Follicular growth and serum estradiol concentration as predictors of in vitro fertilization outcome

Transcription

1 Yale University EliSchlar A Digital Platfrm fr Schlarly Publishing at Yale Yale Medicine Thesis Digital Library Schl f Medicine 1994 Fllicular grwth and serum estradil cncentratin as predictrs f in vitr fertilizatin utcme Nicle Hausman Yale University Fllw this and additinal wrks at: Recmmended Citatin Hausman, Nicle, "Fllicular grwth and serum estradil cncentratin as predictrs f in vitr fertilizatin utcme" (1994). Yale Medicine Thesis Digital Library This Open Access Thesis is brught t yu fr free and pen access by the Schl f Medicine at EliSchlar A Digital Platfrm fr Schlarly Publishing at Yale. It has been accepted fr inclusin in Yale Medicine Thesis Digital Library by an authrized administratr f EliSchlar A Digital Platfrm fr Schlarly Publishing at Yale. Fr mre infrmatin, please cntact elischlar@yale.edu.

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3 HARVEY CUSHING / JOHN HAY WHITNEY MEDICAL LIBRARY MANUSCRIPT THESES Unpublished theses submitted fr the Master's and Dctr's degrees and depsited in the Medical Library are t be used nly with due regard t the rights f the authrs. Bibligraphical references may be nted, but passages must nt be cpied withut permissin f the authrs, and withut prper credit being given in subsequent written r published wrk. This thesis by has been used by the fllwing persns, whse signatures attest their acceptance f the abve restrictins. NAME AND ADDRESS DATE

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5 Digitized by the Internet Archive in 2017 with funding frm The Natinal Endwment fr the Humanities and the Arcadia Fund

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8 FOLLICULAR GROWTH AND SERUM ESTRADIOL CONCENTRATION AS PREDICTORS OF IN VITRO FERTILIZATION OUTCOME A Thesis Submitted t the Yale University Schl f Medicine in Partial Fulfillment f the Requirements fr the Degree f Dctr f Medicine by Nicle Hausman 1994

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10 ACKNOWLEDGEMENT I wuld like t express my gratitude t Dr. David Olive fr his guidance during the curse f this prject, as well as a special thanks t Dr. Antni Duleba fr his invaluable instructin, limitless enthusiasm, and generus cmmitment t all aspects f this wrk. I must als thank my mther, father, and Dug fr their uncnditinal supprt f my endeavrs. Withut their genersity, warmth, and cnfidence, my jurney thrugh medical schl and the cmpletin f this prject wuld never have been pssible.

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12 TABLE OF CONTENTS I. ABSTRACT i H INTRODUCTION A. Infertility: Nature and Magnitude f the Prblem B. Diagnstic Cnsideratins 1. Tubal Factr Infertility 2. Endmetrisis 3. Plycystic Ovary Syndrme 4. Male Factr Infertility 5. Unexplained Infertility C Ovulatin Inductin in IVF 1. Flliculgenesis 2. Clmiphene Citrate 3. Gnadtrpin Releasing Hrmne Agnists D. Factrs Affecting the Outcme f IVF 1. Age 2. Male Factr Infertility 3. Embry Quality 4. Patterns f Estradil Secretin 5. Endmetrial Receptivity 6. Ocyte Quality 7. Number f Ocytes Retrieved 8. Number f Prevulatry Fllicles IE. STATEMENT OF PURPOSE 23 IV. METHODS A. Overview f the Ppulatin B. Ovulatin Inductin Prtcls C. Etilgic Definitins D. Statistical Methds E. Cntributins V. RESULTS A. Ppulatin Distributin B. Outcme f IVF C. Crrelatins D. Influence f the Number f Fllicles n Pregnancy Rate E. Influence f the Estradil Level n Pregnancy Rate F. Distributin f Pregnancy Rates G. Factrs Influencing Pregnancy in Tubal Disease VI. DISCUSSION 36

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14 VH. FIGURES AND TABLES Table 1. Distributin f the Ppulatin by Type f Cycle Figure 1. Distributin f the Ppulatin by Type f Cycle Table 2. Distributin f the Ppulatin by Diagnsis Figure 2. Distributin f the Ppulatin by Diagnsis Table 3. Distributin f Stimulatin Prtcls in the General Ppulatin Table 4. Parameters f IVF by Diagnsis Figure 3. Pregnancy Rates in Individual Diagnstic Grups Figure 4. Number f Fllicles Measuring ^ 12 mm. n the Day f hcg Administratin in Individual Diagnstic Grups Figure 5. Estradil Levels in Individual Diagnstic Grups Table 5. Crrelatin Between the Number f Fllicles ^ 12 mm and Other IVF Parameters Table 6. Crrelatin Between the Number f Ocytes Retrieved and Other IVF Parameters Figure 6. Pregnancy Rate by Diagnsis as a Functin f the Number f Fllicles Measuring ^12 mm n the Day f hcg Administratin Figure 7. Pregnancy Rate by Diagnsis as a Functin f the Estradil Level Measured n the Day f hcg Administratin Table 7. Numbers and Rates f Pregnancies per Number f Fllicles >12 mm Figure 8. Effect f the Number f Fllicles ^12 mm n the Day f hcg Administratin n the Pregnancy Rate Table 8. IVF Parameters by Pregnancy Outcme fr Tubal Disease Ppulatin Figure 9. Number f Transferred Embrys in Pregnant Patients with Tubal Disease Table 9. hmg/fsh Administratin in Pregnant Tubal Disease Patients VIII. REFERENCES

