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Measurement of endometrial tissue blood flow: a novel way to assess uterine receptivity for implantation
FERTILITY AND STERILITY威 VOL. 76, NO. 6, DECEMBER 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Measurement of endometrial tissue blood flow: a novel way to assess uterine receptivity for implantation Masao Jinno, M.D.,a Tsuneo Ozaki, M.D.,a Mitsutoshi Iwashita, M.D.,a Yukio Nakamura, M.D.,a Akihiko Kudo, Ph.D.,b and Hiroshi Hirano, M.D., Ph.D.b,c Department of Obstetrics and Gynecology and Department of Anatomy, School of Medicine, Kyorin University, Mitaka City, Tokyo, Japan
Received March 12, 2001; revised and accepted June 12, 2001. Reprint requests: Masao Jinno, M.D., Department of Obstetrics and Gynecology, School of Medicine, Kyorin University, 6-20-2 Shinkawa, Mitaka City, Tokyo 181-8611, Japan (Fax: 81-422-47-3177; E-mail: jinno@kyorin-u. ac.jp). a Department of Obstetrics and Gynecology, School of Medicine, Kyorin University. b Department of Anatomy, School of Medicine, Kyorin University. c Nittai Jusei Medical College for Judo Therapeutics, Tokyo, Japan. 0015-0282/01/$20.00 PII S0015-0282(01)02897-7
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Objective: To assess endometrial receptivity in terms of endometrial tissue blood flow (ETBF) measured hysterofiberscopically by laser blood-flowmetry, and to examine the technique’s effectiveness in an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) program. Design: A prospective clinical study. Setting(s): IVF program in a university hospital. Patient(s): A total of 75 infertile women with normal menstrual cycles undergoing IVF/ICSI. Intervention(s): ETBF, conventional ultrasonographic, endocrinologic, and histologic parameters for receptivity and immunoreactivity for vascular endothelial growth factor (VEGF) in endometrium were assessed between days 4 and 6 of the luteal phase in a spontaneous menstrual cycle. Then all patients underwent IVF/ICSI. Main Outcome Measure(s): Achievement of clinical pregnancy by IVF/ICSI. Result(s): ETBF, VEGF expression, and the number of embryos were significantly higher in the women who became pregnant than in those who did not. By stepwise multiple logistic regression, significant predictors of pregnancy were the number of embryos and ETBF but not conventional receptivity markers. The rate of pregnancy was significantly higher in women with ETBF values of at least 29 mL/min per 100 grams of tissue than in women with lower values (42 vs. 15% in 36 and 39 women, respectively). ETBF was significantly greater in morphologically normal than abnormal uteri. In normal uteri, ETBF was greatest in the fundus. Correspondingly, in normal uteri 85% of gestational sacs were implanted in the fundus. Conclusion(s): ETBF is superior to conventional parameters for determining endometrial receptivity for implantation. (Fertil Steril威 2001;76:1168 –74. ©2001 by American Society for Reproductive Medicine.) Key Words: Blood flow, endometrium, implantation, endometrial receptivity
Treatment of infertility has become dramatically more effective with advances in assisted reproductive technologies such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Approximately 80% to 90% of IVF/ICSI patients undergo embryo transfer into the uterus following successful fertilization, but only about 25% of them achieve pregnancy (1). This low rate of implantation may involve poor embryo quality and/or impaired uterine capacity for implantation (i.e., limited endometrial receptivity). Although developmental observation provides satisfactory assessment of embryo quality, the conventional methods for assessing endometrial receptivity—including histologic dating of endome-
trium, serum hormone measurements, and ultrasound examination of the uterus— have not provided a clinically useful indication for receptivity in most patients (2). Angiogenesis has a critical role in female reproductive physiology (3). Development of a rich capillary network is necessary for follicular growth and selection as well as for formation of a functioning corpus luteum. Growth of the endometrium and placentation also are accompanied by extensive angiogenesis. Thus, an actively maintained blood supply is an essential requirement for reproductive functions, including normal implantation. Noninvasive and real-time measurement of tissue blood flow by laser blood-flowmetry has been used widely in
various medical fields and are adequately accurate compared with other methods, such as Xenon washout and microsphere methods (4, 5). In this study, we attempted to assess endometrial receptivity in terms of tissue blood flow in endometrium, which we measured directly and noninvasively by laser bloodflowmetry under hysterofiberscopic observation.
FIGURE 1 Hysterofiberscope equipped with a laser blood-flowmeter. The endometrium is illuminated by linearly polarized laser light through the optical fiber probe; scattered light is detected simultaneously by the probe.
MATERIALS AND METHODS Patients and Design of Study A total of 75 infertile women with normal menstrual cycles were included in this prospective clinical trial. Endometrial tissue blood flow (ETBF) was measured hysterofiberscopically by a laser blood-flowmeter between days 4 and 6 of the luteal phase in a spontaneous menstrual cycle. On the same day, Doppler ultrasound study of the uterine artery and serum hormone measurements were performed; for the women who agreed to additional studies, an endometrial biopsy for histological dating and immunohistochemical study performed. Within 4 months after these examinations of endometrial receptivity, all of the patients underwent IVF or ICSI, resulting in embryo transfer to the uterus. Data from the patients who underwent IVF and ICSI were combined because no statistically significant differences in the rate of fertilization, embryonic development, or pregnancy were noted between the two procedures. Pregnancy was diagnosed by ultrasonographic observation of a gestational sac. We then analyzed relationships between the results of evaluations of endometrial receptivity before IVF or ICSI and subsequent achievement of pregnancy. Informed consent was obtained from all the study participants. The study was approved by the Kyorin University ethics committees and institutional review board approval was obtained. No statistically significant difference was observed between the 21 patients who became pregnant and 54 who failed with respect to mean age ⫾ SEM (34.0 ⫾ 0.7 vs. 35.0 ⫾ 0.7 years, respectively) or distribution of causes of infertility: tubal (33% vs. 41%, respectively), male factor (48% vs. 39%, respectively), both (5% vs. 7%, respectively), and unexplained infertilities (14% vs. 13%, respectively). Of 75 women, 70 had morphologically normal uteri, 3 had uterus bicornes unicolles, 1 had submucous myoma in the fundus (26 mm in diameter), and 1 had multiple intramural myomas (30 to 50 mm).
Hysterofiberscopic Measurement of Endometrial Tissue Blood Flow The optical fiber probe of a laser blood-flowmeter (FLON1, Omega Wave, Tokyo, Japan) was inserted into the infusion channel of a hysterofiberscope (HYF-P, Olympus, Tokyo, Japan) through a T-connector (FS-A, Kitazato Supply, Fujinomiya, Japan) up to the tip (Fig. 1). The hysterofiberscope was inserted into the uterine cavity through the FERTILITY & STERILITY威
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
cervical canal while the uterine cavity was expanded by CO2 insufflation through the other inlet of the T-connector. Under hysterofiberscopic observation, the tip of the probe was kept several millimeters apart from the endometrium, which was illuminated through the probe by linearly polarized laser light. Scattered light was detected by the probe. The site of measurement is marked by visible red laser light. Moment-to-moment ETBF was calculated automatically according to the theory of dynamic light scattering (6). ETBF was recorded for 5 seconds. A time-averaged mean ETBF (mETBF) over that interval then was determined. Pulsatility of ETBF was observed, being synchronous with the heartbeat.
