Ari Kim, M.D., Ji Eun Han, M.D., Tae Ki Yoon, M.D., Sang Woo Lyu, M.D., Hyun Ha Seok, M.D., and Hyung Jae Won, M.D.

Transcription

1 Relationship between endometrial and subendometrial blood flow measured by three-dimensional power Doppler ultrasound and pregnancy after intrauterine insemination Ari Kim, M.D., Ji Eun Han, M.D., Tae Ki Yoon, M.D., Sang Woo Lyu, M.D., Hyun Ha Seok, M.D., and Hyung Jae Won, M.D. Department of Obstetrics and Gynecology and Fertility Center, CHA Gangnam Medical Center, CHA University, Seoul, South Korea Objective: To evaluate whether endometrial and subendometrial blood flow parameters measured using threedimensional power Doppler ultrasound (3D PD-US) can predict pregnancy after IUI. Design: Prospective clinical study. Setting: Infertility center in a referral hospital. Patient(s): One hundred six women who underwent ovulation induction and IUI. Intervention(s): A color Doppler ultrasound and a 3D PD-US examination were performed on the day of IUI. Main Outcome Measure(s): Pulsatility index (PI), resistance index (RI), and systolic/diastolic (S/D) ratio of uterine artery, and vascularization index (VI), flow index (FI), and vascularization flow index (VFI) of the endometrium as well as those of subendometrial region. These measurements were analyzed in relation to IUI outcome (pregnant vs. nonpregnant). Result(s): The pregnant group had higher endometrium VI, FI, and VFI scores than the nonpregnant group. In contrast, the subendometrial region VI, FI, and VFI scores did not differ between the groups, nor did the uterine artery PI, RI, and S/D. Pregnancies did not occur when endometrial blood flow had not been detected. Conclusion(s): Three-dimensional PD-US was useful for evaluating endometrial and subendometrial neovascularization in IUI cycles. Endometrial blood flow parameters may be useful predictors for pregnancy. (Fertil Steril Ò 2010;94: Ó2010 by American Society for Reproductive Medicine.) Key Words: Power Doppler, endometrial blood flow, subendometrial blood flow, endometrial receptivity, IUI The endometrium undergoes cyclic changes during the menstrual cycle. In the follicular phase, elevated estrogen levels due to follicular growth lead to proliferative endometrial change. Ovulation induces formation of corpus luteum, which produces P, and elevated P levels cause secretary changes in the endometrium. These hormone-driven endometrial changes provide for optimal implantation conditions. Evaluation of endometrial development and receptivity continues to be a challenge in reproductive medicine. Endometrial development and receptivity cannot be predicted using serum hormone levels, and methods such as histologic and molecular studies are very invasive (1, 2). Ultrasound examination is generally used for evaluation of the endometrium (3, 4). Transvaginal ultrasound may be an ideal tool Received December 10, 2008; revised and accepted March 20, 2009; published online May 22, A.K. has nothing to disclose. J.E.H. has nothing to disclose. T.K.Y. has nothing to disclose. S.W.L. has nothing to disclose. H.H.S. has nothing to disclose. H.J.W. has nothing to disclose. Supported by a grant (A084923) from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea. Reprint requests: Ji Eun Han, M.D., Fertility Center of CHA General Hospital, Department of Obstetrics and Gynecology, College of Medicine, CHA University, Yeoksam, Kangnamgu, Seoul , Korea (FAX: ; ttochil@hanmail.net). for examining endometrial receptivity. Several sonographic parameters have been used to assess uterine receptivity, including endometrial thickness, volume, pattern, and junctional zone contraction, and endometrial, subendometrial, and uterine blood flow (5 8). A number of studies have explored endometrial blood supply as a marker of receptivity (9). Although several studies have reported that endometrial, subendometrial, or uterine blood flow may be predictors for achieving pregnancy during IVF, limited data exist concerning the relationship between endometrial, subendometrial, and uterine blood flow and pregnancy after IUI. The present study evaluated whether endometrial, subendometrial, and uterine blood flow parameters measured using three-dimensional power Doppler ultrasound (3D PD-US) and color Doppler ultrasound can predict pregnancy after IUI. MATERIALS AND METHODS Patient Characteristics The study