HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection: DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells.

DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.

Evaluation of embryo aneuploidy (PGT-A) and endometrial receptivity (ERA) testing in patients with recurrent implantation failure in ICSI cycles.

OBJECTIVE: The objective of a study was to assess the ability of the pre-implantation genetic testing of embryos for aneuploidy (PGT-A) and Endometrial receptivity array (ERA)-alone or in combination to improve the clinical outcomes in intracytoplasmic sperm injection (ICSI) cycles in patients with repeated implantation failure (RIF). METHODS: This was a retrospective study of the 253 cycles with a history of the previous RIF. They were divided into four groups: Group I - frozen embryo transfers without any additional tests or procedures (pure FET), n = 72 cycles; Group II - FET with PGT-A, n = 87; Group III - FET with PGT-A and ERA, n = 72; Group IV - FET with ERA, n = 22. RESULTS: Median age of the entire study group for the females was 35 years. Only Group II (FET + PGT-A) showed statistically significant higher chance in achieving both biochemical (p = .01, OR = 5.5) and clinical pregnancy (p =.049, OR = 2.3), as compared to the Group I (FET with no additional tests). Both Group III and Group IV failed to demonstrate better clinical outcomes as compared to the Group I. CONCLUSIONS: Patients with RIF can benefit from testing for embryo aneuploidy using the PGT-A method, but the ability of the ERA test to improve the clinical outcome in ICSI cycles seems to be rather limited. Although the endometrium cycle is also weakened with age, the contribution of the embryo genetic quality is evidently more important for successful implantation, although in principle both factors reflect the reproductive health.

Prevalence of testosterone deficiency among aging men with and without morbidities.

In this cross-sectional study 1852 men aged 40-70 years attending primary health care were invited to fill out the aging male symptoms (AMS) scale. Out of these, 1222 men were found positive for the AMS and agreed to provide blood samples for the general blood test, lipid profile, glucose levels, and assessment of both total and free testosterone (T) levels. Men were screened for the following morbidities and syndromes: dyslipidemia, arterial hypertension, obesity, type II diabetes, metabolic syndrome, and chronic obstructive pulmonary disease (COPD). Testosterone deficiency was diagnosed if total T ≤ 3.46 ng/mL or free T ≤ 72 pg/mL. Among all 1222 men with positive AMS, decreased blood testosterone levels were detected in 669 men (55%). A total of 402 men were found healthy and 820 men were detected with different morbidities. Out of 669 men with testosterone deficiency, only 2.8% had no co-morbidities and 97.2% were men with co-morbidities. Testosterone levels were found significantly higher among healthy men (median 4.7 ng/mL) as compared to the men with morbidities (median 2.55 ng/mL, p<.001), adjusted for age. Testosterone deficiency was detected in significantly lower proportion of 402 men without co-morbidities as compared to the 820 men with co-morbidities: in 19 men (4.7) and in 650 men (79.3%, p<.05), respectively.

Case of Inherited Partial AZFa Deletion without Impact on Male Fertility.

Male factor infertility accounts for 40-50% of all infertility cases. Deletions of one or more AZF region parts in chromosome Y are one of the most common genetic causes of male infertility. Usually full or partial AZF deletions, including genes involved in spermatogenesis, are associated with spermatogenic failure. Here we report a case of a Caucasian man with partial AZFa region deletion from a couple with secondary infertility. Partial AZFa deletion, involving part of USP9Y gene appears to be benign, as we proved transmission from father to son. According to our results, it is recommended to revise guidelines on markers selected for testing of AZFa region deletion, to be more selective against DDX3Y gene and exclude probably benign microdeletions involving only USP9Y gene.

The application of PGT-A for carriers of balanced structural chromosomal rearrangements.

The aim of this study was to analyze differences in chromosomal aberrations and euploidy in embryos of each translocation type and gender of carrier in the case series of 10 couples with balanced translocations who underwent IVF with embryos trophectoderm (TE) biopsy and PGT-A to detect chromosomal aberrations. This is a Case Series (Retrospective study). In each case, controlled ovarian hyperstimulation, oocyte insemination with intracytoplasmic sperm injection (ICSI) and cultivation gave multiple blastocysts, that underwent trophectoderm (TE) biopsy with PGT-A analysis using aCGH and NGS. Number of total unbalanced translocations compared to the number of sporadic aneuploid embryos was 39.6% to 39.6% (50% to 50% of all 37 aneuploid embryos). The highest euploidy rate was in male carrier group - 26.7% and the lowest in the Robertsonian translocation carrier group - 18.2%. Sporadic aneuploidy - 68.2% was highest in Robertsonian translocation carrier group and lowest in female group - 11.1%. Chromosomal aberrations related to translocation were highest in female carrier group - 77.8% and lowest in Robertsonian translocation carrier group - 13.6%. Our study showed that expectancy of total embryo aneuploidy rates will be higher in carriers, than in people with normal karyotype. The prevalence of chromosomal aberrations related to translocation was 4.5 times higher in Reciprocal carrier group than in Robertsonian translocation carrier group. Among maternal and paternal carrier groups, the embryos from female carriers had the lowest euploidy rate, unbalanced translocation rate 4.7 times higher than in the male carrier group and higher total aneuploidy rates.