Ultrasonographic, Endocrinologic, and Histologic Assessment of Endometrial Receptivity Doppler ultrasound was performed with a Logiq 500 MD unit (Yokogawa, Tokyo, Japan) using a 6-MHz curvilinear transvaginal probe (E721) coupled with pulsed color Doppler equipment. The uterus was imaged in the long axis, and the greatest anteroposterior endometrial dimension was measured. This represents the endometrial thickness, including two layers of endometrium. Next, flow velocity waveforms from the right and left uterine arteries were obtained by pulsed Doppler measurements following color flow mapping. The pulsatility index (PI) for each artery was calculated electronically from a smooth curve fitted to the average waveform over three cardiac cycles, according to the formula PI ⫽ (S ⫺ D)/A, where S is the peak systolic shift, D is the end-diastolic shift, 1169
and A is the mean maximum Doppler-shifted frequency over the cardiac cycle. The mean PI of the right and left uterine arteries was calculated and used as an index of uterine flow impedance. Serum concentrations of 17-estradiol, progesterone, and PRL were measured by radioimmunoassay (Estradiol kit and Progesterone kit, Diagnostic Products, Los Angeles, CA; and Spack-S PRL, Daiichi Radioisotope, Tokyo, Japan). Concentrations of LH and vascular endothelial growth factor (VEGF) in serum were measured by enzyme immunoassay (Immulyze LH, Diagnostic Products, Los Angeles, CA; and Quantikine human VEGF immunoassay, R & D Systems, Minneapolis, MN). Sensitivities of 17-estradiol, progesterone, PRL, LH, and VEGF assays were 37 pmol/L, 0.2 nmol/L, 1.0 ng/mL, 0.7 IU/L, and 30 pg/mL, respectively. Intra-assay and interassay coefficients of variation were 5.6% and 6.8% for 17-estradiol, 5.8% and 7.2% for progesterone, 6.3% and 6.9% for PRL, 6.2% and 5.5% for LH, and 4.5% and 7.0% for VEGF, respectively. An endometrial biopsy was performed using a curette to obtain two pieces of tissue. One piece of endometrial tissue was promptly fixed in formalin and stained with hematoxylin and eosin. Histologic dating of the endometrium was performed according to the Noyes criteria (7). Endometrium was designated in-phase when the histologic date was within 1 day of the chronologic date. The other piece of endometrial tissue was embedded in optimum cutting temperature compound (Tissue-Tek, Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at ⫺80°C until the immunohistochemical analysis of expression of VEGF in the endometrium was performed later.
Endometrial VEGF Expression
Tissues were cryosectioned at a thickness of 4 m and mounted on ␥-aminopropyltriethoxysilane-coated glass slides before fixation with acetone for 10 minutes at ⫺27°C. Sections were washed in 0.1 M phosphate-buffered saline (PBS), pH 7.4, and treated with 5% normal donkey serum (NDS; 017-000-121, Jackson Immunoresearch, West Grove, PA) in PBS for 10 minutes at room temperature. Sections were incubated for 90 minutes at room temperature in a humid chamber with an affinity-purified rabbit polyclonal antibody raised against human VEGF (VEGF A-20, Catalog no. sc-152, Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution by 5% NDS in PBS. Sections were washed in PBS and incubated for 30 minutes at room temperature with indocarbocyanine (Cy3)-labeled donkey anti-rabbit IgG antibody (No. 711-165-152, Jackson Immunoresearch) at a 1:500 dilution by 5% NDS in PBS. Then SYBR Green I (S-7563, Molecular Probes, Eugene, OR) was added at 20 ng/mL to the secondary antibody solution for counterstaining of nuclei. After washing in PBS, specimens were mounted with 10 mM Tris buffered (pH 8.3) 90% glycerol containing 1 1170 Jinno et al.
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g/mL p-phenylenediamine as an antioxidant, coverslipped, and observed under a confocal laser-scanning microscope (MRC-1024, Bio-Rad) equipped with a krypton/argon laser. Normal rabbit ␥-globulin (No. 011-000-002, Jackson Immunoresearch) at a concentration equivalent to that of the primary antibody was used as a negative control. Optical sectioned images of immunofluorescence for VEGF and SYBR Green I counterstaining for nuclei were acquired in a digital format and transferred to a Power Macintosh computer (Apple Computer, Cupertino, CA). By digital image analysis, fluorescence intensity representing VEGF immunohistochemical staining was measured in the area corresponding to the cytoplasm of the glandular epithelial cells of the endometrium (8). Image processing was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health) and also Adobe Photoshop (Adobe Systems). Briefly, the cytoplasmic area of glandular epithelial cells was distinguished from nuclei and extracellular space, and the average brightness of this area was calculated for each image.
Ovarian Stimulation, In Vitro Fertilization, and Intracytoplasmic Sperm Injection Follicular development was stimulated with the long protocol of a GnRH agonist and human menopausal gonadotropin (hMG) regimen. Nasal administration of buserelin acetate (Suprecur; Hoechst, Tokyo, Japan), 900 g per day, was begun on day 4 of the luteal phase preceding the IVF/ICSI cycle, and continued at the same dose until administration of hCG. Daily administration of three ampules of hMG (Pergonal; Teikokuzouki, Tokyo, Japan; 75 IU of LH plus 75 IU of FSH in each ampule) was begun between days 3 and 10 of the follicular phase in the IVF/ICSI cycle. Serum concentrations of 17-estradiol were measured every morning. When dominant follicles reached 14 mm in diameter, the dose of hMG was reduced to 2 ampules per day. Human chorionic gonadotropin (Gonatropin, Teikokuzouki, Tokyo, Japan), 10,000 IU, was administered when one or more follicles were at least 18 mm in diameter and the serum 17-estradiol concentration exceeded 600 pg/mL (conversion factor to SI units, 3.671). Oocytes were collected transvaginally 36 hours after hCG administration. Semen was diluted two times with human tubal fluid (HTF) medium (no. 9962; Irvine Scientific, Irvine, CA) containing 10% patient serum (9). Diluted semen was centrifuged either directly (if semen analysis was normal) or after placement upon two layers of Sil-Select solutions (FertiPro NV, Beemem, Belgium) (if semen analysis was abnormal). Semen analyses were performed according to the World Health Organization criteria (10). The sperm pellet was resuspended in medium and then centrifuged again, after which motile spermatozoa were collected by a swim-up technique. Oocytes were inseminated 2 to 6 hours after their retrieval at a concentration of 100,000 motile spermatozoa per milliliter. Vol. 76, No. 6, December 2001
TABLE 1 Parameters in patients achieving or failing to achieve pregnancy. Parameters Age (years) mETBF-Fa (mL/min/100g tissue) Mean PIc Endometrial thickness (mm) 17-estradiol (pg/mL)d Progesterone (ng/mL)e The ratio of 17-estradiol/progesterone LH (IU/L) PRL (ng/mL) VEGFf in serum (pg/mL) Endometrial VEGFg In-phase/out-of-phase endometrium No. of embryos produced in vitro No. of transferred embryos
Pregnant (n ⫽ 21)
Nonpregnant (n ⫽ 54)
34.0 ⫾ 0.7 (n ⫽ 21) 32.4 ⫾ 2.3b (n ⫽ 21) 2.3 ⫾ 0.1 (n ⫽ 19) 11.2 ⫾ 0.7 (n ⫽ 19) 120 ⫾ 12 (n ⫽ 21) 11.9 ⫾ 1.3 (n ⫽ 21) 13.6 ⫾ 2.2 (n ⫽ 21) 3.4 ⫾ 0.6 (n ⫽ 21) 9.9 ⫾ 1.1 (n ⫽ 21) 206 ⫾ 39 (n ⫽ 20) 40.1 ⫾ 1.4b (n ⫽ 10) 86%/14% (n ⫽ 14) 10.1 ⫾ 1.0b (n ⫽ 21) 4.2 ⫾ 0.3 (n ⫽ 21)
