Department of Obstetrics and Gynaecology, Medical School University of Zagreb, Sveti Duh Hospital, Sveti Duh 64, HR-10000 Zagreb, Croatia
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Abstract |
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Key words: antral follicle number/ovarian stromal blood flow/ovarian volume/stromal area/three-dimensional ultrasound
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Introduction |
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To substantiate this observation, we undertook a three-dimensional (3D) ultrasound prospective study evaluating antral follicle number, ovarian volume measurements, stromal area and ovarian stromal blood flow after pituitary suppression. Pretreatment 3D ultrasound ovarian measurements were compared with subsequent ovulation induction parameters [peak estradiol (E2) on day of HCG administration and number of oocytes] and cycle outcome (fertilization and pregnancy rates).
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Materials and methods |
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The intra- and interobserver coefficients of variation were low (7 and 8% respectively). The study protocol was approved by the local ethics committee and written informed consent was obtained from all subjects.
All patients underwent a standard regime of the GnRH agonist. Triptorelin (Decapeptyl; Ferring, Kiel, Germany) was administered s.c. at a daily dose of 100 µg starting in the mid-luteal phase. After confirmation of pituitary down-regulation (no ovarian cysts >1 cm, endometrial thickness <5 mm and E2 <50 pg/ml) a transvaginal ultrasound scan was performed using an electronic 7.5 MHz transducer with 3D facility (Combison 530D; Kretztechnique, Zipf, Austria).
Using this method, three rotatable perpendicular planes were displayed for each ovary to obtain the largest dimensions. The data set was then stored digitally on an internal disk for subsequent analysis. This ability to store 3D data on a hard disk allowed us to shorten the examination time (25 min).
Detailed analysis of antral follicle number, ovarian volume and stromal area was performed after the patient had gone and lasted 1020 min. The stromal area was measured by meticulously outlining the region of interest. At the same time, the number of antral follicles was carefully obtained. Both measurements were summed up to obtain an individual's total stromal area and total antral follicle number. The volume of the ovary was measured by outlining the areas of multiple parallel sections at different distances from the ovary and was calculated using the trapezoid formula. At least 10 serial slices, 12 mm apart, were taken across the ovary for volume measurement. The actual volume was calculated by the built-in computer program. The volumetric data were then stored digitally on a hard disc (SyQuest; Technology Inc., Fremont, CA, USA) for subsequent analysis. Each ovarian volume was recorded and then summed to obtain an individual's total ovarian volume.
After B-mode analysis, power Doppler imaging was switched on together with the volume mode. To reduce the acquisition time, the volume of the colour box and sweep angle were reduced. The colour frame rate was adjusted as follows: both colour density and colour quality were as low as necessary to obtain a good image, whereas the pulsed repetition frequency was as high as possible to enable the display of targeted flow velocity. The spatial peak temporal average intensity was ~80 mW/cm2. Wall filters (50 Hz) were used to eliminate low frequency signals. The average patient examination time for 3D power Doppler sonography was 3 min. With the use of the lowest line density, the average acquisition time was 22 s (range 1232). At the end of each examination, combined colour and grey scale rendering was performed and quantitative analysis of the blood flow in the outlined area was achieved by implementing the colour histogram mode. The flow index (FI) reflects the intensity of blood flow and was calculated automatically by the built-in computer: FI = weighted colour values/colour values. Since no significant differences were noted between the two sides for ovarian stromal blood flow in any patient, the data were calculated together and the mean measurement of both sides in an individual patient was used for statistical analysis. The subsequent analysis of the power Doppler reformatted sections lasted 510 min.
The standard starting dose of FSH was 24 ampoules (150300 IU of FSH activity) depending on the patient age and basal serum FSH concentration. Follicular growth was monitored with serial ultrasound scans, and the dose of FSH was adjusted according to the follicular response. When the average diameters of the three leading follicles were at least 18 mm, as measured by ultrasound, 10 000 IU of HCG was administered as a single injection.
Transvaginal ultrasound-directed oocyte retrieval was performed ~36 h after HCG administration and embryo transfer took place 5 days after oocyte retrieval in the blastocyst stage. Progesterone pessaries (400 mg twice daily, Utrogestan; Laboratories Besins International, Paris, France) were given as luteal support, starting on the day of embryo transfer and continuing until 16 days thereafter.
