The TP73 Gene Polymorphism (rs4648551, A>G) Is Associated with Diminished Ovarian Reserve

It’s known that the members of the TP53 family are involved in the regulation of female reproduction. Studies in mice showed that the TP73 gene (member of this family) plays a role in the size of follicular pool, ovulation rate and maintenance of genomic stability. In the present study we analyzed data from 605 patients with ≤ 37 years attending their first intracytoplasmic sperm injection (ICSI). The association between the TP73 polymorphism (rs4648551, A>G) and the following parameters related to ovarian reserve, like age, antral follicular count (AFC), anti-Mullerian hormone levels (AMH) and ovarian response prediction index (ORPI) was evaluated. Our results showed an association of the AA genotype with diminished ovarian reserve (AMH <1, AFC ≤9). Women presenting the AA genotype had a 2.0-fold increased risk for having AMH <1 and AFC ≤9 (OR 2.0, 95% CI 1.23-3.31, P = 0.005). Patients presenting AA genotype had the lowest levels of AMH (P = 0.02), the lowest number of antral follicles (P = 0.01) and the lowest ORPI (P = 0.007). Analyzing the alleles, we can see an enrichment of the A allele in the group of diminished ovarian reserve (OR 1.4, 95%CI 1.02-1.83, P = 0.04). To the best of our knowledge, the present study is the first to analyze this polymorphism in humans for assessing the numbers of ovarian follicles and AMH levels and, therefore, the ovarian reserve. Our findings can contribute to the use of this polymorphism as a potential marker of diminished ovarian reserve.     

Inferences of body energy reserves on conception rate of suckled Zebu beef cows subjected to timed artificial insemination followed by natural mating

The influence of body condition score (BCS), rump fat thickness (RFAT), and live weight (LW), and the changes in these parameters during the interval from 165 of prepartum (i.e., 125 days of prior gestation) to 112 postpartum on first service conception and pregnancy rates were investigated in suckled Zebu (Bos indicus) beef cows (n = 266) subjected to timed artificial insemination (TAI) followed by natural mating. The aforementioned parameters were recorded at 165 ± 14 days (mean ± standard error) prepartum (concurrent with the weaning of previous calf), at parturition, and at 42 ± 7 days (at the onset of the synchronization of ovulation protocol), 82 ± 7 days (30 days after TAI), and 112 ± 7 days (60 days after TAI) postpartum. At the start of the breeding season (BS), cows were subjected to a synchronization of ovulation program for TAI. Bulls were placed with cows 10 days after TAI and remained until the end of the study (112 days postpartum). Cows with the highest BCS at parturition had an increased probability of first service conception rate at 60 days after TAI (P = 0.02) and a reduced probability of occurrence of pregnancy loss (P = 0.05). Also, cows had a greater likelihood of conceiving postpartum if they had greater RFAT and BCS at 165 ± 14 days prepartum (P = 0.01 and P = 0.03, respectively) and at parturition (P = 0.0007 and P = 0.003, respectively). Cows that had an increase in RFAT and BCS during the dry period (i.e., interval from weaning of the previous calf to parturition) also had a greater likelihood of conceiving (P = 0.03 and P = 0.06, respectively) during the BS. Among the different time points, RFAT and BCS at parturition had the largest impact on risk of conception during the BS. The LW was a poor predictor of conception during the BS (P = 0.11–0.68) except for LW at 165 ± 14 days prepartum (P = 0.01). Collectively, the findings indicated that the likelihood of conception during the BS was highest in cows that had an improvement in RFAT and BCS during the dry period. Therefore, assuring a good nutritional status in the dry period (BCS ≥ 3.0 at 165 ± 14 days prepartum and ≥3.25 at parturition) is an important aim to optimize the postpartum conception rate of suckled Zebu beef cows subjected to TAI followed by natural mating.     

Seven-Day Mortality Can Be Predicted in Medical Patients by Blood Pressure,Age, Respiratory Rate,Loss of Independence,and Peripheral Oxygen Saturation (the PARIS Score): A Prospective Cohort Study with External Validation

Background

Most existing risk stratification systems predicting mortality in emergency departments or admission units are complex in clinical use or have not been validated to a level where use is considered appropriate. We aimed to develop and validate a simple system that predicts seven-day mortality of acutely admitted medical patients using routinely collected variables obtained within the first minutes after arrival.

