| [Background]Thin endometrium(TE)is defined as the thickness of the endometrium that is below the threshold for obtaining a pregnancy,currently recognized as 6-7 mm.Most studies on the etiology of thin endometrium are now considered to be related to repeated uterine operations,infections,inadequate endometrial blood supply after uterine artery embolization,and possibly to the use of continuous oral contraceptives,clomiphene for ovulation,etc.Endometrial receptivity is the ability of the maternal endometrium to accept embryonic blasts.The endometrium can only accommodate embryonic blasts during the implantation window to complete the implantation process,during which special morphological,physiological and molecular biological changes occur in the endometrium to facilitate embryo implantation.The thin endometrium not only affects the endometrial receptivity and low pregnancy rate,but also increases the occurrence of obstetric complications.How to improve the endometrial receptivity of thin endometrium(moderate to severe uterine adhesions,recurrent thin endometrium in artificial fertility treatment)is a hot and difficult issue in reproductive medicine research today.Vaginal administration of estrogen acts directly on target organs such as the uterus,avoiding gastrointestinal absorption and hepatic first-pass effects,and prevents the conversion of estradiol(E2)to estrone(E1)in the intestine and liver,bringing E2/E1 closer to the physiological ratio while maintaining a stable blood concentration.The vagina is about 8-12 cm long and the folds on the surface not only make the vagina more dilated but also increase the absorption area to 95 cm2.The vagina is surrounded by an arteriovenous network,which allows the drug to be absorbed quickly and easily into the bloodstream through the vaginal epithelium for direct action on target organs such as the uterus.Vaginal administration reduces the incidence of systemic side effects such as breast tenderness,which may increase patient compliance.Vaginal administration of estrogen is gradually being used in the reproductive field for its above advantages,especially in the improvement of difficult cases,such as endometrial repair after separation of severe uterine adhesions with fertility requirements,repeated graft failures,and refractory thin endometrium.Most domestic and foreign studies on vaginal estrogen administration are limited to clinical applications in reproduction,basic research and clinical applications in perimenopausal women,but there are no systematic studies on the use of vaginal estrogen administration in thin endometrium.A summary analysis of the clinical application and mechanisms of action of estrogen vaginal administration would provide a comprehensive understanding of the effects of estrogen vaginal administration on the endometrial environment and tolerability,which would provide critical clinical support to address the fertility challenges of patients with thin endometrium.In this study,we first recruited patients with premature ovarian failure whose endometrium was not affected by their own estrogen to give vaginal and oral administration of estrogen to initially study the effect of vaginal administration of estrogen on the tolerance of human thin endometrium,and then further observed the effect of vaginal administration on the repair of thin endometrium and the possible mechanisms involved through a rat model of thin endometrium.Finally,the effect of vaginal administration of estrogen in clinical practice was observed.This study is divided into four parts,part I:Preliminary study of estrogen and progestin vaginal administration to improve the receptivity of thin endometrium;part Ⅱ:Study of the mechanism related to Wnt pathway in estrogen vaginal administration to improve the endometrial environment and receptivity in a rat model of thin endometrium;part Ⅲ:Clinical application of estrogen vaginal administration after separation of moderate to severe uterine adhesions;part Ⅳ:Clinical application of estrogen vaginal administration in clinical application in the freeze-thaw embryo transfer cycle of thin endometrium.Part Ⅰ.Preliminary study of vaginal administration of estrogen and progestin to improve the receptivity of thin endometrium[Object]To compare the role of vaginal and oral administration of estrogen and progestin in the proliferation and secretory transformation and endometrial receptivity of thin endometrium.[Method]Thirty patients with premature ovarian failure were recruited.Group A was given 2 mg of estradiol/dydrogesterone orally and group B was given 1 mg of estradiol/dydrogesterone vaginally for 28 days in a sequential manner using a randomized number table.The endometrial tissue was aspirated by a disposable uterine tissue aspiration tube and evaluated morphologically and functionally by HE staining,scanning electron microscopy and immunohistochemistry.