Use of contrast enhanced ultrasound in testicular diseases: A comprehensive review

Abstract Background Contrast‐enhanced ultrasound (CEUS) is a sonographic technique that increases the diagnostic accuracy of ultrasound and color Doppler ultrasound (CDUS) when studying testicular abnormalities. However, its role in clinical practice is still debatable because there are no accepted standards regarding how and when this technique should be used for patients with testicular disease. Objectives To perform a nonsystematic review of the current literature to highlight the strength and flaws of performing CEUS and to provide a critical overview of current research evidence on this topic. Materials and methods A thorough search of published peer‐reviewed studies in PubMed was performed using proper keywords. Results Strong enhancement of neoplastic lesions (both benign and malignant) during CEUS aids in differential diagnosis with non‐neoplastic lesions, which usually appears either nonenhanced or enhanced in a manner similar to that of the surrounding parenchyma. CEUS enhancement has a high predictive value in the identification of neoplastic lesions, whereas a similar or complete absence of enhancement may be interpreted as strong evidence of benignity, although there are exceptions. Literature on quantitative analysis is still scarce, though promising, particularly in distinguishing benign from malignant neoplasms. Furthermore, CEUS may be useful in many emergency situations, such as acute scrotum, blunt scrotal trauma, and focal infarction of the testis. Finally, CEUS can help increase the probability of sperm recovery in azoospermic males. Discussion and conclusion CEUS is a safe, easy‐to‐perform, and cost‐effective diagnostic tool that can provide a more accurate diagnosis in testicular lesions and acute scrotal disease. However, further studies with larger cohorts are required to refine the differential diagnosis between benign and malignant neoplasms. Finally, these preliminary results can instigate the development of innovative research on pre‐testicular sperm extraction to increase the chances of sperm recovery.


INTRODUCTION
Testicular ultrasound (US) currently represents a routine and mandatory investigation for patients with scrotal symptoms and is considered the first-line imaging modality in the evaluation of the testis and adjacent structures, in addition to physical examination. Since its introduction, US has become an integral diagnostic tool in clinical settings that has been further enhanced by continuous developments to improve the resolution of US machines and probes. [1][2][3] Although grayscale US, color Doppler US (CDUS), and power Doppler US (PDUS) demonstrate high diagnostic accuracy for detecting most testicular pathologies, interpretation of the acquired images is not well standardized and may rely on the operators' expertise.
In the last few decades, the use of intravascular contrast-enhanced US (CEUS) has grown considerably and has proved to be a useful tool in many diagnostic fields. [14][15][16][17][18] US contrast medium consists of US-detectable microbubbles, which are very small-sized (<10 μm) organic shells that are filled with gas.
A dedicated machine-setting with a low mechanical index (0.05-0.08) is needed to avoid early microbubble destruction. 20 The localization of microbubbles is exclusively intravascular because they are small enough to pass through the lumina of capillaries, yet large enough to prevent extravasation from vessels. Due to their high impedance, they reflect the majority of US waves with a higher echo than the parenchyma. In fact, unlike CDUS and PDUS, CEUS provides a reliable representation of blood perfusion and parenchymal microcirculation in various organs, using intravascular blood tracers. After injection of the contrast medium, two phases are described in organs with a single arterial blood supply 20 : the first is the arterial phase (10-40 s), which shows a progressive enhancement; the second one is the venous phase (30-45 s), which starts after injection and exhibits a plateau followed by a progressive decrease, until the microbubble signal completely disappears.
CEUS offers a number of advantages: it is easy to perform, costeffective, safe, and does not have any harmful effects compared to other complementary imaging methods such as CT and MRI. First, the number of allergic reactions reported is lower than those arising with CT and MRI contrast medium. [21][22][23] The overall reporting rate for all adverse events is 0.125% (only 0.0086% for serious ones) 23 including itching, mild dizziness, moderate hypotension, headache, and nausea which resolved spontaneously. Second, CEUS is neither cardiotoxic nor nephrotoxic and can be safely administered in patients with renal insufficiency because the contrast medium is not excreted via the kidney, but it is cleared by the lungs. 20,24 Consequently, and due to all the other advantages offered, this diagnostic tool can also be used on children. 25,26 Finally, compared to MRI, CEUS offers higher spatial resolution (especially using new high frequency probes, up to 18 MHz), allowing for a dynamic assessment even of smaller lesions.
Over the last years, CEUS has proved to be particularly useful in testicular setting: microbubbles trace normal parenchymal microcirculation and are able to highlight intraparenchymal abnormalities within the testicle. This is particularly useful in the characterization of testicular lesions 7-10,27-33 and acute scrotum. [34][35][36] Recent studies have also focused on the utility of CEUS in evaluating testicular perfusions prior to testicular sperm extraction (TESE) in infertile men. 37,38 However, to date, there are no well-established and accepted standards with respect to how and when this technique should be used when dealing with patients suffering from testicular disease.
The purpose of this study was to perform a comprehensive, upto-date review of the current literature to highlight the strength and flaws of performing CEUS, and to provide a critical overview of current research evidence on this topic to inform and guide clinicians' choices of performing CEUS in certain conditions, and to provide them support in the interpretation of the exam.
A computerized literature search was performed using the following keywords: "CEUS," "testicle," "testicular tumor," "testicular lesion", "seminoma", "Leydig cell tumor", "scrotal trauma," "testicular torsion", and "infertility". Keywords were properly combined with Boolean operators to optimize the search strategy. Typically, the contrast medium is no longer visible after an average of 3-5 min. 20 The entire examination needs to be recorded for subsequent analyses. Recording should be initiated at the end of each contrast-enhancement injection and should be concluded after at least 90 s. The first evaluation that can be performed involves a qualitative analysis: after each injection, it is possible to observe whether the contrast medium enhances the area of interest, and subsequently evaluate the intensity and timing of the uptake (wash-in), and release (washout) of microbubbles compared with parenchyma. The area of interest can be defined as hyper-enhancing (Figure 1), hypo-enhancing, or non-enhancing compared with the surrounding parenchyma, and the wash-in and washout can be defined as faster, similar, or slower than parenchyma. 7,9,[27][28][29]31 However, qualitative analysis is subjective and operator-dependent. In contrast, quantitative analysis using appropriate software generally integrated in the US machine is a significantly less biased approach.

