Assessment of changes in volume and vascularity of the ovaries during the normal menstrual cycle using three-dimensional power Doppler ultrasound

Ligita Jokubkiene, Povilas Sladkevicius, Linas Rovas, Lil Valentin, Assessment of changes in volume and vascularity of the ovaries during the normal menstrual cycle using three-dimensional power Doppler ultrasound, Human Reproduction, Volume 21, Issue 10, 1 October 2006, Pages 2661–2668, https://doi.org/10.1093/humrep/del211

Abstract

BACKGROUND: Our aim was to describe changes in the volume and vascularization of both ovaries, the dominant follicle and the corpus luteum during the normal menstrual cycle using three-dimensional (3D) power Doppler ultrasound. METHODS: Fourteen healthy volunteers underwent serial transvaginal 3D ultrasound examinations of both ovaries on cycle day 2, 3 or 4, then daily from cycle day 9 until follicular rupture and 1, 2, 5, 7 and 12 days after follicular rupture. The volume and vascular indices of the ovaries, the dominant follicle and the corpus luteum were calculated off-line using virtual organ computer-aided analysis (VOCAL TM ) software. RESULTS: The volume of the dominant ovary increased during the follicular phase, decreased after follicular rupture and then increased again during the luteal phase. Vascular indices in the dominant ovary and the dominant follicle/corpus luteum increased during the follicular phase, the vascular flow index (VFI) in the dominant follicle being on average (median) 1.7 times higher on the day before ovulation than 4 days before ovulation (P = 0.003). The vascular indices continued to rise after follicular rupture so that VFI in the corpus luteum was on average (median) 3.1 times higher 7 days after ovulation than in the follicle on the day before ovulation (P = 0.0002). The volume and vascular indices in the non-dominant ovary manifested no unequivocal changes during the menstrual cycle. CONCLUSIONS: Substantial changes occur in volume and vascularization of the dominant ovary during the normal menstrual cycle. 3D power Doppler ultrasound may become a useful tool for assessing pathological changes in the ovaries, for example, in subfertile patients.

Introduction

The ovary is a well-vascularized organ. Angiogenesis is an essential factor for the growth and regression of follicles and the corpus luteum ( Suzuki et al., 1998; Hazzard and Stouffer, 2000). Therefore, the investigation of vascular changes in the ovaries, follicles and corpus luteum may yield important information on normal and pathologic ovarian conditions.

Using ultrasound, we can see changes in the morphology of the ovary, and Doppler ultrasound enables us to estimate vascular changes. Vascular changes as assessed by colour and spectral Doppler ultrasound have been described in the uterus and ovaries during the normal menstrual cycle ( Scholtes et al., 1989; Sladkevicius et al., 1993, 1994; Bourne et al., 1996; Tan et al., 1996) and in the corpus luteum in early pregnancy ( Valentin et al., 1996). The introduction of three-dimensional (3D) power Doppler ultrasound has opened a possibility to assess vascularization in the whole volume of an organ, e.g. the ovary. 3D power Doppler has been used to quantify vascularity in the ovary in the late follicular phase ( Jarvela et al., 2002), to study changes in blood flow in the ovarian stroma with age ( Pan et al., 2002a) and to compare intraovarian vascularization in polycystic ovaries with that in normal ovaries ( Pan et al., 2002b). All published studies involving 3D power Doppler ultrasound examination of the ovaries are cross-sectional ( Jarvela et al., 2002; Pan et al., 2002a,b). To the best of our knowledge, there are no published studies where 3D power Doppler ultrasound has been used to study changes in volume and vascularity in the ovary or follicle/corpus luteum longitudinally during a whole normal menstrual cycle.

The aim of our study is to describe changes in the volume and vascularity of the ovaries, the dominant follicle and corpus luteum during the normal menstrual cycle using 3D power Doppler ultrasound.

Materials and methods

Subjects

The study protocol was approved by the Ethics Committee of the Medical Faculty of Lund University, Sweden. Informed consent was obtained from all participants after the nature of the procedures had been fully explained.