35.0 ⫾ 0.7 (n ⫽ 54) 26.4 ⫾ 1.3 (n ⫽ 54) 2.5 ⫾ 0.1 (n ⫽ 51) 11.1 ⫾ 0.3 (n ⫽ 49) 141 ⫾ 12 (n ⫽ 54) 14.5 ⫾ 1.2 (n ⫽ 54) 11.2 ⫾ 0.9 (n ⫽ 54) 3.0 ⫾ 0.3 (n ⫽ 53) 7.6 ⫾ 0.5 (n ⫽ 54) 191 ⫾ 20 (n ⫽ 48) 36.6 ⫾ 0.9 (n ⫽ 10) 95%/5% (n ⫽ 19) 6.0 ⫾ 0.6 (n ⫽ 54) 3.6 ⫾ 0.2 (n ⫽ 54)
a
Time-averaged mean of endometrial tissue blood flow in the fundus of the uterine cavity. P⬍.05 vs. nonpregnant group (unpaired t-test). c Mean of pulsatility indices of the right and left uterine arteries. d To convert values for 17-estradiol to pmol/L, multiply by 3.671. e To convert values for progesterone to nmol/L, multiply by 3.180. f Vascular endothelial growth factor. g Average VEGF labeling intensity in 256 gradations measured by semiquantitative immunohistochemistry. b
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
Oocytes were considered to be fertilized when two pronuclei were observed at 16 to 18 hours following insemination. Embryos were transferred into the uterus 38 to 48 hours following insemination. The transfer was performed without ultrasound guidance. A 25-mg dose of progesterone was administered daily throughout the luteal phase. ICSI was performed when the partner had severe male infertility with a sperm concentration less than 5 ⫻ 106 per mL and/or sperm motility below 20%. The cells of the cumulus and corona radiata were removed from the oocyte complexes by incubation with hyaluronidase, 50 IU/mL (type IV-S; Sigma, St. Louis, MO) and aspiration of the complexes in and out of pipettes with serially smaller openings decreasing from 300 m to 150 m. A single motile spermatozoon, immobilized by incising its tail with the tip of the injection pipette (10-MIC, Humagen, Charlottesville, VA) was aspirated tail first into the injection pipette. Polyvinylpyrrolidone was not used. The oocyte was fixed on the holding pipette with the polar body at the 12- or 6-o’clock position while the injection pipette was pushed through the zona pellucida and the oolemma at the 3-o’clock position. A spermatozoon was injected into the ooplasm with as small amount of medium as possible. A piezo-micromanipulator (PMAS-CT16, PRIMA, Tokyo, Japan) was used to ensure penetration of the oolemma, and also was used if necessary for detachment of the spermatozoon from the injection pipette. The injected oocytes were washed and incubated in HTF medium with 10% serum. FERTILITY & STERILITY威
Statistical Analysis Data were analyzed using the Student’s t-test, analysis of variance (ANOVA), Fisher’s protected least significant difference (PLSD) test, stepwise multiple logistic regression analysis, 2 test, or Fisher’s exact test, as appropriate. P values less than .05 were considered statistically significant. Results are presented as the mean ⫾ SEM unless otherwise stated.
RESULTS Various parameters were compared between patients who became pregnant and those who failed (Table 1). The mETBF in the fundus of uterine cavity (mETBF-F), expression of VEGF in the endometrium, and numbers of embryos produced in vitro were significantly higher in the pregnant women. No statistically significant differences were observed between pregnant and nonpregnant women in the age, number of transferred embryos, or conventional parameters of endometrial receptivity including ultrasonographic measurements, serum hormone concentrations, and histologic date of the endometrium. In a forward stepwise multiple logistic regression analysis of 10 factors (age, cause of infertility, mETBF-F, mean PI, concentrations of 17-estradiol, progesterone and VEGF in serum, the ratio of 17-estradiol/progesterone concentrations in serum, numbers of embryos produced in vitro, and numbers of embryos transferred), significant predictors of 1171
achievement of pregnancy were only numbers of embryos produced in vitro (P⬍.01) and mETBF-F (P⬍.05). For each additional embryo and each mL/min/100 grams of tissue increase in mETBF-F, the likelifood of pregnancy increased by 1.23-fold and 1.08-fold, respectively (odds ratios, 1.23 and 1.08; 95% confidence intervals, 1.07 to 1.42 and 1.01 to 1.16, respectively). Endometrial VEGF expression and histologic dating of endometrium were not included in this analysis because the number of samples was too small.
FIGURE 2 Immunohistochemical localization of vascular endothelial growth factor (VEGF) in human secretory endometrium. VEGF immunoreactivity, shown as red areas, is confined to glandular epithelial cells, with little detectable immunofluorescence in stromal cells. Immunoreactivity is stronger in (A) a woman who became pregnant than (B) one who did not. Nuclei are shown in green. Scale bar ⫽ 10m.
A receiver-operating characteristics (ROC) curve analysis of mETBF-F for prediction of pregnancy was performed (data not presented). A cutoff value of 29 mL/min/100 grams of tissue of mETBF-F, which corresponds to the nearest point to the coordinates (1-specificity ⫽ 0, sensitivity ⫽ 1) on the ROC curve, was chosen for further analysis. Rate of pregnancy was significantly higher in women with an mETBF-F value of at least 29 than in women with lower mETBF-F values (42 vs. 15% in 36 and 39 women, respectively; P⬍.05, 2 test). Sensitivity and specificity of mETBF-F for prediction of pregnancy were 0.71 and 0.61, respectively. When dichotomized (⬍29 or ⱖ29) values for mETBF-F was used in the same forward stepwise multiple logistic regression analysis as described above, the significant predictors of achievement of pregnancy remained numbers of embryos and mETBF-F (P⬍.01 and ⱕ.05; odds ratios, 1.24 and 5.60; 95% confidence intervals, 1.07 to 1.43 and 1.43 to 22.03, respectively). We detected VEGF immunoreactivity in glandular epithelial cells of the secretory endometrium; it was stronger in the pregnant group than in the nonpregnant group (Fig. 2; see Table 1). Little immunofluorescence was detectable in stromal cells or in cells associated with the microvasculature. Endometrial receptivity-related parameters were compared between structurally normal and abnormal uteri (Table 2); mETBF-F and endometrial thickness were significantly greater and thicker in normal uteri, whereas no statistically significant difference was observed in mean PI or hormonal parameters. The rate of implantation appeared to be impaired in abnormal uteri, although the difference did not attain statistical significance. In 52 women with normal uteri and 4 women with abnormal uteri, mETBF was measured in five regions of the endometrium: the fundus and anterior, posterior, right-lateral, and left-lateral walls (Fig. 3). In normal uteri mETBF was significantly higher in the fundus than in the other regions, but not in abnormal uteri. Correspondingly, transvaginal ultrasonography between 5 and 6 weeks of gestation demonstrated that in normal uteri 22 (85%), 1, 2, 1, and 0 of 26 gestational sacs were implanted in the fundus, anterior, posterior, right-lateral, and left-lateral walls of the endometrium, respectively. A patient with a uterus bicornis unicollis became pregnant and her uterus had 22.5 and 31.3 mL/min/ 100-gram tissue of mETBF in the right and left endometrial 1172 Jinno et al.
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Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
horns, respectively. One gestational sac was observed in the left uterine cavity but was aborted spontaneously at 7 weeks of gestation.