TESTICULAR CEUS TECHNIQUE
Time-intensity curves can be obtained by manually placing a region of interest (ROI) to entirely cover the area to be examined.
Another identical ROI should be placed on the adjacent parenchyma for comparison. 28 Within the ROI, the mean intensity of contrast enhancement can be described as a function of time with timeintensity curves: they are bell-shaped curves that describe an initial uptake phase of the contrast medium (wash-in) up to the maximum peak of intensity, and a subsequent release phase (washout) ( Figure 2).
Wash-in and washout of the contrast agent can be quantified by calculating intensity and temporal parameters (quantitative parameters).

CEUS AND TESTICULAR LESIONS
One of the most successful uses of CEUS reported in the literature involves the differential diagnosis of intratesticular lesions. 33 Males presenting a palpable testis nodule are likely to have malignant germ-cell tumor in > 90% of cases. However, the increased use of testicular US as a diagnostic tool in most andrological pathologies and the recent developments of high-frequency probes in ultrasonography have allowed for an increase in the detection of small, incidental intratesticular lesions that are thought to be benign in > 30% of cases.
Thus, a radical orchiectomy should be considered as overtreatment. 40 The first step for clinicians is to distinguish whether a lesion is neoplastic or non-neoplastic. If a neoplastic lesion is suspected, the second step is to differentiate between benign and malignant testicular tumors (TTs). Clinical history (genetic syndromes, history of cryptorchidism, previous surgery, infertility, previous contralateral tumor, and familiarity for testicular cancer), symptoms (sudden or chronic pain and swelling), and laboratory data (serum tumoral markers) can significantly assist this process. However, in certain cases, the differential diagnosis can still be challenging. During the last decades, CEUS has become a very useful method to improve the characterization of nonpalpable testicular lesions, and its use is recommended by the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) guidelines. 20,41

CEUS in the differential diagnosis between non-neoplastic and neoplastic intratesticular lesions
Non-neoplastic intratesticular lesions include simple cyst, epidermoid cyst, segmental ischemia, abscess, hematomas, post biopsy scars, orchitis, adrenal rest tumors, and sarcoidosis. Their US and CDUS characteristics are reported in Table 1 According to several published studies, neoplastic lesions (both benign and malignant) tend to be strongly enhanced during CEUS, thus facilitating the differential diagnosis with non-neoplastic lesions, which, in general, are either nonenhanced or enhanced in a similar manner to that of the surrounding parenchyma ( Figure 4).
The first study in this field dates back to 2011. 27 The authors prospectively described the feasibility of CEUS in the differential diag- To sum up, lesions that were more enhanced compared to the surrounding parenchyma seem to have a higher predictive value in identifying neoplastic lesions, whereas similar enhancement or its complete absence can be interpreted as strong evidence for benignity. 10,42 However, there are some exceptions, represented by epidermoid cysts

