Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods.

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conventionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indicated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 ± 1.9%), and 1.0 or 1.5 mm/s (56.5 ± 1.5%, 56.7 ± 1.7% respectively), and lowest survival at 0.5 (52.1 ± 1.6%) and 3.0 mm/s (51.4 ± 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and directionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.

Sub-Optimal Paternal Diet at the Time of Mating Disrupts Maternal Adaptations to Pregnancy in the Late Gestation Mouse.

Pregnancy represents a stage during which maternal physiology and homeostatic regulation undergo dramatic change and adaptation. The fundamental purpose of these adaptations is to ensure the survival of her offspring through adequate nutrient provision and an environment that is tolerant to the semi-allogenic foetus. While poor maternal diet during pregnancy is associated with perturbed maternal adaptations during pregnancy, the influence of paternal diet on maternal well-being is less clearly defined. We fed C57BL/6 male mice either a control (CD), low protein diet (LPD), a high fat/sugar Western diet (WD) or the LPD or WD supplemented with methyl donors (MD-LPD and MD-WD, respectively) for a minimum of 8 weeks prior to mating with C57BL/6 females. Mated females were culled at day 17 of gestation for the analysis of maternal metabolic, gut, cardiac and bone health. Paternal diet had minimal influences on maternal serum and hepatic metabolite levels or gut microbiota diversity. However, analysis of the maternal hepatic transcriptome revealed distinct profiles of differential gene expression in response to the diet of the father. Paternal LPD and MD-LPD resulted in differential expression of genes associated with lipid metabolism, transcription, ubiquitin conjugation and immunity in dams, while paternal WD and MD-WD modified the expression of genes associated with ubiquitin conjugation and cardiac morphology. Finally, we observed changes in maternal femur length, volume of trabecular bone, trabecular connectivity, volume of the cortical medullar cavity and thickness of the cortical bone in response to the father's diets. Our current study demonstrates that poor paternal diet at the time of mating can influence the patterns of maternal metabolism and gestation-associated adaptations to her physiology.

FSH receptor gene variants are rarely associated with premature ovarian failure.

FSH receptor (FSHR) gene variants have been associated with premature ovarian failure (POF). Genomic DNA from New Zealand women with POF (n=80) and control women (n=80) was screened for variants in FSHR exons 7 and 10. FSHR exon 7 variants, including the c.566C>T Finnish founder mutation (p.Ala189Val), were not detected. Previously reported FSHR exon 10 polymorphisms were identified in both groups with similar allelic distributions. A novel heterozygous FSHR exon 10 variant c.1411A>T, p.Ile471Phe was observed in one woman with a family history of POF, but not her affected siblings. It is concluded that variants in exons 7 and 10 of FSHR are not frequently associated with the development of POF in the New Zealand population.

Fibroblast growth factor 2 induces the precocious development of endothelial cell networks in bovine luteinising follicular cells.

The transition from follicle to corpus luteum represents a period of intense angiogenesis; however, the exact roles of angiogenic factors during this time remain to be elucidated. Thus, the roles of vascular endothelial growth factor (VEGF) A, fibroblast growth factor (FGF) 2 and LH in controlling angiogenesis were examined in the present study. A novel serum-free luteinising follicular angiogenesis culture system was developed in which progesterone production increased during the first 5 days and was increased by LH (P<0.01). Blockade of signalling from FGF receptors (SU5402; P<0.001) and, to a lesser extent, VEGF receptors (SU1498; P<0.001) decreased the development of endothelial cell (EC) networks. Conversely, FGF2 dose-dependently (P<0.001) induced the precocious transition of undeveloped EC islands into branched networks associated with a twofold increase in the number of branch points (P<0.001). In contrast, VEGFA had no effect on the area of EC networks or the number of branch points. LH had no effect on the area of EC networks, but it marginally increased the number of branch points (P<0.05) and FGF2 production (P<0.001). Surprisingly, progesterone production was decreased by FGF2 (P<0.01) but only on Day 5 of culture. Progesterone production was increased by SU5402 (P<0.001) and decreased by SU1498 (P<0.001). These results demonstrate that FGF and VEGF receptors play a fundamental role in the formation of luteal EC networks in vitro, which includes a novel role for FGF2 in induction of EC sprouting.

