Antral follicle count determinations and measurements of the ovarian volume are easy to perform and inexpensive because ultrasound imaging facilities are readily available (13). In addition, ultrasound imaging is currently the only method that allows a direct assessment of each ovary as a separate entity (21). Ultrasound measurements of follicular numbers were developed as a “test” of reproductive age (22).

With total AFC determination (tAFC), the number of follicles between 2 and 10 mm is counted (21), usually on the third day of the cycle during the early follicular phase (23). The tAFC can be estimated using 2D or 3D ultrasound imaging (1, 16, 23). Recent reports have shown that more information can be obtained from observing the antral follicles than from quantification only (16, 24). The size of the antral follicles can actually be more significant than the total number of follicles and is therefore more predictive of the “functional ovarian reserve” (19). The success of ART treatment strongly relates to the number of antral follicles with a diameter between 2 and 6 mm, in terms of the relation of these follicles to the number of mature oocytes that can be collected. Deb et al. (24) were among the first to determine the AFC using SonoAVC software. With that software, they were able to identify and measure each antral follicle separately, an undertaking that would be very time-consuming and highly operator-dependent using 2D ultrasound imaging. Deb et al.’s studies (16, 24) support the theory that the smaller antral follicles actually reflect the ovarian potential and are most predictive of a response to controlled ovarian stimulation (COS). Following from that, a study by Saumet et al. (25) shows that 3D AFC determination and anti-Müllerian hormone (AMH) determination are equally capable of predicting the ovarian response, whereas 2D AFC determination is inadequate. This result can be explained by the fact that the smaller antral follicles (AFC if ≤6.00 mm) are mostly responsible for AMH secretion.

The ovarian volume can also be measured via transvaginal ultrasound (26) in the early follicular phase before any significant follicular dominance occurs (1, 23, 24). Studies by Lass et al. (3, 26) indicate that patients with very small ovaries (<3.0 cm) have to cancel the treatment cycle more often and have a significantly lower yield of oocytes; therefore, those patients have a lower chance of pregnancy. Ovarian volume measurement is fast and cost-effective, but its predictive value does not exceed that of tAFC (3, 26). In the study by Kupesic and Kurjak (27), total ovarian volume measured with 3D ultrasound imaging also showed a lower predictive value compared with tAFC. This difference can be explained by the fact that the number of smaller follicles (2–6 mm) decreases as a woman’s age increases, and the size of the follicles will slightly compensate for the decrease in tAFC as the age increases (28). Ovarian volume predicts the success of IVF to a lesser extent than tAFC does, but is useful in making a distinction between multifollicular and polycystic ovaries (27).

Assessing follicle size with 2D technology requires the measurement of every follicle in two dimensions and the calculation of the average diameter. This process is time-consuming, and the reliability of the measurements decreases when the number of follicles increases because it is difficult to check whether each follicle was measured once and only once (16). Because ovarian follicles often have complex shapes (29), particularly in stimulated ovaries with “follicular overpopulation” (1, 5, 12), manual determinations of the follicular diameter often offer a weak representation of the follicular volume and might have a negative effect on medical decision-making (17, 30). In these cases, volume measurement will provide more reliable information (1). These more precise results can lead to a higher follicle-to-oocyte ratio and a reduction in the number of immature oocytes (6). Therefore, it is assumed that 3D SonoAVC is closer to biological reality. Raine-Fenning et al. (14) studied 51 women undergoing COH for IVF therapy. The researchers measured the follicular volumes right before oocyte collection. The volumes obtained with SonoAVC were compared with the volume of the manually measured follicular aspirate. The actual follicular diameter was estimated by measuring the follicular volume using an aspirate and applying the formula 4/3πR³ to derive the diameter.

