IL-12 Expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice

Background

Despite advances in surgical aggressiveness and conventional chemotherapy, ovarian cancer remains the most lethal cause of gynecologic cancer mortality; consequently there is a need for new therapeutic agents and innovative treatment paradigms for the treatment of ovarian cancer. Several studies have demonstrated that ovarian cancer is an immunogenic disease and immunotherapy represents a promising and novel approach that has not been completely evaluated in ovarian cancer. Our objective was to evaluate the anti-tumor activity of an oncolytic herpes simplex virus “armed” with murine interleukin-12 and its ability to elicit tumor-specific immune responses. We evaluated the ability of interleukin?12-expressing and control oncolytic herpes simplex virus to kill murine and human ovarian cancer cell lines in vitro. We also administered interleukin?12-expressing oncolytic herpes simplex virus to the peritoneal cavity of mice that had developed spontaneous, metastatic ovarian cancer and determined overall survival and tumor burden at 95 days. We used flow cytometry to quantify the tumor antigen-specific CD8⁺ T cell response in the omentum and peritoneal cavity.

Results

All ovarian cancer cell lines demonstrated susceptibility to oncolytic herpes simplex virus in vitro. Compared to controls, mice treated with interleukin?12-expressing oncolytic herpes simplex virus demonstrated a more robust tumor antigen-specific CD8⁺ T-cell immune response in the omentum (471.6 cells vs 33.1 cells; p?=?0.02) and peritoneal cavity (962.3 cells vs 179.5 cells; p?=?0.05). Compared to controls, mice treated with interleukin?12-expressing oncolytic herpes simplex virus were more likely to control ovarian cancer metastases (81.2 % vs 18.2 %; p?=?0.008) and had a significantly longer overall survival (p?=?0.02). Finally, five of 6 mice treated with interleukin?12-expressing oHSV had no evidence of metastatic tumor when euthanized at 6 months, compared to two of 4 mice treated with sterile phosphate buffer solution.

Conclusion

Our pilot study demonstrates that an interleukin?12-expressing oncolytic herpes simplex virus effectively kills both murine and human ovarian cancer cell lines and promotes tumor antigen-specific CD8⁺ T-cell responses in the peritoneal cavity and omentum, leading to reduced peritoneal metastasis and improved survival in a mouse model.

     

MiR-124 inhibits the migration and invasion of ovarian cancer cells by targeting SphK1

Background

Epithelial ovarian cancer (EOC) is still a major gynecologic problem with poor 5 year survival rate due to distance metastases, despite routine surgery and chemotherapy. The precise underlying molecular mechanisms that trigger EOC migration and invasion are unclear. Recent studies suggest that the expression of microRNAs is widely dysregulated in ovarian cancer; and that they have evolved into tumorigenic processes, including cell proliferation, apoptosis and motility.

Methods

The expression of miR-124 was assessed in clinical ovarian cancer specimens and cell lines using miRNA qRTPCR. The function of miR-124 on cell migration and invasion was confirmed in vitro through wound healing assay and transwell assay. Luciferase reporter assay was used to confirm target associations.

Results

We showed that miR-124 is down-regulated in ovarian cancer specimens as well as in cell lines; and that low-level expression of miR-124 is much lower in highly metastatic ovarian cancer cells and tissues. Meantime, overexpression of miR-124 dramatically inhibits the motility of ovarian cancer cells in vitro and substantially suppresses the protein expression of SphK1, reported as an invasion and metastasis-related gene in human cancers, whose expression is markedly increased in both ovarian cancer cell lines and clinical samples, particularly in two highly metastasis cells, SKOV3-ip and HO8910pm as well as metastatic ovarian tumor tissues. Furthermore, SphK1 is identified as a direct target of miR-124, and knock-down of SphK1 in ovarian cancer cells, SKOV3-ip and HO8910pm, could mimic the inhibition of migration and invasion by miR-124, while re-introduction of SphK1 abrogates the suppression of motility and invasiveness induced by miR-124 in both cell lines.

