Inhibition of Progesterone Secretion by Oestradiol Administered in the Luteal Phase of Assisted Conception Cycles

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1 Inhibitin f Prgesterne Secretin by Oestradil Administered in the Luteal Phase f Assisted Cnceptin Cycles P Y STay, MRCOG*, E A Lentn, PhD** *Department f Obstetrics and Gynaeclgy, University f Malaya, Kuala Lumpur, '"Sheffield Fertility Centre, 26 Glen Rad, Sheffield S7 IRA, United Kingdm Intrductin The rle f supplementing prgesterne during the luteal phase in stimulated in-vitr fertilisatin (IVF) is the subject f numerus publicatins. Hwever, there are relatively little attempts t clarify the significance f estradil fllwing varian stimulatin. Althugh the rle f estradil in endmetrial priming during the fllicular phase is well recgnised, the impact f estradil n the luteal phase in human remains cntrversial. This article was accepted: 10 Nvember 2002 Crrespnding Authr: Paul Y 5 Tay, Department f Obstetrics and Gynaeclgy, University f Malaya, Kuala Lumpur 187

2 A cncmitant decline in plasma sex hrmnes cncentratins was cnstantly bserved in pituitary dwn regulated cycles 1. Such precipitus decline in sterids may be harmful t receptive endmetrium and implantatin, hence luteal supprt was recmmended 2. A study by Sharara and McClamrck (999) assessed 106 prgesterne supplemented IVF cycles with pituitary dwn regulatin 3. Cycles were divided int grups accrding t the rati f estradil cncentratins n the day f HCG administratin t the mid luteal phase. It was fund that the pregnancy rate is significantly lwer if the rati exceeded 5.0 cmpared t thse less than 5.0. They pstulated that the magnitude f decline in estradil cncentratins fllwing varian stimulatin is highly predictive f pregnancy utcme and the rapid fall in estradil fllwing cyte recvery may cmprmise endmetrial integrity. This study has als raised the issue f incrprating estradil int the prgesterne supplement. Oestradil supplementatin has been extended int Clmiphene Citrate (CC)-stimulated IVF cycles. Studies f endmetrium after expsure t CC have shwn histlgical features cnsistent with a hypestrgenic effect 4 and reductin in glandular density and an increase in the number f vaculated cells 5. The deleterius effect f CC n endmetrium maturity can be reverse by 0.2 mg f ethinyl-estradil 4. Hurd et al. (996) have randmised 79 subjects t either receiving n luteal supprt r supprt with bth ral estradil and vaginal prgesterne in CC stimulated IVF cycles. They fund that the latter grup exhibited a significantly higher pregnancy rate 6. This study was perfrmed in an attempt t clarify the impact f estradil supplement n the crpus luteum functin as well as the pregnancy utcme, fllwing varian stimulatin with (GnRH-a and r FSH) r withut (CC and r-fsh) pituitary dwn regulatin. Materials and Methds 118 subjects attending Sheffield Fertility Centre, Sheffield, United Kingdm between Jan 1998 t Jan 1999 were recruited int the study. Nne f the wmen had had previus infertility treatment and with a minimum duratin f infertility f 2 years. The mean age f the subjects was 32.2 ± 4.5 years (range frm 22 t 39) and all f them exhibited basal fllicle stimulating hrmne (FSH) f <10 lull. Subjects were allcated t ne f the tw treatment grups, A and B. Subjects in Grup A (n=63) were stimulated with lng prtcl dwn regulated IVF regime (GnRH-a/r-FSH). Frtypercent f the subjects in this grup had tubal disease, 29% had male factr infertility, 10% had endmetrisis, 19% had vulatry disrder and 2% f the cases were unexplained infertility. Sme f these subjects had mre than ne infertility factr. All subjects in Grup B (n=55) have undergne stimulated intrauterine inseminatin treatment using Clmiphene citrate and r-fsh (CC/r-FSH). All the