Endometrial uNK cell counts do not predict successful implantation in an IVF population.

STUDY QUESTION: Are uterine natural killer (uNK) cell numbers and their distribution relative to endometrial arterioles altered in women with recurrent implantation failure (RIF) compared to women with embryo implantation success (IS)? SUMMARY ANSWER: uNK cell numbers and their distribution relative to endometrial arterioles are not significantly different in women with RIF compared to women in whom embryo implantation occurs successfully following IVF. WHAT IS ALREADY KNOWN: uNK cells are regulators of decidual angiogenesis and spiral arteriole remodelling during early pregnancy. Although some studies have shown that uNK cell numbers may be altered in women with RIF, the methods used to measure uNK cell numbers have proven inconsistent, making reproduction of these results difficult. It is unclear, therefore, whether the results reported so far are reproducible. Moreover, it is not known how uNK cell numbers may impact IVF outcomes. Despite the lack of conclusive evidence, uNK cell numbers are often evaluated as a prognostic criterion in women undergoing assisted reproductive procedures. STUDY DESIGN, SIZE, DURATION: Endometrial pipelle biopsies were collected 6-8 days post-LH surge in natural cycles from women with RIF (n = 14), women with IS (n = 11) and women with potential RIF at the time of the study (PRIF; n = 9) from 2013 to 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: uNK cells (i.e. CD56+ and/or CD16+ phenotypes) and their distribution relative to endometrial arterioles were investigated by standard immunohistochemistry protocols and quantified using Aperio ScanScopeXT images digitized by ImageJ and deconvoluted into binary images for single cell quantification using a Gaussian Blur and Yen algorithm. MAIN RESULTS AND THE ROLE OF CHANCE: There was no significant difference in the cell density of CD56+ or CD16+ uNK cells in women with RIF compared to women with IS or PRIF. There was a higher proportion of uNK cells in the distal regions compared to the regions closest to the arterioles in all patient groups. Further, we identified a significant reduction in uNK cell density in women who had a previous pregnancy compared to those who had not, regardless of their current implantation status. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Spiral arterioles could not always be accurately identified by digital image analysis; therefore, all endometrial arterioles were selected and analysed. Patient numbers for the study were low. However, as the clinical phenotypes of each patient were well defined, and endometrial dating was accurately determined by three independent pathologists, differences between patient groups with respect to the uNK numbers and distribution should have been measurable if uNK cell counts were to be useful as a prognostic marker of RIF. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that CD56+ and CD16+ uNK cell numbers are not significantly different in women with RIF in a typical cohort of women undergoing IVF. Further, prior pregnancy was associated with a significantly reduced number of uNK cells in both the RIF and IS patient groups, suggestive of a long-term pregnancy induced suppression of uNK cells. Combined, these findings do not support the clinical value of using uNK cell numbers as a prognostic indicator of implantation success with IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): Funding for this work was provided by Royal Women's Hospital Foundation. P.P. was supported by an NHMRC Early Career Fellowship [TF 11/14] and W.T.T. was supported by an NHMRC Postgraduate Scholarship [1055814]. The authors do not have any competing interests with this study.

Eosinophil-Associated Gene Pathways but not Eosinophil Numbers are Differentially Regulated between Synchrotron Microbeam Radiation Treatment and Synchrotron Broad-Beam Treatment by 48 Hours Postirradiation.

Synchrotron microbeam radiation treatment (MRT) is a preclinical radiotherapy technique with considerable clinical promise, although some of the underlying radiobiology of MRT is still not well understood. In recently reported studies, it has been suggested that MRT elicits a different tumor immune profile compared to broad-beam treatment (BB). The aim of this study was to investigate the effects of synchrotron MRT and BB on eosinophil-associated gene pathways and eosinophil numbers within and around the tumor in the acute stage, 48 h postirradiation. Balb/C mice were inoculated with EMT6.5 mouse mammary tumors and irradiated with microbeam radiation (112 and 560 Gy) and broad-beam radiation (5 and 9 Gy) at equivalent doses determined from a previous in vitro study. After tumors were collected 24 and 48 h postirradiation, RNA was extracted and quantitative PCR performed to assess eosinophil-associated gene expression. Immunohistochemistry was performed to detect two known markers of eosinophils: eosinophil-associated ribonucleases (EARs) and eosinophil major basic protein (MBP). We identified five genes associated with eosinophil function and recruitment (Ear11, Ccl24, Ccl6, Ccl9 and Ccl11) and all of them, except Ccl11, were differentially regulated in synchrotron microbeam-irradiated tumors compared to broad-beam-irradiated tumors. However, immunohistochemical localization demonstrated no significant differences in the number of EAR- and MBP-positive eosinophils infiltrating the primary tumor after MRT compared to BB. In conclusion, our work demonstrates that the effects of MRT on eosinophil-related gene pathways are different from broad-beam radiation treatment at doses previously demonstrated to be equivalent in an in vitro study. However, a comparison of the microenvironments of tumors, which received MRT and BB, 48 h after exposure showed no difference between them with respect to eosinophil accumulation. These findings contribute to our understanding of the role of differential effects of MRT on the tumor immune response.

