A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

Recent advances in reproductive research in Australia and New Zealand: highlights from the Annual Meeting of the Society for Reproductive Biology, 2022.

In 2022, the Society for Reproductive Biology came together in Christchurch New Zealand (NZ), for its first face-to-face meeting since the global COVID-19 pandemic. The meeting showcased recent advancements in reproductive research across a diverse range of themes relevant to human health and fertility, exotic species conservation, and agricultural breeding practices. Here, we highlight the key advances presented across the main themes of the meeting, including advances in addressing opportunities and challenges in reproductive health related to First Nations people in Australia and NZ; increasing conservation success of exotic species, including ethical management of invasive species; improvements in our understanding of developmental biology, specifically seminal fluid signalling, ovarian development and effects of environmental impacts such as endocrine-disrupting chemicals; and leveraging scientific breakthroughs in reproductive engineering to drive solutions for fertility, including in assisted reproductive technologies in humans and agricultural industries, and for regenerative medicine.

Making human eggs in a dish: are we close?

In the space of 50 years, we have seen incredible achievements in human reproductive medicine. With these leaps forward, it is no wonder that there is a major interest in women's reproductive health research, including extension of reproductive lifespan. Substantial effort is currently being made to address this challenge, including from the commercial sector. In vitro gametogenesis (IVG) in mice is a spectacular breakthrough and has the potential to offer hope to women with intractable infertility. However, with such lofty goals, some reflection may be called for: mastering all of the techniques required for complete and safe IVG in women is likely to be extraordinarily difficult.

Genetic control of typical and atypical sex development.

Sex development relies on the sex-specific action of gene networks to differentiate the bipotential gonads of the growing fetus into testis or ovaries, followed by the differentiation of internal and external genitalia depending on the presence or absence of hormones. Differences in sex development (DSD) arise from congenital alterations during any of these processes, and are classified depending on sex chromosomal constitution as sex chromosome DSD, 46,XY DSD or 46,XX DSD. Understanding the genetics and embryology of typical and atypical sex development is essential for diagnosing, treating and managing DSD. Advances have been made in understanding the genetic causes of DSD over the past 10 years, especially for 46,XY DSD. Additional information is required to better understand ovarian and female development and to identify further genetic causes of 46,XX DSD, besides congenital adrenal hyperplasia. Ongoing research is focused on the discovery of further genes related to typical and atypical sex development and, therefore, on improving diagnosis of DSD.

Somatic FGFR2 is Required for Germ Cell Maintenance in the Mouse Ovary.

During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.

A New Understanding, Guided by Single-Cell Sequencing, of the Establishment and Maintenance of the Ovarian Reserve in Mammals.

BACKGROUND: Oocytes are a finite and non-renewable resource that are maintained in primordial follicle structures. The ovarian reserve is the totality of primordial follicles, present from birth, within the ovary and its establishment, size, and maintenance dictates the duration of the female reproductive lifespan. Understanding the cellular and molecular dynamics relevant to the establishment and maintenance of the reserve provides the first steps necessary for modulating both individual human and animal reproductive health as well as population dynamics. SUMMARY: This review details the key stages of establishment and maintenance of the ovarian reserve, encompassing germ cell nest formation, germ cell nest breakdown, and primordial follicle formation and activation. Furthermore, we spotlight several formative single-cell sequencing studies that have significantly advanced our knowledge of novel molecular regulators of the ovarian reserve, which may improve our ability to modulate female reproductive lifespans. KEY MESSAGES: The application of single-cell sequencing to studies of ovarian development in mammals, especially when leveraging genetic and environmental models, offers significant insights into fertility and its regulation. Moreover, comparative studies looking at key stages in the development of the ovarian reserve across species has the potential to impact not just human fertility, but also conservation biology, invasive species management, and agriculture.

Pathogenicity and virulence of bacterial strains associated with summer mortality in marine mussels (Perna canaliculus).

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus, from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii, and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus, with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host-pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment.

Comparing program supervision with an external RADAR evaluation of quality of care in integrated community case management for childhood illnesses in Mali.

