Early pregnancy bleeding after assisted reproductive technology: a systematic review and secondary data analysis from 320 patients undergoing hormone replacement therapy frozen embryo transfer.

STUDY QUESTION: How common is bleeding in early pregnancy after Hormone Replacement Therapy (HRT) Frozen Embryo Transfer (FET) and does bleeding affect the reproductive outcome? SUMMARY ANSWER: A total of 47% of HRT-FET patients experience bleeding before the eighth week of gestation, however, bleeding does not affect the reproductive outcome. WHAT IS KNOWN ALREADY: Bleeding occurs in 20% of spontaneously conceived pregnancies, although most will proceed to term. However, our knowledge regarding bleeding in early pregnancy after HRT-FET and the reproductive outcome is sparse. STUDY DESIGN, SIZE, DURATION: We performed a systematic review of the existing literature on early pregnancy bleeding after assisted reproductive technology (ART) to evaluate the bleeding prevalence and resulting reproductive outcome in this population. A random-effects proportional meta-analysis was conducted. Subsequently, we performed a prospective cohort study including 320 pregnant patients undergoing HRT-FET and a secondary analysis of the cohort study was performed to evaluate bleeding prevalence and reproductive outcome. The trial was conducted from January 2020 to November 2022 in a public fertility clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: A systematic literature search was performed, using MESH terms and included studies with data from ART patients and with early pregnancy bleeding as a separate outcome. The cohort study included patients with autologous vitrified blastocyst transfer treated in an HRT-FET protocol. In the event of a positive HCG-test, an early pregnancy scan was performed around 8 weeks of gestation. During this visit, patients answered a questionnaire regarding bleeding or spotting and its duration after the positive pregnancy test. The information was verified through medical files, and these were used to obtain information on reproductive outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: The review revealed a total of 12 studies of interest. The studies reported a prevalence of early pregnancy bleeding ranging from 2.1% to 36.2%. The random effects proportional meta-analysis resulted in a pooled effect estimate of the prevalence of early pregnancy bleeding in the ART population of 18.1% (95% CI (10.5; 27.1)). Four of the included studies included data on miscarriage rate following an episode of bleeding. All four studies showed a significantly increased risk of miscarriage in patients with early pregnancy bleeding as compared to patients with no history of bleeding. No studies investigated bleeding after HRT-FET specifically. In our HRT-FET cohort study, we found that a total of 47% (149/320) of patients with a positive pregnancy test experienced bleeding before 8 weeks of gestation. Generally, the bleeding was described as spotting with a median of 2 days (range 0.5-16 days). Out of 149 patients with one or several bleeding episodes, a total of 106 patients (71%) had an ongoing pregnancy at 12 weeks of gestation. In comparison, 171 patients reported no bleeding episodes and a total of 115 (67%) of these patients had an ongoing pregnancy at 12 weeks of gestation. This difference was not significant (P = 0.45). Furthermore there was no difference in the live birth rate between the two groups (P = 0.29). LIMITATIONS, REASONS FOR CAUTION: Most studies included in the review were older and not all studies specified the type of ART. Moreover, the studies were of moderate methodological quality. The patients in the cohort study were treated in a personalized HRT-FET protocol using a rectal supplementary rescue regimen if serum progesterone levels were <35 nmol/l at embryo transfer. The results may not be applicable to other FET protocols, and the present data were based on self-reported symptoms. The systematic review revealed an increased risk of miscarriage following an episode of early pregnancy bleeding. However our cohort study found no such association. This discrepancy can partly be due to the fact, that the four studies in the review only included episodes of heavy bleeding. Also, none of the four studies included data on HRT-FET cycles making them unfit for direct comparison. WIDER IMPLICATIONS OF THE FINDINGS: Episodes of early bleeding during pregnancy are associated with distress for the pregnant woman, especially in a cohort of infertile patients. Our cohort study showed that at least minor bleeding seems to be a common adverse event of early pregnancy after HRT-FET. From the systematic review, it seems that this prevalence is higher than what has previously been described in relation to other types of ART. However, minor bleeding during early pregnancy after HRT-FET does not seem to affect the reproductive outcome. Knowledge regarding the frequent occurrence of bleeding during early pregnancy after HRT-FET and the fact that this should not be used as a prognostic parameter will help the clinician in counselling patients. STUDY FUNDING/COMPETING INTEREST(S): Gedeon Richter Nordic supported this investigator-initiated study with an unrestricted grant as well as study medication (Cyclogest). B.A. has received an unrestricted grant from Gedeon Richter Nordic and Merck and honoraria for lectures from Gedeon Richter, Merck, IBSA, and Marckyrl Pharma. P.H. received honoraria for lectures from Merck, Gedeon Richter, Institut Biochimique SA (IBSA), and Besins as well as unrestricted research grants from Merck, Gedeon Richter, and Institut Biochimique SA (IBSA). The other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: EudraCT no.: 2019-001539-29.

Rectal progesterone administration secures a high ongoing pregnancy rate in a personalized Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) protocol: a prospective interventional study.

