Järvelä, Ilkka Y. MD; Sladkevicius, Povilas MD; Kelly, Simon MD; Ojha, Kamal MD; Campbell, Stuart MD; Nargund, Geeta MD
The ability of the ovary to respond to exogenous gonadotrophin stimulation and to produce several mature oocytes simultaneously is essential for successful in vitro fertilization (IVF). Ovarian responsiveness is highly variable and therefore difficult to predict. Transvaginal ultrasonography has proved to be an easy and noninvasive method to provide essential information on the ovarian responsiveness before the initiation of gonadotrophin stimulation.1–5 Ovarian volume as determined by trans-vaginal ultrasonography seems to correlate with the ovarian reserve. Lass et al6 observed that small ovaries are associated with poor response to human menopausal gonadotropin and a very high cancellation rate during IVF. Similarly, the number of antral follicles seems to correlate with the response to gonadotrophin stimulation.2–5 A very low number of antral follicles seems to be associated with poor response and a high cancellation rate,2–5 whereas a high number of antral follicles seems to predict not only good response but also sometimes an increased risk for ovarian hyperstimulation syndrome.4 It is possible to classify the ovaries according to ultrasonographic features as inactive or active, even before the initiation of gonadotrophin stimulation.2
It seems that follicular blood flow plays a major role during the growth and development of the follicle containing the oocyte.7–11 The follicle acquires a vascular sheet of its own at the antral stage.12 Combining the color Doppler facility in ultrasonography has enabled the detection and measurement of the follicular blood flow. According to two-dimensional color Doppler studies, peak systolic velocity of individual follicles on the day of human chorionic gonadotropin (hCG) injection and egg collection correlates with oocyte recovery,7,8 development potential of the oocyte,10 quality of the embryo,7,9 and even with the pregnancy rate during IVF therapy.11 High stromal peak systolic velocity or low resistance index before the initiation of gonadotrophin stimulation seems to be associated with retrieval of a higher numbers of oocytes.13–15
In two-dimensional color Doppler studies, the information concerning the vascularization and blood flow in the organ is obtained from a single artery lying in a two-dimensional plane, which is subjectively chosen by the researcher. To accurately measure the blood flow velocity, the angle of insonation to the blood vessels should be known. In the ovary the arteries are thin and tortuous, which makes the measurement difficult. A recent technical achievement, three-dimensional power Doppler ultrasonography, is less angle-dependent and enables the mapping and quantifying of the power Doppler signal within the entire volume of interest, basically making it possible to detect the total vascularization and blood flow in the organ.16–22
The aim of the present study was to evaluate whether quantification of power Doppler signals within the entire ovary as obtained with three-dimensional ultrasonography could predict the response to gonadotrophin stimulation in terms of the number of oocytes retrieved and the pregnancy rate. Additionally, the aim was to evaluate the possible differences in three-dimensional power Doppler measurements in ovaries with various responses to stimulation during the IVF treatment cycle.
MATERIALS AND METHODS
We examined 45 consecutive patients attending the Assisted Conception Unit at St. George's Hospital Medical School for IVF treatment between August 2000 and June 2001. Ethical approval was obtained from the ethics committee of the medical school, and each subject gave written informed consent before participating in the study. Women with polycystic ovaries, endometrioma, single ovary, salpingectomy, ovarian cystectomy, or uterine fibroids were excluded from the study. Each patient had her follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels measured between days 1 and 5 of the cycles during the 6 months preceding the study. Only patients with regular menstruation cycle were involved. Some of the patients had also participated in previous studies concerning the three-dimensional ultrasonographic features of the ovaries during the late follicular phase18,19 and the effect of pituitary downregulation.23
A standard long gonadotropin-releasing hormone (GnRH) agonist protocol for IVF therapy was used. Pituitary suppression was obtained with GnRH agonist treatment (buserelin acetate 0.5 mg subcutaneously daily), which was started on cycle day 21 if no pathology in the uterus or ovaries could be observed. Two weeks later, the patient was scanned, and if an endometrium of less than 4 mm and ovaries with only small antral follicles (diameter of 2–5 mm) were detected, the stimulation of ovaries with FSH was initiated. Otherwise the patient continued using GnRH agonist and was scanned again 1 week later. The standard starting dose of FSH was 150–375 IU, depending on the patient's age, previous response, and early follicular phase serum FSH levels. Monitoring of follicular growth was achieved with serial ultrasound scans, and the dose of FSH was adjusted according to follicular response. When two or three leading follicles were at least 18 mm in diameter, hCG injection was administered. If the response to FSH stimulation was low (total number of follicles four or less), the cycle was either converted to intrauterine insemination (if the tubes were patient) or abandoned. The three-dimensional ultrasonographic measurements were performed on the day that pituitary suppression was confirmed and on the day of hCG administration. Clinical pregnancy was determined as a positive pregnancy test and intrauterine gestational sac 3 weeks after embryo transfer.