Sixteen healthy volunteers with regular menstrual cycles participated in our study. The inclusion criteria were age 20–45 years, regular menstrual cycles of 26–32 days with 3–8 days of menstrual bleeding, no more than mild menstrual pain and no cycle disturbances for at least 1 year; no hormonal contraception for the last 2 months; no intrauterine contraceptive device; no previous major gynaecological surgery; normal ovaries at a baseline scan and normal baseline values of FSH and estradiol (E₂) on cycle day 2, 3 or 4. E₂ and FSH were analysed before inclusion by a competitive immunoassay method using Technicon Immuno 1® System (Bayer Corporation, Tarrytown, NY, USA) or UniCel™ DxI 800, Beckman Access® Immunoassay system (Beckman-Coulter, Chaska, MI, USA), with normal baseline values of E₂ being 100–250 and g for 10 min. The serum was separated and frozen at −20°C until analysis in one batch. LH was measured with a chemiluminescent sandwich immunoassay method and progesterone with a chemiluminescent one-step competitive immunoassay. Both analyses were made using UniCel™ DxI 800, Beckman Access® Immunoassay system.

Equipment

All data were acquired using a GE Voluson 730 Expert ultrasound system (GE Healthcare, Zipf, Austria) equipped with a 2.8–10 MHz transvaginal transducer. Identical fixed pre-installed power Doppler ultrasound settings were used in all women: frequency 3–9 MHz, pulse repetition frequency 0.6 kHz, gain −4.0 and wall motion filter ‘low 1’ (40 Hz at pulse repetition frequency 0.6 kHz).

Study design

This is a prospective longitudinal study. All women underwent a baseline transvaginal ultrasound examination on cycle day 2, 3 or 4, then daily from cycle day 9 until follicular rupture and 1, 2, 5, 7 and 12 days after follicular rupture. The examinations were carried out in the late afternoon (4–6 pm) by one of the three examiners (P.S., L.R. and L.J.), the same examiner performing all examinations in the same woman. The results are expressed in relation to presumed ovulation: the last day when the dominant follicle was visible on the ultrasound screen was called day −1, and the following day when a corpus luteum was visible was called day +1. Thus, ovulation was presumed to have occurred between examination days −1 and +1. Sonographic features confirming ovulation were the disappearance of a follicle >15 mm in diameter identified the previous day and the visualization of a corpus luteum instead ( Ritchie, 1986; Hanna et al., 1994). Only those cycles were included where LH peaked on day −1 and where serum progesterone levels were adequate on day +7. The ovary bearing the ovulating follicle/corpus luteum was called the dominant ovary and the contralateral ovary the non-dominant ovary. We called the ovulating follicle the dominant follicle.

The women were examined in the lithotomy position with an empty bladder. The 3D ultrasound probe was introduced into the vagina. Once a satisfactory longitudinal view of the ovary had been obtained, the ovary was centralized within the 3D sector on the screen, and the ultrasound machine was switched into the power Doppler mode. Then, the 3D ultrasound mode was switched on. The woman was asked to remain as still as possible, and a 3D power Doppler data set of the ovary was acquired. The resultant multiplanar display was examined to ensure that the whole ovary had been captured in the volume. Volumes of satisfactory quality and with no artefacts were stored on a hard disk for future analysis.

All analyses of stored ultrasound volumes were done off-line by the first author (L.J.) using a personal computer. The virtual organ computer-aided analysis (VOCAL™) imaging program was used to calculate the volume and vascularity indices of both ovaries and the dominant follicle/corpus luteum. The acquired volumes yielded multiplanar views of the ovaries in the mid-sagittal, transverse and coronal planes. All calculations were done on these multiplanar images. The longitudinal view was used as the reference image. The rotation steps were 30°, resulting in the definition of six contours of the ovary. Ovarian contours were manually drawn in all six sections using the computer mouse. Once all contours had been drawn, the volume of the ovary was calculated automatically. Using the histogram facility of VOCAL™ software, three vascular indices were generated: vascularization index (VI), flow index (FI) and vascularization flow index (VFI). VI is the ratio of colour voxels to all voxels in the region of interest expressed as a percentage. It reflects the density of vessels in the volume analysed. FI is the mean intensity of all the power Doppler voxels in the volume (the sum of weighted colour voxels divided by the number of all colour voxels in the region of interest), and it reflects the number of blood corpuscles flowing in the vessels—i.e. the more blood corpuscles the higher the FI values. VFI is the sum of weighted colour Doppler voxels divided by all voxels in the region of interest. It reflects both the density of vessels and the number of blood corpuscles flowing in the vessels of the volume analysed (Parleitner et al., 1999).