BACKGROUND: Many countries have adopted integrated community case management (iCCM) to reduce mortality among children under five years from common childhood illnesses. The 2016-2020 Malian Red Cross iCCM program trained 441 Community Health Workers (CHWs) to treat malaria, pneumonia, diarrhea, and malnutrition for children under five years of age in six districts. Implementation strength and quality of care (QoC) were assessed through the program's supervision function, using the Malian Ministry of Health's system. OBJECTIVE: This paper compares methods and results of program supervision data and an independent evaluation to assess the effectiveness of program implementation and supervision and inform program improvement. It also presents the benefits and limitations of each method. METHOD: An independent QoC evaluation was conducted using tools developed by the Real Accountability: Data Analysis for Results (RADAR) project, hereafter referred to as the RADAR evaluation. RADAR evaluation data collected in July and August 2018 were compared with program supervision data collected mostly between May and December 2018. RESULTS: The RADAR evaluation provided detailed findings on correct assessment, classification, and treatment per illness, medication type, and dosage. Program supervision combined the findings for all illnesses, medication type, and dosage due to limitations in the data collection process. Six indicators were comparable between both methods. Findings were similar for temperature and mid-upper arm circumference measurements but diverged between program supervision and the RADAR evaluation, respectively, on correct classification for all illnesses (87.1% vs. 65.3%), correct treatment for all illnesses (69.5% vs. 39.8%), correct respiratory rate counting (88.5% vs. 54.7%), and administering the first dose by CHW (75.4% vs. 65.0%). Findings from the RADAR evaluation guided improvements in program supervision. CONCLUSIONS: A robust program supervision system can serve as a credible method to assess QoC. However, a rigorous independent QoC evaluation provides a valuable benchmark to gauge the effectiveness of the supervisory process.

Measuring Quality of Maternal, Neonatal, Child, Reproductive Health and Nutrition Care with tools developed by the RADAR project and tested in Sub Saharan Africa.

Increasing coverage of evidence-based maternal, neonatal, child, reproductive health and nutrition (MNCRHN) programs in low- and middle-income countries has coincided with dramatic improvements in health despite variable quality of implementation. Comprehensive evaluation to inform program improvement requires standardized but adaptable tools, which the Real Accountability, Data Analysis for Results (RADAR) project has developed. To inform selection of tools and methods packages ('packages') to measure program quality of care (QoC), we documented experiences testing the packages, which were developed and adapted based on global and local expertise, and pre- and pilot-testing. We conducted cross-sectional studies in 2018-2019 on the quality of 1) integrated community case management, 2) counseling on maternal, infant, and young child feeding, 3) intrapartum care, and 4) family planning counseling in Mali, Mozambique, Tanzania, and Malawi. Herein we describe package performance and highlight experiences that inform their selection and use. Direct observation packages provided high-quality, immediately applicable results but they required specialized expertise, in-person collection, adequate patient volume, reasonable wait times, and unambiguously 'correct' provision of care. General satisfaction questions from exit interview packages produced unvaryingly positive responses despite variable observed quality of care. Variation increased when questions were more targeted, but findings on caregiver and client's recall of recommendations were more actionable. When interactive, clinical vignettes can capture knowledge of clinical care. But for conditions that can be simulated, like provision of family planning counseling, we could capture provider practice from simulated clients. Clinicians could more easily demonstrate tactile aspects of intrapartum care using observed structured clinical examinations, but this method required storage and transport of the required mannequins. Based on our findings we recommend ten questions upon which evaluators can base package selection. Findings from these packages inform programs and, in the context of comprehensive program evaluation enable us to link programs with impact.

Dataset of differentially expressed genes in mouse P12 testes in response to the loss of ATRX in Sertoli cells.

This dataset represents genes that are dysregulated in the postnatal day 12 (P12) mouse testis when ATRX is specifically inactivated in Sertoli cells (ScAtrxKO mice). The differentially expressed genes included in the dataset may play important roles in the testicular phenotypes observed in the ScAtrxKO mice, which were first reported in our previous work [1]. In fetal ScAtrxKO mice, Sertoli cells undergo apoptosis due to cell cycle defects, resulting in smaller testes with reduced tubule volume [1]. Adult ScAtrxKO mice show a wide range of spermatogenesis defects probably due to a failure of the dysfunctional ATRX protein to interact with the androgen receptor (AR) [1]. ATRX, a chromatin remodeling protein, is widely expressed in the human testis including Sertoli cells [2,3]. In XY individuals, the loss of ATRX leads to ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome associated with a wide range of genital abnormalities such as hypospadias, ambiguous genitalia, and small testes with reduced tubule volume [4], [5], [6], [7], [8]. Our dataset contributes towards understanding the mechanism underlying ATRX regulation of testis development and spermatogenesis.