Blood sampling for serum FSH and LH was performed on day 3 of the menstrual cycle, while E2 measurements were taken on the day of pituitary suppression and on HCG administration day. Serum FSH and LH levels were measured by microparticle enzyme immunoassay (Abbott AXSYM reagent pack; Abbott Diagnostics, Abbott Park, IL, USA) and serum E2 levels were assayed using a commercially available chemiluminescent immunoassay (Abbott). The intra- and inter-assay coefficients of variation were 4.2 and 7.2% for FSH, 4.5 and 7.5% for LH and 5.0 and 7.5% for E2 respectively.
All of the following outcome variables (total antral follicle number, total ovarian volume, total ovarian stromal area and mean FI of the ovarian stromal blood flow) were correlated with peak serum E2 concentration (measured in all patients on the day of HCG administration), number of oocytes retrieved, number of embryos resulting from the cycle and number of clinical pregnancies. Pregnancy was defined as the occurrence of a positive ß-HCG (>10 IU) value on day 12 after embryo transfer and a second higher value 2 days later. Only pregnancies reaching ß-HCG values >100 IU were considered for evaluation.
Statistical analysis
All data are expressed as median (range). Comparison between two outcomes (pregnant and non-pregnant) was carried out by the non-parametric MannWhitney test. A P-value (two-tailed) < 0.05 was considered statistically significant.
Correlation was assessed by the Spearman's rank method and correlation coefficients were determined for all measures and outcome variables. At the end of the study, logistic regression analysis was performed using the following predictors: age, E2 on day of HCG administration, total antral follicle number, total ovarian volume, total ovarian stromal area and mean ovarian stromal FI.
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Results |
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Spearman's correlation coefficients were determined for each measure and outcome variable. The total number of aspirated oocytes correlated well (P < 0.01) with total antral follicle number (r = 0.98), total ovarian volume (r = 0.89), total ovarian stromal area (r = 0.89) and mean ovarian stromal FI (r = 0.08) (Table II). Antral follicle count and stromal FI were found to have a high degree of interaction with one another. When we took into consideration the ovaries with a comparable number of follicles and stromal vascularity, we found the following results: patients with less than five antral follicles had a mean FI of 10.6, patients with an antral follicle count of 510 had a mean FI of 12.1, and patients with more than 10 antral follicles had a mean FI of 12.9. Therefore, patients with higher antral follicle count presented higher stromal vascularity due to higher need for perfusion.
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Discussion |
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Tomas et al. and Chang et al. were the first to report on prediction of the ovarian response by counting the number of antral follicles (Tomas et al., 1997; Chang et al., 1998
). In 166 women undergoing their first IVF cycle after pituitary down-regulation, it was concluded that the number of antral follicles present before ovarian stimulation was a better predictor of the ovarian response than the ovarian volume or age alone (Tomas et al., 1997
). The authors revealed that the number of antral follicles was correlated with the number of recovered oocytes, while the ovarian volume was correlated with the number of antral follicles before the stimulation, but not with the number of oocytes. Chang et al. studied 130 women in 149 IVF cycles after they failed six cycles of infertility treatment including ovarian stimulation with or without intrauterine insemination (Chang et al., 1998
). They found a highly significant correlation between the antral follicle number and the number of oocytes. A higher chance of cycle cancellation, a lower E2 concentration and a need for higher gonadotrophin dosage was detected in cycles with less than three antral follicles. Scheffer et al. evaluated antral follicle counts by transvaginal ultrasonography in relation to age in women with proven natural fertility (Scheffer et al., 1999
). They found that the antral follicle count showed the clearest correlation with age, and a mean yearly decline of 4.8% before the age of 37 years, compared with 11.7% thereafter. The number of small antral follicles in both ovaries, as measured by ultrasound, is clearly related to reproductive age and could well reflect the size of the remaining primordial follicle pool.
The second most important predictor of IVF outcome in our study was ovarian stromal FI. Zaidi et al. were the first to show that there was a relationship between ovarian stromal blood flow velocity and ovarian follicular response (Zaidi et al., 1995). They measured the ovarian stromal peak systolic velocity (PSV) in the early follicular phase and showed that poor responders had low ovarian blood flow PSV. Engmann et al. found that in patients with normal basal FSH concentrations, the mean ovarian stromal PSV on the day of pituitary suppression was a better predictor of ovarian responsiveness than age (Engmann et al., 1999
).
