Serum Vitamin D status is associated with increased blastocyst development rate in women undergoing IVF.

RESEARCH QUESTION: To determine the relationship between vitamin D (VitD) status and embryological, clinical pregnancy and live birth outcomes in women undergoing IVF. DESIGN: Cross-sectional, observational study conducted at a university-affiliated private IVF clinic. A total of 287 women underwent 287 IVF cycles and received a fresh embryo transfer. Patients had their serum 25-hydroxyvitamin D2/D3 (VitD) determined on the day of oocyte retrieval, which was analysed in relation to blastocyst development rate, clinical pregnancy and live birth outcomes. RESULTS: In stepwise, multivariable logistic regression models, increases in blastocyst development rate, number and quality, along with embryo cryopreservation and utilization rates were associated with women with a sufficient VitD status (≥20 ng/ml). For a single increase in the number of blastocysts generated per cycle or embryos cryopreserved per cycle, the likelihood for the patient to be VitD sufficient was increased by 32% (odds ratio [OR] 1.32, 95% confidence interval [CI] 1.10-1.58, P = 0.002 and OR 1.33, 95% CI 1.10-1.60, P = 0.004, respectively). Clinical pregnancy (40.7% versus 30.8%, P = 0.086) and live birth rates (32.9% versus 25.8%, P = 0.195) in the sufficient VitD group versus the insufficient group were not significantly different and VitD sufficiency was not significantly associated with these outcomes. CONCLUSION: A strong relationship was observed between blastocyst development and VitD sufficiency. However, there was no association between VitD and clinical pregnancy or live birth outcomes. Further larger studies are needed to investigate whether the observed effect on blastocyst development may have downstream implications on subsequent clinical pregnancy or live birth rates, and on a potential mechanism where sufficient VitD concentrations are linked to improved IVF outcomes.

Reticulon-1 and Reduced Migration toward Chemoattractants by Macrophages Differentiated from the Bone Marrow of Ultraviolet-Irradiated and Ultraviolet-Chimeric Mice.

The ability of macrophages to respond to chemoattractants and inflammatory signals is important for their migration to sites of inflammation and immune activity and for host responses to infection. Macrophages differentiated from the bone marrow (BM) of UV-irradiated mice, even after activation with LPS, migrated inefficiently toward CSF-1 and CCL2. When BM cells were harvested from UV-irradiated mice and transplanted into naive mice, the recipient mice (UV-chimeric) had reduced accumulation of elicited monocytes/macrophages in the peritoneal cavity in response to inflammatory thioglycollate or alum. Macrophages differentiating from the BM of UV-chimeric mice also had an inherent reduced ability to migrate toward chemoattractants in vitro, even after LPS activation. Microarray analysis identified reduced reticulon-1 mRNA expressed in macrophages differentiated from the BM of UV-chimeric mice. By using an anti-reticulon-1 Ab, a role for reticulon-1 in macrophage migration toward both CSF-1 and CCL2 was confirmed. Reticulon-1 subcellular localization to the periphery after exposure to CSF-1 for 2.5 min was shown by immunofluorescence microscopy. The proposal that reduced reticulon-1 is responsible for the poor inherent ability of macrophages to respond to chemokine gradients was supported by Western blotting. In summary, skin exposure to erythemal UV radiation can modulate macrophage progenitors in the BM such that their differentiated progeny respond inefficiently to signals to accumulate at sites of inflammation and immunity.

Oxidative stress pathways in pancreatic ß-cells and insulin-sensitive cells and tissues: importance to cell metabolism, function, and dysfunction.

It is now accepted that nutrient abundance in the blood, especially glucose, leads to the generation of reactive oxygen species (ROS), ultimately leading to increased oxidative stress in a variety of tissues. In the absence of an appropriate compensatory response from antioxidant mechanisms, the cell, or indeed the tissue, becomes overwhelmed by oxidative stress, leading to the activation of intracellular stress-associated pathways. Activation of the same or similar pathways also appears to play a role in mediating insulin resistance, impaired insulin secretion, and late diabetic complications. The ability of antioxidants to protect against the oxidative stress induced by hyperglycemia and elevated free fatty acid (FFA) levels in vitro suggests a causative role of oxidative stress in mediating the latter clinical conditions. In this review, we describe common biochemical processes associated with oxidative stress driven by hyperglycemia and/or elevated FFA and the resulting clinical outcomes: ß-cell dysfunction and peripheral tissue insulin resistance.

