The study of the normal changes of uterine and ovarian blood flow during the menstrual and ovarian cycle and the mechanisms controlling the modifi-

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1 FERTILITY AND STERILITY Copyright e 1993 The Amerian Fertility Soiety Printed on aid-free paper in U.S.A. Assessment of ovarian and uterine blood flow by transvaginal olor Doppler in ovarian-stimulated women: orrelation with the number of folliles and steroid hormone levels Zeev Weiner, M.D. Israel Thaler, M.D. Jaob Levron, M.D. Nathan Lewit, M.D. Joseph Itskovitz-Eldor, M.D., D.S.t Department of Obstetris and Gyneology, Rambam Medial Center, and Faulty of Mediine, Tehnion-Israel Institute of Tehnology, Haifa, Israel Objetive: To study the hanges that our in vasular resistane to flow in the utero-ovarian irulation and their orrelation with the number of folliles and steroid hormone levels in patients undergoing ovarian stimulation with gonadotropin. Design: In a prospetive study, the impedane to flow in the intraovarian and uterine vessels was measured by means of transvaginal olor flow Doppler imaging throughout the yle. Setting: University-based IVF program. Patients: Eleven infertile patients undergoing ovarian stimulation with gonadotropin in preparation for IVF and ET. Main Outome Measures: Intraovarian, uterine, and aruate artery blood flow veloity waveforms, folliular growth, and serum E2 and P onentrations throughout the yle. Results: Pulsatility index (PI) of the intraovarian blood vessels and uterine artery dereased gradually during the folliular and luteal phase. The PI of the aruate artery did not hange signifiantly. The PI of the intraovarian blood vessels orrelated with the number of folliles (> 15 mm; day of hcg). Serum E2 onentrations but not P demonstrated negative linear orrelation with the PI of the intraovarian vessels and uterine artery. Conlusions: Indued yles are assoiated with dereased impedane to blood flow in the uteroovarian irulation. Intraovarian PI orrelates negatively at eah stage of the yle with the eventual number of preovulatory folliles. Little or no hanges are observed when the response to ovarian stimulation is poor. Fertil SterilI993;59:743-9 Key Words: Color flow Doppler, ovarian blood flow, uterine blood flow, follile, ovarian stimulation, in vitro fertilization Regulation of ovarian and uterine blood flow is an important aspet of reprodutive physiology. Changes in the vasulature of the yling ovary are rapid and profound. Previous studies have suggested that blood flow to individual folliles may play an Reeived August 26, 1992; revised and aepted Deember 22, * Supported in part by grant HL from the National Institutes of Health, Bethesda, Maryland. t Reprint requests: Joseph Itskovitz-Eldor, M.D., D.S., Department of Obstetris and Gyneology, Rambam Medial Center, Haifa 31096, Israel. instrumental role in the seletion and maturation of the dominant follile in the primates (1). Other studies in laboratory and domesti animals indiate a funtional relationship between blood flow and steroid output within the ovarian follile and orpus luteum (CL) (2, 3). Extensive hanges in uterine vasulature also our throughout the menstrual yle. The uterine irulation appears to be under the influene of estrogen (E) and P (4). The study of the normal hanges of uterine and ovarian blood flow during the menstrual and ovarian yle and the mehanisms ontrolling the modifi- Weiner et ai. Ovarian and uterine blood flow 743

