Facilitated Asynchronous Online Learning to Build Public Health Strategic Skills.

CONTEXT: The COVID-19 pandemic spurred significant government investments for hiring public health workers. There are clear opportunities to help build capacities among both current and incoming public health workers, closing well-elucidated skill gaps. OBJECTIVE: To report on the development process, methods used, and outcomes seen from a point-in-time public health workforce capacity-building initiative, Public Health Essentials (PHE) . DESIGN: Capacity-building outcomes evaluation using pre/postintervention measures. SETTING: The United States. PARTICIPANTS: A total of 512 learners working in roles (government or adjacent to) that support public health. INTERVENTION: PHE, a cohort-based facilitated asynchronous online course comprising 5 units, 18 modules, 54 learning outcomes, and 266 teaching and applied assessment elements designed to build public health strategic skills. MAIN OUTCOME MEASURES: Two outputs and 3 outcomes were used to assess and improve progress in achieving our goal of building generalizable and transferrable public health ability and confidence among diverse public health workers: Use of PHE , PHE completion rate , Learner competence , Change in self-assessed ability , and Benefits of PHE. RESULTS: From September 2021 to December 2022, 4 agencies used PHE for fellowship training or employee capacity building. Some 79% (n = 512) of learners completed the training, demonstrating competence in all 54 areas assessed by expert course facilitators. Of those, 79% (n = 321) completed both optional pre- and post-PHE surveys, reporting statistically significant gains in all strategic skill domains assessed (n = 9), regardless of demographics and public health experience. Learners gained new skills and knowledge (92%), developed a better understanding of public health (86%), and broadened their public health skill base (84%). A majority can apply the knowledge and skills gained directly to their work (94%), which benefits their team (92%), and have increased their confidence as public health practitioners (49%). CONCLUSIONS: PHE can significantly improve learners' ability across 9 strategic skill areas in as few as 15 weeks, regardless of their demographics, training, or experience.

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm.

Acrosome exocytosis (AE), in which the sperm's single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Segmental differentiation of the murine epididymis: identification of segment-specific, GM1-enriched vesicles and regulation by luminal fluid factors†.

The murine epididymis has 10 distinct segments that provide the opportunity to identify compartmentalized cell physiological mechanisms underlying sperm maturation. However, despite the essential role of the epididymis in reproduction, remarkably little is known about segment-specific functions of this organ. Here, we investigate the dramatic segmental localization of the ganglioside GM1, a glycosphingolipid already known to play key roles in sperm capacitation and acrosome exocytosis. Frozen tissue sections of epididymides from adult mice were treated with the binding subunit of cholera toxin conjugated to AlexaFluor 488 to label GM1. We report that GM1-enriched vesicles were found exclusively in principal and clear cells of segment 2. These vesicles were also restricted to the lumen of segment 2 and did not appear to flow with the sperm into segment 3, within the limits of detection by confocal microscopy. Interestingly, this segment-specific presence was altered in several azoospermic mouse models and in wild-type mice after efferent duct ligation. These findings indicate that a lumicrine factor, itself dependent on spermatogenesis, controls this segmental differentiation. The RNA sequencing results confirmed global de-differentiation of the proximal epididymal segments in response to efferent duct ligation. Additionally, GM1 localization on the surface of the sperm head increased as sperm transit through segment 2 and have contact with the GM1-enriched vesicles. This is the first report of segment-specific vesicles and their role in enriching sperm with GM1, a glycosphingolipid known to be critical for sperm function, providing key insights into the segment-specific physiology and function of the epididymis.

Defects in sperm capacitation/fertilizing ability are equally prevalent across ages in men seeking fertility assistance.