subjects this grup had unexplained infertility. They had vulatry cycles (cnfirmed by a recent vulatry mid luteal phase prgesterne measurement), bilateral patent tubes (cnfirmed by either a recent laparscpy r hystersalpinggram) and nrmal semen analysis (semen.cncentratins ~ 20x10 6 /ml, mtility (grade I + II) ( 30% and nrmal frms ~ 25%). The lcal ethics review cmmittee apprved this study and all subjects gave infrmed cnsent. Treatment prtcls Stimulated IVF (Grup A; GnRH-a/r-FSH) cnsisted f dwn regulatin with gnadtrphin releasing hrmne agnist (GnRH-a) t achieve cmplete pituitary desensitisatin befre starting varian stimulatin. GnRH-a (Suprefact, Shire Pharmaceuticals Ltd., Hants SP10 5RG, UK) was administered daily starting 7 days befre the expected perid and cntinued fr days in 188

3 Inhibitin f Prgesterne Secretin by Oestradil Administered in the luteal Phase f Assisted Cnceptin Cycles ttal. The subjects were then given 150 r 225 IU/day f recmbinant FSH (Gnal-F, Ares-Sern Ltd., Lndn WIN 1AF, UK). Fllicle develpment was mnitred by estradil measurement and serial trans-vaginal ultrasngraphy (US) (Cmbisn 310, Kretztechnik, AG, Austria). HCG 1O,000IU (Prfasi; Ares-Sern Ltd., Lndn WIN 1AF, UK) was administered subcutaneusly when ~ 2 fllicles measuring ~ 18mm in diameter were present. Transvaginal egg cllectin under ultrasnic guidance tk place hurs later. GnRH-a was discntinued n the day f HCG administratin. Ocyte(s) were inseminated and cultured accrding t cnventinal IVF technique. When fertilisatin and cleavage ccurred, a maximum f three embrys were transferred in-uter 2 t 3 days after egg cllectin. The excess embrys were crypreserved. Stimulated intrauterine inseminatin (Grup B; CC/r-FSH) invlved using 100mg f CC per day fr 5 days, starting n cycle day 4 (day 0 is the first day f the perid). This was fllwed by 75 IU f FSH (Gnal-F, Ares-Sern Ltd., Lndn WIN lap, UK), given daily, starting n cycle &iy 7. Treatment was mnitred by daily measurement f estradil and LH, starting frm day 9 f the cycle, tgether with alternate day vaginal ultrasund scans (Cmbisn 310, Kretztechnik, AG, Austria). When tw t fur fllicles measuring ~ 18 mm were present and circulating E2 was ~ 2000 pmlll, IU f HCG was administered fllwed by intrauterine inseminatin hurs later. Luteal supprt regimes Subjects underging stimulated IVF (Grup A; GnRH-a/r-FSH) and stimulated intrauterine inseminatin (Grup B; CC/r-FSH) cycles were allcated randmly t the ne f the tw luteal supprt regimes n the day f embry transfer r inseminatin. 1. Prgesterne supplement nly: 200 mg pessary (Cyclgest, Hechst UK Ltd., Hechst Huse, Salisbury Rad, Hunslw, Middlesex) administered vaginally twice daily. 2. Prgesterne supplement (as described abve) tgether with 2mg per day f ral estradil valerate (E2V). All luteal supplements were administered between hurs, cmmenced n luteal phase (LP) day 1, the day after egg cllectin r intrauterine inseminatin. Definitins Psitive HCG is defined as the detectin f plasma HCG cncentratins f ~ 10 lull by day 14 fllowing egg cllectin. A rise in plasma HCG cncentratins between 14 and 18 days after egg cllectin is als classed as psitive HCG. Clinical pregnancy is defined as fetal heart activity that is visualised n ultrasund. Length flutealphase is the number f days frm the time f egg cllectin (in IVF cycles) r intrauterine inseminatin (in vulatin -inductin cycles) t the first day f menstrual bleeding. Hrmnal assays Bld samples were cllected between hurs n luteal days 8 and 12 nly. All bld samples were cllected and stred at C until the day f assay. Serum estradil (E2) and prgesterne (P) were measured and the samples belnging t the same patient were assayed in the same run t reduce inter-assay variability. Plasma estradil was measured using a cmmercially available micrd-particle enzyme immunassay (MEIA) system [IMx assay technlgy, Abbtt Labratries, UK]. The sensitivity f this system is 91.8pmlil. The intraand inter-assay variatin was % and % respectively. Plasma prgesterne cncentratins were measured using a high affinity mnclnal antibdy in a co'mpetitive enzyme immunassay system with magnetic phase separatin [Serzyme immun-assay system, BIODATA Diagnstics, Italy]. The system has a sensitivity f less than 0.48 nmlil. Its intra-assay variatin was % and inter-assay variatin was %. 189

