Fertility preservation service for children and young adults at high risk of infertility; the hub and spoke model.

PURPOSE: To evaluate the evolution of fertility preservation surgery in children and young adults at high risk of infertility from a single centre to a networked 'Hub and Spoke' service. METHODS: A case note review of patients referred for ovarian or testicular cryopreservation between Jan 2013 and Dec 2023. Demographic data, procurement numbers, and site of procurement were collected. Specialist feedback was obtained to identify the challenges faced. RESULTS: Over time, the number of referrals increased from 4 to 349 patients per year with the number of Spoke centres rising to 36 ovarian and 16 testicular. In 2013-2014; 100% of procurement was ovarian as compared to 2023; 51% ovarian, 49% testicular. Of the 395 referrals in 2021, 81% (n = 319) went on to have procurement and storage of tissue. Between 2013 and 2016, 96% of cases were performed at the Hub. In 2023, 53/349 (15%) cases were performed at the Hub with the remaining 296 (85%) procured at Spoke sites. Surgical issues such as access to theatre, variation and availability of surgical equipment, thermal injury to ovarian tissue and variation in the size of the testicular specimen were identified. CONCLUSION: The Hub and Spoke model successfully delivers treatment to patients close to home as safely possible within their local treatment centre.

COVID-19 therapeutics for the pregnant patient.

SARS-CoV-2 infection can cause severe disease among pregnant persons. Pregnant persons were not included in initial studies of therapeutics for COVID-19, but cumulative experience demonstrates that most are safe for pregnant persons and the fetus, and effective for prevention or treatment of severe COVID-19.

Anin vitrothree-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues.

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of anin vitroculture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for thein vitromaintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 µm; the mean number of organoids was 5.57 ± 1.60/105µm2on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression ofC-KITbut reduced the expression ofGFRα1at day 28 when compared to those without hormone supplements(p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishingin vitrospermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.

Determining the optimal time interval between sample acquisition and cryopreservation when processing immature testicular tissue to preserve fertility.

The cryopreservation of immature testicular tissue (ITT) prior to gonadotoxic therapy is crucial for fertility preservation in prepubertal boys with cancer. However, the optimal holding time between tissue collection and cryopreservation has yet to be elucidated. Using the bovine model, we investigated four holding times (1, 6, 24, and 48 h) for ITTs before cryopreservation. Biopsies from two-week-old calves were stored in transport medium and cryopreserved following a standard slow-freezing clinical protocol. Thawed samples were then assessed for viability, morphology, and gene expression by haematoxylin and eosin (H&E) staining, immunohistochemistry and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Analysis failed to identify any significant changes in cell viability when compared between the different groups. Sertoli (Vimentin+) and proliferating cells (Ki67+) were well-preserved. The expression of genes related to germ cells, spermatogenesis (STRA8, PLZF, GFRα-1, C-KIT, THY1, UCHL-1, NANOG, OCT-4, CREM), and apoptosis (HSP70-2) remained stable over 48 h. However, seminiferous cord detachment increased significantly in the 48-h group (p < 0.05), with associated cord and SSC shrinkage. Collectively, our analyses indicate that bovine ITTs can be stored for up to 48 h prior to cryopreservation with no impact on cell viability and the expression levels of key genes. However, to preserve the morphology of frozen-thawed tissue, the ideal processing time would be within 24 h. Testicular tissues obtained from patients for fertility preservation often need to be transported over long distances to be cryopreserved in specialist centres. Our findings highlight the importance of determining optimal tissue transport times to ensure tissue quality in cryopreservation.

Testing the effect of PAR1 inhibitors on Plasmodium falciparum-induced loss of endothelial cell barrier function.