Methods and Findings

This observational prospective cohort study used three independent cohorts at the medical admission units at a regional teaching hospital and a tertiary university hospital and included all adult (≥15 years) patients. Multivariable logistic regression analysis was used to identify the clinical variables that best predicted the endpoint. From this, we developed a simplified model that can be calculated without specialized tools or loss of predictive ability. The outcome was defined as seven-day all-cause mortality. 76 patients (2.5%) met the endpoint in the development cohort, 57 (2.0%) in the first validation cohort, and 111 (4.3%) in the second. Systolic blood Pressure, Age, Respiratory rate, loss of Independence, and peripheral oxygen Saturation were associated with the endpoint (full model). Based on this, we developed a simple score (range 0–5), ie, the PARIS score, by dichotomizing the variables. The ability to identify patients at increased risk (discriminatory power and calibration) was excellent for all three cohorts using both models. For patients with a PARIS score ≥3, sensitivity was 62.5–74.0%, specificity 85.9–91.1%, positive predictive value 11.2–17.5%, and negative predictive value 98.3–99.3%. Patients with a score ≤1 had a low mortality (≤1%); with 2, intermediate mortality (2–5%); and ≥3, high mortality (≥10%).

Conclusions

Seven-day mortality can be predicted upon admission with high sensitivity and specificity and excellent negative predictive values.     

Suitability of antral follicle counts and computer-assisted analysis of ultrasonographic and magnetic resonance images for estimating follicular reserve in porcine,ovine and bovine ovaries ex situ

This study was conducted to determine if correlations exist between the numbers of microscopic follicles comprising ovarian follicular reserve (OFR) and antral follicle counts (AFCs), and to assess the usefulness of computerized analyses of ovarian ultrasonograms and magnetic resonance (MR) images for estimating OFR in excised porcine, ovine and bovine ovaries. As a pre-requisite to these analyses, we characterized and compared ovarian cortical histomorhpology and follicle populations in the three species varying in prolificacy and overall reproductive longevity, and hence the total number of microscopic and antral follicles. Ultrasonographic and MR images were obtained at the scanner settings optimized to provide opposing contrasts between antral follicles and the ovarian stroma. Commercially available ImageProPlus® analytical software was used to calculate numerical pixel values (NPVs) and pixel heterogeneity (standard deviation of the pixel values) along the computer-generated lines (4–6) placed in the area corresponding to the ovarian cortex. The numbers of primordial (r = 0.38, P < 0.01) and intermediate follicles (r = 0.37, P < 0.01) were correlated with the numbers of antral follicles in bovine ovarian sections. The numbers of primordial (r = 0.28, P < 0.05), intermediate (r = 0.31, P < 0.01) and primary follicles (r = 0.27, P < 0.05) correlated directly with mean NPVs of the ultrasonographic ovarian images in cattle. There was a negative correlation between primary follicle numbers and NPVs of MR images (3D FAST-SPOILED GRADIENT ECHO) of the porcine ovarian cortex (r = −0.31, P < 0.05). To summarize, the numbers of primordial and intermediate follicles could only be estimated from AFCs in cows. Using ultrasound NPVs, the numbers of primordial, intermediate and primary follicles could be directly estimated in bovine ovaries and the quantitative image attributes of MR images were useful for quantifying porcine primary follicles. The bovine ovarian model is compatible with human situation and hence future studies should be undertaken to ascertain the usefulness of AFCs and ultrasonographic image analyses for estimating OFR in women.     

Fixed-time AI protocols replacing eCG with a single dose of FSH were less effective in stimulating follicular growth, ovulation, and fertility in suckled-anestrus Nelore beef cows

The aim of the present study was to evaluate the effects of a single treatment with FSH on diameter of the largest follicle and on conception rates of suckled Bos indicus beef cows submitted to timed artificial insemination (TAI). Four hundred fifty-six suckled anestrous Nelore beef cows at 30-60 days postpartum were assigned to treatments. At the first day of the estrous synchronization protocol (Day 0), all cows received a progesterone-releasing intravaginal device plus 2mg of estradiol benzoate. On Day 8, cows were assigned to blocks according to the diameter of the largest follicle and then allocated to one of three treatment groups (Control, FSH, or eCG) within each block. Simultaneously to progesterone device withdrawal on Day 8, cows in the eCG treatment group (n=150) received 300 IU of eCG and cows in FSH treatment group (n=153) received 10mg of FSH, and Control cows (n=153) did not receive any additional treatment. Additional treatments with 150 μg of cloprostenol and 1mg of estradiol cypionate (EC) were also administered concurrently to progesterone device removal in all cows on Day 8. Two days later (D10), TAI and ovarian ultrasonic examinations to evaluate follicle size were performed in all cows. On Day 12, a subset of cows (n=389) were submitted a second ultrasonic exam to confirm ovulation. Final follicular growth (mm/day) was less (P=0.006) in both Control (0.95±0.11) and in FSH-treated cows (0.90±0.10) than in eCG-treated cows (1.40±0.13). Interestingly, there was a treatment-by-BCS interaction in ovulation results (P=0.03), in which, eCG treatment increased percentage of cows having ovulations with a lesser BCS. Similarly, there was a treatment-by-BCS interaction for conception (P=0.04), where the eCG treatment increased fertility in cows with a lesser BCS. In conclusion, FSH failed to stimulate final follicular growth, ovulation, and conception rate in sucked-anestrous beef cows submitted to TAI as effectively as eCG. However, physiological effects of eCG seem to be more evident in cows with a lesser BCS.     