[Result]1.Hormone level measurementBlood E2 levels were significantly higher in the vaginal administration group than in the oral administration group on days 14 and 21(P<0.001);FSH and LH levels decreased more in the vaginal administration group than in the pre-dose group(P<0.05).On the 14th day of administration,E2 levels in endometrial tissue were significantly higher in the vaginal administration group than in the oral administration group and the normal control group at ovulation(P<0.001).2.Ultrasonic test resultsThe endometrial thickness was significantly higher in the vaginal group than in the oral group on days 14 and 21(10.3 vs.7.5 mm;9.5 vs.7.8 mm;P<0.001).On day 14 of administration,endometrial spiral artery blood flow assay showed that endometrial blood flow parameters PI and RI were lower in the vaginal group than in the oral group(P<0.05).3.Routine morphological examination of endothelial tissue by HE stainingAt 14 days of administration,the number of endometrial glands increased in the vaginal group compared with the oral group,and the lumen was enlarged and the interstitial stroma was edematous,showing mid-to late-proliferative changes,while the endometrial glandular lumen area was smaller in the oral group,showing mid-proliferative changes.At 21 days of administration,the lumen of endometrial glands in the vaginal group was enlarged,the parietal pulp was highly secreted,and the stromal cells were large and round,showing mid-secretory changes,while the nucleus of endometrial glandular epithelial cells in the oral group was located in the middle of the cells,and the typical subnuclear vacuole structure was seen;the stromal cells were slightly edematous,showing early secretory changes.Comparing the results of image acquisition by NIS-Elements image analysis system in both groups showed that the glandular perimeter,total glandular area,glandular epithelial cell area,mean height of glandular epithelium,glandular lumen area,glandular percentage,glandular epithelium and glandular lumen percentage of endometrium were higher in the vaginal administration group than in the oral administration group at 14 and 21 days of administration(P<0.05).It is suggested that compared with oral administration,estrogen and progestin vaginal administration can better promote the proliferation and secretory transformation of the endometrium and more closely resemble the endometrial tissue changes of the normal physiological cycle.4.Comparison of the expression levels of endometrial receptivity factors ER,PR,LIF,lntegrinβ3,MMP9 and VEGF between the two groupsThe endothelial glandular and stromal ER expression levels were higher in the vaginal administration group than in the oral administration group at 14 and 21 days of administration(P<0.05).At 14 days of administration,the PR expression levels of endometrial glands and stroma in the vaginal group were lower than those in the oral group,and the difference was not statistically significant(P>0.05).At 21 days of administration,the PR expression level of endometrial glands in the vaginal group was lower than that in the oral group,and the PR expression level of stroma was higher than that in the oral group,but the difference was not statistically significant(P>0.05).At 14 days of administration,the stromal VEGF expression level was higher in the vaginal administration group than in the oral administration group(P<0.05).There was no significant difference in the expression levels of LIF,Integrinβ3,and MMP9 in the luminal epithelium,glandular epithelium,and stroma between the two groups(P>0.05).At 21 days of administration,the luminal epithelial LIF,Integrinβ3,MMP9,and VEGF expression levels were higher in the vaginal administration group than in the oral administration group,but only the difference in LIF expression levels was statistically significant(P<0.05).5.Comparison of pinopodes endometrial tissue between the two groupsAt 21 days of administration,no pinopodes in the mature stage were observed in either group.In the vaginal group,the number of developing pinopodes on the cell surface was higher than that in the oral group(P=0.025),with 12 developing pinopodes,no pinopodes and only 3 microvilli observed in the vaginal group;5 developing pinopodes and 10 no pinopodes were observed in the oral group.