Michele Cummings

Cytokines in ovarian folliculogenesis, oocyte maturation and luteinisation

Cytokines are key regulators of ovarian physiology, particularly in relation to folliculogenesis and ovulation, where they contribute to creating an environment supporting follicle selection and growth. Their manifold functions include regulating cellular proliferation/differentiation, follicular survival/atresia, and oocyte maturation. Several cytokines, such as TGF‐β‐superfamily members, are involved at all stages of folliculogenesis while the production of others is stage‐dependent. This review draws upon evidence from both human and animal models to highlight the species‐specific roles at each milestone of follicular development. Given these pivotal roles and their ease of detection in follicular fluid, cytokines have been considered as attractive biomarkers of oocyte maturational status and of successful assisted reproductive outcome. Despite this, our understanding of cytokines and their interactions remains incomplete, and is still frequently limited to overly simplistic descriptions of their interrelationships. Given our increased appreciation of cytokine activity in complex and highly regulated networks, we put forward the case for using Bayesian modelling approaches to describe their hierarchical relationships in order to predict causal physiological interactions in vivo. Mol. Reprod. Dev. 81: 284–314, 2014.

A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.

Age-Related Biological Features of Germ Cell Tumors

Germ cell tumors (GCTs) are rare but clinically and pathologically diverse tumors that occur in an extensive range of age groups, from children to older adults and which include both seminomatous and nonseminomatous tumors. Current clinical management for both male and female teenagers and young adults (TYAs) with GCTs remains inconsistent, alternating between pediatric and adult multidisciplinary oncology teams, based on locally defined age cutoffs. Therefore, we reviewed available literature to determine the biological similarities and differences between GCTs in young children (0–12 years), TYAs (13–24 years), and older adults (>24 years). GCTs arising in pediatric and adult populations in general showed marked molecular biological differences within identical histological subtypes, whereas there was a distinct paucity of available data for GCTs in the TYA population. These findings highlight that clinical management based simply on chronological age may be inappropriate for TYA and suggests that the optimal future management of GCTs should consider specific molecular biological factors in addition to clinical parameters in the context of patient‐specific age group rather than medical specialty.

Glyceraldehyde-3-phosphate Dehydrogenase is an Inappropriate Housekeeping Gene for Normalising Gene Expression in Sepsis

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been used as a default reference gene in quantitative mRNA profiling experiments. However, its expression reportedly varies in response to a range of pathophysiological variables (inflammation, oxidative stress, hyperinsulinaemia, hypoxia) which feature prominently in sepsis. We therefore assessed the applicability of using GAPDH as a reference gene for expression studies in sepsis compared to other housekeeping genes (succinate dehydrogenase complex subunit A (SDHA), hypoxanthine phosphoribosyltransferase (HPRT)-1). Severe sepsis resulted in a 42.4-fold increase in median GAPDH expression (P < 0.001), whereas median HPRT expression was raised more modestly (2.9-fold; P < 0.001), and there was no significant difference in SDHA expression between sepsis and control patients. HPRT was identified by NormFinder to be the most stably expressed single gene. In order to assess the impact of this variability on data interpretation, interleukin (IL)-10 expression was normalised separately to GAPDH and to the geometric mean of HPRT and SDHA. In the former case, there was no significant difference in IL-10 expression between controls and septic patients, whilst in the latter, a significant 8.5-fold increase in median IL-10 expression was noted (P < 0.001). GAPDH is thus an unreliable housekeeping gene for normalising gene expression in sepsis which should be replaced by alternative, validated reference genes.

Bayesian modeling suggests that IL-12 (p40), IL-13 and MCP-1 drive murine cytokine networks in vivo

BackgroundCytokine-hormone network deregulations underpin pathologies ranging from autoimmune disorders to cancer, but our understanding of these networks in physiological/pathophysiological states remains patchy. We employed Bayesian networks to analyze cytokine-hormone interactions in vivo using murine lactation as a dynamic, physiological model system.ResultsCirculatory levels of estrogen, progesterone, prolactin and twenty-three cytokines were profiled in post partum mice with/without pups. The resultant networks were very robust and assembled about structural hubs, with evidence that interleukin (IL)-12 (p40), IL-13 and monocyte chemoattractant protein (MCP)-1 were the primary drivers of network behavior. Network structural conservation across physiological scenarios coupled with the successful empirical validation of our approach suggested that in silico network perturbations can predict in vivo qualitative responses. In silico perturbation of network components also captured biological features of cytokine interactions (antagonism, synergy, redundancy).ConclusionThese findings highlight the potential of network-based approaches in identifying novel cytokine pharmacological targets and in predicting the effects of their exogenous manipulation in inflammatory/immune disorders.

The prognostic significance of tumour-stroma ratio in endometrial carcinoma

BackgroundHigh tumour stromal content has been found to predict adverse clinical outcome in a range of epithelial tumours. The aim of this study was to assess the prognostic significance of tumour-stroma ratio (TSR) in endometrial adenocarcinomas and investigate its relationship with other clinicopathological parameters.MethodsClinicopathological and 5-year follow-up data were obtained for a retrospective series of endometrial adenocarcinoma patients (n = 400). TSR was measured using a morphometric approach (point counting) on digitised histologic hysterectomy specimens. Inter-observer agreement was determined using Cohen’s Kappa statistic. TSR cut-offs were optimised using log-rank functions and prognostic significance of TSR on overall survival (OS) and disease-free survival (DFS) were determined using Cox Proportional Hazards regression analysis and Kaplan-Meier curves generated. Associations of TSR with other clinicopathological parameters were determined using non-parametric tests followed by Holm-Bonferroni correction for multiple comparisons.ResultsTSR as a continuous variable associated with worse OS (P = 0.034) in univariable Cox-regression analysis. Using the optimal cut-off TSR value of 1.3, TSR-high (i.e. low stroma) was associated with worse OS (HR = 2.51; 95 % CI = 1.22–5.12; P = 0.021) and DFS (HR = 2.19; 95 % CI = 1.15–4.17; P = 0.017) in univariable analysis. However, TSR did not have independent prognostic significance in multivariable analysis, when adjusted for known prognostic variables. A highly significant association was found between TSR and tumour grade (P < 0.001) and lymphovascular space invasion (P < 0.001), both of which had independent prognostic significance in this study population.ConclusionsLow tumour stromal content associates with both poor outcome and with other adverse prognostic indicators in endometrial cancer, although it is not independently prognostic. These findings contrast with studies on many - although not all - cancers and suggest that the biology of tumour-stroma interactions may differ amongst cancer types.

