Decreasing Bilirubin Serum Tests in Healthy Newborns During Birth Hospitalization.

OBJECTIVES: Substantial variability exists in hyperbilirubinemia screening and monitoring leading to unnecessary total serum bilirubin (TSB) testing in healthy newborns. We aimed to assess the impact of value-care interventions to decrease the monthly TSB testing rate per 100 patient-days among healthy newborns in our Mother-Baby Unit by 30% by June 2022. METHODS: We formed a multidisciplinary team to review the current practice for ordering TSB among housestaff in our Mother-Baby Unit. We identified several themes: variation in clinical practice, fear of hyperbilirubinemia, and desire to act for high-intermediate risk bilirubin levels. The interventions consisted of obtaining faculty buy-in, redesigning the hyperbilirubinemia pathway, educating staff on high value-care, producing an instructional video, and prompting staff to incorporate a bilirubin risk assessment via smart phrases in our electronic health record. The primary outcome was the monthly TSB testing rate per 100 patient-days. Universal predischarge bilirubin screening, length of stay, phototherapy rates, and readmission rates were chosen as balancing measures. RESULTS: The monthly rate of TSB testing was reduced from 51 to 26.3 TSB per 100 patient-days, representing a 48% reduction. This improvement was sustained for 12 months. The percentage of infants with at least 1 TSB measurement during birth hospitalization decreased from 48% to 30%. Predischarge bilirubin screening, length of stay, and readmission rates were unchanged. CONCLUSIONS: Our quality improvement initiative led to a significant reduction in the monthly TSB testing per 100 patient-days in healthy newborns without evidence of harm.

Preexposure Prophylaxis for HIV Prevention in a Rural Southern Clinic.

OBJECTIVES: Little is known about implementing preexposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) in the rural US South, where most HIV infections occur. To investigate how to better reach the at-risk population in rural eastern North Carolina (ENC), the characteristics and clinical courses of those receiving PrEP from our clinic were reviewed and analyzed. METHODS: A retrospective study was used to describe the characteristics and outcomes of PrEP participants in rural ENC. RESULTS: Thirty-five patients were included whose median age was 37 years (range 22-69). The majority of patients tolerated PrEP well and continued the medication, with only 6 (17%) discontinuations; the reasons for discontinuing included partner mistrust, intolerance to medication, and pregnancy. Patients who initiated PrEP in ENC were more likely to be insured (89%), White (66%), and male (69%). Average travel time to the clinic was 34 minutes (range 2-123 minutes) and the average travel distance was 23 mi (range 1-93 mi). CONCLUSIONS: The study results were noteworthy given that in North Carolina, young Black men have the highest estimated rates of HIV infection. The reasons behind this discrepancy are likely multifactorial, and this study highlights the need for future programs and research to make PrEP more widely accessible to the at-risk population in this rural ENC community.

Molecular identification of Aedes triseriatus and Aedes hendersoni by a novel duplex polymerase chain reaction assay.

Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.

Cluster analysis reveals a binary effect of storage on boar sperm motility function.

Storage of liquid-preserved boar spermatozoa is associated with a loss of fertilising ability of the preserved spermatozoa, which standard semen parameters barely reflect. Monitoring responses to molecular effectors of sperm function (e.g. bicarbonate) has proven to be a more sensitive approach to investigating storage effects. Bicarbonate not only initiates capacitation in spermatozoa, but also induces motility activation. This occurs at ejaculation, but also happens throughout passage through the oviduct. In the present study we tested whether the specific response of boar sperm subpopulations to bicarbonate, as assessed by motility activation, is altered with the duration of storage in vitro. Three ejaculates from each of seven boars were diluted in Beltsville thawing solution and stored at 17°C. Only minor changes in the parameters of diluted semen were revealed over a period of 72h storage. For assessment of bicarbonate responses, subsamples of diluted spermatozoa were centrifuged through a discontinuous Percoll gradient after 12, 24 and 72h storage. Subsequently, spermatozoa were incubated in two Ca2+-free variants of Tyrode's medium either without (TyrControl) or with (TyrBic) 15mM bicarbonate, and computer-aided sperm analysis motility measurements were made. Cluster analysis of imaging data from motile spermatozoa revealed the presence of five major sperm subpopulations with distinct motility characteristics, differing between TyrBic and TyrControl at any given time (P<0.001). Although there was an increasing loss of motility function in both media, bicarbonate induced an increase in a 'fast linear' cohort of spermatozoa in TyrBic regardless of storage (66.4% at 12h and 63.9% at 72h). These results imply a binary pattern in response of sperm motility function descriptors to storage: although the quantitative descriptor (percentage of motile spermatozoa) declines in washed semen samples, the qualitative descriptor (percentage of spermatozoa stimulated into fast linear motion by bicarbonate) is sustained independent of the duration of storage.

Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis.

The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability.

Control of bull sperm cell volume during epididymal maturation.