RESEARCH QUESTION: How do capacitation ability, measured by Cap-Score™, and traditional semen analysis measures (volume, concentration, motility) change with age in men questioning their fertility (MQF)? DESIGN: Cap-Score and semen analysis measures were obtained from MQF (n = 2652; multicentric design: 35 reproductive endocrinologist prescribers, n = 16 clinics). Morphology was not included due to differences among clinics. A Mann-Whitney test was used to compare Cap-Scores between MQF and men with known recent paternity (n = 76). The following age groups were constructed for MQF: 20-24, 25-29, 30-34, 35-39, 40-44, 45-49 and 50+. Associations between semen analysis, Cap-Score and age groups were evaluated using mixed-model analysis of variance to identify possible influence of Cap-Score collection kit type (n = 763 collected at home; n = 1889 collected at clinics). RESULTS: MQF had reduced capacitation ability (mean ± SE; 29.25 ± 0.15 versus 35.34 ± 0.88; P < 0.001). No change in Cap-Score (P = 0.916) or concentration (P = 0.926) was detected with age group. In contrast, both volume (P = 0.008) and % motility (P < 0.001) declined with age. CONCLUSIONS: Men presenting because of difficulties in generating pregnancy showed equivalent reductions in capacitation ability regardless of age. In contrast, motility and volume declined with age. These data suggest that capacitation ability is a more sensitive indicator of male fertility across age groups than traditional semen analysis and should not be reserved for older men. Importantly, these data do not address whether sperm fertilizing ability declines in the general population as men age. Instead, they indicate that if men are having difficulty conceiving, no matter what their age, then defects in sperm fertilizing ability are equally likely to be the cause.

Case-control study to identify risk factors for SARS-CoV-2 infection among university students in the northeastern USA.

Curbing the coronavirus disease 2019 (COVID-19) pandemic requires a thorough understanding of risk factors for transmission of SARS-CoV-2, the etiologic agent. Institutions of higher education present unique challenges for controlling disease spread because of features inherent to these settings. Our objective was to determine risk factors for SARS-CoV-2 infection among a university student population in the northeastern USA during the spring and fall 2021 semesters, using the case-control study design. Cases were defined as students with a newly diagnosed SARS-CoV-2 infection detected either through the robust PCR-based surveillance testing program on campus or through healthcare testing if symptoms compatible with COVID-19 were present. Controls were defined as students with negative SARS-CoV-2 status, based on consistently negative PCR results at the time of selection. A comprehensive questionnaire was administered to each student enrolled in the study, covering a broad range of campus life activities. A total of 446 cases and 1,185 controls were included in this study. Multivariable logistic regression analysis showed that recent party attendance (adjusted OR = 2.3, p < .0001), recently visiting a bar (aOR = 1.6, p = .007), living in a campus residence hall (aOR = 1.6, p = .001), fraternity/sorority membership (aOR = 1.8, p = .002), and recent travel (aOR = 1.3, p = .04) were associated with being a COVID-19 case. Having an on-campus job was negatively associated with being a COVID-19 case (aOR = 0.6, p = .0003). Among cases, the most commonly reported symptoms were cough (43.9%), fatigue (38.1%) and sore throat (30.3%). These findings can be used to inform the development of COVID-19 mitigation strategies and public health outreach efforts in university settings, thus reducing SARS-CoV-2 transmission among students and helping to preserve the vital education and research missions of these institutions.

Integrated Surveillance System for Controlling COVID-19 on a University Campus, 2020‒2021.

To minimize the impacts of COVID-19 and to keep campus open, Cornell University's Ithaca, NY, campus implemented a comprehensive process to monitor COVID-19 spread, support prevention practices, and assess early warning indicators linked to knowledge, behaviors, and attitudes of campus community members. The integrated surveillance approach informed leadership and allowed for prompt adjustments to university policies and practices through evidence-based decisions. This approach enhanced healthy behaviors and promoted the well-being and safety of all community members. (Am J Public Health. 2022;112(7):980-984. https://doi.org/10.2105/AJPH.2022.306838).

A genetically targeted sensor reveals spatial and temporal dynamics of acrosomal calcium and sperm acrosome exocytosis.

Secretion of the acrosome, a single vesicle located rostrally in the head of a mammalian sperm, through a process known as "acrosome exocytosis" (AE), is essential for fertilization. However, the mechanisms leading to and regulating this complex process are controversial. In particular, poor understanding of Ca2+ dynamics between sperm subcellular compartments and regulation of membrane fusion mechanisms have led to competing models of AE. Here, we developed a transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE) to investigate the spatial and temporal Ca2+ dynamics in AE in live sperm. AcroSensE combines a genetically encoded Ca2+ indicator (GCaMP) fused with an mCherry indicator to spatiotemporally resolve acrosomal Ca2+ rise (ACR) and membrane fusion events, enabling real-time study of AE. We found that ACR is dependent on extracellular Ca2+ and that ACR precedes AE. In addition, we show that there are intermediate steps in ACR and that AE correlates better with the ACR rate rather than absolute Ca2+ amount. Finally, we demonstrate that ACR and membrane fusion progression kinetics and spatial patterns differ with different stimuli and that sites of initiation of ACR and sites of membrane fusion do not always correspond. These findings support a model involving functionally redundant pathways that enable a highly regulated, multistep AE in heterogeneous sperm populations, unlike the previously proposed "acrosome reaction" model.