DISCUSSION Uterine blood flow may be estimated by Doppler ultrasound analysis as uterine artery blood flow volume. However, such flow volume analysis often is difficult and inaccurate because the results depend on the angle of insonation, accurate measurement of vessel diameter, tortuosity of the Vol. 76, No. 6, December 2001
TABLE 2 Endometrial receptivity parameters in normal and abnormal uteri. Parameters Age (years) mETBF-Fa (mL/min/100g tissue) Mean PIc Endometrial thickness (mm) 17-estradiol (pg/mL)d Progesterone (ng/mL)e No. of embryos produced in vitro Implantation ratef
Normal uteri (n ⫽ 70)
Abnormal uteri (n ⫽ 5)
34.7 ⫾ 0.5 (n ⫽ 70) 28.7 ⫾ 1.2b (n ⫽ 70) 2.5 ⫾ 0.1 (n ⫽ 65) 11.3 ⫾ 0.3b (n ⫽ 64) 138 ⫾ 10 (n ⫽ 70) 14.0 ⫾ 1.0 (n ⫽ 70) 7.1 ⫾ 0.6 (n ⫽ 70) 12% (n ⫽ 258)
36.0 ⫾ 2.1 (n ⫽ 5) 19.4 ⫾ 3.5 (n ⫽ 5) 2.1 ⫾ 0.2 (n ⫽ 5) 8.8 ⫾ 1.0 (n ⫽ 4) 93 ⫾ 14 (n ⫽ 5) 10.2 ⫾ 2.3 (n ⫽ 5) 8.0 ⫾ 2.8 (n ⫽ 5) 3.6% (n ⫽ 28)
a
Time-averaged mean of endometrial tissue blood flow in the fundus of the uterine cavity. P⬍.05 vs. abnormal uteri (unpaired t-test). c Mean of pulsatility indices of the right and left uterine arteries. d To convert values for 17-estradiol to pmol/L, multiply by 3.671. e To convert values for progesterone to nmol/L, multiply by 3.180. f Percentage of gestational sacs per transferred embryo. b
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
vessel, and the analytic power of instrumentation (11). Instead, PI, resistance index (RI), and waveform analysis, which detect downstream impedance to blood flow and are independent of the angle of insonation, are used widely to estimate blood flow volume indirectly (2, 11). Absence of all diastolic flow or of early diastolic flow in uterine artery blood flow waveforms has been associated with repeated failure of implantation in patients undergoing
FIGURE 3 Time-averaged mean of endometrial tissue blow flow (mETBF) in the fundus (F) and anterior (A), posterior (P), right-lateral (R), and left-lateral (L) walls of the endometrium in normal and abnormal uteri. * ⫽ P⬍.0001 vs. A/P/R/L group (ANOVA and Fisher’s PLSD).
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
FERTILITY & STERILITY威
IVF (12). Uterine artery PI and RI were significantly lower in women who conceived by IVF than in those who did not conceive (13–15). Significantly lower rates of implantation per transferred embryo (14) and pregnancy per cycle (14, 15) were observed in IVF patients with a uterine artery PI of 3.0 or more than those with a PI less than 3.0. Accumulating recent reports, however, have demonstrated similar PI values in pregnant and nonpregnant IVF/ICSI patients (16 –18), and a lack of statistically significant differences in rates of implantation and pregnancy between women with low (1.00 to 1.99), medium (2.00 to 2.99) and high (ⱖ3.00) PI (16, 18). Taken together, available data indicate a wide range of overlap in uterine artery PI values between pregnant and nonpregnant cycles, making clinical judgment concerning the uterine receptivity difficult in most cases (2, 11, 19). Only a remarkably high vascular impedance (PI ⱖ3.3 to 4.0 and/or absence of diastolic flow) may predict impaired implantation (2, 11, 19); such a high impedance is detected in only 9% to 26% of nonpregnant cycles (2, 15, 20). In the present study, mETBF, a novel parameter of endometrial blood perfusion, was a significant predictor of achievement of pregnancy by IVF/ICSI while, as in most previous reports, uterine artery PI was not. Several reasons may account for this. The mETBF is a direct measurement of blood flow volume in the endometrium, the actual site where implantation occurs. On the other hand, uterine artery PI is a parameter indirectly reflecting blood flow volume in the entire uterus, which includes blood supply not only to the endometrium but also to the myometrium and epimetrium. Moreover, collateral communication exists between uterine and ovarian blood vessels, which may further obscure the relationship between uterine artery PI and actual blood flow in the endometrium. Consistently, a poor correlation (r ⫽ 1173
0.24) has been demonstrated between PIs in the uterine and spiral arteries (11). We observed that 85% of gestational sacs in normal uteri were implanted in the fundic endometrium, which had the highest mETBF among uterine regions (see Fig. 3). Embryos may implant selectively at the endometrial site with most abundant blood flow, or alternatively, only embryos implanted at the endometrial site with the most blood flow can survive to the stage where a gestational sac is seen by ultrasonography. In any cases, abundant blood flow at the implantation site appears necessary for embryonic growth. This agrees with another of our present observations, that abnormal uteri with a tendency for impaired endometrial receptivity had deficient blood flow in the fundus. The unique ability of mETBF to provide site-specific evaluation of endometrial blood flow may contribute to functional diagnosis and decision making concerning surgical correction of abnormal uteri. VEGF plays a pivotal role in regulation of physiologic and pathologic angiogenesis (21) and also is believed to be a paracrine regulator of the effects of ovarian steroids on endometrial angiogenesis (3). During the menstrual cycle, the expression of VEGF mRNA and protein in the endometrium was greatest in the secretory phase (22, 23), a time when endometrium has the greatest angiogenic activity (24). Consistent with our present observation, strong immunoreactivity of VEGF was detected in the glandular epithelial cells of the secretory endometrium but weak immunoreactivity in stromal cells (22, 25). Our present study shows for the first time that stronger expression of endometrial VEGF is related with successful implantation, although a larger number of patients should be examined to confirm our observation. The role of endometrial VEGF in the implantation remains to be clarified.
3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14.
15. 16.
17. 18.
19.
ETBF reflects endometrial receptivity more closely than do conventional receptivity markers, including ultrasonographic parameters, serum hormone concentrations, and histologic date of endometrium. In addition, ETBF can be measured easily, quickly, and noninvasively in the outpatient clinic. The possibility that the unique capacity of ETBF for site-selective evaluation of endometrial blood flow can help in clinical management of abnormal uteri awaits further investigation. References
21. 22. 23.
24.
1. de Mouzon J, Lancaster P. World collaborative report on in vitro fertilization, preliminary data for 1995. J Assist Reprod Genet 1997; 14(suppl):250S– 65S. 2. Friedler S, Schenker JG, Herman A, Lewin A. The role of ultrasonography in the evaluation of endometrial receptivity following assisted
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20.