[Conclusion]1.Absorption of estrogen through the vaginal mucosa after vaginal administration,with significantly higher peripheral blood estradiol levels and significantly higher local endometrial tissue estradiol levels,suggesting more efficient estrogen absorption.2.After vaginal administration of estradiol 1 mg,endometrial thickness increased significantly,endometrial hemodynamics improved significantly,and stromal ER and VEGF expression were significantly upregulated,suggesting that vaginal administration of low dose estradiol can fully transform the endometrium from ultrathin amenorrheic state endometrium to proliferating mid-to late-stage endometrium.3.After 7 days of vaginal addition of dydrogesterone,the endometrial morphology was transformed into the secretory phase endometrium,and the expression of endometrial ER and PR was down-regulated,and the expression of endometrial receptivity factors(LIF,Integrinβ3,MMP9,VEGF)was up-regulated to varying degrees,and the development of pinopodes on the surface of endometrial cells was significantly improved,which confirmed that vaginal administration of dydrogesterone could effectively promote the transformation of the endometrium from the proliferative phase to the secretory phase and the corresponding change in endometrial tolerance.4.Vaginal administration of estradiol/dydrogesterone improves the tolerance of thin endometrium better than oral administration.Part Ⅱ:Mechanisms related to the improvement of endometrial environment and receptivity in a rat model of thin endometrium by vaginal administration of estrogen[Object]A rat model of thin endometrium was constructed to observe the effects of estrogen vaginal administration on endometrial thickness,glandular proliferation and vascular regeneration and the regulatory role of Wnt/β-catenin signaling pathway in endometrial vascular regeneration,and to further search for Wnt genes in the Wnt family that specifically affect this regulatory role.[Method]1.Sixty-four SD female rats were selected from the motility cycle and divided into blank control group,model control group,model+low-dose estrogen vaginal administration group and model+high-dose estrogen vaginal administration group,16 rats in each group.Except for the blank control group,the remaining three groups were constructed by damaging the right uterus with 95%ethanol to construct a rat thin endometrial model.Eight days after surgery,estradiol was given vaginally at a dose of 0.09 mg/kg.d(low-dose group,equivalent to 1 mg estradiol for 60 kg adults)and 0.18 mg/kg.d(high-dose group,equivalent to 2mg estradiol for 60 kg adults)for 12 days.8 rats in each group were executed after 12 days,and serum and uterine tissue E2 levels and HE staining were measured.The morphological changes of endometrial tissues were observed by HE staining;the expression of Wnt3a,β-catenin,VEGFA,MMP9,CD31,CK18,CK19 and Vimentin in endometrial tissues of rats was analyzed by immunohistochemistry;the expression of Wnt3a,Wnt4,Wnt5a,Wnt6,β-catenin,VEGFA,MMP9 and Vimentin was quantified by RT-PCR.The expression of Wnt3a,Wnt5a,β-catenin,VEGFA and MMP9 in the uterine tissue specimens of the rats was detected by Western-blot.Rats in each group were caged with male rats at a ratio of 2:1 on the 16th day after the operation,and the rats were sacrificed 11-14 days after the co-cage to count the number of gestational sacs.2.Human endometrial stromal cells(hESCs)were cultured in vitro and the expression changes of downstream molecules β-catenin and VEGFA were observed by RT-PCR technique and Western-blot after adding estrogen and parallel SiRNA interference with Wnt3a.[Result]1.Serum and endometrial tissue E2 levels were significantly increased after estrogen vaginal administration compared with the blank control and model groups(P<0.01).Serum and endometrial tissue E2 levels were significantly higher in the high-dose administration group compared to the low-dose administration group(P<0.05).Endometrial thickness and morphology:compared with the blank control group,the model group had sparse glands and blood vessels,significantly fewer stromal cells,and significantly thinner endometrium(P<0.05);compared with the model group,the estrogen administration group had more stromal cells,more glands and blood vessels,and thicker endometrium(P<0.05),with no significant differences between the low-dose and high-dose groups.Immunohistochemical assay:compared with the blank control group,the expression of CD31,CK18,CK19,and Vimentin in the endometrium of the model group was decreased(P<0.05);compared with the model group,the expression of CD31,CK18,CK19,and Vimentin in the estrogen administration group was increased(P<0.05),and there was no significant difference between the low-dose and high-dose groups.RT-PCR assay:β-catenin,VEGFA,and MMP9 mRNA levels were decreased in the model group compared with the blank control group(P<0.05);while β-catenin,VEGFA,and MMP9 mRNA levels were increased in the estrogen administration group compared with the model group(P<0.05);there was an increase in the high-dose group compared with the low-dose group(P>0.05).There were no significant differences in WNT4 and WNT6 mRNA contents in each group;while WNT3a and WNT5a mRNA contents were decreased compared with the model group;WNT3a and WNT5a mRNA contents were increased in the estrogen-administered group compared with the model group.Western-blot assay:compared with the blank control group,Wnt3a,Wnt5a,β-catenin,VEGFA,and MMP9 protein levels were decreased in the model group(P<0.05);while