Multicentric, prospective observational data show sperm capacitation predicts male fertility, and cohort comparison reveals a high prevalence of impaired capacitation in men questioning their fertility.

RESEARCH QUESTIONS: Can a previously defined relationship between sperm capacitation and the probability of a man generating pregnancy within three cycles, prospectively predict male fertility in diverse clinical settings? A second study asked, what is the prevalence of impaired sperm fertilizing ability in men questioning their fertility (MQF), and does this relate to traditional semen analysis metrics? DESIGN: In the multicentric, prospective observational study, data (n = 128; six clinics) were analysed to test a published relationship between the percentage of fertilization-competent, capacitated spermatozoa (Cap-Score) and probability of generating pregnancy (PGP) within three cycles of intrauterine insemination. Logistic regression of total pregnancy outcomes (n = 252) assessed fit. In the cohort comparison, Cap-Scores of MQF (n = 2155; 22 clinics) were compared with those of 76 fertile men. RESULTS: New outcomes (n = 128) were rank-ordered by Cap-Score and divided into quintiles (25-26 per group); chi-squared testing revealed no difference between predicted and observed pregnancies (P = 0.809). Total outcomes (n = 252; 128 new + 124 previous) were pooled and the model recalculated, yielding an improved fit (P < 0.001). Applying the Akaike information criterion found that the optimal model used Cap-Score alone. Cap-Scores were performed on 2155 men (with semen analysis data available for 1948). To compare fertilizing ability, men were binned by PGP (≤19%, 20-29%, 30-39%, 40-49%, 50-59%, ≥60%). Distributions of PGP and the corresponding Cap-Scores were significantly lower in MQF versus fertile men (P < 0.001). Notably, 64% of MQF with normal volume, concentration and motility (757/1183) had PGP of 39% or less (Cap-Scores ≤31), versus 25% of fertile men. CONCLUSIONS: Sperm capacitation prospectively predicted male fertility. Impaired capacitation affects many MQF with normal semen analysis results, informing diagnosis versus idiopathic infertility.

Localisation and function of glucose transporter GLUT1 in chicken (Gallus gallus domesticus) spermatozoa: relationship between ATP production pathways and flagellar motility.

Glucose plays an important role in sperm flagellar motility and fertility via glycolysis and oxidative phosphorylation, although the primary mechanisms for ATP generation vary between species. The glucose transporter 1 (GLUT1) is a high-affinity isoform and a major glucose transporter in mammalian spermatozoa. However, in avian spermatozoa, the glucose metabolic pathways are poorly characterised. This study demonstrates that GLUT1 plays a major role in glucose-mediated motility of chicken spermatozoa. Using specific antibodies and ligand, we found that GLUT1 was specifically localised to the midpiece. Sperm motility analysis showed that glucose supported sperm movement during incubation for 0-80min. However, this was abolished by the addition of a GLUT1 inhibitor, concomitant with a substantial decrease in glucose uptake and ATP production, followed by elevated mitochondrial activity in response to glucose addition. More potent inhibition of ATP production and mitochondrial activity was observed in response to treatment with uncouplers of oxidative phosphorylation. Because mitochondrial inhibition only reduced a subset of sperm movements, we investigated the localisation of the glycolytic pathway and showed glyceraldehyde-3-phosphate dehydrogenase and hexokinase I at the midpiece and principal piece of the flagellum. The results of this study provide new insights into the mechanisms involved in ATP production pathways in avian spermatozoa.

Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog.

Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.

Activin promotes growth and antral cavity expansion in the dog ovarian follicle.