CEUS in the differential diagnosis between benign and malignant neoplastic lesions
TTs are rare neoplasms that account for approximately 1-1.5% of all human cancers. However, TTs represent the most common neoplasm in males between 15 and 44 years who are in full reproductive age. 47 TT can be primarily distinguished into germ-cell tumors and nongerm-cell tumors. 48 Germ-cell tumors are almost always malignant, whereas non-germ-cell tumors are most commonly stromal tumors with a benign behavior. Recent evidence has shown that the frequency of stromal tumors is probably underestimated, particularly when they are small. In fact, according to recent series, the incidence could be significantly higher (3-22%) 28,31,40,[49][50][51] than the one reported in previous research studies. In certain cases, malignant tumors can also undergo regression, necrosis, and scarring while spreading with distant metastasis, as forBOT. 51 Finally, although rarer, tumors of lymphatic or hematopoietic origin with intratesticular localization should also be described. 30,52 Conventional US demonstrates high sensitivity for TT detection, yet this diagnostic method offers low specificity in differentiating benign from malignant lesions. 53 The appearance of TTs in US may change according to histology. A classification of TTs 48  According to previous reports, increased vascularization (with arborization and branches) has been considered to be a malignant tumor characteristic. 54,55 Nonetheless, vascularization is not specific for malignant diagnosis because it can also be increased in stromal tumors, 40 focal orchitis, 10 TARTs, 56 or benign mesenchymal tumors such as capillary hemangioma 57-59 and leiomyoma. 60 Particularly, LCTs can appear as having a more intense blood flow than seminomas. 9,29 For this reason, distinguishing a malignant tumor from a benign neoplasm between incidental lesions is a significantly challenging task, particularly for small, hypoechoic, and well-vascularized masses with regular margins. In particular, LCTs and seminomas can be very F I G U R E 5 Epidermoid cyst. B-mode US demonstrates a well-circumscribed, solid, mixed-reflectivity lesion with high-reflectivity "onion-skin" peripheral rims. Contrast-enhanced US demonstrates a clear lack of enhancement within the lesion similar on nonenhanced US. 61,62 Performing an accurate and careful differential diagnosis is imperative because both benign and malignant tumors have a very different clinical course. Patients suspected of benign lesions can be addressed to tissue-sparing surgery enucleation or, in selected cases, to clinical and US strict surveillance, thus preserving the testicle instead of performing total orchiectomy, 63,64 which is suggested in case of malignancies.
In this perspective, CEUS could represent as an additional and effective tool. To date, there have been only a few prospective studies that tried to evaluate whether the use of CEUS could help in the differential diagnosis between benign and malignant TTs. 7,28,29,31 Results are promising, however, the available data are not always in agreement, and the majority of reports are based on qualitative rather than a more objective quantitative assessment.
As reported earlier, hyperenhancement is the most common feature observed in TT on CEUS. 28,29,31 The current literature underlines that benign lesions are characterized by lower enhancement; however, in these reports, both neoplastic and non-neoplastic lesions are included in the benign group. 7,10,32 It must be considered, though, that some malignant lesions have an architecture that does not allow the uptake of the contrast medium. In fact, this is the case in BOT, where malignant cells are rapidly replaced by fibrotic tissue. 28 As already pointed out, qualitative analysis can guide the clinician's evaluation. However, this process is based heavily upon the operator's experience; thus, the subjective interpretation of US images can be biased. In contrast, quantitative analysis can provide more objective data, yet research in this field is still substantially limited, and studies  it should be emphasized that the authors of this study included both neoplastic and non-neoplastic among benign lesions. 7

