经皮雌激素与口服雌激素对内膜的影响

Preparation of cycles for cryopreservation transfers using estradiol patches and Crinone 18%vaginal gel is effective and doesnot need any monitoringC.Banz a ,A.Katalinic b ,S.Al-Hasani a ,A.S.Seelig a ,J.M.Weiss a ,K.Diedrich a ,M.Ludwig a,*aDepartment of Gynecology and Obstetrics,University Hospital,Ratzeburger Allee 160,23538Lu¨beck,Germany bDepartment of Social Medicine and Department of Cancer Epidemiology,Medical University of Lu¨beck,Beckergrube 43-47,23552Lu¨beck,Germany Received 30April 2001;accepted 21December 2001AbstractSupernumary pronucleated stage oocytes (PN)can be cryopreserved and later transferred in spontaneous,stimulated or artificial cycles.Inthis study,we re-evaluated 342artificial cycles with a transdermal estradiol release system (Estraderm TTS 1001)in combination with a vaginal progesterone delivery system (Crinone 18%).Endometrial thickness and serum estradiol on day 14were correlated with clinical and ongoing pregnancy rates.Endometrial thickness between 7and 15mm did not relate to significantly different pregnancy rates.The estradiol serum level did not predict success.In conclusion,this method of endometrial preparation is comfortable for patients and monitoring is unnecessary.#2002Elsevier Science Ireland Ltd.All rights reserved.Keywords:Cryopreservation;IVF;ICSI;Cetrorelix;Progesterone vaginal gel1.IntroductionCryopreservation of excess 2pronuclear (2PN)oocytes plays an important role in IVF (in vitro fertilization),allowing another transfer of frozen-thawed PN oocytes or embryos after mildly stimulated or artificial cycle [1,6].Since cryo-preservation of embryos is not allowed in Germany,due to the regulations of the Embryo Protection Law (Embryo-nenschutzgesetz),the cryopreservation of 2PN oocytes is the only way to offer patients this advantage in treatment [7].There are different possibilities to prepare patients for a transfer of frozen-thawed embryos,for example,a mild protocol using clomiphencitrat,gonadotropines or a combi-nation of gonadotropins and GnRH agonists (Gelety and Surrey 1993).The disadvantages of this method are the injections,a strict monitoring and high costs.Recently,we could publish good results with the prepara-tion of the endometrium in artificial cycles by treatment with an estrogen-gestagen supplementation [3].In this article,we re-analyzed 342of these cycles of cryopreservation transfers with regard to the pregnancy ratein dependence on the endometrial thickness and oestrodial serum levels,to evaluate whether or not measurement of these parameters allows a prediction of pregnancy.2.Material and methodsThe study population consisted of 342consecutive infer-tile couples with a tubal and/or severe male factor infertility who underwent conventional IVF or ICSI (intracytoplasmic sperm injection)treatment and in addition to having a fresh embryo transfer,had supernumerary 2PN oocytes cryopre-served.All these patients were prepared for a frozen transfer using the same preparation scheme (Fig.1).All patients with that scheme,who underwent a transfer in the time period from January 1998up to June 2000were included in this analysis.2.1.Controlled ovarian stimulation in the fresh cycle Controlled ovarian stimulation in the collecting cycle was done as previously described either by the long agonist or multiple dose antagonist protocol [5,9].Only triptoreline (Decapeptyl Gyn Depot 1,Ferring Arzneimittel GmbH,Kiel,Germany)and cetrorelix (Cetrotide 10.25mg,SeronoEuropean Journal of Obstetrics &Gynecology andReproductive Biology 103(2002)43–47*Corresponding author.Tel.:þ49-451-5002158;fax:þ49-451-5059334.E-mail address:ludwig_m@-online.de (M.Ludwig).0301-2115/02/$–see front matter #2002Elsevier Science Ireland Ltd.All rights reserved.PII:S 0301-2115(02)00004-0International S.A.,Geneva,Switzerland)were used in the long agonist and multiple dose antagonist protocol,respec-tively.As gonadotropins either hMG (Menogon 1,Ferring Arzneimittel GmbH,Kiel,Germany)or recFSH (Gonal F 751,Serono International S.A.,Geneva,Switzerland)were used.Independently of the stimulation protocol ovula-tion was induced by administration of 10.000IU hCG (Choragon 1,Ferring Arzneimittel GmbH,Kiel,Germany)when the leading follicles reached a diameter of at least 18mm.A vaginal ultrasound-guided puncture of the folli-cles for oocyte retrieval was performed 36h after ovulation induction.2.2.IVF ,ICSI,freezing and thawing procedures2.2.1.IVF and ICSI were routinely performed as previously described [2]The freezing solution consisted of Ham ’s F-10(Bio-chrom,Berlin,Germany)supplemented with 20%human umbilical cord serum using 1,2-propanediol (PROH)and sucrose as cryoprotectants at concentrations of 1.5and 0.1mol,respectively [1].Pronucleate stage oocytes were equilibrated in two steps (1.5mol PROH,1.5mol PROH and 0.1mol sucrose)at room temperature for 10min each.Up to three 2PN were transferred with medium to each ministraw (CTE,Erlangen,Germany).For cryopreservation the CTE-880biological freezer (Cryo-Technik,Erlangen,Germany),working with the open freezing system and with self-seeding procedure was used.The ministraws were cooled slowly,from room temperature to À338C.After holding them at this temperature for 30min,they were plunged directly into liquid nitrogen for storage.For thaw-ing,the ministaws were transferred immediately for 30s in a 308C water bath.After this,the cryoprotectants were diluted in four steps,using 1.0mol PROH and 0.2mol sucrose,0.5mol PROH and 0.2mol sucrose,0.2mol sucrose,and finally Ham ’s F-10medium alone,each for 5min.Pronuclear oocytes were cultured in Ham ’s F-10forup to 2–3h and then inspected for survival under both a stereomicroscope (50Âmagni fication)and an inverted microscope (200–400Âmagni fication).Pronuclear stage oocytes were considered to have sur-vived the freezing-thawing procedure and were appropriate for intrauterine transfer if they had retained their pre-freeze morphology with no obvious damage to the zona pellucida or oolemma and the cytoplasm was clear and had re-expanded to its original volume after re-hydration.Embryo transfer was performed after a 24h period culture at clea-vage stage.Up to three cleaving embryos were transferred.2.2.2.Preparation of the cryopreservation cyclesFor the preparation of the cryopreservation transfers,an arti ficial cycle with a transdermal therapeutic system was used for estradiol release (Estraderm TTS 1001,Novartis,Wehr,Germany)in combination with a targeted drug deliv-ery system for vaginal progesteron release (Crinone 18%vaginal gel,Serono International S.A.,Geneva,Switzerland)(Fig.1).Patients started transdermal 17b -estradiol treatment on cycle day 1.One clinical monitoring was done on day 14to measure endometrial thickness by transvaginal ultra-sound.The same day,a blood sample for estradiol,proges-terone and luteinizing hormone (LH)serum levels was taken.Embryo transfer was performed on the third day of progesterone treatment (day 17).2.2.3.Measurement of estradiol levelsThe Elecsys Estradiol II testing procedure (Roche,Basel,Switzerland)was used for estradiol measurement.Clinical pregnancies were de fined by the presence of positive fetal heartbeats.Ongoing pregnancies were those,which went on for more than 12weeks of gestation.2.3.StatisticsMeans for unpaired samples were compared using the Mann –Whitney U -test.Survival,implantation,andpregnancyFig.1.Preparation scheme for cryopreservation transfers using Estraderm TTS 1001patches and Crinone 1US$8vaginal gel.Shown are the days of the spontaneous menstrual cycle [4].44 C.Banz et al./European Journal of Obstetrics &Gynecology and Reproductive Biology 103(2002)43–47rates were compared using a w2-test and Fisher’s exact-test if appropriate.P<0:05was considered statistically significant.3.ResultsA total of286patients treated with the long protocol and either