Understanding regulators of folliculogenesis remains limited in the domestic dog (Canis familiaris), which challenges our ability to develop in vitro follicle culture systems for canid genome rescue efforts. Here, we investigated the influence of activin on dog follicle development and survival, oocyte quality, and FSH receptor expression in culture. Preantral (150 - ≤230 µm diameter), early antral (231 - ≤330 µm), and antral (>330-550 µm) stage follicles were encapsulated in a fibrin-alginate hydrogel with 0, 100, or 200 ng/ml rhActivin plus 0, 0.1, 1, or 10 µg/ml FSH for 12 or 21 d of in vitro culture. All follicle groups increased in diameter (P < 0.05) with activin acting synergistically with FSH to improve (P < 0.05) growth and antral cavity expansion (to >630 µm) in early antral and antral cohorts. This complementary effect was not linked to changes in FSHR mRNA expression (P > 0.05). Although not influencing (P > 0.05) follicle survival or transzonal projection (TZP) density in shorter term 12 d culture, activin in the presence of 1 ng/ml FSH maintained TZP density from the 12-21 d interval. Activin also increased oocyte diameter and improved nuclear integrity compared to un-supplemented controls. These results indicate that activin acts synergistically with FSH to promote growth and antral cavity expansion of the dog follicle in vitro, information useful to formulating an effective culture microenvironment for this species.

Small-scale egg production centres increase children's egg consumption in rural Zambia.

Animal source foods can efficiently enhance dietary quality, but they remain inaccessible and unaffordable for many women and young children in remote, low-income communities. We piloted an intervention in which 20 groups established egg production centres (EPCs) in their rural Zambian communities to increase the availability of eggs in the local food system. In a repeated cross-sectional design over 1 year (midline [4 months after the start of egg production] and endline [11 months]), we evaluated programme impact on household egg acquisition within those communities and on egg consumption and height-for-age z score (HAZ) among young children (6-36 months) using multilevel linear, logistic, and truncated negative binomial regression techniques. At midline, households in project areas were significantly more likely to consume eggs than those in control areas (OR 2.08, 95% CI [1.56, 2.78]), particularly those located within 250 m of the EPC. Similarly, children living in project communities were significantly more likely to consume eggs at midline than those in control areas (OR 5.53, 95% CI [2.90, 10.58]). Although increased over baseline, egg acquisition and consumption decreased by endline because of depressed egg production over time. There was no impact on children's HAZ, likely because of the short follow-up time and relatively modest "dose" of egg consumption. Although productivity can be improved, the EPC programme offers a novel approach to improving access to eggs in rural communities, and optimization of the production practices and marketing is needed to ensure that egg consumption translates to improved dietary quality, growth, and health.

Cap-Score™ prospectively predicts probability of pregnancy.

Semen analysis (SA) poorly predicts male fertility, because it does not assess sperm fertilizing ability. The percentage of capacitated sperm determined by GM1 localization ("Cap-Score™"), differs between cohorts of fertile and potentially infertile men, and retrospectively, between men conceiving or failing to conceive by intrauterine insemination (IUI). Here, we prospectively tested whether Cap-Score can predict male fertility with the outcome being clinical pregnancy within ≤3 IUI cycles. Cap-Score and SA were performed (n = 208) with outcomes initially available for 91 men. Men were predicted to have either low (n = 47) or high (n = 44) chance of generating pregnancy using previously-defined Cap-Score reference ranges. Absolute and cumulative pregnancy rates were reduced in men predicted to have low pregnancy rates versus high ([absolute: 10.6% vs. 29.5%; p = 0.04]; [cumulative: 4.3% vs. 18.2%, 9.9% vs. 29.1%, and 14.0% vs. 32.8% for cycles 1-3; n = 91, 64, and 41; p = 0.02]). Only Cap-Score, not male/female age or SA results, differed significantly between outcome groups. Logistic regression evaluated Cap-Score and SA results relative to the probability of generating pregnancy (PGP) for men who were successful in, or completed, three IUI cycles (n = 57). Cap-Score was significantly related to PGP (p = 0.01). The model fit was then tested with 67 additional patients (n = 124; five clinics); the equation changed minimally, but fit improved (p < 0.001; margin of error: 4%). The Akaike Information Criterion found the best model used Cap-Score as the only predictor. These data show that Cap-Score provides a practical, predictive assessment of male fertility, with applications in assisted reproduction and treatment of male infertility.

Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility.

