P4-ATPase endosomal recycling relies on multiple retromer-dependent localization signals.

Type IV P-type ATPases (P4-ATPases) are lipid flippases that generate an asymmetric membrane organization essential for cell viability. The five budding yeast P4-ATPases traffic between the Golgi complex, plasma membrane, and endosomes but how they are recycled from the endolysosomal system to the Golgi complex is poorly understood. In this study, we find that P4-ATPase endosomal recycling is primarily driven by the retromer complex and the F-box protein Rcy1. Defects in P4-ATPase recycling result in their mislocalization to the vacuole and a substantial loss of membrane asymmetry. The P4-ATPases contain multiple predicted retromer sorting signals, and the characterization of these signals in Dnf1 and Dnf2 led to the identification of a novel retromer-dependent signal, IPM[ST] that acts redundantly with predicted motifs. Together, these results emphasize the importance of endosomal recycling for the functional localization of P4-ATPase and membrane organization.

Stem-Cell, Shockwave, and Platelet Rich Plasma Therapy for the Treatment of Erectile Dysfunction and Peyronie's Disease: A Survey of Clinics Across the USA.

OBJECTIVE: To identify clinics offering off-label therapies for erectile dysfunction (ED) and Peyronie's disease (Pyd) including stem cell, platelet-rich plasma (PRP), and shockwave therapy and to determine the transparency they provided to patients inquiring about these treatment modalities. METHODS: Clinics were identified in different regions in the US using a systematic search on online website directories and were approached by asking a series of standardized questions regarding the cost of treatment, duration of therapy, the medical staff involved, and patient outcome data. A total of 26 clinics were surveyed for stem cell therapy, 26 for PRP treatment, and 27 for shockwave therapy. RESULTS: Of the 79 clinics contacted, 93.7% provided some answers to the questions we asked, with a majority offering treatments for both ED and Pyd. The cost of treatment varied widely between clinics. The average cost per stem cell therapy injection was $5291, PRP per injection was $1336, and shockwave therapy per session was $413. A physician was involved in 67% of treatments, and only 6 of 79 clinics reported that a urologist was involved. Over 75% of the clinics reported patient satisfaction following treatment. Durability of benefits to patients ranged from months to years according to the clinics' reports. CONCLUSION: Our data not only demonstrate the widespread use of off-label therapies for ED and PyD across the United States but also the lack of scientific data to support the claims made to patients. This study highlights the need for more oversight and standardization in novel regenerative therapies for ED and PyD.

An interaction between ß'-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling.

The essential COPI coat mediates retrieval of transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (binding of coatomer, cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.

Quantification of Golgi Protein Mislocalization to the Budding Yeast Vacuole.

The localization of proteins to the Golgi complex is a dynamic process requiring sorting signals in the cytosolic domains of resident Golgi proteins and retrograde vesicular trafficking. Disruptions in these signals or in the retrograde pathways often lead to mislocalization of Golgi proteins to the vacuole in budding yeast. The extent of vacuolar mislocalization can be quantified through colocalization of GFP-tagged Golgi proteins with fluorescent dyes that mark either the vacuole limiting membrane or the vacuole lumen. Manders' colocalization coefficient (MCC) is a useful tool for quantifying the degree of colocalization. However, the dilution of fluorescence signal intensity that occurs when GFP-tagged Golgi proteins mislocalize to the much larger vacuole is problematic for thresholding the images prior to calculating the MCC. In this chapter, we describe the use of Multi-Otsu thresholding in ImageJ to quantify the degree of GFP-tagged protein mislocalization to the vacuole. Furthermore, these methods can be applied to other colocalization events within the cell.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of ß'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

Advanced Paternal Age and Sperm DNA Fragmentation: A Systematic Review.

PURPOSE: Male ageing is often associated with defective sperm DNA remodeling mechanisms that result in poorly packaged chromatin and a decreased ability to repair DNA strand breaks. However, the impact of advanced paternal age on DNA fragmentation remains inconclusive. The aim of the present systematic review was to investigate the impact of advancing paternal age (APA) on DNA fragmentation. MATERIALS AND METHODS: We conducted a thorough search of listed publications in Scopus, PubMed, and EMBASE, in accordance with the PRISMA guidelines. RESULTS: We identified 3,120 articles, of which nineteen were selected for qualitative analysis, resulting in a sample of 40,668 men. Of the 19 articles evaluating the impact of APA on DFI% (DNA fragmentation Index) included, 4 were on Normozoospermic and subfertile men, 3 on normozoospermic, Oligoasthenoteratozoospermic and Teratozoospermic, 6 on fertile and infertile men, 4 on just infertile men, and 2 evaluated a general population. Seventeen of the ninrnteen studies demonstrated APA's effect and impact on DFI%. CONCLUSIONS: Although there was no universal definition for APA, the present review suggests that older age is associated with increased DFI. In elderly men with normal semen parameters, further studies should be performed to assess the clinical implications of DFI, as a conventional semen analysis can often fail to detect an etiology for infertility.