hMG(n¼194)or recFSH(n¼92)as well as56 patients treated with the multiple dose antagonist protocol with either hMG(n¼16)or recFSH(n¼40)were included in this analysis.All had supernumerary2PN oocytes cryopreserved and transferred later on after thawing (Table1).The overall clinical pregnancy rate was15.5% in the freeze-thaw cycles,as assessed by transvaginal sono-graphy documenting fetal heart activity.In the long protocol group the clinical pregnancy rate was15.4%(hMG)and 13.1%(recFSH).In the multiple dose antagonist protocol group pregnancy rates of25.0%(hMG)and17.5%(recFSH) were achieved.The overall ongoing pregnancy rate was 11.7%.No statistically significant differences could be seen between the four groups(Table1).The data of the endometrial thickness on day14was available in228patients(67%).Patients who became preg-nant after the transfer had an endometrial thickness of 10:17Æ1:78mm,those who did not had an endometrial thickness of10:40Æ2:24mm(Table2).The estradiol serum level on day14was available of325patients(95%).The estradiol serum level was289:46Æ146:97and307:87Æ200:75pg/ml in pregnant and non-pregnant patients,respec-tively.There were no statistically significant differences between the two groups.To further evaluate,whether there is a group of patients, who might profit from a monitoring during the preparation period before the transfer,we divided patients infive groups according to different endometrial thickness on day14of preparation:<7mm(n¼4),7–9mm(n¼78),10–12mm (n¼116),13–15mm(n¼23)and>15mm(n¼7) (Fig.2).The rate of clinical pregnancies per embryo transfer showed no significant difference in the three groups with an endometrial thickness of7–9mm(19.2%),10–12mm (14.7%)and13–15mm(17.4%).In the patients with an endometrial thickness<7mm or>15mm,no pregnancies were achieved.However,these two groups included only4.8%of the patients.A similar result was seen by comparison of the estradiol serum level on day14of preparation(Fig.3).Four groups of patients were separately analyzed:estradiol<150pg/ml (n¼52),150–299pg/ml(n¼142),300–450pg/ml(n¼85)and>450pg/ml(n¼46).With a clinical pregnancy rate per embryo transfer of15.4,16.9,15.3and15.2%,respec-tively,there was no significant difference observed between the four groups.Table1Cycles included in the analysis aStimulation protocol n(%)Clinicalpregnancies(%)b Ongoing pregnancies (%)cCetrotide1/recFSH40(11.7)7(17.5)7(17.5) Cetrotide1/hMG16(4.7)4(25.0)3(18.8) GnRH agonist/recFSH92(26.9)12(13.0)8(8.7) GnRH agonist/hMG194(56.7)30(15.5)22(11.3) Total342(100.0)53(15.5)40(11.7)a There was no significant difference between the four different stimulation protocols,from which the pronuclear stage oocytes resulted.b Those pregnancies with positive fetal heartbeats on ultrasound.c Those pregnancies,which went on for more than12weeks of gestation.Table2Endometrial thickness and serum estradiol values in patients,who became pregnant after transfer of previously frozen pronuclear stage oocytes,and those who did not aParameters n b(%)PregnantpatientsNon-pregnantpatients Endometrium onday14(mm)228(67)c10.17Æ1.7810.40Æ2.24Estradiol onday14(pg/ml)325(95)c289.46Æ146.97307.87Æ200.75a Data are meanÆS.D.,if not otherwise defined.b Since this was a retrospective analysis,not all data were present for all patients.c Percentage of all patients,who are included in theanalysis.Fig.2.Prediction of pregnancy by measurement of endometrial thickness in artificial cycles for cryopreservation transfers.There were4,78,116,23,and7 patients in the groups of<7,7–9,10–12,13–15and>15mm.In these groups0,15,17,4,and0patients,respectively,became pregnant.No significant differences were observed between7and15mm.Endometrial thickness was available in228of the342patients.C.Banz et al./European Journal of Obstetrics&Gynecology and Reproductive Biology103(2002)43–4745Furthermore,no correlation between estradiol serum levels and thickness of the endometium could be