recruited a total of 158 cycles of 149 nulliparous women from the ovulation induction and IUI program of the Fertility Center of CHA Medical University (Seoul, Korea) between May 2008 and June The inclusion /$36.00 Fertility and Sterility â Vol. 94, No. 2, July doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 criteria were that couples had no known infertility factors and that women were administered clomiphene citrate only for ovulation induction. To identify male factor infertility, all male partners underwent physical examination and semen analysis before IUI in the urology department of CHA General Hospital. Patients were excluded upon evidence of endometriosis, ovarian cyst, tubal obstruction, severe adenomyosis, uterine fibroid, endometrial polyp, severe pelvic adhesion, and uterine anomaly according to ultrasound and hysterosalpingography findings. Also excluded were cases in which there was low ultrasound resolution due to obesity or severe bowel movements, given that this may affect power Doppler transmission to the endometrium. All patients were included only once to avoid selection bias. The former cycle of consecutive IUI was selected for this study. Patients underwent no therapeutic intervention except routine procedures. The study was approved by the institutional review board of CHA Medical University, and each woman gave informed verbal consent. Ovulation Induction and IUI Procedures Ovulation induction began on the third day of the menstrual cycle and continued for 5 days, with 100 mg of clomiphene citrate administered after confirmation of no pathologic condition by conventional transvaginal ultrasound. On day of the menstrual cycle, transvaginal ultrasound was performed for monitoring follicular and endometrial development, and was then repeated every 1 to 2 days to decide the dose and time of hcg administration. When two or three follicles measured approximately R18 mm in diameter, hcg (5,000 or 10,000 IU) was administered. The hcg dose depended on the number of mature follicles: the dose was 5,000 IU if there were more than five follicles larger than 17 mm. Approximately 36 hours after hcg administration, the rupture of follicles was confirmed by transvaginal ultrasound, and then a single IUI was performed. A semen specimen was obtained by masturbation and collected in a sterile container. It was processed with a mixed media of Ham s F-10, (Gibco Invitrogen, Carlsbad, CA), penicillin/ streptomycin (Gibco Invitrogen) and serum protein substitute (SAGE media; Cooper Surgical, Trumbull, CT). During washing and 30 minutes of swim-up, the sperm specimen was centrifuged twice at 1,500 rpm for 5 minutes. World Health Organization values were referred to for interpreting semen quality (10). The cervix was exposed using a bivalve speculum and the cervical mucus cleaned with a cotton dressing. The fully processed sperm specimen was drawn into an artificial insemination catheter (Wallace; Smiths Medical International, Hythe, Kent, United Kingdom) and then instilled into the upper portion of the uterine cavity. After insemination, the patient rested in a supine position for approximately 15 minutes. Qualitative hcg urine tests were performing 14 days after insemination to determine pregnancy. A clinical pregnancy was then confirmed by transvaginal ultrasound scanning of the gestational sac. Luteal support was based on an endometrial thickness of 8 mm. Ultrasound Investigation Routine serial ultrasound examination was used to confirmation ovulation. Adjunctive 3D PD-US (Accuvix XQ; Medison, Seoul, Korea) and color Doppler evaluation using a vaginal 3D probe were used to evaluate endometrial, subendometrial, and uterine vascularity on the day of IUI during assessment. All 3D PD-US scans and Doppler evaluations were carried out by one investigator to avoid interobserver variation. The results of the adjunctive ultrasound assessment did not affect subsequent clinical management procedures. With color Doppler in the two-dimensional mode, flow velocity waveforms were obtained from the ascending main branch of the uterine artery on the left and right sides of the cervix in a longitudinal plane. The cursor of the Doppler was positioned to where the vessel with good color signals was identified on the screen. The pulsatility index (PI), resistance index (RI), and systolic/diastolic (S/D) ratio of