Effect of insulin-like growth factor system on luteinising angiogenesis.

Preovulatory follicle growth and the luteal transition requires intense angiogenesis. This enables progesterone production to increase sufficiently to support a pregnancy. Inadequate follicular or luteal vascularisation can lead to reduced ovarian function and thus compromise fertility. Insulin-like growth factor 1 (IGF1) and IGF2 regulate multiple ovarian processes and are key links between an animal's reproductive and metabolic status. This study investigated the role that the IGF system plays in regulating luteinising follicular endothelial cell (EC) networks and progesterone production in vitro. Bovine luteinising follicular angiogenesis cultures were treated with 1) LR3-IGF1 (10 or 100ng/ml) under basal and angiogenic-stimulated conditions or 2) IGF1 receptor inhibitor (picropodophyllin (PPP); 1µM) in the presence or absence of LR3-IGF1, IGF2, or combined LR3-IGF1+IGF2 (10ng/ml). EC networks were quantified by von Willebrand factor immunohistochemistry. Progesterone production was analysed by ELISA and cell proliferation was determined by MTT assay. LR3-IGF1 had limited effects on EC growth parameters, whilst PPP (p<0.001) markedly reduced EC growth parameters (by 60-70%). Cell proliferation was slightly increased (by 3-5%) by LR3-IGF1 (p<0.001). LR3-IGF1 had variable effects on progesterone production, whilst PPP reduced progesterone concentration (p<0.001) with or without LR3-IGF1 or IGF2 alone or in combination. IGF1 was detected in cell conditioned media and was increased by LH (50ng/ml) (p<0.001). In conclusion, exogenous IGF1 and IGF2 had minimal effects on luteinising follicular angiogenesis and progesterone production, but the inhibitory effect of the IGFR1 inhibitor (PPP) suggests that IGF1 receptor signalling is critical for the development of EC networks and progesterone production in luteinising follicular cells.

Fibroblast growth factor 2 is a key determinant of vascular sprouting during bovine luteal angiogenesis.

Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and coordination of this complex process remain unknown. Thus, the temporal and spatial patterns of endothelial network formation were determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6 h to 9 days). Endothelial cells, as determined by von Willebrand factor immunohistochemistry, initially grew in cell islands (days 0-3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3-6), and after 9 days in culture had formed extensive intricate networks. Mixed populations of luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0-3, 3-6 or 6-9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3-6 vs controls (mean reduction 81%; P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3-6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.

How can mating systems inform future biobanking strategies? An illustration using two Indonesian bovids, banteng (Bos javanicus) and lowland anoa (Bubalus depressicornis).

Storing cryopreserved spermatozoa in a genome resource bank safeguards against the loss of heterozygosity in endangered species and provides opportunities to reincorporate genes into populations through the application of assisted reproductive technologies. The aim of this study was to demonstrate the effect of breeding strategy on ejaculate characteristics to illustrate how this information may be used to select appropriate methods for the storage and use of cryopreserved sperm. In the present study, ejaculates from a polygynous bovid, banteng (Bos javanicus), were characterized (motility 72.7 ± 4.3%; total sperm count 2,702 ± 764 ×106 sperm; morphologically normal sperm 87.9 ± 3.0%), as well as ejaculates from a monogamous bovid, lowland anoa (Bubalus depressicornis; motility 47.5 ± 5.4%; total sperm count 279 ± 84 ×106 sperm; morphologically normal sperm 69.0 ± 6.1%). As banteng produce an ejaculate with characteristics similar to domestic cattle, translating assisted reproductive technologies from domestic cattle is feasible. By contrast, lowland anoa produce smaller quantities of sperm with a higher prevalence of morphologically abnormal sperm; thus, alternative protocols, optimized for the storage and use of ejaculates containing lower quantities of sperm, is necessary. Sperm tail length was more conserved in banteng (CV 2.7%) than lowland anoa (CV 6.4%) and could be due to differences in levels of sperm competition between species. Additionally, the use of three different diluents (Biladyl, TES-Tris yolk buffer, and whole milk) were investigated for banteng sperm cryopreservation. Sperm cryopreserved in Biladyl and whole milk diluents produced significantly higher post-thaw survival parameters then TES-Tris yolk buffer.

