| Objective:Success implantation depends on invasive blastocyst,acceptive endometrium and their synchronization in IVF.A key factor of successful implantation in frozen-thawed embryo transfer cycle is endometrial receptivity with equal embryo quality.Currently,endometrial preparation regimens include nature cycle,ovulation induction cycle and artificial cycle with or without a gonadotrophin releasing hormone agonist.Because of its possibility to reduce cycle cancellation rate and improve pregnancy outcome,the protocol using a GnRH agonist followed by estrogens and progesterone is widely used in frozen-thawed embryo transfer.Besides its well-known endocrine function as an important regulator of the HPO axis,recent studies showed that GnRH may directly regulate human reproductive activity by GnRHR expression in other female reproductive tissue such as ovary and endometrium,especially during the luteal phase.Leukemia inhibitory factor is one of the most accepted markers.Pinopodes,are known as morphologic marker of endometial receptivity,their expression is associated with the “implantation window”.Comparison of different regimens in published papers focused on implantation rate,pregnancy rate and live birth rate.data for endometrial receptivity of different regimens,are little.The aim of the current study was to establish models of different endometrial preparation regimens by using estrogen,progesterone and gonadotropin releasing hormone agonist with SD rats and to explore the influence of GnRHa on endometrial receptivity by detecting the expression of LIF,pinopodes and thickness of endometrium at the transformation day.Methods:1 General situation assessment of SD ratsThe rats were monitored closely every day,body weight,diet and activities were measured every 4 days.2 The establishment of different endometrial preparation regimens model of SD ratsReproductive cycle of rats were identified by daily vaginal smear cytology for at least two cycles,and ultrasonographic examination was performed to quantify endometrial thickness at the same time.A total of 60 non-pregnant female rats with normal reproductive cycle were randomly assigned to four groups(n=15 per group)as follows: nature cycle group(NC),hormone replacement therapy group(HRT),high-dose GnRHa and hormone replacement therapy group(HDG-HRT)and,low-dose GnRHa and hormone replacement therapy group(LOG-HRT).NC group rats were mated with male rats the first estrus 28 days after 0.2ml sterile water was injected intramuscularly,meanwhile the thickness of the endometrium was mearsured.The morning detection of vaginal plug was recorded at day 0 and barbiturate-anesthetized rats were killed for uterus at day 4.Rats in HRT group,HDG-HRP group and LDG-HRT group were injected with 0.2ml sterile water,0.4mg/kg(0.2ml)and 0.2mg/kg(0.2ml)Diphereline respectively in diestrus,followed by progynova 0.42 mg/kg by gavage every day.6mg/kg of progesterone was added while mated with male rats when the endometrial thickness reached estrus level(2.82mm).And all the rats were killed for uterus at day 4 of pregnancy.3 Quantification of endometrial thickness of SD ratsUltrasound examination was performed by Philips IU 22 with high frequency 30 MHZ linear array probe to quantify the thickness of endometrium.The thickest endometrium of the rats’ two uteruses was detected and averaged.Timing of endometrium measurement was as follows: rats of NC group were detected at estrus and disestrus.Endometrial thickness was measured every day from the day Prognova was given to the day of transformation.When GnRHa was administrated in HDG-HRT group and LDG-HRT group,ultrasound examination was performed every 7 days until the 28 th day and then every day from the day Prognova was given to the day of transformation.4 Analysis of endometrial receptivity marker of SD ratsThe endometrial morphology of four groups was observed with HE staining after the samples collected by CKX-41 inverted microscope.Glands numbers of each cross-section were counted and MPIAS-500 image analysis system was used to measure areas of glands.SP immunohistochemical technique was used to detect the expression of LIF in rat endometrium.S-3500 N scanning electron microscope was used to observe the expression of