UV Irradiation of Skin Enhances Glycolytic Flux and Reduces Migration Capabilities in Bone Marrow-Differentiated Dendritic Cells.

A systemic immunosuppression follows UV irradiation of the skin of humans and mice. In this study, dendritic cells (DCs) differentiating from the bone marrow (BM) of UV-irradiated mice had a reduced ability to migrate toward the chemokine (C-C motif) ligand 21. Fewer DCs also accumulated in the peritoneal cavity of UV-chimeric mice (ie, mice transplanted with BM from UV-irradiated mice) after injection of an inflammatory stimulus into that site. We hypothesized that different metabolic states underpin altered DC motility. Compared with DCs from the BM of nonirradiated mice, those from UV-irradiated mice produced more lactate, consumed more glucose, and had greater glycolytic flux in a bioenergetics stress test. Greater expression of 3-hydroxyanthranilate 3,4-dioxygenase was identified as a potential contributor to increased glycolysis. Inhibition of 3-hydroxyanthranilate 3,4-dioxygenase by 6-chloro-dl-tryptophan prevented both increased lactate production and reduced migration toward chemokine (C-C motif) ligand 21 by DCs differentiated from BM of UV-irradiated mice. UV-induced prostaglandin E2 has been implicated as an intermediary in the effects of UV radiation on BM cells. DCs differentiating from BM cells pulsed in vitro for 2 hours with dimethyl prostaglandin E2 were functionally similar to those from the BM of UV-irradiated mice. Reduced migration of DCs to lymph nodes associated with increased glycolytic flux may contribute to their reduced ability to initiate new immune responses in UV-irradiated mice.

Finding a place for corifollitropin within the PIVET FSH dosing algorithms.

PIVET recombinant FSH (rFSH) dosing algorithms have been designed for rFSH injection pens, providing optimal pregnancy and live birth productivity rates whilst minimizing risk and occurrence of ovarian hyperstimulation syndrome (OHSS). Recently, long-acting recombinant gonadotrophin corifollitropin (Elonva) was approved for use in assisted reproduction, and welcomed by patients as the single injection allowed ovarian stimulation over 7 days without need for multiple injections. Consequently, another rFSH dosing algorithm was devised to incorporate Elonva, and these cycles were compared to standard rFSH agents, Gonal-f and Puregon. Initiated Elonva cycles (n = 165) were compared with 972 cycles initiated with standard rFSH. Elonva replaced standard rFSH dosages across the 200-400 IU range, but provided equivalent oocyte retrieval numbers and live birth outcomes. Elonva is considered risky for women whose antral follicle count is ≥20 follicles, and was inadvertently administered contra-protocol in 19 cycles with ≥20 follicles. However, while oocyte retrieval numbers were higher, raising risk for OHSS, no actual cases ensued. Taken together, this indicated that Elonva was equivalent to standard rFSH stimulation, and consequently has been added to the rFSH algorithms for medium to lower antral follicle counts and represented by green colour coding in the existing PIVET algorithmic charts.

Molecular actions of vitamin D in reproductive cell biology

Vitamin D (VitD) is an important secosteroid and has attracted attention in several areas of research due to common VitD deficiency in the population, and its potential to regulate molecular pathways related to chronic and inflammatory diseases. VitD metabolites and the VitD receptor (VDR) influence many tissues including those of the reproductive system. VDR expression has been demonstrated in various cell types of the male reproductive tract, including spermatozoa and germ cells, and in female reproductive tissues including the ovaries, placenta and endometrium. However, the molecular role of VitD signalling and metabolism in reproductive function have not been fully established. Consequently, the aim of this work is to review current metabolic and molecular aspects of the VitD­VDR axis in reproductive medicine and to propose the direction of future research. Specifically, the influence of VitD on sperm motility, calcium handling, capacitation, acrosin reaction and lipid metabolism is examined. In addition, we will also discuss the effect of VitD on sex hormone secretion and receptor expression in primary granulosa cells, along with the impact on cytokine production in trophoblast cells. The review concludes with a discussion of the recent developments in VitD­VDR signalling specifically related to altered cellular bioenergetics, which is an emerging concept in the field of reproductive medicine.

Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: Metabolic state of cancer cells.

Malignant mesothelioma (MM) is an aggressive cancer, characterized by rapid progression, along with late metastasis and poor patient prognosis. It is resistant to many forms of standard anti-cancer treatment. In this study, we determined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt pathway inhibitor, on cancer cell proliferation and metabolism using the JU77 mesothelioma cell line. Treatment with sFRP4 (250 pg/ml) resulted in a significant reduction of cell proliferation. The addition of the Wnt activator Wnt3a (250 pg/ml) or sFRP4 had no significant effect on ATP production and glucose utilisation in JU77 cells at both the 24 and 48 h time points examined. We also examined their effect on Akt and Glycogen synthase kinase-3 beta (GSK3ß) phosphorylation, which are both important components of Wnt signalling and glucose metabolism. We found that protein phosphorylation of Akt and GSK3ß varied over the 24h and 48 h time points, with constitutive phosphorylation of Akt at serine 473 (pAkt) decreasing to its most significant level when treated with Wnt3a+sFRP4 at the 24h time point. A significant reduction in the level of Cytochrome c oxidase was observed at the 48 h time point, when sFRP4 and Wnt3a were added in combination. We conclude that sFRP4 may function, in part, to reduce/alter cancer cell metabolism, which may lead to sensitisation of cancer cells to chemotherapeutics, or even cell death.

Higher ß-HCG concentrations and higher birthweights ensue from single vitrified embryo transfers.

To examine the effect of cryopreservation on developmental potential of human embryos, this study compared quantitative ß-HCG concentrations at pregnancy test after IVF-fresh embryo transfer (IVF-ET) with those arising after frozen embryo transfer (FET). It also tracked outcomes of singleton pregnancies resulting from single-embryo transfers that resulted in singleton live births (n = 869; with 417 derived from IVF-ET and 452 from FET). The initial serum ß-HCG concentration indicating successful implantation was measured along with the birthweight of the ensuing infants. With testing at equivalent luteal phase lengths, the median pregnancy test ß-HCG was significantly higher following FET compared with fresh IVF-ET (844.5 IU/l versus 369 IU/l; P < 0.001). Despite no significant difference in the average period of gestation (38 weeks 5 days for both groups), the mean birthweight of infants born following FET was significantly heavier by 161 g (3370 g versus 3209 g; P < 0.001). Furthermore, more infants exceeded 4000 g (P < 0.001) for FET although there was no significant difference for the macrosomic category (≥4500 g). We concluded that FET programme embryos lead to infants with equivalent (if not better) developmental potential compared with IVF-ET, demonstrated by higher pregnancy ß-HCG concentrations and ensuing birthweights.

The potential regulatory role of vitamin D in the bioenergetics of inflammation.

PURPOSE OF REVIEW: The extraskeletal health benefits of vitamin D still need scientific endorsement. Obesity and related chronic diseases are pathogenically linked by inflammation, which carries a considerable energetic cost. Recent techniques for the determination of the bioenergetic demand of inflammation, offer an avenue to cement the regulatory role of vitamin D in this process. RECENT FINDINGS: Nuclear vitamin D receptors may be translocated into mitochondria of certain cell types, opening up a pathway for direct action on cellular bioenergetics. Classical M1 (inflammatory)/M2(anti-inflammatory) phenotypes can vary with the clinical context. M2 macrophages do not always depend on oxidative metabolism/fatty acid oxidation. Newer methodologies offer real-time bioenergetic measurements that can be used as an index of metabolic health. SUMMARY: Vitamin D may prove to be a therapeutic agent for inflammation of chronic disease and understanding its role in cellular bioenergetics may offer a diagnostic/prognostic indicator of its action.

The impact of cryopreservation on human peripheral blood leucocyte bioenergetics.