compared with the model group,Wnt3a,Wnt5a,β-catenin,VEGFA,and MMP9 protein levels were increased in the estrogen-administered group(P<0.05),with slightly increased levels in the high-dose group compared with the low-dose group.Among them,Wnt3a protein levels were significantly higher than Wnt5a protein levels(P<0.05).Immunohistochemical assay:Wnt3a,β-catenin,VEGFA,and MMP9 expression were decreased in the model group compared with the blank control group(P<0.05);Wnt3a,VEGFA,β-catenin,and MMP9 expression were upregulated in the estrogen administration group(P<0.05);the upregulation was higher in the high-dose group compared with the low-dose,but there was no statistical difference.Pregnancy test:pregnancy results on days 11-14 of gestation showed a significant increase in the mean number of gestational sacs on the injured side in both the blank control and estrogen-administered groups compared with the injured side in the model group(P<0.05),with no significant difference in the number of gestational sacs on the uninjured side.The number of gestational sacs was increased in the high-dose administration group compared with the low-dose administration group,but it was not statistically significant(P>0.05).2.Cellular assay:RT-PCR assay:human endometrial stromal cells(hESCs)were cultured in vitro,and Wnt3a,β-catenin,and VEGFA mRNA contents were increased to different degrees after the addition of estrogen(P<0.05);the expression of Wnt3a was significantly lower in the group with and without estrogen after siRNA transfection compared with the group without transfection(P<0.05).Western-blot test:The expression of Wn3a,β-catenin,and VEGFA protein increased to different degrees after the addition of estrogen(P<0.05),and the expression of Wnt3a,β-catenin,and VEGFA protein increased to different degrees after the addition of estrogen(P<0.05).The expression of Wnt3a was significantly lower in the group with and without estrogen after siRNA transfection compared with the group without transfection(P<0.05),indicating successful interference;the expression of downstream target genes β-catenin and VEGFA protein were significantly lower in the transfected group(P<0.05).[Conclusion]1.Vaginal administration of estrogen was able to increase E2 levels in serum and endometrial tissue of rats with thin endometrium,increase endometrial thickness,number of glands and blood vessels,increase CD31,CK18,CK19,Vimentin expression in endometrial tissue and finally increase the number of gestational sacs in rats.2.Vaginal administration of estrogen can upregulate the expression of VEGFA and MMP9 in the endometrium of rats with thin endometrium through the Wnt/β-catenin pathway and promote the repair of thin endometrium,which may be regulated through WNT3a in the WNT family.Part Ⅲ Clinical application of estrogen vaginal administration after separation of moderate to severe uterine adhesions[Object]Observation of clinical effect of estrogen vaginal administration for endometrial repair after separation of moderate to severe uterine adhesions[Method]Women with fertility requirements were identified as having moderate to severe hysteroscopic adhesions.The patients were given oral estradiol 4 mg/d for 7 days immediately after the operation,and after 7 days were randomly divided into two groups:group A was given estradiol 4 mg/d orally for 14 days,followed by dydrogesterone 20 mg for 14 days;group B was given estrogen vaginally 1 mg/d for 14 consecutive days,followed by dydrogesterone 10 mg for 14 consecutive days.The regimen of the previous cycle was continued on day 4 after the onset of menstruation for 2 consecutive months.In the third cycle,changes in menstrual flow were monitored and endometrial thickness and endometrial blood flow typing were measured by vaginal ultrasound at 14 days of administration.During the follow-up,the endometrial status was assessed according to the change in menstrual flow and vaginal ultrasound,and a second detachment was performed if necessary.Conception was guided at the appropriate time according to the recovery of the endometrium,and pregnancy was recorded at 24 months of follow-up.The changes in endometrial thickness,serum estradiol level and clinical pregnancy rate before and after treatment were compared between the two groups.