Conclusions

Our studies suggest a protective role of miR-124 in inhibition of migration and invasion in the molecular etiology of ovarian cancer, and a potentially novel application of miR-124 in the regulation of migration and invasion in EOC.

     

Targeting of mesenchymal stem cells to ovarian tumors via an artificial receptor

Background

Mesenchymal Progenitor/Stem Cells (MSC) respond to homing cues providing an important mechanism to deliver therapeutics to sites of injury and tumors. This property has been confirmed by many investigators, however, the efficiency of tumor homing needs to be improved for effective therapeutic delivery. We investigated the feasibility of enhancing MSC tumor targeting by expressing an artificial tumor-binding receptor on the MSC surface.

Methods

Human MSC expressing an artificial receptor that binds to erbB2, a tumor cell marker, were obtained by transduction with genetically modified adenoviral vectors encoding an artificial receptor (MSC-AR). MSC-AR properties were tested in vitro in cell binding assays and in vivo using two model systems: transient transgenic mice that express human erbB2 in the lungs and ovarian xenograft tumor model. The levels of luciferase-labeled MSCs in erbB2-expressing targeted sites were evaluated by measuring luciferase activity using luciferase assay and imaging.

Results

The expression of AR enhanced binding of MSC-AR to erbB2-expressing cells in vitro, compared to unmodified MSCs. Furthermore, we have tested the properties of erbB2-targeted MSCs in vivo and demonstrated an increased retention of MSC-AR in lungs expressing erbB2. We have also confirmed increased numbers of erbB2-targeted MSCs in ovarian tumors, compared to unmodified MSC. The kinetic of tumor targeting by ip injected MSC was also investigated.

Conclusion

These data demonstrate that targeting abilities of MSCs can be enhanced via introduction of artificial receptors. The application of this strategy for tumor cell-based delivery could increase a number of cell carriers in tumors and enhance efficacy of cell-based therapy.

     

Comparison of stage III mucinous and serous ovarian cancer: a case-control study

Background

The purpose of this case-control study was to compare the prognoses of women with stage III mucinous ovarian carcinoma (MOC) who received maximal or optimal cytoreduction followed by paclitaxel plus carboplatin chemotherapy to those of women with stage III serous epithelial ovarian cancer (EOC) treated in the similar manner.

Methods

We performed a multicenter, retrospective review to identify patients with stage III MOC at seven gynecologic oncology departments in Turkey. Eighty-one women with MOC were included. Each case was matched to two women with stage III serous EOC in terms of age, tumor grade, substage of disease, and extent of residual disease. Survival estimates were measured using Kaplan-Meier plots. Variables predictive of outcome were analyzed using Cox regression models.

Results

With a median follow-up of 54 months, the median progression-free survival (PFS) for women with stage III MOC was 18.0 months (95% CI; 13.8–22.1, SE: 2.13) compared to 29.0 months (95% CI; 24.04–33.95, SE: 2.52) in the serous group (p?=?0.19). The 5-year overall survival rate of the MOC group was significantly lower than that of the serous EOC group (44.9% vs. 66.3%, respectively; p?<?0.001). For the entire cohort, presence of multiple peritoneal implants (Hazard ratio [HR] 2.39; 95% confidence interval [CI], 1.38–4.14, p?=?0.002) and mucinous histology (HR 2.28; 95% CI, 1.53–3.40, p?<?0.001) were identified as independent predictors of decreased OS.

Conclusion

Patients with MOC seem to be 2.3 times more likely to die of their tumors when compared to women with serous EOC.

     

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.

     

Freeze-all cycle for all normal responders?

Purpose

The purpose of this study is to evaluate the freeze-all strategy in subgroups of normal responders, to assess whether this strategy is beneficial regardless of ovarian response, and to evaluate the possibility of implementing an individualized embryo transfer (iET) based on ovarian response.