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16 1 I. ABSTRACT FOLLICULAR GROWTH AND SERUM ESTRADIOL CONCENTRATION AS PREDICTORS OF IN VITRO FERTILIZATION OUTCOME. Nicle Hausman, Antni J. Duleba, and David L. Olive, Sectin f Reprductive Endcrinlgy, Department f Obstetrics and Gyneclgy, Yale University Schl f Medicine, New Haven, Cnnecticut. The utcme f in vitr fertilizatin and embry transfer (IVF-ET) is influenced by multiple factrs. Studies perfrmed in ppulatins with unspecified etilgies f infertility have suggested that the number f cytes retrieved during a cycle f IVF may inversely crrelate with subsequent fertilizatin and chance f pregnancy. The purpse f this study was t examine the relatinships between pretransfer parameters in IVF-ET and the subsequent ccurrence f pregnancy in patients assigned t individual diagnstic grups, with particular fcus n patients with tubal disease. The study evaluated 466 stimulated cycles which prceeded t cyte retrievals. In patients with tubal disease, the number f fllicles measuring at least 12 mm n the day f human chrinic gnadtrpin (hcg) administratin crrelated negatively with pregnancy rates: pregnancy rates were 28.3% fr thse with < 7 fllicles and 8.7% fr thse with > 7 fllicles (P = 0.014). Cmparable findings were bserved when crrelating pregnancy rates with estradil level n the day f hcg administratin: pregnancy rates were 28.9% fr thse with estradil ^ 1,000 pg/ml and 13.3% fr thse with estradil > 1,000 pg/ml (P = 0.049). In cntrast, the ppsite trends were nted in patients with plycystic vary syndrme and unexplained infertility, with

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18 11 increasing pregnancy rates at bth higher numbers f fllicles and higher levels f estradil. These findings demnstrate that varian respnse is a predictr f the success f IVF-ET in tubal disease patients, and that vulatin inductin prtcls may need t be individualized fr each diagnstic grup.

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20 1 IL INTRODUCTION A. Infertility: Nature and Magnitude f the Prblem Cnsidering the cmplexity f the reprductive prcess, it is remarkable that 80% f cuples achieve cnceptin within ne year f attempting pregnancy. Of the ppulatin attempting t cnceive, a spectrum f fertility exists, extending frm highly fertile cuples t thse with relative infertility. Infertility is usually defined as the inability f a cuple practicing frequent intercurse withut cntraceptin t cnceive a child within ne year. It affects 15 t 20% f cuples r apprximately seven millin peple f reprductive age in the United States,^ and may be due t multiple etilgies in ne r bth partners. In fact, apprximately 20% f infertile cuples have mre than ne majr cause (male factr, vulatry dysfunctin, r tubalperitneal disease) fr their inability t cnceive. Withut therapy, spntaneus intrauterine pregnancies d ccur in infertile cuples, albeit at a substantially decreased rate; thus, treatments are pragmatically aimed at shrtening the time t and increasing the likelihd f cnceptin. With a thrugh evaluatin f the infertile cuple and applicatin f current treatments shrt f in vitr fertilizatin (IVF), 50 t 60% can be expected t cnceive.^ Fr infertile cuples experiencing failure f ther available treatments, IVF with embry transfer (ET) remains the last resrt, with apprximately 20% f thse cuples underging the prcedure ultimately establishing a pregnancy. B. Diagnstic Cnsideratins The precise incidence f the etilgic factrs invlved in infertility varies with the ppulatin studied. As an apprximatin, 15 t 20% f infertility cases are due t vulatry dysfunctin, 30 t 40% are due t pelvic

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22 2 factrs (endmetrisis, adhesins, r tubal disease), 30 t 40% are due t male factrs (abnrmal semen parameters), 5 t 10% are due t abnrmal spermcervical mucus penetratin r antisperm antibdies, and 10 t 15% are due t unexplained infertility, fr which n cause can be elucidated using currently available tests.^ 1. Tubal Factr Infertility During the past decade, the incidence f infertility caused by damage t the viduct has increased, mst prbably due t an increased incidence f salpingitis. Obstructin f the viduct may ccur at either the prximal r distal prtin, and may ccasinally invlve bth regins simultaneusly. The extent and lcatin f the intrinsic and extrinsic tubal disease shuld be accurately determined by bth hystersalpinggraphy and laparscpy. The prgnsis fr cnceptin after surgical tubal recnstructin depends heavily n the severity f tubal damage, with very lw intrauterine pregnancy rates demnstrated in wmen with extensive pre-surgical bstructin. In these wmen, in vitr fertilizatin prvides the mst efficacius interventin.^ In the case f distal tubal bstructin, the hystersalpinggram (HSG) aids in determining whether the tubal bstructin is cmplete r partial, the diameter f the hydrsalpinx (accumulatin f serus fluid in the fallpian tube), and the appearance f the rugae. Beynd this, laparscpy is usually necessary t determine the actual size f the hydrsalpinx, the thickness f the viduct wall after distensin with dye, and the presence r extent f pelvic adhesins. As previusly stated, the prgnsis fr cnceptin fllwing distal tubal recnstructin crrelates strngly with the extent f tubal damage present befre surgery. If the fimbriae appear anatmically nrmal, but phimtic with nly partial cclusin by adhesins, remval f

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24 3 the adhesins by a fimbriplasty prcedure results in pst-surgical cnceptin rates n the rder f 60 %.^ If the distal end is cmpletely ccluded and a salpingstmy (the creatin f an artificial pening in a fallpian tube clsed by inflammatin) is necessary, cnceptin rates fllwing recnstructin drp t 30% with apprximately 25% f these being tubal pregnancies.^ As such, it is nly lgical that a psitive crrelatin exists between the degree f distal tubal damage prir t recnstructive surgery and the incidence f ectpic pregnancies fllwing surgery.^ If n dye reaches the viduct during perfrmance f an HSG, the diagnsis f prximal tubal bstructin must be cnsidered. Prximal tubal bstructin is mst cmmnly due t damage resulting frm infectin, but may ccasinally be caused by endmetrisis. Spasm f the uter-tubal junctin may ccur during the HSG and shuld als be cnsidered in the differential diagnsis. Micrsurgical tubcrnual reanastmsis in wmen with prximal tubal damage has resulted in term pregnancy rates f apprximately 50% and ectpic rates less than 10%.^ As a rule, if cnceptin des nt ccur within 6 t 12 mnths f surgical recnstructin and a repeat HSG reveals recurrent tubal bstructin, a secnd surgical prcedure is nt undertaken, due t dismal fllw-up pregnancy rates. These patients are subsequently referred fr in vitr fertilizatin.^ 2. Endmetrisis Althugh endmetrisis is a benign disease, it ften becmes prgressive and chrnic, with lcal infiltratin and wide disseminatin. By definitin, endmetrisis is the presence and grwth f the glands and strma f the uterine lining in an aberrant lcatin. Althugh the age-