veri fied.4.DiscussionIn this analysis,we have evaluated 342patients,who underwent an embryo transfer after freezing-thawing of 2PN oocytes.For endometrial preparation an arti ficial cycle with estradiol patches (Estraderm TTS 1001)and vaginal progesterone gel (Crinone 18%vaginal gel)was used.After re-evaluating the available data,we could show,that preg-nancy rate does not depend on the estradiol serum level on day 14of the cycle.The endometrial thickness is not predictive for a pregnancy,despite it is lower than 7mm or higher than 15mm,which is a rare case.With this measurement no pregnancies could be observed.However,less than 5%of patients fell within that group,which makes statistical analysis unreliable.It was found out that monitoring for the prediction of a pregnancy during this way of endometrial preparation is not effective and therefore not necessary.Regarding the role of the endometrium during assisted reproduction cycles and implantation,not only the endo-metrial thickness,but also the sonographic pattern is impor-tant for the evaluation of the chance to conceive.The trilamilar pattern is said to have a high predictive value for implantation and an ongoing pregnancy.Fanchin et al.[4]have recently con firmed this observation by a computer-ized endometrium analysis.They could also show,that with a lower rate of echogenicity of the endometrium the implan-tation rate increased.We did not document endometrial pattern according to a standardized method and therefore could not re-analyze these data additionally.Pregnancy rate after transfer of cryopreserved pronu-cleated oocytes have been reported to be in the range of 7.7–27%[1,6].The results of the series,which is reported here,therefore is in the normal range (15.5%clinical pregnancy rate;11.7%ongoing pregnancy rate).Estradiol can be administered orally,as injection or as a transdermal system.The latter is quite useful,has low side effects and provides high levels of circulating estradiollevels for endometrium preparation.This way is easy and can be done by the patients themselves.In arti ficial cycles,transfer of frozen-thawed pronucleated oocytes is usually performed on day 2or 3of progesteron supplement,which is started on cycle day 14or 15in addition to estrogen treatment.Three different effective routes of progesterone replacement are available:oral,intramuscular (i.m.)and vaginal.Oral micronized proges-teron is only effective at higher doses,but is absorbed variably and metabolized by the liver (first pass effect).Due to unphysiological metabolites this route has a high rate of unwanted side effects.The i.m.injections of progesteron are safe and effective,but uncomfortable for the patient,since it makes involvement of a third party necessary,is painful and cannot be done by the patient herself.Vaginal suppositories with progesteron are effective in embryo transfer cycles [8].Crinone 18%vaginal gel (Serono International S.A.,Geneva,Switzerland),a preparation that contains 90mg natural progesterone,is available in an applicator specially developed for vaginal administration once a day.Various studies have shown that 90mg administered once daily seems to be at least as effective as 600mg Utrogest 1for luteal phase support in the course of IVF treatment.The essential difference between progesterone capsules or suppositories and Crinone 18%vaginal gel is that the former contains an oil emulsion,while the latter is an oil-in-water emulsion on a polycarbophil base.Polycarbophil provides adhesive properties and ensures that the preparation adheres to the vaginal epithelium.The oil-in-water emulsion guar-antees the continuous release of progesterone directly from the aqueous phase to the vagina and uterus.In a prospective,randomized study,126patients received luteal supplementation in IVF or IVF/ICSI cycles,adminis-tered by the vaginal route,with either two capsules of Utrogest 1three times a day (600mg per