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reproductive treatments: a critical review. Hum Reprod Update 1996; 2:323–35. Gordon JD, Shifren JL, Foulk RA, Taylor RN, Jaffe RB. Angiogenesis in the human female reproductive tract. Obstet Gynecol Surv 1995;50: 688 –97. Engelhart M, Kristensen JK. Evaluation of cutaneous blood flow responses by xenon washout and a laser-Doppler flowmeter. J Invest Dermatol 1983;80:12–15. Kashima S, Oka S, Ishikawa J, Ohsawa T, Hiki Y. Measurement of tissue blood volume in a model system and in the canine intestine by dynamic light scattering. Lasers Life Sci 1994;6:79 –90. Kashima S. Non-contact laser tissue blood flow measurement using polarization to reduce the specular reflection artefact. Opt Laser Technol 1994;26:169 –75. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950;1:3–25. Kudo A, Hirano H. The practical side of image processing with the use of a personal computer: automation of the analyzing processes employing NIH Image and Adobe Photoshop with Power Macintosh Computers. Acta Histochem Cytochem 1999;32:229 –33. Jinno M. Comparison of media used for human in vitro fertilization and embryo transfer programs: a new method of serum preparation. Acta Obstet Gynaecol Jpn 1986;38:102–10. World Health Organization. WHO laboratory manual for examination of human semen and semen-cervical mucus interaction. 3rd ed. Cambridge: Cambridge University Press; 1987:1– 64. Dickey RP. Doppler ultrasound investigation of uterine and ovarian blood flow in infertility and early pregnancy. Hum Reprod Update 1997;3:467–503. Goswamy RK, Williams G, Steptoe PC. Decreased uterine perfusion: a cause of infertility. Hum Reprod 1988;3:955–9. Sterzik K, Grab D, Sasse V, Hutter W, Rosenbusch B, Terinde R. Doppler sonographic findings and their correlation with implantation in an in vitro fertilization program. Fertil Steril 1989;52:825– 8. Steer CV, Campbell S, Tan SL, Crayford T, Mills C, Mason BA, Collins WP. The use of transvaginal color flow imaging after in vitro fertilization to identify optimum uterine conditions before embryo transfer. Fertil Steril 1992;57:372– 6. Cacciatore B, Simberg N, Fusaro P, Tiitinen A. Transvaginal Doppler study of uterine artery blood flow in in vitro fertilization-embryo transfer cycles. Fertil Steril 1996;66:130 – 4. 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;10:688 –93. Tekay A, Martikainen H, Jouppila P. Comparison of uterine blood flow characteristics between spontaneous and stimulated cycles before embryo transfer. Hum Reprod 1996;11:364 – 8. Aytoz A, Ubaldi F, Tournaye H, Nagy ZP, Steirteghem AV, Devroey P. The predictive value of uterine artery blood flow measurements for uterine receptivity in an intracytoplasmic sperm injection program. Fertil Steril 1997;68:935–7. Tekay A, Martikainen H, Jouppila P. The clinical value of transvaginal colour Doppler ultrasound in assisted reproductive technology procedures. Hum Reprod 1996;11:1589 –91. Coulam CB, Bustillo M, Soenksen DM, Britten S. Ultrasonographic predictors of implantation after assisted reproduction. Fertil Steril 1994; 62:1004 –10. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4 –25. Torry DS, Holt VJ, Keenan JA, Harris G, Caudle MR, Torry RJ. Vascular endothelial growth factor expression in cycling human endometrium. Fertil Steril 1996;66:72– 80. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, Jaffe RB, Taylor RN. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996;81:3112– 8. Rogers P, Abberton K, Susil B. Endothelial cell migratory signal produced by human endometrium during the menstrual cycle. Hum Reprod 1992;7:1061– 6. Li XF, Gregory J, Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium. Growth Factors 1994;11: 277– 82.
Vol. 76, No. 6, December 2001
Measurement of endometrial tissue blood flow: a novel way to assess uterine receptivity for implantation Masao Jinno, M.D.,a Tsuneo Ozaki, M.D.,a Mitsutoshi Iwashita, M.D.,a Yukio Nakamura, M.D.,a Akihiko Kudo, Ph.D.,b and Hiroshi Hirano, M.D., Ph.D.b,c Department of Obstetrics and Gynecology and Department of Anatomy, School of Medicine, Kyorin University, Mitaka City, Tokyo, Japan
Received March 12, 2001; revised and accepted June 12, 2001. Reprint requests: Masao Jinno, M.D., Department of Obstetrics and Gynecology, School of Medicine, Kyorin University, 6-20-2 Shinkawa, Mitaka City, Tokyo 181-8611, Japan (Fax: 81-422-47-3177; E-mail: jinno@kyorin-u. ac.jp). a Department of Obstetrics and Gynecology, School of Medicine, Kyorin University. b Department of Anatomy, School of Medicine, Kyorin University. c Nittai Jusei Medical College for Judo Therapeutics, Tokyo, Japan. 0015-0282/01/$20.00 PII S0015-0282(01)02897-7
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Objective: To assess endometrial receptivity in terms of endometrial tissue blood flow (ETBF) measured hysterofiberscopically by laser blood-flowmetry, and to examine the technique’s effectiveness in an in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) program. Design: A prospective clinical study. Setting(s): IVF program in a university hospital. Patient(s): A total of 75 infertile women with normal menstrual cycles undergoing IVF/ICSI. Intervention(s): ETBF, conventional ultrasonographic, endocrinologic, and histologic parameters for receptivity and immunoreactivity for vascular endothelial growth factor (VEGF) in endometrium were assessed between days 4 and 6 of the luteal phase in a spontaneous menstrual cycle. Then all patients underwent IVF/ICSI. Main Outcome Measure(s): Achievement of clinical pregnancy by IVF/ICSI. Result(s): ETBF, VEGF expression, and the number of embryos were significantly higher in the women who became pregnant than in those who did not. By stepwise multiple logistic regression, significant predictors of pregnancy were the number of embryos and ETBF but not conventional receptivity markers. The rate of pregnancy was significantly higher in women with ETBF values of at least 29 mL/min per 100 grams of tissue than in women with lower values (42 vs. 15% in 36 and 39 women, respectively). ETBF was significantly greater in morphologically normal than abnormal uteri. In normal uteri, ETBF was greatest in the fundus. Correspondingly, in normal uteri 85% of gestational sacs were implanted in the fundus. Conclusion(s): ETBF is superior to conventional parameters for determining endometrial receptivity for implantation. (Fertil Steril威 2001;76:1168 –74. ©2001 by American Society for Reproductive Medicine.) Key Words: Blood flow, endometrium, implantation, endometrial receptivity
Treatment of infertility has become dramatically more effective with advances in assisted reproductive technologies such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Approximately 80% to 90% of IVF/ICSI patients undergo embryo transfer into the uterus following successful fertilization, but only about 25% of them achieve pregnancy (1). This low rate of implantation may involve poor embryo quality and/or impaired uterine capacity for implantation (i.e., limited endometrial receptivity). Although developmental observation provides satisfactory assessment of embryo quality, the conventional methods for assessing endometrial receptivity—including histologic dating of endome-
trium, serum hormone measurements, and ultrasound examination of the uterus— have not provided a clinically useful indication for receptivity in most patients (2). Angiogenesis has a critical role in female reproductive physiology (3). Development of a rich capillary network is necessary for follicular growth and selection as well as for formation of a functioning corpus luteum. Growth of the endometrium and placentation also are accompanied by extensive angiogenesis. Thus, an actively maintained blood supply is an essential requirement for reproductive functions, including normal implantation. Noninvasive and real-time measurement of tissue blood flow by laser blood-flowmetry has been used widely in
various medical fields and are adequately accurate compared with other methods, such as Xenon washout and microsphere methods (4, 5). In this study, we attempted to assess endometrial receptivity in terms of tissue blood flow in endometrium, which we measured directly and noninvasively by laser bloodflowmetry under hysterofiberscopic observation.
FIGURE 1 Hysterofiberscope equipped with a laser blood-flowmeter. The endometrium is illuminated by linearly polarized laser light through the optical fiber probe; scattered light is detected simultaneously by the probe.
MATERIALS AND METHODS Patients and Design of Study A total of 75 infertile women with normal menstrual cycles were included in this prospective clinical trial. Endometrial tissue blood flow (ETBF) was measured hysterofiberscopically by a laser blood-flowmeter between days 4 and 6 of the luteal phase in a spontaneous menstrual cycle. On the same day, Doppler ultrasound study of the uterine artery and serum hormone measurements were performed; for the women who agreed to additional studies, an endometrial biopsy for histological dating and immunohistochemical study performed. Within 4 months after these examinations of endometrial receptivity, all of the patients underwent IVF or ICSI, resulting in embryo transfer to the uterus. Data from the patients who underwent IVF and ICSI were combined because no statistically significant differences in the rate of fertilization, embryonic development, or pregnancy were noted between the two procedures. Pregnancy was diagnosed by ultrasonographic observation of a gestational sac. We then analyzed relationships between the results of evaluations of endometrial receptivity before IVF or ICSI and subsequent achievement of pregnancy. Informed consent was obtained from all the study participants. The study was approved by the Kyorin University ethics committees and institutional review board approval was obtained. No statistically significant difference was observed between the 21 patients who became pregnant and 54 who failed with respect to mean age ⫾ SEM (34.0 ⫾ 0.7 vs. 35.0 ⫾ 0.7 years, respectively) or distribution of causes of infertility: tubal (33% vs. 41%, respectively), male factor (48% vs. 39%, respectively), both (5% vs. 7%, respectively), and unexplained infertilities (14% vs. 13%, respectively). Of 75 women, 70 had morphologically normal uteri, 3 had uterus bicornes unicolles, 1 had submucous myoma in the fundus (26 mm in diameter), and 1 had multiple intramural myomas (30 to 50 mm).