Acute scrotal pain
Acute scrotal pain is a common urological emergency that requires a prompt diagnosis to determine the most appropriate treatment approach. Pain can be due to several causes, including epididymoorchitis, testicular torsion, testis' appendix or epididymis torsion, intratesticular abscess, focal infarction, neoplasm, and trauma. 36,68 At first, diagnosis of clinical and medical history, associated with symptoms and biochemical assessment, is mandatory. Indeed, patients with testicular torsion usually present with symptoms of severe acute unilateral scrotal pain, nausea, and vomiting. 69 US diagnostic images pertaining to testicular torsion are characterized by the absence of intratesticular blood flow at CD evaluation 70 (Figure 7). In other cases, symptoms and clinical presentation might be similar among all causes of acute scrotal pain, whereas physical examination and laboratory evaluation may often not be exhaustive. 36 Thus, CDUS could be helpful in investigating the underlying pain etiology. 68,70 Epididymitis and orchitis can be diagnosed by CDUS as a result of their typical clinical features. More specifically, epididymitis appears as an enlarged epididymis with distinct inflammatory signs such as increased vascularity CD and hydrocele. 70 In contrast, orchitis is represented by an enlarged testis with decreased echogenicity and increased vascularity at CD. 70 Other lesions such as abscess, hematoma, and infarction appear as hypoechoic lesions with absent vascularity (Figure 8), 70  trauma. This was particularly true for small lesions. 35 Also, CEUS could efficiently help depict fracture lines which could usually not be seen using grayscale US. 16 Another interesting field of application of CEUS in acute scrotal pain involves focal testicular infarction. In CDUS examination, testicular infarction typically appears as an avascular wedge-shaped hypoechoic lesion. 73 However, segmental testicular infarction can be round and resembling a TT, 74 and sometimes presents a rim enhancement, probably due to granulation tissue in response to ischemic processes. 34 In such cases, the patient's clinical history can help the clinician in the differential diagnosis process (Figure 9).
In a retrospective study, 20 men with acute scrotal pain, suspected of testicular infarction, were examined with CEUS. Compared with CDUS, CEUS facilitated improved lesion conspicuity, leading to the identification and recognition of ischemic lobules. 34 Moreover, a perilesional rim enhancement was also identified by CEUS, which may represent a specific sign of subacute segmental testicular infarction. 34,75 The ability of CEUS in assessing the complete absence of vascularization with a rim enhancement has been used to distinguish a testicular hematoma 76 or segmental testicular infarction 77-79 from a tumor, in asymptomatic patients. Out of all the causes of scrotal pain, the use of CEUS has also been described in the diagnosis of spontaneous spermatic vein thrombosis, a rare condition which can lead to testicular pain and testicular swelling. 80

Infertility
Nowadays, approximately 10% of infertile males are affected by nonobstructive azoospermia (NOA), which is characterized by a complete absence of spermatozoa in the seminal fluid due to minimal or no spermatogenesis. 81 In some cases, the only therapeutic option in these in areas with high tissue perfusion. 85 The high accuracy of CEUS in assessing blood perfusion and microvascular architecture of the testes may suggest that this technique could help increase the probability of sperm retrieval.
To the best of our knowledge, a case report described for the first time that sperm quality and quantity depend on tissue perfusion within the testis. 86 Since then, only two recent studies have focused on determining the usefulness of CEUS in infertility, by investigating whether it could be used to predict the success rate of testicular sperm retrieval techniques in infertile men. 37 These findings suggest that spermatogenesis is not uniformly distributed throughout the testis because sperm quality is better in areas with high tissue perfusion. Moreover, the subsequent ROC analysis showed that W-in ≤27 s, TTP ≤45 s, and PI ≥11 dB in the selected area could be considered the best cut-off values for predicting positive sperm retrieval. 37 In contrast, Xue et al. did not observe significant differences in the success rates of SR between the major and minor perfused areas in the 46 nonobstructive azoospermic patients examined. However, TESA had a very little chance of success in patients with NOA in case of: decreased intensity of the main perfusion area (defined as decreased intensity within 30 s after reaching the peak for both the main perfusion area and whole testis) with values < 8.6 dB; TTP of the whole testis > 9.0 s; slope-in of the whole testis < 1.7 dB/s. Therefore, these quan-titative CEUS features could have a negative predictive value on sperm retrieval. 38

CONCLUSIONS
In conclusion, the literature underlines that CEUS is a safe, easy-toperform, and cost-effective diagnostic tool that is able to provide an accurate diagnosis in testicular lesions and in acute scrotal diseases when US findings are unclear. CEUS can increase diagnostic confidence levels, particularly in less experienced investigators. Therefore, CEUS should be proposed in every case where US diagnosis remains inconclusive, namely in the differential diagnosis of small testicular lesions to facilitate greater confidence in terms of selecting the appropriate patient intervention. Lesion enhancement indeed seems to have a high predictive value in the identification of neoplastic lesions. Similarly, the complete absence of enhancement can be interpreted as strong evidence for benignity, although some exceptions must be carefully considered. Literature on quantitative analysis is still scanty, particularly when distinguishing benign from malignant neoplasms. Further studies with larger cohorts are definitively required to refine the differential diagnosis between benign and malignant neoplasms. CEUS can also play an essential role in cases of acute scrotum, by excluding infarction and trauma, when testicular torsion cannot be defined. Finally, these interesting preliminary results can instigate the development of innovative research studies on pre-TESE testicular perfusion to increase the chances of sperm recovery.

CONFLICT OF INTEREST
The authors declare no conflict of interest regarding the publication of this article.

AUTHOR CONTRIBUTION
All authors contributed to the conception and design of the review.