the uterine artery were calculated electronically. There was almost no difference in uterine PI, RI, and S/D ratio between the left and right sides. When a longitudinal view of the uterus was obtained, the power Doppler mode was turned on. The area of interest was the endometrium and subendometrial region within 5 mm of the echogenic endometrial borders. The settings were as follows: frame average, 5; balance, 16; sensitivity, 15; scale, 0.6 khz; filter, 1; density, low; and gray, 60. The 3D mode was then activated and the area of interest adjusted. Three-dimensional volume data were obtained using an automatic sweep with the angle set to 90 to ensure that a complete uterine volume encompassing the entire subendometrium was included. The patient and 3D vaginal probe remained as still as possible during volume acquisition. The resultant multiplanar display was examined to ensure that the area of interest had been captured in its entirety. The built-in VOCAL (Virtual Organ Computer-Aided Analysis) software for the 3D power Doppler histogram was used in the analysis to measure indices of blood flow. The manual mode of the VOCAL Contour Editor was used to cover the whole 3D volume of the endometrium with a 15 rotation step. Twelve endometrial slices were obtained outlining the endometrium at the myoendometrial junction from the fundus to the internal os (Fig. 1). The three vascular indices calculated automatically in this way were the vascularization index (VI), flow index (FI), and vascularization flow index (VFI). The VI, FI, and VFI of the subendometrial region, which was considered to be within 5 mm from the endometrial border, were obtained by editing in shell imaging (Fig. 1). Statistical Analysis All results are presented as mean and SD values or as median and range, according to the distribution. Normal distribution was identified using the Kolmogorov-Smirnov test. For normally distributed variables, comparisons between two groups were carried out with Student s t-test, whereas the Mann- Whitney U test was used to analyze non normally distributed 748 Kim et al. Endometrial blood flow in IUI Vol. 94, No. 2, July 2010

3 FIGURE 1 Three-dimensional power Doppler images generated using VOCAL software. (A) Endometrial and (B) subendometrial blood flow parameters on the day of IUI. nonpregnant groups. Of these characteristics, mean age was the only one to differ between the groups, with the pregnant group being younger. Both groups were similar in terms of uterine PI, RI, and S/D ratio and in terms of diastolic notch or diastolic absence in the uterine artery (Table 2). Table 2 summarizes the endometrial and subendometrial blood flow indices in both groups. Endometrial blood flows were found to be lower in nonpregnant patients compared with pregnant patients. Endometrial VI, FI, and VFI were all higher in the pregnant group, whereas subendometrial VI, FI, and VFI were similar for the two groups. Endometrial blood flow was absent in 18 patients, all of whom were in the nonpregnant group, and 3 of these patients showed absent subendometrial blood flow. In the pregnant group there were no cases of absent endometrial blood flow or absent subendometrial blood flow. Endometrial blood flow showed partial correlation with patient age, whereas subendometrial blood flow did not (Table 3). We analyzed the receiver operating characteristic curve of endometrial and subendometrial blood flow indices to assess their predictive value for pregnancy. The area under the curve was significant for the endometrial VI, FI, and VFI but not for the subendometrial VI, FI, and VFI (Table 3). The best predictive rate was achieved for values of endometrial VI R0.205 (sensitivity 66.7%, specificity 68.7%), endometrial FI R (sensitivity 76.2%, specificity 60.2%), and endometrial VFI R0.045 (sensitivity 61.9%, specificity 73.5%). data. Correlations were estimated using Pearson s correlation coefficient. The receiver operating characteristic curve was applied to determine the predictive value of the endometrial parameters. The significance level for all analyses was P<.05. Statistical analysis was performed using commercial software (SPSS; Chicago, IL). RESULTS Of a total recruitment of 158 cycles from 149 women, 106 women were ultimately included in the study after application of the exclusion criteria. Of those, one patient had a tubal