day)(Dr.Kade,Berlin,Germany)or 90mg Crinone 18%(Serono Interna-tional S.A.,Geneva,Switzerland)once daily [8].There were no differences between the groups either demographically or in relation to the method of stimulation used.The ongoing pregnancy rates of 24.7%in the Crinone 18%group and 17.0%in the Utrogest 1group were comparably high,andFig.3.Prediction of pregnancy by measurement of estradiol serum levels in artificial cycles for cryopreservation transfers.There were 52,142,85,and 46patients in the groups of <150,150–299,300–450,and >450pg/ml estradiol.In these groups 8,24,13,and 7patients,respectively,became pregnant.No significant differences were observed between the four groups.Levels of estradiol were available in 325of the 342patients.46 C.Banz et al./European Journal of Obstetrics &Gynecology and Reproductive Biology 103(2002)43–47were not significantly different.However,using standar-dized questionnaires,there was a significantly higher com-fort for the patients documented,who used Crinone18% instead of Utrogest1.Therefore,Crinone18%seems to be the way of choice for luteal phase supplementation.5.ConclusionIn the present study,we could show,that the Estraderm TTS1001/Crinone18%protocol is a simple and effective preparation for cryopreservation transfers.It is convenient for the patients,because no injections and monitoring are necessary.The possibility of an ovarian hyperstimulation syndrome(OHSS)a major drawback and risk factor in ovarian stimulation is excluded.Therefore by this method additional pregnancies can be achieved after minimal burden for the patient.In our mind,this should be the method of choice for endometrium preparation for this indication.References[1]Al-Hasani S,Ludwig M,Gagsteiger F,et parison ofcryopreservation of supernumerary pronuclear human oocytes ob-tained after intracytoplasmic sperm injection(ICSI)and after conventional in-vitro fertilization.Hum Reprod1996;11:604–47.[2]Al-Hasani S,Ludwig M,Karabulut O,et al.Results of intracytoplasmicsperm injection(ICSI)using microprocessor controlled Transferman Eppendorf Manipulator system.J Middle East Fertil Soc1999;4:41–4.[3]Bals-Pratsch M,Al-Hasani S,Schopper B,et al.A simple,inexpensiveand effective artificial cycle with exogenous transdermal oestradiol and vaginal progesterone for the transfer of cryopreserved pronu-cleated human oocytes in women with normal cycles(in process citation).Hum Reprod1999;14(1):222–30.[4]Fanchin R,Righini C,Olivennes F,Taieb J,De Ziegler D,Frydman R,et puterized assessment of endometrial echogenicity:clues to the endometrial effects of premature progesterone elevation.Fertil Steril1999;71:174–81.[5]Felberbaum RE,Albano C,Ludwig M,et al.Controlled ovarianstimulation for assisted reproduction with hMG and concomitant midcycle administration of the LHRH-antagonist Cetrorelix(Cetro-tide1)according to the multiple dose protocol-results of a prospective non-controlled phase III study.Hum Reprod2000;15:1015–20. [6]Ludwig M,Al-Hasani S,Felberbaum R,Diedrich K.New aspects ofcryopreservation of oocytes and embryos in assisted reproduction and future perspectives(in process citation).Hum Reprod1999;14(1):162–85.[7]Ludwig M,Diedrich K.Regulation of assisted reproductive technol-ogy:the German experience.In:Brinsden PR,editor.A textbook of in vitro fertilization and assisted reproduction.2nd ed.New York: Parthenon Publishing Group Inc.,1999,p.431–4.[8]Ludwig M,Schwartz P,Babahan B,Katalinic A,Ku¨pker W,Felberbaum R,Al-Hasani S,Diedrich K,Luteal phase support using either Crinone18%or Utrogest1:results of a prospective, randomized study.Eur J Obstet Gynecol Reprod Biol2002,(in press).[9]Ludwig M,Felberbaum RE,Diedrich K.Interactions of GnRHanalogues and gonadotrophins.In:Adashi EY,Baird DT,Crosignani PG,editors.Gonadotrophins and fertility.1st ed.Rome:Christengraf, 1999,p.125–43.C.Banz et al./European Journal of Obstetrics&Gynecology and Reproductive Biology103(2002)43–4747。