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26 4 specific incidence r prevalence f endmetrisis can nly be apprximated, it is believed that the prevalence f the disease has been increasing ver the past 25 years. Estimates reveal that endmetrisis is present in 5 t 15% f lapartmies perfrmed n wmen f reprductive age, while the prevalence increases t 30 t 45% in thse wh are infertile. Hetertpic endmetrial tissue grws under the cyclic influence f varian hrmnes, thus explaining nset and symptms during the reprductive years. Mst cmmnly, patients with endmetrisis are wmen in their mid-30's, nulliparus, invluntarily infertile, with symptms f dysmenrrhea and pelvic pain. The etilgy f endmetrisis, while nt accurately elucidated, has been hypthesized t invlve many factrs, including retrgrade menstruatin, vascular disseminatin, metaplasia, genetic predispsitin, immunlgic defects, and hrmnal influences. N single thery adequately explains the many manifestatins f the disease.^ The underlying pathphysilgy prducing infertility in wmen with endmetrisis remains largely unknwn, althugh a relatinship between the severity f the disease and infertility has been established. Olive et al. reprted that apprximately 65% f wmen with mild endmetrisis cnceived withut treatment, while nly 25% and 0% f wmen with mderate and severe endmetrisis, respectively, became pregnant with expectant management alne.^ In the presence f mderate r severe disease with extensive adhesins invlving the viducts, the causal relatinship between endmetrisis and infertility seems clear. Hwever, in patients with minimal r mild disease, a causal relatinship between infertility and endmetrisis has nt yet been established.^

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28 5 3. Plycystic Ovary Syndrme The mst cmmn cause f chrnic anvulatin is the plycystic vary syndrme (PCO), hyperandrgenism a and cmplex endcrine anvulatin, disrder invlving cnsisting dysfunctin f f the hypthalamus, pituitary, varies, adrenals, and peripheral adipse tissues. One f the hallmarks f PCO is the relative elevatin f luteinizing hrmne (LH) ver fllicle-stimulating hrmne (FSH), with exaggerated pulse levels f LH in assciatin with lw nrmal levels f FSH. Explanatins fr the relative disparity f LH t FSH have invked hypthalamic-pituitary invlvement with a central disturbance in the frequency and amplitude f gnadtrpin releasing hrmne (GnRH). As such, factrs that affect GnRH secretin must als be cnsidered in the pathgenesis f PCO, including endgenus piids, neurlgic factrs, and prlactin/ A strng assciatin between galactrrhea, hyperprlactinemia, and PCO has lng been recgnized. Many reprts have suggested that hyperprlactinemia in PCO is caused by the inhibitry effect f increased estrgen levels n the dpaminergic system/ Supprt f this cncept is demnstrated, in part, by a sustained reductin in circulating LH levels achieved in PCO patients administered dpamine/ Further studies have shwn a synchrny between endgenus pulses f LH and prlactin/ with brmcriptine (a dpamine agnist) induced reductins f prlactin and LH secretin in PCO patients/ Mrever, hyperprlactinemic wmen demnstrate significantly higher levels f dihydrepiandrsterne-sulfate (DHEAS) than nrmal cntrls, with treatment f the elevated prlactin resulting in DHEAS reductins/ Bilateral plycystic varies represent the sine qua nn f PCO. Typically, fllicles in the varies are arrested in fllicular develpment.

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30 6 measure apprximately tw t eight mm in diameter and appear as a "string f pearls" lcated beneath a smth, glistening capsule. In cntrast with nrm-vulatry patients, fllicles in PCO patients are usually fund t be in varying stages f grwth and atresia, withut prductin f a dminant fllicle/ Ericksn et al., demnstrated that arrested fllicular develpment in PCO was nt due t an inherent defect in varian steridgenesis. In this study, the fllicles f PCO patients were capable f prducing nrmal levels f estrgen frm the precursr andrstenedine in the presence f apprpriate levels f gnadtrpin secretin r the additin f exgenus FSH/0 Plycystic vary syndrme ften presents with excessive hirsutism and hyperandrgenism. The adrenal gland, as a prducer f apprximately 50% f peripheral andrstenedine (the remaining 50% being f varian rigin), may be significantly invlved in the pathgenesis f a prprtin f PCO patients. Cngenital r acquired blcks in the andrgen steridgenesis pathway may lead t elevatins in levels f precursr hrmnes with andrgenizing ptential, resulting in hirsutism, acne, ligmenrrhea, infertility, and ther findings similar t thse in PCO. Hwever, mst investigatrs agree that this PCO-like appearance in cngenital adrenal hyperplasia is distinct frm "classical" PCO. The adrenal gland is nrmally regulated by a tight feedback lp invlving the hypthalamus and pituitary. As such, the assciatin between adrencrtictrpic hrmne (ACTH) and PCO has becme a subject f much interest/ Althugh PCO patients usually have higher levels f LH, dihydrepiandrsterne (DHEA), DHEAS, teststerne, and andrstenedine than healthy wmen, ACTH levels are similar in bth grups. ^ This finding intrduces the pssibility that adrenal andrgen prductin in PCO patients may be due t altered adrenal respnsiveness t ACTH, rather than t alteratins in ACTH prductin/

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32 7 Of great imprtance in the pathgenesis f PCO in many patients are the peripheral extraglandular surces f circulating estrgens. Althugh the majrity f estrgens riginate frm the varies and adrenal glands, armatizatin f andrgens t weaker estrgens ccurs in the skin, brain, and adipse tissue. Because PCO patients are ften verweight, the increased peripheral armatizatin f elevated andrgens may lead t excessive levels f estrgens. The increased estradil levels have, in turn, been shwn t crrelate with bth high LH/FSH ratis^ and reductins in hypthalamic dpamine levels in wmen with PCO.^ The mst cmmn medicatin used in the vulatin inductin f patients with PCO is clmiphene citrate (CC; Clmid), a partial agnist f estrgen. Using CC, apprximately 80 t 90% f PCO patients achieve vulatin, with 40 t 50% f these wmen ultimately cnceiving. In patients with pr vulatry respnse t CC, the treatment f chice is usually human menpausal gnadtrpin (hmg)/ Ovulatin inductin in patients with PCO is assciated with an increased risk f multiple births and varian hyperstimulatin syndrme (characterized by massive varian enlargement and third-space fluid accumulatin), pssibly due t the presence f multiple small fllicles and elevated LH and estrgen levels. Therefre, in PCO patients, pregnancy rates are maximized and cmplicatin rates minimized by administering gnadtrpins in small dses ver a lnger perid f time than in the vulatin inductin f ther infertility patients/ 4. Male Factr Infertility In apprximately 30 t 40% percent f infertile cuples, male factrs are at least partially respnsible fr the inability t cnceive/^ In ne third f