A randomised controlled trial to evaluate the impact of indoor living space on dairy cow production, reproduction and behaviour.

As a global society, we have a duty to provide suitable care and conditions for farmed livestock to protect animal welfare and ensure the sustainability of our food supply. The suitability and biological impacts of housing conditions for intensively farmed animals is a complex and emotive subject, yet poorly researched, meaning quantitative evidence to inform policy and legislation is lacking. Most dairy cows globally are housed for some duration during the year, largely when climatic conditions are unfavourable. However, the impact on biology, productivity and welfare of even the most basic housing requirement, the quantity of living space, remains unknown. We conducted a long-term (1-year), randomised controlled trial (CONSORT 10 guidelines) to investigate the impact of increased living space (6.5 m2 vs 3 m2 per animal) on critical aspects of cow biology, behaviour and productivity. Adult Holstein dairy cows (n = 150) were continuously and randomly allocated to a high or control living space group with all other aspects of housing remaining identical between groups. Compared to cows in the control living space group, cows with increased space produced more milk per 305d lactation (primiparous: 12,235 L vs 11,592 L, P < 0.01; multiparous: 14,746 L vs 14,644 L, P < 0.01) but took longer to become pregnant after calving (primiparous: 155 d vs 83 d, P = 0.025; multiparous: 133 d vs 109 d). In terms of behaviour, cows with more living space spent significantly more time in lying areas (65 min/d difference; high space group: 12.43 h/day, 95% CI = 11.70-13.29; control space group: 11.42 h/day, 95% CI = 10.73-12.12) and significantly less time in passageways (64 min/d), suggesting enhanced welfare when more space was provided. A key physiological difference between groups was that cows with more space spent longer ruminating each day. This is the first long term study in dairy cows to demonstrate that increased living space results in meaningful benefits in terms of productivity and behaviour and suggests that the interplay between farmed animals and their housed environment plays an important role in the concepts of welfare and sustainability of dairy farming.

Maternal protein restriction affects fetal ovary development in sheep.

Maternal malnutrition has important developmental consequences for the foetus. Indeed, adverse fetal ovarian development could have lifelong impact, with potentially reduced ovarian reserve and fertility of the offspring. This study investigated the effect of maternal protein restriction on germ cell and blood vessel development in the fetal sheep ovary. Ewes were fed control (n = 7) or low protein (n = 8) diets (17.0 g vs 8.7 g crude protein/MJ metabolizable energy) from conception to day 65 of gestation (gd65). On gd65, fetal ovaries were subjected to histological and immunohistochemical analysis to quantify germ cells (OCT4, VASA, DAZL), proliferation (Ki67), apoptosis (caspase 3) and vascularisation (CD31). Protein restriction reduced the fetal ovary weight (P < 0.05) but had no effect on fetal weight (P > 0.05). The density of germ cells was unaffected by maternal diet (P > 0.05). In the ovarian cortex, OCT4+ve cells were more abundant than DAZL+ve (P < 0.001) and VASA+ve cells (P < 0.001). The numbers, density and estimated total weight of OCT4, DAZL, and VASA+ve cells within the ovigerous cords were similar in both dietary groups (P > 0.05). Similarly, maternal protein restriction had no effect on germ cell proliferation or apoptotic indices (P > 0.05) and the number, area and perimeter of medullary blood vessels and degree of microvascularisation in the cortex (P > 0.05). In conclusion, maternal protein restriction decreased ovarian weight despite not affecting germ cell developmental progress, proliferation, apoptosis, or ovarian vascularity. This suggests that reduced maternal protein has the potential to regulate ovarian development in the offspring. LAY SUMMARY: Variations in a mother's diet during pregnancy can influence her offspring's growth and might cause fertility problems in the offspring in later life. We investigated whether reducing the protein fed to sheep during early pregnancy affects their daughters' ovaries. We then compared our findings to the offspring of sheep on a complete diet. We measured ovary size and estimated the number of germ cells (cells that become eggs) they contained. We used cell markers to assess potential changes in the pattern of germ cell growth, division, and death, and how the ovarian blood supply had developed. We found that protein restriction reduced ovary size. However, the pattern of germ cell development, growth, or death was not altered by poor diet and blood vessels were also unaffected. This suggests that maternal diet can change ovarian development by an unknown mechanism and might reduce future fertility in their offspring.

