Plasma miRNAs as biomarkers for endometriosis.

STUDY QUESTION: Can plasma miRNAs be used for the non-invasive diagnosis of endometriosis in infertile women? SUMMARY ANSWER: miRNA-based diagnostic models for endometriosis failed the test of independent validation. WHAT IS KNOWN ALREADY: Circulating miRNAs have been described to be differentially expressed in patients with endometriosis compared with women without endometriosis, suggesting that they could be used for the non-invasive diagnosis of endometriosis. However, these studies have shown limited consistency or conflicting results, and no miRNA-based diagnostic test has been validated in an independent patient cohort. STUDY DESIGN, SIZE, DURATION: We performed genome-wide miRNA expression profiling by small RNA sequencing to identify a set of plasma miRNAs with discriminative potential between patients with and without endometriosis. Expression of this set of miRNAs was confirmed by RT-qPCR. Diagnostic models were built using multivariate logistic regression with stepwise feature selection. In a final step, the models were tested for validation in an independent patient cohort. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Plasma of all patients was available in the biobank of the Leuven Endometriosis Centre of Excellence. Biomarker discovery and model development were performed in a discovery cohort of 120 patients (controls = 38, endometriosis = 82), and models were tested for validation in an independent cohort of 90 patients (controls = 30, endometriosis = 60). RNA was extracted with the miRNeasy Plasma Kit. Genome-wide miRNA expression analysis was done by small RNA sequencing using the NEBNext small RNA library prep kit and the NextSeq 500 System. cDNA synthesis and qPCR were performed using the Qiagen miScript technology. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a set of 42 miRNAs with discriminative power between patients with and without endometriosis based on genome-wide miRNA expression profiling. Expression of 41 miRNAs was confirmed by RT-qPCR, and 3 diagnostic models were built. Only the model for minimal-mild endometriosis (Model 2: hsa-miR-125b-5p, hsa-miR-28-5p and hsa-miR-29a-3p) had diagnostic power above chance performance in the independent validation (AUC = 60%) with an acceptable sensitivity (78%) but poor specificity (37%). LIMITATIONS, REASONS FOR CAUTION: The diagnostic models were built and tested for validation in two patient cohorts from a single tertiary endometriosis centre. Further validation tests in large cohorts with patients from multiple endometriosis centres are needed. WIDER IMPLICATION OF THE FINDINGS: Our study supports a possible biological link between certain miRNAs and endometriosis, but the potential of these miRNAs as clinically useful biomarkers is questionable in women with infertility. Large studies in well-described patient cohorts, with rigorous methodology for miRNA expression analysis, sufficient statistical power and an independent validation step, are necessary to answer the question of whether miRNAs can be used as diagnostics markers for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by a grant from the Research Foundation - Flanders (FWO). A.V., D.F.O. and D.P. are PhD fellows from the FWO. T.D. is vice president and Head of Global Medical Affairs Fertility, Research and Development, Merck KGaA, Darmstadt, Germany. He is also a professor in Reproductive Medicine and Biology at the Department of Development and Regeneration, Group Biomedical Sciences, KU Leuven (University of Leuven), Belgium and an adjunct professor at the Department of Obstetrics and Gynecology in the University of Yale, New Haven, USA. Neither his corporate role nor his academic roles represent a conflict of interest with respect to the work done by him for this study. The other co-authors have no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Sheep as maintenance host for Leptospira interrogans serovar hardjo subtype hardjobovis.

Transmission of Leptospira interrogans serovar hardjo subtype hardjobovis from naturally infected sheep to uninfected sheep and calves was studied. A microscopic agglutination test and ELISA were used to determine specific antibody responses in serum. Polymerase chain reaction was used to detect bacterial shedding in urine. Six sheep were derived from a dairy farm where cows were infected with L hardjobovis. Three of these sheep were seropositive for L hardjobovis, and 1 also shed leptospires in the urine. The other 2 sheep shed leptospires in the urine 7 days after the first observation date. The 6 sheep were placed on an isolated pasture together with a second group of 6 noninfected sheep. During the observation period of 140 days, 1 sheep of the second group became infected with L hardjobovis. On 5 consecutive days, a urine mixture from the 4 infected sheep was sprayed on the heads of 4 noninfected calves. Within 56 days, all calves that had been sprayed with urine shed L hardjobovis in the urine and became seropositive for L hardjobovis.