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles. Cysteine dioxygenase (CDO) is a critical enzyme for taurine synthesis. A previous study reported that male CDO-/- mice exhibit idiopathic infertility, prompting us to investigate the functions of CDO in male fertility. Immunoblotting and quantitative reverse transcription-polymerase chain reaction analysis of epididymal segments showed that androgen-dependent CDO expression was highest in the caput epididymidis. CDO-/- mouse sperm demonstrated a severe lack of in vitro fertilization ability. Acrosome exocytosis and tyrosine phosphorylation profiles in response to stimuli were normal, suggesting normal functioning of pathways associated with capacitation. CDO-/- sperm had a slight increase in head abnormalities. Taurine and hypotaurine concentrations in CDO-/- sperm decreased in the epididymal intraluminal fluid and sperm cytosol. We found no evidence of antioxidant protection against lipid peroxidation. However, CDO-/- sperm exhibited severe defects in volume regulation, swelling in response to the relatively hypo-osmotic conditions found in the female reproductive tract. Our findings suggest that epididymal CDO plays a key role in post-testicular sperm maturation, enabling sperm to osmoregulate as they transition from the male to the female reproductive tract, and provide new understanding of the compartmentalized functions of the epididymis.

Timing of sperm capacitation varies reproducibly among men.

Sperm must mature functionally in the process of capacitation to become able to fertilize. Capacitation depends on membrane lipid changes, and can be quantitatively assessed by redistribution of the ganglioside GM1 , the basis of the Cap-Score™ sperm function test. Here, differences in Cap-Score were compared among and within men at two time points. Ejaculates were liquefied, washed, and incubated for 3 hr under capacitating (Cap) conditions, then fixed and analyzed immediately (Day0); after being incubated 3 hr under Cap conditions then maintained 22-24 hr in fix (Day1-fix); or after 22-24 hr incubation under Cap conditions prior to fixation (Day1). In all cases, a light fixative previously shown to allow membrane lipid movements was used. Day1-fix and Day1 Cap-Scores were greater than Day0 (p < 0.001; n = 25), whereas Day1-fix and Day1 Cap-Scores were equivalent (p = 0.43; n = 25). In 123 samples from 52 fertile men, Cap-Score increased more than 1SD (7.7; calculated previously from a fertile cohort) from Day0 to Day1-fix in 44% (54/123) of the samples. To test whether timing of capacitation was consistent within an individual, 52 samples from 11 fertile men were classified into either "early" or "late" capacitation groups. The average capacitation group concordance within a donor was 81%. Median absolute deviation (MAD; in Cap-Score units) was used to assess the tightness of clustering of the difference from Day0 to Day1-fix within individuals. The average (2.21) and median (1.98) MAD confirmed consistency within individuals. Together, these data show that the timing of capacitation differed among men and was consistent within men.

Examining the association between livestock ownership typologies and child nutrition in the Luangwa Valley, Zambia.

OBJECTIVE: To investigate the association between livestock ownership and dietary diversity, animal-source food consumption, height-for-age z-score, and stunting among children living in wildlife "buffer zones" of Zambia's Luangwa Valley using a novel livestock typology approach. METHODS: We conducted a cross-sectional study of 838 children aged 6-36 months. Households were categorized into typologies based on the types and numbers of animals owned, ranging from no livestock to large numbers of mixed livestock. We used multilevel mixed-effects linear and logistic regression to examine the association between livestock typologies and four nutrition-related outcomes of interest. Results were compared with analyses using more common binary and count measures of livestock ownership. RESULTS: No measure of livestock ownership was significantly associated with children's odds of animal-source food consumption, child height-for-age z-score, or stunting odds. Livestock ownership Type 2 (having a small number of poultry) was surprisingly associated with decreased child dietary diversity (ß = -0.477; p<0.01) relative to owning no livestock. Similarly, in comparison models, chicken ownership was negatively associated with dietary diversity (ß = -0.320; p<0.01), but increasing numbers of chickens were positively associated with dietary diversity (ß = 0.022; p<0.01). Notably, neither child dietary diversity nor animal-source food consumption was significantly associated with height, perhaps due to unusually high prevalences of morbidities. CONCLUSIONS: Our novel typologies methodology allowed for an efficient and a more in-depth examination of the differential impact of livestock ownership patterns compared to typical binary or count measures of livestock ownership. We found that these patterns were not positively associated with child nutrition outcomes in this context. Development and conservation programs focusing on livestock must carefully consider the complex, context-specific relationship between livestock ownership and nutrition outcomes-including how livestock are utilized by the target population-when attempting to use livestock as a means of improving child nutrition.

Validation of a laboratory-developed test of human sperm capacitation.