Improvement of Oligonucleotide Probe Design Criteria for Functional Gene Microarrays in Environmental Applications

To optimize oligonucleotide probe design criteria, PCR products with different similarities to probes were hybridized to a functional gene microarray designed to detect homologous genes from different organisms. In contrast to more restrictive probe designs based on a single criterion, simultaneous consideration of the percent similarity (≤90%), the length of identical sequence stretches (≤20 bases), and the binding free energy (≥−35 kcal mol⁻¹) was found to be predictive of probe specificity.     

A Predictive Model for Selecting Patients with HCV Genotype 3 Chronic Infection with a High Probability of Sustained Virological Response to Peginterferon Alfa-2a/Ribavirin

Background

Access to direct-acting antiviral agents (DAAs) is restricted in some settings; thus, the European Association for the Study of the Liver recommends dual peginterferon/ribavirin (PegIFN/RBV) therapy wherever DAAs are unavailable. HCV genotype (GT) 3 infection is now the most difficult genotype to eradicate and PegIFN/RBV remains an effective option. The goal of this study was to devise a simple predictive score to identify GT3 patients with a high probability of achieving a sustained virologic response (SVR) with PegIFN alfa-2a/RBV therapy.

Methods

Relationships between baseline characteristics and SVR were explored by multiple logistic regression models and used to develop a simple scoring system to predict SVR using data from 1239 treatment-naive GT3 patients who received PegIFN alfa-2a/RBV for 24 weeks in two large observational cohort studies.

Results

The score was validated using a database of 473 patients. Scores were assigned for six factors as follows: age (years) (≤40: 2 points; >40 but ≤55: 1); bodyweight (kg) (<70: 2; ≥70 but <90: 1); no cirrhosis/transition to cirrhosis (2); ALT ≤2.5 x ULN (1); platelets (10⁹/L) (>200: 2; ≥100 but <200: 1); HCV RNA (<400,000 IU/mL: 1). The points are summed to arrive at a score ranging from 0‒10 where higher scores indicate higher chances of SVR; 141, 123, 203, 249, 232, and 218 patients had total scores of 0‒4, 5, 6, 7, 8, and 9–10, respectively, among whom SVR rates were 45%, 62%, 72%, 76%, 84%, and 89%. Among 622 patients who had scores of 6‒10 and HCV RNA <50 IU/mL by treatment week 4 the SVR rate was 86% (532/622).

Conclusions

A simple baseline scoring system involving age, bodyweight, cirrhosis status, ALT level, platelet count and HCV RNA level can be used to identify treatment-naive Caucasian patients with HCV GT3 infection with a high probability of SVR with PegIFN alfa-2a/RBV therapy.     

Strategies to improve fertility in Bos indicus postpubertal heifers and nonlactating cows submitted to fixed-time artificial insemination

Two experiments were designed to evaluate strategies to increase fertility of Bos indicus postpubertal heifers and nonlactating cows submitted to a fixed-time artificial insemination (TAI) protocol consisting of an intravaginal device containing 1.9 g of progesterone (CIDR) insertion + estradiol benzoate on Day 0, CIDR withdrawal + estradiol cypionate on Day 9, and TAI on Day 11. In Experiment 1, heifers (n = 1153) received a new or an 18-d previously used CIDR and, on Day 9, prostaglandin F_2α (PGF_2α) + 0, 200, or 300 IU equine chorionic gonadotropin (eCG). Heifers treated with a new CIDR had greater (least squares means ± SEM) serum concentration of progesterone on Day 9 (3.06 ± 0.09 ng/mL vs. 2.53 ± 0.09 ng/mL; P < 0.05) and a smaller follicle at TAI (11.61 ± 0.11 mm vs. 12.05 ± 0.12 mm; P < 0.05). Heifers with smaller follicles at TAI had lesser serum progesterone concentrations on Day 18 and reduced rates of ovulation, conception, and pregnancy (P < 0.05). Treatment with eCG improved (P < 0.05) follicle diameter at TAI (11.50 ± 0.10 mm, 11.90 ± 0.11 mm, and 12.00 ± 0.10 mm for 0, 100, and 200 IU, respectively), serum progesterone concentration on Day 18 (2.77 ± 0.11 ng/mL, 3.81 ± 0.11 ng/mL, and 4.87 ± 0.11 ng/mL), and rates of ovulation (83.8%, 88.5%, and 94.3%) and pregnancy (41.3%, 47.0%, and 46.7%). In Experiment 2, nonlactating Nelore cows (n = 702) received PGF_2α treatment on Days 7 or 9 and, on Day 9, 0 or 300 IU eCG. Cows receiving PGF_2α on Day 7 had lesser serum progesterone concentrations on Day 9 (3.05 ± 0.21 ng/mL vs. 4.58 ± 0.21 ng/mL; P < 0.05), a larger follicle at TAI (11.54 ± 0.21 mm vs. 10.84 ± 0.21 mm; P < 0.05), and improved (P < 0.05) rates of ovulation (85.4% vs. 77.0%), conception (60.9% vs. 47.2%), and pregnancy (52.0% vs. 36.4%). Treatment with eCG improved (P < 0.05) serum progesterone concentration on Day 18 (3.24 ± 0.14 ng/mL vs. 4.55 ± 0.14 ng/mL) and the rates of ovulation (72.4% vs. 90.0%) and pregnancy (37.5% vs. 50.8%). In conclusion, giving PGF_2α earlier in the protocol in nonlactating cows and eCG treatment in postpubertal heifers and nonlactating cows improved fertility in response to a TAI (progesterone + estradiol) protocol.     