Hysterofiberscopic Measurement of Endometrial Tissue Blood Flow The optical fiber probe of a laser blood-flowmeter (FLON1, Omega Wave, Tokyo, Japan) was inserted into the infusion channel of a hysterofiberscope (HYF-P, Olympus, Tokyo, Japan) through a T-connector (FS-A, Kitazato Supply, Fujinomiya, Japan) up to the tip (Fig. 1). The hysterofiberscope was inserted into the uterine cavity through the FERTILITY & STERILITY威
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
cervical canal while the uterine cavity was expanded by CO2 insufflation through the other inlet of the T-connector. Under hysterofiberscopic observation, the tip of the probe was kept several millimeters apart from the endometrium, which was illuminated through the probe by linearly polarized laser light. Scattered light was detected by the probe. The site of measurement is marked by visible red laser light. Moment-to-moment ETBF was calculated automatically according to the theory of dynamic light scattering (6). ETBF was recorded for 5 seconds. A time-averaged mean ETBF (mETBF) over that interval then was determined. Pulsatility of ETBF was observed, being synchronous with the heartbeat.
Ultrasonographic, Endocrinologic, and Histologic Assessment of Endometrial Receptivity Doppler ultrasound was performed with a Logiq 500 MD unit (Yokogawa, Tokyo, Japan) using a 6-MHz curvilinear transvaginal probe (E721) coupled with pulsed color Doppler equipment. The uterus was imaged in the long axis, and the greatest anteroposterior endometrial dimension was measured. This represents the endometrial thickness, including two layers of endometrium. Next, flow velocity waveforms from the right and left uterine arteries were obtained by pulsed Doppler measurements following color flow mapping. The pulsatility index (PI) for each artery was calculated electronically from a smooth curve fitted to the average waveform over three cardiac cycles, according to the formula PI ⫽ (S ⫺ D)/A, where S is the peak systolic shift, D is the end-diastolic shift, 1169
and A is the mean maximum Doppler-shifted frequency over the cardiac cycle. The mean PI of the right and left uterine arteries was calculated and used as an index of uterine flow impedance. Serum concentrations of 17-estradiol, progesterone, and PRL were measured by radioimmunoassay (Estradiol kit and Progesterone kit, Diagnostic Products, Los Angeles, CA; and Spack-S PRL, Daiichi Radioisotope, Tokyo, Japan). Concentrations of LH and vascular endothelial growth factor (VEGF) in serum were measured by enzyme immunoassay (Immulyze LH, Diagnostic Products, Los Angeles, CA; and Quantikine human VEGF immunoassay, R & D Systems, Minneapolis, MN). Sensitivities of 17-estradiol, progesterone, PRL, LH, and VEGF assays were 37 pmol/L, 0.2 nmol/L, 1.0 ng/mL, 0.7 IU/L, and 30 pg/mL, respectively. Intra-assay and interassay coefficients of variation were 5.6% and 6.8% for 17-estradiol, 5.8% and 7.2% for progesterone, 6.3% and 6.9% for PRL, 6.2% and 5.5% for LH, and 4.5% and 7.0% for VEGF, respectively. An endometrial biopsy was performed using a curette to obtain two pieces of tissue. One piece of endometrial tissue was promptly fixed in formalin and stained with hematoxylin and eosin. Histologic dating of the endometrium was performed according to the Noyes criteria (7). Endometrium was designated in-phase when the histologic date was within 1 day of the chronologic date. The other piece of endometrial tissue was embedded in optimum cutting temperature compound (Tissue-Tek, Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at ⫺80°C until the immunohistochemical analysis of expression of VEGF in the endometrium was performed later.
Endometrial VEGF Expression
Tissues were cryosectioned at a thickness of 4 m and mounted on ␥-aminopropyltriethoxysilane-coated glass slides before fixation with acetone for 10 minutes at ⫺27°C. Sections were washed in 0.1 M phosphate-buffered saline (PBS), pH 7.4, and treated with 5% normal donkey serum (NDS; 017-000-121, Jackson Immunoresearch, West Grove, PA) in PBS for 10 minutes at room temperature. Sections were incubated for 90 minutes at room temperature in a humid chamber with an affinity-purified rabbit polyclonal antibody raised against human VEGF (VEGF A-20, Catalog no. sc-152, Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution by 5% NDS in PBS. Sections were washed in PBS and incubated for 30 minutes at room temperature with indocarbocyanine (Cy3)-labeled donkey anti-rabbit IgG antibody (No. 711-165-152, Jackson Immunoresearch) at a 1:500 dilution by 5% NDS in PBS. Then SYBR Green I (S-7563, Molecular Probes, Eugene, OR) was added at 20 ng/mL to the secondary antibody solution for counterstaining of nuclei. After washing in PBS, specimens were mounted with 10 mM Tris buffered (pH 8.3) 90% glycerol containing 1 1170 Jinno et al.
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g/mL p-phenylenediamine as an antioxidant, coverslipped, and observed under a confocal laser-scanning microscope (MRC-1024, Bio-Rad) equipped with a krypton/argon laser. Normal rabbit ␥-globulin (No. 011-000-002, Jackson Immunoresearch) at a concentration equivalent to that of the primary antibody was used as a negative control. Optical sectioned images of immunofluorescence for VEGF and SYBR Green I counterstaining for nuclei were acquired in a digital format and transferred to a Power Macintosh computer (Apple Computer, Cupertino, CA). By digital image analysis, fluorescence intensity representing VEGF immunohistochemical staining was measured in the area corresponding to the cytoplasm of the glandular epithelial cells of the endometrium (8). Image processing was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health) and also Adobe Photoshop (Adobe Systems). Briefly, the cytoplasmic area of glandular epithelial cells was distinguished from nuclei and extracellular space, and the average brightness of this area was calculated for each image.