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34 8 infertile cuples, male-factr abnrmalities are detected in the man alne, while in an additinal 20% f cases, etilgic factrs are diagnsed in bth the man and wman. As part f a standard evaluatin, a semen analysis shuld be undertaken in all cuples presenting with infertility. Althugh there is wide variatin between patients and repeat samples frm the same patients in terms f vlume, number, and mtility f sperm, nrmal guidelines fr each f these parameters are well established.^ Typical abnrmalities detected by semen analysis are lw sperm cncentratin (ligspermia), decreased mtility (asthenspermia), decreased nrmal frms (teratspermia), and increased semen viscsity. ^ If an abnrmality is fund, a repeat analysis shuld be perfrmed tw t three mnths later in rder t determine the actual presence r absence f a male factr. It is cnsidered clinically inapprpriate t designate a male patient as infertile based upn a single semen analysis.^ Examinatin f the male with abnrmal semen analyses may reveal a variccele r ductal bstructin (bth f which are surgically crrectable), an immunlgic disrder, a hrmnal disturbance, r after thrugh negative evaluatin, an idipathic etilgy. When treatment f the male partner is unsuccessful, inseminatin with split ejaculates, intrauterine inseminatin with washed sperm, r cmbinatins f these mdalities with vulatin inductin may be effective. In cases f severe sperm abnrmalities, cnventinal IVF is ften useful, given its ability t cmbine high cncentratins f mtile sperm with cytes in a small vlume f medium, ptimizing the pssibility f fertilizatin. Fertilizatin rates are unifrmly reduced in male factr infertility. Althugh embry transfer rates are apprximately 50%, pregnancy rates per transfer are similar t thse fr ther diagnstic etilgies f infertility.^

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36 9 5. Unexplained Infertility The term "unexplained infertility" is reserved fr cuples experiencing infertility f greater than tw years' duratin in the face f a nrmal, cmplete evaluatin, including dcumentatin f nrmal vulatin, nrmal semen parameters, nrmal sperm-mucus interactin and pst-cital test, and nrmal uterus and tubes by HSG and laparscpy.^ A study cnducted by Barnea et al. demnstrated that amng patients with unexplained infertility diagnsed by such an evaluatin, the average duratin f infertility was 39 mnths, with 34% achieving pregnancy within six mnths f study registratin and 80% within five years. Many explanatins fr the etilgy f unexplained infertility have been pstulated, including changes in the "varian reserve" and ther ccult abnrmalities. An increase in the prprtin f cuples assigned t the unexplained categry f infertility may, in part, be a cnsequence f changing childbearing behavir, with mre wmen delaying childbearing t lder ages.'7 With lder chrnlgical female age may cme alteratins in the varian respnse, as indicated by a study perfrmed n 51 wmen aged 35 r lder with unexplained infertility. In this study, a clmiphene challenge test was used t assess fertility, with wmen subsequently divided int tw grups: thse with diminished varian reserve (with pst-challenge FSH levels elevated t a mean f 39 miu/ml) and thse with adequate reserve (with pst-challenge mean FSH levels f 12 miu/ml). Treatment cnsisting f vulatin inductin, timed intercurse, r inseminatin resulted in pregnancy in 42% f wmen with adequate reserve, while nly 5% f thse with diminished reserve became pregnant. ^ Other explanatins fr unexplained infertility may include ccult dysfunctin f vum prductin and release, defective crpus luteum functin, defects in sperm transprt and

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38 10 functin (with therwise nrmal semen parameters), subtle uterine r tubal defects including dysfunctinal vum pick-up and transprt, "silent" infectin in either the seminal fluid r the female genital tract (chlamydia, mycplasma, and ureaplasma being cmmnly assciated with subfertility), and immunlgic infertility. ^ Treatment fr true unexplained infertility, as cnfirmed by a mre detailed evaluatin (ultrasund evaluatin f fllicular dynamics, sperm functin assays, evaluatin f subtle tubal, uterine, and pelvic factrs, tests fr ccult infectius agents, and immunlgic testing) usually includes vulatin inductin and assisted reprductive technlgy in the frm f gamete intrafallpian transfer (GIFT) r IVF C. Ovulatin Inductin in IVF The early use f supervulatin cycles in IVF established that success was principally determined by the number f embrys transferred int the uterine cavity. In essence, the replacement f three r mre healthy embrys ptimized the likelihd f achieving an nging pregnancy. Replacement f mre than three embrys increases the likelihd f multiple pregnancies while nt increasing the pregnancy rate.-^o Given this infrmatin, it has becme usual practice t recver atleast fur r five gd quality cytes in rder t be reasnably sure f the replacement f three healthy embrys.^ Althugh natural cycle IVF may be f value in a limited number f patients, it has been predminantly superseded by supervulatin cycles fr assisted reprductin in an attempt t maximize the generatin f large numbers f mature cytes. What fllws is a review f nrmal flliculgenesis in the unstimulated cycle and an verview f the vulatin inductin prcedures cmmnly used in IVF.

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40 11 1. Flliculgenesis Ovarian functin in the nrmal r stimulated cycle rests upn cmmunicatin, f either a stimulatry r inhibitry nature, between the hypthalamus, pituitary, and vary. During supraphysilgic stimulatin f the varies fr in vitr fertilizatin, the pharmaclgy f exgenus hrmne therapy markedly alters the relatinships between these endcrine rgans, affecting the dynamics f fllicular develpment.^ During the embrynic perid, primrdial germ cell ppulatins are established that will later give rise t cytes. Just prir t birth, the tw t fur millin primary cytes frmed during fetal life reach prphase f the first meitic divisin. The primrdial fllicle then cnsists f this primary cyte cvered by a single layer f granulsa cells.a prtin f the primrdial fllicles begin t grw r underg atresia as sn as they are frmed, while thers enter a quiescent state until puberty.^ Reactivatin f quiescent primrdial fllicles, fllicular develpment, and cyte atresia appear t punctuate a cntinuus prcess in the vary, beginning immediately after the first fllicles frm and prgressing until the end f the reprductive perid.^ Hdgen prpses a tw-tiered varian mechanism perative in fllicular develpment. At the first tier, specific factrs, bth intravarian and extravarian, gvern the prgressive diminutin f the chrt f develping fllicles t the vulatry quta f each cycle. This regulatin f chrt size nly perates, hwever, when circulating gnadtrpins (FSH and LH) exceed minimal tnic levels. At the secnd tier, varian steridal and nnsteridal hrmnes negatively feed back t inhibit gnadtrpin secretin, thus cntrlling circulating gnadtrpins at apprpriate tnic levels. The first and secnd tiers f the prpsed mechanism are intimately