Impact of the SARS-CoV-2 virus on male reproductive health.

The coronavirus 2019 (COVID-19) pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 160 million infections and 3.5 million deaths globally. Men are disproportionately affected by COVID-19, having more severe disease with higher mortality rates than women. Androgens have been implicated as the underlying cause for more severe disease, as the androgen receptor has been noted to upregulate the cell surface receptors that mediate viral cell entry and infection. Unfortunately, despite testosterone's potential role in COVID-19 prognosis, androgen deprivation therapy is neither protective nor a treatment for COVID-19. Interestingly, the male reproductive organs have been found to be vulnerable in moderate to severe illness, leading to reports of erectile dysfunction and orchitis. COVID-19 viral particles have been identified in penile and testis tissue, both in live patients who recovered from COVID-19 and post mortem in men who succumbed to the disease. Although sexual transmission remains unlikely in recovered men, moderate to severe COVID-19 infection can lead to germ cell and Leydig cell depletion, leading to decreased spermatogenesis and male hypogonadism. The objective of this review is to describe the impact of SARS-CoV-2 on male reproductive health. There are still many unanswered questions as to the specific underlying mechanisms by which COVID-19 impacts male reproductive organs and the long-term sequelae of SARS-CoV-2 on male reproductive health.

Current Treatment Modalities Targeting Tumor Microenvironment in Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) is responsible for significant cancer-related morbidity and mortality following local treatment failure in men. The initial stages of PCa are typically managed with a combination of surgical resection and/or androgen deprivation therapy (ADT). Unfortunately, a significant proportion of PCa continues to progress despite being at castrate levels of testosterone (<50 ng/dl), at which point it is coined castration-resistant prostate cancer (CRPC). In recent years, many novel therapeutics and drug combinations have been created for CRPC patients. These include immune checkpoint inhibitors, chemokine receptor antagonists, steroidogenic enzyme inhibition, and novel tyrosine kinase inhibitors as well as combinations of drugs. The selection of the most appropriate therapy depends on several factors like stage of the disease, age of the patient, metastasis, functional status, and response towards previous therapies. Here, we review the current state of the literature regarding treatment modalities, focusing on the treatment recommendations per the American Urological Association (AUA), recent clinical trials, and their limitations. An accurate and reliable overview of the strengths and limitations of PCa therapeutics could also allow personalized therapeutic interventions against PCa.

Stimulant-Induced Pituitary Failure and Reversible Azoospermia.

Attention-deficit hyperactivity disorder (ADHD) is a commonly diagnosed disorder that is managed with stimulant medications, which function by increasing the levels of dopamine in the brain. Excess dopamine has been known to affect several body systems, including the endocrine system. This case presents male factor infertility caused by a negative interaction between excess dopamine and the endocrine system, inducing pituitary failure, which led to azoospermia. The patient and her partner presented to the fertility clinic for evaluation after one year of failing to conceive. The patient's partner had been treated throughout the conception of their first three pregnancies for ADHD with methylphenidate (Ritalin) for many years; however, eight months prior to presentation at the clinic, the partner had been switched to amphetamine-dextroamphetamine (Adderall) for treatment of ADHD. A fertility evaluation revealed azoospermia which was confirmed via two separate semen analyses two weeks apart. In addition, the patient's total testosterone, prolactin, luteinizing hormone, and follicle-stimulating hormone were below normal limits. A normal semen analysis was obtained after a five-month withdrawal of amphetamine-dextroamphetamine, which was followed by a naturally conceived pregnancy. The possibility of pre-testicular azoospermia caused by medication-induced pituitary failure should be considered in males prescribed stimulant medication who are seeking to reproduce.

Elevated sperm DNA fragmentation does not predict recurrent implantation failure.