Evaluation of vaginal electrical resistance as an indicator of follicular maturity and suitability for timed artificial insemination in beef cows subjected to a synchronization of ovulation protocol

Objectives were to test the hypothesis that vaginal electrical resistance (VER) could be used to identify cows without a large (<10mm) follicle at timed-AI (TAI) following a synchronization of ovulation protocol and thus serve as a prospective decision aid for determining cows that should not be inseminated. Brahman x Hereford (F1) females (n=233) were synchronized with the CO-Synch+CIDR protocol that consisted of a controlled internal drug release (CIDR) insert and i.m. injection of GnRH (GnRH-1; 100 microg) on day 0, removal of CIDR and i.m. injection of prostaglandin F2alpha (PGF; 25mg) on day 7, and i.m. injection of GnRH (GnRH-2, 100 microg) and TAI 66h after CIDR removal (day 10). Vaginal electrical resistance was determined with a commercially available device (Ovascan; Animark Inc., Aurora, CO) at days 0, 7, and 10. Transrectal ultrasonography was used on day 10 to assess ovarian morphology at TAI in all cattle and in a subset of females (n=98) on days 0 and 7. Mean (+/-S.E.M.) age, body condition score (BCS), BW and days postpartum were 7.2+/-0.3 years, 5.2+/-0.1, 538+/-5.3kg, and 77+/-1.1 days, respectively. Mean VER (Omega) was greatest (101.4+/-0.8) on day 0 and declined (P<0.01) to 95.2+/-0.8 and 82.0+/-0.8 Omega, respectively, on days 7 and 10. Mean diameter of the largest follicle and VER values in females conceiving after TAI differed (P=0.05) from those that did not conceive. Mean VER on days 7 and 10 and VER difference (VER on day 10 minus VER on day 7) did not differ between females with a small (<10mm) or large (>or=10mm) follicle at TAI. Timed-AI pregnancy rate was greater (P<0.01) for females with large follicles (43%) than those with small follicles (22%). Vaginal electrical resistance difference values, categorized as negative (<0 Omega) or neutral/positive (>or=0 Omega), did not differ between females that conceived to TAI compared with those that did not. We conclude that VER measurements, as used in the present study, are not adequately sensitive to differentiate between females with and without a large follicle and thus are unable to serve as a prospective decision aid for determining suitability for TAI after synchronization.     

Pre-Conception Dyslipidemia Is Associated with Development of Preeclampsia and Gestational Diabetes Mellitus

IntroductionThe association between glucose intolerance, elevated blood pressure and abnormal lipid levels is well established and comprises the basis of metabolic syndrome pathophysiology. We hypothesize that abnormal preconception lipid levels are associated with the increased risk of severe pregnancy complications such as preeclampsia and gestational diabetes mellitus.MethodsWe included all singleton deliveries (n = 27,721) of women without known cardiovascular morbidity and preeclampsia and gestational diabetes mellitus during previous pregnancies. Association between preconception low high density lipoprotein cholesterol (HDLc level≤50 mg/dL), high triglycerides (level≥150 mg/dL) and the primary outcome (composite of gestational diabetes mellitus/or preeclampsia) was assessed using Generalized Estimation Equations.ResultsPrimary outcome of preeclampsia and/or gestational diabetes was observed in a total of 3,243 subjects (11.7%). Elevated triglycerides and low HDLc were independently associated with the primary outcome: with odds ratio (OR) of 1.61 (95% CI 1.29–2.01) and OR = 1.33 (95% CI 1.09–1.63), respectively, after adjusting for maternal age, weight, blood pressure, repeated abortions, fertility treatments and fasting glucose. There was an interaction between the effects of HDLc≤50 mg/dL and triglycerides≥150 mg/dL with an OR of 2.69 (95% CI 1.73–4.19).ConclusionsOur analysis showed an increased rate of preeclampsia and/or gestational diabetes in women with low HDLc and high triglycerides values prior to conception. In view of the severity of these pregnancy complications, we believe this finding warrants a routine screening for the abnormal lipid profile among women of a child-bearing age.     