pinopodes.Pinopodes score was gained as follows: fifty fields of endometrial epithelium were examined per sample at 3,500 magnification(the apical surface of 250 cells can be visualized per field: 250×50=12,500 cells visualized per sample).The total number of pinopodes seen in all 50 fields was added up and used as the final sample score.In uteri collected after embryo implantation,endometrial epithelium was scored in areas between implantation sites.5 Statistical methods: Data were evaluated using SPSS17.0 statistical software and expressed as mean ± standard deviation.The data were analyzed by one-way ANOVA followed by LSD method and t test.The difference was considered significant if P < 0.05.Results: 1 General situation assessment of SD ratsHypoestrogenic symptoms,such as increased activities and reduced food intake,appeared 5 days after the administration of GnRHa in HDG-HRT group and LDG-HRT group.On day 14 of pituitary suppression,food-intake of rats in HDG-HRT group and LDG-HRT group were less than that of rats in NC group,but no significant difference was found(P=0.010,P=0.012).And weight of rats in these two groups decreased,yet no significant difference while compared with NC group.Hypoestrogenic symptoms disappeared and food-intake increased when estrogen was administrated.Food-intake and body weight in the four groups showed no difference when executed(P=0.010,P=0.010).2 Vaginal smear results of SD ratsVaginal cells changed periodically,about 4 days a cycle.The sequence of the estrus stages is: proestrus,estrus,metestrus and disestrus.The results showed that the major cell type of proestrus which lasted about 9 to 18 hours was nucleated epithelial.Occasionally,some keratinized epithelium appeared in the sample.The number of keratinized epithelium reached a peak at the one-day estrus.Leukocytes,keratinocytes and nucleated epithelial can be observed during metestrus for about 10 to 14 hours.At the stage of diestrus,a large number of Leukocytes and a few epithelial cells and mucus showed up.The number of keratinizing epithelium increased gradually after the administration of progynova by gavage in HRT group,HDG-HRT group and LDG-HRT group.Cell types stopped changing 5 days after pituitary suppression in HDG-HRT group and LDG-HRT group.In conclusion,cells of vaginal smear was the same in HRT group,HDG-HRT group and LDG-HRT group with that cells at estrus in NC group after administration of progynova.3 Thickness of endometrium measured by ultrasonic apparatus 3.1 Endometrial thickness of different reproductive stages of SD ratsThe thickness of endometrium changed periodically during the reproductive cycle.The sequence of the stages is: proestrus,estrus,metestrus and disestrus.Thickness of endometrium of different stages are as follows: 2.76±0.08 mm,2.82±0.10 mm,2.78±0.09 mm,2.70±0.07 mm.Compared with that at preoestrus,metestrus and diestrus,Endometrial thickness at estrus was much larger(P=0.009,P=0.000,P=0.000).Endometrial thickness was the smallest at diestrus,and there’s no significant difference between diestrus and preoestrus(P=0.100)while significant difference was found when compared with that of metestrus.No significant difference was found between preoestrus and metestrus(P=0.160).3.2 Endometrial thickness of SD rats in different groups 2.2.1 Endometrial thickness of SD rats in the two pituitary groupsDuring the first day to the 28 th day,endometrial thickness declined gradually after pituitary suppression.There was a significant difference between the two pituitary suppression groups and control group(P=0.000,P=0.000).And endometrial thickness in HDG-HRT group was statistically smaller than that in LDG-HRT group at the 7th day of pituitary suppression(1.28±0.05 mm,1.53±0.1mm,P=0.000).But no significant difference was observed between the two groups at the day 14,21 and 28 of pituitary suppression(P=0.120,P=0.051,P=0.105).3.2.2 Dosage and treatment time of medicationNo exogenous hormones was used in NC group and rats in HRT group were free of GnRHa.The estrogen treatment time was shorter in HRT group when compared with HDG-HRT group and LDG-HRT group(P = 0.000,P = 0.000).Compared with LDG-HRT group,it was longer in HDG-HRT group,but no significant difference was seen(P=0.068).Comparison