Quantitative And Functional Changes Of Peripheral Natural Killer Cells And Regulatory T Cells In Women With Reproductive Failure After Artificial Insemination With Donor Sperm

Artificial insemination with donor sperm (AID) as the primary empirical treatment for uncured male infertility is world-wide used. However, the pregnancy rate per AID cycle was no more than 20% and the delivery rate reduced to less than 13% per insemination. Most of the women undergoing AID treatment experienced reproductive failure, including multiple insemination failure, biochemical pregnancy loss and sporadic miscarriage or embryo arrest. The underlying cause and precise mechanism remain unknown.Considerable evidence indicated that natural killer (NK) cells and regulatory T (Treg) cells played important roles in successful pregnancy. The dysregulation of NK and Treg cells, either in proportion (%) or function has been associated with some reproductive pathologies, such as recurrent spontaneous abortions (RSA), infertility and pre-eclampsia. Renewed interest has been found in recent observations that certain parameters of NK and Treg cells were changed in women with multiple implantation failures undergoing in-vitro fertilization (IVF) treatment. What's more, some parameters of NK and Treg cells were found to predict the outcome of the next pregnancy in women with reproductive failure. To the best of our knowledge, there was no report on the alterations of NK and Treg cells parameters in reproductive failure of other ARTs like AID.In attempting to answer this question, the present study analyzed the quantitative and functional differences of pre-conceptual peripheral NK and Treg cell between women with reproductive failures after AID cycle and normal controls with successful delivery. By use of Receiver operating characteristic (ROC) curve assay, we evaluated the predictive value of NK and Treg cell parameters, and identified a prospective cutoff with maximal sensitivity and specificity.PartⅠQuantitative and Functional Changes of Peripheral Natural Killer Cells in Women with Reproductive Failure after Artificial Insemination with Donor SpermObjective To determine if peripheral NK cell numbers and NK cell cytotoxicity (NKCC) are altered in women with reproductive failures undergoing AID treatment.Methods Seventy-five patients experiencing reproductive failure after AID cycles were recruited in this study. They experienced at least 4 times failures with negative pregnancy test (Group I, n = 25), biochemical pregnancy loss (Group II, n = 25), or underwent embryo growth arrest or miscarriage in first trimester (Group III, n = 25). Twenty healthy non-pregnant women with successful delivery were selected as controls. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation in late follicular phase blood samples. NK cell subsets within lymphocytes were evaluated by three-color flow cytometry and NKCC was evaluated after PBMC co-cultured with K562 target cells by two-color flow cytometry.Results The percentage of CD56~+ NK cells was increased in Group II and Group III compared with the controls (P < 0.05). The percentage of CD56~+CD16~+ NK cells in all study groups was higher than that in the controls (P < 0.05). However, there was no statistically significant difference in the percentage of CD56~+CD16- NK cells between the study and control groups (P > 0.05). The elevated percentage of NKCC was also detected in the peripheral blood of study groups compared with the controls (P < 0.01).Conclusion Our results implicated that the increase of peripheral CD56~+, CD56~+CD16~+ cytotoxic NK cell subsets and NKCC was related to the incidence of AID failures. PartⅡQuantitative and Functional Changes of Peripheral Regulatory Cells in Women with Reproductive Failure after Artificial Insemination with Donor SpermObjective To determine if peripheral Treg cell numbers and proportion of functional phenotype are altered in women with reproductive failures undergoing AID treatment.Methods Sixty patients experiencing reproductive failure after AID cycles were recruited in this study. They experienced at least 4 times failures with negative pregnancy test (Group I, n = 20), biochemical pregnancy loss (Group II, n = 20), or embryo disintegration or miscarriage in first trimester (Group III, n = 20). Twenty healthy non-pregnant women with one successful delivery were selected as normal controls. PBMC were isolated by density gradient centrifugation in the late follicular and luteal phase blood samples. Treg cell numbers and proportion of functional phenotype within CD4~+ cells were evaluated by three-colour flow cytometry.Results A significantly decreased proportion of CD4~+CD25~+FOXP3~+ functional phenotype in CD4~+ cells was detected in the peripheral blood of study groups in late follicular phase compared with normal controls (P < 0.01). And a significantly increased proportion of CD4~+CD25~+FOXP3~+ functional phenotype was detected in late follicular phase compared to that in late luteal phase in normal controls (P < 0.01). However, no significant difference was observed in the proportion of functional phenotype during the menstrual cycle in the study groups Also, there was no significant difference in the percentage of CD4~+CD25~+ Treg cells between the study and control groups.Conclusion Decreased proportion of functional Treg cell phenotype in peripheral blood may relate to AID failure. The incidence of decreased proportion of functional Treg phenotype may be interpreted by the deficiency of functional Treg phenotype expansion in menstrual cycle. Objective To evaluate the predictive value of NK and Treg cell parameters in predicting AID failure and find the best predictor.Methods The NK and Treg parameters which was found to contribute to the incidence of AID failure in part I and II, like the percentages of CD56~+, CD56~+CD16~+ NK subsets, NKCC and proportion of CD4~+CD25~+FOXP3~+ in CD4~+ cells were analyzed by receiver operating characteristic (ROC) curve. Area under curves (AUC), sensitivity and specificity of each value were calculated and further assessed potential biomarkers to predict AID failure.Results According to ROC analysis, AUC of CD56~+, CD56~+CD16~+, NKCC and CD4~+CD25~+FOXP3~+ percentage were 0.76, 0.77, 0.82 and 0.77 respectively. And CD56~+ percentage > 16%, CD56~+CD16~+ percentage > 12%, NKCC > 10% and CD4~+CD25~+FOXP3~+ proportion < 2% were highly sensitive and specific for estimating AID failure. Furthermore, NKCC > 10% was the best predictor.Conclusion The percentages of CD56~+, CD56~+CD16~+ NK subsets, NKCC and CD4~+CD25~+FOXP3~+ in CD4~+ cells were potential biomarkers for AID failure. NKCC can best predict AID failure.