Raine-Fenning et al. (12) analyzed 224 follicles with an average follicular volume of 3.7 mL. SonoAVC measurements were very close to the actual follicular volumes, with an average difference of 0.04 mL (±0.25 mL). The study also showed that the 95% confidence interval is lower with SonoAVC compared with calculations based on one 2D-measured diameter (0.98 versus 5.88, respectively), which proves that SonoAVC is reliable. This study (12, 14) was the first to state that SonoAVC made the most accurate estimations of the follicular diameter compared with the actual follicular diameter. In this study, the d_v and MFD were of equal value. As in the in vitro studies (31, 32), these measurements seem to mildly underestimate the actual follicular diameter (maximum of 2.17 and 2.86 mm, respectively). Lamazou et al. (15) evaluated 27 women undergoing COS for IVF and compared the follicular volumes measured with SonoAVC to the actual volumes (based on the follicular fluid aspirate) of 72 follicles. The median actual volume was 3.20 mL (0.80–10.20 mL), compared with 3.25 mL (0.98–8.63 mL) for the volumes measured with SonoAVC. In another study, Raine-Fenning et al. (33) studied 100 follicles measured on the day of oocyte collection. Automatic volume measurements were compared with measurements of the follicular aspirate. Thus, the results showed that SonoAVC estimates the volume of a follicle most accurately using d_v results, with values that were almost identical to the estimated diameter based on calculations from the actual follicular volume (3.82 ± 3.03 versus 3.89 ± 3.07 mL, respectively). Automatic MFD calculations were less accurate (3.43 ± 2.75 mL) but still more precise than volume estimations of follicular diameters measured with manual 2D. Furthermore, the volume estimations made with manually measured diameters were more correct when 3D technology was used than when conventional 2D technology was used. In a similar study by Raine-Fenning et al. (34), five follicles of every diameter within a range from 11 to 22 mm were measured with SonoAVC using 2D manual measurements, 3D manual measurements and automatic volume measurements. In this case, the d_v values were also the most accurate ones.

Salama et al. (29, 35) studied 15 women undergoing IVF with embryo transfer in a cycle with a monodominant follicle. They measured the size of the dominant follicle before oocyte collection using SonoAVC software and performed the measurements three times. No significant difference was demonstrated between the three measurements using SonoAVC (3.57, 2.41–8.19 mL; 3.71, 2.49–8.90 mL; 4.07, 3.12–8.16 mL) and the actual follicular volume (3.60, 2.90–8.00 mL).

All of the studies published to date have independently shown that an obvious connection exists between follicular volumes calculated with SonoAVC and the actual follicular volumes obtained for follicular aspirate. Most of the studies showed that d_v values provided the most accurate result, but that MFD values provide more accurate results than manually measured 2D values do.

A significant question when comparing the two measurement techniques is how different the measurements are allowed to be without causing problems in clinical practice. Bland and Altman (36) investigated a way to measure the probability that a new technology differs from the old one. This method is the gold standard for comparing two measurement techniques.

Raine-Fenning et al. (6) studied 89 women undergoing IVF treatment and measured the number and average diameter of the follicles in both ovaries on day 10 using both 2D ultrasound imaging and SonoAVC with stored 3D volumes. The follicles were divided into different categories based on average diameter (≥10, ≥14, or ≥ 18 mm). A large degree of correlation existed between the two measurement methods (r = 0.84).

The average difference between both measurement techniques was less than 1 follicle in each category, demonstrating this technique provides very reliable results. Ata et al. (5, 37) studied 100 women undergoing COH after at least 5 days of stimulation. The average difference in the number of follicles measuring 14–17 and >18 mm between the 2D, SonoAVC-MFD, and SonoAVC-d_v measurements of the dominant follicle was less than 1 mm. In this study, SonoAVC-d_v resulted in slightly higher results than 2D manual measurements (on average, 1.3 additional follicles in the 10–13 mm category). On average, the difference in measurements decreased as the follicle size increased. The MFD measurements showed the same trend. On average, in follicles measuring 10–13 mm, more follicles (1.1) were detected with the SonoAVC technology. In this study, we also noted that the differences between both technologies increase when a woman has several follicles. Therefore, SonoAVC proves to create smaller d_v measurements because on average, the technology detects more follicles in the smaller categories. In comparison, on average, the technology detects fewer follicles in the largest category. Ata et al. (5), however, suggest that it is unlikely that the minor differences between the measurements would affect the clinical outcome. Deutch et al. (38) concluded after their study that no difference existed between the manually measured MFDs and the MFDs and d_vs calculated using SonoAVC. In another study, Deutch et al. (31) studied 347 follicles in 14 women undergoing COH. They found the best correlation between the manually measured MFD and the d_v calculated with SonoAVC, with a correlation coefficient of 0.99. Rodriguez-Fuentes et al. (17) analyzed 92 ovaries in 58 women undergoing IVF treatment. They observed a significant difference in measurement results between conventional 2D technology and 3D technology with SonoAVC in 49% of ovaries (n = 45, P < 0.05). However, when they distinguished between good-quality and moderate- to poor-quality images, they observed proper correlation between 2D and SonoAVC in 62.3% of the good images (n = 33) versus 35.9% of the poor images (n = 14). In the study by Deb et al. (19), SonoAVC observed a significantly lower number of antral follicles than 2D ultrasound imaging did, both in tAFC and when the follicles were divided into five groups according to size. In the study, 24 women ≤40 years old who were undergoing ART were studied. After measurements of the AFC using the 2D mode, a 3D image was made of each ovary and SonoAVC was applied. The tAFC was significantly lower with SonoAVC (17.16 ± 9.71 versus 19.89 ± 10.33, respectively; P < 0.001). This result can demonstrate both an actual difference and a fundamental difference in the measuring techniques. Furthermore, the study showed that SonoAVC could demonstrate approximately half of the expected number of follicles with a diameter of 1–2 mm, while conventional 2D technologies were unsuccessful in registering one single follicle with these measurements. This difference most likely reflects the resolution limitations of the ultrasound imaging, but it can also be explained by the subjective nature of 2D ultrasound imaging. We noted the least similarity between the two measurement techniques for the antral follicles with a diameter of 3.0–4.99 mm. If the size of the follicles that contribute the tAFC is more important than the absolute size of the complete population – as suggested earlier – these findings have significant implications for clinical practice.