STUDY QUESTION: Can supplementation with rectal administration of progesterone secure high ongoing pregnancy rates (OPRs) in patients with low serum progesterone (P4) on the day of blastocyst transfer (ET)? SUMMARY ANSWER: Rectally administered progesterone commencing on the ET day secures high OPRs in patients with serum P4 levels below 35 nmol/l (11 ng/ml). WHAT IS KNOWN ALREADY: Low serum P4 levels at peri-implantation in Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) cycles impact reproductive outcomes negatively. However, studies have shown that patients with low P4 after a standard vaginal progesterone treatment can obtain live birth rates (LBRs) comparable to patients with optimal P4 levels if they receive additionalsubcutaneous progesterone, starting around the day of blastocyst transfer. In contrast, increasing vaginal progesterone supplementation in low serum P4 patients does not increase LBR. Another route of administration rarely used in ART is the rectal route, despite the fact that progesterone is well absorbed and serum P4 levels reach a maximum level after ∼2 h. STUDY DESIGN, SIZE, DURATION: This prospective interventional study included a cohort of 488 HRT-FET cycles, in which a total of 374 patients had serum P4 levels ≥35 nmol/l (11 ng/ml) at ET, and 114 patients had serum P4 levels <35 nmol/l (11 ng/ml). The study was conducted from January 2020 to November 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients underwent HRT-FET in a public Fertility Clinic, and endometrial preparation included oral oestradiol (6 mg/24 h), followed by vaginal micronized progesterone, 400 mg/12 h. Blastocyst transfer and P4 measurements were performed on the sixth day of progesterone administration. In patients with serum P4 <35 nmol/l (11 ng/ml), 'rescue' was performed by rectal administration of progesterone (400 mg/12 h) starting that same day. In pregnant patients, rectal administration continued until Week 8 of gestation, and oestradiol and vaginal progesterone treatment continued until Week 10 of gestation. MAIN RESULTS AND THE ROLE OF CHANCE: Among 488 HRT-FET single blastocyst transfers, the mean age of the patients at oocyte retrieval (OR) was 30.9 ± 4.6 years and the mean BMI at ET 25.1 ± 3.5 kg/m2. The mean serum P4 level after vaginal progesterone administration on the day of ET was 48.9 ± 21.0 nmol/l (15.4 ± 6.6 ng/ml), and a total of 23% (114/488) of the patients had a serum P4 level lower than 35 nmol/l (11 ng/ml). The overall, positive hCG rate, clinical pregnancy rate, OPR week 12, and total pregnancy loss rate were 66% (320/488), 54% (265/488), 45% (221/488), and 31% (99/320), respectively. There was no significant difference in either OPR week 12 or total pregnancy loss rate between patients with P4 ≥35 nmol/l (11 ng/ml) and patients with P4 <35 nmol/l, who received rescue in terms of rectally administered progesterone, 45% versus 46%, P = 0.77 and 30% versus 34%, P = 0.53, respectively. OPR did not differ whether patients had initially low P4 and rectal rescue or were above the P4 cut-off. Logistic regression analysis showed that only age at OR and blastocyst scoring correlated with OPR week 12, independently of other factors like BMI and vitrification day of blastocysts (Day 5 or 6). LIMITATIONS, REASONS FOR CAUTION: In this study, vaginal micronized progesterone pessaries, a solid pessary with progesterone suspended in vegetable hard fat, were used vaginally as well as rectally. It is unknown whether other vaginal progesterone products, such as capsules, gel, or tablet, could be used rectally with the same rescue effect. WIDER IMPLICATIONS OF THE FINDINGS: A substantial part of HRT-FET patients receiving vaginal progesterone treatment has lowserum P4. Adding rectally administered progesterone in these patients increases the reproductive outcome. Importantly, rectal progesterone administration is considered convenient, and progesterone pessaries are easy to administer rectally and of low cost. STUDY FUNDING/COMPETING INTEREST(S): Gedeon Richter Nordic supported the study with an unrestricted grant as well as study medication. B.A. has received unrestricted grant from Gedeon Richter Nordic and Merck and honoraria for lectures from Gedeon Richter, Merck, IBSA and Marckyrl Pharma. P.H. has received honoraria for lectures from Gedeon Richter, Merck, IBSA and U.S.K. has received grant from Gedeon Richter Nordic, IBSA and Merck for studies outside this work and honoraria for teaching from Merck and Thillotts Pharma AB and conference expenses covered by Merck. The other co-authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER (25): EudraCT no.: 2019-001539-29.

Invited review: The effect of milk feeding practices on dairy calf behavior, health, and performance-A systematic review.

