| Part Ⅰ:The optimum number of oocytes in IVF treatmentObjective:It has been reported that there is a strong association between oocyte number and in vitro fertilization-embryo transfer (IVF-ET) outcome.However, the association between the number of eggs retrieved and the live birth rate (LBR) both per fresh cycle and cumulatively per stimulation cycle are not known yet. The purpose of this study is to explore the relationship between oocyte number and LBR following first IVF treatment cycles.Method:A retrospective cohort study was performed. All patients aged18-34years with normal menstrual cycles and were stimulated with a long protocol at the Reproductive Medicine Center of Anhui Provincial Hospital, P.R. China from April2007to December2011were identified and reviewed. In our study, the patients’ own oocytes were used, and fresh or frozen-thawed embryos were transferred without pre-implantation genetic screening for embryo aneuploidies. For the purpose of this study, cycles involving egg donation, egg sharing, egg cryopreservation and frozen egg thawing were excluded from the analysis. Patients who underwent natural cycles IVF/intra cytoplasmic sperm injection (ICSI) or who had not become pregnant but still had frozen embryos left were also excluded from our study. All patients were undergoing their first IVF cycle. Thus,2455patients with one complete treatment cycle were included in this study.Univariate analysis was conducted to identify confounding factors that predict live birth outcome. Patients were categorized into four groups according to the number of oocytes retrieved:A(0-5), B (6-10), C (10-15), D (≥16)oocytes. Important aspects of the treatment outcome including fertilization rate, embryo transferable, fresh cycle LBR per started cycle and per transfer cycle, cumulative LBR, abortion rate and moderate-severe ovarian hyperstimulation syndrome (OHSS) rate were analyzed. Logistic regression analyses were conducted to identify independent correlates between each possible confounding factor especially oocyte number and live birth outcome after adjusting for other confounders that were identified in our univariate analysis.Result:2455FVF/ICSI long protocol cycles from our unit were analyzed in this study, and1333cycles (54.30%) result in live births. Women who achieved live births were significantly younger (P=0.001) and required a smaller amount of daily and total gonadotropins (P<0.001, P<0.05). However, the duration of ovarian stimulation in live birth patients was significantly longer than their no live birth counterparts (P<0.001). There were no significant differences in the duration of infertility, insemination method, type of infertility, diagnosis of infertility between live birth and non-live birth cycles. The number of oocytes retrieved, fertilization rate and embryos transferable were significantly higher in live birth cycles (P<0.001). On the contrary, the average number of fresh embryos transferred in live birth patients was significantly fewer than non-live birth patients (P<0.05).When divided by oocyte number, there was a negative association between age and oocytes yield (P<0.001). The fertilization rate decreased when more oocytes were retrieved (P<0.001).However, the embryos transferable increased with oocytes number (P<0.001). In343cycles (13.97%), fresh embryo transfer was not done for the following reasons:no oocytes retrieved (9cycles [0.37%]), failed fertilization (26cycles [1.06%]), no embryo transferable (17cycles [0.69%]), and all embryos cryopreservation because of the OHSS or other reasons (290cycles [11.81%]).The whole embryo cryopreservation rate for avoiding moderate-severe OHSS increase with oocyte number (P<0.01). The fresh cycle live birth per started cycle increased with oocytes number up to group B-C (6-15oocytes group) and then decreased (P<0.01) because of the highly whole embryo cryopreservation rate for avoiding moderate-severe OHSS. The fresh cycle LBR per transfer cycle and cumulative LBR per started cycle increased with oocyte number, from38.69%and35.00%of group A to49.07%and67.10%of group D (P<0.01,0.005). Also, the incidence of moderate-severe OHSS increased with the oocyte number (P<0.01). There was no significant difference in the abortion rate among the patient groups (P>0.05).In the logistic regression analyses:the0-5oocyte category was used as a reference. After adjustment for age, duration of stimulation, total dose of gonadotrophins and fertilization rate, the adjusted ORs for fresh embryo live birth per started cycle increased from1.823(1.395-2.381) in the6-10oocyte category to2.142(1.609-2.851) in the11-15oocyte category. However, the adjusted ORs of fresh embryo live birth decreased in the≥6oocyte category, being1.918(1.376-2.672). The duration of stimulation, total dose of gonadotrophins and fertilization rate had a significant effect on fresh cycle LBR. But for age, the effect was not significant. For the cumulative live births per started cycle, when adjusted for age, the duration of stimulation, total dose of gonadotrophins and fertilization rate, the adjusted ORs for cumulative live birth were2.232(1.708-2.918) in the6-10oocyte category,3.323(2.492-4.432) in the11-15oocyte category and5.805(4.125-8.170) in≥16oocyte category. Thus, the odds of cumulative live birth appeared to significantly increase with the ovarian response. The duration of stimulation and fertilization rate also had a significant effect on the cumulative LBR.Conclusion:There