Transforming growth factor-ß1 disrupts angiogenesis during the follicular-luteal transition through the Smad-serpin family E member 1 (SERPINE1)/serpin family B member 5 (SERPINB5) signalling pathway in the cow.

Intense angiogenesis is critical for the development of the corpus luteum and is tightly regulated by numerous factors. However, the exact role transforming growth factor-ß1 (TGFB1) plays during this follicular-luteal transition remains unclear. This study hypothesised that TGFB1, acting through TGFB receptor 1 (TGFBR1) and Smad2/3 signalling, would suppress angiogenesis during the follicular-luteal transition. Using a serum-free luteinising follicular angiogenesis culture system, TGFB1 (1 and 10ngmL-1 ) markedly disrupted the formation of capillary-like structures, reducing the endothelial cell network area and the number of branch points (P <0.001 compared with control). Furthermore, TGFB1 activated canonical Smad signalling and inhibited endothelial nitric oxide synthase (NOS3 ) mRNA expression, but upregulated latent TGFB-binding protein and TGFBR1 , serpin family E member 1 (SERPINE1 ) and serpin family B member 5 (SERPINB5 ) mRNA expression. SB431542, a TGFBR1 inhibitor, reversed the TGFB1-induced upregulation of SERPINE1 and SERPINB5 . In addition, TGFB1 reduced progesterone synthesis by decreasing the expression of steroidogenic acute regulatory protein (STAR ), cytochrome P450 family 11 subfamily A member 1 (CYP11A1 ) and 3ß-hydroxysteroid dehydrogenase (HSD3B1 ) expression. These results show that TGFB1 regulates NOS3 , SERPINE1 and SERPINB5 expression via TGFBR1 and Smad2/3 signalling and this could be the mechanism by which TGFB1 suppresses endothelial networks. Thereby, TGFB1 may provide critical homeostatic control of angiogenesis during the follicular-luteal transition. The findings of this study reveal the molecular mechanisms underlying the actions of TGFB1 in early luteinisation, which may lead to novel therapeutic strategies to reverse luteal inadequacy.

Detrimental effects of uterine disease and lipopolysaccharide on luteal angiogenesis.

Reproductive tract inflammatory disease (RTID) commonly occurs after the traumatic events of parturition and adversely affects follicular function. This study is the first to describe the cellular and steroidogenic characteristics of corpora lutea from cattle with RTID and the effects of pathogen-associated molecular patterns (PAMPs) on luteal angiogenesis and function in vitro. Luteal weight (P < 0.05) and progesterone content (P < 0.05) were reduced (1.2-fold) in cows with RTID, accompanied by reduced CYP11A (P < 0.05), HSD3B (P < 0.01) and STAR (P < 0.01) protein expression. Immunohistochemistry revealed that luteal vascularity (VWF) and pericyte (ACTA2) coverage were >3-fold lower in RTID cows (P < 0.05). To link these observations to bacterial infection and determine specificity of action, a physiologically relevant luteal angiogenesis culture system examined the effects of PAMPs on endothelial cell (EC) network formation and progesterone production, in the presence of pro-angiogenic factors. Luteal EC networks were reduced ≤95% (P < 0.05) by lipopolysaccharide (LPS, toll-like receptor (TLR) 4 agonist) but not by TLR2 agonists lipoteichoic acid or peptidoglycan. Conversely, progesterone production and steroidogenic protein expression were unaffected by PAMPs (P > 0.05). Moreover, the adverse effect of LPS on luteal EC networks was dose-dependent and effective from 1 ng/mL (P < 0.05), while few EC networks were present above 10 ng/mL LPS (P < 0.001). LPS reduced proliferation (P < 0.05) and increased apoptosis of EC (P < 0.001). The specific TLR4 inhibitor TAK242 reversed the effects of LPS on EC networks. In conclusion, luteal vasculature is adversely sensitive to LPS acting via TLR4, therefore ovarian exposure to LPS from any Gram-negative bacterial infection will profoundly influence subsequent reproductive potential.