Methods

This was an observational, cohort study performed in a private IVF center. A total of 938 IVF cycles were included in this study. The patients were submitted to controlled ovarian stimulation (COS) with a gonadotropin-releasing hormone (GnRH) antagonist protocol and a cleavage-stage day 3 embryo transfer. We performed a comparison of outcomes between the fresh embryo transfer (n?=?523) and the freeze-all cycles (n?=?415). The analysis was performed in two subgroups of patients based on the number of retrieved oocytes: Group 1 (4–9 oocytes) and Group 2 (10–15 oocytes).

Result(s)

In Group 1 (4–9 retrieved oocytes), the implantation rates (IR) were 17.9 and 20.5% (P?=?0.259) in the fresh and freeze-all group, respectively; the ongoing pregnancy rates (OPR) were 31 and 33% (P?=?0.577) in the fresh and freeze-all group, respectively. In Group 2 (10–15 oocytes), the IR were 22.1 and 30.1% (P?=?0.028) and the OPR were 34 and 47% (P?=?0.021) in the fresh and freeze-all groups, respectively.

Conclusion(s)

Although the freeze-all policy may be related to better in vitro fertilization (IVF) outcomes in normal responders, these potential advantages decrease with worsening ovarian response. Patients with poorer ovarian response do not benefit from the freeze-all strategy.

     

Outcome of gestational surrogacy according to IVF protocol

Purpose

Surrogacy remains the only option for having a biologic child for a unique population of women with severe medical conditions. However, no study has looked at surrogacy outcome as a result of the type of ovarian stimulation of the intended mother [controlled ovarian stimulation (COH), modified natural cycle (MNC), and in vitro maturation (IVM)] for oocyte retrieval.

Methods

This is a retrospective study, including all intended mothers and gestational carriers in a tertiary, university affiliated, medical center, from 1998 to 2016.

Results

Fifty-two women underwent 252 oocyte retrieval cycles. The pregnancy outcome of 212 embryo transfer cycles (64 gestational carriers) was reviewed according to the origin of the embryo. The number of retrieved oocytes was significantly higher following COH (n?=?132) compared with IVM (n?=?58) and MNC cycles (n?=?62) (p?=?0.013 and p?<?0.0001, respectively). Pregnancy rates for embryos transferred according to each protocol were similar. All pregnancies that ended in live births when oocytes from IVM cycles were used derived from transfers of retrieved mature and mixed mature and immature oocytes. Pregnancies that involved embryos derived solely from immature oocytes that further matured in vitro and were transferred to gestational carriers were unsuccessful.

Conclusions

MNC protocol is a good option to achieve pregnancy for intended mothers using gestational surrogacy who have contraindications to COH. The yield of IVM cycles in which immature oocytes are retrieved is inconclusive.

     

The role of complement components C1q,MBL and C1 inhibitor in pathogenesis of endometriosis

Purpose

The purpose of the work was to evaluate possible associations between the complement components C1q, mannose-binding lectin (MBL) and C1 inhibitor (C1INH) with pathogenesis of endometriosis.

Methods

Concentrations of C1q, MBL and C1INH were measured by ELISA in peritoneal fluid (PF) in 80 women with or without endometriosis.

Results

Significantly higher PF levels of C1q, MBL and C1INH in women with endometriosis compared to control group were observed (p?<?0.0001). A higher concentration of the studied parameter was found in PF of women at the early stage of the disease, as compared to women with advanced endometriosis (p?<?0.0001).

Conclusions

Our research suggests that in the peritoneal cavity in women with endometriosis there are abnormal regulations of both the classical and lectin pathways of the complement system. This can suggest impairments in purification of peritoneal cavity from ectopic endometrial cells and augmented local inflammation in endometriosis patients.

     

Oviductal glycoprotein 1 (OVGP1) is expressed by endometrial epithelium that regulates receptivity and trophoblast adhesion

Purpose

To study the regulation and functions of oviductal glycoprotein 1 (OVGP1) in endometrial epithelial cells.