pregnancy. Twenty-one clinical pregnancies resulted from the IUI procedure. These patients were classified as the pregnant group, and the other 84 patients were classified as the nonpregnant group. The pregnancy rate was 19.8% per IUI. Table 1 summarizes the clinical characteristics, including age, endometrial thickness, endometrial volume, days of IUI, hcg dose, interval between IUI and hcg injection, number of follicles, and luteal support for the pregnant and the DISCUSSION Successful implantation depends on a close interaction between the blastocyst and the receptive endometrium (11). Uterine receptivity refers to a state when endometrium allows a blastocyst to attach, penetrate, and induce changes in the stroma, which results in the so-called process of implantation (9). Although it seems that a favorable endometrial milieu is necessary for successful implantation, the nature of such a milieu is poorly understood (9, 12). Endometrial vasculature has been considered to play an important role in the early endometrial response to blastocyst implantation, and vascular changes may affect uterine receptivity (13). Endometrial microvascularization significantly increases during early follicular and early luteal phases, suggesting that endometrial vasculature affects endometrial growth and implantation (14). Blood flow to the endometrium is from the radial artery, which divides after passing through the myometrial endometrial junction. Thus, uterine blood flow is assumed to reflect the blood flow toward the endometrium (3). Results from previous Doppler studies on the uterine artery and endometrial blood supply have not always concurred (6, 15). Whereas some investigators found that spiral artery PI was lower in pregnant cycles as compared with nonpregnant cycles, others disagreed (16, 17). Some investigators found that uterine artery RI or PI were similar in conceptual and nonconceptual Fertility and Sterility â 749

4 TABLE 1 Clinical characteristics of the pregnant and nonpregnant groups. Parameter Pregnant group (n [ 21) Nonpregnant group (n [ 84) P value Age (y) a Endometrial thickness (mm) a Endometrial volume (ml) a Days of IUI a Dosage of hcg (IU) b 18 (85.7%) 65 (77.4%).420 c Interval of IUI from hcg (h) a No of ruptured follicles d a No of unruptured follicles (R15 mm) d a Luteal support 12 (57.1%) 34 (40.5%).134 c Note: Values are mean SD. a Student s t-test. b Dosage of hcg was 10,000 IU. c Chi-square test. d Estimated on the day of IUI. cycles in patients undergoing similar ovarian stimulation (18). As a conflicting issue, the Doppler study showed that pregnant and nonpregnant groups were similar in terms of the uterine artery in the present study. The quality and quantity of the spiral artery in the endometrium may be influenced by the uterine artery, but Doppler studies of uterine arteries do not reflect the actual blood flow to the endometrium, because the main portion of uterus is the myometrium, to which there are collateral circulations (19). Endometrial blood flow can now be evaluated noninvasively using color and power Doppler ultrasound. Power Doppler imaging is generally superior to color Doppler imaging for detecting low-velocity flows and visualizing small vessels (20). The combination of 3D ultrasound and power Doppler imaging has become a clinically feasible, fast, simple, and reproducible means of examining the blood supply to the whole endometrium and subendometrial region (21). The use of 3D ultrasound with power Doppler can also provide several vascular parameters. Vascularization index is a relative value that is a ratio of the number of color voxels TABLE 2 Uterine vascularity indices and endometrial and subendometrial 3D power Doppler indices for the pregnant and nonpregnant groups on the day of IUI. Parameter Pregnant group (n [ 21) Nonpregnant group (n [ 84) P value PI a RI a S/D ratio a Diastolic notch 21 (100%) 77 (91.7%).340 b Diastolic absent 0 (0%) 13 (15.5%).065 b Endometrial VI (%) c Endometrial FI (0 100) c Endometrial VFI (0 100) c Subendometrial VI (%) c Subendometrial FI (0 100) a Subendometrial VFI (0 100) c Note: Values are mean SD. a Student s t-test. b Fisher s exact test. c Mann-Whitney U test. 750 Kim et al. Endometrial blood flow in IUI Vol. 94, No. 2, July 2010