The aim of this systematic review was to summarize the literature assessing the effects of milk feeding practices on behavior, health, and performance on dairy calves. Peer-reviewed, published articles, written in English, directly comparing the effects of milk allowance, milk feeding methods, or milk feeding frequency on dairy calves were eligible for inclusion. Outcome measures could include sucking behavior, sucking on a teat (nutritive sucking, non-nutritive sucking on a teat), abnormal sucking behavior (non-nutritive sucking on pen fixtures, other oral behaviors, or cross-sucking), signs of hunger (vocalizations or unrewarded visits at the milk feeder), activity (lying time or locomotor play), feeding behavior (milk intake, starter intake, milk meal duration, or starter meal duration), growth (body weight or average daily gain), and health (occurrence of diarrhea, respiratory disease, or mortality). We conducted 2 targeted searches using Web of Science and PubMed to identify key literature. The resulting articles underwent a 2-step screening process. This process resulted in a final sample of 94 studies. The majority of studies investigated milk allowance (n = 69). Feeding higher milk allowances had a positive or desirable effect on growth, reduced signs of hunger, and increased locomotor play behavior during the preweaning period, whereas starter intake was reduced. Studies addressing health pointed to no effect of milk allowance, with no consistent evidence indicating that higher milk allowances result in diarrhea. Studies addressing milk feeding methods (n = 14) found that feeding milk by teat reduced cross-sucking and other abnormal oral behaviors. However, results on the effect of access to a dry teat were few and mixed. Milk feeding frequency (n = 14 studies) appeared to have little effect on feed intakes and growth; however, there is some evidence that calves with lower feeding frequency experience hunger. Overall, findings strongly suggest feeding higher volumes of milk using a teat; however, further work is needed to determine the optimal feeding frequency for dairy calves.

Assessing effects of dietary and milking frequency changes and injection of cabergoline during dry-off on hunger in dairy cows using 2 feed-thwarting tests.

We investigated the single and combined effects of 2 feeding levels (normal lactation diet vs. energy-reduced diet, both fed for ad libitum intake) and 2 daily milking frequencies (twice vs. once) during 1 wk before the dry-off day (d 0), as well as an intramuscular injection of either a dopamine agonist (cabergoline; Velactis, Ceva Santé Animale; labeled for use only with abrupt dry-off, e.g., no reduction in feeding level or milking frequency before the last milking) or saline after the last milking on d 0 on the feeding motivation of clinically healthy, loose-housed, pregnant, lactating Holstein cows. From d 0, all cows were fed the same dry-cow diet for ad libitum intake. Cows were subjected to 2 feed-thwarting tests, a test in the home pen using their diets (test A: d -6, -1, and 1; during 35 min when the feed bins were filled, but locked) and another test carried out in an adjacent pen in which access to concentrate provided in a familiar plastic box was blocked by a wire-mesh lid (test B: d -5 and 2). In test A, we recorded how often cows attempted to feed per 35 min, whether cows vocalized during the 35-min period, and latency to feed within 300 s after feed bins were unlocked. In test B, we recorded latency to approach either of 2 familiar boxes (the wire-mesh box and an identical open box with a small portion of concentrate) within 600 s and how often cows directed behaviors toward the wire-mesh box (number of occurences/5 min). On d -6 (test A), no clear differences in feeding motivation among treatments were found. On d -5 and -1, cows fed the energy-reduced diet displayed a higher probability of vocalizing (test A), were more than 50% quicker to feed (test A), were approximately 5× quicker to approach a box (test B), and directed 60% more behavior toward the wire-mesh box (test B) than cows fed the normal diet. Moreover, cows fed the energy-reduced diet attempted to feed approximately 75% more on d -1 compared with d -6 (test A). On d 2 (test B), cows previously fed the normal diet directed 40% more behavior toward the wire-mesh box than cows previously fed the energy-reduced diet. Reducing feeding level, either before or on the dry-off day, resulted in consistently increased feeding motivation, interpreted as a sign of hunger. No clear effects of change in milking frequency, singly or combined with reduced diet energy density, on feeding motivation were found before d 0. Whereas, on d 2, cows previously milked twice daily were quicker to approach a box than cows previously milked once daily. Cows injected with cabergoline attempted to feed more, but showed lower probability of vocalizing compared with saline-injected cows (d 1; test A), irrespective of treatment before d 0. The effects of cabergoline on feeding motivation are not easily interpreted and warrant further investigation. From a hunger perspective, reducing milking frequency rather than diet energy density seems to be a less negative management to reduce milk production before dry-off.

Breast-contour preserving procedures for early-stage breast cancer: a population-based study of the trends, variation in practice and predictive characteristics in Denmark and the Netherlands.

PURPOSE: Breast-contour preservation (BCP) is possible for most women treated for early-stage breast cancer. BCP can be defined as primary breast-conserving treatment (BCT), neoadjuvant chemotherapy (NAC) followed by BCT and immediate postmastectomy breast reconstruction (IBR). This study provides insight in current BCP strategies in Denmark and the Netherlands and aims to identify opportunities for improvement within both countries. METHODS: A total of 92,881 patients with early-stage breast cancer who were operated in Denmark and the Netherlands between 2012 and 2017 were selected from the Danish Breast Cancer Group and the Dutch National Breast Cancer Audit databases. BCP procedures and predictive factors were analyzed within and between both countries. RESULTS: BCP was achieved in 76.7% (n = 16,355) of the Danish and in 74.5% (n = 53,328) of the Dutch patients. While BCP rate did not change significantly over time in Denmark (p = 0.250), a significant increase in BCP rate from 69.5% in 2012 to 78.5% in 2017 (p < 0.001) was observed in the Netherlands. In both countries, variation in BCP rates between hospitals decreased over time. NAC followed by BCT and postmastectomy IBR was substantially more often used in the Netherlands compared to Denmark, specifically in patients younger than 50 years. CONCLUSIONS: In more than 75% of all Danish and Dutch patients, surgically treated for early-stage breast cancer, the breast-contour was preserved. The different use of BCP strategies within Denmark and the Netherlands and the differences observed between hospitals in both countries emphasize the need for more (inter)national consensus on treatment modalities.