Background: Sequestration and cytoadherence of Plasmodium falciparum-infected erythrocytes (IE) to microvascular endothelium alters endothelial barrier function and plays a role in the pathogenesis of severe malaria. Binding of IE is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) and the PfEMP1 variants that binds to endothelial protein C receptor (EPCR) have, in particular, been associated with the dysregulation of the coagulation/inflammation pathways in endothelial cells. This has prompted speculation about the role of protease-activated receptor-1 (PAR1) activation and signalling in causing endothelial activation and loss of barrier function in cerebral malaria. Methods: We used a co-culture of primary human brain microvascular endothelial cells (HBMEC) with P. falciparum material, recombinant PfEMP1 or lysates from IE, and measured barrier function by trans endothelial electrical resistance (TEER).  A selection of PAR1 inhibitors was tested for their ability to reverse the P. falciparum and thrombin induced decrease in barrier function. Results: An initial screen in the presence of recombinant PfEMP1 identified a few inhibitors that were able to reduce the rapid thrombin-induced barrier disruption even when activated protein C (aPC) was unable to do so. However, in the IE lysate co-culture system we identified a mechanism that slowly reduces barrier function and which is insensitive to PAR1 inhibitors. Conclusions: The selected PAR1 inhibitors were able to reverse the disruption of barrier function by thrombin but did not reverse the IE lysate induced disruption of barrier function, implicating a different PAR1-independent mechanism.  These findings have implications for the design of adjunct therapies to reduce brain swelling in cerebral malaria.

Combined quadriceps lengthening (using an external ring fixator) and patellar tendon reconstruction (using a tendoachilles allograft) in a case of chronic patellar tendon rupture: A case report.

BACKGROUND: Patellar tendon rupture is an infrequent and debilitating condition, which if left untreated, is complicated by quadriceps contracture and patella alta. This results in reduced function of the knee extensor mechanism including extension lag, reduced range of motion (ROM), chronic knee pain, and frequent falls. Early primary repair has good results in most cases and is performed by opposing and suturing the tendon ends. However, when there is a delay between rupture and repair or in case of a re-rupture, primary repair techniques may not work due to retraction of the extensor mechanism. Several treatment modalities have been proposed in such cases, but there is no clear consensus. METHODOLOGY: We present the case of a 39-year-old male with chronic rupture of patellar tendon that was reconstructed with a two-stage technique. In the first stage, a ring-fixator was applied to stretch the quadriceps muscle and the second stage consisted of reconstruction of the patellar tendon with a tendoachilles allograft. This procedure was performed in two stages five weeks apart and almost four years after the initial injury. RESULTS: Results were excellent at three years' follow-up with the patient achieving full extension, a ROM of 0-110° and 85% quadriceps strength. There was significant improvement in his pain, mobility and quadriceps bulk. CONCLUSION: To our knowledge, this treatment has not previously been described in the literature. We present this case as a proven treatment option for treatment of chronic patellar tendon injuries with extensor mechanism shortening.

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria.

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84 nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.

The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants.

BACKGROUND: The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers. METHODS: Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant. RESULTS: Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set (n = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in BRCA1 or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants - associated with cancer, cardiac or neurodegenerative phenotypes - remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual. INTERPRETATION: Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care.

Corrigendum: Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

This corrects the article DOI: 10.1038/srep23458.

Genetic Counselors' Experiences and Interest in Telegenetics and Remote Counseling.

In 2009, the National Society of Genetic Counselors Service (NSGC) Delivery Model Task Force defined genetic counseling service delivery models including telephone (genetic counseling provided remotely by telephone) and telegenetics (counseling provided remotely using videoconferencing). Little is known about the experience of genetic counselors practicing telemedicine in the USA. We sought to evaluate perceived satisfaction, advantages, disadvantages, and barriers to the practice and implementation of telegenetics by practicing genetic counselors. A 21-question online survey was distributed via the NSGC's member directory. Descriptive statistics and a thematic analysis were used to analyze data. A total of 344 surveys were completed of which 235 (68.3%) respondents had delivered genetic counseling via telemedicine and 109 (36.6%) had not. Overall genetic counseling providers who had provided telegenetics were satisfied or very satisfied with their position (91%) and those who were not performing telegenetics were at least slightly interested in a telehealth position (92%).The most common appealing reasons for working in or wanting to work in telemedicine included an innovative approach to healthcare delivery, aspects of remote positions such as the ability to work from home, and flexibility of hours. Unappealing characteristics of telemedicine included the inability to see nonverbals, limited psychosocial counseling, and limited social interaction with colleague that is associated with remote positions. Barriers to implementation of telegenetics were noted by 53% of respondents with the largest barrier being billing and reimbursement. The results of this work suggest that telegenetics service organizations could consider increasing social interactions, attempting to use the preferred method of care (video) to increase ability to see nonverbals, offering flexible work hours, and allowing time to address psychosocial issues as they arise in consultations.