[Result]A total of 98 patients were included in this study.History of uterine operation accounted for 98.98%(97/98),including 73 cases with history of miscarriage clearance,of which 34 cases were cleared for missed abortion.Comparison of the efficacy of the two groups:92 cases(93.88%of the total number of patients)had improved menstrual flow after surgery,with a higher percentage of improvement in the vaginal group(97.96%)than in the oral group(89.8%),but the difference was not statistically significant(P>0.05).The endometrial thickness and the increase of the endometrium were higher in the vaginal group than in the oral group at 3 months after separation of intrauterine adhesions,and the difference was statistically significant(P<0.05).Endometrial blood flow typing:there was no significant difference between the two groups before surgery(P>0.05),but the endometrial blood flow typing in the vaginal group was better than that in the oral group 3 months after surgery(P<0.05).There were 56 clinical pregnancies at 24 months postoperative follow-up,with an overall clinical pregnancy rate of 57.14%.The clinical pregnancy rate was higher in the vaginal group(65.31%,32/49)than in the oral group(48.98%,24/49),but the difference was not statistically significant(P>0.05).[Conclusion]1.History of uterine operations is an absolute factor in the occurrence of uterine adhesions,with abortion accounting for the highest percentage,especially post-abortion clearance after missed abortion and multiple abortions.2.The use of estrogen vaginally administered to repair the endometrium after moderate to severe cavity adhesions can effectively improve endometrial thickness,endometrial blood perfusion,increase menstrual flow,and improve pregnancy rate to some extent,which is an effective measure to promote endometrial repair and prevent recurrence after separation of moderate to severe cavity adhesions.Part Ⅳ Clinical application of estradiol vaginal administration in thin endometrial freeze-thaw embryo transfer cycles[Object]Retrospective analysis of the clinical effects of vaginal and oral administration of estradiol in thin endometrial freeze-thaw embryo transfer cycles[Method]Retrospective analysis of patients undergoing IVF/ICSI.Patients with controlled ovulation cycles with endometrial thickness<7 mm on HCG day who cancelled transplantation and underwent FET hormone replacement therapy were studied.The hormone replacement therapy regimen was divided into an oral administration group and a vaginal administration group.In the oral group,oral estradiol was administered at 4 mg/d from day 3 of menstruation,and in the vaginal group,oral estradiol was administered at 4 mg/d from day 3 of menstruation,and the dose was reduced to 2 mg/d at the end of menstruation,and vaginal estradiol was added at 1 mg/d.If the endometrial thickness was less than 7 mm measured by vaginal ultrasound after 10 days of administration,the duration of administration was extended to a maximum of 20 days.After satisfactory endometrial growth,progesterone was added to convert the endometrium and serum E2 levels were measured by blood sampling on the day of endometrial conversion.Resuscitation of oogenesis/blastocysts was performed on day 3/5 of progesterone administration,respectively,and embryo transfer was performed on the day following resuscitation.Blood HCG levels were measured 14 days after embryo transfer;ultrasonography was performed 28-30 days after transfer to determine clinical pregnancy;the birth of the fetus was followed up after the expected date of delivery.Statistical analysis was performed to compare age,height and body mass index,years of infertility,type of infertility,indication for pregnancy,COH regimen,IVF/ICSI method,number of previous transfers and cancelled cycles,duration and dose of estrogen before progesterone conversion,estrogen level,mean number of embryos transferred,percentage of blastocyst cycles,biochemical pregnancy rate,clinical pregnancy rate,embryo implantation rate,miscarriage rate The rate of ectopic pregnancy,preterm birth rate,live birth rate,obstetric complications,etc.[Result]Eligible patients 134 were included,including 84 cases in the oral group and 50 cases in the vaginal group.There was no statistically significant difference in general iInformation between the two groups(P>0.05).The number of previous transplant cycles and previous cancelled cycles was significantly higher in the vaginal group than in the oral group(P<0.05).Endometrial thickness was significantly thicker in both the vaginal and oral administration groups,and the thickness of endometrial thickening was higher in the vaginal than in the oral group(P<0.05).The blood E2 level on the day of endometrial transformation was significantly higher in the vaginal group than in the oral group(P<0.05).The biochemical pregnancy rate and clinical pregnancy rate were higher in the vaginal group than in the oral group(P<0.05);there was no significant difference in the spontaneous abortion rate and live birth rate between the two groups(P>0.05).[Conclusion]1.Vaginal administration of estradiol increases endometrial thickness and improves pregnancy outcomes in patients with refractory thin endometrium.2.When preparing the endometrium with hormone replacement cycles in patients with refractory thin endometrium,vaginal administration of estradiol is an optional and effective treatment modality to promote endometrial growth and improve pregnancy outcomes. |
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