According to the ultrasonographic appearance of the ovaries after pituitary suppression, the patients were divided into two groups: one with low ovarian reserve and the other with normal ovarian reserve. If the total sum of antral follicles with a mean diameter of 2–5 mm in both ovaries was less than five, the patient was categorized as having low ovarian reserve; if the sum was five or more, the patient had normal ovarian reserve.2
The three-dimensional power Doppler ultrasonographic examinations were performed with Kretz Combison 530D Voluson (Kretztechnik-Medison, Zipf, Austria). The volume acquisition and data analysis has been described in detail in earlier publications.18,19,23,24 In addition to volume assessment and mean grey value, three indices quantifying the power Doppler signal were determined (Figure 1): vascularization index, flow index, and vascularization flow index.
Vascularization index measures the ratio of color voxels to all the voxels in the defined volume, represents the vessels in the tissue, and is expressed as a percentage. Flow index, the mean value of the color voxels, represents the average intensity of flow (range 0–100). Vascularization flow index is the mean color value in all the voxels in the defined volume and is a feature of both vascularization and flow (range 0–100). The mean grey value in the grey voxels expresses the mean echogenicity or brightness of the defined volume (range 0–100).
We have previously assessed the intraobserver and interobserver variability in ovarian volume, color index, and grey index measurements by transvaginal three-dimensional power Doppler ultrasonography.24 The intraobserver correlation coefficient for volume measurements was 1.0, for vascularization index 0.89, for flow index 0.82, for vascularization flow index 0.90, and for mean gray value 0.91. The size of the study was estimated according to possible differences in vascularization index between the groups. According to our earlier results concerning reproducibility,24 the difference in vascularization index was set to 5% (estimated standard deviation [SD] = 4), which with 80% power gave a group size of 11 patients.
Statistical analysis was performed with SPSS 11.5 (SPSS Inc., Chicago, IL). Departure from a normal distribution was assessed with the Kolmogorov-Smirnov test. Paired t tests were used for normally distributed data and the Wilcoxon signed-rank test for skewed data. Correlation was estimated with Pearson correlation co-efficient. Proportions were compared by χ2 test. P < .05 was considered significant. All values given are means (SD).
According to the criteria used, 12 of 45 patients studied were classified as having low ovarian reserve and the remaining 33 as having normal ovarian reserve. The groups did not differ from each other in terms of age or pretreatment FSH and LH levels (Table 1).
The total gonadotrophin dosage required during ovarian stimulation was equal in the groups (Table 1). Six of 12 cycles in the low-ovarian-reserve group and one of 33 cycles in the normal-ovarian-reserve group were abandoned or converted into intrauterine insemination. The mean number of oocytes collected was 6.7 in the low-ovarian-reserve group and 11.1 in the normal-ovarian-reserve group, the difference not being statistically significant. In the cycles in which the oocyte retrieval and embryo transfer were performed, the number of embryos transferred was equal between the groups. No statistical difference in the clinical pregnancy rate was detected (Table 1).
Because no differences in any of the three-dimensional parameters could be detected between right and left ovary, the mean values were used for subsequent analysis.
Scanning on the day of pituitary downregulation showed that low- and normal-ovarian-reserve groups did not differ from each other in terms of ovarian volume, vascularization index, flow index, vascularization flow index, or mean gray value (Table 2).
Gonadotrophin stimulation induced a statistically significant increase in the ovarian volume and decrease in the mean gray value level (Table 2). In the normal-ovarian-reserve group, there was an increase in the vascularization index, flow index, and vascularization flow index values, but no statistically significant rise was demonstrated in low-ovarian-reserve group.
After stimulation, the ovaries in the low-ovarian-reserve group were smaller and had lower vascularization index, flow index, and vascularization flow index levels than the ones in the normal-ovarian-reserve group (Table 2). No differences could be detected in the mean gray value between the groups.
The number of oocytes retrieved correlated with antral follicle count (R = .458, P = .004), with ovarian volumes both after pituitary downregulation (R = .388, P < .016) and after gonadotrophin stimulation (R = .649, P < .001). Patient age did not correlate with antral follicle count, ovarian volume, or any other of the three-dimensional indices. The antral follicle count correlated with ovarian volume (R = .545, P < .001) after pituitary downregulation, and volume (R = .598, P < .001), vascularization index (R = .314, P = .043), flow index (R = .447, P = .003), and vascularization flow index (R = .316, P = .042) after gonadotrophin stimulation.
Comparison between those conceiving and those not conceiving revealed that the groups did not differ from each other after pituitary suppression (Table 3). After gonadotrophin stimulation, the vascularization index and the vascularization flow index were higher in the conceiving group. No differences existed in the number of antral follicles, oocytes retrieved, or embryos transferred (Table 3).
The aim in the present study was to evaluate the impact of three-dimensional power Doppler ultrasonographic analysis of ovaries after pituitary suppression to predict the ovarian response during IVF therapy. The main response was chosen to be the number of oocytes retrieved and pregnancy achieved. We also evaluated the effect of gonadotrophin stimulation on the ovarian volume and vascularization during IVF therapy in two groups differing from each other by the antral follicle count after pituitary suppression.
