Establishing a reproductive biorepository for basic and translational research: experience developing the reproductive subjects registry and sample repository.

PURPOSE: To report experience designing and establishing a reproductive registry and sample biorepository and to describe initial subject characteristics and biospecimens. METHODS: Beginning in December 2017, patients presenting for reproductive care at the University of Michigan were approached for study enrollment. Following consent, subjects completed detailed reproductive and health questionnaires. A variety of reproductive specimens and tissues were collected and processed for multiple downstream applications. RESULTS: Subject enrollment began in December of 2017. There are currently 1798 subjects enrolled. Female participants report a variety of reproductive disorders. Available samples include semen, sperm, follicular fluid, granulosa cells, immature oocytes, ovarian and uterine tissue, and blood samples. CONCLUSION: We report the successful establishment of a reproductive registry and sample biorepository. Furthermore, we describe methods for collection and storage of a variety of reproductive tissue processed for multiple downstream translational applications.

Immunosuppressive Myeloid Cells' Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy.

Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM.

A Review of Animal Models Investigating the Reproductive Effects of Gender-Affirming Hormone Therapy.

Gender-affirming hormone therapy (GAHT) is an important component in the process of transitioning for many transgender and gender-diverse (TGD) individuals. Multiple medical organizations recommend fertility preservation counseling prior to initiation of GAHT; however, there remains little high-quality data regarding the impact of GAHT on fertility and reproductive function. A PubMed literature review was performed using Boolean search operators linking keywords or phrases such as "mouse", "rat", "primate", "animal model", "transgender", "gender", "estrogen", "testosterone", "fertility", and "fertility preservation". Recent research has produced a number of animal models of GAHT that utilize similar hormonal regimens and produce similar phenotypic results to those used and observed in human patients. Specific to testosterone(T)-containing GAHT, animals demonstrate loss of menstrual cyclicity with therapy, resumption of menses on cessation of therapy, suppression of gonadotropin levels, and physical changes such as clitoromegaly. Models mimicking GAHT for transmasculine individuals in the peripubertal period demonstrate that pretreatment with GnRHa therapy does not modify the effects of subsequent T administration, which were similar to those described in adult models. Both models suggest promising potential for future fertility with cessation of T. With estradiol (E)-containing GAHT, animals exhibit decreased size of testicles, epididymis, and seminal vesicles, as well as ongoing production of spermatocytes, and seminiferous tubule vacuolization. Given the ethical challenges of conducting human studies in this area, high-fidelity animal models represent a promising opportunity for investigation and could eventually transform clinical counseling about the necessity of fertility preservation. Future studies should better delineate the interactions (if any exist) between treatment attributes such as dosing and duration with the extent of reversibility of reproductive perturbations. The development of models of peripubertal feminizing GAHT is an additional area for future work.

A review of fertility preservation in patients with breast cancer.

Breast cancer remains the most common cancer diagnosed in women and causes more lost disability-adjusted life years (DALYs) than any other cancer worldwide; however, improvements in therapies have led to increased survival and therefore a new focus on quality of life following treatment. Fertility is an important concern among cancer survivors of reproductive age. The purpose of this article is to contextualize the importance of oncofertility services for women with breast cancer and review options for fertility preservation, including oocyte/embryo cryopreservation, GnRH agonist therapy, and ovarian tissue cryopreservation. We also discuss special considerations for preimplantation genetic testing for women with germline pathogenic mutations associated with breast cancer, as well as issues related to endocrine therapy. Finally, we review barriers to accessing fertility preservation services, including cost of treatment and lack of referral to reproductive care providers or fertility preservation programs.

Assisted reproductive technology: considerations for the nonheterosexual population and single parents.

This study aimed to discuss fertility concerns unique to the lesbian, gay, bisexual, transgender, queer, plus and single-parent-to-be populations and review special considerations regarding the evaluation and treatment of these patients relevant to the practicing reproductive medicine provider. The use of assisted reproductive technology has rapidly increased over the past 50 years. Given these trends, providers can expect a greater diversity of patients making use of these technologies. Both the lesbian, gay, bisexual, transgender, queer, plus community and single parents-to-be represent understudied and important populations who often require the use of assisted reproductive technology to build their families. The American Society for Reproductive Medicine advocates for equitable treatment of patients regardless of sexual orientation and partner status, and health care providers working in the fertility field should be comfortable and confident in assessing the needs of and providing care to these populations.

Outcomes of Fertility Preservation Consults for Women at Risk for Primary Ovarian Insufficiency Due to History of Cancer Treatment or Mosaic Turner Syndrome.