With 2D ultrasound imaging, the investigator gradually rotates the transvaginal probe to scan each ovary while he/she identifies each follicle first and then measures its dimensions (39). This method is both time-consuming and uncomfortable for the patient (Table 2). However, a 3D examination only requires a quick view of each ovary while the probe registers the ultrasound data. The ovarian follicles can be counted and measured offline later. Various studies have evaluated the time required to apply SonoAVC.

Raine-Fenning et al. (6) were the first to investigate time gained in stimulated cycles. In their study, 89 patients underwent an ultrasound scan on the 10th day of stimulation. The authors established that the ultrasound examination time required using the automatic 3D method was significantly shorter (P < 0.001) than examinations using conventional 2D technology (39.00 ± 6.00 versus 236.10 ± 57.07 s, respectively). The 3D technology required additional time for data analysis (141.50 ± 64.13 s), but the total time of 180.50 s (±63.55 s) was still significantly shorter than that associated with 2D technology use (P < 0.01). In all cases in the study, the ultrasound examination using 3D technology could be performed in <1 min, while the 2D manual measurement required approximately one additional minute.

Rodriguez-Fuentes et al. (17, 40) studied 58 women undergoing IVF and performed the ultrasound scan on the day of human chorionic gonadotropin (hCG) injection. They established that examinations of patients with >10 follicles lasted 9.6 min on average using conventional 2D technology, compared with 5.6 min using automatic monitoring. The examination was 7.6 min shorter on average for the patient with SonoAVC software. For the investigator, SonoAVC led to 4 min of time gained per patient. The study by Deb et al. (19) also showed that the average time of the automatic analysis using SonoAVC was significantly shorter than when 2D ultrasound monitoring was used (132.05 ± 56.23 and 324.47 ± 162.22 s, respectively; P < 0.001). The automatic analysis always required post-processing. The average time was 41.06 ± 11.12 s before the measurement and 90.99 ± 45.11 s before post-processing. In the study by Sherbahn et al. (41), a difference of 0.6 min on average was recorded (5.6 min for SonoAVC measurements and 6.2 min for 2D manual measurements). Deutch et al. (31, 42) also registered the time required to measure all follicles in an ovarian volume using both manual 2D measurements and SonoAVC. The differences in the number of follicles per ovary led to a large range of results. The time required for SonoAVC was significantly shorter than that required for manual measurements [133 s (range 73–271 s) versus 361 s (range 129–878 s), respectively; P < 0.001]. This led to savings of 3.8 min per ovary or 7.6 min per patient. With SonoAVC measurements, the post-processing took up the largest portion of time. Another study by Deutch et al. (38) only examined the measurement time (without analysis and post-processing). That study noted that the automatic software required 5.8 s on average to determine the diameter of all follicles, compared with 56.8 s for the manual method (P < 0.001).

Decreased operator dependence results in more reproducible folliculometry, as shown by low intraobserver and interobserver variability (Table 3).

