Utility of color Doppler indices of dominant follicular

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1 Ultrasound Obstet Gynecol 2002; 20: Utility of color Doppler indices of dominant follicular Blackwell Science, Ltd blood flow for prediction of clinical factors in in vitro fertilization-embryo transfer cycles T. OZAKI*, K. HATA*, H. XIE*, K. TAKAHASHI* and K. MIYAZAKI* *Department of Obstetrics and Gynecology, Shimane Medical University, Izumo, Japan KEYWORDS: Color Doppler indices, Dominant follicle, IVF-ET, Peak systolic velocity ABSTRACT Objective To investigate the relationship between color Doppler indices of dominant follicular blood flow and clinical factors in in vitro fertilization-embryo transfer cycles. Subjects and methods This was a prospective study involving 26 patients completing a total of 33 in vitro fertilization cycles. Dominant follicular blood flow indices, peak systolic velocities, the resistance index and the pulsatility index were evaluated using transvaginal color Doppler. The indices were compared to the clinical outcomes of in vitro fertilizationembryo transfer. Results There was a significant correlation between dominant follicular peak systolic velocities and the number of oocytes retrieved, as well as the number of mature oocytes obtained. There was no significant correlation between dominant follicular resistance index or pulsatility index and the number of follicles > 10 mm in diameter, the number of oocytes retrieved or the number of mature oocytes. There were no significant differences between dominant follicular peak systolic velocities, resistance index or pulsatility index, and fertilization rate or the ratio of good quality embryos. However, significant differences were found between the number of oocytes retrieved, as well as the number of mature oocytes for those patients in which the peak systolic velocity was below 25 cm/s. Conclusions Doppler assessment of dominant follicle blood flow alone is useful for predicting the number of retrievable oocytes. However, morphological quality of the embryo produced or the pregnancy rate cannot be predicted by this method. INTRODUCTION The maturity of oocytes and the morphological quality of embryos are important factors determining the outcome of an in vitro fertilization (IVF) program. Programmed superovulation protocols now provide a convenient and effective means of scheduling and organizing a clinical IVF program, allowing oocyte retrievals to be performed on specific days of the week. A standard protocol that can be used to maximize the number of oocytes recovered uses a gonadotropin-releasing hormone (GnRH) agonist to down-regulate the endogenous hormone activity, followed by ovarian stimulation with human menopausal gonadotropin (hmg). Cycle monitoring during the stimulation protocol is very important for retrieval of multiple mature oocytes. Ultrasound assessment and measurement of plasma estradiol (E 2 ) levels are generally the methods used for cycle monitoring. Most IVF units use the diameter of the leading follicle as assessed by transvaginal ultrasound, with or without the concentration of plasma E 2, as an index of follicular maturity. However, the size of the follicle is an imprecise guide to follicular maturity 1. Recently, transvaginal ultrasonography with color and pulsed Doppler ultrasound has enabled the assessment of vascular changes in the ovary during the menstrual cycle 2 7. Many authors have observed that the color Doppler indices were correlated with oocyte recovery 8 10, fertilization rate 8, developmental potential of the oocyte 11,12, quality of the conceptus 8,13 and pregnancy rate 10,13. These investigators assessed the blood flow of all follicles individually. However, it is a time-consuming process to examine all follicles by color Doppler ultrasound. A more convenient method is necessary for cycle monitoring in the IVF clinic. The aim of the present study was to confirm the utility of dominant follicular blood flow assessment for predicting the retrievable number and maturity of all oocytes, morphological quality of embryos and pregnancy, after IVF-embryo transfer (ET). Correspondence: Dr Kohkichi Hata, Department of Obstetrics and Gynecology, Shimane Medical University, 89-1, Enya-cho, Izumo , Japan ( hata31@shimane-med.ac.jp) Accepted ORIGINAL PAPER

2 MATERIALS AND METHODS Patients Twenty-six patients with tubal infertility and a normal semen analysis were studied. All patients had completed their 33 IVF cycles in which embryo transfer was also performed during the period from 1999 to 2001 at Shimane Medical University Hospital. Patient mean (standard deviation (SD)) age was 32.4 (4.8) years (range, years). All patients had regular menstrual cycles of days. All patients had normal basal levels of serum luteinizing hormone (4.4 ± 0.5 miu/ ml), follicle stimulating hormone (FSH) (8.1 ± 0.9 miu/ml), E 2 (32.1 ± 2.6 pg/ml) and prolactin (17.6 ± 1.6 ng/ml). Treatment protocol for IVF-ET Multiple follicular stimulation, cycle monitoring and oocyte retrieval were performed using standard protocols. All women received a GnRH analog protocol of 400 µg/day of nafarelin acetate nasal spray (Nasanyl ; Yamanouchi, Tokyo, Japan) starting in the midluteal phase of the previous cycle. Gonadotropic stimulation of the ovaries was begun when the serum E 2 levels declined to < 30 pg/ml and the serum FSH levels declined to < 10 