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42 12 cnnected, such that changes in the cmpnents f ne tier may drastically alter activities f the secnd. Fr example, it is hypthesized that if circulating gnadtrpin levels are far abve the tnic set-pint, then first tier varian mechanisms becme impaired, resulting in supervulatin. Therefre, multiple fllicular develpment ccurs as a result f bth increased gnadtrpin availability and the verriding f early fllicle selectin mechanisms.^ During each cycle, primrdial fllicles are "recruited" and depart frm the resting pl t underg a well-characterized pattern f develpment,^ termed by Schwartz the "trajectry f fllicle grwth".^ Grwing fllicles are vulnerable t atresia at any pint alng this "trajectry", making recruitment an bligatry, but decidedly preliminary, step in the selectin f a dminant fllicle frm the develping chrt.-^ Under the influence f FSH in the natural cycle, the number f granulsa cells in the primrdial fllicle increases and the fllicle matures int a primary r preantral fllicle. As the number f granulsa cells increases, the prductin and secretin f estradil frm these cells increases, representing the first sign f functinal maturatin.^' ^4 Estradil, in turn, stimulates preantral fllicle grwth, prevents fllicular atresia, and ptentiates the effects f FSH n the granulsa cells. The fllicle destined t becme dminant secretes the greatest amunt f estradil; in fact, almst 80% f the apprximately 500 mg f estradil prduced daily just prir t vulatin riginates frm the dminant fllicle. The elevated estradil levels increase the density f FSH receptrs n the fllicle surface, ultimately causing a surge in the mittic activity f the granulsa cells. Mrever, the rising cncentratin f estradil exerts a negative feedback effect n the release f FSH frm the pituitary, halting the develpment f the less FSH

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44 13 sensitive nn-dminant fllicles. The dminant fllicle, in cntrast, cntinues t develp due nt nly t its increased highly vascularized theca cells, allwing mre FSH t reach the receptrs, but als t its increased density f FSH receptrs. As the granulsa cells prliferate, LH receptrs appear n their surface membranes in respnse t FSH and estradil; when LH binds t the receptrs, granulsa cell prliferatin ceases and the cells begin t secrete prgesterne. 23 in essence, the fllicle destined t vulate plays a pivtal rle in regulating the size f the vulatry quta, inhibiting the develpment f ther fllicles in the chrt, and becming "dminant" abut a week prir t vulatin. Interestingly enugh, this dminant fllicle cntinues t thrive in a physilgic milieu it has itself made inhspitable fr thers.^ Just prir t vulatin, the dminant fllicle attains, n average, a mean diameter f apprximately 19.5 mm, with a range f 18 t 25 mm. The maximal size f the dminant fllicle can vary amng different wmen, as well as in the same wman in different cycles. Rapidly rising estradil levels, in cmbinatin with the small increase in prgesterne prduced by the dminant fllicle, serve as the signal t the hypthalamic-pituitary axis that the fllicle has matured sufficiently fr vulatin. At midcycle, the substantial estradil increase stimulates LH secretin and the small prevulatry prgesterne increase stimulates FSH secretin, culminating in an LH and FSH surge. The midcycle LH surge is respnsible fr initiating the vulatry prcess, germinal vesicle disruptin, and cmpletin f metaphase I f meisis. LH stimulates synthesis f prstaglandins and prtelytic enzymes, bth f which aid in fllicular rupture; FSH stimulates prductin f plasmingen activatr, prducing plasmin t detach the cumulus frm the granulsa cells, assisting egg extrusin during fllicular rupture. It has been

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46 14 determined that vulatin ccurs apprximately 24 hurs after the estradil peak, 12 t 16 hurs after the LH peak in serum, and abut 32 hurs after the initial elevatin in LH level. Once the cyte is extruded during fllicle rupture, the crpus luteum frms and prduces prgesterne under the influence f LH. After vulatin, the increasing levels f estradil and prgesterne exert a negative feedback effect n FSH and LH, respectively.^ Althugh the precise mechanisms f flliculgenesis during vulatin inductin have still nt been fully elucidated, it is generally pstulated that fllicular recruitment ccurs in much the same fashin as it des during the natural cycle. The mst ntable difference between stimulated and nn- stimulated cycles remains, quite simply, the sheer number f fllicles recruited fr vulatin, with higher yields fr stimulated cycles. 2. Clmiphene Citrate Until recently, the mst frequent varian stimulatin regimen used in assisted cnceptin was the cmbinatin f clmiphene citrate and exgenus gnadtrpins (hmg). The purpse f such a regimen is t verride the estrgen-driven negative feedback n endgenus gnadtrpin secretin that ccurs in a natural cycle. In essence, CC causes a mild hypersecretin f pituitary gnadtrpins, thus stimulating the recruitment f a number f small fllicles. Exgenus gnadtrpins may be administered thereafter t sustain the grwth f this chrt f recruited fllicles, achieving multiple synchrnus fllicular develpment. Multiple fllicular develpment causes increased estradil levels, resulting in psitive feedback elevatins in LH. The best success rates with the CC/hMG cmbinatin have been achieved with mnitring fr the endgenus LH surge, which may ccur in the late fllicular phase. If the endgenus LH surge remains undetected, vulatin