The analysis of the dominant follicle started when it was ≥15 mm in diameter (mean of three orthogonal diameters). The contours of the dominant follicle were drawn manually following its inner wall. The contours were drawn in six sections, wherefrom the volume of the follicle was calculated automatically as described earlier for volume calculation of the whole ovary. Then, a 2-mm shell outside and parallel to the manually drawn follicular contour was generated automatically by the VOCAL™ software. A 2-mm shell was chosen to cover the whole ultrasonographically visible vascularized wall of the dominant follicle. The sum of the volume of the follicle and that of the shell was considered to represent the volume of the dominant follicle. Vascular indices of the dominant follicle were calculated in the 2-mm shell of the follicle (Figure 1a). The contours of the corpus luteum were drawn manually following the outer contour of the thick colour ring surrounding it. Vascular indices of the corpus luteum were calculated in a 3-mm shell automatically generated by the VOCAL™ software inside the manually drawn contour of the corpus luteum (Figure 1b). The 3‐mm shell was chosen to cover the whole colour ring surrounding the corpus luteum. We assumed the vascular indices in the shell of the dominant follicle/corpus luteum to reflect the vascularization of the whole follicle/corpus luteum.

Multiplanar display of the dominant ovary obtained by three-dimensional ultrasound (a) 1 day before follicular rupture (day −1) and (b) 7 days after follicular rupture (day +7). Both images (a) and (b) are from the same woman. Longitudinal section through the dominant ovary is shown in the upper left quadrant, transverse section is shown in the upper right quadrant and coronal section in the lower left quadrant. The resultant vascular tree of the dominant follicle/corpus luteum is shown in the lower right quadrant.

Reproducibility of measurements of volume and vascular indices

To determine the intra-observer reproducibility of the calculation of volumes and flow indices, one observer (L.J.) analysed the same volume twice ∼48 h apart, being unaware of the results of the first analysis when performing the second one. To determine the inter-observer reproducibility, a second observer (P.S.)—unaware of the results of the first observer—analysed once the same volumes as the first observer. The results of the second observer were compared with those of the first analysis of the first observer. The volumes to be analysed in the reproducibility study were selected from our datasheet as follows: each woman contributed one volume from her non-dominant ovary and another from her dominant ovary, the volumes being selected so that each examination day (–12 to +12) was represented in the study sample; each woman also contributed two or three volumes of their dominant follicle and two or three volumes of their corpus luteum so that each day of the follicular phase (days −5 to −1) and each day of the luteal phase (days +1 to +12) were represented in the study sample.

Statistical analysis

The intra-observer reproducibility was expressed as the difference between two measurement results obtained by the same observer. The differences between measured values were plotted against the mean of the two measurements to assess the relationship between the difference and the magnitude of the measurements ( Bland and Altman, 1986). The limits of agreement (mean difference ± 2 SD) were calculated as described by Bland and Altman (1986). Systematic bias between the first and second analyses was determined by calculating the 95% confidence interval (CI) of the mean difference (mean difference ± 2 SEM). If zero lay within this interval, no bias was assumed to exist between the first and second measurements. The inter-observer reproducibility was calculated using the same methods as described earlier for the intra-observer reproducibility. The intra- and inter-observer reproducibilities were also expressed as intra- and inter-class correlation coefficients (intra- and inter-CCs) ( Shrout and Fleiss, 1979), variance components being estimated using two-way random analysis of variance (absolute agreement).