Ovarian Stimulation, In Vitro Fertilization, and Intracytoplasmic Sperm Injection Follicular development was stimulated with the long protocol of a GnRH agonist and human menopausal gonadotropin (hMG) regimen. Nasal administration of buserelin acetate (Suprecur; Hoechst, Tokyo, Japan), 900 g per day, was begun on day 4 of the luteal phase preceding the IVF/ICSI cycle, and continued at the same dose until administration of hCG. Daily administration of three ampules of hMG (Pergonal; Teikokuzouki, Tokyo, Japan; 75 IU of LH plus 75 IU of FSH in each ampule) was begun between days 3 and 10 of the follicular phase in the IVF/ICSI cycle. Serum concentrations of 17-estradiol were measured every morning. When dominant follicles reached 14 mm in diameter, the dose of hMG was reduced to 2 ampules per day. Human chorionic gonadotropin (Gonatropin, Teikokuzouki, Tokyo, Japan), 10,000 IU, was administered when one or more follicles were at least 18 mm in diameter and the serum 17-estradiol concentration exceeded 600 pg/mL (conversion factor to SI units, 3.671). Oocytes were collected transvaginally 36 hours after hCG administration. Semen was diluted two times with human tubal fluid (HTF) medium (no. 9962; Irvine Scientific, Irvine, CA) containing 10% patient serum (9). Diluted semen was centrifuged either directly (if semen analysis was normal) or after placement upon two layers of Sil-Select solutions (FertiPro NV, Beemem, Belgium) (if semen analysis was abnormal). Semen analyses were performed according to the World Health Organization criteria (10). The sperm pellet was resuspended in medium and then centrifuged again, after which motile spermatozoa were collected by a swim-up technique. Oocytes were inseminated 2 to 6 hours after their retrieval at a concentration of 100,000 motile spermatozoa per milliliter. Vol. 76, No. 6, December 2001
TABLE 1 Parameters in patients achieving or failing to achieve pregnancy. Parameters Age (years) mETBF-Fa (mL/min/100g tissue) Mean PIc Endometrial thickness (mm) 17-estradiol (pg/mL)d Progesterone (ng/mL)e The ratio of 17-estradiol/progesterone LH (IU/L) PRL (ng/mL) VEGFf in serum (pg/mL) Endometrial VEGFg In-phase/out-of-phase endometrium No. of embryos produced in vitro No. of transferred embryos
Pregnant (n ⫽ 21)
Nonpregnant (n ⫽ 54)
34.0 ⫾ 0.7 (n ⫽ 21) 32.4 ⫾ 2.3b (n ⫽ 21) 2.3 ⫾ 0.1 (n ⫽ 19) 11.2 ⫾ 0.7 (n ⫽ 19) 120 ⫾ 12 (n ⫽ 21) 11.9 ⫾ 1.3 (n ⫽ 21) 13.6 ⫾ 2.2 (n ⫽ 21) 3.4 ⫾ 0.6 (n ⫽ 21) 9.9 ⫾ 1.1 (n ⫽ 21) 206 ⫾ 39 (n ⫽ 20) 40.1 ⫾ 1.4b (n ⫽ 10) 86%/14% (n ⫽ 14) 10.1 ⫾ 1.0b (n ⫽ 21) 4.2 ⫾ 0.3 (n ⫽ 21)
35.0 ⫾ 0.7 (n ⫽ 54) 26.4 ⫾ 1.3 (n ⫽ 54) 2.5 ⫾ 0.1 (n ⫽ 51) 11.1 ⫾ 0.3 (n ⫽ 49) 141 ⫾ 12 (n ⫽ 54) 14.5 ⫾ 1.2 (n ⫽ 54) 11.2 ⫾ 0.9 (n ⫽ 54) 3.0 ⫾ 0.3 (n ⫽ 53) 7.6 ⫾ 0.5 (n ⫽ 54) 191 ⫾ 20 (n ⫽ 48) 36.6 ⫾ 0.9 (n ⫽ 10) 95%/5% (n ⫽ 19) 6.0 ⫾ 0.6 (n ⫽ 54) 3.6 ⫾ 0.2 (n ⫽ 54)
a
Time-averaged mean of endometrial tissue blood flow in the fundus of the uterine cavity. P⬍.05 vs. nonpregnant group (unpaired t-test). c Mean of pulsatility indices of the right and left uterine arteries. d To convert values for 17-estradiol to pmol/L, multiply by 3.671. e To convert values for progesterone to nmol/L, multiply by 3.180. f Vascular endothelial growth factor. g Average VEGF labeling intensity in 256 gradations measured by semiquantitative immunohistochemistry. b
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
Oocytes were considered to be fertilized when two pronuclei were observed at 16 to 18 hours following insemination. Embryos were transferred into the uterus 38 to 48 hours following insemination. The transfer was performed without ultrasound guidance. A 25-mg dose of progesterone was administered daily throughout the luteal phase. ICSI was performed when the partner had severe male infertility with a sperm concentration less than 5 ⫻ 106 per mL and/or sperm motility below 20%. The cells of the cumulus and corona radiata were removed from the oocyte complexes by incubation with hyaluronidase, 50 IU/mL (type IV-S; Sigma, St. Louis, MO) and aspiration of the complexes in and out of pipettes with serially smaller openings decreasing from 300 m to 150 m. A single motile spermatozoon, immobilized by incising its tail with the tip of the injection pipette (10-MIC, Humagen, Charlottesville, VA) was aspirated tail first into the injection pipette. Polyvinylpyrrolidone was not used. The oocyte was fixed on the holding pipette with the polar body at the 12- or 6-o’clock position while the injection pipette was pushed through the zona pellucida and the oolemma at the 3-o’clock position. A spermatozoon was injected into the ooplasm with as small amount of medium as possible. A piezo-micromanipulator (PMAS-CT16, PRIMA, Tokyo, Japan) was used to ensure penetration of the oolemma, and also was used if necessary for detachment of the spermatozoon from the injection pipette. The injected oocytes were washed and incubated in HTF medium with 10% serum. FERTILITY & STERILITY威
Statistical Analysis Data were analyzed using the Student’s t-test, analysis of variance (ANOVA), Fisher’s protected least significant difference (PLSD) test, stepwise multiple logistic regression analysis, 2 test, or Fisher’s exact test, as appropriate. P values less than .05 were considered statistically significant. Results are presented as the mean ⫾ SEM unless otherwise stated.
RESULTS Various parameters were compared between patients who became pregnant and those who failed (Table 1). The mETBF in the fundus of uterine cavity (mETBF-F), expression of VEGF in the endometrium, and numbers of embryos produced in vitro were significantly higher in the pregnant women. No statistically significant differences were observed between pregnant and nonpregnant women in the age, number of transferred embryos, or conventional parameters of endometrial receptivity including ultrasonographic measurements, serum hormone concentrations, and histologic date of the endometrium. In a forward stepwise multiple logistic regression analysis of 10 factors (age, cause of infertility, mETBF-F, mean PI, concentrations of 17-estradiol, progesterone and VEGF in serum, the ratio of 17-estradiol/progesterone concentrations in serum, numbers of embryos produced in vitro, and numbers of embryos transferred), significant predictors of 1171
achievement of pregnancy were only numbers of embryos produced in vitro (P⬍.01) and mETBF-F (P⬍.05). For each additional embryo and each mL/min/100 grams of tissue increase in mETBF-F, the likelifood of pregnancy increased by 1.23-fold and 1.08-fold, respectively (odds ratios, 1.23 and 1.08; 95% confidence intervals, 1.07 to 1.42 and 1.01 to 1.16, respectively). Endometrial VEGF expression and histologic dating of endometrium were not included in this analysis because the number of samples was too small.
FIGURE 2 Immunohistochemical localization of vascular endothelial growth factor (VEGF) in human secretory endometrium. VEGF immunoreactivity, shown as red areas, is confined to glandular epithelial cells, with little detectable immunofluorescence in stromal cells. Immunoreactivity is stronger in (A) a woman who became pregnant than (B) one who did not. Nuclei are shown in green. Scale bar ⫽ 10m.
A receiver-operating characteristics (ROC) curve analysis of mETBF-F for prediction of pregnancy was performed (data not presented). A cutoff value of 29 mL/min/100 grams of tissue of mETBF-F, which corresponds to the nearest point to the coordinates (1-specificity ⫽ 0, sensitivity ⫽ 1) on the ROC curve, was chosen for further analysis. Rate of pregnancy was significantly higher in women with an mETBF-F value of at least 29 than in women with lower mETBF-F values (42 vs. 15% in 36 and 39 women, respectively; P⬍.05, 2 test). Sensitivity and specificity of mETBF-F for prediction of pregnancy were 0.71 and 0.61, respectively. When dichotomized (⬍29 or ⱖ29) values for mETBF-F was used in the same forward stepwise multiple logistic regression analysis as described above, the significant predictors of achievement of pregnancy remained numbers of embryos and mETBF-F (P⬍.01 and ⱕ.05; odds ratios, 1.24 and 5.60; 95% confidence intervals, 1.07 to 1.43 and 1.43 to 22.03, respectively). We detected VEGF immunoreactivity in glandular epithelial cells of the secretory endometrium; it was stronger in the pregnant group than in the nonpregnant group (Fig. 2; see Table 1). Little immunofluorescence was detectable in stromal cells or in cells associated with the microvasculature. Endometrial receptivity-related parameters were compared between structurally normal and abnormal uteri (Table 2); mETBF-F and endometrial thickness were significantly greater and thicker in normal uteri, whereas no statistically significant difference was observed in mean PI or hormonal parameters. The rate of implantation appeared to be impaired in abnormal uteri, although the difference did not attain statistical significance. In 52 women with normal uteri and 4 women with abnormal uteri, mETBF was measured in five regions of the endometrium: the fundus and anterior, posterior, right-lateral, and left-lateral walls (Fig. 3). In normal uteri mETBF was significantly higher in the fundus than in the other regions, but not in abnormal uteri. Correspondingly, transvaginal ultrasonography between 5 and 6 weeks of gestation demonstrated that in normal uteri 22 (85%), 1, 2, 1, and 0 of 26 gestational sacs were implanted in the fundus, anterior, posterior, right-lateral, and left-lateral walls of the endometrium, respectively. A patient with a uterus bicornis unicollis became pregnant and her uterus had 22.5 and 31.3 mL/min/ 100-gram tissue of mETBF in the right and left endometrial 1172 Jinno et al.