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48 15 prir t cyte recvery r the cllectin f pstmature cytes may ccur. The ccurrence f the LH surge is nt predictable by fllicle size nce the leading fllicle exceeds 15 mm in diameter, by the abslute level f estrgen, r by the rate f estrgen rise. If the LH surge is successfully detected, used t time cyte recvery, and supprted by the administratin f human chrinic gnadtrpin (hcg), the patient's clinical perfrmance thrugh an assisted cnceptin cycle is ptimized. The criteria fr the administratin f hcg fr vulatin inductin in CC/hMG cycles are the detectin f the spntaneus LH surge r ne r mre fllicles reaching mm in diameter.if hcg is given t late, the mst advanced fllicles may yield pstmature (r fragmented) eggs f lw ptential viability; cnversely, if hcg is injected t early, the eggs may be immature.^ Ocyte recvery is timed apprximately 32 hurs after 5,000-10,000 IU f hcg is administered.^ Early results with CC/hMG demnstrated a clinical pregnancy rate f 35% fllwing the transfer f three embrys, clinical pregnancy rates per transfer f 25%, and a "take-hme baby rate" f apprximately 15%. A cmparisn f patients wh became pregnant after embry transfer and thse whse embrys failed t implant revealed that patients wh established a clinical pregnancy had bth significantly lwer urinary LH utput in the 2 days prir t vulatin inductin and lwer plasma LH levels in the late fllicular phase. Mre specifically, higher late fllicular phase LH levels crrelated cmprmised with cyte quality and fitness, reduced implantatin rate, and early pregnancy lss. The degree f gnadtrpin hypersecretin induced with clmiphene appears t vary between individuals and between cycles within the same individual.^ The cnsequences f high late fllicular phase LH levels may be explained by cnsideratin f the mechanism f cyte maturatin and

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50 16 varian paracrinlgy. One hypthesis states that the natural midcycle LH surge prmtes the degradatin f the gap-junctinal cmmunicatin system in the granulsa cell layers, thus impeding the passage f cyte maturatin inhibitr(s) frm the fllicle wall t the cyte.^ A secnd hypthesis pstulates that the LH surge stimulates the expressin f maturatininducing factrs by the granulsa cells.^ it has been shwn that high basal LH cncentratins, as are present in individuals with PCO, may have direct negative influences upn cyte viability in varian stimulatin fr IVf30,31/ althugh ther evidence has failed t generate supprt f this Gnadtrpin Releasing Hrmne Agnists In patients wh shw inapprpriately elevated levels f LH during supervulatin with CC/hMG, imprvements in fllicular recruitment, fertilizatin rates, and pregnancy rates can be achieved thrugh an inhibitin f LH by administratin f gnadtrpin releasing hrmne (GnRH) agnists. Upn first expsure t the GnRH agnist, a massive release f the pituitary gnadtrpins LH and FSH ccurs fr apprximately tw t fur days, fllwed by the requisite feedback inhibitin f LH synthesis and depletin f its readily releasable frm. The membrane-bund GnRH agnist stimulates receptr internalizatin and degradatin, thus decreasing receptr numbers; mrever, the cntinued ccupatin f the pituitary gnadtrpin receptrs maintains a lwered sensitivity t endgenus GnRH. The cnsequence f prlnged GnRH agnist use is lwered circulating levels f LH (and FSH, t a smaller degree) as well as inhibitin f vulatin. After cessatin f GnRH analgue administratin, nrmal pituitary functin is nly recvered as de nv receptr synthesis ccurs. As the gnadtrpin

51

52 17 receptrs becme sensitive t endgenus GnRH, circulating levels f LH and FSH are restred.^ Ovulatin inductin strategies cmbining GnRH analgues and exgenus gnadtrpins (hmg r pure FSH) fall int three basic categries: lng, shrt, and ultra-shrt prtcls. In the lng prtcl, a GnRH agnist is administered fr a perid f 8 t 21 days, beginning in the early t mid-luteal phase f the preceding cycle, achieving a temprary state f hypgnadtrpic hypgnadism. Once desensitizatin f the pituitary gnadtrphs t endgenus GnRH is cmpleted, the GnRH agnist dse is reduced t a maintenance level, prlnging varian quiescence and suppressin f circulating gnadtrpins while preventing an endgenus LH surge. Adequate supervulatin may then be achieved by the daily administratin f exgenus gnadtrpins, with apprximately 10 days f stimulatin being adequate fr sufficient vulatin inductin and estrgen priming f the endmetrium. Due t the suppressin f endgenus LH secretin, luteinizatin rarely ccurs until an vulatin-inducing dse f hcg is given. 277 The shrt and ultra-shrt regimes bth make use f the initial release r "flare" phase f GnRH agnist actin, stimulating fllicular recruitment and early develpment^ and reducing the amunt f gnadtrpin required.^ As the pituitary gnadtrphs becme desensitized by prlnged GnRH agnist administratin and hypsecretin f endgenus gnadtrpins ccurs, cntinued fllicular grwth is supprted by exgenus gnadtrpins. In rder t take advantage f the release phase, bth shrt prtcls must cmmence in the early fllicular phase, by day tw r three f menstruatin. It is imprtant t nte, hwever, that the nset f menses des nt unifrmly crrelate with the cmplete demise f the crpus luteum

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54 18 frm the previus cycled As such, the crpus luteum may be "rescued" by the endgenus gnadtrpin release fllwing agnist administratin, causing elevated levels f prgesterne thrugh the fllicular phase at a time when the endmetrium shuld be prliferating withut expsure t prgestatinal influences.^ The use f GnRH agnists, in cmbinatin with exgenus gnadtrpins, significantly increases the chance f pregnancy when cmpared t treatment with clmiphene citrate cmbined with hmg. LH suppressin in the late fllicular phase results in imprved cyte and embry quality.^ D. Factrs Affecting the Outcme f IVF 1. Age The utcme f in vitr fertilizatin and embry transfer (IVF-ET) is influenced by multiple factrs. Accrding t a prspective study undertaken by Hughes et al., the single factr mst significantly detrimental t pregnancy rates in IVF was advancing female age, with a linear decline beginning at 25 years.34 Studies evaluating the effect f increasing female age n IVF utcme have yielded varying estimates f pregnancy rates, with wmen aged 40 years r mre achieving cnceptin in anywhere frm 3.8 %34 t 23%35 f cycles. Mrever, patients in the higher age grup demnstrate a decreased ability t maintain pregnancies, with spntaneus abrtin rates bserved t be as high as 60%.35 It is generally accepted that aging female infertility patients have cnsiderably wrse IVF utcmes than yunger diagnsis-matched patients.