2 ations of blood flow to the reprodutive organs in the human has been hampered by the inability to monitor pelvi vasular hemodynamis noninvasively. The Doppler duplex system, whih ombines gray-sale images and pulsed Doppler, offers the opportunity to study noninvasively blood flow patterns in the reprodutive organs. Uterine and ovarian artery blood flow veloity waveforms were obtained transabdominally (5-7) or transvaginally (8-11) at different stages of the normal or stimulated menstrual yle. The reent introdution of transvaginal ultrasonography with olor blood flow imaging failitates the detetion of small vessels in the uteroovarian irulation and the measurements of impedane to flow in this vasular tree (12). This tehnique has been used for the study of neovasularization (angiogenesis) of pelvi tumors (13, 14). Angiogenesis also ours in the growing ovarian follile and the developing CL. The pattern of ovarian and uterine flow veloity waveforms in the normal menstrual yle has been reently desribed (15,16). In this study, we have used the transvaginal olor Doppler imaging to measure vasular resistane to flow in the ovary and uterus in patients treated with gonadotropins in preparation for IVF. Changes in blood flow were orrelated with the number of developing folliles and steroid hormone levels. MATERIALS AND METHODS Patients and Protools Eleven patients from our IVF program were inluded in the study. The patients' ages ranged from 25 to 39 years with a mean age of 36.5 years. The indiations for IVF were mehanial infertility in 6 patients and unexplained infertility in 5 patients. For ovarian stimulation, patients were injeted with two ampules of FSH (Metrodin; Teva Pharmaeutial Industries, Petah-Tikva, Israel) and two ampules of hmg (Pergonal; Teva Pharmaeutial Industries) on days 3 and 4 of the yle. This was followed by injetion of two to three ampules of hmg on subsequent days. Patients were monitored during the folliular phase with daily determination of serum E2 and P and with transvaginal ultrasonographi sanning of ovarian folliles. Human horioni gonadotropins (10,000 IU hcg, Chorigon; Teva Pharmaeutial Industries) was administered when at least one follile had reahed a diameter of 16 mm, and transvaginal ovum pik-up was performed 36 hours later. Cleaved embryos were transferred to the uterus 42 to 48 hours after insemina- tion. The luteal phase was supported by 2,500 IU of hcg injeted on days 2, 5, and 8 after follile aspiration. Hormonal Assays Serum E2 and P were assayed using a ommerially available RIA kit (Coat-A-Count; Diagnosti Produt Corporation, Los Angeles, CA). The intraassay and interassay oeffiients of variation were 6% and 10% for E2 and 8% and 11 % for P. Ultrasound (US) and Doppler Examination Transvaginal ultrasonography (SSD 680; Aloka, Tokyo, Japan), with 5-MHz transduer, was used for the assessment of folliular development. Measurements of folliular size were performed daily, beginning on day 5 ofthe yle until the day of ooyte retrieval. Doppler flow measurements of the intraovarian vessels were performed transvaginally (SSD 680; Aloka Color Doppler) with 5-MHz pulsed Doppler system. The examinations were performed by one examiner (Z.W.) with intraobserver variability of 7%. A 100-Hz filter was used to eliminate low frequeny signals originating from vessel wall movements. The spatial peak temporal average intensity at the maximum amplitude and minimum gate width did not exeed 100 m W /m2. By using olor flow imaging, olor signals were identified around the folliles and also in the ovarian stroma when folliles were not observed. Uterine and aruate artery blood flow veloity waveforms were also measured by transvaginal olor flow Doppler. The asending branh of the uterine artery was insonated lateral to the orpus of the uterus. The aruate artery was identified within the uterine wall. The basi priniples of the olor signals and their olor oding are desribed elsewhere (17). When olor signals were obtained, blood flow veloity waveforms were reorded by plaing the sample volume over the olored area and entering the pulsed Doppler mode. Pulsatility index (PI), defined as the differene between peak-systoli and end-diastoli flow veloity divided by the mean flow veloity, was alulated. The values of PI are inversely orrelated with blood flow. Frequently, several blood vessels were deteted inside the ovaries but only the one with the lowest PI was seleted for Doppler measurements. We piked up the vessels representing the lowest downstream impedane, assuming that these are the branhes supplying diretly the developing folliles or CL. No signifiant differene between the PI of left and right 744 Weiner et ai. Ovarian and uterine blood flow Fertility and Sterility