4 Statistic analysis Hrmnal data were first lgarithmically transfrmed t nrmalise the distributins and then gemetric means and 95% cnfidence intervals calculated. Statistical analysis such as X 2 and Students' t-test were emplyed where apprpriate. This was perfrmed with SPSS (Versin 10) fr Windws. P value f less than 0.05 indicates statistical significance. Results Individual variatin t varian stimulatin in bth Grups A and B resulted in a wide range f cnfidence intervals fr plasma estradil cncentratins n luteal day 8. Despite the additin f E2V, n significant difference was seen in plasma estradil cncentratins n luteal day 8 when cmpared t thse withut any E2V supplementatin in bth stimulated IVF and inseminatin grups (Figure 1). The presence f crpra lutea, cnsequence t varian stimulatin, led t a supra-physilgical amunt f estradil in the circulatin. Hence, the small cntributin f E2V was masked by the enrmus estradil prductin in the mid luteal phase. Hwever, as the endgenus estradil secretin fell during the late luteal phase (Day 12) in nn-cnceptin cycles, plasma estradil cncentratins became significantly higher fr the grup receiving E2V (P < 0.05) (Figure 1). The inclusin f E2V suppressed endgenus prgesterne secretin in patients in bth Grups A and B, leading t lwer cncentratins n luteal days 8 and 12. Statistical analysis between the grups supplemented with r withut E2V reached significant value (P < 0.05) (Figure 1). Interestingly, the negative impact f E2V can be vercme by achieving a successful implantatin. Expnentially rising HCG derived frm the embry(s) prmptly rescued the crpra lutea and n further difference was evidenced between plasma prgesterne cncentratins n day 12 (Figure 2). In the nn-cnceptin cycles, the length f the luteal phase was 14.9 ± 1.8 and 15.6 ± 1.5 in the Grup A and 14.7 ± 1.7 and 15.4 ± 1.5 in Grup B in subjects supplemented with Cyclgest alne r in cmbinatin with E2V respectively. There is n statistical difference in the duratin between these grups. Tables I and II shw the clinical parameters and pregnancy rates in wmen supplemented with Cyclgest r E2V in Grups A and B respectively. N significant difference in psitive HCG and clinical pregnancy rates between the tw supplemented grups in subjects underging assisted cnceptin treatment using CC r GnRH-a (X 2, P> 0.05). Table I: Cmparisn between different parameters and pregnancy rate fr wmen supplemented with Cyclgest alne r in cmbinatin with E2V in stimulated IVF cycles. Cyclgest Cyclgest and E2V N. f cycles Age (years) 33 ± 6 35 ± 4 Gnadtrphin used (Amps) 25.9 ± ± 10.3 Duratin f stimulatin (days) 12.5 ± ± 2.0 Peak estradil (pml/ml) * 7174 ( ) 5089 ( ) N. cytes /patient 10.2 ± ± 7.2 Fertilisatin rate (%) 69 ± ± 16 N. embrys transfer 2.6 ± ± 0.5 Psitive HCG (%) 10/35 (28) 7/28 (25) Clinical pre~nancy (%) 7/35 (20) 5/28 (18) All values are means ± SD except * denting gemetric mean and 95% cnfident intervals. 190