In this study, we sought to determine whether sperm DNA fragmentation (DFI%) and high DNA stainability (HDS%) evaluated by sperm chromatin structure assay (SCSA) predict recurrent implantation failure (RIF) or pregnancy rate. A retrospective study was performed of consecutive cycles of ICSI treatment from 2009 to 2018. A total of 386 couples that underwent 1,216 frozen embryo transfer (FET) cycles were analysed. Mean female and male age was 34 ± 3.6 years and 37.3 ± 6.6 years, respectively, and a median total motile sperm count (TMSC) was 43.5 [9.9-105.5] million. Overall median DFI% and HDS% was 12 [7.1-18.9] and 9.6 [6.5-14.4] respectively. On multivariable analysis, DFI% and HDS% were not associated with RIF (DFI%: OR = 1.01, 95% CI: 0.98-1.04, p = .414; HDS%: OR = 0.97, 95% CI: 0.94-1.01, p = .107) or IVF success, defined as clinical pregnancy (DFI%: OR = 1.00, 95% CI: 0.99-1.01, p = .641; HDS%: OR = 1.01, 95% CI: 0.99-1.02, p = .565). We found that neither DFI% or HDS%, as assessed by SCSA, were predictive of RIF or pregnancy rate. This finding suggests that sperm DNA fragmentation does not predict RIF or pregnancy rate.

Androgenization in Klinefelter syndrome: Clinical spectrum from infancy through young adulthood.

Klinefelter syndrome (KS) is an uncommon chromosomal disorder in males that has a variable clinical appearance. Classic KS involves an extra X chromosome, (47, XXY), although other variations may exist, including a milder mosaic form as well as multiple extra sex chromosomes with more dramatic phenotypes. KS is underdiagnosed, especially pre-pubertally, owing to a paucity of concrete clinical signs; however, diagnostic rates increase during and after puberty, as the consequences of hypergonadotropic hypogonadism begin to manifest. Testicular failure causing decreased circulating testosterone (T) and germ cell depletion, a hallmark feature in KS, commonly begins shortly after the onset of puberty and leads to the most commonly recognized KS traits: small testes, azoospermia, gynecomastia, decreased facial and pubic hair. While many KS men maintain adequate T levels leading up to young adulthood, some may have lower T levels at an earlier age leading to varied levels of androgenization and clinical KS features. At certain critical time points, absent or decreased T may alter the development of normal male reproductive organs, external genitalia, development of secondary sexual characteristics and spermatogenesis. Testicular failure in utero may lead to ambiguous genitalia, cryptorchidism and/or hypospadias, all of which depend on fetal T production. In the neonatal period and childhood, decreased T levels during the mini-puberty of infancy may negatively impact germ cell differentiation and male neuropsychological development. Finally, decreased T during pubertal and young adulthood can lead to decreased virilization during puberty, eunuchoid skeleton and decreased spermatogenesis. Depending on the timing of the testicular failure, a reproductive window of sperm production may exist to achieve paternity for KS men. The presence or absence of clinical characteristics reflecting decreased androgenization provides an insight to the relative testicular function during these developmental time points for those with KS and contributes to variability within the syndrome.

Evaluation of SARS-CoV-2 in Human Semen and Effect on Total Sperm Number: A Prospective Observational Study.

PURPOSE: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a surge of research to help better understand the breadth of possible sequelae. However, little is known regarding the impact on semen parameters and fertility potential. We sought to investigate for presence of viral RNA in semen of men with SARS-CoV-2 infection and to evaluate its effect on semen parameters in ejaculate. MATERIALS AND METHODS: We prospectively recruited thirty men diagnosed with acute SARS-CoV-2 infection using real-time reverse transcriptase polymerase chain reaction (RT-PCR) of pharyngeal swab specimens. Semen samples were collected from each individual using mailed kits. Follow-up semen samples were done with mailed kits or in-person in office setting. Semen analysis and PCR was performed after samples were received. RESULTS: Thirty semen samples from recovered men were obtained 11-64 days after testing positive for SAR-CoV-2 infection. The median duration between positive SAR-CoV-2 test and semen collection was 37 days (interquartile range [IQR]=23). The median total sperm number (TSN) in ejaculate was 12.5 million (IQR=52.1). When compared with age-matched SARS-CoV-2(-) men, TSN was lower among SARS-CoV-2(+) men (p=0.0024). Five men completed a follow-up sperm analysis (median 3 months) and had a median TSN of 18 million (IQR=21.6). No RNA was detected by means of RT-PCR in the semen in 16 samples tested. CONCLUSIONS: SARS-CoV-2 infection, though not detected in semen of recovered men, can affect TSN in ejaculate in the acute setting. Whether SARS-CoV-2 can affect spermatogenic function long-term remains to be evaluated.