Adolescents and young adults (AYAs) at risk of primary ovarian insufficiency (POI) often request fertility preservation consultation. We report consult/treatment outcomes for 21 cancer survivors and 3 mosaic Turner syndrome (TS) patients (mean age 21.6 at consult, 3 with POI). Ten AYAs (9 survivors, 1 mosaic TS) attempted ovarian stimulation; 4 cancelled for poor response. Of completed cycles, mean 3.8 mature oocytes were retrieved, with mean anti-Müllerian hormone 0.653 ng/mL. Ovarian stimulation for mosaic TS AYA and survivors is possible, even with diminished ovarian reserve. Further study is needed to establish guidelines for patient selection, treatment timing, and stimulation protocols.

INX-08189, a phosphoramidate prodrug of 6-O-methyl-2'-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties.

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Surgery Residents' Experiences With Seriously-Ill and Dying Patients: An Opportunity to Improve Palliative and End-of-Life Care.

OBJECTIVE: To describe how and when surgery residents provided primary palliative care and engaged specialty palliative care services. DESIGN: Phase I consisted of a previously validated survey instrument supplemented with additional questions. We then conducted semistructured interviews with a subset of the survey respondents (Phase II). Using thematic analysis, we characterized surgery residents' perceptions of palliative care delivery among surgical patients. SETTING: General surgery residency programs across the state of Michigan. PARTICIPANTS: General surgery residents across the state of Michigan. All residents in participating programs were invited to complete the survey in Phase I. Phase II consisted of a subset of the survey respondents who underwent semistructured interviews. Interview respondents were sampled to reflect the overall surveyed group. RESULTS: Among 119 survey respondents (response rate 70%), all had encountered a palliative care specialist but only 58.8% had been taught when to consult or to refer to palliative care. Survey respondents reported on a multitude of barriers within the clinician, patient and family, and systemic domains. Interviews expanded on survey findings and 4 influential factors of palliative care delivery emerged: (1) Resident Education and Training; (2) Resident Attitudes Toward Palliative Care; (3) Knowledge of Palliative Care; and (4) Training within a Surgical Culture. CONCLUSIONS: This study reveals how surgery resident training and experiences impact palliative and end-of-life care for surgical patients at teaching institutions. Knowledge of how and when residents are providing primary palliative care and engaging with palliative care services will inform future knowledge and behavioral interventions for trainees who often provide care for patients nearing the end of life.

Urinary markers aiding in the detection and risk stratification of prostate cancer.

The purpose of this review is to highlight the role of existing and promising urinary biomarkers for the detection and prognostication of prostate cancer (PCa). A number of novel urinary biomarkers have been introduced into the clinical space, which in combination with clinical variables, have demonstrated an increased ability to select patients for biopsy and identify men at risk of harboring clinically significant PCa. Though a number of assays require further validation, initial data is promising and forthcoming results will ultimately determine their clinical utility and commercial availability. For the past 30 years, first-line screening for PCa has relied on measurement of serum prostate-specific antigen (PSA) levels and the results from a digital rectal exam (DRE). A large body of evidence from the last 3 decades indicates that these screening methods are problematic, and often inadequate for detecting clinically significant PCa. Extensive efforts have recently been made to identify and commercialize novel PCa biomarkers for more effective detection of PCa, either alone or in combination with current screening methods. This review article highlights problems with current screening standards, and discusses 6 urinary biomarker assays in terms of their ability to detect and risk-stratify PCa: prostate cancer antigen 3 (PCA3), TMPRSS2-ERG, second chromosome locus associated with prostate-1 (SChLAP1), ExoDx, SelectMDx, and Michigan Prostate Score (MiPS).

Localized Inflammatory Myofibroblastic Tumor Involving the Genitourinary System: Adolescent Case Series and Review.

We describe four adolescent cases of inflammatory myofibroblastic tumor involving the genitourinary system. Two patients with masses of the urinary bladder presented with gross hematuria. The third patient presented with left flank pain and a mass encasing the left ureter causing hydronephrosis. The fourth patient presented with a painless, growing palpable mass of the left hemiscrotum. Currently, no standards exist for the management of inflammatory myofibroblastic tumors. Herein, we discuss the work-up and treatment approaches taken in each case.

Induction of bivalent immune responses by expression of dengue virus type 1 and type 2 antigens from a single complex adenoviral vector.