Sperm must undergo capacitation to become fertilization competent. Here we validated that monosialotetrahexosylganglioside (GM1 ) localization patterns, which were assessed in the Cap-Score™ Sperm Function Test, reflect a capacitated state in human sperm. First, we defined patterns representing sperm that do or do not respond to stimuli for capacitation. Sperm with "capacitated" patterns had exposed acrosomal carbohydrates and underwent acrosome exocytosis in response to calcium ionophore (A23187). Precision was evaluated by percent change of the Cap-Score measured for 50, 100, 150, and 200 sperm. Changes of 11%, 6%, and 5% were observed (n ≥ 23); therefore, we counted ≥150 sperm per condition. Variance within and between readers was evaluated using 20 stitched image files generated from unique ejaculates. Two trained readers randomly resampled each image 20 times, reporting an average standard deviation of 3 Cap-Score units and coefficient of variation of 13% when rescoring samples, with no difference between readers. Semen liquefaction times ≤2 hr and mechanical liquefaction with Pasteur or wide-orifice transfer pipettes did not alter Cap-Score values. However, liquefaction with chymotrypsin (p = 0.002) and bromelain (p = 0.049) reduced response to capacitating stimuli and induced membrane damage, while counterintuitively improving sperm motility. Together, these data validate the Cap-Score assay for the intended purpose of providing information on sperm capacitation and male fertility. In addition to its clinical utility as a diagnostic tool, this test of sperm function can reveal the impact of common practices of semen handling on the ability of sperm to respond to capacitation stimuli.

Localization patterns of the ganglioside GM1 in human sperm are indicative of male fertility and independent of traditional semen measures.

Semen analysis lacks a functional component and best identifies extreme cases of infertility. The ganglioside GM1 is known to have functional roles during capacitation and acrosome exocytosis. Here, we assessed whether GM1 localization patterns (Cap-Score™) correspond with male fertility in different settings: Study 1 involved couples pursuing assisted reproduction in a tertiary care fertility clinic, while Study 2 involved men with known fertility versus those questioning their fertility at a local urology center. In Study 1, we examined various thresholds versus clinical history for 42 patients; 13 had Cap-Scores ≥39.5%, with 12 of these (92.3%) achieving clinical pregnancy by natural conception or ≤3 intrauterine insemination cycles. Of the 29 patients scoring <39.5%, only six (20.7%) attained clinical pregnancy by natural conception or ≤3 intrauterine insemination cycles. In Study 2, Cap-Scores were obtained from 76 fertile men (Cohort 1, pregnant partner or recent father) and compared to 122 men seeking fertility assessment (Cohort 2). Cap-Score values were normally distributed in Cohort 1, with 13.2% having Cap-Scores more than one standard deviation below the mean (35.3 ± 7.7%). Significantly, more men in Cohort 2 had Cap-Scores greater than one standard deviation below the normal mean (33.6%; p = 0.001). Minimal/no relationship was found between Cap-Score and sperm concentration, morphology, or motility. Together, these data demonstrate that Cap-Score provides novel, clinically relevant insights into sperm function and male fertility that complement traditional semen analysis. Furthermore, the data provide normal reference ranges for fertile men that can help clinicians counsel couples toward the most appropriate fertility treatment.

Follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of pre- and early antral dog follicles.

Understanding stage-specific requirements of mammalian folliculogenesis is limited in the domestic dog. The present study examined the effects of two potential regulators of dog follicle growth and survival in vitro, namely the original stage of the follicle (i.e. preantral (≤230µm diameter) vs early antral (diameter from >230 to ≤330µm) and FSH and/or LH concentrations. After isolation and alginate encapsulation, follicles were cultured in 0, 1, 10 or 100µgmL-1 FSH and 0, 1 or 10ngmL-1 LH for 20 days. Regardless of stage, FSH promoted growth, but LH did the same only in the absence of FSH. Production of 17ß-oestradiol and progesterone was detectable, indicating theca cell activity. The greatest growth occurred in preantral (mean (± s.d.) 61.4±25.9%) versus antral (42.6±20.3%) follicles, but neither developmental stage nor gonadotropin affected survival. Antrum detection was minimal due, in part, to antral collapse, and oocytes exhibited an increasingly pale appearance and chromatin degeneration over time. The results demonstrate that pre- and early antral stage dog follicles encapsulated in alginate grow significantly in vitro. However, because FSH and LH alone or in combination fail to promote antrum development, the next step is identifying factors that enhance antral expansion.