Clinical Update on Home Testing for Male Fertility.

Male factor infertility accounts for about 50% of the incidence of infertility in couples. In current practice, the men must attend a clinic or hospital facility to provide a semen analysis, which is key to the diagnosis of the male reproductive potential. However, many men are often embarrassed with the process and conventional semen analysis requires complex, labor intensive inspection with a microscope. To mitigate these problems, one of the solutions can be at-home semen analysis. In this review we examine the literature of currently available at home semen analysis test kits, describe their limitations, and compare them to the conventional lab-based methods.

Curcumin Supplementation and Endometrial Lining: Examining the Role and Pathophysiology of Use During Frozen-Thawed Embryo Transfer.

Curcumin is a commonly used herbal supplement purported for its antioxidant, anti-inflammatory, and antineoplastic properties. The effects of curcumin supplementation on endometrial lining have been proposed; however, endometrial preparation in the case of frozen-thawed embryo transfer (FET) has not been established. This case series references two scenarios where turmeric was ingested by the patient, and endometrial thickness was subsequently reduced disrupting the FET cycle. Throughout this case series, curcumin's possible interactions with the uterine lining are summarized. Additionally, these cases highlight the importance of physicians' awareness of taking a full history of any herbal remedies or supplements in addition to prescription or over-the-counter medications taken when undergoing treatment for controlled FET cycles or in-vitro fertilization (IVF). To our knowledge, no studies to date have investigated this relationship.

A cross-sectional comparison of secondary polycythemia in testosterone-deficient men treated with nasal testosterone gel vs. intramuscular testosterone cypionate.

INTRODUCTION: Secondary polycythemia is a known adverse effect of testosterone replacement therapy (TRT). Different testosterone formulations are available, with significantly different half-lives, which have varying influences on the development of secondary polycythemia. Herein, we compared the prevalence of secondary polycythemia in testosterone-deficient men treated with intranasal testosterone gel (Natesto®) vs. intramuscular testosterone cypionate (TC) therapy. METHODS: We performed a cross-sectional analysis of secondary polycythemia (hematocrit [Hct] ≥54%) in men who received TRT. We included a total of 60 men: 30 men who received Natesto (4.5% testosterone gel [tid, 5.5 mg/nostril, 11 mg/dose, 33 mg/day]), and 30 who received TC (between 0.5 and 1.0 mL or 100-200 mg intramuscularly weekly). A univariable and multiple regression analysis was performed considering last Hct measurement as the main outcome. The analyzed variables included were age, body mass index (BMI), smoking history, treatment group, and testosterone levels on followup. RESULTS: We identified polycythemia (Hct ≥54%) in 10% (3/30) of men who received TC. Additionally, in men treated with TC, 33.3% (10/30) had a Hct ≥50% during therapy. None of the men who received Natesto had a Hct ≥50% during therapy. On multivariable linear regression analysis, we demonstrated that the use of TC increased Hct by 3.24% (95% confidence interval [CI] 0.74-5.73%, p=0.012) compared to Natesto. CONCLUSIONS: The prevalence of polycythemia in men treated with Natesto was markedly lower compared to the men who received TC therapy.

A prospective study analyzing both inflation and deflation preference for commonly available inflatable penile prostheses.

Despite popularity, satisfaction rates of inflatable penile prosthesis (IPP) use can be improved by evaluating the ability to operate devices in the preoperative setting. The purpose of this study was to prospectively analyze the preference of three commonly available IPPs. In total, 125 IPP-naïve men 60 years of age or older were prospectively recruited from an outpatient Urology clinic from June 2019 to January 2020. A questionnaire standardized to all encounters was utilized to collect demographics, selected medical information, and key pinch strength. Participants were then asked to rank three models in terms of preference (from 1 to 3, 1 representing most preferred) for each inflation and deflation in a double-blinded manner. Statistical analysis was performed using ANOVA, a Chi-square test and multivariable logistical regression analysis. The results demonstrated preference for Coloplast Titan (44%) for inflation, and preference for AMS 700 (40%) for deflation. Men who preferred the Coloplast Titan inflation had a lower chance of preferring the AMS 700 MS deflation (OR = 0.29; p = 0.010) and Coloplast Titan Touch deflation (OR = 0.27; p = 0.012). Preference for Coloplast Titan was weakly associated with participant history of coronary artery disease (OR = 5.96, p = 0.006) and osteoarthritis (OR = 3.04, p = 0.044). Neither key pinch strength nor age was associated with preference for a particular model. IPP-naïve men over 60 years favor Coloplast Titan for inflation and AMS 700 for deflation, and men who preferred the Coloplast Titan for inflation were less likely to choose the AMS 700 MS or Coloplast Titan Touch for deflation. Further studies should aim to confirm these findings.