FGF2 is crucial for the development of bovine luteal endothelial networks in vitro.

The development of the corpus luteum requires angiogenesis, and involves the complex interplay between factors such as vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). However, the relative role of these factors remains to be elucidated. This study used a new physiologically relevant mixed luteal cell culture system to test the hypotheses that: a) FGF2 and VEGFA are critical for bovine luteal angiogenesis; and b) local luteal PDGF signalling stimulates the formation of endothelial networks. Cells were treated with receptor tyrosine kinase inhibitors against VEGFA (SU1498), FGF2 (SU5402) or PDGF (AG1295) activity. After 9 days in culture, endothelial cells were immunostained for von Willebrand factor (VWF) and quantified by image analysis. Highly organised intricate endothelial networks were formed in the presence of exogenous VEGFA and FGF2. The inhibition of FGF2 activity reduced the total area of VWF staining versus controls (>95%; P<0.001). Inhibition of VEGF and PDGF activity reduced the endothelial network formation by more than 60 and 75% respectively (P<0.05). Progesterone production increased in all treatments from day 1 to 7 (P<0.001), and was unaffected by FGF2 or PDGF receptor kinase inhibition (P>0.05), but was reduced by the VEGF receptor inhibitor on days 5 and 7 (P<0.001). In conclusion, this study confirmed that VEGF signalling regulates both bovine luteal angiogenesis and progesterone production. However, FGF2 was crucial for luteal endothelial network formation. Also, for the first time, this study showed that local luteal PDGF activity regulates bovine luteal endothelial network formation in vitro.

A high incidence of polymorphic CYP2C19 variants in archival blood samples from Papua New Guinea.

There is considerable inter-ethnic variability in the incidence of CYP2C19 genetic poor metabolisers ( var / var ). About 3 per cent of Caucasians are CYP2C19 var/var . By contrast, an extremely high incidence (70 per cent) is observed in the Melanesian island of Vanuatu. The colonisation of the Pacific Islands is believed to have involved migration through Papua New Guinea (PNG), and hence a high incidence may also be expected in this population. The reported incidence in PNG was only 36 per cent, however. PNG is a country of extensive ethnic diversity, and the incidence of the CYP2C19 var/var in other regional populations of PNG is currently not established. In this study, restriction fragment length polymorphism-polymerase chain reaction analysis of archival blood serum samples was used to determine the prevalence of the CYP2C19*2 and *3 variant alleles in three different ethnic and geographically isolated populations of PNG. In the largest population studied (Iruna), the frequency of both variant CYP2C19 alleles was high (0.37 and 0.34, respectively). Specifically, the frequency of the CYP2C19*3 allele was significantly higher than in the PNG (East Sepik) population reported previously (0.34 vs 0.16; p < 0.0001). In the Iruna population, 48.9 per cent of the samples were homozygous variants for CYP2C19*2 or *3 , which although higher was not statistically different from the East Sepik population (36 per cent). The results of this study indicated that other regional populations of PNG also have a relatively high incidence of the CYP2C19 genetic polymorphism compared with Caucasian populations. The high incidence reported in Vanuatu, however, may be due to genetic drift rather than a PNG founder population, as the Vanuatu population is dominated by the CYP2C19*2 allele, with a lower contribution from the *3 allelic variant.

Evaluation of progestogen supplementation for luteal phase support in fresh in vitro fertilization cycles.