Rates of re-excision and conversion to mastectomy after breast-conserving surgery with or without oncoplastic surgery: a nationwide population-based study.

BACKGROUND: There is no consensus regarding the impact of oncoplastic surgery (OPS) on rates of re-excision and conversion to mastectomy following breast-conserving surgery (BCS). Here these two outcomes after BCS and OPS were compared in a nationwide population-based setting. METHODS: In Denmark, all OPS is registered and categorized into volume displacement, volume reduction or volume replacement. Patients who underwent BCS or OPS between 2012 and 2018 were selected from the Danish Breast Cancer Group database. Multivariable analyses were performed to adjust for confounders, and propensity score matching to limit potential confounding by indication bias. RESULTS: A total of 13 185 patients (72·5 per cent) underwent BCS and 5003 (27·5 per cent) OPS. Volume displacement was used in 4171 patients (83·4 per cent), volume reduction in 679 (13·6 per cent) and volume replacement in 153 (3·1 per cent). Re-excision rates were 15·6 and 14·1 per cent after BCS and OPS respectively. After adjusting for confounders, patients were less likely to have a re-excision following OPS than BCS (odds ratio (OR) 0·80, 95 per cent c.i. 0·72 to 0·88), specifically after volume displacement and reduction. The rate of conversion to mastectomy was similar after OPS and BCS (3·2 versus 3·7 per cent; P = 0·105), but with a lower risk in adjusted analysis (OR 0·69, 0·58 to 0·84), specifically after volume displacement and reduction procedures. Findings were similar after propensity score matching. CONCLUSION: A modest decrease in re-excision rate and less frequent conversion to mastectomy were observed after OPS compared with BCS.

ANTECEDENTES: No existe consenso con respecto al impacto de la cirugía oncoplástica (oncoplastic surgery, OPS) en las tasas de re-exéresis y conversión a mastectomía tras la cirugía conservadora de la mama (breast conserving surgery, BCS). En este estudio se compararon los dos resultados después de BCS y OPS en una base de datos poblacional de ámbito nacional. MÉTODOS: En Dinamarca, todas las OPS se registran y clasifican en procedimientos que incluyen 1) desplazamiento (o remodelación) de volumen, 2) reducción de volumen o 3) restitución de volumen. Todas las pacientes sometidas a BCS o OPS entre 2012 y 2018 fueron seleccionadas de la base de datos del Grupo de Cáncer de Mama Danés. Se realizaron análisis multivariables para ajustar por factores de confusión y un emparejamiento por puntaje de propensión para limitar las variables de confusión potenciales por el sesgo en la indicación. RESULTADOS: Un total de 13.185 (72,5%) pacientes fueron sometidos a BCS y 5.003 (27,5%) a OPS. Se utilizó el desplazamiento de volumen en 4.171 (83,4%) pacientes, reducción de volumen en 679 (13,6%) pacientes y restitución de volumen en 153 (3,1%) pacientes. Las tasas de re-exéresis fueron del 15,6% y 14,1% tras BCS y OPS, respectivamente. Tras ajustar por factores de confusión, las pacientes tuvieron menos probabilidad (razón de oportunidades, odds ratio, OR 0,80, i.c. del 95%. 0,72-0,88) de requerir re-exéresis tras OPS, específicamente después de desplazamiento y reducción de volumen, en comparación con BCS. La tasa de conversión a mastectomía fue inferior (3,2% versus 3,7%, P = 0,04) y menos probable (OR 0,71, i.c. del 95% 0,58-0,87) tras OPS, específicamente en el desplazamiento y reducción de volumen, en comparación con BCS. Los resultados fueron similares después del emparejamiento por puntuación de propensión. CONCLUSIÓN: Se observó un descenso modesto en las tasas de re-exéresis y una menor frecuencia de conversiones a mastectomía después de la cirugía oncoplástica cuando se comparó con la cirugía conservadora de la mama.

Evaluation of tumor-infiltrating lymphocytes and association with prognosis in BRCA-mutated breast cancer.

BACKGROUND: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (<10%), intermediate (10-59%) and high (≥60%). Prognostic associations of sTils were evaluated as a continuous variable in univariate and multivariate models. Only follow-up time beyond date of BRCA mutation test was included. RESULTS: A large proportion had high sTils (27% in the full cohort, 36% in BRCA1-mutated, and 44% in ER negative breast cancers). Higher sTils were associated with BRCA1, ER negative breast cancer, high histological grade and medullary histology. In combined analysis for BRCA1 and BRCA2-mutated breast cancers, increasing sTils in 10% intervals were significantly associated with OS (HR 0.92, 95% CI 0.84-1.00, p = .05). For each 10% increment of sTils in BRCA1 breast cancers, a 10% reduction of mortality (adjusted HR 0.90 95% CI 0.81-0.99, p = .03) and a 13% reduction in risk of DFS-event (HR 0.87 95% CI 0.76-1.00, p = .05) was observed even after adjustment for ER status. No significant association with survival was of observed in the BRCA2 subgroup. Test for interaction of sTils and BRCA status was not statistically significant (p = .3). CONCLUSIONS: Breast cancer patients with a germline BRCA mutation had higher sTils than previously reported in sporadic breast cancers, and sTils were associated with favorable survival among BRCA carriers.

