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Improved in vitro development of cloned bovine embryos using S-adenosylhomocysteine, a non-toxic epigenetic modifying reagent

Jafari S, Hosseini MS, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.

Mol. Reprod. Dev., 2011 Aug;78(8):576-84.
PMID: 21721066 DOI: 10.1002/mrd.21344

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.
Fulltext Epigenetic modification does not determine the time of POU5F1 transcription activation in cloned bovine embryos

Jafari S, Hosseini SM, Hajian M, Forouzanfar M, Jafarpour F, Abedi P, et al.

J Assist Reprod Genet, 2011 Nov;28(11):1119-27.
PMID: 22020531 DOI: 10.1007/s10815-011-9638-1

To investigate the effect of epigenetic modification on pattern, time and capacity of transcription activation of POU5F1, the key marker of pluripotency, in cloned bovine embryos.
Fulltext Post-Vasectomy Semen Analysis: Optimizing Laboratory Procedures and Test Interpretation through a Clinical Audit and Global Survey of Practices

Agarwal A, Gupta S, Sharma RK, Finelli R, Kuroda S, Vij SC, et al.

World J Mens Health, 2022 Jul;40(3):425-441.
PMID: 35021311 DOI: 10.5534/wjmh.210191

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.

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