMC4255635  PMID: 25424877
Ovarian cancer; RECQL1; siRNA; Apoptosis
15.  The impact of endometrioma and laparoscopic cystectomy on ovarian reserve and the exploration of related factors assessed by serum anti-Mullerian hormone: a prospective cohort study 
Background
To evaluate the impact of the presence of endometrioma and laparoscopic cystectomy on ovarian reserve as assessed by serum anti-Müllerian hormone (AMH) level. In addition, factors related to the decline in ovarian reserve were analyzed.
Methods
From June 2013 to January 2014, we prospectively included 40 women with endometriomas as the study group (group A), 36 women with tubal factor infertilities as control group 1 (group B) and 22 women with the other benign ovarian cysts as control group 2 (group C). The women with ovarian cysts underwent laparoscopic cystectomy. Serum AMH levels were determined preoperatively and at 1 month after surgery.
Results
The endometrioma group had lower AMH levels (1.53 ± 1.37 ng/ml) compared with the other benign ovarian cyst group (2.20 ± 1.23 ng/ml) and the tubal factor infertility group (2.82 ± 1.74 ng/ml). The rate of serum AMH decline 1 month after surgery in the endometrioma group (0.62 ± 0.35) was larger than the decline in the other benign ovarian cyst group (0.32 ± 0.30). The preoperative AMH level showed a significant correlation with patient age (group A, r = −0.32; group B, r = −0.54; group C, r = −0.71); there was a statistically significant correlation between the rate of serum AMH decline and endometrioma diameter as well as with the preoperative serum AMH level. In addition, the rate of serum AMH decline was larger for bilateral endometriomas than for unilateral endometriomas, but there was no similar correlation in the other benign ovarian cyst group. The rate of AMH decline after surgery in the subgroup of >7 cm was significantly higher than in the subgroup of ≤7 cm.
Conclusions
Ovarian endometriomas per se may damage ovarian reserve, and cystectomy of endometriomas may cause greater damage to ovarian reserve compared with other benign ovarian cysts. The operation-related damage to the ovarian reserve was positively related to whether the endometriomas were bilateral, as well as cyst size (especially for cysts >7 cm), but was negatively related to the preoperative serum AMH level. Age was a negative factor that affected the ovarian reserve.
doi:10.1186/s13048-014-0108-0
PMCID: PMC4255637  PMID: 25424986
Anti-Müllerian hormone; Endometrioma; Ovarian cystectomy
16.  Interleukin-2 and SOCS-1 proteins involvement in the pathophysiology of severe ovarian hyperstimulation syndrome-a preliminary proof of concept 
Background
Ovarian hyperstimulation syndrome (OHSS), is characterized by marked ovarian enlargement and acute third space fluid sequestration that almost always develops after hCG administration or in early pregnancy. OHSS is similar to vascular leak syndrome (VLS), which may be attributable to the massive increase in systemic inflammatory cytokines. In the present pilot exploratory case series, we sought to evaluate interleukin (IL)-2 and suppressor of cytokine signaling (SOCS)-1 expressions in the peripheral blood mononuclear cells (PBMCs) of patients suffering from severe ovarian hypertimulation syndrome (OHSS), and to examine whether their expressions differ when compared to PBMCs originated from normal early pregnant women (without OHSS).
Methods
Interleukin-2 and SOCS-1 mRNA expressions were examined in PBMCs of 5 women who were hospitalized due to severe OHSS (OHSS group) and 5 women with early IVF pregnancies and without OHSS (control group).
Results
Interleukin-2 mRNA levels in PBMCs were significantly higher in the OHSS as compared to the control groups. Moreover, while SOCS-1 mRNA levels were non-significantly lower, the ratio between IL-2 and SOCS-1 mRNA levels was significantly higher in the OHSS, as compared to the control group.
Conclusions
The inflammatory response to hCG, leading to dysregulation of Il-2 expression and SOCS activation, might be the culprit of OHSS. Additional large prospective studies are required to elucidate the effect of hCG on patients’ inherited inflammatory cascades, which may help discriminating those at risk to develop severe OHSS from those who are not.
doi:10.1186/s13048-014-0106-2
PMCID: PMC4255649  PMID: 25424734
OHSS; Inflammation; Cytokines; Interleukin-2; SOCS; hCG; Pregnancy
17.  Anti-IGF-1R monoclonal antibody inhibits the carcinogenicity activity of acquired trastuzumab-resistant SKOV3 
Background
Antibody resistance, not only de novo but also acquired cases, usually exists and is related with lower survival rate and high risk of recurrence. Reversing the resistance often results in better clinical therapeutic effect. Previously, we established a trastuzumab-resistant ovarian cancer cell line, named as SKOV3-T, with lower HER2 and induced higher IGF-1R expression level to keep cell survival.