5 Inhibitin f Prgesterne Secretin by Oestradil Administered in the luteal Phase f Assisted Cnceptin Cycles Table II: Cmparisn between the clinical parameters and pregnancy rate fr wmen supplemented with Cyclgest alne r in cmbinatin with E2V in stimulated inseminatin cycles. Cyclgest Cyclgest and E2V N. fcycles Age (years) 29 ± ± 3.4 Gnadtrphin used (Amps) 5.2 ± ± 3.2 Peak estradil (pml/ml) 1759 ( ) 2083 ( ) N. fllicles ~18 mm 2.4 ± ± 1.3 Psitive HCG (%) 7/30 (23) 5/25 (20) Clinical pregnancy (%) 5/30 (17) 3/25 (12) All values are means ± SD except' denatinggemetric mean and 95% cnfident intervals ġ 1500 i :s 1000 " ~ f 500 J L--...J 8 Grup A Stimulated IVF: Nn pregnant cycles 12 8 Days frm egg cllectin * I 200 l! 150I 5"" ~ 50 J < )!.. :s 1500 :10.j ":s 1000 "7) Ė ~ 0. 0 f----"-- GrupS Intrauterine inseminatin: Nn pregnant cycles Days frm intrauterine inseminatin * 250$'! 200 l!,g ~ II 100 e t 50 ~ a J Legends Fig. 1: Plasma estradil and prgesterne cncentratins (Gemetric means :!: 95% cnfidence intervals) n days 8 and 12 fllwing egg cllectin r intrauterine inseminatin in stimulated nn-cnceptin cycles supplemented with prgesterne nly ( 0 ) r bth prgesterne and Oestradil Valerate (Ill). Pairs that are significantly diherent (P < 0.05) are shwn by asterisks. 191

6 E' 3000 '0!In C.S! 'tv 2000 i CII g 8 : 1000 'ti ift E= '-- 1i: 8 Grup A Stimulated IVF: Pregnant cycles 12 8 Days frm egg cllectin ~ E.s-.. c: 300 t 8 c: c: e J!l [ ~.. a: 5' 3000!III CII l:! u "5 :a 1000 i e 0 + L. GrupS Intrauterine inseminatin : pregnant cycles Days frm intrauterine inseminatin 12 I t! 300 ~ i g 200 ~ c:ei 400 =: 100 e0 m Fig. 2: Plasma estradil and prgesterne cncentratins (Gemetric means:!: 95% cnfidence intervals) n days 8 and 12 fllwing egg cllectin r intrauterine inseminatin in stimulated cnceptin cycles supplemented with prgesterne nly ( D ) r bth prgesterne and Oestradil Valerate ( ). Pairs that are significantly different (P.< 0.05) are shwn by asterisks. e <II.!! ll Discussin Prgesterne is the mst imprtant sex hrmne fr human implantatin, while estradil may play a permissive, but nt an essential rle 7. Therefre, when the luteal supprt is used in varian stimulatin, prgesterne is frequently used alne and estrgen is usually mitted. This apprach fails t recgnize a ptential rle fr estrgen during the peri-implantatin perid. A recent study by Sharara and McClamrck (999) has recgnised the significance f luteal estradil in pregnancy utcme 3. A greater degree f decline in estradil levels during the mid luteal phase fllwing GnRH-a/HMG cycles was shwn t 192

7 Inhibitin f Prgesterne Secretin by Oestradil Administered in the luteal Phase f Assisted Cnceptin Cycles significantly assciate with higher pregnancy lsses arid lwer implantatin rate. Therefre, further studies int the rle f luteal estradil supplementatin are warranted. With the supplement f prgesterne in the spntaneus cycles, a 2 t 3 fld rise in cncentratin was bserved 1. Hwever, the additin f estradil valerate int the supplemental regime immediately induced a fall in prgesterne cncentratins. This bservatin suggests that a physilgical quantity f exgenus estradil can adversely affect crpus luteum functin. The mechanism f estradilrelated inhibitin f prgesterne secretin is nt entirely clear and the sites at which estradil act needs clarificatin. One pssible mechanism is that exgenus estradil acts indirectly upn the hypthalam-hypphyseal axis t inhibit LH secretin 8, thus denying the crpus luteum f necessary lutetrphic supprt. Evidence fr such an effect is prvided by Schnmaker et al. (1981) 9 They demnstrated that a small increase in peripheral estradil prduced by a subcutaneus estradil implant in rhesus mnkeys culd lead t LH suppressin and prematute lutelysis in the absence f any increase in estradil within the crpus luteum. In subjects pssessing an intact hypthalamicpituitary varian feedback system, it is prbable that the estradil-related suppressin in pituitary LH secretin might lead t abnrmal crpus luteum functin. Hwever, similar mechanism cannt be