Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

X-Box Binding Protein 1 Is Essential for the Anti-Oxidant Defense and Cell Survival in the Retinal Pigment Epithelium

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD

A Meta-analysis of Multiple Myeloma Risk Regions in African and European Ancestry Populations Identifies Putatively Functional Loci

Genome-wide association studies (GWAS) in European populations have identified genetic risk variants associated with multiple myeloma (MM)

Post-vasectomy semen analysis: Optimizing laboratory procedures and test interpretation through a clinical audit and global survey of practices

Purpose: The success of vasectomy is determined by the outcome of a post-vasectomy semen analysis (PVSA). This article describes a step-by-step procedure to perform PVSA accurately, report data from patients who underwent post vasectomy semen analysis between 2015 and 2021 experience, along with results from an international online survey on clinical practice. Materials and Methods: We present a detailed step-by-step protocol for performing and interpretating PVSA testing, along with recommendations for proficiency testing, competency assessment for performing PVSA, and clinical and laboratory scenarios. Moreover, we conducted an analysis of 1,114 PVSA performed at the Cleveland Clinic’s Andrology Laboratory and an online survey to understand clinician responses to the PVSA results in various countries. Results: Results from our clinical experience showed that 92.1% of patients passed PVSA, with 7.9% being further tested. A total of 78 experts from 19 countries participated in the survey, and the majority reported to use time from vasectomy rather than the number of ejaculations as criterion to request PVSA. A high percentage of responders reported permitting unprotected intercourse only if PVSA samples show azoospermia while, in the presence of few non-motile sperm, the majority of responders suggested using alternative contraception, followed by another PVSA. In the presence of motile sperm, the majority of participants asked for further PVSA testing. Repeat vasectomy was mainly recommended if motile sperm were observed after multiple PVSA’s. A large percentage reported to recommend a second PVSA due to the possibility of legal actions. Conclusions: Our results highlighted varying clinical practices around the globe, with controversy over the significance of non-motile sperm in the PVSA sample. Our data suggest that less stringent AUA guidelines would help improve test compliance. A large longitudinal multi-center study would clarify various doubts related to timing and interpretation of PVSA and would also help us to understand, and perhaps predict, recanalization and the potential for future failure of a vasectomy.American Center for Reproductive Medicin

The Evolving Landscape of Male Varicocele Pathophysiology in the Era of Multi-Omics: A Narrative Review of the Current Literature

Male-factor infertility is implicated in over half of the millions of cases of infertility worldwide, and varicoceles are the most common correctable cause of male-factor infertility. The pathophysiologic mechanism for varicoceles is complex and next-generation technologies offer promising insights into the molecular underpinnings of this condition. In this narrative review, we highlight historical and contemporary paradigms associated with varicoceles, with an emphasis on the biological underpinnings of this disease. Specifically, we review the literature describing the underlying causes of varicoceles, discuss the molecular and cellular mechanisms causing pathological changes in some (but not all) men, and highlight key articles regarding the next-generation analyses (e.g., transcriptome, epigenome, proteome, and microbiome) being applied to better understand the condition and its treatment. These data demonstrate an ongoing evolution of the knowledge of varicoceles and the potential for improved personalized care in the future for men with this condition

The microbiome of the infertile male

Contrary to historic dogma, many tissues and organs in the human body contain a resident population of bacteria, fungi, and viruses collectively known as the microbiome. The microbiome plays a role in both homeostatic symbiosis and also pathogenic dysbiosis in a wide array of diseases. Our understanding of the relationship between the microbiome and male factor infertility is in its infancy but is slowly evolving. Recent literature indicates that semen (and likely the testis) is not sterile and contains a distinct microbiome, and these changes in its composition are associated with alterations in semen quality and fertility status. Preliminary investigation indicates that manipulating the human microbiome may have implications in improving semen parameters and fertility. In this review, we describe relationships between the microbiome and the genitourinary system, discuss the prior work on the relationship among bacteriospermia, leukocytospermia and male factor infertility, and summarize the current literature utilizing 16s rRNA-based next-generation sequencing on the seminal and testicular microbiome. We explore the specific microbial taxa implicated in various aspects of spermatic dysfunction and introduce preliminary evidence for therapeutic approaches to alter the microbiome and improve fertility status

Clinical outcomes of cryptozoospermic patients undergoing surgical sperm retrieval

IntroductionCryptozoospermia is defined by the World Health Organization (WHO) as the presence of isolated sperm cell in the ejaculate only identified after an extended microscopic search or after being pelleted. Although the number of spermatozoa is usually sufficient for intracytoplasmic sperm injection (ICSI), ICSI fails due to poor sperm quality in some cases. Contention remains regarding whether testicular sperm offers any advantage in this unique situation. At our tertiary referral center, we will offer patients a surgical sperm retrieval via conventional or microdissection testicular sperm extraction (microTESE) for men with cryptozoospermia and failed ICSI, or where ejaculated specimens are immotile or insufficient for ICSI. In this study, we sought to describe our experience and evaluate the predictors of success in cryptozoospermic patients who had microTESE at our center.MethodsWe retrospectively reviewed our electronic medical records for all patients with cryptozoospermia who underwent microTESE between 2007- 2021 for failed ICSI with ejaculated sperm or sperm quality deemed to be of insufficient quality for ICSI (e.g., nonmotile sperm). We evaluated demographics, preoperative lab results, pathology results, sperm retrieval rate (SRR) and ICSI outcomes.Results28 cryptozoospermic patients were identified. These patients underwent 37 unique microTESE. 22 of these men had failed previous ICSI treatment with ejaculated sperm, while the other 6 patients had ejaculated sperm with non-suitable quality for ICSI. None had genetic abnormalities. Successful retrieval of motile sperm suitable for ICSI was achieved in in 30 micro TESE procedures (SRR: 81.0%).14 out of 28 patients (50%) who underwent embryo transfer had positive pregnancy result, and 12/28 patients (42.8%) had successful live birth. The most common pathological pattern was hypospermatogenesis found in 65.3% (17/26). Fibrosis pathology was significantly higher in the negative pregnancy group. There were no postoperative complications noted.DisscussionThe use of testicular sperm in cryptozoospermic men with failed prior ICSI using ejaculated sperm has a high rate of pregnancy and live birth. While still controversial, our results suggest that surgical sperm retrieval is a viable option for these men with minimal risk of complications