Waist Circumference,Not Body Mass Index,Is Associated with Renal Function Decline in Korean Population: Hallym Aging Study

Background

Prospective investigation of obesity and renal function decline in Asia is sparse. We examined the associations of body mass index (BMI) and waist circumference (WC) with renal function decline in a prospective study of Korean population.

Methods

A total of 454 participants who had baseline estimated glomerular filtration rate (eGFR) levels of more than 60 mL/min/1.73 m² in Hallym Aging Study (HAS) were included and followed for 6 years. Renal function decline was defined as follows: (1) an eGFR decline ≥3 mL/min/1.73 m²/year (n = 82 cases); (2) an eGFR decrease of 20% or greater (n = 87 cases) at follow-up; (3) an eGFR decrease of 20% greater at follow-up or eGFR decline ≥3 mL/min/1.73 m²/year (n = 91 cases); and (4) an eGFR <60 mL/min/1.73 m² at follow-up (n = 54 cases). eGFR was determined based on the Modification of Diet in Renal Disease (MDRD) Study equation. Multivariate logistic regression model was used to determine the association between obesity and renal function decline.

Results

We found that central obesity was associated with faster renal function decline. Comparing WC of >95 cm in men or >90 cm in women with ≤90 cm in men or ≤85 cm in women, ORs (95% CIs) ranged from 2.31 (1.14–4.69) to 2.78 (1.19–6.50) for the 4 definitions of renal function decline (all p-values for trend <0.05). Waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) also was associated with renal function decline. There was no significant association of BMI with renal function decline.

Conclusions

Central obesity, but not BMI, is associated with faster renal function decline in Korean population. Our results provide important evidence that simple measurement of central fat deposition rather than BMI could predict decline in renal function in Korean population.     

Genetic markers of ovarian follicle number and menopause in women of multiple ethnicities

Oocyte loss has a significant impact on fertility and somatic health. Yet, we know little about factors that impact this process. We sought to identify genetic variants associated with ovarian reserve (oocyte number as measured by antral follicle count, AFC). Based on recently published genome-wide scans that identified loci associated with age of menopause, we also sought to test our hypothesis that follicle number and menopausal age share underlying genetic associations. We analyzed menopause-related variants for association with follicle number in an independent population of approximately 450 reproductive-aged women of European and African ancestry; these women were assessed for AFC, anthropometric, clinical, and lifestyle factors. One SNP strongly associated with later menopausal age in Caucasian women (+1.07?±?0.11?years) in previous work was also associated with higher follicle counts in Caucasians (+2.79?±?1.67 follicles) in our study. This variant is within the Minichromosome Maintenance Complex Component 8 (MCM8) gene, which we found was expressed within oocytes in follicles of the human ovary. In genome-wide scans of AFC, we also identified one marginally genome-wide and several nominally significant SNPs within several other genes associated with follicle number in both ethnic groups. Further, there were overlapping variants associated with multiple ovarian reserve markers (AFC, serum hormone levels, menopausal age). This study provides the first evidence for direct genetic associations underlying both follicle number and menopause and identifies novel candidate genes. Genetic variants associated with ovarian reserve may facilitate discovery of genetic markers to predict reproductive health and lifespan in women.     

Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle

Methods to predict numbers of healthy oocytes in the ovaries of young adults could have important diagnostic relevance in family planning and animal agriculture. We have observed that peak antral follicle count (AFC) determined by serial ovarian ultrasonography during follicular waves is very highly reproducible within individual young adult cattle, despite 7-fold variation among animals. Herein, we tested the hypothesis that AFC is positively associated with the number of morphologically healthy oocytes and follicles in ovaries and with serum concentrations of anti-Müllerian hormone (AMH), an indirect marker for number of healthy follicles and oocytes in ovaries. In the present study, age-matched young adult cattle (12-18 mo old) were subjected to serial ultrasonography to identify animals with a consistently high (> or =25 follicles that were > or =3 mm in diameter) or low (< or =15 follicles) AFC during follicular waves. Differences in serum AMH concentrations, ovary weight, and number of morphologically healthy and atretic follicles and oocytes were determined. The phenotypic classifications of cattle based on AFC during follicular waves or AMH concentrations both predict reliably the relative number of morphologically healthy follicles and oocytes in ovaries of age-matched young adult cattle.     