Author Correction: Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Potent Antimalarial 2-Pyrazolyl Quinolone bc 1 (Qi) Inhibitors with Improved Drug-like Properties.

A series of 2-pyrazolyl quinolones has been designed and synthesized in 5-7 steps to optimize for both in vitro antimalarial potency and various in vitro drug metabolism and pharmacokinetics (DMPK) features. The most potent compounds display no cross-resistance with multidrug resistant parasite strains (W2) compared to drug sensitive strains (3D7), with IC50 (concentration of drug required to achieve half maximal growth suppression) values in the range of 15-33 nM. Furthermore, members of the series retain moderate activity against the atovaquone-resistant parasite isolate (TM90C2B). The described 2-pyrazoyl series displays improved DMPK properties, including improved aqueous solubility compared to previously reported quinolone series and acceptable safety margin through in vitro cytotoxicity assessment. The 2-pyrazolyl quinolones are believed to bind to the ubiquinone-reducing Qi site of the parasite bc 1 complex, which is supported by crystallographic studies of bovine cytochrome bc 1 complex.

Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis.

Elimination of filariasis requires a macrofilaricide treatment that can be delivered within a 7-day period. Here we have identified a synergy between the anthelmintic albendazole (ABZ) and drugs depleting the filarial endosymbiont Wolbachia, a proven macrofilaricide target, which reduces treatment from several weeks to 7 days in preclinical models. ABZ had negligible effects on Wolbachia but synergized with minocycline or rifampicin (RIF) to deplete symbionts, block embryogenesis, and stop microfilariae production. Greater than 99% Wolbachia depletion following 7-day combination of RIF+ABZ also led to accelerated macrofilaricidal activity. Thus, we provide preclinical proof-of-concept of treatment shortening using antibiotic+ABZ combinations to deliver anti-Wolbachia sterilizing and macrofilaricidal effects. Our data are of immediate public health importance as RIF+ABZ are registered drugs and thus immediately implementable to deliver a 1-wk macrofilaricide. They also suggest that novel, more potent anti-Wolbachia drugs under development may be capable of delivering further treatment shortening, to days rather than weeks, if combined with benzimidazoles.

Validation of microbiological testing in cardiovascular tissue banks: results of a quality round trial.

OBJECTIVES: Surgeons needing human cardiovascular tissue for implantation in their patients are confronted with cardiovascular tissue banks that use different methods to identify and decontaminate micro-organisms. To elucidate these differences, we compared the quality of processing methods in 20 tissue banks and 1 reference laboratory. We did this to validate the results for accepting or rejecting tissue. We included the decontamination methods used and the influence of antibiotic cocktails and residues with results and controls. The minor details of the processes were not included. METHODS: To compare the outcomes of microbiological testing and decontamination methods of heart valve allografts in cardiovascular tissue banks, an international quality round was organized. Twenty cardiovascular tissue banks participated in this quality round. The quality round method was validated first and consisted of sending purposely contaminated human heart valve tissue samples with known micro-organisms to the participants. The participants identified the micro-organisms using their local decontamination methods. RESULTS: Seventeen of the 20 participants correctly identified the micro-organisms; if these samples were heart valves to be released for implantation, 3 of the 20 participants would have decided to accept their result for release. Decontamination was shown not to be effective in 13 tissue banks because of growth of the organisms after decontamination. Articles in the literature revealed that antibiotics are effective at 36°C and not, or less so, at 2-8°C. The decontamination procedure, if it is validated, will ensure that the tissue contains no known micro-organisms. CONCLUSIONS: This study demonstrates that the quality round method of sending contaminated tissues and assessing the results of the microbiological cultures is an effective way of validating the processes of tissue banks. Only when harmonization, based on validated methods, has been achieved, will surgeons be able to fully rely on the methods used and have confidence in the consistent sterility of the tissue grafts. Tissue banks should validate their methods so that all stakeholders can trust the outcomes.

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.

Does personal genome testing drive service utilization in an adult preventive medicine clinic?