Methods

Expression of OVGP1 in mouse endometrium during pregnancy and in the endometrial epithelial cell line (Ishikawa) was studied by immunofluorescence, Western blotting, and RT-PCR. Regulation of OVGP1 in response to ovarian steroids and human chorionic gonadotropin (hCG) was studied by real-time RT-PCR. OVGP1 expression was knockdown in Ishikawa cells by shRNA, and expression of receptivity associated genes was studied by real-time RT-PCR. Adhesion of trophoblast cell line (JAr) was studied by in vitro adhesion assays.

Results

OVGP1 was localized exclusively in the luminal epithelial cells of mouse endometrium at the time of embryo implantation. Along with estrogen and progesterone, hCG induced the expression of OVGP1 in Ishikawa cells. Knockdown of OVGP1 in Ishikawa cells reduced mRNA expression of ITGAV, ITGB3, ITGA5, HOXA10, LIF, and IL15; it increased the expression of HOXA11, MMP9, TIMP1, and TIMP3. Supernatants derived from OVGP1 knockdown Ishikawa cells reduced the adhesiveness of JAr cells in vitro. Expression of OVGP1 mRNA was found to be significantly lowered in the endometrium of women with recurrent implantation failure.

Conclusion

OVGP1 is specifically induced in the luminal epithelium at the time of embryo implantation where it regulates receptivity-related genes and aids in trophoblast adhesion.

     

Activated ovarian endothelial cells promote early follicular development and survival

Background

New data suggests that endothelial cells (ECs) elaborate essential “angiocrine factors”. The aim of this study is to investigate the role of activated ovarian endothelial cells in early in-vitro follicular development.

Methods

Mouse ovarian ECs were isolated using magnetic cell sorting or by FACS and cultured in serum free media. After a constitutive activation of the Akt pathway was initiated, early follicles (50–150 um) were mechanically isolated from 8-day-old mice and co-cultured with these activated ovarian endothelial cells (AOEC) (n = 32), gel (n = 24) or within matrigel (n = 27) in serum free media for 14 days. Follicular growth, survival and function were assessed.

Results

After 6 passages, flow cytometry showed 93% of cells grown in serum-free culture were VE-cadherin positive, CD-31 positive and CD 45 negative, matching the known EC profile. Beginning on day 4 of culture, we observed significantly higher follicular and oocyte growth rates in follicles co-cultured with AOECs compared with follicles on gel or matrigel. After 14 days of culture, 73% of primary follicles and 83% of secondary follicles co-cultured with AOEC survived, whereas the majority of follicles cultured on gel or matrigel underwent atresia.

Conclusions

This is the first report of successful isolation and culture of ovarian ECs. We suggest that co-culture with activated ovarian ECs promotes early follicular development and survival. This model is a novel platform for the in vitro maturation of early follicles and for the future exploration of endothelial-follicular communication.

Capsule

In vitro development of early follicles necessitates a complex interplay of growth factors and signals required for development. Endothelial cells (ECs) may elaborate essential “angiocrine factors” involved in organ regeneration. We demonstrate that co-culture with ovarian ECs enables culture of primary and early secondary mouse ovarian follicles.

     

Detrimental effect of maternal and post-weaning high-fat diet on the reproductive function in the adult female offspring rat: roles of insulin-like growth factor 2 and the ovarian circadian clock

Purpose

We evaluate the impact of maternal and post-weaning high-fat (HF) diet on ovarian follicular population, steroidogenesis, and gene expression with a focus on the circadian clock system and insulin-like growth factor 2 (Igf2) in adult offspring ovaries, and to elucidate whether a maternal and post-weaning diet confers similar risks.

Methods

Virgin Sprague-Dawley rats were fed with normal chow (C) diet or HF diet for 5 weeks before mating, during gestation, and lactation. Female offspring were fed with the C or HF diet from weaning to 6 months of age, resulting in four study groups (n = 6 per group): C/C, C/HF, HF/C, and HF/HF.