Methods
IGF-1R was identified important for SKOV3-T growth. Then, a novel anti-IGF-1R monoclonal antibody, named as LMAb1, was used to inhibit SKOV3-T in cell growth/proliferation, migration, clone formation and in vivo carcinogenicity.
Results
In both in vitro and in vivo assays, LMAb1 showed effective anti-tumor function, especially when being used in combination with trastuzumab, which was beneficial to longer survival time of mice as well as smaller tumor. It was also confirmed preliminarily that the mechanism of antibody might be to inhibit the activation of IGF-1R and downstream MAPK, AKT pathway transduction.
Conclusion
We achieved satisfactory anti-tumor activity using trastuzumab plus LMAb1 in trastuzumab-resistant ovarian cancer model. In similar cases, not only acquired but also de novo, good curative effect might be achieved using combined antibody therapy strategies.
doi:10.1186/s13048-014-0103-5
PMCID: PMC4260252  PMID: 25424625
IGF-1R; Monoclonal antibody; Acquired resistant; Trastuzumab; Ovarian cancer
18.  Urinary interleukin-1β levels among gynecological patients 
Background
Early detection of epithelial ovarian cancer (OC) is necessary to overcome the high mortality rate of late stage diagnosis; and, examining the molecular changes that occur at early disease onset may provide new strategies for OC detection. Since the deregulation of inflammatory mediators can contribute to OC development, the purpose of this pilot study was to determine whether elevated urinary levels of Interleukin-1beta (IL-1 beta) are associated with OC and associated clinical parameters.
Methods
Urinary and serum levels of IL-1 beta were analyzed by ELISA from a patient cohort consisting of healthy women (N = 10), women with ovarian benign disease (N = 23), women with OC (N = 32), women with other benign gynecological conditions (N = 22), and women with other gynecological cancers (N = 6).
Results
Average urinary IL-1 beta levels tended to be elevated in ovarian benign (1.26 pg/ml) and OC (1.57 pg/ml) patient samples compared to healthy individuals (0.36 pg/ml). Among patients with benign disease, urinary IL-1β levels were statistically higher in patients with benign inflammatory gynecologic disease compared to patients with non-inflammatory benign disease. Interestingly, urinary IL-1 beta levels tended to be 3-6x greater in patients with benign ovarian disease or OC as well as with a concomitant family history of ovarian and/or breast cancer compared to similar patients without a family history of ovarian and/or breast cancer. Lastly, there was a pattern of increased urinary IL-1 beta with increasing body mass index (BMI); patients with a normal BMI averaged urinary IL-1 beta levels of 0.92 pg/ml, overweight BMI averaged urinary IL-1 beta levels of 1.72 pg/ml, and obese BMI averaged urinary IL-1 beta levels of 5.26 pg/ml.
Conclusions
This pilot study revealed that urinary levels of IL-1 beta are elevated in patients with epithelial OC supporting the thought that inflammation might be associated with cancer progression. Consequently, further studies of urinary IL-1 beta and the identification of an inflammatory profile specific to OC development may be beneficial to reduce the mortality associated with this disease.
doi:10.1186/s13048-014-0104-4
PMCID: PMC4247195  PMID: 25403235
Interleukin-1 beta; Obesity; Ovarian cancer; Urine; Body mass index
19.  Uterine mast cell tumor: a clinical and cytohistopathological study 
Background
Mast cells are one of the characteristic factors in angiogenesis, growth, and metastatic spread of tumors. Further studies are suggested to determine the type of these cells which might be useful in the assessment of biological nature of the tumor and its future treatment modality. Few studies have evaluated mast cell infiltration in visceral tumors, especially uterine tumors.
Case presentation
In this study, age, sex, death rate, and histologic patterns were in agreement with those of previous reports on canine mast cell tumors. Cytopathology assays are widely used to prognosticate canine uterine mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the uterine. The anisocytosis and anisocytosis and giant cells were present in the tumor. Furthermore, the tumor had nuclear atypia with scattered multinucleated cells and prominent nucleoli and tumor were classified as poorly granulated. Under microscopic examination, we observed diffuse infiltration and proliferation of tumor cells from the uterine different area and the infiltrative characteristics and distribution patterns of neoplastic cells were observed. This tumor consisted of sheets and cords of uniform round cells with discrete cytoplasmic margins. Microscopically, the neoplastic masses were poorly-demarcated and lacked capsules and tumor cell usually showed a distinct cell boundary. Nevertheless, the neoplastic cells were located between collagen bundles forming small clusters and sheets and had large, centrally located, round to ovoid nuclei. In addition, eosinophils were scattered among the mast cells at the periphery of the masses. The presence of eosinophils and the observation, at high magnification, of cells with cytoplasmic metachromatic granules.