of cumulative dosages of estrogen were as follows: HRT group were lower than HDG-HRT group and LDG-HRT group statistically(P=0.000,P=0.000);HDG-HRT is higher than that of LDG-HRT group(P=0.048).Dosage and treatment time of progesterone were the same in the three artificial groups.3.2.3 Endometrial thickness of SD rats at the secretory transformation dayEndometrial thickness of rats in the four groups at the transformation day were as below: 2.81±0.16 mm,2.83±0.2mm,2.83±0.3mm and 2.83±0.4mm.It was the smallest in NC group when compared with HRT group,HDG-HRT group and LDG-HRT group,but no statistical difference was found among the four groups.4 Expression of endometrial receptivity marker in different groups 4.1 Histological evaluation of SD rats endometriumNumbers of uterine glands in NC group,HRT group,HDG-HRT group LDG-HRT group were 9.7±1.6,9.8±1.5,11.1±1.7,10.8±1.4 and areas of glands in the four groups were 1870.07 ± 162.93μm2,1834.87 ±154.07μm2,2078.76±161.16μm2,2010.88±178.99μm2 Respectively.As for NC group and HRT group,uterine gland number and area were the not different significantly(P=0.814,P=0.560).When compared with NC group,numbers of uterine glands were larger in HDG-HRT group LDG-HRT group(P=0.016,P=0.049)and areas were lager as well(P=0.001,P=0.000).Numbers in HRT group was smaller than HDG-HRT group and LDG-HRT group(P=0.028,P=0.041)and so too do areas(P=0.000,P=0.000).Neither uterine glands number nor areas in HDG-HRT group and LDG-HRT group had a significant different.Based on data above and observation of the morphology of endometrium under light microscope,when compared with NC group and HRT group,glands in HDG-HRT group and LDG-HRT group were larger as for numbers and areas,irregular in shape and more sections.4.2 Expression of LIF of the four groupsH-score of LIF in NC group,HRT group,HDG-HRT group and LDG-HRT group were 2.59±0.38,2.58±0.24,2.70±0.42,2.72±0.65.LIF expression was lower in HRT group than that in NC group,whereas there was no difference between the two groups(P=0.657).It was higher in HDG-HRT group than that in LDG-HRT group,however no statistical difference existed between them(P=0.460).Compared with NC group,the expression of LIF in HDG-HRT group and LDG-HRT group was statistically higher(P=0.001,P=0.019).It was lower in HRT group when compared with HDG-HRT group and LDG-HRT group(P=0.000,P=0.000).4.3 Morphology of pinopodes in the four groupsIn NC group,pinopodes were fully developed and well-stacked with smooth surface.Cells with little microvilli covered the rest of the surface.In HRT group,pinopodes were less developed and varied in size.And surface of the pinopodes were not so smooth.In HDG-HRT group and LDG-HRT,a pronounced cell bulging appeared with smooth surface and the rest of the endometrial surface was covered by cells with little microvilli.In conclusion,pinopodes in HRT group and NC group could be identified as follows: it was obvious that the majority former were less developed and the most pinopodes of the latter group were plump.When compared with NC group,pinopodes in HDG-HRT group and LDG-HRT were more developed with smoother surface and shorter microvilli.4.4 Pinopodes score of the four groupsIn NC group,the pinopodes were fully developed and well-stacked with smooth surface.Cells with microvilli covered the rest of the surface.In HRT group,pinopodes were less developed and varied in size.And surface of the pinopodes was not so smooth.In HDG-HRT group and LDG-HRT,a pronounced cell bulging appeared with smooth surface and the rest of the endometrial surface was covered by microvilli.In conclusion,pinopodes in HDG-HRT group and LDG-HRT group were more developed and smoother when compared with that in NC group and HRT group(P=0.440).Conclusion:1 Hormone replacement therapy cycle and pituitary suppression combined with hormone replace therapy cycle model of rats were successfully established in this study.2 Histological morphology and pinopodes were more developed and H-score of LIF and pinopodes score are higher in HDG-HRT group and LDG-HRT group than in NC group and HRT group.GnRHa can improve endometrial receptivity of SD rats.3 Low dosage of GnRHa is as effective as high dosage which can be adjusted according to clinical situation. |