Possible correlations were assessed using Spearman’s rank correlation coefficient. The statistical significance of differences in results between cycle days was determined using Wilcoxon-signed rank test. Two-tailed P-values are given, a P-value

Statistical calculations were performed using StatView™, version 5 (SAS Institute, Berkely, CA, USA, 1999) except for the calculations of inter- and intra-CCs, the latter being calculated using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA, version 12.0.1, 2003).

Results

Two women were excluded from our study—one because of unruptured follicle and the other because of short luteal phase and low serum progesterone in the mid-luteal phase. The 14 menstrual cycles included were normal, with normal baseline E₂ and FSH values (median E₂ 115 pmol/l, range 75–166 and 134 pmol/l, range 125–247; median FSH 4.45 IU/l, range 4–8.2 and 8.4 IU/l, range 4.9–9.4, depending on the method used), sonographic criteria of ovulation (follicle ≥15 mm replaced by a corpus luteum), an LH peak on day −1 and adequate serum progesterone levels on day +7 (median LH at peak 45.8 IU/l, range 23.6–106.8; median mid-luteal serum progesterone 44.8 nmol/l, range 30–80.9). The mean age of the 14 women studied was 28 ± 5.2 years (range 24–44). Nine of them were nulliparous.

Reproducibility results are summarized in Tables I and II. Neither intra-observer nor inter-observer differences changed with the magnitude of measurement values. There were no systematic differences between the first and second measurements of the first observer with two exceptions: slightly higher values for the volume and FI of the shell of the corpus luteum were obtained in the second analysis. Mean intra-observer differences were small, limits of agreement were narrow and all intra-CC values were ≥0.94. There were no systematic differences between the two observers with three exceptions: the second observer obtained slightly smaller follicular shell volumes and slightly higher VI values and slightly lower FI values in the shell of the corpus luteum than the first observer. All inter-CC values were high (one being 0.77, all the others being >0.90).

Intra-observer reproducibility of the analysis of volume and vascular indices of the ovaries, the dominant follicle and corpus luteum

CI, confidence interval; FI, flow index; intra-CC, intra-class correlation coefficient; VFI, vascularization flow index; VI, vascularization index.

Intra-observer reproducibility of the analysis of volume and vascular indices of the ovaries, the dominant follicle and corpus luteum

CI, confidence interval; FI, flow index; intra-CC, intra-class correlation coefficient; VFI, vascularization flow index; VI, vascularization index.

Inter-observer reproducibility of analysis of volume and vascular indices of the ovaries, the dominant follicle and corpus luteum

CI, confidence interval; FI, flow index; inter-CC, inter-class correlation coefficient; VFI, vascularization flow index; VI, vascularization index.

Inter-observer reproducibility of analysis of volume and vascular indices of the ovaries, the dominant follicle and corpus luteum

CI, confidence interval; FI, flow index; inter-CC, inter-class correlation coefficient; VFI, vascularization flow index; VI, vascularization index.

Volume changes in the dominant ovary and dominant follicle/corpus luteum were virtually identical in all women. The volume increased during the follicular phase, decreased immediately after follicular rupture and then increased again to remain unchanged from 2 to 12 days after follicular rupture (Figures 2 and 3, Table III). The volume of the non-dominant ovary did not change during the menstrual cycle (Figure 2).

Changes in the volume of the dominant and non-dominant ovary during the menstrual cycle. The filled diamonds represent the dominant ovary and the open triangles the non-dominant ovary. The open triangles are shown slightly to the right of the filled diamonds, but measurements were taken on the same day in both ovaries. Median, 10th and 90th percentiles are shown. The figures in brackets denote the number of women examined—the upper row for the dominant ovary and the lower for the non-dominant ovary. Days −12 to −1 represent the number of days before follicular rupture, and days +1 to +12 represent the number of days after follicular rupture. Because of differences in the length of the follicular phase, day −12 represents days −17 to −9.

Changes in the volume of the dominant follicle/corpus luteum during the menstrual cycle. (a) Median, 10th and 90th percentiles are shown. The figures in brackets denote the number of women examined. (b) Individual curves demonstrating that the pattern of change was virtually identical in all women. Days −5 to −1 represent the number of days before follicular rupture, and days +1 to +12 represent the number of days after follicular rupture.

