Ultrasound evaluation of patients with male accessory gland inflammation: a pictorial review

Abstract Male accessory gland infection/inflammation (MAGI) is a major cause of male infertility. The importance of ultrasound evaluation in these patients is highly controversial, although evidence of its relevance has increased in recent years. Ultrasound criteria are an important element for confirming the clinical diagnosis. Furthermore, they help to localize the anatomical site or sites of the inflammatory process and in assessing its extension which, in turn, have different consequences on the seminological and symptomatic aspects. This article summarizes the clinical interpretations related to ultrasound aspects in patients with MAGI and the possible effects on the seminological, microbiological, endocrinologic, urological, sexological, and internist aspects.


| INTRODUC TI ON
Since the first case of coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was reported in Wuhan, China, it has rapidly spread and affected more than 21 million people worldwide as of 17 August 2020. 1 SARS-CoV-2 uses angiotensin-converting enzyme II (ACE2) to enter host cells, similar to SARS-CoV, which emerged 18 years ago. 2 COVID-19 induces respiratory-predominant multiorgan dysfunction, including myocardial, renal, enteric and hepatic dysfunction, which coincides with the tissue expression of ACE2. 3 Meanwhile, several studies have shown that ACE2 is expressed in human testes (eg spermatogonia, Leydig cells and Sertoli cells), 4,5 suggesting that the testes may be another organ affected by COVID-19.
Numerous viruses have been detected in human semen. 6 Viruses may persist in semen and last longer in seminal fluid than in other body fluids due to the immune privilege of the testes and the contribution of the blood-testes barrier to resistance to therapeutic agents. 7,8 Semen may also have higher loads of viruses, such as Zika virus, than blood. 9,10 Therefore, the testes may act as a reservoir of virus, which may cause imprecise evaluation of viral clearance in patients. Viruses, including Zika virus, Ebola virus, cytomegalovirus and human immunodeficiency virus (HIV), have been isolated from semen and can be sexually transmitted. 6,11,12 Furthermore, some viruses (eg HIV, Zika virus, herpes simplex virus (HSV) and human papillomavirus) can adhere to or be internalized by spermatozoa, 7,13 which may pose a risk for embryonic infection and cause adverse reproductive outcomes.
On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35,37 while the pathological characteristics in survivors remain unknown. In this review, we summarize the current research focusing on the effects of COVID-19 on male reproduction from the following 3 aspects: detection of SARS-CoV-2 in the male reproductive tract, determination of the impact of COVID-19 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.  morphostructural congenital anomalies; (f) hemospermia; (g) disease recurrence in patients with previous prostatectomy. 5 The European Association of Urology (EAU) guidelines suggest the use of TRUS only for patients with a suspicion of obstructive azoospermia and, in particular, when the lack of spermatozoa in the ejaculate is associated with a low volume.
US evaluation of the epididymal tract should be limited to patients with signs indicative of obstruction. These include dilation of the rete testis, cystic dilation of the cephalic tract, suspected absence of the vas deferens. 1 In particular, Pezzella and colleagues showed that when the longitudinal diameter of the epididymal cephalic tract is >0.85 mm associated with serum FSH levels <7.8 IU/ ml, it is suggestive of obstructive azoospermia (sensitivity 58.8%; specificity 91.4%). 6 A recent study conducted by the European Academy of Andrology (EAA) confirmed that the dilation of the head and tail of the epididymis is associated with the presence of acute and chronic inflammation, distal obstruction, and with a positive MAR test, suggesting an association of this finding with alteration of the blood-epididymal barrier. 7 A systematic review has shown that the TRUS evaluation of epididymal and of the prostatevesicular region is useful in the clinical evaluation of patients with chronic inflammation of these anatomical sites suggesting the use of TRUS in the characterization of MAGI. 8 Moreover, a US scan allows more accurate classification of MAGI by identifying the number of glands involved in the inflammatory process as well as its extension. Accordingly, MAGI can be classified in uncomplicated (prostatitis alone) and complicated (prostate-vesiculitis and prostate-vesiculo-epididymitis), and into unilateral or bilateral forms. This classification correlates with the outcome of fertility since complicated and bilateral forms have a worse impact on sperm parameters. 2 Previously, we have reported US criteria suggestive for prostatitis, prostate-vesiculitis), PVE (prostate-vesiculo-epididymitis) ( Table 1). 9 In particular, the diagnostic sensitivity and specificity of US examination increases as the number of US signs found increases.

TA B L E 1 Ultrasound criteria of MAGI 9
Prostatitis is suspected in the presence of >2 of the following ultrasonographic signs: • asymmetry of the gland volume; • areas of low echogenicity; • areas of high echogenicity; • dilatation of periprostatic venous plexus; • single or multiple areas of acinar ectasia; • area/s of moderate increased of vascularity (focal or multiple).
Epididymitis is suspected in the presence of >2 of the following ultrasonographic signs: • increase in size of the head (cranio-caudal diameter >12 mm) and/ or of the tail (cranio-caudal diameter >6 mm) (mono or bilateral); • presence of multiple microcystis in the head and/or tail (mono or bilateral); • low echogenicity or high echogenicity (mono or bilateral); • large hydrocele mono or bilateral; • enlargement in superior part of the cephalic tract and superior/ inferior part ratio >1; • unchanged anteroposterior diameter of tail after ejaculation. On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.