3 uterine artery was observed, and therefore the average value of both arteries was used. Measurements were performed only when five idential waveforms were obtained onseutively. The latter were reorded on a olor video printer (SSZ 700E; Aloka, Tokyo, Japan). Doppler measurements were obtained on five oasions during the yle: day 3 (early folliular phase), day 7 (midfolliular phase), the day of ooytes retrieval (late folliular phase), the day of ET (early luteal phase), and 14 days after ooyte retrieval (late luteal phase). Statistial Analyses Student's t-test with Bonferroni's orretion for multiple measurements (18) was used to evaluate the hanges in PI of the intraovarian and uterine vessels throughout the yle. Correlations between PI, number of folliles (>15 mm; day of hcg), and serum hormonal levels were determined by regression analysis. RESULTS Changes in the intraovarian and uterine vasular resistane to flow throughout the yle are shown C' A o "t: 1 ;;:. 5 1"- "-1 T' T \ l B : i ;!!; ::::l D L Er MF LFEL LL : ts. -.s x w o z A _---_-- o J 456 B _---_--- o 2 J 5 6 O.BOO 0.400±-O----2:---J--4--5::::::...,6 D ,...::::...,6 E _--_- o 2 J 5 6 No. OF FOLLICLES Figure 1 The intraovarian (A) and uterine artery (B) PI throughout the yle in 11 patients undergoing ovarian stimulation with gonadotropin in preparation for IVF and ET. Values are means ± SE. EF, early folliular; MF, midfolliular; LF, late folliular; EL, early luteal; LL, late luteal. * P < 0.05, signifiantly different from the values at the early folliular phase. Figure 2 Intraovarian PI and its orrelation with the number of folliles (>15 mm; day of heg). (A), Early folliular phase (r = 0.67, P < 0.001); (B), midfolliu1ar phase (r = 0.74, P < 0.001); (C), late folliular phase (r = 0.81, P < 0.001); (D), early luteal phase (r = 0.86, P < ); (E), late luteal phase (r = 0.75, P < 0.001). Weiner et al. Ovarian and uterine blood flow 745

4 ..-. ::. A X! li!: 5 :;) Q :: B D.\:! o 0 :=. li!: <>---0" 5 3 o-----o---.o-!: 41 :;) Q. EF MF LF EL LL Figure 3 Intraovarian PI. (A), The impedane to flow in eight patients who had more than two folliles (> 15 mm; day of heg) in eah ovary. The dashed lines represent measurements obtained from two women who oneived after ET. (B), PI in three women in whom the impedane to blood flow did not hange throughout the yle. In patient 2, impedane to flow dereased only at the early luteal phase. Patients 1 and 3 had one follile and one ooyte that did not fertilize. In patient 2, three ooytes were retrieved and fertilized. in Figures 1A and B. Pulsatility index of the intraovarian blood vessels dereased throughout the folliular phase and remained low at the early luteal phase. No signifiant differene was found between the PI of the right and left ovaries. However, the PI orrelated with the number of folliles (> 15 mm; day of hcg) even during the early folliular phase when folliular reruitment and development had just started (Fig. 2A to E). When onsidering eah patient separately (Fig. 3A and B), no derease in intraovarian PI during the folliular phase was observed in three ases (Fig. 3B). These patients had the lowest number of preovulatory folliles (::::;3). In two of these three patients, only one ooyte, whih was not fertilized, had been retrieved. In the third patient, the PI of the intraovarian blood vessels dereased only between the late folliular phase and the early luteal phase. In this patient, three ooytes were retrieved and fertilized. Two patients shown in Figure 3A oneived after ET. In these patients the intraovarian PI vessels remained low at the late luteal