Assessing provider performance of intrapartum care using simulated encounters and clinical vignettes: A comparison study from Tanzania.

OBJECTIVE: To compare clinical vignettes and objective structured clinical examinations (OSCE) as methods for assessing the quality of intrapartum care among skilled providers in rural primary-level health facilities in Tanzania. METHODS: Cross-sectional study conducted at six health facilities in the Simiyu region of Tanzania. Providers were assessed using OSCE and clinical vignettes in spontaneous delivery, neonatal resuscitation, and management of postpartum hemorrhage. Trained researchers used a structured clinical checklist. The frequencies of items are presented as percentages and the agreement of the methods of assessment are reported using kappa statistics (high: kappa > 0.80, moderate: kappa = 0.60-0.80, low: kappa < 0.60). RESULTS: Most healthcare providers were female (60.7%), registered nurses by training (29.0%), and worked in a dispensary (56.1%), with an average age of 33 years and an average of 7.4 years of experience in their respective professions. Five items had high agreement between OSCE and clinical vignettes: postpartum vital signs every 15 min, oxytocin within 1 min of birth, diagnosis of postpartum hemorrhage, elevating legs of the mother, and deciding on manual compression of the uterus. CONCLUSION: OSCE and clinical vignettes should be viewed as complimentary to one another in the assessment of provider knowledge and skill, with priority given to OSCE, particularly in intrapartum care.

Transcriptomic profiling of neonatal mouse granulosa cells reveals new insights into primordial follicle activation†.

The dormant population of ovarian primordial follicles is determined at birth and serves as the reservoir for future female fertility. Yet our understanding of the molecular, biochemical, and cellular processes underpinning primordial follicle activation remains limited. The survival of primordial follicles relies on the correct complement and morphology of granulosa cells, which provide signaling factors essential for oocyte and follicular survival. To investigate the contribution of granulosa cells in the primordial-to-primary follicle transition, gene expression profiles of granulosa cells undergoing early differentiation were assessed in a murine model. Ovaries from C57Bl/6 mice were enzymatically dissociated at time-points spanning the initial wave of primordial follicle activation. Post-natal day (PND) 1 ovaries yielded primordial granulosa cells, and PND4 ovaries yielded a mixed population of primordial and primary granulosa cells. The comparative transcriptome of granulosa cells at these time-points was generated via Illumina NextSeq 500 system, which identified 131 significantly differentially expressed transcripts. The differential expression of eight of the transcripts was confirmed by RT-qPCR. Following biological network mapping via Ingenuity Pathway Analysis, the functional expression of the protein products of three of the differentially expressed genes, namely FRZB, POD1, and ZFX, was investigated with in-situ immunolocalization in PND4 mouse ovaries was investigated. Finally, evidence was provided that Wnt pathway antagonist, secreted frizzled-related protein 3 (FRZB), interacts with a suppressor of primordial follicle activation WNT3A and may be involved in promoting primordial follicle activation. This study highlights the dynamic changes in gene expression of granulosa cells during primordial follicle activation and provides evidence for a renewed focus into the Wnt signaling pathway's role in primordial follicle activation.

Investigating the biochemical effects of heat stress and sample quenching approach on the metabolic profiling of abalone (Haliotis iris).

