ABSTRACT
Problem Many studies have demonstrated the negative impact of high rates of NK cytotoxicity (NKc) on reproductive processes, but there is no agreement as to the appropriateness and feasibility of using the NKc for routine diagnostic in IVF patients. This study conducted a retrospective analysis of embryo transfer (ET) success rates and live birth rates (LBR) in patients with different NKc values. Method of study 1854 cycles of ET were selected and divided into three groups according to NKc levels, and randomized by anamnesis and age: normal (nNKc, n = 871), elevated (eNKc, n = 759), and reduced NKc (rNKc, n = 123). ET with donors' embryos (n = 101) were analyzed separately. NKc-to-K562 was measured in PBMC (peripheral blood mononuclear cells) by flow cytometry before ET. The patients did not obtain any additional treatments. Results Patients with eNKc, in addition to having reduced clinical pregnancy rates (OR1.59, p < 0.0001), had increased levels of subsequent pregnancy failures (OR2.545, p < 0.0001) when compared to nNKc patients. As a result, patients with eNKc had almost half the LBR than patients with nNKc (OR2.2, p < 0.0001). In patients with rNKc, LBR was also lowered. eNKc was equally unfavorable for implantation and delivery in cryo- or fresh cycles. Markedly, eNKc was much more unfavorable for reproduction than slightly elevated NKc. The donor's embryos were implanted irrespective of the recipient's NKc levels, but the later stages of pregnancy were worse in patients with eNKc. Conclusions Our findings highlighted the negative impact of high levels of NK cytotoxicity on pregnancy outcomes.
ABSTRACT
BACKGROUND: The mitogen-activated protein kinase (MAPK) pathway is functionally generic and critical in maintaining physiological homeostasis and normal tissue development. This pathway is under tight regulation, which is in part mediated by dual-specific phosphatases (DUSPs), which dephosphorylate serine, threonine, and tyrosine residues of the ERK family of proteins. DUSP5 is of high clinical interest because of mutations we identified in this protein in patients with vascular anomalies. Unlike other DUSPs, DUSP5 has unique specificity toward substrate pERK1/2. Using molecular docking and simulation strategies, we previously showed that DUSP5 has two pockets, which are utilized in a specific fashion to facilitate specificity toward catalysis of its substrate pERK1/2. Remarkably, most DUSPs share high similarity in their catalytic sites. Studying the catalytic domain of DUSP5 and identifying amino acid residues that are important for dephosphorylating pERK1/2 could be critical in developing small molecules for therapies targeting DUSP5. RESULTS: In this study, we utilized computational modeling to identify and predict the importance of two conserved amino acid residues, H262 and S270, in the DUSP5 catalytic site. Modeling studies predicted that catalytic activity of DUSP5 would be altered if these critical conserved residues were mutated. We next generated independent Glutathione-S-Transferase (GST)-tagged full-length DUSP5 mutant proteins carrying specific mutations H262F and S270A in the phosphatase domain. Biochemical analysis was performed on these purified proteins, and consistent with our computational prediction, we observed altered enzyme activity kinetic profiles for both mutants with a synthetic small molecule substrate (pNPP) and the physiological relevant substrate (pERK) when compared to wild type GST-DUSP5 protein. CONCLUSION: Our molecular modeling and biochemical studies combined demonstrate that enzymatic activity of phosphatases can be manipulated by mutating specific conserved amino acid residues in the catalytic site (phosphatase domain). This strategy could facilitate generation of small molecules that will serve as agonists/antagonists of DUSP5 activity.