There are approximately 100 million new cases of dengue (DEN) virus infection each year. Infection can result in illness ranging from a mild fever to hemorrhaging, shock, or even death. There are four serotypes of dengue virus (DEN1-4), and immunity to one serotype does not cross protect from infection with other serotypes. Currently there are no approved vaccines for dengue fever. In this report, we describe the construction of a bivalent dengue virus vaccine using a complex recombinant adenovirus approach to express multiple genes of DEN1 and DEN2 serotypes. In vaccinated mice, this vector induced humoral immune responses against all four dengue serotypes as measured by enzyme-linked immunosorbent assay. However, the neutralizing antibody responses were specific for DEN1 and DEN2 serotypes. Expansion of this vaccine development platform towards the DEN3 and DEN4 serotypes can lead towards the development of an adenovirus-based tetravalent dengue vaccine.

Preclinical Efficacy and Safety Profile of Allometrically Scaled Doses of Doxycycline Used to Turn "On" Therapeutic Transgene Expression from High-Capacity Adenoviral Vectors in a Glioma Model.

Glioblastoma multiforme (GBM) is the most commonly occurring primary brain cancer in adults, in whom its highly infiltrative cells prevent total surgical resection, often leading to tumor recurrence and patient death. Our group has discovered a gene therapy approach for GBM that utilizes high-capacity "gutless" adenoviral vectors encoding regulatable therapeutic transgenes. The herpes simplex type 1-thymidine kinase (TK) actively kills dividing tumor cells in the brain when in the presence of the prodrug, ganciclovir (GCV), whereas the FMS-like tyrosine kinase 3 ligand (Flt3L) is an immune-stimulatory molecule under tight regulation by a tetracycline-inducible "Tet-On" activation system that induces anti-GBM immunity. As a prelude to a phase I clinical trial, we evaluated the safety and efficacy of Food and Drug Administration (FDA)-approved doses of the tetracycline doxycycline (DOX) allometrically scaled for rats. DOX initiates the expression of Flt3L, which has been shown to recruit dendritic cells to the brain tumor microenvironment-an integral first step in the development of antitumor immunity. The data revealed a highly safe profile surrounding these human-equivalent doses of DOX under an identical therapeutic window as proposed in the clinical trial. This was confirmed through a neuropathological analysis, liver and kidney histopathology, detection of neutralizing antibodies, and systemic toxicities in the blood. Interestingly, we observed a significant survival advantage in rats with GBM receiving the 300 mg/day equivalent dosage of DOX versus the 200 mg/day equivalent. Additionally, rats rejected "recurrent" brain tumor threats implanted 90 days after their primary brain tumors. We also show that DOX detection within the plasma can be an indicator of optimal dosing of DOX to attain therapeutic levels. This work has significant clinical relevance for an ongoing phase I clinical trial in humans with primary GBM and for other therapeutic approaches using Tet-On transactivation system in humans.

Phosphoramidate ProTides of 2'-C-methylguanosine as highly potent inhibitors of hepatitis C virus. Study of their in vitro and in vivo properties.

Hepatitis C virus infection constitutes a serious health problem in need of more effective therapies. Nucleoside analogues with improved exposure, efficacy, and selectivity are recognized as likely key components of future HCV therapy. 2'-C-Methylguanosine triphosphate has been known as a potent inhibitor of HCV RNA polymerase for some time, but the parent nucleoside is only moderately active due to poor intracellular phosphorylation. We herein report the application of phosphoramidate ProTide technology to bypass the rate-limiting initial phosphorylation of this nucleoside. Over 30 novel ProTides are reported, with variations in the aryl, ester, and amino acid regions. l-Alanine compounds are recognized as potent and selective inhibitors of HCV in replicon assay but lack rodent plasma stability despite considerable ester variation. Amino acid variation retaining the lead benzyl ester moiety gives an increase in rodent stability but at the cost of potency. Finally l-valine esters with ester variation lead to potent, stable compounds. Pharmacokinetic studies on these agents in the mouse reveal liver exposure to the bioactive triphosphate species following single oral dosing. Systemic exposure of the ProTide and parent nucleoside are low, indicating possible low toxicity in vivo, while liver concentrations of the active species may be predictive of efficacy in the clinic. This represents one of the most thorough cross-species studies of ProTides to date.

Multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against H5N1 avian influenza viruses.