Conclusion
Based on these findings, a diagnosis of poorly-differentiated mast cell tumor was made and data histologic grading was available for tumor. Neoplasm was poorly differentiated or gradeIII.
doi:10.1186/s13048-014-0105-3
PMCID: PMC4243290  PMID: 25376173
Mact cell tumor; Histopathology; Cytology; Uterine; Diagnosis
20.  Notch signaling in serous ovarian cancer 
Ovarian cancer is the most lethal of all gynecologic malignancies because women commonly present with advanced stage disease and develop chemotherapy refractory tumors. While cytoreductive surgery followed by platinum based chemotherapy are initially effective, ovarian tumors have a high propensity to recur highlighting the distinct need for novel therapeutics to improve outcomes for affected women. The Notch signaling pathway plays an established role in embryologic development and deregulation of this signaling cascade has been linked to many cancers. Recent genomic profiling of serous ovarian carcinoma revealed that Notch pathway alterations are among the most prevalent detected genomic changes. A growing body of scientific literature has confirmed heightened Notch signaling activity in ovarian carcinoma, and has utilized in vitro and in vivo models to suggest that targeting this pathway with gamma secretase inhibitors (GSIs) leads to anti-tumor effects. While it is currently unknown if Notch pathway inhibition can offer clinical benefit to women with ovarian cancer, several GSIs are currently in phase I and II trials across many disease sites including ovary. This review will provide background on Notch pathway function and will focus on the pre-clinical literature that links altered Notch signaling to ovarian cancer progression.
doi:10.1186/s13048-014-0095-1
PMCID: PMC4228063  PMID: 25366565
Ovarian serous carcinoma; Notch; Gamma secretase inhibitor; Patient derived xenograft models
21.  Malignant mixed germ cell tumour of ovary- an unusual combination and review of literature 
Abstract
Mixed germ cell tumours of the ovary are malignant neoplasms of the ovary comprising of two or more types of germ cell components. Most of the malignant mixed germ cell tumours consists of dysgerminoma accompanied by endodermal sinus tumours, immature teratoma or choriocarcinoma. There are only few case reports of mixed germ cell tumours with different combinations of malignant components.
We report a very rare case of mixed germ cell tumours consisted of malignant components of endodermal sinus tumour, emryonal carcinoma, and benign component of teratomatuos and trophoblastic differentiation. This is the first case report in the literature with both benign and malignant component of type described to best of our knowledge.
Patient was an 18 year old girl, who presented with pain abdomen, abdominal mass and irregular bleeding. Ultrasound and CT scan showed a huge mass with solid and cystic component. Tumour markers i.e alpha feto- protein (AFP), human chorionic gonadotropin (hCG), lactate dehydrogenate (LDH) and Ca-125 were raised. We performed fertility sparing surgery by preserving one ovary, tube and uterus.
Conclusion
Malingnant mixed germ cell tumours of ovary are highly aggressive neoplasm and early intervention and fertility sparing surgery is required for any adolescent girl presenting with rapidly enlarging pelvic mass.
doi:10.1186/s13048-014-0091-5
PMCID: PMC4236473  PMID: 25366470
Malignant mixed germ cell tumour; Endodermal sinus tumour; Teratoma; Embryonal cell carcinoma
22.  Ultrastructural changes in oocytes during folliculogenesis in domestic mammals 
The ultrastructural analysis of oocytes and ovarian follicles has been used to evaluate the effects of assisted reproductive techniques, such as cryopreservation or in vitro oocyte maturation. It also benefits the understanding of such complex mechanisms that occur during folliculogenesis. From the beginning of primordial follicles growth until oocyte maturation in preovulatory follicles oocyte cytoplasmic organelles undergo dynamic alterations that reflect physiological changes and development. This review aims to make a retrospective survey of the relevant features of follicles and oocytes ultrastructure, highlighting the differences between mammalian species, specially the domestic ones.
doi:10.1186/s13048-014-0102-6
PMCID: PMC4224757  PMID: 25358389
Preantral follicle; Cortical granules; Zona pellucida; Lipid droplets
23.  Borderline Brenner tumor of the ovary: a case report with immunohistochemical and molecular study 
Background
Borderline Brenner tumor of the ovary is a rare entity characterized by papillary structures with a fibro-vascular core, covered by a transitional epithelium, and by the absence of stromal infiltration. It is associated, by definition, with a benign component of Brenner tumor.
Case
We report a case of a 68-year-old woman, with a right ovarian mass, whose morphology and immuno-profile were consistent with the diagnosis of a borderline Brenner tumor. Immunohistochemistry carried out on selected markers may help to formulate the diagnosis, more than the molecular analyses.