According to our results, after pituitary downregulation there were two ultrasonographic parameters that predicted the ovarian stimulation response in terms of number of oocytes retrieved. The parameters were the total antral follicle count and the mean ovarian volume. After gonadotrophin stimulation, the ovarian volume correlated with the number of oocytes subsequently. None of the indices quantifying the power Doppler signal in the ovary was able to predict the response.
Our results confirm the earlier finding that antral follicle count can be used to predict the ovarian response to gonadotrophin stimulation.2–5 In our study, 50% of cycles in the low-ovarian-reserve group were abandoned or converted into intrauterine insemination, which is consistent with reports from Ng et al4 and Chang et al,3 who found a higher cycle cancellation rate in patients with low antral follicle count.
Ovarian volume proved to be a significant factor in predicting the ovarian response. Earlier studies in which two-dimensional ultrasonography was used have shown that total ovarian volume is a significant predictor of the success in assisted reproductive techniques.6,25 Recent studies with three-dimensional ultrasonography have confirmed the finding.20,22 Small volume seems to reflect the diminished capacity of the ovary to respond to gonadotrophin stimulation and is associated with a high cancellation rate during IVF.6
The indices quantifying the power Doppler signal in the ovaries after pituitary suppression did not correlate with the number of oocytes retrieved, which suggests that these indices cannot be used as predictors to gonadotrophin stimulation. Earlier studies with two-dimensional color Doppler have shown that high stromal peak systolic velocity or low resistance index before the initiation of gonadotrophin stimulation is associated with higher numbers of oocytes being retrieved.13–15 Kupesic et al,20,22 using three-dimensional power Doppler ultrasonography, observed that the mean flow index—which represents the average color intensity of the color voxels—after pituitary suppression correlated with the number of oocytes retrieved and was even predictable for IVF outcome in terms of conception. They proposed that the intensity of ovarian stromal blood flow seems to predict increased delivery of gonadotropins to target cells for stimulation of follicular growth.22 In their study, the determination of flow index was restricted to part of the ovarian stroma and vascularization index, vascularization flow index, and mean gray value were not assessed.20 Our results are not in accordance with the earlier two-dimensional and three-dimensional Doppler studies, probably because in our study the power Doppler signal was quantified for the first time within the entire ovary and not only in a subjectively chosen vessel or restricted volume.
Gonadotrophin stimulation induced an increase in the ovarian volume and a decrease in mean gray value in both groups. The growth of several follicles at the same time enlarges the total ovarian volume, causing a concomitant decrease in mean gray value because the gray-scale voxel value for the follicular fluid is low. We have previously shown that mean gray value is lower in the dominant ovary carrying the leading follicle compared with the nondominant ovary,19 which is consistent with the present finding.
We observed an increase in ovarian vascularization index, flow index, and vascularization flow index after gonadotrophin stimulation in patients with normal ovarian reserve. No such change took place in patients with low ovarian reserve. It seems that the ovaries with a low count of antral follicles are incapable of significantly increasing the blood flow in response to gonadotrophin stimulation, or even if this increase occurred it was not strong enough to be detected by the methods described. According to our results, the number of antral follicles correlated with the vascularization index, flow index, and vascularization flow index values after gonadotrophin stimulation, which suggests that the increase in vascularization and blood flow originates mainly from the individual follicles. The discrimination between stromal and follicular vascularization in a stimulated ovary is troublesome, and therefore the proportion of increase in stromal vascularization is difficult to estimate with the method we used here. Earlier two-dimensional color Doppler studies have shown that gonadotrophin stimulation seems to induce a rise in stromal blood flow velocity, which is accompanied by a concomitant increase in serum vascular endothelial growth factor concentration.26,27 Vascular endothelial growth factor is an endothelial cell mitogen with potent angiogenic properties, and it has been shown to correlate with perifollicular blood flow as measured by two-dimensional color and pulsed Doppler ultrasonography.10 It might be that normal ovaries respond to gonadotrophin stimulation by increasing vascular endothelial growth factor production, which is not as apparent in the ovaries with low antral follicle count.
In relation to clinical pregnancy, the patients who conceived had higher vascularization index and vascularization flow index levels after gonadotrophin stimulation than the nonconceiving patients, which suggests increased vascularization in the ovaries. No differences existed in the number of antral follicles, oocytes retrieved, or embryos transferred between the groups. It might be that the oocytes collected in the conceiving cycles were of better quality, because it has been shown that adequate blood supply to the developing follicles is essential in producing chromosomally intact oocytes.10 It is possible that better-quality oocytes have resulted in better-quality embryos and finally in conception.
According to our results, the use of three-dimensional power Doppler ultrasonography showed that antral follicle count and ovarian volume seem to predict the ovarian responsiveness to gonadotrophin stimulation during IVF treatment. We could not, however, confirm that indices quantifying the power Doppler signals after pituitary downregulation would be of any value when estimating the ovarian response. In addition, it seems that gonadotrophin stimulation–induced change in ovarian vascularization and blood flow is related to the antral follicle count and might even have an impact in terms of conception.
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