The effect of transition cow housing on lying and feeding behavior in Holstein dairy cows.

The present study investigated the effect of straw yard housing during the dry period and 2 d of additional maternity pen housing postcalving on lying and feeding behavior and calving difficulty in Holstein dairy cows. In this study, 122 multiparous cows were moved to either a straw yard or into freestall housing 4 wk before their expected calving date. Cows that had been housed in straw yards stayed in the maternity pen for an additional 2 d after their calving day, but cows that had been housed in freestalls were moved to the general lactation group the morning after calving. Lying time, lying bouts, feeding time, number of feeder visits, feed intake, feeding rate, and assisted calvings were recorded. Observations were divided into 2 periods: precalving (the 4-wk dry period before calving) and postcalving (the day of calving and the 2 d after). During the precalving period, cows housed in straw yards showed a higher number of lying bouts but no difference in lying time compared with cows housed in freestalls. Cows that were housed longer in the straw-bedded maternity pen postcalving spent more time lying during the 2 d postcalving and had a higher number of lying bouts on the day of calving than cows moved to the freestall area on the day postcalving. Additionally, cows that were housed longer in the maternity pen had a slower feeding rate and longer total feeding time during the 2 d after calving than cows with a shorter stay in the maternity pen. We found no difference in the number of assisted calvings. This study suggests that straw yard housing during the dry period may facilitate the transition between standing and lying. Furthermore, the extended stay in the maternity pen postcalving increased lying time, the number of lying bouts, and feeding time, but decreased feeding rate compared with cows that were moved to the general lactation group on the day postcalving. These results suggest potential recovery benefits with an extended stay in a maternity pen postcalving. However, further studies are needed to separate the effects of housing in the dry period and the effects of an extended housing in individual maternity pens.

Clinical and molecular characterization of BRCA-associated breast cancer: results from the DBCG.

BACKGROUND: In breast cancer (BC) patients a cancer predisposing BRCA1/2 mutation is associated with adverse tumor characteristics, risk assessment and treatment allocation. We aimed to estimate overall- (OS) and disease-free survival (DFS) according to tumor characteristics and treatment among women who within two years of definitive surgery for primary BC were shown to carry a mutation in BRCA1/2 . MATERIAL AND METHODS: From the clinical database of the Danish Breast Cancer Group we included 141 BRCA1 and 96 BRCA2 BC patients. Estrogen receptor and HER2 status were centrally reviewed on paraffin-embedded tumor tissue. Information on risk reducing surgery was obtained from the Danish Pathology and Patient Registries and included as time-dependent variables in Cox proportional hazard models. RESULTS: Ten-year OS and DFS for BRCA1 BC patients were 78% (95% CI 69-85) and 74% (95% CI 64-81). Ten-year OS and DFS for BRCA2 BC were 88% (95% CI 78-94) and 84% (95% CI 74-91). BRCA1 BC patients as compared to BRCA2 BC patients had a higher risk of BC relapse or non-breast cancer within ten years of follow-up, independent of ER status (adjusted HR 2.78 95% CI 1.28-6.05, p = .01), but BRCA mutation was not associated with OS (adjusted HR 1.98, 95% CI 0.87-4.52, p = .10). In multivariate analysis, including both BRCA1 and BRCA2 carriers, no chemotherapy was associated with a higher risk of death (adjusted OS HR 3.58, 95% CI 1.29-9.97, p = .01) and risk reducing contralateral mastectomy (RRCM) was associated with a significantly reduced risk of death (adjusted OS HR 0.42, 95% CI =0.21-0.84, p = .01). CONCLUSION: Difference in OS between BRCA1 and BRCA2 BC patients could be ascribed to tumor-biology. BRCA1 BC patients may have a shorter ten-year DFS than BRCA2 BC patients. Chemotherapy and risk reducing contralateral mastectomy reduce mortality for both BRCA1 and BRCA2 BC patients.

Prognostic and predictive importance of the estrogen receptor coactivator AIB1 in a randomized trial comparing adjuvant letrozole and tamoxifen therapy in postmenopausal breast cancer: the Danish cohort of BIG 1-98.