phase. The appearane of blood vessels within the ovaries hanged throughout the yle. The number and size 746 Weiner et al. Ovarian and uterine blood flow of blood vessels that ould be deteted inside the ovaries inreased with folliular development. In the late folliular phase (day of ooyte retrieval), most of the disernable intraovarian blood vessels were arranged around one or a few of the leading preovulatory folliles (Fig. 4A). Similarly, during the luteal phase blood vessels were prominent around the CL (Fig. 4B). In one patient, in whom only one ooyte was retrieved but not fertilized, blood vessels around the follile ould just be deteted and the PI was high (Fig. 4C). The PI of the uterine artery (Fig. 4D) dereased during the folliular and early luteal phase and returned to near basal levels during the late luteal phase (Fig. 1B). A linear orrelation was found between the PI of the intraovarian vessels and uterine artery (r = 0.65, P < ). The PI of the aruate arteries was 1.71 ± 0.09 (day 3 of the yle), lower than the uterine artery and did not hange signifiantly throughout the yle. Early folliular phase serum E2 level was 49 ± 12 pgjml (180 ± 44 pmoljl) and inreased to a peak level of 1,662 ± 314 pgjml (6,100 ± 1,153 pmoljl) the morning after hcg injetion. Serum E2 onentrations demonstrated a negative linear orrelation with the PI of the intraovarian blood vessels (r = -0.63, P < ) (Fig. 5A) and uterine artery (r = -0.51, P < 0.001) (Fig. 5B). Serum P levels did not orrelate with the PI of the intraovarian blood vessels (r = -0.28, P> 0.05) or the uterine artery (r = -0.25, P > 0.05). DISCUSSION The results from the present study demonstrate a funtional relationship between utero-ovarian blood flow, folliular development, and E output from the ovary. A lower intraovarian PI, implying redued downstream impedane and inreased blood flow was observed on day 3 of the yle in patients who eventually developed multiple folliles (more than 3 preovulatory folliles) after ovarian stimulation. In these women, intraovarian PI further dereased throughout the folliular phase and luteal phase in parallel with folliular growth and rising irulating levels ofe2. No derease in intraovarian PI was observed in patients who demonstrated a poor response to ovarian stimulation. Our results suggest but do not prove that vasularization of the folliles may play a role in their maturation from early folliular phase onward. Inreased vasularity may result in preferential delivery of gonadotropi hormones or other growth fa- Fertility and Sterility

5 Figure 4 Patterns of blood flow in the utero-ovarian irulation. (A), Intraovarian arterial waveform arising from vessels supplying a preovulatory follile in the late folliular phase. Note the high diastoli flow (t). (B), Inreased diastoli flow arising from a vessel supplying a CL. (C), Intraovarian waveform arising from an ovary with one follile. The ooyte retrieved from this follile did not fertilize. Note the nearly absent diastoli flow (t). (D), Typial arterial waveform arising from the asending branh of the uterine artery observed laterally to the uterus (U). tors or substrates required for steroidogenesis to individual folliles who are destined to be reruited and mature. This hypothesis is supported by histomorphometri studies in the rhesus monkey that have shown a distintly higher vasularization in the thea of dominant folliles as ompared with that of smaller folliles (1). It is also possible, however, that the inreased vasularity reflets a response to speifi signals for angiogenesis or aelerated metabolism in the growing folliles and is not the primary ause of their reruitment. Autoradiographi and morphometri studies suggest that folliular growth is followed by an adaptive inrease in the volume of the perifolliular apillary network (19). The development of this network depends on the growth of the stratum granulosum, and the apillary area in the thea interna