Results

Ovaries from offspring exposed to post-weaning HF diet (i.e., the C/HF and HF/HF groups) had a decrease in small follicle numbers, but with similar numbers of antral follicles and corpora lutea. Offspring from HF-fed dams (i.e., the HF/C and HF/HF groups) had increased plasma estradiol concentrations and decreased luteinizing hormone levels at 6 months of age. In addition, Igf2 and each of the circadian rhythm core genes Clock, Per1, Per2, and Per3 were increased in the ovaries of offspring exposed to maternal HF diet (both HF/C and HF/HF groups).

Conclusions

Maternal and post-weaning HF diet programs the reproductive profile of the female offspring in adult life through different manners. Post-weaning HF intake resulted in the reduction of small follicles in adulthood, whereas maternal HF diet had long-term deleterious consequences on female offspring steroidogenesis and coincided with alteration of the upregulation of the imprinted gene Igf2 and changes in ovarian circadian rhythms.

     

Primordial follicle activation in the ovary of Ames dwarf mice

Background

The insulin receptor substrate 1 (IRS1), phosphoinositide 3-kinase (Pi3k), protein kinase B (Akt1), Forkhead Box O3a (FOXO3a) pathway is directly involved in aging and ovarian activation of follicle growth. Therefore, the aim of this work was to measure the expression of genes related to the ovarian pathway for activation of primordial follicles and FOXO3a protein phosphorylation between young and old female Ames dwarf (df/df) and normal (N) mice.

Methods

For this study ovaries from N (n?=?10) and df/df (n?=?10) female mice were collected at 5–6 months of age and at 21–22 months of age. For immunohistochemistry ovaries from 12 month-old and df/df mice were used.

Results

The expression of Irs1, Pi3k, Akt1, mammalian target of rapamycin (Mtor), suppressor of cytokine signaling ?2 (Socs2), Socs3 was lower (P?<?0.05) in older than younger N mice and not different (P?>?0.05) between young and old df/df mice. The expression of Foxo3a was also lower (P?<?0.05) in old than younger N and df/df mice and was higher (P?<?0.05) in old df/df than N mice. Expression of Amh was lower (P?<?0.05) in old than young N and df/df mice and was higher (P?=?0.0009) in df/df than N mice. Imunnostaining for p-FOXO3 was lower in df/df than N mice (P?<?0.001), although FOXO3 immunostaining was not different (P?>?0.05) between df/df and N mice.

Conclusions

In sum, the present study indicates that lower expression of Irs1, Socs2, Socs3, Akt1, Pi3k, Mtor and Foxo3a mRNA in the ovaries of older mice of both genotypes is associated to a reduced ovarian activity revealed by lower expression of Amh mRNA. At the same time, ovaries of old df/df mice maintained higher expression of Foxo3a mRNA, which was associated to higher ovarian activity. We have shown that df/df females have a lower level of p-FOXO3 in oocytes from primordial/primary follicles, an important activator of follicular growth. Therefore, this study strongly indicates that Prop1^df mutation causes delayed ovarian aging.

     

Breast cancer patients’ satisfaction with individual therapy goals and treatment in a standardized integrative medicine consultancy service

Purpose

Mutations in the CDH1 gene are linked both to diffuse gastric cancer and invasive lobular carcinoma (ILC). A high mutation rate is found in families fulfilling the diagnostic criteria for hereditary diffuse gastric cancer. Aim of this study was to clarify whether or not there is a significant contribution of CDH1 mutations in hereditary breast-/ovarian cancer (HBOC).

Methods

Ninety-seven unrelated probands fulfilling the diagnostic criteria for HBOC (96 affected, 1 unaffected) but tested negative for pathogenic BRCA1/2 mutations were screened for CDH1 mutations by denaturing high performance liquid chromatography (DHPLC) and subsequent Sanger sequencing of suspicious and positive DHPLC results.