PURPOSE: To evaluate the estrogen receptor coactivator amplified in breast cancer 1 (AIB1) as a prognostic marker, as well as a predictive marker for response to adjuvant tamoxifen and/or aromatase inhibitors, in early estrogen receptor-positive breast cancer. METHOD: AIB1 was analyzed with immunohistochemistry in tissue microarrays of the Danish subcohort (N = 1396) of the International Breast Cancer Study Group's trial BIG 1-98 (randomization between adjuvant tamoxifen versus letrozole versus the sequence of the two drugs). RESULTS: Forty-six percent of the tumors had a high AIB1 expression. In line with previous studies, AIB1 correlated to a more aggressive tumor-phenotype (HER2 amplification and a high malignancy grade). High AIB1 also correlated to higher estrogen receptor expression (80-100 vs. 1-79%), and ductal histological type. High AIB1 expression was associated with a poor disease-free survival (univariable: hazard ratio 1.35, 95% confidence interval 1.12-1.63. Multivariable: hazard ratio 1.29, 95% confidence interval 1.06-1.58) and overall survival (univariable: hazard ratio 1.34, 95% confidence interval 1.07-1.68. Multivariable: hazard ratio 1.25, 95% confidence interval 0.99-1.60). HER2 did not seem to modify the prognostic effect of AIB1. No difference in treatment effect between tamoxifen and letrozole in relation to AIB1 was found. CONCLUSIONS: In a subset of the large international randomized trial BIG 1-98, we confirm AIB1 to be a strong prognostic factor in early breast cancer. Hence, although tumor AIB1 expression does not seem to be useful for the choice of tamoxifen versus an aromatase inhibitor in postmenopausal endocrine-responsive breast cancer, AIB1 is an interesting target for new anti-cancer therapies and further investigations of this biomarker is warranted.

External quality assessment for molecular typing of Salmonella 2013-2015: performance of the European national public health reference laboratories.

We report the results of three consecutive External Quality Assessments (EQAs) for molecular subtyping of Salmonella to assess the performance of the European national public health reference laboratories (NPHRLs). The EQA included the molecular typing methods used for European enhanced surveillance of human Salmonella infections: pulsed field gel electrophoresis (PFGE), including gel analysis by the use of the software BioNumerics, and 5-locus multiple locus variable number of tandem repeat analysis (MLVA) for serovar Typhimurium. The participation in the PFGE laboratory part was higher (27/35) than in the gel analysis (19/35) and MLVA (15/35), suggestive of the need for capacity building in methods requiring specialized equipment (MLVA) or software (gel analysis). The majority (25/27) of the participating NPHRLs produced inter-laboratory comparable PFGE gel(s). Two laboratories continued to produce low-quality gels and should have additional technical assistance in the future. In particular, two gel quality evaluation parameters, measuring "image acquisition and running conditions" and "bands", were identified to cause gel quality problems throughout the EQAs. Despite the high number of laboratories participating in the PFGE laboratory part, the participation in gel analysis was low, although increasing. In the MLVA part, the NPHRLs correctly assigned 96% (405/420) allelic profiles according to the nomenclature. In conclusion, the EQAs identified critical parameters for unsuccessful performance and helped to offer assistance to those laboratories that needed it most. The assessments supported the development of quality in molecular typing and promoted the harmonization of subtyping methods used for EU/EEA-wide surveillance of human Salmonella infections.

Dairy cows with prolonged calving seek additional isolation.

In modern calving facilities, dairy cows either calve in a group pen or are moved to a separate individual pen when calving is imminent. In practice, cows are often moved too close to calving, which poses a health risk to cow and calf. Thus, a need exists for new calving facility designs and management practices that better align with the motivations of the cow. This study examined dairy cow preferences for individual calving pens by offering 3 different levels of isolation (tall and narrow, low and wide, and tall and wide) by analyzing the association between precalving behavior, choice of degree of isolation, and the progress of calving. The hypotheses were that cows would prefer the highest level of isolation when giving birth, and that calving in a high level of isolation would be associated with less restlessness and a shorter calving duration. Contrary to these hypotheses, no specific preference between degrees of isolation or difference in calving behavior in the different calving pens was found. However, cows experiencing a longer calving duration chose to calve in the most secluded calving pen (tall and wide). These results cannot determine cause and effect, but may suggest that interactions between motivation for isolation seeking and calving behavior exist.

Short communication: Calving site selection of multiparous, group-housed dairy cows is influenced by site of a previous calving.

A calving cow and her newborn calf appear to have an attracting effect on periparturient cows, which may potentially influence the functionality of future motivation-based calving pen designs. In this pilot study we examined whether calving site selection of group-housed Holstein dairy cows was affected by the site of a previous calving. Ten multiparous cows moved to 1 of 2 group pens 11 (range = 4-27) d before calving were included. Each pen consisted of an open area (9 × 9 m) connected to 6 secluded areas (4.5 × 3 m each), where cows could move freely between all areas. Time of calving, location of the breaking of the amniotic sac, as well as the place of birth were recorded. In all but 1 case cows calved within a distance of 1 cow length from where the previous calving took place, suggesting that the cows did not select calving site at random. These preliminary observations indicate that choice of calving site may be affected by the site of a previous calving, potentially explained by the presence of amniotic fluids.

Effect of sand and rubber surface on the lying behavior of lame dairy cows in hospital pens.