inreases with growing follile size. After the LH surge there is a linear inrease in the apillary ross-setional area of the thea interna; beause there is no orresponding inrease in the number of blood vessels, this inrease in apillary area (and blood flow) was attributed to vasodilation (20). The intraovarian PI was low in the early luteal phase, after hcg injetion, and then inreased toward the baseline in the late luteal phase. It remained low in two women who oneived after ET. The low PI in the early luteal phase is onsistent with the rapid neovasularization that takes plae, in the wall of the developing CL and the extremely high rate of blood flow in this tissue (21). Our data indiate a funtional relationship between ovarian blood flow and E output but not P output. There is a great deal of ontroversy related Vol. 59, No.4, April1993 Weiner et al. Ovarian and uterine blood flow 747

6 :: A D." w. 0 ;!; ::::; '. :;) n 'i' 5..s " 4 B 2- \ 0 ;!; 3 -' 2 ::l n SERUM ESTRADIOL LEVELS [pg/ml] Figure 5 The relationship between PI and serum E2 levels. (A), Intraovarian vessels (r = -0.63; P < ); (B), Uterine artery (r = -0.51, P < 0.001). to the funtional relationship between luteal blood flow and luteal steroidogenesis. Studies in the rabbit suggest that blood flow in the CL is not autely dependent on normal rates of steroidogenesis (21). Infusion of E2 inreases ovarian stromal blood flow but fails to alter the high level of blood flow in the CL (22), supporting the onept that the blood vessels in the CL are maximally dilated and inapable of autoregulation. One of the diffiulties in assessing the ovarian irulation is related to the numerous blood vessels identified by olor Doppler within the ovarian stroma and around the folliles or CL. In the present study, Doppler measurements were obtained from the blood vessels within the ovarian stroma that demonstrated the lowest resistane to flow. This approah does not take into aount the number and distribution of the blood vessels within the ovary. An index that would reflet the number of blood vessels in a predefined area (23) or mapping blood vessels within the ovarian stroma or around the folliles or CL ould better haraterize ovarian vasularity and hemodynamis and their relationship to ovarian funtion. The resistane to flow in the uterine artery delined throughout the yle and demonstrated a negative linear orrelation with rising serum E2 levels. This derease in vasular resistane may be re- lated to the diret vasodilatory effet of irulating E2 on the uterine artery (4, 24) but other mehanisms ould also be involved. As expeted, the PI in the aruate arteries was lower than in the asending uterine artery, but it did not hange signifiantly throughout the yle. One possible explanation is related to the fat that it is diffiult to obtain repeated Doppler measurements from the same aruate artery throughout the yle. The PI in the aruate arteries may vary onsiderably from vessel to vessel beause of the large variability in size and diameter of these vessels (25). If indeed the blood flow in the aruate arteries did not hange while uterine blood flow inreased, this would imply that blood flow was shunted away from the uterine irulation toward the ovaries, through the utero-ovarian arterial arade, possibly to provide the inreased demand for blood supply to the ovaries ontaining the multiple folliles or CL. In summary, our study demonstrates a funtional relationship between utero-ovarian hemodynamis and folliular development. The linial appliability of Doppler US for noninvasive evaluation of the utero-ovarian irulation, its usefulness in monitoring folliular development, and the predition of suessful outome in patients undergoing ovarian stimulation with gonadotropins has yet to be established. REFERENCES 1. Zeleznik AJ, Shuler HM, Reihert LE Jr. Gonadotropinbinding sites in the rhesus monkey ovary: role of the vasulature in the seletive distribution of