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56 19 2. Male Factr Infertility Studies perfrmed n patients with male factr infertility have shwn a distinct relatinship between sperm factrs and IVF-ET utcme. Sperm mtility and mrphlgy, cncentratin f sperm in the inseminatin medium, and sperm vitality have all crrelated with fertilizatin rate.^6/ Embry Quality Althugh definitins f embry quality and embry grading systems differ amng IVF centers, bth mrphlgic appearance and cleavage rate have been clearly crrelated with pregnancy rate. Puissant et al. defined a semi-quantitative, nn-invasive methd fr scring embrys based upn the quantity f anucleate fragments expelled by the embrys at cleavage and the embry develpment rate. Healthy appearing embrys were mre ften assciated with pregnancy, and in particular, with multiple pregnancy.^ 4. Patterns f Estradil Secretin Pregnancies resulting frm IVF-ET cycles have been assciated with wide ranges f estradil values. There is cnsiderable cntrversy regarding apprpriate levels f estradil during the varius phases f IVF. As shwn by Jnes et al., wmen classified as high, nrmal, r lw respnders with respect t estradil levels shwed nly small differences in pregnancy rates. It was therefre cncluded that the abslute estradil level was generally a pr predictr f success in IVF.^9 Hwever, in a study cnducted by Dr et al., changes in the daily pattern f estradil during the peri-hcg perid were fund t be a reliable predictr f utcme in cycles f wmen stimulated with CC/hMG r hmg alne. Althugh there were n differences in the mean daily fllicular phase estradil levels between the pregnant and cntrl grups, failure f fertilizatin was assciated with significantly lwer estradil

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58 20 levels during the days just prir t and fllwing the administratin f hcg. In fact, 96% f the wmen in the study wh achieved cnceptin demnstrated either rising r plateauing estradil levels n the day fllwing hcg (seen in nly 62.5% f cntrl cycles), while 37.5% f the wmen in the cntrl, nn-pregnant grup had decreasing estradil levels during the same time perid. If declining estradil is interpreted as indicative f fllicular atresia, then it is plausible that lwer estradil levels in the pst-hcg perid culd result in a prer quality cyte and a crrespndingly diminished likelihd f fertilizatin and cnceptin.^ In cntrast t the Dr et al. study, Hwies et al. shwed that plasma levels f estradil fur and six days after cyte recvery during CC/hMG stimulated cycles were significantly higher in patients wh did nt becme pregnant than in the pregnant grup,^ supprting the pssibility that elevated estradil levels after hcg administratin may be detrimental t establishing a pregnancy. 5. Endmetrial Receptivity The establishment f pregnancy thrugh the use f IVF depends nt nly n the generatin f multiple embrys f gd quality, but als upn the presence f a receptive endmetrium t allw and maintain embrynic implantatin. While stimulating adequate numbers f cytes fr fertilizatin, vulatin inductin simultaneusly causes the prductin f supraphysilgic levels f estradil and prgesterne frm the multiple fllicles and subsequent crpra lutea. The extrardinarily high sterid levels encuntered in the early luteal phase during varian stimulatin must undubtedly influence the quality and receptivity f the endmetrium. In fact, sme studies suggest that the balance r rati f sterid hrmnes, and their subsequent influence n the endmetrium, may be aberrant in wmen

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60 21 wh fail t implant.^ Alng these same lines, cnflicting discussin abunds in the literature regarding the necessity fr r detriment f high prgesterne utput during the luteal phase f stimulated cycles. Supplementatin f the luteal phase with exgenus prgesterne and stimulatin f endgenus prgesterne prductin by the crpus luteum with hcg are ppular practices in IVF, indicating the belief that higher luteal phase prgesterne may psitively impact endmetrial quality and implantatin success.studies perfrmed t date, hwever, have nt cnsistently demnstrated that luteal phase supprt increases subsequent pregnancy rates. 6. Ocyte Quality The quality f the cyte btained in an IVF prgram has been shwn t be ne f the keys t determining cyte maturity and embry quality, bth f which influence nrmal fertilizatin and subsequent develpment. Premature r pstmature inseminatin f cytes have been demnstrated t result in a higher incidence f failed r abnrmal fertilizatin.^ Despite this, the ptimal diameter f the prevulatry fllicle has nt been precisely determined. Mst likely, the ptimal cyte measures between 18 and 20 mm in diameter befre vulatin and 26 t 28 mm sn after vulatin begins, arund the time f cyte retrieval.^ At the time f fllicular puncture, the retrieval f smaller fllicles frequently ccurs. It has been shwn, hwever, that cytes derived frm large fllicles are mre efficient with respect t resumptin f meisis and fertilizing ability. In a study by Bmsel-Helmreich et al., n nuclear maturatin was fund in va f fllicles measuring less than 16 mm, highlighting the delay in maturatin amng smaller fllicles.^

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62 22 7. Number f Ocytes Retrieved Many studies have examined the relatinship between the number f cytes retrieved during a cycle f IVF and subsequent fertilizatin and utcme f embry transfer. Pellicer et al. demnstrated that the fertilizatin rate was significantly decreased in grups f patients prducing 11 r mre cytes in cmparisn with patients prducing smaller numbers f cytes at the time f cllectin.-^ Testart et al. have als fund a lwer fertilizatin and pregnancy rate when the number f fllicles increases, with the prprtin f nrmal embrys per recvered cyte inversely related t the degree f varian respnse.-^ In anther study by Sharma et al., the clinical pregnancy rate per embry transfer shwed a linear rise when up t 5 cytes were cllected, with the rate plateauing at higher cyte numbers. Mrever, this study demnstrated that the success f implantatin (defined as the number f gestatinal sacs + number f embrys transferred x 100) was highest when 7 t 9 cytes were retrieved and mre than 60% f these fertilized; when 10 r mre cytes were cllected, the implantatin rate revealed a prgressive decline, even thugh the fertilizatin rate remained abve 60%.^7 8. Number f Prevulatry Fllicles The relatinship between the number f fllicles and the ccurrence and quality f pregnancy has als received attentin. Testart et al. demnstrated the influence f varian respnse n cyte suitability fr transfer in cycles with the transfer f ne, tw, r three embrys. In this study, the number f punctured fllicles, recvered cytes, and cleaved embrys had n significant effect n the success f embry transfer in patients wh received ne, tw, r three embrys, althugh the prprtin f nging