Fertility-associated antigen on Nelore bull sperm and reproductive outcomes following first-service fixed-time AI of Nelore cows and heifers

The objective was to determine whether the presence of fertility-associated antigen (FAA) on sperm collected from Nelore (Bos indicus) bulls can be used to assess potential fertility of sperm for use at first-service fixed-time AI (TAI). Six Nelore bulls were selected based on FAA status (FAA-negative: N = 3; FAA-positive: N = 3) and the ability to produce neat semen with ≥ 70% morphologically normal sperm and 60% estimated progressive motility before cryopreservation. In Experiment 1, suckled multiparous Nelore cows (N = 835) were evaluated for body condition score (BCS) and received an intravaginal progesterone device (CIDR) and 2.0 mg of estradiol benzoate (Day 0). On Day 9 the CIDR was removed, 12.5 mg of PGF_2α and 0.5 mg of estradiol cypionate were administered, and calves were removed for 48 h. All cows received TAI on Day 11 (48 h after CIDR removal). Pregnancy per TAI (P/TAI) was not different between FAA-positive and FAA-negative bulls (41.5% vs. 39.3%, respectively). There was an effect of AI technician on P/TAI (36.0% vs. 43.9%; P < 0.05) and BCS tended to affect P/TAI (P = 0.09), as cows with BCS ≥ 2.75 were 1.4 times more likely to become pregnant compared with cows with BCS < 2.75. In Experiment 2, nulliparous Nelore heifers (N = 617) were evaluated for BCS and received a CIDR and estradiol benzoate (2.0 mg) on Day 0. On Day 7, all heifers received PGF_2α (12.5 mg). On Day 9, CIDR inserts were removed and all heifers received estradiol cypionate (0.6 mg) and 200 IU eCG. All heifers received TAI on Day 11 (48 h after CIDR removal). Pregnancy/TAI was different (P = 0.04) between FAA-positive and FAA-negative bulls (33.7% vs. 40.7%, respectively). Presence of FAA on sperm was unsuccessful in assessing the potential fertility of sperm for use in TAI.     

Effects of excess metabolizable protein on ovarian function and circulating amino acids of beef cows: 1. Excessive supply from corn gluten meal or soybean meal

In the dairy industry, excess dietary CP is consistently correlated with decreased conception rates. However, the source from which excess CP is derived and how it affects reproductive function in beef cattle is largely undefined. The objective of this experiment was to determine the effects of feeding excess metabolizable protein (MP) from feedstuffs differing in rumen degradability on ovulatory follicular dynamics, subsequent corpus luteum (CL) development, steroid hormone production and circulating amino acids (AA) in beef cows. Non-pregnant, non-lactating mature beef cows (n=18) were assigned to 1 of 2 isonitrogenous diets (150% of MP requirements) designed to maintain similar BW and body condition score (BCS) between treatments. Diets consisted of ad libitum corn stalks supplemented with corn gluten meal (moderate rumen undegradable protein (RUP); CGM) or soybean meal (low RUP; SBM). After a 20-day supplement adaptation period, cows were synchronized for ovulation. After 10 days of synchronization, gonadotropin releasing hormone (GnRH) was administered to reset ovarian follicular growth. Starting at GnRH administration and daily thereafter until spontaneous ovulation, transrectal ultrasonography was used to diagram ovarian follicular growth, and blood samples were collected for hormone, metabolite and AA analyses. After 7 days of visual detection of estrus, CL size was determined via ultrasound. Data were analyzed using the MIXED procedures of SAS. As designed, cow BW and BCS were not different (P⩾0.33). Ovulatory follicular wavelength, antral follicle count, ovulatory follicle size at dominance and duration of dominance were not different (P>0.13) between treatments. Cows supplemented with CGM had greater post-dominance ovulatory follicle growth, larger dominant follicles at spontaneous luteolysis, shorter proestrus, and larger ovulatory follicles (P⩽0.03) than SBM cows. No differences (P⩾0.44) in peak estradiol, ratio of estradiol to ovulatory follicle volume, or plasma urea nitrogen were observed. While CL volume and the ratio of progesterone to CL volume were not affected by treatment (P⩾0.24), CGM treated cows tended to have decreased (P=0.07) circulating progesterone 7 days post-estrus compared with SBM cows. Although total circulating plasma AA concentration did not differ (P=0.70) between treatments, CGM cows had greater phenylalanine (P=0.03) and tended to have greater leucine concentrations (P=0.07) than SBM cows. In summary, these data illustrate that excess MP when supplemented to cows consuming a low quality forage may differentially impact ovarian function depending on ruminal degradability of the protein source.     