There are legitimate concerns that the highly pathogenic H5N1 avian influenza virus could adapt for human-to-human transmission and cause a pandemic similar to the 1918 "Spanish flu" that killed 50 million people worldwide. We have developed pandemic influenza vaccines by incorporating multiple antigens from both avian and Spanish influenza viruses into complex recombinant adenovirus vectors. In vaccinated mice, these vaccines induced strong humoral and cellular immune responses against pandemic influenza virus antigens, and protected vaccinated mice against lethal H5N1 virus challenge. These results indicate that this multi-antigen, broadly protective vaccine may serve as a safer and more effective approach than traditional methods for development of a pandemic influenza vaccine.

Two complex, adenovirus-based vaccines that together induce immune responses to all four dengue virus serotypes.

Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.

Complex adenovirus-mediated expression of West Nile virus C, PreM, E, and NS1 proteins induces both humoral and cellular immune responses.

West Nile Virus (WNV), a member of the family Flaviviridae, was first identified in Africa in 1937. In recent years, it has spread into Europe and North America. The clinical manifestations of WNV infection range from mild febrile symptoms to fatal encephalitis. Two genetic lineages (lineages I and II) are recognized; lineage II is associated with mild disease, while lineage I has been associated with severe disease, including encephalitis. WNV has now spread across North America, significantly affecting both public and veterinary health. In the efforts to develop an effective vaccine against all genetic variants of WNV, we have studied the feasibility of inducing both neutralizing and cellular immune responses by de novo synthesis of WNV antigens using a complex adenoviral vaccine (CAdVax) vector. By expressing multiple WNV proteins from a single vaccine vector, we were able to induce both humoral and cellular immune responses in vaccinated mice. Neutralization assays demonstrated that the antibodies were broadly neutralizing against both lineages of WNV, with a significant preference for the homologous lineage II virus. The results from this study show that multiple antigens synthesized de novo from a CAdVax vector are capable of inducing both humoral and cellular immune responses against WNV and that a multiantigen approach may provide broad protection against multiple genetic variants of WNV.

Development of a cAdVax-based bivalent ebola virus vaccine that induces immune responses against both the Sudan and Zaire species of Ebola virus.

Ebola virus (EBOV) causes a severe hemorrhagic fever for which there are currently no vaccines or effective treatments. While lethal human outbreaks have so far been restricted to sub-Saharan Africa, the potential exploitation of EBOV as a biological weapon cannot be ignored. Two species of EBOV, Sudan ebolavirus (SEBOV) and Zaire ebolavirus (ZEBOV), have been responsible for all of the deadly human outbreaks resulting from this virus. Therefore, it is important to develop a vaccine that can prevent infection by both lethal species. Here, we describe the bivalent cAdVaxE(GPs/z) vaccine, which includes the SEBOV glycoprotein (GP) and ZEBOV GP genes together in a single complex adenovirus-based vaccine (cAdVax) vector. Vaccination of mice with the bivalent cAdVaxE(GPs/z) vaccine led to efficient induction of EBOV-specific antibody and cell-mediated immune responses to both species of EBOV. In addition, the cAdVax technology demonstrated induction of a 100% protective immune response in mice, as all vaccinated C57BL/6 and BALB/c mice survived challenge with a lethal dose of ZEBOV (30,000 times the 50% lethal dose). This study demonstrates the potential efficacy of a bivalent EBOV vaccine based on a cAdVax vaccine vector design.

Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges.

The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guinea pigs. Significantly, guinea pigs vaccinated with at least 5 x 10(7) pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV.

De novo syntheses of Marburg virus antigens from adenovirus vectors induce potent humoral and cellular immune responses.

Marburg virus (MARV) is an African filovirus that causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, there are no MARV vaccines or therapies approved for human use. We hypothesized that developing a vaccine that induces a de novo synthesis of MARV antigens in vivo will lead to strong induction of both a humoral and cell-mediated immune response against MARV. Here, we develop and characterize three novel gene-based vaccine candidates which express the viral glycoprotein (GP) from either the Ci67, Ravn or Musoke strain of MARV. Immunization of mice with complex adenovirus (Ad)-based vaccine candidates (cAdVax vaccines), led to efficient production of both antibodies and cytotoxic T lymphocytes (CTL) specific to Musoke strain GP and Ci67 strain GP, respectively. Antibody responses were also shown to be cross-reactive across the MARV strains, but not cross-reactive to Ebola virus, a related filovirus. Additionally, three 1 x 10(8)pfu doses of vaccine vector were demonstrated to be safe in mice, as this did not lead to any detectable toxicity in liver or spleen. These promising results indicate that a cAdVax-based vaccine could be effective for induction of both humoral and cell-mediated immune responses to multiple strains of the Marburg virus.