5 TABLE 3 Analysis of 3D power Doppler endometrial parameters for predicting pregnancy in IUI cycles. ROC curve (n [ 105) Parameter Area under curve 95% CI P value Correlation with age (P a ) Endometrial VI Endometrial FI Endometrial VFI Subendometrial VI Subendometrial FI Subendometrial VFI Note: ROC ¼ receiver operating characteristic; CI ¼ confidence interval. a Pearson correlation test. to the total number of voxels and is believed to indicate the presence of blood vessels in the area of interest. Flow index, the mean power Doppler signal intensity inside the area, is considered to indicate the average intensity of flow. Vascularization flow index is a combination of vascularity and flow intensity (22). Raine-Fenning et al. (23) reported that women with unexplained subfertility demonstrate a significant reduction in endometrial and subendometrial vascular perfusion, as defined by 3D PD-US before ovulation. Endometrial and subendometrial blood flow assessed by 3D PD-US vary not only during the menstrual cycle but also cycle by cycle (24). Previous studies comparing natural and stimulated cycles had conflicting opinions about increases or decreases in endometrial and subendometrial blood flow (16, 19, 25). Whereas some studies suggest that endometrial or subendometrial vascularity indices may be predictive factors for pregnancy in IVF (16, 25), others disagree (4, 26). The present study is the first to examine endometrial and subendometrial blood flows using 3D PD-US to determine their relationship with pregnancy after IUI. Our study involved only ovarian-stimulated cycles and found that the pregnant group had higher endometrial indices of vascularization, flow, and vascularization flow on the day of IUI compared with the nonpregnant group. These results are consistent with those of Chien et al. (3). Although blood flow parameters measured using 3D PD-US are relative values, we found that an endometrial VI R0.205, FI R12.415, and VFI R0.045 were predictive of pregnancy in IUI patients. The data suggest that increased blood flow in the endometrium can reflect the favorable endometrial receptivity. Of the nonvascular parameters, age was the only clinical factor that differed between the pregnant and nonpregnant groups. Age is known to be a major factor influencing fertility in women (27). The present study found that younger age was a positive factor for pregnancy after IUI. We also analyzed the correlation between endometrial and subendometrial blood flow and age. A previous study reported that age had an effect on endometrial and subendometrial vascular perfusion in fertile women (24). In our study of infertile women undergoing ovulation induction and IUI, we found that endometrial FI was the only index that correlated with age; it was a negative correlation, with a Pearson correlation coefficient of Although other endometrial and subendometrial indices also seemed to correlate negatively with age, statistical analysis showed that these did not reach significance. Similar findings can be due to iatrogenic ovarian stimulation and elevated estrogen levels. In conclusion, 3D PD-US was a useful and effective method for assessing endometrial blood flow in IUI cycles. Good endometrial blood flow was associated with pregnancy, which is indicative of endometrial receptivity. No such correlations existed between subendometrial blood flow and pregnancy. Endometrial blood flow was negatively affected by age, as is pregnancy. The present findings are likely to be useful for developing prognostic protocols for women undergoing IUI stimulated by clomiphene citrate for pregnancy. REFERENCES 1. Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo implantation. Hum Reprod Update 2006;12: Esmailzadeh S, Faramarzi M. Endometrial thickness and pregnancy outcome after intrauterine insemination. Fertil Steril 2007;88: Chien LW, Au HK, Chen PL, Xiao J, Tzeng CR. Assessment of uterine receptivity by the endometrial-subendometrial blood flow distribution pattern in women undergoing in vitro fertilization-embryo transfer. Fertil Steril 2002;78: Ng EH, Chan CC, Tang OS, Yeung WS, Ho PC. The role of endometrial and subendometrial blood flows measured by three-dimensional power Doppler ultrasound in the prediction of pregnancy during IVF treatment. Hum Reprod 2006;21: Zaidi J, Campbell S, Pittrof R, Tan SL. Endometrial thickness, morphology, vascular penetration and velocimetry in predicting implantation in an in vitro fertilization program. Ultrasound Obstet 1995;6: Fertility and Sterility â 751