[Serological study of the occurrence of L. hardjo in sheep in The Netherlands]. / Een serologisch onderzoek naar het voorkomen van L. hardjo bij schapen in Nederland.

Sheep (3918) from 137 farms in the regions of North-, West- and Mid-Netherlands and Gelderland were serologically investigated for the presence of antibodies against Leptospira hardjo. Antibodies were detected in 3.3% of the sheep. There were large regional differences with respect to both the percentage of positive sheep and the percentage of positive flocks. All sera from sheep in Gelderland were negative. In West- en Mid-Netherlands there were 0.9 and 6.5% positive sera, respectively, and 19.4 and 32.7% positive flocks. The percentage of positive sheep per positive flocks varied from 1 tot 51.6. Serological positive sheep were from farms with and without cattle. On farms with cattle, there was no clear relationship between serologically sheep and the presence of hardjo antibodies in cattle. It thus seems plausible that sheep can be infected with hardjo independently of cattle.

[Udder infections with coagulase-negative staphylococci in freshly calved heifers]. / Uierinfecties met coagulase negatieve stafylococcen (CNS) bij nieuwmelkte vaarzen.

To determine the prevalence of udder infections in fresh calved heifers all quarters of 98 heifers on eleven commercial farms were sampled weekly during the first eight weeks of lactation. The samples during the first four weeks of this period were used for bacteriological examination and in all samples a somatic cell count was made. The prevalence of S. aureus after calving in these heifers was 12.5%. Coagulase-negative staphylococci (CNS) were isolated from half of the heifers. The predominant coagulase-negative staphylococcus species were S. xylosis and S. hyicus. The CNS, with exception of S. hyicus, had a very small effect on the cell count.

[Salmonella dublin on dairy farms in the North of the Netherlands]. / Salmonella dublin op melkveebedrijven in Noord-Nederland.

During the last century many calves and cows with Salmonella dublin infections are diagnosed in the North of the Netherlands, especially in the province of Friesland. In this paper are retrospectively traced the problems by paratyphoid infections in cattle, from 1919 till now, the diagnosis of which was made at the Animal Health Service in Friesland and North-Netherlands. Over the decennia a decrease is noticed for the percentage of necropsies of calves with S. dublin infections, although some irregular peaks appear. However since 1988 an increase is observed in dairy farms with S. dublin problems, revealing high mortality in calves, abortions and sick cows.

[Leptospirosis in cattle; milker's fever in cattle farmers]. / Leptospirose bij runderen; melkerskoorts bij de veehouders.

In this paper the symptomatology, epidemiology, and diagnosis of Leptospira interrogans serovar hardjo infections in cattle are reviewed. The possibilities on monitoring and control of this disease in both foreign countries and the Netherlands are discussed. Special attention is paid to the zoonotic aspects of the infection (dairy fever).

[The occurrence of hardjo-positive dairy cows in the Northern Netherlands. A prospective serological survey]. / Het voorkomen van hardjo-positieve melkrunderen in Noord-Nederland. Een oriënterend serologisch onderzoek.

More than 5000 lactating cows from 82 farms, in the northern part of the Netherlands, were serologically examined for antibodies to Leptospira hardjo. Hardjo-positive cows were found on 52% of the farms with a mean incidence of 23% positive animals. Considerable differences were observed in the percentages hardjo-positive cows per region (7-30%), as well as in the percentages positive cows per positive farm (22-46%). With the exception of one region (14%) the percentages of hardjo-positive farms per region hardly differed (average 58%). In addition to the animal survey 488 sera of men working and/or living on cattle-farms were also examined. Twenty-six persons were hardjo-positive. Most of these persons (circa 90%) milked regularly. All seropositive persons were found on farms with hardjo-positive cattle. Results from an inquiry on circa 200 farms indicated, that hardjo-infections probably occur with a higher frequency on larger farms. Finally the results from this survey confirm the observation in the literature, that symptoms of hardjo-infections are more frequently found in the autumn.

[Bacterial count of milk, pin-points and udder health in dairying herds]. / Kiemgetal van melk, 'pinpoints' en de uiergezondheid op bedrijven.