Circulating immune cells are considered a source for biomarkers in health and disease, since they are exposed to nutritional, metabolic and immunological stimuli in the vasculature. Cryopreservation of leucocytes is routinely used for long-term storage and determination of phenotypic/functional changes at a later date. Exploring the role of bioenergetics and mitochondrial (dys)function in leucocytes is often examined by using freshly isolated cells. The aim of the pilot study described herein was to assess leucocyte bioenergetics in cryopreserved cells. Leucocytes were isolated from whole blood, counted and frozen in liquid nitrogen (LN2) for a period of 3 months. Cells were thawed at regular intervals and bioenergetic analysis performed using the Seahorse XFe96 flux analyser. Cryogenic storage reduced cell viability by 20%, but cell bioenergetic responses were largely intact for up to 1 month storage in LN2. However, after 1 month storage, mitochondrial function was impaired as reflected by decreasing basal respiration, ATP production, maximum (MAX) respiration, reserve capacity and coupling efficiency. Conversely, glycolytic activity was increased after 1 month, most notably the enhanced glycolytic response to 25 mM glucose without any change in glycolytic capacity. Finally, calculation of bioenergetic health index (BHI) demonstrated that this potential diagnostic parameter was sensitive to cryopreservation. The present study has demonstrated for the first time that cryopreservation of primary immune cells modified their metabolism in a time-dependent fashion, indicated by attenuated aerobic respiration and enhanced glycolytic activity. Taken together, we recommend caution in the interpretation of bioenergetic responses or BHI in cryopreserved samples.

Mid-luteal serum progesterone concentrations govern implantation rates for cryopreserved embryo transfers conducted under hormone replacement.

This study explores the relevance of mid-luteal serum hormonal concentrations in cryopreserved embryo transfer cycles conducted under hormone replacement therapy (HRT) control and which involved single-embryo transfer (SET) of 529 vitrified blastocysts. Widely ranging mid-luteal oestradiol and progesterone concentrations ensued from the unique HRT regimen. Oestradiol had no influence on clinical pregnancy or live birth rates, but an optimal progesterone range between 70 and 99 nmol/l (P < 0.005) was identified in this study. Concentrations of progesterone below 50 nmol/l and above 99 nmol/l were associated with decreased implantation rates. There was no clear interaction between oestradiol and progesterone concentrations but embryo quality grading did show a significant influence on outcomes (P < 0.001 and P = 0.002 for clinical pregnancy and live birth rates, respectively). Multiple comparison analysis showed that the progesterone effect was influential regardless of embryo grading, body mass index or the woman's age, either at vitrification or at cryopreserved embryo transfer. The results support the argument that careful monitoring of serum progesterone concentrations in HRT-cryopreserved embryo transfer is warranted and that further studies should explore pessary adjustments to optimize concentrations for individual women to enhance implantation rates.

Inflammation and Oxidative Stress: The Molecular Connectivity between Insulin Resistance, Obesity, and Alzheimer's Disease.

Type 2 diabetes (T2DM), Alzheimer's disease (AD), and insulin resistance are age-related conditions and increased prevalence is of public concern. Recent research has provided evidence that insulin resistance and impaired insulin signalling may be a contributory factor to the progression of diabetes, dementia, and other neurological disorders. Alzheimer's disease (AD) is the most common subtype of dementia. Reduced release (for T2DM) and decreased action of insulin are central to the development and progression of both T2DM and AD. A literature search was conducted to identify molecular commonalities between obesity, diabetes, and AD. Insulin resistance affects many tissues and organs, either through impaired insulin signalling or through aberrant changes in both glucose and lipid (cholesterol and triacylglycerol) metabolism and concentrations in the blood. Although epidemiological and biological evidence has highlighted an increased incidence of cognitive decline and AD in patients with T2DM, the common molecular basis of cell and tissue dysfunction is rapidly gaining recognition. As a cause or consequence, the chronic inflammatory response and oxidative stress associated with T2DM, amyloid-ß (Aß) protein accumulation, and mitochondrial dysfunction link T2DM and AD.

The Concept of Growth Hormone Deficiency Affecting Clinical Prognosis in IVF.