Mature spermatozoa have a mechanism by which they can reduce cellular swelling caused by hypo-osmotic stress. The development of this ability during epididymal maturation in the bull was investigated. Caput and cauda sperm preparations were exposed to various osmotic stresses at 38 degrees C and measurements of cell volume made by electronic cell sizing. (1) Epididymal spermatozoa recovered and incubated in a medium isotonic with caudal epididymal plasma (360 mOsm kg(-1)) showed better viability and better volume regulatory ability than those incubated in a medium isotonic with seminal plasma (300 mOsm kg(-1)) or in seminal plasma itself. (2) Preparations of both caput and cauda spermatozoa, isolated in a medium isotonic with caudal epididymal plasma, contained two volumetric subpopulations, unrelated to the presence or absence of attached cytoplasmic droplets. (3) The cell volume of both subpopulations of caput spermatozoa was always greater than that of the corresponding cauda spermatozoa subpopulations. (4) After exposure to hypotonic challenge, both caput and cauda spermatozoa were able to reduce their relative volumes, demonstrating that both immature and mature cells are able to express regulatory volume decrease under physiological conditions. (5) When spermatozoa were incubated in chloride- or sodium-free media, although two subpopulations remained present, the volume of the caput sperm populations decreased to that of their counterparts in cauda sperm preparations. It is concluded that immature caput spermatozoa are capable of regulating their volume in a similar fashion to mature cauda spermatozoa but are less able to control their isotonic volume, probably due to poorly controlled sodium and chloride ion transport.

Fibronectin type II-module proteins in the bovine genital tract and their putative role in cell volume control during sperm maturation.

The male reproductive tract of ungulates contains two protein families bearing tandemly arranged fibronectin II (Fn2) modules; one (small Fn2 proteins) bears two modules (e.g. BSP-A1/2), the other (long Fn2 proteins) bears four (e.g. epididymal sperm-binding protein 1 (ELSPBP1)). While it is well known that small Fn2 proteins are present in bull semen, nothing is known about long Fn2 proteins. In the present study, the presence of ELSPBP1 proteins in the bull epididymis and their association with maturing spermatozoa were investigated using a specific antibody against canine ELSPBP1. Analysis of western blots showed ELSPBP1 to be present in the caput, corpus and cauda regions of the epididymis. The protein, which bound phosphorylcholine (PC) strongly, appeared to associate with the spermatozoa during maturation because it was absent from caput spermatozoa but present on cauda spermatozoa. Immunocytochemistry of cauda spermatozoa showed the protein to be bound to the post-acrosomal and midpiece regions. ELSPBP1 could not be detected on freshly ejaculated spermatozoa but was revealed after a capacitating treatment. Our previous studies have shown differences between bovine caput and cauda spermatozoa in terms of their ability to control cell volume. Because of the close homology of BSP-A1/2 PC binding regions with Fn2 regions in ELSPBP1, BSP-A1/2 was used as a model to investigate the effect of a PC-binding Fn2 protein on cell volume control. While the protein had no effect on cauda spermatozoa, it caused caput spermatozoa to swell more in response to hypotonic stress, similarly to untreated cauda spermatozoa.

Bicarbonate-induced membrane processing in sperm capacitation.

During capacitation, major changes take place in the sperm plasma membrane so as to render it fusogenic and responsive to zona pellucida glycoproteins. However, the mechanisms involved have not been defined. As bicarbonate is known to be the key component that induces capacitation, we have investigated the bicarbonate-dependent changes in the boar sperm's plasma membrane architecture. We have discovered that bicarbonate induces a rapid collapse of phospholipid transverse asymmetry, exposing phosphatidylethanolamine and phosphatidylserine at the outer surface of the lipid bilayer. The collapse, which is reversible, is brought about as a result of activation of the phospholipid scramblase that exchanges phospholipids in a non-specific fashion between the two leaflets of the lipid bilayer. The activation takes place via a cyclic AMP-protein kinase A-dependent pathway and is initiated via stimulation of the so-called 'soluble' adenylyl cyclase in the sperm cell by bicarbonate. As a result of the collapse and the concurrent increase in phospholipid exchange, removal of cholesterol by albumin is facilitated (perhaps due to increased lipid packing disorder). This finding is in conflict with earlier surmises that cholesterol loss precedes activation of the cyclic AMP-protein kinase A axis. We have noted that not all cells in a given sperm population show rapid changes in response to bicarbonate stimulation; samples from individual boars also differ in their response. Maturation differences between cells have been found to play an important role in such functional heterogeneity.

Bicarbonate stimulation of boar sperm motility via a protein kinase A-dependent pathway: between-cell and between-ejaculate differences are not due to deficiencies in protein kinase A activation.

Because poorly motile sperm samples can often be stimulated by treatments that increase intracellular levels of cyclic adenosine monophosphate (cAMP), it has been supposed that such samples are unable to maintain an adequate supply of the cyclic nucleotide with which to activate protein kinase A (PKA). To investigate this hypothesis, we incubated boar sperm samples with bicarbonate (a stimulator of adenylyl cyclase) and compared its effect with that of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (cBIMPS, a highly permeable and stable cAMP analog). Videomicroscopy assessment of motility was followed by computer analysis of the sperm tracks to produce motility descriptor values for many individual cells in each sample, whence "cluster" analysis of these data identified groups of spermatozoa that differed in motility characteristics. Bicarbonate stimulation of motility was characterized by an increase in the linearity (LIN) and progressive velocity of part of the sperm population only. The size of this "fast linear" subpopulation varied considerably between ejaculates. However, treatment with cBIMPS did not induce significantly more "fast linear" sperm than treatment with bicarbonate. In further experiments investigating the role of protein kinases in motility control, bicarbonate stimulation was greatly inhibited by H89 (a specific inhibitor of PKA), whereas GF109203X and lavendustin A (inhibitors of protein kinase C [PKC] and protein tyrosine kinase [PTK], respectively) had essentially no effect. While inclusion of the protein phosphatase inhibitor calyculin stimulated motility, it failed to increase the overall percentage of "fast linear sperm" induced by bicarbonate. We conclude that intersperm and interejaculate differences in boar sperm motility are not due to inadequacy in cAMP supply or to ineffective PKA activity.