Influence of different hormonal regimens on endometrial microvascular density and VEGF expression in women suffering from breakthrough bleeding.

BACKGROUND: The aim of this study was to quantify blood vessel density (BVD) and immunoreactive vascular endothelial growth factor (VEGF) levels in endometrial biopsies taken from women suffering breakthrough bleeding (BTB) under different exogenous hormonal regimes. METHODS: Endometrial biopsies from women in Melbourne with BTB were divided into four groups: combined-continuous hormone therapy (HT) (estrogen and progestin taken daily), cyclical HT (daily estrogen with progestin for 14 days each cycle), progestin-only, or no HT. Subjects from Barcelona were using the Mirena intrauterine levonorgestrel-releasing system for contraceptive purposes, with menstrual diaries for classification into four groups (amenorrhea, infrequent, regular and prolonged). Control biopsies from Melbourne were included in the study. Endometrial samples were immunostained for VEGF and blood vessel localization using an antibody to CD34. RESULTS: Results showed that BVD was significantly reduced in the progestin-only treated group compared with the other three treatment groups (P = 0.028). In addition, all four Mirena BTB groups had significantly reduced BVD compared with controls. Considerable heterogeneity was observed in VEGF immunostaining within and between individual samples with no major differences between HT or Mirena. CONCLUSION: These results provide strong evidence that unopposed progestins reduce endometrial BVD and that there is no link between VEGF immunostaining and BVD or BTB.

Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos.

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.

Myometrial microvascular endothelial cells express oxytocin receptor.

OBJECTIVE: 1. To establish whether microvascular endothelial cells from the human myometrium (MMECs) express oxytocin receptor, and to compare its expression levels relative to HUVECs. 2. To verify an up-regulation of oxytocin receptor expression in MMECs as a result of vascular endothelial growth factor (VEGF), which had been found in a previous study. DESIGN: Laboratory scientific study. SETTING: University department. POPULATION OR SAMPLE: Myometrial biopsies from 12 hysterectomy specimens. METHODS: MMECs and HUVECs were established in vitro. Immunohistochemistry of in vitro cultures was performed to investigate protein expression of the oxytocin receptor. Semi-quantitative RT-PCR and Northern blots were performed to examine the presence and relative abundance of oxytocin receptor mRNA in MMECs and HUVECs, and in both cell types with and without VEGF. Total RNA from oxytocin acetate (100 nmol/L) and vehicle stimulated endothelial cell cultures was used to examine gene expression differences on a 10.5K cDNA microarray. MAIN OUTCOME MEASURES: Oxytocin receptor mRNA and protein; gene expression anlysis. RESULTS: Oxytocin receptor mRNA and protein was present in MMECs. The level of expression was the same as for the HUVECs, but much less than the pregnant myometrium. No effect on gene expression could be demonstrated by gene expression microarray following 10 hours of oxytocin stimulation. Twenty-four hours of VEGF stimulation did not significantly alter oxytocin receptor mRNA expression in MMECs or HUVECs. CONCLUSION: The myometrial microvasculature expresses oxytocin receptor. This finding means that oxytocin may exert some of its effects on the myometrial microvasculature. No evidence could be found for a transcriptional effect of oxytocin stimulation in this study, but further work on the role of the myometrial vessel oxytocin receptor is needed.

Bladder microvasculature in women with interstitial cystitis.

PURPOSE: A cardinal cystoscopic finding in women with interstitial cystitis is mucosal small vessel hemorrhage or glomerulations after hydrodistention. We quantified and compared microvascular density and endothelial proliferation in the bladder biopsies of women with interstitial cystitis and a control group of women who were undergoing incontinence or prolapse surgery. MATERIALS AND METHODS: We performed computer assisted image analysis and immunohistochemical studies to compare differences in the blood vessel count, and proportional area in the bladder suburothelium and deeper submucosa of bladder biopsies of 52 women, including 26 with interstitial cystitis. Routine light microscopy features were examined and correlated with microvascular density. RESULTS: In the bladder biopsies of women with interstitial cystitis there was a lower blood vessel count (p = 0.01), and a lower proportion of the total image consisted of blood vessel wall (p = 0.03) in the suburothelium than in control biopsies. We noted no difference in the blood vessel count of the deeper submucosa or in the degree of endothelial cell proliferation. Suburothelial blood vessel differences correlated with the degree of histological change, such as edema, inflammatory infiltrate and vascular congestion. CONCLUSIONS: We found decreased microvascular density in the suburothelium but not in the deeper submucosa in bladder biopsies of women with interstitial cystitis.