human horioni gonadotropin to the preovulatory follile. Endorinology 1981;109: Niswender GD, Reimers TJ, Diekman MA, Nett TM. Blood flow: a mediator of ovarian funtion. Bioi Reprod 1976;14: Damber JE, Cajander S, Gafvels M, Selstam G. Blood flow hanges and vasular appearane in preovulatory folliles and orpora lutea in immature, pregnant mare's serum gonadotropin-treated rats. Bioi Reprod 1987;37: Greiss FC Jr, Anderson SG. Effet of ovarian hormones on the uterine vasular bed. Am J Obstet Gyneol 1970;107: Taylor KJW, Burns PN, Wells PNT, Conway DI, Hull MGR. Ultrasound Doppler flow studies of the ovarian and uterine arteries. Br J Obstet Gynaeol 1985;92: Goswamy RK, Steptoe PC. Doppler ultrasound studies of the uterine artery in spontaneous ovarian yles. Hum Reprod 1988;3: Long MG, Boultbee JE, Hanson ME, Begent RHJ. Doppler time veloity waveform studies of the uterine artery and uterus. Br J Obstet Gynaeol 1989;96: Sholtes MCW, Wladimiroff JW, van Rijen HJM, Hop WCJ. Uterine and ovarian flow veloity waveforms in the normal 748 Weiner et al. Ovarian and uterine blood flow Fertility and Sterility

7 menstrual yle: a transvaginal Doppler study. Fertil Steril 1989;52: Baber RJ, MSweeney MB, Gill RW, Porter RN, Piker RH, Warren PS, et al. Transvaginal pulsed Doppler ultrasound assessment of blood flow to the orpus luteum in IVF patients following embryo transfer. Br J Obstet Gynaeol 1988;95: Hata K, Hata T, Senoh D, Makihara K, Aoki S, Takamiya 0, et al. Changes in ovarian arterial ompliane during the human menstrual yle assessed by Doppler ultrasound. Br J Obstet GynaeoI1990;97: Deutinger J, Reinthaller A, Bernashek G. Transvaginal pulsed Doppler measurement of blood flow veloity in the ovarian arteries during yle stimulation and after follile punture. Fertil Steril1989;51: Fleisher AC. Ultrasound imaging-2000: assessment of uteroovarian blood flow with transvaginal olor Doppler sonography; potential linial appliations in infertility. Fertil Steril 1991;55: Bourne TH, Campbell S, Steer CV, Whitehead MI, Collins WP. Transvaginal olor flow imaging: a possible new sreening tehnique for ovarian aner. Br Med J 1989;299: Weiner Z, Thaler I, Bek D, Rottem S, Deutsh M, Brandes JM. Differentiating malignant from benign ovarian tumors with transvaginal olor flow imaging. Obstet GyneoI1992;79: Steer CV, Campbell S, Pampiglione JS, Kingsland CR, Mason BA, Collins WP. Transvaginal olour flow imaging of the uterine arteries during the ovarian and menstrual yles. Hum Reprod 1990;5: Bourne TW, Jurkovi D, Waterstone J, Campbell S, Collins WP. Intrafolliular blood flow during human ovulation. Ultrasound Obstet Gyneol 1991;1: Kurjak A, Jurkovi D, Alfirevi Z, Zalud I. Transvaginal olor flow imaging. J Clin Ultrasound 1990;18: Wallenstein S, Zuker CL, Fleiss JL. Some statistial method useful in irulation researh. Cir Res 1980;47: Kranzfelder D, Maurer-Shultze B. Development of the perifolliular apillary network. Autoradiographi and morphometri studies in the rabbit ovary. Eur J Obstet Gyneol Reprod Bioi 1989;30: Cavender JL, Murdoh W J. Morphologial studies of the miroirulatory system of periovulatory ovine folliles. BioI Reprod 1988;39: Wiltbank MC, Gallagher KP, Christensen AK, Brabe RK, Keyes PL. Physiologial and immunoytohemial evidene for a new onept of blood flow regulation in the orpus luteum. BioI Reprod 1990;42: Wiltbank MC, Gallagher KP, Dysko RC, Keyes L. Regulation of blood flow to the rabbit orpus luteum: effets of estradiol and human horioni gonadotropin. Endorinology 1989;124: Cosgrove DO, Bamber JC, Davey JB, MKinna A, Sinnett HD. Color Doppler signals from breast tumors. Radiology 1990;176: de Ziegler D, Bessis R, Frydman R. Vasular resistane of uterine arteries: physiologial effets of estradiol and progesterone. Fertil Steril 1991;55: Itskovitz J, Lindenbaum ES, Brandes JM. Arterial anastomosis in the pregnant human uterus. Obstet GyneoI1980;55: Weiner et al. Ovarian and uterine blood flow 749