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64 23 pregnancies decreased when the number f fllicles increased. Mrever, a higher cyte cleavage rate was demnstrated amng patients with ne t fur fllicles (termed a "mderate" respnse) than amng thse prducing five r mre fllicles.^ in a study cnducted by Frman et al., the pregnancy rate in a ppulatin cmbining all infertility diagnses was shwn t decrease frm 52% with <, 2 fllicles measuring mre than 14 mm in diameter n the day f hcg administratin t 33% with 3 t 4 fllicles and 20% with 9 r mre fllicles detected (PcO.Ol). Surprisingly, the authrs f this study cncluded that the number f fllicles measuring greater than 14 mm at the time f hcg did nt crrelate with pregnancy rate, althugh the data suggest therwise.^ Of the studies cited here, it is apparent that a relatinship exists between the number f prevulatry fllicles and the utcme f ET, bserved bth as a trend^s and as a statistically significant result. 49 Hwever, it is essential t nte that these crrelatins were demnstrated in ppulatins cmbining patients with many etilgies f infertility. Whether the relatinship between vulatry respnse and pregnancy rate is brne ut in grups f patients with specific diagnses has nt yet been determined. III. STATEMENT OF PURPOSE The purpse f the study prpsed here is an examinatin f the relatinships between pretransfer parameters in IVF-ET and the subsequent ccurrence f pregnancy in patients assigned t individual diagnstic grups, with particular fcus n patients with tubal disease.

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66 IV. METHODS A. Overview f the Ppulatin The study evaluated 505 randmly selected cycles f IVF perfrmed between February 1984 and Nvember Cases used in the study included thse with successful varian respnse, fertilizatin, and embry transfer, as well as thse in which n fertilizatin r transfer f retrieved cytes ccurred. The nly cycles mitted during chart review were thse drpped befre adequate varian stimulatin culd be cmpleted due t pr respnse (few fllicles, lw estradil), r an exaggerated respnse t stimulatin (excessive fllicular develpment, high estradil, risk f varian hyperstimulatin syndrme). B. Ovulatin Inductin Prtcls Cntrlled varian hyperstimulatin was primarily achieved with human menpausal gnadtrpin (hmg; Pergnal) frmulated as ampules cntaining 75 IU each f FSH and LH, r with pure FSH cntaining 75 IU f FSH alne (pure FSH/Metrdin). In the majrity f cycles included in the study, hmg r pure FSH was used either alne r in cmbinatin with each ther and/r with a GnRH agnist (Luprn). The remainder f cases were stimulated using Clmid prtcls, either alne r in cmbinatin with hmg. The daily dses f hmg/fsh administered t each patient per cycle varied and were adjusted accrding t diagnsis, flliculgenic respnse, and past cycle experience. Mnitring bichemically. was perfrmed bth ultrasngraphically and Baseline ultrasund scans were perfrmed befre hmg/fsh administratin t detect varian cysts and fibrids, as well as t ensure wnmm wsmm

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68 adequate varian suppressin. Mnitring beynd this baseline scan was then individualized, with subsequent endvaginal ultrasund exams and estradil levels dictating changes in management. On the day f hcg administratin, each patient had an estradil and prgesterne level drawn and an endvaginal ultrasund scan perfrmed. The standard criteria fr the administratin f hcg (10,000 IU) included ultrasngraphic detectin f at least 2 fllicles measuring greater than 16 mm in diameter, and an estradil level nt exceeding 3,000 pg/ml. Ocyte retrievals were carried ut apprximately 32 hurs after hcg administratin. N cytes were excluded frm the inseminatin prtcl. All cytes were examined 16 t 18 hurs after inseminatin and again at 48 hurs, just prir t embry transfer; if, at these times, cyte cleavage r tw prnuclei were visualized, fertilizatin was cnsidered t have ccurred. Upn examinatin, embrys were scred t indicate quality, maturity, and suitability fr transfer. The embry scring system used in cycles included in this study was defined as fllws: grade 1 was assigned t unfragmented embrys with regular blastmeres; grade 2 was assigned t embrys with minr fragments and regular blastmeres; remaining embrys with evidence f irregular blastmeres r mre severe fragmentatin were assigned a grade f 3. Embrys were transferred 2 days after cyte retrieval. Variable numbers f embrys were transferred and frzen in each cycle, based upn the number and quality f cytes and embrys prduced, as well as upn the patient's age, past reprductive histry, and plans fr fetal reductin in the case f multiple gestatin. Plasma radiimmunassays t detect embrynic hcg secretin were perfrmed n the twelfth day after embry transfer. Clinical pregnancy was dcumented by detectin f fetal heart activity n ultrasund scan.

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70 26 C. Etilgic Definitins Patients were cnsidered t have tubal factr infertility if the fallpian tubes were shwn t be blcked, either unilaterally r bilaterally, by HSG r laparscpy. evident All cases f severe tubal scarring r encasement in adhesins upn laparscpy were accmpanied by abnrmal hystersalpinggrams, and as such, were easily interpretable as tubal factr infertility. Patients with recurrent ectpic pregnancies and pelvic adhesins diagnsed by laparscpy, with r withut HSG dcumentatin f definitive blckage, were als cnsidered t have tubal disease. Patients were determined t have "pure" tubal disease in the presence f a nrmal semen analysis, regular vulatins dcumented by a histry f regular cycles, psitive LH predictr kits, r biphasic basal bdy temperature (BBT) charts, and the absence f endmetrisis and PCO. The diagnsis f plycystic vary syndrme was made based upn a cmbinatin f a number f factrs, including a histry f lig- r anvulatin, an ultrasund scan revealing enlarged varies with multiple small fllicles, hirsutism, and hyperandrgenism indicated by increased DHEA, DHEAS, and free teststerne levels. Based n the ratinale that the presence f PCO dminated the clinical picture f patients with this disrder, patients were assigned t the PCO diagnstic grup with r withut the presence f ther etilgies f infertility. Patients diagnsed with endmetrisis were designated as such based upn laparscpic dcumentatin f the presence r absence f ectpic endmetrial implants, categrized accrding t the American Fertility Sciety classificatin.50 Stage I f the classificatin was designated as minimal endmetrisis, stage II as mild, stage m as mderate, and stage IV as severe, fr the purpses f this study. Patients gruped in the "pure" endmetrisis