Changes in volume and vascular indices in the dominant ovary and the dominant follicle/corpus luteum during the normal menstrual cycle

Comparison of results between days −4 and −1.

Comparison of results between days −1 and +7.

Changes in volume and vascular indices in the dominant ovary and the dominant follicle/corpus luteum during the normal menstrual cycle

Comparison of results between days −4 and −1.

Comparison of results between days −1 and +7.

The individual patterns of change in vascular indices of the dominant ovary and dominant follicle/corpus luteum were less uniform than the volume changes of the dominant ovary, but the following patterns were discerned in all women: the vascular indices in the dominant ovary and dominant follicle increased during the follicular phase. For example, VFI in the dominant follicle was on average 1.7 times higher on day −1 than on day −4. Blood flow indices continued to rise after follicular rupture so that indices were higher in the luteal phase than in the follicular phase. For example, VFI in the corpus luteum on day +7 was on average 3.1 times higher than that in the dominant follicle on day −1. Blood flow indices in the dominant ovary and corpus luteum did not differ significantly between days +7 and +12. In the non-dominant ovary, blood flow indices manifested no unequivocal changes during the menstrual cycle. Changes in blood flow indices in the ovaries, the dominant follicle and corpus luteum are shown in Figures 4 and 5 and in Table III.

Changes in (a) vascularization index (VI), (b) flow index (FI) and (c) vascularization flow index (VFI) in the dominant (filled diamonds) and non-dominant (open triangle) ovary during the menstrual cycle. The open triangles are shown slightly to the right of the filled diamonds, but measurements were taken on the same day in both ovaries. Median, 10th and 90th percentiles are shown. The figures in brackets denote the number of women examined—the upper row for the dominant ovary and the lower for the non-dominant ovary. Days −12 to −1 represent the number of days before follicular rupture, and days +1 to +12 represent the number of days after follicular rupture. Because of differences in the length of the follicular phase, day −12 represents days −17 to −9.

Changes in vascular indices in the dominant follicle/corpus luteum during the menstrual cycle. The filled diamonds represent flow index (FI), the open triangles the vascularization index (VI) and the filled circles the vascularization flow index (VFI). The symbols are slightly displaced in relation to each other to make it possible to distinguish between results of VI, FI and VFI, but all measurements were taken at the same time. Median, 10th and 90th percentiles are shown. The figures in brackets denote the number of women examined. Days −5 to −1 represent the number of days before follicular rupture, and days +1 to +12 represent the number of days after follicular rupture.

There were no statistically significant correlations between the volume and vascular indices of any of the ovaries or the dominant follicle on day −1 and LH levels on day −1 (Spearman’s rho ranged from −0.49 to 0.29) or between the volume and vascular indices of any of the ovaries or of the corpus luteum on day +7 and progesterone levels on day +7 (Spearman’s rho ranged from −0.42 to 0.27).

Discussion

Our results suggest that the volume and blood flow of the dominant ovary change markedly during the normal menstrual cycle, whereas in the non-dominant ovary, no unequivocal changes occur. The volume of the dominant ovary and the dominant follicle increases throughout the follicular phase, decreases shortly after follicular rupture and then increases again. Vascular indices increase during the follicular phase and continue to rise after follicular rupture so that values are higher in the luteal phase than in the follicular phase. It is reasonable to believe that the changes in vascular indices parallel true changes in vascularization. Increasing 3D power Doppler vascular indices in the dominant ovary/dominant follicle in the late follicular phase and especially after follicular rupture can be explained by physiological vascular changes. The granulosa layer of the follicle is avascular until the time of ovulation ( Hazzard and Stouffer, 2000), but the newly formed corpus luteum contains developing capillaries in its luteinized granulosa cell layer ( Suzuki et al., 1998). The amount of blood vessels and the volume of granulosa cells in the corpus luteum increase until 7 days after follicular rupture, and blood vessels in the corpus luteum do not become less abundant until in the follicular phase of the following menstrual cycle ( Gaytan et al., 1999). The increasing amount of angiogenic factors in follicular fluid, e.g. vascular endothelial growth factor (VEGF), promotes new vessel formation in the follicles and corpus luteum ( Anasti et al., 1998). Despite LH being a major regulator of angiogenesis in the ovary ( Stouffer et al., 2001), we found no correlation between the LH values and vascular indices on the day of the LH surge (i.e. 1 day before ovulation). This lack of a direct correlation is likely to be explained by LH mediating its effect via other angiogenic factors, e.g. VEGF ( Geva and Jaffe, 2000; Hazzard and Stouffer, 2000). Good vascularization is thought to be important for egg and embryo quality ( Nargund et al., 1996; Coulam et al., 1999; Bhal et al., 2001; Vlaisavljevic et al., 2003). We also found no correlation between progesterone levels and blood flow indices in the dominant ovary/corpus luteum in the mid-luteal phase. Progesterone levels are supposed to reflect the function of the corpus luteum, but blood flow indices might not reflect progesterone production in the corpus luteum.