A Flow Cytometry Method for Rapid Detection and Enumeration of Total Bacteria in Milk

Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (≤1-h) FCM assay based on enzymatic clearing of milk to determine total bacteria in milk. When bacteria were added to ultra-heat-treated milk, a good correlation (r ≥ 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (P < 0.01) and a good agreement (r = 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was ≤10⁴ bacteria ml of milk⁻¹. This limit is below the level of detection required to satisfy legislation in many countries and states.     

High Risk of Under-Grading and -Staging in Prostate Cancer Patients Eligible for Active Surveillance

BackgroundActive surveillance (AS) is increasingly offered to patients with low risk prostate cancer. The present study was conducted to evaluate the risk of tumor under-grading and -staging for AS eligibility. Moreover, we analyzed possible biomarkers for predicting more unfavorable final tumor histology.Methods197 patients who underwent radical prostatectomy (RPE) but would have met the EAU (European Association of Urology) criteria for AS (PSA<10 ng/ml, biopsy GS ≤6, ≤2 cancer-positive biopsy cores with ≤50% of tumor in any core and clinical stage ≤T2a) were included in the study. These AS inclusion parameters were correlated to the final histology of the RPE specimens. The impact of preoperative PSA level (low PSA ≤4 ng/ml vs. intermediate PSA of >4–10 ng/ml), PSA density (<15 vs. ≥ 15 ng/ml) and the number of positive biopsy cores (1 vs. 2 positive cores) on predicting upgrading and final adverse histology of the RPE specimens was analyzed in uni- and multivariate analyses. Moreover, clinical courses of undergraded patients were assessed.ResultsIn our patient cohort 41.1% were found under-graded in the biopsy (final histology 40.1% GS7, 1% GS8). Preoperative PSA levels, PSA density or the number of positive cores were not predictive for worse final pathological findings including GS >6, extraprostatic extension and positive resection margin (R1) or correlated significantly with up-grading and/or extraprostatic extension in a multivariate model. Only R1 resections were predictable by combining intermediate PSA levels with two positive biopsy cores (p = 0.004). Sub-analyses showed that the number of biopsy cores (10 vs. 15 biopsy cores) had no influence on above mentioned results on predicting biopsy undergrading. Clinical courses of patients showed that 19.9% of patients had a biochemical relapse after RPE, among all of them were undergraded in the initial biopsy.ConclusionIn summary, this study shows that a multitude of patients fulfilling the criteria for AS are under-diagnosed. The use of preoperative PSA levels, PSA density and the number of positive cores were not predictable for undergrading in the present patient collective.     

Seasonal fluctuations in the response of Italian Mediterranean buffaloes to synchronization of ovulation and timed artificial insemination

A comprehensive study of the efficiency of synchronization of ovulation and timed artificial insemination (TAI) was undertaken in a large group of Italian Mediterranean buffaloes at a commercial dairy. A total of 2791 synchronization protocols were carried out on 857 animals over 3 years. Of these protocols, 823 (29.5%) did not proceed beyond Day 7 (due to the absence of a vascularized CL) and 620 (22.2%) were discontinued on Day 10 (due to the absence of follicles >1.0 cm and tonic uteri); hence, 1443 (51.7%) protocols did not progress to TAI. Data were analyzed for four periods: P1, transition to spring (from breeding season to low breeding season); P2, low breeding season; P3, transition to fall (low breeding season to breeding season); and P4, breeding season. No differences were found among the four periods in terms of the proportion of protocols that did not result in TAI. Of the 857 buffaloes, 660 (77%) conceived and delivered a calf. The average number of TAI per pregnancy was 2.1 and ranged from 1.9 to 2.3 across years. Logistic regression analysis showed that buffaloes that calved during P3 had a higher odds ratio for pregnancy (1.380; P < 0.05) than buffaloes that calved in other periods. Pregnancy was also influenced by the calving to service period (odds ratio = 0.977; P < 0.01) and the pregnancy per AI (P/AI) at successive TAI (odds ratio = 1.480; P < 0.01). The pregnancy per AI at the first TAI (424/857, 49.5%) was greater (P < 0.01) than in subsequent TAI. The occurrence of late embryonic mortality (between Days 27 and 45 after TAI) was similar among the four periods. These findings indicated that there are distinct seasonal differences in the response of Italian Mediterranean buffaloes to synchronization and TAI.     