applied t dwn regulated IVF cycles whereby very little LH is present in the circulatin thrughut the entire luteal phase lo In an envirnment with lw LH cncentratins, single vulatry dse f HCG given prir t egg cllectin is the nly stimulus fr supprting crpra lutea functin 11. The levels f HCG are nt influenced by exgenus estradil. Frm these bservatins, it seems likely that estradil has a systemic as well jls a lcal effect n the varies. Injectin f estradil int the vary cntaining the crpus luteum induces premature luteal regressin in rhesus mnkeys 11. Under in vitr experimental cnditins, estradil can act directly upn human 12 and mnkey granulsa and theca cells 13 t inhibit bth basal and gnadtrphin stimulated prgesterne prductin. It has been 'suggested that estradil may act thrugh a shrt lp feedback paracrine actin and playa vital rganizing rle in andrgen and prgesterne prductin 12. Several studies have suggested a lutelytic rle fr exgenus estradil n the crpus luteum, as evidence by a reductin in prgesterne secretin and the nset f premature menstruatin 11. Because f the supplementatin with prgesterne, mst f the subjects in this study did nt experience premature menstruatin. The prgesterne supplement seems t be the key that ensures ptimal priming and maintenance the endmetrium in its' functinal state'. Mrever, in this study, the crpus luteum retained its full 'functinal capacity' as it was readily rescued by human chrinic gnadtrphin (HCG) after embry implantatin with prductin f apprpriate amunts f prgesterne (data nt shwn). This indicates that, althugh estradil can render the crpus luteum less active, it des nt induce a functinal destructin f the crpus luteum. Relatively high amunts f estradil seen n day 8 mask the presence f exgenus E2V in bth stimulated and natural cycles and reflect the small amunt f E2V that was used in this study. Nt surprisingly, the exgenus estradil had a minimal impact and failed t prevent the decline in estradil levels during the mid luteal phase. One way' f crrecting this decline is t increase the dses f E2V, but this wuld nt seem wise because it may further exacerbate the suppressive effect f estradil n the functin f crpus luteum. Anther apprach t crrect the defect is t use human chrinic gnadtrphin (HCG) as this restres the cncentratins f bth estrgen and prgesterne in the mid luteal phase The argument against the rutine use f HCG is the higher incidence f varian hyperstimulatin syndrme. Shham and Schachter (1996) have 193

8 suggested that the actual cncentratins f plasma estradil d nt affect the quality f endmetrial receptivity, prviding that a certain threshld level is exceeded 8. Hurd et al. (1996) have analysed 79 CC stimulated IVF cycles and have fund that luteal supplement f estradil and vaginal prgesterne imprved pregnancy utcme in cmparisn t thse subjects receiving n luteal supprt 6. Hwever, their data have t be interpreted with cautin, as the questin f whether the estradil, r the prgesterne r a cmbinatin f bth is respnsible fr the increase pregnancy rate remains unanswered. In this study, it is wrth nting that there is n statistical difference in the pregnancy rates between the grups supplemented with r withut EZV. This finding is in agreement with previus studies n CC 16 and GnRH-a cycles 17. An imprtant pint t be stressed is that the small number f subjects in this study may lead t the insufficient pwer f the study. Therefre, the pssibility remains that the results f these trials, including the current ne, are false negative and a meta-analysis in the future may vercme this prblem. Hwever, judgment f meta-analysis t has t be interpreted with cautin, as publicatin bias can distrt the end result 18. In cnclusin, the inclusin f exgenus estradil in the frm f estradil valerate