OBJECTIVE: To evaluate the effectiveness of progestogen supplementation in improving clinical pregnancy rates in women undergoing fresh IVF cycles and to compare different routes, start times, durations, and estrogen coadministration regimen. DESIGN: Comprehensive systematic review and meta-analysis. SETTING: University. PATIENT(S): Women undergoing fresh IVF cycles who did and did not receive progestogen supplementation. INTERVENTION(S): Summary odds ratios (ORs) were calculated by binomial logistic regression. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates. RESULT(S): Eighty-two articles (26,726 women) were included. Clinical pregnancy rates were increased by IM (OR = 4.57), vaginal (OR = 3.34), SC (OR = 3.36), or oral (OR = 2.57) progestogen supplementation versus no treatment. The greatest benefit was observed when progestogens were supplemented IM versus vaginally (OR = 1.37). The optimal time to commence administration was between oocyte retrieval and ET (OR = 1.31), with oocyte retrieval +1 day being most beneficial. Coadministration of estrogen had no benefit (OR = 1.33), whether progestogens were coadministered vaginally or IM. Clinical pregnancy rates were equivalent when progestogen supplementation was ceased after ≤3 weeks or continued for up to 12 weeks (OR = 1.06). CONCLUSION(S): This broad-ranging meta-analysis highlights the need to reevaluate current clinical practice. The use of progestogens in fresh IVF cycles is substantially beneficial to clinical pregnancy. Critically, the use of IM progestogens should not be dismissed, as it yielded the greatest clinical pregnancy rates. Pregnancy success was impacted by initiation of therapy, with 1 day after oocyte retrieval being optimal. There is little evidence to support coadministration of estrogen or prolonging progestogen treatment beyond 3 weeks.

Gene expression profiling of breast tumours from New Zealand patients.

AIMS: New Zealand has one of the highest rates of breast cancer incidence in the world. We investigated the gene expression profiles of breast tumours from New Zealand patients, compared them to gene expression profiles of international breast cancer cohorts and identified any associations between altered gene expression and the clinicopathological features of the tumours. METHODS: Affymetrix microarrays were used to measure the gene expression profiles of 106 breast tumours from New Zealand patients. Gene expression data from six international breast cancer cohorts were collated, and all the gene expression data were analysed using standard bioinformatic and statistical tools. RESULTS: Gene expression profiles associated with tumour ER and ERBB2 status, molecular subtype and selected gene expression signatures within the New Zealand cohort were consistent with those found in international cohorts. Significant differences in clinicopathological features such as tumour grade, tumour size and lymph node status were also observed between the New Zealand and international cohorts. CONCLUSIONS: Gene expression profiles, which are a sensitive indicator of tumour biology, showed no clear difference between breast tumours from New Zealand patients and those from non-New Zealand patients. This suggests that other factors may contribute to the high and increasing breast cancer incidence in New Zealand compared to international populations.

Multimodal Assessment of Estrogen Receptor mRNA Profiles to Quantify Estrogen Pathway Activity in Breast Tumors.

BACKGROUND: Molecular markers have transformed our understanding of the heterogeneity of breast cancer and have allowed the identification of genomic profiles of estrogen receptor (ER)-α signaling. However, our understanding of the transcriptional profiles of ER signaling remains inadequate. Therefore, we sought to identify the genomic indicators of ER pathway activity that could supplement traditional immunohistochemical (IHC) assessments of ER status to better understand ER signaling in the breast tumors of individual patients. MATERIALS AND METHODS: We reduced ESR1 (gene encoding the ER-α protein) mRNA levels using small interfering RNA in ER+ MCF7 breast cancer cells and assayed for transcriptional changes using Affymetrix HG U133 Plus 2.0 arrays. We also compared 1034 ER+ and ER- breast tumors from publicly available microarray data. The principal components of ER activity generated from these analyses and from other published estrogen signatures were compared with ESR1 expression, ER-α IHC, and patient survival. RESULTS: Genes differentially expressed in both analyses were associated with ER-α IHC and ESR1 mRNA expression. They were also significantly enriched for estrogen-driven molecular pathways associated with ESR1, cyclin D1 (CCND1), MYC (v-myc avian myelocytomatosis viral oncogene homolog), and NFKB (nuclear factor kappa B). Despite their differing constituent genes, the principal components generated from these new analyses and from previously published ER-associated gene lists were all associated with each other and with the survival of patients with breast cancer treated with endocrine therapies. CONCLUSION: A biomarker of ER-α pathway activity, generated using ESR1-responsive mRNAs in MCF7 cells, when used alongside ER-α IHC and ESR1 mRNA expression, could provide a method for further stratification of patients and add insight into ER pathway activity in these patients.

Luteal angiogenesis and its control.