Anatomy, Development, Histology, and Normal Function of the Ovary

The ovary is an unique organ which fulfils a plethora of physiological functions ranging from its well-recognized roles in steroid hormone biosynthesis, oocyte production and the support of early pregnancy through to its rather less well-known involvement in the regulation of growth, behaviour and immune function in response to an array of endogenous and environmental cues. While multiple cytokine-mediated interactions between the granulosa/theca cell compartments and the oocyte of developing follicles are central to the coordination of ovarian cyclicity, its systemic effects are instead attributable to exquisitely well-honed interactions between ovarian follicles, the hypothalamus and pituitary implicating an array of peptide and steroid hormones. During the follicular phase, primordial follicles are called upon to initiate a process of follicular development which culminates in ovulation. However, the release of an oocyte belies the fact that the postovulatory follicle has yet another role to play in the luteal phase. Therein, it adopts a new keystone position in supporting the establishment and maintenance of pregnancy through its production of progesterone. Once the ovary has exhausted its reserve of follicles throughout reproductive life, many of its functions are lost as women enter the menopause. This Chapter aims to explore the ovary’s developmental origins and functional roles to highlight the complexity of this remarkable product of reproductive evolution.

Integrated eicosanoid lipidomics and gene expression reveal decreased prostaglandin catabolism and increased 5-lipoxygenase expression in aggressive subtypes of endometrial cancer: Eicosanoid metabolism in endometrial cancer

Eicosanoids comprise a diverse group of bioactive lipids which orchestrate inflammation, immunity, and tissue homeostasis, and whose dysregulation has been implicated in carcinogenesis. Among the various eicosanoid metabolic pathways, studies of their role in endometrial cancer (EC) have very much been confined to the COX‐2 pathway. This study aimed to determine changes in epithelial eicosanoid metabolic gene expression in endometrial carcinogenesis; to integrate these with eicosanoid profiles in matched clinical specimens; and, finally, to investigate the prognostic value of candidate eicosanoid metabolic enzymes. Eicosanoids and related mediators were profiled using liquid chromatography–tandem mass spectrometry in fresh frozen normal, hyperplastic, and cancerous (types I and II) endometrial specimens (n = 192). Sample‐matched epithelia were isolated by laser capture microdissection and whole genome expression analysis was performed using microarrays. Integration of eicosanoid and gene expression data showed that the accepted paradigm of increased COX‐2‐mediated prostaglandin production does not apply in EC carcinogenesis. Instead, there was evidence for decreased PGE2/PGF2α inactivation via 15‐hydroxyprostaglandin dehydrogenase (HPGD) in type II ECs. Increased expression of 5‐lipoxygenase (ALOX5) mRNA was also identified in type II ECs, together with proportional increases in its product, 5‐hydroxyeicosatetraenoic acid (5‐HETE). Decreased HPGD and elevated ALOX5 mRNA expression were associated with adverse outcome, which was confirmed by immunohistochemical tissue microarray analysis of an independent series of EC specimens (n = 419). While neither COX‐1 nor COX‐2 protein expression had prognostic value, low HPGD combined with high ALOX5 expression was associated with the worst overall and progression‐free survival. These findings highlight HPGD and ALOX5 as potential therapeutic targets in aggressive EC subtypes. Copyright

Laser Capture Microdissection and Isolation of High-Quality RNA from Frozen Endometrial Tissue.

Laser capture microdissection (LCM) allows expression profiling of specific cell populations within tissues. However, isolation of high-quality RNA from laser capture microdissected frozen tissue is beset by problems arising from intrinsic tissue RNase activity. Herein, we describe an optimized staining/LCM/RNA extraction protocol developed for the isolation of epithelial RNA from frozen tissue sections using human endometrial cancer as a model tissue. This method combines excellent, reproducible visualization of tissue morphology with the isolation of high-integrity RNA suitable for downstream applications such as expression microarray analysis. We present quantitative and qualitative RNA data obtained from >200 endometrial epithelial samples (normal, hyperplastic, and cancerous), where 92% of samples had RIN values of 7 and above and highlight common pitfalls faced by investigators. This method should also be broadly applicable to a range of other tissue types.

Cytokine Networks in the Ovary

Although our understanding of the role of cytokines in ovarian physiology is incomplete, it is increasingly recognized that these mediators are responsible for all stages of folliculogenesis, the dialog between the maturing oocyte and its somatic cell complement, ovulation, luteinization, and the regulation of intraovarian immune effector cell function. It is therefore unsurprising that cytokine network perturbations are a central feature of ovarian pathologies as diverse as polycystic ovary syndrome, ovarian hyperstimulation syndrome, premature ovarian insufficiency, endometriosis, and ovarian cancer. Meeting the challenge of developing novel modeling modalities for these networks offers the promise of painting a more informative picture of their interactions and underpins the development of novel, targeted therapies for the treatment of ovarian disorders.