Deb et al. (13) investigated 55 women with subfertility based on FSH levels <15 IU/L. Ultrasound scans were performed between days 2 and 5 of the menstrual cycle. The number of follicles in each ovary with a diameter between 2 and 10 mm was counted to determine the tAFC in that ovary. Three different methods were used to measure the number of follicles: 2D images in real-time (2D-RTE), 3D images in multiplanar view (3D-MPV), and 3D images in combination with SonoAVC software, whereby a distinction was made between those without (sAVC-AA) or with post-processing (sAVC-PP). These three methods were performed independently by two investigators. The median tAFCs measured using 2D-RTE, 3D-MPV, sAVC-AA, and sAVC-PP were 18 (10.56–26.81), 16.5 (10.25–26.75), 5 (3.37–9.18), and 15.5 (10.25–24.75), respectively. The initial tAFC investigated using SonoAVC without post-processing was missing a number of follicles, which is noticeable in its significantly lower median tAFC compared with all other methods (P < 0.001). The median tAFC after post-processing increased to 15.5, but remained significantly lower than the amounts found using 2D-RTE (P = 0.006) and 3D-MPV (P = 0.028). Calculations using Bland–Altman plots demonstrate that measurements using sAVC-PP are more reliable than measurements using 3D-MPV and 2D-RTE technologies are. Furthermore, with SonoAVC software, every data set was examined twice by the same investigator to assess the intraobserver variability. SonoAVC also provided the most reliable results after post-processing. Salama et al. (29) obtained an intraclass correlation coefficient (ICC) of 0.97 for the SonoAVC calculations in their study, indicating proper reproducibility. However, the manual measurements led to a lower ICC (<0.80). A study by Deutch et al. (38) also demonstrated that the intraobserver variability is significantly higher (P < 0.05) with the manual measurement of follicular diameters than with the automatic measurement using SonoAVC. The study by Bouhanna et al. (43) of 22 women in a monofollicular cycle also demonstrates that both the intra- (ICC = 0.96; 0.94–0.98) and interobserver (ICC = 0.94; 0.90–0.99) reliability of SonoAVC is higher than that of 2D manual measurements (ICC = 0.62; 0.45–0.79 and ICC = 0.83; 0.73–0.94, respectively).

A major problem with ART is the differences in pregnancy rates not just between different patients, but also between different centers. We notice differences between hospitals that cannot be explained by patient characteristics or regulations alone (44). Consequently, it seems plausible that the standardization of procedures could result in improved ART results. SonoAVC offers the possibility of standardizing follicular measurements so that scans of the ovaries can be performed by different investigators (5, 29). In addition, standardization opens the channel to more multicentre research (14).

Another advantage is the possibility of implementing a quality control system for follicular measurements. In practice, with 2D measurements, no data about follicular measurements other than the results can be kept. However, with SonoAVC, it is possible to store ovarian volumes. Such storage allows random checks to be performed on stored volumes and makes a quality control system available (5). Training programs show that a learning curve of 19 to 38 procedures is necessary to obtain proficiency in 3D SonoAVC (45). In addition, with SonoAVC, the quality standards drawn up by the European Union can be complied with because of the shorter examination time and the objective follicular diameter measurement (46).

Meseguer et al. (44) are convinced that the introduction of robotics in the ART laboratory can offer an advantage. Robotics can standardize various procedures and prevent interindividual differences. The new SonoAVC software offers a step in that direction.

Another important development that can be introduced via follicular volume calculations is the new volume-based criteria for hCG administration (5). Follicular volume calculation can provide a better indicator of oocyte maturation and increase the production of mature oocytes (5). The study by Salha et al. (47) shows this association is not simple. The authors were unable to relate the follicular volume to follicular readiness for fertilization and the possibility that the embryo will implant, despite the fact that in their study, embryo implantation ratios appeared to have decreased with follicular volumes of ≤1.0 or >5 mL. Rodriguez-Fuentes et al. (17) evaluated the relationship between the follicular volumes on the day of hCG administration and the number of mature oocytes obtained. The researchers wanted to establish cut-off values for the more exact timing of the hCG administration. In the study, 98.6% of the follicles ≥0.6 mL led to mature oocytes, while all other studied volumes overestimated or underestimated the number of mature oocytes. Because 3D measurements provide a better representation of the actual follicular size, the criteria for hCG in ovulation induction cycles, currently based on 2D technology, must be redefined. Murtinger et al. (46) established that during the last ultrasound scan, the average follicular diameter was lower with 3D (13.1 follicles) than with 2D imaging (15.1 follicles). This difference can be explained by the fact that 3D SonoAVC measurements measure more small follicles. That characteristic obviously raises the question of where the limit should be set to calculate the average. Because the values obtained with 2D and 3D are not sufficiently comparable in this regard, new criteria should be implemented that specifically match the more accurate measurements of SonoAVC. It is obvious that this new technology requires a complete revision of the criteria for hCG administration (30). Notwithstanding the fact that different studies dispute the improvement of the clinical result, the renewal of the criteria seems of interest. It was also very recently shown that the number of mature oocytes can be reliably predicted using volume measurements and that a distinction can even be made between different stimulation protocols (48).