The main shortcoming of the previous studies on ovarian stromal blood flow is that they used PSV as a main predictor. The accurate measurement of blood flow velocity requires knowledge of the angle of insonation to the blood vessels analysed. Ovarian vessels inside the ovarian stroma are thin and tortuous and therefore it is impossible to obtain accurately the angle between the ultrasound beam and intra-ovarian vessels. This potential limitation leads to subjective measurements and depends on experience of the sonographer, since he/she should search for the highest velocity of the ovarian stromal vessels. Our data indicate that ovarian stromal blood flow obtained by 3D power Doppler imaging after pituitary suppression truly reflects total ovarian stromal blood flow. Using the colour histogram mode, we assessed the intensity of ovarian stromal blood flow, which seems to be predictive of increased delivery of gonadotrophins to target cells for stimulation of follicular growth. In addition to the fact that more oocytes are expected to be retrieved in aspiration procedures, better quality of the oocytes and embryos may lead to higher pregnancy rates.
Patients with a depleted ovarian reserve assessed by 3D ultrasound may require an increased starting dosage of gonadotrophins in order to improve follicular response (Ben-Rafael and Feldberg, 1993), although some studies failed to show any beneficial effect of increasing the dosage of gonadotrophins in poor responders (Stadmauer et al., 1994
; Laud et al., 1996
). We believe that some angiogenic factors could potentially be used for improving ovarian responsiveness and IVF outcome in patients with diminished ovarian blood flow. It is possible that deficient intra-ovarian vascularity may serve as the initial marker of reduced ovarian reserve which precedes an increased FSH level and reduction of the ovarian volume (Engmann et al., 1999
). If this is so, exogeneous angiogenic factors may be used to improve ovarian blood flow and prevent the onset of ovarian failure.
Syrop et al. demonstrated that ovarian volume, as determined by transvaginal ultrasonography, is another predictor of ovarian response to ovulation induction and clinical pregnancy rates (Syrop et al., 1995). Decreased total ovarian volume and the volume of the smallest ovary were significant variables predicting peak E2 and number of the oocytes and embryos. Lass et al. recommend that ovarian volume should be measured by transvaginal scan before ovulation induction in all patients, regardless of age, and the stimulation protocol planned accordingly (Lass et al., 1997
). They suggest that women who have a mean ovarian volume of <3 cm3 have a very high chance of failure to respond to exogenously applied stimuli. Wu et al. found that 3D ultrasound facilitates determination of the ovarian volume in patients with and without polycystic ovarian disease (Wu et al., 1998
). Furthermore, volume of the ovary assessed by 3D ultrasound correlates better with direct measurement of the surgical specimen than that obtained by 2D ultrasound (Bonilla-Musoles et al., 1995
; Kyei-Mensah et al., 1996
).
In our selected group of patients, total ovarian volume and total stromal area assessed by 3D ultrasound were less predictive of IVF outcome than total antral follicle number and ovarian stromal vascularity. It seems that some patients may have diminished ovarian reserves without evident changes in ovarian volume. Although ovarian volume and stromal area measurements are useful to distinguish between multifollicular and polycystic ovaries (Wu et al., 1998), they are less significant predictors of IVF outcome in patients with normal basal serum FSH and LH concentrations.
Some investigators (Gougeon, 1996; Pellicer et al., 1998
) suggest that the loss of resting follicles up to 30 years of age is due to atresia; thereafter, this loss is due principally to the entrance of resting follicles into the growth phase, with the decay rate of resting follicles accelerating from 38 years onward. If this hypothesis is correct, evaluation of the follicles in the growing phase may be an acceptable way to measure the decay rate of resting follicles. In this context, 3D ultrasound extends the boundaries of conventional ultrasonographic methods enabling more objective assessments of ovarian morphology and volume.
Using this method the examination time is short, and does not increase the patient's discomfort with serial ultrasound ovarian and endometrial monitoring. For the first time it becomes possible to assess pelvic structures of an infertile patient in three separate volume acquisitions (uterus, left and right adnexa). The total examination time is short and the patient can leave the examination room once the volumes have been stored and all further investigations can be performed without the presence of the patient. Retrospective evaluation of the ovarian measurements enables precise correlation with response to stimulating drugs and IVF outcome. In addition to ovarian measurements, endometrial volume measurements and assessment of endometrial perfusion by 3D power Doppler histogram on the day of embryo transfer could be used for determination of uterine receptivity (Kupesic et al., 2001). Therefore, it is believed that 3D ultrasound ovarian and endometrial measurements may become simple additional tests which could predict response to stimulating drugs, degree of endometrial receptivity and outcome of assisted reproductive techniques.
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Acknowledgements |
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Notes |
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References |
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Submitted on August 3, 2001; accepted on November 6, 2001.