The current understanding of human growth hormone (hGH; here GH) action is that the molecule is a 191-amino acid, single-chain polypeptide that is synthesized, stored and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. It can be classified as a protein (comprising more than 50 amino acids) but true proteins have tertiary and quaternary chains creating a more complex structure, hence GH is usually classified as a polypeptide. GH is normally secreted at night during sleep and promotes skeletal, visceral and general body growth through the action of somatomedins or IGFs, notably IGF-1. In some tissues, GH action is directed via specific receptors GHRs; these are most abundant in liver, adipose and muscle tissues but have also been shown in granulosa cells, testicular tissues and on the oocyte, as well as in glandular cells of the luteal phase endometrium and decidua; such findings being recent and minimally researched to now. Following engagement with its receptor, the transduction process activates multiple signaling proteins. These all lead to extensive metabolic and mitogenic (growth promoting) responses. Clinically, GH is known to have an important role in pubertal development and is a key hormone for the vigor associated with adolescence and early adult life stages but has a faded presence and role for later adulthood, beyond age 30 years, and is minimally detected in advanced age, beyond 40 years. In association with the rapidly increasing trend for delaying reproduction beyond age 35 years, GH is being widely researched now as a potential adjuvant for infertility treatment in this group who, studies consistently show, have a poorer prognosis than younger females when relying on autologous oocytes. The idea that the age-related reduction in fertility prognosis is a feature of growth hormone deficiency is supported by our studies showing an elevated binding protein IGFBP-3/IGF-1 ratio and this can be reduced to a normal range (matching younger, good prognosis women) by the administration of GH as an adjuvant.

Growth Hormone and Insulin-Like Growth Factor Action in Reproductive Tissues.

The role of growth hormone (GH) in human fertility is widely debated with some studies demonstrating improvements in oocyte yield, enhanced embryo quality, and in some cases increased live births with concomitant decreases in miscarriage rates. However, the basic biological mechanisms leading to these clinical differences are not well-understood. GH and the closely-related insulin-like growth factor (IGF) promote body growth and development via action on key metabolic organs including the liver, skeletal muscle, and bone. In addition, their expression and that of their complementary receptors have also been detected in various reproductive tissues including the oocyte, granulosa, and testicular cells. Therefore, the GH/IGF axis may directly regulate female and male gamete development, their quality, and ultimately competence for implantation. The ability of GH and IGF to modulate key signal transduction pathways such as the MAP kinase/ERK, Jak/STAT, and the PI3K/Akt pathway along with the subsequent effects on cell division and steroidogenesis indicates that these growth factors are centrally located to alter cell fate during proliferation and survival. In this review, we will explore the function of GH and IGF in regulating normal ovarian and testicular physiology, while also investigating the effects on cell signal transduction pathways with subsequent changes in cell proliferation and steroidogenesis. The aim is to clarify the role of GH in human fertility from a molecular and biochemical point of view.

Method Protocols for Metabolic and Functional Analysis of the BRIN-BD11 ß-Cell Line: A Preclinical Model for Type 2 Diabetes.

In type 2 diabetes, prolonged dysregulation of signalling and ß-cell metabolic control leads to ß-cell dysfunction, and is increasingly associated with abnormal metabolic states which disrupt normal cellular physiology. Utilization of appropriate ß-cell models enables a systematic approach to understand the impact of perturbations to the biological system. The BRIN-BD11 ß-cell line is a useful, pre-clinical cell model for ß-cell dysfunction associated with type 2 diabetes, among other metabolic disorders. The present chapter describes detection and analysis of ß-cell dysfunction with respect to changes in bioenergetics and metabolism, generation of intracellular reactive oxygen species, and acute and chronic insulin secretion in the BRIN-BD11 cell line.

Live birth outcomes of vitrified embryos generated under growth hormone stimulation are improved for women categorized as poor-prognosis.