Personal genome testing (PGT) that assesses risk for common diseases may influence the use of preventive health services, but outcome data are limited. We aimed to assess health service utilization following PGT. We conducted a retrospective matched cohort study at an adult health clinic. Medical records of clients who pursued PGT at their comprehensive health assessment (CHA) over a 1-year period (N = 388) were reviewed and compared to age- and sex-matched clients who underwent CHA but not PGT (N = 388). We measured condition-specific health services used post CHA up to two subsequent visits while accounting for confounding factors (e.g., family history, health status, and age). A relatively equal number of post CHA services were used by clients who pursued PGT and those who did not pursue PGT (52% and 48%, respectively). Overall and across the majority of conditions examined, clients' service utilization was significantly associated with health status, e.g., clients identified as "at risk" on CHA for heart attack used 2.86 times more services than clients not at risk. Pursuing PGT was not significantly associated with increased use of services post CHA overall or for most of the conditions examined. Our data demonstrate that health status rather than pursuing PGT is the strongest driver of service utilization in this population. Overall, pursuit of PGT and PGT results does not appear to significantly drive the utilization of downstream health services.

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.

Maternal Lopinavir/Ritonavir Is Associated with Fewer Adverse Events in Infants than Nelfinavir or Atazanavir.

Combination antiretroviral therapy (cART) is successfully used for prevention of perinatal HIV transmission. To investigate safety, we compared adverse events (AE) among infants exposed to different maternal cART regimens. We reviewed 158 HIV-uninfected infants born between 1997 and 2009, using logistic regression to model grade ≥1 AE and grade ≥3 AE as a function of maternal cART and confounding variables (preterm, C-section, illicit drug use, race, ethnicity, infant antiretrovirals, and maternal viremia). Frequently used cART regimens included zidovudine (63%), lamivudine (80%), ritonavir-boosted lopinavir (37%), nelfinavir (26%), and atazanavir (10%). At birth, anemia occurred in 13/140 infants (9%), neutropenia in 27/107 (25%), thrombocytopenia in 5/133 (4%), and liver enzyme elevation in 21/130 (16%). Corresponding rates of AE at 4 weeks were 59/141 (42%), 54/130 (42%), 3/137 (2%), and 3/104 (3%), respectively. Serious AE (grade ≥ 3) exceeded 2% only for neutropenia (13% at birth; 9% at 4 weeks). Compared with infants exposed to maternal lopinavir/ritonavir, infants exposed to nelfinavir and atazanavir had a 5-fold and 4-fold higher incidence of AE at birth, respectively. In conclusion, hematologic and hepatic AE were frequent, but rarely serious. In this predominantly protease inhibitor-treated population, lopinavir/ritonavir was associated with the lowest rate of infant AE.

Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

Lymphatic filariasis and onchocerciasis are parasitic helminth diseases, which cause severe morbidities such as elephantiasis, skin disease and blindness, presenting a major public health burden in endemic communities. The anti-Wolbachia consortium (A·WOL: http://www.a-wol.com/) has identified a number of registered antibiotics that target the endosymbiotic bacterium, Wolbachia, delivering macrofilaricidal activity. Here we use pharmacokinetics/pharmacodynamics (PK/PD) analysis to rationally develop an anti-Wolbachia chemotherapy by linking drug exposure to pharmacological effect. We compare the pharmacokinetics and anti-Wolbachia efficacy in a murine Brugia malayi model of minocycline versus doxycycline. Doxycycline exhibits superior PK in comparison to minocycline resulting in a 3-fold greater exposure in SCID mice. Monte-Carlo simulations confirmed that a bi-daily 25-40 mg/Kg regimen is bioequivalent to a clinically effective 100-200 mg/day dose for these tetracyclines. Pharmacodynamic studies showed that minocycline depletes Wolbachia more effectively than doxycycline (99.51% vs. 90.35%) after 28 day 25 mg/Kg bid regimens with a more potent block in microfilarial production. PK/PD analysis predicts that minocycline would be expected to be 1.7 fold more effective than doxycycline in man despite lower exposure in our infection models. Our findings warrant onward clinical investigations to examine the clinical efficacy of minocycline treatment regimens against lymphatic filariasis and onchocerciasis.