The pin-point colonies in bacterial counts of bulk milk were examined for the causative agents of mastitis. Udder health in herds showing unduly high bacterial counts as a result of pin-points was found to be reduced. There was no relationship between the organism isolated from the pin-points and the most important udder pathogen in the herd. The dairy farmers should be informed of the presence of pin-points when the bacterial counts are too high because of their presence.

Laminitis and interdigital dermatitis and heel horn erosion. A European perspective.

Laminitis is one of the most important claw disorders in dairy herds. Nutrition, calving, burdening of the lateral claw of the rear feet, and hereditary susceptibility are all contributing factors. Interdigital dermatitis in cattle may be a result of infection by Bacteroides nodosus and Fusobacterium necrophorum. If this infection becomes chronic, heel horn erosion is its consequence.

[Formalin foot baths in the prevention of interdigital dermatitis in cattle]. / Formalinebaden ter bestrijding van de stinkpootinfectie bij rundvee.

Formalin (= approximately 35 per cent formaldehyde) foot baths are used with variable success to prevent interdigital dermatitis, eczema of the interdigital skin accompanied by secondary destruction of the bulb horn. Experimental studies were done in vivo and in vitro to determine the significance of the temperature and concentration of the formalin solution and the method of bathing (standing in a bath for sixty minutes or walking through a bath, in which case each foot is immersed twice in the solution). The results show that a low temperature of the bathing solution (10 degrees C), which theoretically produces poor disinfection (Table 3), does not necessarily imply inadequate disinfection in actual practice (Table 4) as the solution will adhere to the interdigital skin for about thirty minutes (Table 2) and very rapidly will acquire the temperature of the skin (20 degrees-30 degrees C, Table 1). Therefore contact between the interdigital skin and grass, mud, dung and so on has to be prevented for the first thirty minutes after bathing. The more intensive contact between formalin and skin in a stationary foot bath offers advantages over the pass-through bath (Table 4). When a 3 percent formalin solution in a pass-through bath fails to produce satisfactory results, a 5 percent solution may be used. This concentration will result in more effective disinfection (Table 4); higher concentrations may cause skin lesions. Repeated daily disinfection does not produce further reduction of the bacterial flora on the interdigital skin to a significant degree (Table 5). In addition to the disinfecting effect, the hardening effect on the interdigital skin may be of importance.

[Incidence of lesions of the sole (author's transl)]. / Het optreden van zoollaesies bju melkvee.

Lesions of the sole, ranging in severity from discolouration to ulceration of the sole and undermining of the horn of the wall and sole, are common in heifers under the conditions prevailing in the Netherlands. In view of the similarity of these lesions in the two lateral claws of the hind-feet of the same animal, both as regards the localization on the sole and the severity of the lesions as well as the time of onset, these lesions cannot be solely attributed to mechanical injury. Also in view of the fact that they are associated with the time of calving, they may be regarded as symptoms of chromic laminitis. Housing (loose housing versus trying stalls) and the season of calving (housing period versus grazing period) are factors in the incidence of lesions of the sole.

[Nutrition as a possible factor in the pathogenesis of ulcers of the sole in cattle (author's transl)]. / Een mogelijke invloed van de voeding op het optreden van zoolzweren bij het rund.

Two groups of eight dairy cows, one fed a high (6 kg.) and one fed a low (1 kg.) level of concentrates before calving were compared with regard to the incidence of haemorrhage in the horny sole of the hind claws. These lesions are considered to be a symptom of subclinical laminitis and a possible preliminary stage of ulcer of the sole. In both groups, more as well as more severe lesions were observed within five to eight weeks after calving than there were two weeks prior to parturition. After calving, the incidence of these lesions was significantly higher in the 'high concentrate group' than it was in the 'low concentrate group'.

[Studies on intersucking in dairy cattle (author's transl)]. / Een onderzoek naar melkzuigen bij runderen.

Intersucking occurred on nearly 50 per cent of the farms in the Province of Utrecht. The incidence was not affected by housing systems, the size of the herd and the surface area available to each cow in cubicle stalls. It is unlikely that the animals imitate intersucking by others. Intersucking often occurs prior to evening milking, the sucking animal usually forming a steady pair with the suckled cow. Of four A.I. bulls studied, one had a significantly higher proportion of intersucking heifers among his offspring than the others. Some other factors possibly affecting the incidence of intersucking, such as the rearing system, composition of the ration and the space allotted to each cow for feeding were not studied because of insufficient information or inadequate variation of the material.