miu/ml. From day 1 to day 3 of ovarian stimulation, 150 or 300 IU of FSH (Fertinorm P ; Serono Japan, Tokyo, Japan) was administered. Beginning on day 4, 150 IU or 300 IU of hmg (Humegon ; Organon, Tokyo, Japan) was administered based on the ovarian response. The criteria for human chorionic gonadotropin (hcg) administration (HCG Mochida ; Mochida, Tokyo, Japan) was the presence of follicles > 18 mm in diameter and serum E 2 levels > 200 pg/ml per the number of follicles > 15 mm in diameter. Venous blood samples for E 2 levels were drawn on the day of hcg administration. Nafarelin treatment was discontinued on the day of hcg administration. Oocyte aspiration was performed with a needle (Hakko, Tokyo, Japan) under transvaginal ultrasonographic guidance 36 h after hcg administration. All oocyte cumulus complexes were assessed for morphological maturity (immature or mature) by noting the volume, density and condition of the surrounding coronal and cumulus cells 14,15. Three embryos, which cleaved into 4 8 blastomeres, were transferred to each subject on day 2 or 3. All embryos were assessed for morphological grade according to Veeck s pre-embryo grading system (from I, good to V, poor) 16 and good quality embryos were defined as grade I or II. An additional 50 mg of progesterone (Luteum, Teikoku, Tokyo, Japan) was administered by intramuscular injection on alternate days beginning on the day of follicular aspiration and 3000 IU of hcg (HCG Mochida ) was administered 3 and 7 days after oocyte aspiration. was always below 95 mw/cm 2. All scans were performed by the same operator and confirmed by a colleague. We first monitored follicles in women experiencing superovulation at stimulation day 6 or 7. We defined the dominant follicle as the largest follicle at the first follicular assessment. Subsequently, we reviewed follicular size daily to determine whether the dominant follicle was growing smoothly. The first examination with transvaginal color Doppler imaging and pulsed Doppler spectral analysis was performed on the day of, but before, hcg administration. Perifollicular blood flow of a dominant follicle was examined for the presence of color signals indicative of vascularity (Figure 1). Care was taken to ensure that signals assigned to a particular follicle were clearly separate from the walls of an adjoining follicle. A pulsed Doppler range gate was placed over the vessels of interest. Flow velocity waveforms that were obtained from those vessels were used for spectral analysis. The highest peak systolic velocity (PSV) was recorded from the flow velocity waveform. Blood flow impedance was expressed as the resistance index (RI) and the pulsatility index (PI). This was calculated from curves fitted to the flow velocity waveform over three cycles according to the formula: RI = (S D)/S and PI = (S D)/M, where S is the peak systolic shifted frequency, D is the maximum end-diastolic frequency and M is the time-averaged maximum frequency over the whole cardiac cycle. Venous blood samples for hormonal measurements were obtained on the day of hcg administration. All the samples were drawn close to the time of the ultrasound examination ( h) and serum levels of E 2 were measured. Appropriate university hospital committee permission was obtained for the sample-collection protocol. Assays Serum levels of hormones were measured using direct radioimmunoassays for E 2 (DPC Estradiol Kit, Diagnostic Products Corporation, Los Angeles, CA, USA). The intra-assay and interassay coefficients of variation were 5.0% and 4.5%, respectively. Serum E 2 levels are expressed in pg/ml. Transvaginal ultrasonography An Aloka color Doppler SSD 5000 scanner (Aloka, Tokyo, Japan) equipped with a 5-MHz frequency transvaginal transducer was used for blood flow analysis. The spatial peak temporal average intensity for B-mode and Doppler imaging Figure 1 Ultrasound image of blood flow around a dominant follicle. The flow velocity waveform from the vessel defined by the pulsed Doppler range gate is shown. Ultrasound in Obstetrics and Gynecology 593

3 Statistical analysis The data are expressed as the mean ± standard error (SE). The statistical analyses were carried out with the aid of a Macintosh computer using the Statview SE statistical program (Abacus Concepts Inc., Berkeley, CA, USA). The statistical significance of the differences between the clinical characteristics of the two groups of women was tested with the chi-square and Mann Whitney U-tests. The association between color Doppler indices and clinical factors of IVF-ET was assessed using linear regression analysis. P < 0.05 was considered statistically significant. RESULTS There was a significant correlation between dominant follicular PSV and the number of follicles > 10 mm (r = 0.94, P < 0.01), the number of oocytes retrieved (r = 0.73, P < 0.01) (Figure 2), as well as the number of mature oocytes (r = 0.72, P < 0.01) (Figure 3). However, there was no significant correlation between dominant follicular RI or PI and the number of follicles > 10 mm in diameter, the number of oocytes retrieved or the number of mature oocytes. There was a significant correlation between the number of oocytes retrieved and serum E 2 levels on the day of hcg administration (r = 0.65, P < 0.05). There was a significant correlation between dominant follicular PSV and serum E 2 levels on the day of hcg administration (r = 0.62, P < 