We have found no published studies where 3D ultrasound was used to study changes in ovarian volume and vascularity during a whole normal menstrual cycle. All currently available 3D ultrasound studies were conducted in women of couples with subfertility problems ( Jarvela et al., 2002; Vlaisavljevic et al., 2003). By and large, our results agree with those of 2D ultrasound studies of changes in ovarian volume during the normal menstrual cycle ( Bakos et al., 1994) and with those of 2D Doppler ultrasound studies of ovarian vascularization during the normal menstrual cycle ( Sladkevicius et al., 1993; Tan et al., 1996; Ojha et al., 2003). Volumes calculated using 3D ultrasound are more accurate than those calculated using a mathematical formula including diameters measured by 2D ultrasound ( Bonilla-Musoles et al., 1995; Amer et al., 2003). In 2D spectral Doppler ultrasound studies, blood flow velocity is measured in one or possibly a few subjectively selected vessels. With 3D ultrasound, vascularity indices in a whole organ are calculated. It seems likely that the results of 3D power Doppler ultrasound examinations better reflect true vascular changes in an organ than those of 2D colour or spectral Doppler ultrasound examinations. This is supported by the changes in vascularity in the present study being more distinct than those in a study (with almost identical design) where we used 2D Doppler ultrasound ( Sladkevicius et al., 1993).

The inter- and intra-observer reproducibilities of calculations of ovarian volume and ovarian vascular indices using 3D ultrasound have been shown to be acceptable ( Kyei-Mensah et al., 1996; Jarvela et al., 2003; Raine-Fenning et al., 2003a,b, 2004). Our results agree. To the best of our knowledge, there are no publications describing the reproducibility of the calculations of 3D power Doppler vascular indices in the wall of the follicle and corpus luteum, but we found these to be reproducible. Despite this, 3D power Doppler ultrasound with the calculation of flow indices using the VOCAL™ software must be regarded as a rather crude technique for studying blood flow, and small changes and differences may well go undetected. Technical factors may influence the results, e.g. the distance between the ovary and ultrasound transducer, whether an ultrasound beam hits a vessel during systole or diastole and the angle of insonation. Even though power Doppler is said to be angle independent ( Martinoli et al., 1998), this is not entirely true. An ultrasound beam hitting the bloodstream in a vessel under a 90° angle will generate no Doppler shift.

3D power Doppler is a new method of studying vascularization. It might better reflect vascular changes than 2D colour or power Doppler, because vascular changes in a whole organ can be assessed, not only as changes in blood flow velocity in one or a few vessels. However, our fundamental knowledge about vascular changes taking place in the ovaries during the menstrual cycle has not inherently changed through the introduction of 3D power Doppler. What 3D power Doppler might add is more precision, if one wants to study these changes. Our results may serve as a guideline as to what constitutes normal findings. There is potential for the 3D technique to become a useful tool for studying pathological vascular changes associated with ovarian dysfunction.

This study was supported by the Swedish Medical Research Council (grant numbers K2001-72X-11605-06A, K2002-72X-11605-07B and K2006-73X-11605-11-3), two governmental grants (Landstingsfinansierad regional forskning (Region Skåne) and ALF-medel) and funds administered by Malmö University Hospital.