Evaluation of 18F-PSMA-1007 PET/CT in prostate cancer patients with biochemical recurrence after radical prostatectomy

PurposeProstate-specific membrane antigen (PSMA) ligands targeting has shown promising results in staging of prostate cancer (PCa). The aim of present study was to evaluate the value of ¹⁸F-PSMA-1007 PET/CT in PCa patients with biochemical recurrence.Methods71 patients with PCa after radical prostatectomy (RP) were included in the present study. Median prostate-specific antigen (PSA) level was 1.27 ng/mL (range 0.01–67.40 ng/mL, n = 69). All patients underwent whole-body PET/CT imaging after injection of 333±38 MBq ¹⁸F-PSMA-1007. The distribution of PSMA-positive lesions was assessed. The influence of PSA level, androgen deprivation therapy and primary Gleason score on PSMA-positive finding and uptake of ¹⁸F-PSMA-1007 were evaluated.Results56 (79%) patients showed at least one pathological finding on ¹⁸F-PSMA-1007 PET/CT. The rates of positive scans were 50%, 80%, 100%, 100% among patients with PSA levels ≤0.5, 0.51–1.0, 1.1–2.0 and >2.0 ng/mL, respectively. The median Gleason score was 8 (range 7–10), and higher Gleason score (≤7 vs. ≥8) leads to higher detection rates (58.3% (14/24) vs. 88.9% (32/36), P = 0.006). The median SUVmax of positive findings in patients with PSA levels ≤0.5, 0.51–1.0, 1.1–2.0 and >2.0 ng/mL were 4.51, 4.27, 11.50 and 14.08, respectively. The median SUVmax in patients with PSA level >2.0 ng/mL was significantly higher than that in patients with PSA ≤2.0 ng/mL (14.08 vs. 6.13, P<0.001).Conclusion¹⁸F-PSMA-1007 PET/CT demonstrated a high detection rate for patients with a raised PSA level after radical prostatectomy even in patients with extremely low PSA level (eg. PSA level ≤0.5 ng/mL), which was essential for further clinical management for PCa patients.     

Timed artificial insemination plus heat II: gonadorelin injection in cows with low estrus expression scores increased pregnancy in progesterone/estradiol-based protocol

The use of tail chalk and estrus/heat expression scores (HEATSC) evaluation is instrumental in identifying cows with greater estrus expression and greater artificial insemination pregnancy rates (P/AI) in cows submitted to timed artificial insemination (TAI), and cows with low or no estrus expression present lower P/AI. It was intended in this study to improve the pregnancy rates in TAI for Bos indicus beef cows, and gonadotrophin-releasing hormone (GnRH) injection was hypothesized to increase pregnancy rates in a TAI program for cows submitted to progesterone–estradiol-based protocols with low or no estrus expression, evaluated by HEATSC. Cows (n= 2284) received a progesterone device and 2 mg estradiol benzoate, after 8 days the device was removed and 1 mg estradiol cypionate, 150 μg of d-cloprostenol and 300 IU equine chorionic gonadotropin was administered. All cows were marked with chalk and HEATSC evaluated (scales 1 to 3) at TAI performed on day 10. Animals with HEATSC1 and HEATSC2 (n= 937) received 100 μg de gonadorelin (GNRH group; n= 470), or 1 ml saline (Control group; n= 467), and cows with HEATSC3 (named HEAT group; n= 1347) received no additional treatment. The larger dominant follicle, evaluated on day 8and at TAI (day 10), was greater in HEAT group (P= 0.0145 and P <0.001, respectively). Corpus luteum (CL) area and progesterone concentration was evaluated on day 17, and CL area was larger in HEAT group, intermediary in Control and lower in GnRH group (Control= 2.68 cm², GnRH= 2.37 cm², HEAT group= 3.07 cm², P <0.001). Greater progesterone concentrations were found in HEAT group than in Control and GnRH groups (Control= 4.74 ng/ml, GnRH= 4.29 ng/ml, HEAT group= 6.08 ng/ml, P<0.001). There was a difference in ovulation rate, greater in HEAT group than GnRH and Control groups (Control= 72.5%; GnRH= 81.25%; HEAT group= 90.71%; P= 0.0024). Artificial insemination pregnancy rates was greater in HEAT group (57.09% (769/1347) than in Control and GNRH groups, with positive effect of GnRH injection at the time of TAI in P/AI (Control= 36.18% (169/467), GnRH= 45.95% (216/470); P<0.0001). In conclusion, GnRH application in cows with low HEATSC (1 and 2) is a simple strategy, requiring no changes in TAI management to increase pregnancy rates in postpartum beef cows submitted to progesterone–estradiol-based TAI protocols, without reaching, however, the pregnancy rates of cows that demonstrate high estrus expression at the TAI.