Electronic supplementary material
The online version of this article (doi:10.1186/s13048-014-0101-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13048-014-0101-7
PMCID: PMC4226905  PMID: 25358264
Brenner tumors; Immuno-profile; Molecular analyses
24.  Aquaporin 5 expression is altered in ovarian tumors and ascites-derived ovarian tumor cells in the chicken model of ovarian tumor 
Background
Aquaporin 5 (AQP5), a member of the aquaporin family of transmembrane channel proteins, is involved in water transport and cellular proliferation in various tumors. The objective of this study was to determine cellular localization of aquaporin 5 (AQP5) in the ovarian tumors of chicken, a preclinical model for human ovarian tumor and to determine if AQP5 mRNA and protein expression levels in cancerous chicken ovaries and in ascites-derived chicken ovarian cancer (COVCAR) cell lines are different from normal ovaries and normal ovarian surface epithelial (NOSE) cells, respectively.
Methods
Immunohistochemical staining was performed to determine the localization of AQP5-immunoreactive (ir) cells in normal and cancerous ovaries. To determine AQP5 mRNA and protein concentrations in cancerous ovaries and COVCAR cell lines, quantitative real time PCR and Western blotting analysis were performed, respectively. Student’s t-test was performed to compare the levels of AQP5 mRNA or protein in cancerous ovaries and COVCAR cell lines with that of normal ovaries and NOSE cells, respectively.
Results
AQP5-ir cells were localized in granulosa and theca layers of normal ovarian follicles whereas cancerous ovaries showed AQP5 immunostaining in the surface epithelium, fibroblast cells of the stroma, and in the cells lining tumor cysts and acini. AQP5 mRNA concentration were significantly lesser while AQP5 protein concentrations were significantly greater in cancerous ovaries compared to that in normal ovaries (P < 0.05). Whereas AQP5 mRNA concentrations were significantly greater while AQP5 protein concentrations were lesser (P < 0.05) in COVCAR cell lines compared with that in NOSE cells.
Conclusion
AQP5 is differentially expressed in ovarian tumor and in COVCAR cell lines suggesting a potential involvement of AQP5 in ovarian tumorigenesis, metastasis, and survival of ovarian tumor cells in ascites.
doi:10.1186/s13048-014-0099-x
PMCID: PMC4213468  PMID: 25344048
Water channel transmembrane protein; Malignancy; Metastasis; Invasiveness; Epithelial ovarian tumor
25.  Expression and molecular consequences of inhibition of estrogen receptors in granulosa cells of bovine follicles 
Background
Estradiol (E2) receptors mediate E2 effects on cell proliferation and apoptosis under normal and pathological conditions. However, the mechanisms involved in E2 signaling are not completely understood. The objectives in this study were to evaluate the expression of estrogen receptors (ESRs) during follicular selection in cattle, and the effect of intrafollicular injection of fulvestrant (an antagonist of ESRs) on follicular development and transcript abundance in granulosa cells.
Methods
Granulosa cells were obtained from the two largest follicles around follicular deviation, after FSH treatment and after intrafollicular injection of fulvestrant. Ovarian follicular dynamics monitored by ultrasonography and quantitative real time PCR were used to validate the in vivo model and investigate the effects of FSH supplementation or ESR blockade on mRNA expression of estradiol-related genes.
Results
ESR1 and ESR2 were expressed in granulosa cells of both dominant (F1) and subordinate (F2) follicles, but their transcripts levels were higher in F1 than F2 after follicular deviation. FSH treatment maintained mRNA levels of both ESR1 and ESR2 in F2 follicles at similar levels observed in F1 follicles. Intrafollicular injection of 100 μM fulvestrant inhibited follicular growth and decreased CYP19A1 mRNA levels. Transcript levels for both ESR1 and ESR2 were not affected by fulvestrant injection. Analyses of FSH-regulated genes revealed that ESRs inhibition in the dominant follicle decreased the transcript levels of the GJA1 but not those of PRKAR2B, MRO or LRP11 genes.
Conclusions
Our findings indicate that: both ESR1 and ESR2 are regulated during follicular deviation and dominance in cattle and in response to FSH treatment, and ESRs are required for normal gene expression and development of the dominant follicle. Furthermore, we have validated an in vivo model to study estrogen signaling during follicular development that allows paracrine signaling between different follicular cells in a physiological endocrine environment.
doi:10.1186/s13048-014-0096-0
PMCID: PMC4210628  PMID: 25339519
Estrogen receptors; Follicular deviation; Fulvestrant; Intrafollicular injection; Bovine

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