Results

In total, we found two potentially pathogenic CDH1 alterations, c.1774G > A, pAla592Thr, and c.2512 A > G, p.Ser838Gly, classified as variants of unknown significance according to ClinVar. In addition, we detected a high number of known CDH1 polymorphisms (n = 62), some of them more frequent in patients with lobular (55%) than in those with invasive ductal carcinoma (27%).

Conclusion

Although none of the probands studied carried a clearly pathogenic CDH1 mutation, CDH1 could be considered a potential breast cancer gene, esp. for ILC worth including it in the NGS (next generation sequencing) HBOC panel.

     

Temporal expression of cumulus cell marker genes during in vitro maturation and oocyte developmental competence

Purpose

Cumulus cells (CC) play important roles in oocyte development and cumulus expressed genes can be used as markers for oocyte quality. This study aimed to investigate temporal changes in the expression of cumulus marker genes during oocyte maturation as possible biomarkers of embryo developmental competence in ovine.

Methods

Gene expression was assessed in the CC of the BCB+ (developmentally competent) and BCB- (developmentally poor) oocytes at 0, 12, and 24 h of in vitro maturation (IVM). Further, the association between the temporal cumulus gene expression and in vitro oocyte and embryo development was assessed.

Results

The maturation and blastocyst formation rates were found significantly greater for the BCB+ than the BCB- oocytes. At the 0 h of IVM, a significant upregulation in the expression of PTGS2, STAR, SDC2, LHR, FGF2, BCL2, IL7RA, HSPA1A, and IFNT was observed in the CC of the poor (BCB-) as compared to the competent (BCB+) oocytes. In contrast, it was observed that as maturation progressed, the cumulus expression of most of the favorable genes was reduced and was found significantly downregulated at the completion of IVM in the poor as compared to the competent oocytes.

Conclusions

The study revealed noticeable differences in the cumulus gene expression profile at different stages of IVM between ovine oocytes of differential developmental ability. The results indicated that the loss of cumulus gene expression along the maturation period in the poor oocytes was related to their intrinsic poor quality in the ovarian follicle.

     

Seminal plasma <Emphasis Type="Italic">HSPA2</Emphasis> mRNA content is associated with semen quality

Purpose

Cell-free mRNAs (cfmRNAs) were quantitatively measured in human seminal plasma and its relationship with semen quality was investigated.

Methods

Herein, a prospectively, controlled investigation was performed to study seminal plasma HSPA2 and uPA cfmRNA alterations between 21 asthenozoospermic patients and 16 normozoospermic individuals. Standard semen analysis was performed and seminal plasma cfmRNAs content was measured by real-time quantitative PCR. In addition, the regression analysis between seminal plasma cfmRNAs expression and semen parameters was performed.

Results

Seminal plasma HSPA2, but not uPA cfmRNA indicated significant difference between normozoospermia and asthenozoospermia men (P?=?0.02444 and 0.07811, respectively). Negative correlation between HSPA2 cfmRNA and sperm motility (R ²?=?0.213, P?=?0.004) as well as sperm concentration (R ²?=?0.133, P?=?0.026) were revealed. However, no correlation was found between seminal plasma uPA cfmRNA content and semen parameters.

Conclusions

Our data suggest that seminal plasma HSPA2 cfmRNA is different between asthenozoospermic and normozoospermic individuals and it might be an indicator for semen quality.

     

Endometrial thickness influences neonatal birth weight in pregnancies with obstetric complications achieved after fresh IVF–ICSI cycles

Purpose

Pregnancy-associated complications, duration of gestation and parity are well-known predictors of neonatal birth weight. Assisted reproductive technology (ART) affects neonatal birth weight as well. Endometrial thickness as measured on the day of HCG triggering may therefore impact on the neonatal birth weight.

Methods

The data of 764 singleton deliveries achieved after fresh transfer between November 1997 and 2014 were collected retrospectively with the intention to analyze the relationship of maternal and neonatal characteristics with endometrial thickness and the possible predictive value of endometrial thickness on neonatal birth weight.