is a strong relationship between the number of oocytes and fresh as well as cumulative LBR following first IVF treatment. For young women, high ovarian response did not compromise IVF outcome. We anticipate these studies may lead to the development of more efficacious ovarian stimulation therapies for IVF. However in the initial fresh IVF cycle, a high ovarian response was associated with a high rate of cycle cancellation due to the risk of OHSS. The optimal number of oocytes for achieving the best chance of live birth in the first IVF cycle, and even higher chances of live birth in cumulative cycles, is somewhere between6and15oocytes. Part Ⅱ:Decidual and peripheral blood TCRαβ+CD3+CD4-CD8-double negative regulatory T cells in early pregnancy subjects and unexplained recurrent spontaneous abortion patientsObjective:To investigate the proportional and function changes of TCRαβ+CD3+CD4-CD8-double negative regulatory T cells (DN Tregs) in peripheral blood and maternal-fetal interface in unexplained recurrent spontaneous abortion (URSA) and normal pregnant (NP) women, and explore the probably role of human DN Tregs in the occurance of URSA. Also, the proportional changes of these cells in peripheral blood after lymphocyte therapy in URSA patients were investigated to find the rationale for this treatment. The results of this study is to explore the probable role that DN Tregs may play in the induction of maternal-fetal immunological tolerance and to find a new treatment target for URSA.Method:Heparinized venous blood was obtained from non-pregnant healthy women and women in normal early pregnancy. Similarly, heparinized venous blood was also obtained from patients with unexplained recurrent spontaneous early abortion after diagnosis and before curettage and URSA patients received alloimmunization. treatment. Peripheral blood mononuclear cells (PBMCs) were separated from venous blood by Ficoll-Hypaque gradients. Decidual samples were obtained from patients with induced abortion, representing early pregnant deciduas, and from patients with spontaneous abortion, representing abortion samples. The decidual mononuclear cells (leukocytes) were purified by the Ficoll-Hypaque method after mechanical disruption and filtration through a32-μm nylon mesh. The proportion of DN Tregs in CD3+T cells of each group were determined by flow cytometry. DN Tregs were isolated from PBMC and decidual mononuclear cells via FACS separation. DN Tregs were activated by beads coated with anti-CD3and anti-CD28antibodies in vitro. Lymphocytes suspensions were isolated from venous blood of each patient.After stained with CFSE, lymphocytes were cocultured with anti-CD3/CD28-coated beads in the presence or absence of alloactivated DN Tregs for5days. Proliferation of cells was determined by flow cytometry. Data were analyzed with CELL Quest software and proliferation rates (the propotion of CFSEdim cells in CD4+or CD8+T cells) were calculated.Result:The propotion of TCR-αβ+CD3+CD4-CD8-DN Tregs in the CD3+cell population were compared between decidual lymphocytes and peripheral blood lymphocytes. The proportions of DN Tregs in decidua were significantly higher than that in peripheral blood both in URSA patients (1.82%±0.85%vs0.92%±0.68%, P <0.01) and normal early pregnant women (5.26%±3.58%vs1.56±1.07%, P<0.01). In peripheral blood lymphocytes, the proportion of DN Tregs did not differ significantly between normal early pregnant and non-pregnant women (1.56%±1.07%vs1.21%±0.63%, P=0.217). However, proportion of peripheral blood αβTCR+CD3+CD4-CD8-T (DN Treg) cells of URSA patients differ significantly from non-pregnant women (0.92%±0.68%vsl.56%±1.07%, P=0.009). Also, proportion of DN Tregs in deicidal were significantly lower in URSA patients than normal early pregnant women (1.82±0.85%vs5.26±3.58%, P=0.011). Result of in vitro suppression assays show that for URSA patients, DN Tregs in peripheral blood lymphocytes and decidua have a suppressive effect on allo-CD4+and allo-CD8+T cell proliferation. Also, DN Tregs in decidua of normal early pregnant women have a suppressive effect on allo-CD4+and allo-CD8+T cell proliferation. The suppression effect of DN Tregs in decidua between the two groups did not differ significantly.54URSA patients had received immunotherapy.32patients achieved a successful pregnancy with17patients already delieverd,8patients pregnant for21-35week,7patients pregnant for12～20week.7patients had not achieving pregnancy.15patients reaborted after pregnancy. The successful rate after this therapy is59.2%. After allogeneic lymphocyte therapy, the proportion of DN Tregs increase, but the difference was not significant (P=0.151). However, for32patients achieving a successful pregnancy, the percentage of DN Tregs in peripheral blood had been enhanced after allogeneic lymphocyte therapy (P<0.05). While for the15patients reaborted after lymphocyte therapy, the proportion of DN Tregs in peripheral blood did not differed significantly.Conclusion:Decidua may be one of the favorite tissue localization of DN Tregs in human in case of pregnancy. Human DN Tregs may play an important role in the induction of maternal-fetal immunological tolerance, the propotion decrease of DN Tregs in peripheral blood lymphocytes and decidua may be associated with the occurrance of URSA. Enhancement of the percentage of DN Tregs in peripheral blood may be one of the rationales for this treatment. |
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