Angiogenesis, the formation of new blood vessels from preexisting ones, is critical to luteal structure and function. In addition, it is a complex and tightly regulated process. Not only does rapid and extensive angiogenesis occur to provide the corpus luteum with an unusually high blood flow and support its high metabolic rate, but in the absence of pregnancy, the luteal vasculature must rapidly regress to enable the next cycle of ovarian activity. This review describes a number of key endogenous stimulatory and inhibitory factors, which act in a delicate balance to regulate luteal angiogenesis and ultimately luteal function. In vitro luteal angiogenesis cultures have demonstrated critical roles for fibroblast growth factor 2 (FGF2) in endothelial cell proliferation and sprouting, although other factors such as vascular endothelial growth factor A (VEGFA) and platelet-derived growth factor were important modulators in the control of luteal angiogenesis. Post-transcriptional regulation by small non-coding microRNAs is also likely to play a central role in the regulation of luteal angiogenesis. Appropriate luteal angiogenesis requires the coordinated activity of numerous factors expressed by several cell types at different times, and this review will also describe the role of perivascular pericytes and the importance of vascular maturation and stability. It is hoped that a better understanding of the critical processes underlying the transition from follicle to corpus luteum and subsequent luteal development will benefit the management of luteal function in the future.

SPACA3 gene variants in a New Zealand cohort of infertile and fertile couples.

SPRASA (also referred to as SLLP1) is a protein identified in the acrosome of human sperm and encoded by the gene SPACA3. SPRASA is associated with sperm-oocyte recognition and binding, and may play a role in fertility. In order to determine whether variants in the SPACA3 gene are associated with human infertility, we undertook a genetic analysis of 102 infertile and 104 fertile couples. Three gene variants were identified using PCR-based DNA sequencing; 1) an insertion of TGC within a quadruple tri-nucleotide (TGC) repeat region in the 5' untranslated region (UTR) (g.-22TGC(4_5), 2) a guanine to adenosine transition at position 239 (c.239G>A) resulting in a non-synonymous amino acid substitution from cysteine to tyrosine (p.C80Y) at position 80 in the putative transmembrane region, and 3) a novel nucleotide variant (c.691G>C) located in the 3'UTR. A functional effect of the g.-22TGC (4_5) was confirmed by a luciferase expression assay, while the effects of the variants c.239G>A and c.691G>C were predicted using in silico analysis. Although the frequencies of these variants were not significantly different between the infertile and fertile populations, we present evidence that the variants could affect the expression levels or function of SPRASA, thereby affecting a couple's fertility. Larger populations, especially individuals/couples with unexplained infertility, need to be screened for these variants to validate a relationship with fertility.

Investigating the association between inhibin alpha gene promoter polymorphisms and premature ovarian failure.

OBJECTIVE: To determine whether variants in the promoter region of the inhibin alpha gene (INHA) are associated with premature ovarian failure (POF). DESIGN: Mutational analysis of the INHA gene promoter in women with POF. SETTING: Academic institution. PATIENT(S): Patients with POF (n = 194) and controls (n = 162) from New Zealand and Slovenia. INTERVENTION(S): Peripheral blood samples were screened for known polymorphisms in the INHA promoter (c.-16C-->T, c.-124A-->G, and an imperfect TG repeat at approximately -300 base pairs). Genotyping was performed by restriction fragment length polymorphism, forced restriction fragment length polymorphism, and nondenaturing high-performance liquid chromatography analysis. MAIN OUTCOME MEASURE(S): Genotypic status of INHA promoter polymorphisms. RESULT(S): Significant differences in INHA promoter allele frequencies were observed between POF patient populations and controls. Significant reductions in allele frequency were observed for the -16T allele (New Zealand POF) and -124G allele (total POF) and for INHA promoter haplotypes C (New Zealand POF) and D (Slovenian POF). CONCLUSION(S): We conclude that INHA promoter variants are associated with the development of POF.

The genetic basis of premature ovarian failure.

Premature ovarian failure (POF) is a common condition, affecting approximately 1:100 women. It is characterised by amenorrhea, hypoestrogenism, and elevated gonadotrophin levels in women under the age of 40. It is often an unexpected and distressing diagnosis, which coincides with infertility and menopausal symptoms. There is a well recognised genetic basis to the development of POF. Our laboratory has identified several candidate genes associated with POF.