Housing lame cows in designated hospital pens with a soft surface may lessen the pain the animals feel when lying and changing position. This study investigated the effect of the lying surface on the behavior of lame cows in hospital pens. Thirty-two lame dairy cows were kept in individual hospital pens, provided with either 30-cm deep-bedded sand or 24-mm rubber mats during 24 h in a crossover design. On each surface, the lying behavior of each cow was recorded during 18 h. On deep-bedded sand, cows lay down more and changed position more often than when housed on the rubber surface. Furthermore, a shorter duration of lying down and getting up movements and a shorter duration of lying intention movements were observed. These results suggest that lame dairy cows are more reluctant to change position on rubber compared with sand, and that sand is more comfortable to lie on. Thus, deep bedding such as sand may provide better lying comfort for lame cows than an unbedded rubber surface.

Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture.

The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine toxigenic culture. In total, 300 faecal samples from Danish hospitalised patients with diarrhoea were included consecutively. Culture was performed in duplicate (routine and 'expanded toxigenic culture': prolonged and/or re-culture) and genotypic toxin profiling by polymerase chain reaction (PCR), PCR ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene C. difficile and PCRFast C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p < 0.05) and significantly more robust to inhibition compared to PCRFast (p < 0.001). Duplicate 'expanded toxigenic culture' increased the culture-positive rate by 29% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95%), and in-house PCR displayed 100% correct identification of PCR ribotypes 066 and 078. Furthermore, the presence of the PCR enhancer bovine serum albumin (BSA) was found to be related to high sensitivity and low inhibition rate. Rapid laboratory diagnosis of toxigenic C. difficile by RT PCR was accurate.

Pair housing and enhanced milk allowance increase play behavior and improve performance in dairy calves.

To investigate the effects of social housing and milk feeding on play behavior and performance, 48 Holstein-Friesian male and female calves were either individually or pair housed in straw-bedded pens (3.0 m × 4.5 m). Half of the calves in each housing treatment were fed a standard milk allowance (5 L/d from d 3 to 42); the other calves were fed an enhanced milk allowance (9 L/d from d 3 to 28 and 5 L/d from d 29 to 42). All calves were abruptly weaned on d 43. Play behavior was recorded for 48 h beginning on d 15, 29, and 43. Variables were square root-transformed before analysis, but back-transformed values are given. On d 15 the duration of locomotor play behavior was greater among enhanced- than standard-fed calves [15.2 vs. 10.8 (± 1.0) corresponding to 231 vs. 117 s/24 h]; this behavior decreased when milk allowance in the enhanced treatment was reduced on d 29. Across ages, the duration of locomotor play behavior was greater among individually housed than pair-housed calves [10.9 vs. 8.2 (± 0.6) corresponding to 119 vs. 67 s/24 h], but the total duration of play did not differ between individual and pair-housed calves [130 (45-295) s/24 h; median and interquartile range]. Only pair-housed calves could perform social play behavior (play fighting), and the duration of this was greater on d 15 and 29 than on d 43 [8.3, 10.7 and 3.5 (± 1.3) corresponding to 69, 115 and 12 s/24 h for d 15, 29, and 43]. Among calves on enhanced milk, pair-housed animals had a greater concentrate intake than individually housed calves (840 vs. 530 ± 110 g/d); for calves on standard milk, we detected no effect of housing [990 and 1,090 (± 110) g/d]. Among calves on enhanced milk, pair-housed calves had greater body weight gain than individually housed calves [990 vs. 850 (± 40) g/d]. For calves on standard milk, we observed no effect of pair versus individual housing [810 and 840 (± 40) g/d]. In conclusion, greater durations of play behavior in enhanced-fed calves suggest an animal welfare benefit of this feeding method. Enhanced-fed calves consumed less concentrate, but pair housing stimulated concentrate intake in these calves, suggesting that pair housing and enhanced feeding should be used together.

The effect of housing on calving behavior and calf vitality in Holstein and Jersey dairy cows.

The present study investigated how calving behavior and calf vitality in Holstein and Jersey dairy cows were affected by housing during the final 4 wk precalving. One hundred twenty-one cows (36 primiparous and 85 multiparous) were moved either to a group pen with deep straw bedding or into freestall housing 4 wk before the expected calving date. Individual straw-bedded maternity pens were placed adjacent to the straw-bedded group pens, and cows were moved to the maternity pens before calving. Cows that spent more than 12 h in the maternity pen before calving and calved unassisted were included in this study (39 multiparous cows and 15 primiparous cows). Dams were observed from 6 h before calving until 6 h after calving. The time from the onset of rhythmical abdominal contractions (defined as the onset of stage II labor), the time from a visible amniotic sac, and the time from visible calf feet until the birth of the calf were recorded. Furthermore, the cows' latency to stand up after birth was recorded. Calves were observed during the first 6 h after birth and the latency to first standing attempt, to first successful standing, to first suckle attempt, and to first successful suckling were recorded. Cows previously housed in straw pens expelled the calf faster once the calves' feet were visible compared with cows previously housed in freestalls. Multiparous cows stood sooner and licked their calf sooner after birth compared with primiparous cows. Jersey calves of cows previously housed in straw pens also stood up and suckled their dams sooner compared with Jersey calves of cows previously housed in freestalls. Holstein cows previously housed in straw pens tended to stand up sooner compared with Holstein cows previously housed in freestalls. These results suggest that a longer period of housing on deep-bedded straw compared with freestalls with mattresses before calving may facilitate the calving process, whereas the effect on calf vitality needs further investigation.