t a luteal supplement fr assisted cnceptin des nt dismiss nr induce a functinal destructin f the crpus luteum, but suppresses secretin f prgesterne by the crpus luteum and is unlikely t be beneficial t the prcess f implantatin. Acknwledgements The authrs liked t thank Infertility Research Trust fr funding this study. IF II 11I11 IIIIPI 1. Tay, PYS, Lentn EA. Prgesterne prfiles in pregnant, nne pregnant, natural and stimulated NF cycles with and withut luteal supprt. Med. J. Mal. 2002; 57 (2): Burgain, c., Smitz, ]., Camus, M., Erard, P., Devrey, P., Van Steirteghem, A. c., Klppel, G. Human endmetrial maturatin is markedly imprved after luteal supplementatin f gnadtrphin - releasing hrmne analgue/ human menpausal gnadtrphin stimulated cycles. Hum. Reprd. 1994; 9: Sharara, F. 1., McClarnrck, H. D. Rati f estradil cncentratin n the day f human chrinic gnadtrphin administratin t mid-luteal estradil cncentratin is predictive f in-vitr fertilizatin utcme. Hum. Reprd. 1999; 11: Dnfer V. Cstabile L Gerli S. Papale E. Marelli G. Di Renz Gc. Lw dse f ethinyl estradil can reverse the antiestrgenic effects f clmiphene citrate n endmetrium. Gynecl. Obstet. Invest. 2001; 51(2): Sereepapng W. Suwajanakrn S. Triratanachat S. Sampatanukul P. Pruksanannda K. Bnkasemsanti W. Reinprayn D. Effects f clmiphene citrate n the endmetrium f regularly cycling wmen. Fertil. Steril. 2000; 73(2): Hurd, W. W., Randlph, J. F., Christman, G. M., Ansbacher, R, Menge, A. c., Gell, J. S. Luteal phase with bth estradil and prgesterne after clmiphene citrate stimulatin r in vitr fertilisatin. Fertil. Steril. 1996; 4:

9 Inhibitin f Prgesterne Secretin by Oestradil Administered in the luteal Phase f Assisted Cnceptin Cycles 7. Ghsh, D., Sengupta, J. Endmetrial receptivity fr implantatin. Hum. Reprd. 1995; 1: Shham, Z., Schachter, M. Estrgen bisynthesisregulatin, actin, remte effects, and value f mnitring in varian stimulatin cycles. Fertil.. Steril. 1996; 65: Schnmaker, J. N.; Victery, W., Karcsh, F. J. A receptive perid fr estradil-induced lutelysis in the rhesus mnkeys. Endcrinlgy 1981; 108: Valbuena, D., Pellicer, A., Guanes, P., Remhi, ]., Simn, C. Effect f disruptin versus cntinuatin f gnadtrphin - releasing agnist after human chrinic gnadtrphin administratin n crpus luteum functin in patients underging vulatin inductin fr in - vitr fertilisatin. Hum. Reprd. 1997; 12: Karsch, F. ]., Suttn, G. P. An intra-varian site fr the lutelytic actin f estrgen in the rhesus mnkeys. Endcrinlgy 1976; 98: Gilling-Smith, C, Willis, D. S., Franks, S. Oestradil feedback stimulatin f andrgen bisynthesis by human theca cells. Hum. Reprd. 1997; 8: Stuffer, R. 1. Crpus luteum functin and dysfunctin. Curr Opin. Obstet. Gynecl. 1990; 3: Glan, A., Herman, A., Sffer, Y, Bukvsky, I., Caspi, E., Rn-El, R. Human chrinic gnadtrphin is a better luteal supprt than prgesterne in ultrashrt gnadtrphin releasing hrmne agnist/mentrphin in-vitr fertilisatin cycles. Hum. Reprd. 1993; 9: Mchtar, M. H., Hgerzeil, H. v., Ml, B. W. J. Prgesterne alne versus prgesterne cmbined with HCG as luteal supprt in GnRH-a/HMG induced IVF cycles: a randmised clinical trial. Hum. Reprd. 1996; 8: Lewin, A., Benshushan, A., Mezker, E., Yanai, N., Schenker, J. G., Gshen, R. The rle f estrgen supprt during the luteal phase f in' vitr fertilisatin embry transplant cycles: a cmparative study between prgesterne alne and estrgen and prgesterne supprt. Fertil. Steril. 1994; 1: Smitz, ]., Devrey, P., Camus, M., Deschacht, ]., Khan, I., Staessen, c., Van Waesberghe, 1., Wisant, A., Van Steirteghem, A. C. The luteal phase and early pregnancy after cmbined GnRHagnist/HMG treatment fr supervulatin in IVF r GIFT. Hum. Reprd. 1988; 5: Egger, M., Smith, G. Misleading meta-analysis. Br. Med. J. 1988; 310;