6 6. Remohi J, Ardiles G, Garcia-Velasco JA, Gaitan P, Simon C, Pellicer A. Endometrial thickness and serum oestradiol concentrations as predictors of outcome in oocyte donation. Hum Reprod 1997;12: Chien LW, Lee WS, Au HK, Tzeng CR. Assessment of changes in uteroovarian arterial impedance during the peri-implantation period by Doppler sonography in women undergoing assisted reproduction. Ultrasound Obstet 2004;23: Killick SR. Ultrasound and the receptivity of the endometrium. Reprod Biomed Online 2007;15: Alcazar JL. Three-dimensional ultrasound assessment of endometrial receptivity: a review. Reprod Biol Endocrinol 2006;9: World Health Organization. [Laboratory manual of the WHO for the examination of human semen and sperm-cervical mucus interaction]. Annali dell Istituto Superiore di Sanita 2001;37:I XII, Ng EH, Chan CC, Tang OS, Yeung WS, Ho PC. The role of endometrial and subendometrial vascularity measured by three-dimensional power Doppler ultrasound in the prediction of pregnancy during frozen-thawed embryo transfer cycles. Hum Reprod 2006;21: Horcajadas JA, Riesewijk A, Domınguez F, Cervero A, Pellicer A, Simon C. Determinants of endometrial receptivity. Ann N Y Acad Sci 2004;1034: Rogers PA. Structure and function of endometrial blood vessels. Hum Reprod Update 1996;2: Gannon BJ, Carati CJ, Verco CJ. Endometrial perfusion across the normal human menstrual cycle assessed by laser Doppler fluxmetry. Hum Reprod 1997;12: Dickey RP. Doppler ultrasound investigation of uterine and ovarian blood flow in infertility and early pregnancy. Hum Reprod Update 1997;3: Kupesic S, Bekavac I, Bjelos D, Kurjak A. Assessment of endometrial receptivity by transvaginal color Doppler and three-dimensional power Doppler ultrasonography in patients undergoing in vitro fertilization procedures. J Ultrasound Med 2001;20: Schild RL, Knobloch C, Dorn C, Fimmers R, van der Ven H, Hansmann M. Endometrial receptivity in an in vitro fertilization program as assessed by spiral artery blood flow, endometrial thickness, endometrial volume, and uterine artery blood flow. Fertil Steril 2001;75: Tekay A, Martikainen H, Jouppila P. Blood flow changes in uterine and ovarian vasculature, and predictive value of transvaginal pulsed colour Doppler ultrasonography in an in-vitro fertilization programme. Hum Reprod 1995;10: Ng EH, Chan CC, Tang OS, Yeung WS, Ho PC. Relationship between uterine blood flow and endometrial and subendometrial blood flows during stimulated and natural cycles. Fertil Steril 2006;85: Guerriero S, Ajossa S, Lai MP, Risalvato A, Paoletti AM, Melis GB. Clinical applications of colour Doppler energy imaging in the female reproductive tract and pregnancy. Hum Reprod Update 1999;5: Merce LT, Barco MJ, Bau S, Troyano J. Are endometrial parameters by three-dimensional ultrasound and power Doppler angiography related to in vitro fertilization/embryo transfer outcome? Fertil Steril 2008;89: Pairleitner H, Steiner H, Hasenoehrl G, Staudach A. Three-dimensional power Doppler sonography: imaging and quantifying blood flow and vascularization. Ultrasound Obstet Gynecol 1999;14: Raine-Fenning NJ, Campbell BK, Kendall NR, Clewes JS, Johnson IR. Endometrial and subendometrial perfusion are impaired in women with unexplained subfertility. Hum Reprod 2004;19: Raine-Fenning NJ, Campbell BK, Kendall NR, Clewes JS, Johnson IR. Quantifying the changes in endometrial vascularity throughout the normal menstrual cycle with three-dimensional power Doppler angiography. Hum Reprod 2004;19: Wu HM, Chiang CH, Huang HY, Chao AS, Wang HS, Soong YK. Detection of the subendometrial vascularization flow index by three-dimensional ultrasound may be useful for predicting the pregnancy rate for patients undergoing in vitro fertilization-embryo transfer. Fertil Steril 2003;79: Jarvela IY, Sladkevicius P, Kelly S, Ojha K, Campbell S, Nargund G. Evaluation of endometrial receptivity during in-vitro fertilization using three-dimensional power Doppler ultrasound. Ultrasound Obstet 2005;26: Committee on Gynecologic Practice of American College of Obstetricians and Gynecologists; Practice Committee of American Society for Reproductive Medicine. Age-related fertility decline: a committee opinion. Fertil Steril 2008;90: Kim et al. Endometrial blood flow in IUI Vol. 94, No. 2, July 2010