Results

Higher maternal age (p < 0.001), diminished ovarian reserve (p < 0.001), endometriosis (p = 0.008) and hypogonadotropic hypogonadism (p < 0.001) predicted thin endometrium. Neonatal birth weight (p = 0.004), longer duration of pregnancy (p = 0.008), parity (p = 0.026) and higher maternal BMI (p = 0.003) were correlated significantly with the degree of endometrial proliferation. Endometrial thickness strongly predicted neonatal birth weight (p = 0.004). After adjusting regression analysis for maternal age and BMI, parity, neonatal gender and pregnancy duration, endometrial thickness remained predictive for neonatal birth weight in pregnancies with obstetric complications (p = 0.017). In uneventful pregnancies duration and parity are determinants of neonatal birth weight.

Conclusions

Our findings suggest that endometrial thickness is an additional ART-related factor influencing neonatal birth weight. This finding should be confirmed in large cohort studies.

     

Elevated RON protein expression in endometriosis and disease-associated ovarian cancers

Background

Recepteur d’origine nantais (RON) protein expression has been demonstrated to correlate with tumor progression, metastasis, and prognosis, and its mRNA expression increases in deeply infiltrating endometriotic lesions. However, it remains unclear whether RON protein expression also increases in endometriotic lesions, and may be a risk factor of malignant transformation in endometriotic lesions.

Methods

The protein expression of RON in control (n = 19), eutopic (n = 16), and ectopic (n = 51) endometria, as well as in endometriosis-associated ovarian cancers (EAOC, n = 16) was determined by immunohistochemical (IHC) staining.

Results

Endometriotic lesions expressed low levels of RON protein, but no RON protein expression appeared in matched eutopic or control endometrium. EAOC exhibited high levels of RON protein. The frequency and IHC score of RON protein expression were both significantly higher in EAOC [100.0% (14/14), 5.37 ± 0.74] than those in endometriotic lesions [51.0% (26/51), 2.15 ± 1.12; P = 0.002, 0.001]. Multivariate analysis of covariance only revealed a correlation of RON protein expression and EAOC (P = 0.006), but no correlations of RON protein expression and clinical parameters (P > 0.05).

Conclusions

These obtained results suggest that increased RON expression might be involved in the pathogenesis of endometriosis and disease-associated ovarian cancers.

     

Is the aging human ovary still ticking?: Expression of clock-genes in luteinized granulosa cells of young and older women

Background

It has been shown – mostly in animal models - that circadian clock genes are expressed in granulosa cells and in corpora luteum and might be essential for the ovulatory process and steroidogenesis.

Objective

We sought to investigate which circadian clock genes exist in human granulosa cells and whether their expression and activity decrease during aging of the ovary.

Study design

Human luteinized granulosa cells were isolated from young (age 18–33) and older (age 39–45) patients who underwent in-vitro fertilization treatment. Levels of clock genes expression were measured in these cells 36?h after human chorionic gonadotropin stimulation.

Methods

Human luteinized granulosa cells were isolated from follicular fluid during oocyte retrieval. The mRNA expression levels of the circadian genes CRY1, CRY2, PER1, PER2, CLOCK, ARNTL, ARNTL2, and NPAS2 were analyzed by quantitative polymerase chain reaction.

Results

We found that the circadian genes CRY1, CRY2, PER1, PER2, CLOCK, ARNTL, ARNTL2, and NPAS2, are expressed in cultured human luteinized granulosa cells. Among these genes, there was a general trend of decreased expression in cells from older women but it reached statistical significance only for PER1 and CLOCK genes (fold change of 0.27?±?0.14; p?=?0.03 and 0.29?±?0.16; p?=?0.05, respectively).

Conclusions

This preliminary report indicates that molecular circadian clock genes exist in human luteinized granulosa cells. There is a decreased expression of some of these genes in older women. This decline may partially explain the decreased fertility and steroidogenesis of reproductive aging.