The Effect of Longer-Acting vs Shorter-Acting Testosterone Therapy on Follicle Stimulating Hormone and Luteinizing Hormone.

INTRODUCTION: Testosterone (T) replacement therapy causes suppression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) that can lead to decrease in semen parameters and possible infertility. Different T formulations may have varying suppression on FSH and LH. OBJECTIVE: To study whether shorter-acting T (multiple daily dosing) has less suppression on FSH and LH serum levels compared with longer-acting T (transdermal gel, injectable). METHODS: A systematic literature search was conducted by following the protocol based on Preferred Reporting Items for Systematic Reviews and Meta-Analysis protocols. We comprehensively reviewed the literature by systematically searching manuscripts indexed in PubMed from 1995 to March 13, 2019 to identify studies reporting changes in FSH and LH in hypogonadal men treated with exogenous T which evaluated the effect of exogenous T on FSH and LH. RESULTS: A total of 8 studies reported the effect of T on FSH and LH in 793 hypogonadal men: 2 used long-acting injectables (enanthate or undecanoate) in a total of 16 men, 5 used intermediate-acting daily topical gels or patches in a total of 471 men, and 1 used short-acting intranasal T (125 µL/nostril, twice a day or three times a day) in 306 men. Long-acting injectables decreased FSH by 86.3%, intermediate-acting daily gels/patches decreased FSH by 60.2%, and short-acting intranasal gel decreased FSH by 37.8%. Long-acting injectables decreased LH by 71.8%, intermediate-acting daily gels/patches decreased LH by 59.2%, and short-acting intranasal gel decreased LH by 47.3%. CONCLUSIONS: Our findings suggest that short-acting T preparations do not decrease serum FSH or LH to the same extent as longer-acting transdermal gels and injectables. However, further clinical trial data are necessary to determine whether the effect of short-acting TRT on gonadotropins translates into similar changes in semen parameters and fertility. Masterson TA, Turner D, Vo D, et al. The Effect of Longer-Acting vs Shorter-Acting Testosterone Therapy on Follicle Stimulating Hormone and Luteinizing Hormone. Sex Med Rev 2021;9:143-148.

Sperm DNA fragmentation index and high DNA stainability do not influence pregnancy success after intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the ability of sperm DNA fragmentation index (DFI%) and high DNA stainability (HDS%) to influence the chance of achieving pregnancy in couples undergoing intracytoplasmic sperm injection (ICSI) cycles. DESIGN: A retrospective study evaluating couples that underwent an ICSI cycle between 2009 - 2018. SETTING: High-volume reproductive center. PATIENTS: Consecutive couples who underwent an ICSI cycle and had a semen analysis with subsequent DFI% and HDS% testing, evaluated by Sperm Chromatin Structure Assay (SCSA). INTERVENTIONS: Measurement of DFI% and HDS% prior to ICSI cycle. MAIN OUTCOME MEASURES: To determine whether DFI% or HDS% of sperm was predictive of the number of ICSI cycles until the first clinical intrauterine pregnancy. RESULTS: A total of 550 couples who underwent 1050 ICSI cycles were analyzed. Of those, a total of 330 couples achieved pregnancy. As expected, in couples that achieved pregnancy, females were younger (33.7 ± 3.6 years vs 35.3 ± 3.4 years; p < 0.001) and underwent fewer cycles (2 [1-2] vs 2 [1-3]; p =0.001). Importantly, the DFI% and HDS% were similar between couples who achieved pregnancy (DFI% = 12.9 [8-20]; HDS% = 9.3 [6.1-14.6]) and couples who did not (DFI% =12.2 [7.1-20.2]; HDS% = 9.1 [6.7-14]). A multivariable-adjusted analysis evaluating female age at the first cycle was negatively associated with pregnancy (OR = 0.827, 95% CI: 0.778 - 0.879; p < 0.001). CONCLUSIONS: Neither DFI nor HDS at baseline influence the chances of a couple to achieve pregnancy after ICSI. Increased female age and couples who underwent more ICSI cycles were associated with lower chances of achieving pregnancy.