Preferences of lame cows for type of surface and level of social contact in hospital pens.

To investigate preferences of lame cows for flooring and level of social contact, 37 lame, lactating dairy cows (diagnosed with sole ulcer or white line disease) were housed individually for 6 d in experimental hospital pens, where they could choose between 2 equally sized areas (6m × 4.5m) with either deep-bedded sand or a rubber surface. On both surfaces, cows could choose between 2 equally sized areas either near or away from heifers in a neighboring group pen. Cows spent more time lying on the deep-bedded sand than on the rubber surface (870 vs. 71min/d), whereas they spent less time upright (standing or walking) on the sand than on the rubber surface (180 vs. 319min/d). In addition, cows spent less time self-grooming on the sand than on the rubber surface (2.2 vs. 4.7% of time spent upright). With regard to level of social contact, cows spent more time near the neighboring heifers than away from them; this was true both while lying (565 vs. 374min/d) and upright (276 vs. 223min/d). Self-grooming was seen significantly more near neighboring heifers than away from them (4.8 vs. 3.3% of time spent upright). When lying, cows more often positioned themselves in areas of the pen where they could maintain visual contact with neighboring heifers. Lame cows with sole ulcers or white line disease preferred deep-bedded sand for lying, and preferred to perform self-grooming while on the rubber surface. Similarly, they preferred to lie and to perform self-grooming while positioned near animals in a neighboring pen. These results suggest that provision of a deep-bedded lying area in hospital pens is important to the welfare of lame cows. We found no evidence of isolation-seeking behavior in animals with these diagnoses (and no systemic symptoms) while they were kept in individual hospital pens.

A predictive tool to estimate the risk of axillary metastases in breast cancer patients with negative axillary ultrasound.

BACKGROUND: Sentinel node biopsy (SNB) is the "gold standard" in axillary staging in clinically node-negative breast cancer patients. However, axillary treatment is undergoing a paradigm shift and studies are being conducted on whether SNB may be omitted in low-risk patients. The purpose of this study was to evaluate the risk factors for axillary metastases in breast cancer patients with negative preoperative axillary ultrasound. METHODS: A total of 1,395 consecutive patients with invasive breast cancer and SNB formed the original patient series. A univariate analysis was conducted to assess risk factors for axillary metastases. Binary logistic regression analysis was conducted to form a predictive model based on the risk factors. The predictive model was first validated internally in a patient series of 566 further patients and then externally in a patient series of 2,463 patients from four other centers. All statistical tests were two-sided. RESULTS: A total of 426 of the 1,395 (30.5 %) patients in the original patient series had axillary lymph node metastases. Histological size (P < 0.001), multifocality (P < 0.001), lymphovascular invasion (P < 0.001), and palpability of the primary tumor (P < 0.001) were included in the predictive model. Internal validation of the model produced an area under the receiver operating characteristics curve (AUC) of 0.731 and external validation an AUC of 0.79. CONCLUSIONS: We present a predictive model to assess the patient-specific probability of axillary lymph node metastases in patients with clinically node-negative breast cancer. The model performs well in internal and external validation. The model needs to be validated in each center before application to clinical use.

Dairy cows seek isolation at calving and when ill.

Dairy cows are typically gregarious, but isolate themselves in the hours before calving when kept on pasture. Self-isolation is also a common behavior of ill animals. The objectives of this study were to determine if dairy cows would (1) isolate to calve when housed indoors in an individual maternity pen and (2) continue to isolate when ill after calving. We selected individuals from a pool of 79 multiparous Holstein dairy cows based on inclusion criteria created to address each objective. Cows were moved from a group pen to 1 of 10 adjacent maternity pens. Half of these individual pens were partially covered with plywood, creating a secluded corner as well as a window that provided visual access to the group pen. The other individual pens were uncovered on all sides. For our first objective, we selected 39 cows that were moved into the maternity pens >8h before calving (partially covered: n=19; uncovered: n=20). For our second objective, we selected 18 cows housed in the partially covered pens: 9 cows with high rectal temperature after calving and signs of an infectious disease (mastitis, metritis, pneumonia, or some combination), and 9 healthy cows paired with ill cows based on the amount of time they spent in the maternity pen before calving. Ten-minute scan sampling was used to record the location and lying time from 6h before to 72 h after calving. Individual feed intake was measured after calving. Binomial tests were used to determine if cows in both pen types were more likely to calve in the corner or window side of the pen. Repeated-measures ANOVA were used to determine if cows used the corner more as calving approached and if ill cows spent more time lying or more time in the corner compared with healthy cows in the 72 h after calving. Cows in the uncovered pens were equally likely to calve on both sides of the pen (10 vs. 10), but 79% of cows in the partially covered pens calved on the corner side of the pen (15 vs. 4). Cows in the partially covered pens used the corner side of the pen more in the 1h before and after calving compared with those housed in the uncovered pens. Ill cows housed in the partially covered pens ate less, tended to spend more time lying down, and spent more time in the corner of the pen compared with healthy cows. These results indicate that periparturient dairy cows seek seclusion to calve and when ill, which suggests that adding a secluded area to maternity and hospital pens may be beneficial.