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Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
horns, respectively. One gestational sac was observed in the left uterine cavity but was aborted spontaneously at 7 weeks of gestation.
DISCUSSION Uterine blood flow may be estimated by Doppler ultrasound analysis as uterine artery blood flow volume. However, such flow volume analysis often is difficult and inaccurate because the results depend on the angle of insonation, accurate measurement of vessel diameter, tortuosity of the Vol. 76, No. 6, December 2001
TABLE 2 Endometrial receptivity parameters in normal and abnormal uteri. Parameters Age (years) mETBF-Fa (mL/min/100g tissue) Mean PIc Endometrial thickness (mm) 17-estradiol (pg/mL)d Progesterone (ng/mL)e No. of embryos produced in vitro Implantation ratef
Normal uteri (n ⫽ 70)
Abnormal uteri (n ⫽ 5)
34.7 ⫾ 0.5 (n ⫽ 70) 28.7 ⫾ 1.2b (n ⫽ 70) 2.5 ⫾ 0.1 (n ⫽ 65) 11.3 ⫾ 0.3b (n ⫽ 64) 138 ⫾ 10 (n ⫽ 70) 14.0 ⫾ 1.0 (n ⫽ 70) 7.1 ⫾ 0.6 (n ⫽ 70) 12% (n ⫽ 258)
36.0 ⫾ 2.1 (n ⫽ 5) 19.4 ⫾ 3.5 (n ⫽ 5) 2.1 ⫾ 0.2 (n ⫽ 5) 8.8 ⫾ 1.0 (n ⫽ 4) 93 ⫾ 14 (n ⫽ 5) 10.2 ⫾ 2.3 (n ⫽ 5) 8.0 ⫾ 2.8 (n ⫽ 5) 3.6% (n ⫽ 28)
a
Time-averaged mean of endometrial tissue blood flow in the fundus of the uterine cavity. P⬍.05 vs. abnormal uteri (unpaired t-test). c Mean of pulsatility indices of the right and left uterine arteries. d To convert values for 17-estradiol to pmol/L, multiply by 3.671. e To convert values for progesterone to nmol/L, multiply by 3.180. f Percentage of gestational sacs per transferred embryo. b
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
vessel, and the analytic power of instrumentation (11). Instead, PI, resistance index (RI), and waveform analysis, which detect downstream impedance to blood flow and are independent of the angle of insonation, are used widely to estimate blood flow volume indirectly (2, 11). Absence of all diastolic flow or of early diastolic flow in uterine artery blood flow waveforms has been associated with repeated failure of implantation in patients undergoing
FIGURE 3 Time-averaged mean of endometrial tissue blow flow (mETBF) in the fundus (F) and anterior (A), posterior (P), right-lateral (R), and left-lateral (L) walls of the endometrium in normal and abnormal uteri. * ⫽ P⬍.0001 vs. A/P/R/L group (ANOVA and Fisher’s PLSD).
Jinno. Endometrial blood flow and receptivity. Fertil Steril 2001.
FERTILITY & STERILITY威
IVF (12). Uterine artery PI and RI were significantly lower in women who conceived by IVF than in those who did not conceive (13–15). Significantly lower rates of implantation per transferred embryo (14) and pregnancy per cycle (14, 15) were observed in IVF patients with a uterine artery PI of 3.0 or more than those with a PI less than 3.0. Accumulating recent reports, however, have demonstrated similar PI values in pregnant and nonpregnant IVF/ICSI patients (16 –18), and a lack of statistically significant differences in rates of implantation and pregnancy between women with low (1.00 to 1.99), medium (2.00 to 2.99) and high (ⱖ3.00) PI (16, 18). Taken together, available data indicate a wide range of overlap in uterine artery PI values between pregnant and nonpregnant cycles, making clinical judgment concerning the uterine receptivity difficult in most cases (2, 11, 19). Only a remarkably high vascular impedance (PI ⱖ3.3 to 4.0 and/or absence of diastolic flow) may predict impaired implantation (2, 11, 19); such a high impedance is detected in only 9% to 26% of nonpregnant cycles (2, 15, 20). In the present study, mETBF, a novel parameter of endometrial blood perfusion, was a significant predictor of achievement of pregnancy by IVF/ICSI while, as in most previous reports, uterine artery PI was not. Several reasons may account for this. The mETBF is a direct measurement of blood flow volume in the endometrium, the actual site where implantation occurs. On the other hand, uterine artery PI is a parameter indirectly reflecting blood flow volume in the entire uterus, which includes blood supply not only to the endometrium but also to the myometrium and epimetrium. Moreover, collateral communication exists between uterine and ovarian blood vessels, which may further obscure the relationship between uterine artery PI and actual blood flow in the endometrium. Consistently, a poor correlation (r ⫽ 1173
0.24) has been demonstrated between PIs in the uterine and spiral arteries (11). We observed that 85% of gestational sacs in normal uteri were implanted in the fundic endometrium, which had the highest mETBF among uterine regions (see Fig. 3). Embryos may implant selectively at the endometrial site with most abundant blood flow, or alternatively, only embryos implanted at the endometrial site with the most blood flow can survive to the stage where a gestational sac is seen by ultrasonography. In any cases, abundant blood flow at the implantation site appears necessary for embryonic growth. This agrees with another of our present observations, that abnormal uteri with a tendency for impaired endometrial receptivity had deficient blood flow in the fundus. The unique ability of mETBF to provide site-specific evaluation of endometrial blood flow may contribute to functional diagnosis and decision making concerning surgical correction of abnormal uteri. VEGF plays a pivotal role in regulation of physiologic and pathologic angiogenesis (21) and also is believed to be a paracrine regulator of the effects of ovarian steroids on endometrial angiogenesis (3). During the menstrual cycle, the expression of VEGF mRNA and protein in the endometrium was greatest in the secretory phase (22, 23), a time when endometrium has the greatest angiogenic activity (24). Consistent with our present observation, strong immunoreactivity of VEGF was detected in the glandular epithelial cells of the secretory endometrium but weak immunoreactivity in stromal cells (22, 25). Our present study shows for the first time that stronger expression of endometrial VEGF is related with successful implantation, although a larger number of patients should be examined to confirm our observation. The role of endometrial VEGF in the implantation remains to be clarified.
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19.
ETBF reflects endometrial receptivity more closely than do conventional receptivity markers, including ultrasonographic parameters, serum hormone concentrations, and histologic date of endometrium. In addition, ETBF can be measured easily, quickly, and noninvasively in the outpatient clinic. The possibility that the unique capacity of ETBF for site-selective evaluation of endometrial blood flow can help in clinical management of abnormal uteri awaits further investigation. References
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Vol. 76, No. 6, December 2001