OBJECTIVE: To determine the clinical pregnancy (CP) and live birth (LB) rates arising from frozen embryo transfers (FETs) that had been generated under the influence of in vitro fertilization (IVF) adjuvants given to women categorized as poor-prognosis. METHODS: A registered, single-center, retrospective study. A total of 1,119 patients with first FETs cycle include 310 patients with poor prognosis (109 treated with growth hormone [GH], (+)GH group vs. 201 treated with dehydroepiandrosterone, (-)GH group) and 809 patients with good prognosis (as control, (-)Adj (Good) group). RESULTS: The poor-prognosis women were significantly older, with a lower ovarian reserve than the (-)Adj (Good) group, and demonstrated lower chances of CP (p<0.005) and LB (p<0.005). After adjusting for confounders, the chances of both CP and LB in the (+)GH group were not significantly different from those in the (-)Adj (Good) group, indicating that the poor-prognosis patients given GH had similar outcomes to those with a good prognosis. Furthermore, the likelihood of LB was significantly higher for poor-prognosis women given GH than for those who did not receive GH (p<0.028). This was further confirmed in age-matched analyses. CONCLUSION: The embryos cryopreserved from fresh IVF cycles in which adjuvant GH had been administered to women classified as poor-prognosis showed a significant 2.7-fold higher LB rate in subsequent FET cycles than a matched poor-prognosis group. The women with a poor prognosis who were treated with GH had LB outcomes equivalent to those with a good prognosis. We therefore postulate that GH improves some aspect of oocyte quality that confers improved competency for implantation.

DHEA Supplementation Confers No Additional Benefit to that of Growth Hormone on Pregnancy and Live Birth Rates in IVF Patients Categorized as Poor Prognosis.

BACKGROUND: In vitro fertilization (IVF) patients receive various adjuvant therapies to enhance success rates, but the true benefit is actively debated. Growth hormone (GH) and dehydroepiandrosterone (DHEA) supplementation were assessed in women undergoing fresh IVF transfer cycles and categorized as poor prognosis from five criteria. METHODS: Data were retrospectively analyzed from 626 women undergoing 626 IVF cycles, where they received no adjuvant, GH alone, or GH-DHEA in combination. A small group received DHEA alone. The utilization of adjuvants was decided between the attending clinician and the patient depending on various factors including cost. RESULTS: Despite patients being significantly older with lower ovarian reserve, live birth rates were significantly greater with GH alone (18.6%) and with GH-DHEA (13.0%) in comparison to those with no adjuvant (p < 0.003). No significant difference was observed between the GH groups (p = 0.181). Overall, patient age, quality of the transferred embryo, and GH treatment were the only significant independent predictors of live birth chance. Following adjustment for patient age, antral follicle count, and quality of transferred embryo, GH alone and GH-DHEA led to a 7.1-fold and 5.6-fold increase in live birth chance, respectively (p < 0.000). CONCLUSION: These data indicated that GH adjuvant may support more live births, particularly in younger women, and importantly, the positive effects of GH treatment were still observed even if DHEA was also used in combination. However, supplementation with DHEA did not indicate any potentiating benefit or modify the effects of GH treatment. Due to the retrospective design, and the risk of a selection bias, caution is advised in the interpretation of the data.

The Influence of Breast Tumour-Derived Factors and Wnt Antagonism on the Transformation of Adipose-Derived Mesenchymal Stem Cells into Tumour-Associated Fibroblasts.

Within the tumour stroma, a heterogeneous population of cell types reciprocally regulates cell proliferation, which considerably affects the progression of the disease. In this study, using tumour conditioned medium (TCM) derived from breast tumour cell lines - MCF7 and MDA MB 231, we have demonstrated the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into tumour-associated fibroblasts (TAFs). Since the Wnt signalling pathway is a key signalling pathway driving breast tumour growth, the effect of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) was also examined. The response of ADSCs to TCM and sFRP4 treatments was determined by using cell viability assay to determine the changes in ADSC viability, immunofluorescence for mesenchymal markers, glucose uptake assay, and glycolysis stress test using the Seahorse Extracellular Flux analyser to determine the glycolytic activity of ADSCs. ADSCs have been shown to acquire a hyper-proliferative state, significantly increasing their number upon short-term and long-term exposure to TCM. Changes have also been observed in the expression of key mesenchymal markers as well as in the metabolic state of ADSCs. SFRP4 significantly inhibited the differentiation of ADSCs into TAFs by reducing cell growth as well as mesenchymal marker expression (cell line-dependent). However, sFRP4 did not induce further significant changes to the altered metabolic phenotype of ADSCs following TCM exposure. Altogether, this study suggests that the breast tumour milieu may transform ADSCs into a tumour-supportive phenotype, which can be altered by Wnt antagonism, but is independent of metabolic changes.