INTRODUCTION: Ocean temperatures have been consistently increasing due to climate change, and the frequency of heatwave events on shellfish quality is a growing concern worldwide. Typically, shellfish growing areas are in remote or difficult to access locations which makes in-field sampling and sample preservation of shellfish heat stress difficult. As such, there is a need to investigate in-field sampling approaches that facilitate the study of heat stress in shellfish. OBJECTIVES: This study aims to apply a gas chromatography-mass spectrometry (GC-MS) based metabolomics approach to examine molecular mechanisms of heat stress responses in shellfish using abalone as a model, and compare the effects of different quenching protocols on abalone metabolic profiles. METHODS: Twenty adult Haliotis iris abalone were exposed to two temperatures (14 °C and 24 °C) for 24 h. Then, haemolymph and muscle tissues of each animal were sampled and quenched with 4 different protocols (liquid nitrogen, dry ice, cold methanol solution and normal ice) which were analyzed via GC-MS for central carbon metabolites. RESULTS: The effects of different quenching protocols were only observed in muscle tissues in which the cold methanol solution and normal ice caused some changes in the observed metabolic profiles, compared to dry ice and liquid nitrogen. Abalone muscle tissues were less affected by thermal stress than haemolymph. There were 10 and 46 compounds significantly influenced by thermal stress in muscle and haemolymph, respectively. The changes of these metabolite signatures indicate oxidative damage, disturbance of amino acid and fatty acid metabolism, and a shift from aerobic metabolism to anaerobic pathways. CONCLUSIONS: The study provided insights into the heat response of abalone, which could be useful for understanding the effects of marine heatwaves and summer mortality events on abalone. Dry ice appeared to be a suitable protocol, and safer in-field alternative to liquid nitrogen, for quenching of abalone tissues.

Targeted provider education and pre-visit planning increase rates of formal depression screening in childhood-onset SLE.

BACKGROUND: Despite being at high risk for depression, patients with childhood-onset systemic lupus erythematosus (c-SLE) are infrequently and inconsistently screened for depression by their pediatric rheumatologists. We aimed to systematically increase rates of formal depression screening for c-SLE patients in an academic Pediatric Rheumatology clinic. METHODS: Our multi-disciplinary quality improvement (QI) team used electronic health record (EHR) documentation to retroactively calculate baseline rates of documented depression screening using the Patient Health Questionnaire-9 (PHQ-9). We then engaged key stakeholders to develop a clinical workflow for formal depression screening in the clinic. We also provided education to providers regarding mental health disorders in c-SLE, with an emphasis on prevalence, screening methods, and management of positive screens. We then used the Plan-Do-Study Act (PDSA) method of QI to systematically evaluate and adjust our process in real time. The primary outcome was the percentage of patients with c-SLE seen per month who had a documented PHQ-9 screening within the past year. RESULTS: The percentage of children with documented PHQ-9 results ranged from 0 to 4.5 % at baseline to 91.0 % within 12 months of project initiation. By the end of the project, monthly screening rates greater than 80 % has been sustained for 10 months. As a result of these efforts, twenty-seven (48.2 %) patients with at least mild depressive symptoms were identified while seven (12.5 %) with thoughts of self-harm were referred to appropriate mental health resources. CONCLUSIONS: Routine formal depression screening is feasible in a busy subspecialty clinic. Using QI methods, rates of formal depression screening among children with c-SLE were increased from an average of 3.3 % per month to a sustained monthly rate of greater than 80 %. Individuals with depressive symptoms and/or thoughts of self-harm were identified and referred to appropriate mental health resources.

Dartanan: Prototype evaluations of a serious game to engage children in the calibration of their hearing aid functionalities.

INTRODUCTION: It is notoriously difficult to obtain a perfect fitting of hearing aids (HAs) for children as they often struggle to understand their hearing loss well enough to discuss the fitting adequately with their audiologist. Dartanan is an 'edutainment' game developed to help children understand the functions of their HA in different sound contexts. Dartanan also has elements of a leisure game for all children, in order to create an inclusive activity. METHODS: Game prototypes were evaluated during two formative evaluations and a summative evaluation. In total 106 children with and without hearing loss in Italy, Spain and the UK played Dartanan. A built-in virtual HA enabled children with hearing loss to use headphones to play.Results and conclusions: During the formative stages, feedback was discussed during focus groups on factors such as the audiological aspects, the extent to which children learned about HA functions, accessibility and usability, and this feedback was presented to the developers. After redevelopment, a summative evaluation was performed using an online survey. It was concluded that the game had met the goals of helping children understand their HA functionalities and providing an inclusive activity. User-evaluations were crucial in the development of the app into a useful and useable service.

Validity of using mobile phone surveys to evaluate community health worker program in Mali.