0.01) (Figure 4). There were no significant differences between dominant follicular RI or PI and serum E 2 levels. There was no significant correlation between dominant follicular PSV, RI or PI and fertilization rate. There was no significant correlation between dominant follicular PSV, RI or PI and the ratio of good quality embryos. Figure 3 The association between the number of mature oocytes and peak systolic velocity of perifollicular blood flow of a dominant follicle on the day of, but before, human chorionic gonadotropin administration (r = 0.72, P < 0.01, n = 33). Figure 4 The association between serum estradiol levels and peak systolic velocity of perifollicular blood flow of a dominant follicle on the day of, but before, human chorionic gonadotropin administration (r = 0.62, P < 0.01, n = 33). Dominant follicular PSV was > 25 cm/s in 21 cycles. We found a significant correlation between the number of oocytes retrieved, or the number of mature oocytes, for those subjects whose PSV was below 25 cm/s. However, there were no significant differences in fertilization rate or the ratio of good quality embryos between the two groups (Table 1). Figure 2 The association between the number of retrieved oocytes and peak systolic velocity of perifollicular blood flow of a dominant follicle on the day of, but before, human chorionic gonadotropin administration (r = 0.73, P < 0.01, n = 33). DISCUSSION Previous studies have shown that follicular Doppler indices can predict the results after IVF-ET The PSVs of individual 594 Ultrasound in Obstetrics and Gynecology

4 Table 1 Comparison of clinical data and follicular peak systolic velocity (PSV) around dominant follicle PSV around dominant follicle (cm/s) 25 < 25 P Number of follicles > 10 mm 14.1 ± ± 1.1 < Number of retrieved oocytes 11.4 ± ± 0.7 < Number of mature oocytes 10.4 ± ± 0.6 < Fertilization rate 69.9 ± ± 6.0 NS Ratio of good quality embryos 59.9 ± ± 9.0 NS NS, not significant. follicles among women undergoing IVF have been shown to correlate with oocyte recovery 8,12, fertilization rate 9 development potential of the oocyte 11 and quality of the preimplantation embryo 8,13. However, to examine all follicles by color Doppler ultrasound is a time-consuming process. We consider the lengthy transvaginal examination to be stressful for patients. In the present study, we examined only dominant follicular blood flow and assessed whether this can replace the results of individual follicular examination. The results obtained demonstrate that the number of oocytes recovered and the number of mature oocytes were significantly correlated with the dominant follicular PSV. The results were comparable with previous studies 8. However, other factors, such as the fertilization rate and quality of the preimplantation embryo, did not correlate significantly with the dominant follicular PSV. All IVF cycles in this study were ovarian hyperstimulated cycles. The other mature follicles in the ovary, from which many oocytes could be retrieved, surrounded a dominant follicle. We considered that the dominant follicular blood flow could be affected by the other surrounding mature follicles and, consequently, be reflected in the number of retrieved oocytes. A previous study reported that the percentage of the thecal cell layer occupied by blood vessels in the dominant follicle was significantly greater than that of other smaller follicles 17. Thus, we considered that the dominant follicle is more sensitive to hmg, and the vasculature in the dominant follicle strongly reflects the stimulation with hmg. However, in order to predict the development potential of the retrieved oocyte, individual follicular assessment might be needed. In the present study, dominant follicular RI and PI had no correlation with oocyte retrieval and fertilization. A similar tendency was demonstrated in follicular examinations by other investigators 8,10. Nargund et al. 8 reported that, unlike PSV, measurement of follicular PI was not clinically useful. We believe that the lack of correlation of the dominant follicular vascular impedance with clinical outcomes in the IVF cycle reflects previous studies where follicular blood flow in individual follicles was assessed. Our results showed there was a significant correlation between the serum E 2 levels on the day of hcg administration in the follicular phase and dominant follicular Doppler PSV. From the results, we suggest angiogenesis around the dominant follicle might be affected by serum E 2 levels. However, we believe that angiogenesis around the developing follicle is affected not only by serum E 2, but also by other factors. Unfortunately, we were unable to investigate intrafollicular hormone/oxygenation or angiogenic factors of dominant follicles to substantiate the possible angiogenesis. The potential relevance of angiogenic factors in reproductive and ovarian physiology, and the mechanisms of angiogenesis, have been reviewed in detail elsewhere In summary, the dominant follicular PSV was significantly correlated with the number of retrieved oocytes, as well as the number of mature oocytes. From these results, we might be able to predict the number of retrieved oocytes by assessing Doppler blood flow of the dominant follicle alone. However, we could not predict morphological quality of the embryo produced, or the pregnancy rate. 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