Ultrasound imaging of ovarian follicles

The ovarian follicle at ultrasound: is it a circle or a sphere?

The measurement of ovarian follicle size is often inaccurate, since the calculation is made considering the follicle as a two-dimensional structure and not a three-dimensional structure, which it is really. Follicles rarely have a spherical shape and frequently they are elliptical, especially when there is follicular overcrowding in a hyper-stimulated ovary. Although there is a high degree of correlation between the mean diameter and volume of a follicle, follicular volume calculated from the mean diameter has been shown to be less accurate, especially when the follicle is ellipsoid.

The advent of three-dimensional ultrasound allowed us to study and measure the volume, so that the unit that makes the image is no longer pixels (defined by the x and y axes), but the voxel (defined by axes x, y and z ); accordingly, the measurement will be more reliable and reproducible because it’s closer to reality.

The development of three dimensional equipment allows the acquisition of volume data using a volumetric transducer, storage of the acquired volume data, reconstruction of the volume image and simultaneous viewing of all three orthogonal planes. The ability to visualize the oblique or coronal plane allows accurate volume measurements, especially of irregularly shaped objects, because individual variations in structure can be accurately tracked during the measuring process. These measurements are therefore reliable and highly reproducible.

Follicular counts using 3D technique, failed to break through into clinical practice in reproductive medicine clinics, because it requires more time than the usual two-dimensional technique, and because there are no studies that demonstrate a statistically significant difference between measurements performed in 2D and those performed in 3D. The real revolution in ultrasounds performed in reproductive medicine clinics was the automated follicular count, or better defined as SonoAVC (sonography-based automated count volume). It is a software application on a 3D ultrasound platform, which allows follicles present in a given ovarian volume to be identified automatically, not manually, and their size to be calculated. In addition, to complete volumetric analysis, the SonoAVC allows you to work on the volume acquired in post processing; for example, the missing follicles can be added, and the echo hypoechoic structures mistakenly identified as follicles may be removed. (Figure 1).

As previously mentioned, the “conditio sine qua non” for the software application is the 3D platform, which allows multi planar acquisition and manual selection of the region of interest (ROI) by moving a box in which the maximum proportion of the ovary fits. The frame selected by the ROI must be large enough to allow the entire ovary to be analyzed. Once this has occurred, SonoAVC software is applied to the dataset. The automatic analysis lasts approximately 6s; afterwards, the individual follicles are represented in different colours (Figure 2), including their absolute and relative measurements. Next, the user scrolls through the ovarian volume and deselects any non-ovarian follicles. Increasing separation will increase the software’s ability to differentiate each individual follicle. Furthermore, increasing or decreasing the growth will increase or decrease the volume of each selected follicle. Once optimization of the volume has been completed for both the left and the right ovary, the ovarian data is added to the report.

Figure 1
Follicular measurement using sonoAVC, muliplanar view

Figure 2
sonoAVC, render mode

The measurement of follicles in an IVF setting

The antral follicular count (AFC)

Several studies have been published on the correlation between the number of antral follicles (defined as the total number of follicles with dimensions ranging between 2 and 5 mm, or between 2 and 10 mm, on both ovaries) and the ovarian response during in vitro fertilization programs. Antral follicles counts may also be related to the onset of the menopausal transition and this indicates that it is closely linked to the quantitative aspect of ovarian reserve.

Every year, millions of AFC measurements must be performed all over the world (mainly in IVF clinics), but it still remains difficult from a scientific point of view to determine the exact performance of this marker due to some unresolved issues. There are no definitive guidelines defining exactly the practical rules for identification, correct visualization and counting of follicles. What’s more, in many articles, authors do not report exactly what dimensions they refer to when they count antral follicles and, very often, different values are considered by different authors (2-5 mm, 2-10mm, 5-10mm). At the same time, despite the few methodological certainties, there was a brave attempt by some authors who wanted to define the normal range amongts the general population. The establishment of a normogram of AFC values is the first step to counsel patients on a scientific basis. In our clinic, we tried to optimize AFC with “internal” “personal” guidelines, using a probe program with default settings adjusted to provide the best two-dimensional (2D) gray-scale image. These setting are as follows: gain, −5; speckle reduction imaging (SRI), 2; CrossXBeam CRI (Compound Resolution Imaging), 3; CRI filter, high; enhance, 2; reject,25; and harmonics, high (Figure 3)

Figure 3
AFC-best setting

Ovarian follicle tracking during ovarian stimulation

Follicular tracking is the identification of follicles responsive to the gonadotropin stimulation. Follicular recruitment and growth are typically monitored by transvaginal ultrasound, which is used to identify the follicle and to display its maximum diameter.

There are no standards for follicle tracking. Some observers rely on a single “best” estimate, while others use two or three measurements in one or more planes and then calculate an average diameter.

Often the follicles have complex/irregular shapes, particularly, in stimulated ovaries, and manual determination of follicular diameters can be inaccurate because the measured diameters are not real ones.

2D-US imaging, in fact, makes the assumption that an object has a regular shape and uses the measurement of two axes as surrogates to estimate its true size: this may be inaccurate for follicles that have uneven and irregular shapes, like those developing during COS. Accurate follicular monitoring of Controlled Ovarian Stimulation (COS) by transvaginal ultrasound is considered important for the success of human in vitro fertilization. Measuring the mean diameter of multiple follicles with 2D-US has an intra and inter-observer variation that has been estimated to be around 20%, when several follicles are contemporaneously present inside the ovary. The question is whether the 2D-US is still the best method for carrying out the follicular tracking during ovarian stimulation.

A new opportunity to get useful insights about this topic could be presently offered by the 3D-US scanning. Volume measurement will provide more reliable information; more precise results can lead a reduction in the number of immature oocytes. It has been shown that 3D Sono AVC is closer to biological reality. The authors analyzed follicular volume manually (obtained with 2D technique), follicular volume (obtained with 3D technique) and the amount of follicular fluid obtained to pick up. The measurement of the follicles with sonoAVC has been shown to be the one closest to the real follicular volume, with an average difference of 0,04ml (± 0.25 ml). Published data show that Sono AVC offers the possibility of standardizing follicular measurements so that ovarian scans can be performed by different investigators, with the same results, overcoming the lack of standardization in US technique that represents an important limitation of 2D-US. In addition, the reduced intra- and inter-operator variability in the measurement of ovarian follicles may increase the quality of medical assistance in telemedicine (when patients undergo monitoring in medical offices other than the IVF center where they will undergo oocyte retrieval) and this may reduce the well-known variability in the AFC between different centers.

Figure 4
2D Follicular tracking

Figure 5
Follicular tracking using sonoAVC, muliplanar view


Investigation of ovarian follicles is now improved thanks to new ultrasound tools. Sono AVC provides automatic measurements of follicular diameter that are more accurate than those estimated manually. Consequently, measurements are standardized, which facilitates comparison and therefore multicenter research. Using follicular volume measured with sono AVC as the measure of follicular growth may improve treatment outcome over that achieved with conventional monitoring with follicular diameter. Better knowledge in this area could be helpful to optimize IVF outcome, by refining COS protocols and obtain high quality oocytes. It would be desirable that, the simplicity and accuracy of the sono AVC is make it more easily the transition from 2D to 3D, especially in the evaluation of irregular structures as the follicles; and the real challenge in Reproductive Medicine will be no longer speaking of follicular diameter but of follicular volume.

Antonio La Marca MD PhD

Clinica Eugin Modena, Italy


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