BACKGROUND: The monitoring and evaluation of public health programs based on traditional face-to-face interviews in hard-to-reach and unstable regions present many challenges. Mobile phone-based methods are considered to be an effective alternative, but the validity of mobile phone-based data for assessing implementation strength has not been sufficiently studied yet. Nested within an evaluation project for an integrated community case management (iCCM) and family planning program in Mali, this study aimed to assess the validity of a mobile phone-based health provider survey to measure the implementation strength of this program. METHODS: From July to August 2018, a cross-sectional survey was conducted among the community health workers (ASCs) from six rural districts working with the iCCM and family planning program. ASCs were first reached to complete the mobile phone-based survey; within a week, ASCs were visited in their communities to complete the in-person survey. Both surveys used identical implementation strength tools to collect data on program activities related to iCCM and family planning. Sensitivity and specificity were calculated for each implementation strength indicator collected from the phone-based survey, with the in-person survey as the gold standard. A threshold of ≥ 80% for sensitivity and specificity was considered adequate for evaluation purposes. RESULTS: Of the 157 ASCs interviewed by mobile phone, 115 (73.2%) were reached in person. Most of the training (2/2 indicators), supervision (2/3), treatment/modern contraceptive supply (9/9), and reporting (3/3) indicators reached the 80% threshold for sensitivity, while only one supervision indicator and one supply indicator reached 80% for specificity. In contrast, most of the stock-out indicators (8/9) reached 80% for specificity, while only two indicators reached the threshold for sensitivity. CONCLUSIONS: The validity of mobile phone-based data was adequate for general training, supervision, and supply indicators for iCCM and family planning. With sufficient mobile phone coverage and reliable mobile network connection, mobile phone-based surveys are useful as an alternative for data collection to assess the implementation strength of general activities in hard-to-reach areas.

Establishing and maintaining fertility: the importance of cell cycle arrest.

Development of the ovary or testis is required to establish reproductive competence. Gonad development relies on key cell fate decisions that occur early in embryonic development and are actively maintained. During gonad development, both germ cells and somatic cells proliferate extensively, a process facilitated by cell cycle regulation. This review focuses on the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) in mouse gonad development. We particularly highlight recent single-cell RNA sequencing studies that show the heterogeneity of cyclin-dependent kinase inhibitors. This diversity highlights new roles for cell cycle inhibitors in controlling and maintaining female fertility.

Improving eye screening practice among pediatric rheumatology patients receiving hydroxychloroquine.

OBJECTIVE: Hydroxychloroquine (HCQ) is commonly used in the treatment of various autoimmune diseases related to its many benefits and favorable safety profile. Although HCQ retinopathy was considered to be uncommon, a prevalence of 7.5% was described in a recent study making early detection critical. The most updated screening guidelines by the American Academy of Ophthalmology were published in 2016; however, it lacked pediatric-specific recommendations and the overall compliance with screening guidelines was poor in previous studies. We developed a quality improvement (QI) initiative aiming to create institutional screening recommendations. Additionally, to increase eye screening in pediatric rheumatology clinic for patients receiving HCQ from 65% to 85% in 12 months and to sustain that rate for at least 6 months. METHODS: We formed a multidisciplinary team of pediatric rheumatologists and ophthalmologists, clinical pharmacist, clinic nurses, QI specialist, quality data technician and administrative staff. We included patients receiving HCQ and who were evaluated at Nationwide Children's Hospital rheumatology clinic. A key driver diagram was formulated to identify barriers to compliance and determine possible interventions. Main interventions included summarizing screening guidelines in a step by step algorithm, increasing awareness of these guidelines among patients and providers, improving collaboration and communication with ophthalmologists, and initiating pre-visit planning. RESULTS: Baseline performance data included 164 patients. Fifty-four (33%) of those patients were at high risk for HCQ retinopathy. Of them, 50% were on HCQ dose of >5 mg/kg/day and 31.5% had been taking HCQ for ≥5 years. Two center line shifts were noticed over the course of the project. The target of 85% compliance was reached in February 2019 and was sustained until December 2019. CONCLUSIONS: Our study highlights the importance of interdisciplinary communication to increase awareness of screening guidelines among medical providers and patients. Pre-visit planning played a major role in identifying patients and opportunities for optimizing eye screening in patients at risk for HCQ retinopathy. Collaboration between rheumatologists and ophthalmologists is crucial in managing patients on HCQ. The implementation of same-day eye screening allowed this collaboration to be more efficient. Future efforts are being directed at monitoring and improving utilization of the effective interventions.