| Objective: To observe the expression of endocannabinoid system(ECS)on endometrial implantation window phase of unexplained infertility(UI) andstudy the effect of endocannabinoid anandamide(AEA) on leukemiainhibitory factor(LIF) level in endometrial, so as to explore the mechanism ofthe ECS on endometrial implantation window phase of UI.Methods: A prospective trial was performed on the patients hospitalizedin the Assisted Reproductive Center, Second Hospital of Hebei MedicalUniversity from March 2014 to December 2014. 28 Patients’ data was selectedto form the UI group with the following criteria: Age<38years; regularmenstrual cycle(25-35d), spontaneously ovulation; laparoscope or HSGprompt bilateral tubal patency; no pregnant after intrauterine insemination(IUI) at least 3 cycles; excluded significant uterine, endocrine, immune andmale factor infertility, no endometriosis symptom on clinical; have not usedhormone drugs for nearly three months. The control group was formed with 32 patients at the same period who were due to male factor or tubal occlusion(except hydrosalpinx) factor undergoing IVF/ICSI in our center. Inclusioncriteria: Age<38years; pregnancy history; regular menstrual cycle(25-35d);spontaneously ovulation; normal basic hormones levels; have not usedhormone drugs for nearly three months. Exclusion criteria: polycystic ovarysyndrome, endometriosis, hysteromyoma, uterine endometrial polyps andother diseases. First, in order to confirm the ovulation day, both of the twogroups accepted ovulation detection by vaginal ultrasound. Then, 7-9 daysafter ovulation, the serum progesterone levels, endometrial thickness,endometrial morphology and the expression of endometrial LIF, cannabinoidtype 1(CB1), and FAAH of the two groups were studied. Third, culturehuman endometrial adenocarcinoma cell RL95-2. After AEA, AM251(CB1receptor antagonist) and AM630(CB2 receptor antagonist) were added in themedium separately, the changes of LIF levels in medium supernatants werestudied. At last, the effects of endocannabinoid system on endometrialimplantation window phase were analyzed, and its action mechanism wasspeculated.Results: There was no difference between two groups in the BMI, age,duration of infertility, number of basal follicles, blood-based FSH levels,blood-based LH levels, basal E2 levels. There was no significant differencebetween the two groups in the duration of stimulation, dosage ofgonadotrophin, number of follicles above 16 mm in ovaries, oocytes retrieved,fertilization rate and Cleavage rate. However, the difference in embryoimplantation rate(21.74% vs. 56.25%) and Embryos available rate(43.79% vs.67.38%) between the two groups(P<0.05) were significant. The UI group waslower than that in control group; there was significant difference in the freshcycle clinical pregnancy rate(33.33% vs. 78.57%) between the two groups(P<0.05), the fresh cycle clinical pregnancy rate of UI group was lower thanthat in control group. There was no difference between UI group and controlgroup in serum progesterone levels on midluteal phase, serum estrogen levelson HCG day, serum progesterone levels on HCG day. The statisticaldifference in endometrial thickness on midluteal phase(10.43±1.34 mm vs.11.87±1.36mm) and transplant day(11.14±1.03 mm vs. 12.00±1.25mm)between the two groups was significant(P<0.05), the endometrial thicknesson two periods of UI group was lower than that in control group. Theexpression of endometrial CB1(IOD value) of UI group increased slightlycompared with the control group(136.79±8.49 vs. 102.86±40.73) at midlutealphase, but the difference between the two groups was not statisticallysignificant. The expression of endometrial FAAH(95.08±30.13 vs.137.43±29.49) and LIF(134.5±41.4 vs. 134.5±41.4) of UI group decreasedsignificantly compared with the control group at midluteal phase, there wassignificant difference in these indicators between the two groups(P<0.05).The data form cultured human endometrial adenocarcinoma cell RL95-2 invitro indicated that, there were differences in LIF levels between each groupafter added 1μmol/L, 5μmol/L, 10μmol/L and 50μmol/L AEA into the culturemedium at 24 h, 48 h, 72 h respectively, and the difference was statisticallysignificant(F=605.381, P<0.05). There was no difference between 5μmol/L48 h group and 10μmol/L 48 h group in LIF levels, and no significantdifference was found in LIF levels between1μmol/L 72 h group and 10μmol/L72 h group, the difference was found in the other groups were statisticallysignificant(P<0.05). With the increased of AEA concentration in 24 h and 48 hgroups, LIF levels was ascending. In 72 h groups, the LIF level of 5μmol/LAEA group increased significantly compared with 1μmol/L AEA group(2003.87±37.85ng/L vs. 1948.20±21.81ng/L), the LIF level of 10μmol/L AEAgroup decreased significantly compared with 5μmol/L AEA group(1916.60±14.05ng/L vs. 2003.87±37.85ng/L), but LIF level of 50μmol/L AEAgroup was lower than the above three kinds of AEA concentrations, and thedifferences were statistically significant(P<0.05). After AM251 and AM630 were added to the culture medium at the same concentration(5μmol/L), LIFlevels showed that differences were statistically significant(F=930.121,P<0.05). There were no difference between AM251 group and AM630 groupin LIF levels, there were no difference between AEA+AM251 group andAEA+AM630 group in LIF levels. Compared with the AEA group, LIF levelsin AEA+AM251 group and AEA+AM630 group were lower, the differencewas statistically significant(P<0.05). LIF level of AM251 group was lowerthan AEA+AM251 group(1358.04±18.42ng/L vs. 1510.37±16.77ng/L), andthe differences were significant(P<0.05). The LIF level of AM630 groupdecreased significantly compared with AEA+AM630 group(1353.73±20.64ng/L vs. 1508.20±21.50ng/L)(P<0.05). But each groupcompared with the control group, LIF levels were higher than the controlgroup, the difference was statistically significant(P<0.05).Conclusions: The results were reached that the expressions of FAAH andLIF on endometrial implantation window phase of UI were lower than thesecondary infertilities, while the expression of CB1 receptor tended to rise.The implantation rate and fresh cycle pregnancy rate of UI group decreasedsignificantly compared with control group. The implantation window phase ofendometrial environment was simulated by cultured RL95-2 in vitro. With theincrease of the concentration or action time of AFA, LIF levels wereascending. However, they began to go down when the concentration or theaction time of AEA reached a certain value. LIF levels decreased after addingCB1 or CB2 receptor antagonists. The results were reached that a certainconcentration of AEA has a positive effect on implantation, while the highdoses of it may have an adverse effect on it. Meanwhile, CB1 and CB2 receptors may be involved in its regulation. All the results above indicated thatendometrial receptivity of UI may be affected by abnormal expression of ECS,which reduces the pregnancy rate. |
PDF Full Text Request