TA B L E 2
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35,37 while the pathological characteristics in survivors remain unknown. In this review, we summarize the current research focusing on the effects of COVID-19 on male reproduction from the following 3 aspects: detection of SARS-CoV-2 in the male reproductive tract, determination of the impact of COVID-19 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| RE SULTS AND D ISCUSS I ON
After reviewing the studies retrieved from the database, citations and references were added based on a review of the title or abstract ( Figure 1). Fourteen studies were eligible and were included in this study, with 12 studies detecting SARS-CoV-2 in the male reproductive tract, 3 determining the impact of COVID-19 on sperm quality and 3 exploring pathological changes in the testes of COVID-19 patients.

| Detection of COVID-19 in the male reproductive tract
Twelve studies investigated the presence of SARS-CoV-2 in the male reproductive tract (eg semen, prostatic secretion or testicular tissue) and are shown in Table 1. Most studies were cross-sectional in design and included mainly Chinese subjects. In brief, ten of 12 studies reported that none of the participants had SARS-CoV-2 RNA | 3 LA VIGNERA Et AL.

| L ABOR ATORY A S PEC T
The inflammatory process that occurs in MAGI correlates with the production of various cytokines that promote the production of oxygen free radicals (ROS), perpetuating the inflammatory process and thus further increasing sperm damage. Interleukin-6 (IL-6), interleukin-8 (IL-8), tumor-necrosis factorα (TNFα), and interleukin-1β (IL1β) are among the main cytokines that correlate with leukocytospermia and chronic prostatitis or chronic pelvic pain syndrome (CP/CPPS). Accordingly, these cytokines have been proposed as markers of MAGI. 12  do not differ between patients with mono or bilateral inflammatory (amicrobic) prostate-vesicular-epididymitis, confirming the importance of US evaluation. 15 The harmful effects of the inflammatory process that occurs in patients with MAGI on sperm quality are present not only on conventional sperm parameters but also on biofunctional ones. In fact, we have shown that 150 patients with MAGI have not only lower seminal fluid volume, sperm concentration, total sperm count, percentage of spermatozoa with normal forms, and progressive motility compared with controls, but also had a higher percentage of spermatozoa with low mitochondrial membrane potential (MMP), phosphatidyl-serine (PS) externalization (a marker of apoptosis), and sperm DNA fragmentation (SDF), and a decreased percentage of alive spermatozoa (evaluated by annexin V/PI assay) than controls, without significant differences between patients with inflammatory or microbic form. 16  show US signs suggestive of testicular abnormalities, such as low testicular volume, testicular inhomogeneity or hypoechogenicity, or epididymal tail inhomogeneity. Instead, spermatozoa brighter to the PI staining are the sum of viable and non-viable spermatozoa, highly oxidized. The increased SDF of PI brighter spermatozoa correlates with the presence of US prostate signs of inflammation, such as macrocalcifications, hyperemia, and the increase of parenchymal peak systolic velocity, suggesting that DNA fragmentation in this sperm population largely originates downstream of the epididymis. 17 Finally, US evaluation also correlates with the degree of semen viscosity. The prevalence of hyperviscosity in subfertile patients was estimated around 26.2% and correlates with worse sperm parameters, in particular sperm motility and inflammation. 18 The progressive anatomical extension of inflammation assessed by US is associated with a proportional increase of the viscosity of the seminal fluid measured centipoise, confirming that prostate-vesicularepididymitis has a higher detrimental effect on sperm parameters than prostate-vesiculitis and the latter in turn more than prostatitis alone. 19

| MICROB IOLOG IC AL A S PEC TS
The presence of prostate microcalcifications, macrocalcifica- On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35   ROS production and leukocytospermia persist even after three antibiotic courses, reinforcing the need for more aggressive treatments in these patients. 23 HPV is another microorganism responsible for infertility and MAGI. In fact, HPV-DNA can be found in the spermatozoa of 2-31% of the general male population and 10-35% of men undergoing assisted reproductive technique (ART). In a previous study, we found a prevalence of HPV in 20.8% of patients with inflammatory MAGI and 28.8% of microbial MAGI compared to 10% of controls suggesting that viral DNA testing should be done in these patients. 24  with the other groups. Accordingly, US evaluation represents a diagnostic element that helps to confirm this diagnostic hypothesis. 25 Furthermore, the persistence of HPV in the seminal fluid of patients with MAGI, in particular for infections with oncogenic genotypes, also correlates with the persistence of US signs of inflammation. 26 TRUS could also improve the specificity of the bacteriological examination performed on prostatic secretion obtained after massage (US integrated Meares and Stamey test). 27 Indeed, thanks to US guidance, the physician could perform the massage on the parenchymal areas with acinar ectasia (Figures 1 and 2) and/or microcalcifications/macrocalcifications (Figure 3) that are often the sites hosting microbial agents. 28 Finally, the microbiological evaluation is required for patients with leukocytospermia (leukocyte concentration >1 million/ml). 29 However, the presence of US criteria suggestive for persistent

| ENDOCRINOLOG IC AL A S PEC TS
In the experimental model, hypogonadism represents a risk factor for the progression of prostatic inflammation. 30 However also F I G U R E 1 Areas of acinar ectasia within the prostate parenchyma On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| RE SULTS AND D ISCUSS I ON
After reviewing the studies retrieved from the database, citations and references were added based on a review of the title or abstract

| Detection of COVID-19 in the male reproductive tract
Twelve studies investigated the presence of SARS-CoV-2 in the male reproductive tract (eg semen, prostatic secretion or testicular tissue) and are shown in Table 1. Most studies were cross-sectional in design and included mainly Chinese subjects. In brief, ten of 12 studies reported that none of the participants had SARS-CoV-2 RNA in clinical practice, lower concentrations of total testosterone are associated with a higher frequency of prostatitis-like symptoms and, in particular, with severe LUTS, such as reduced maximal flow rate (<10 ml/s) and higher post-void residual urine volume (at least 100 ml). 31 This is probably due to the anti-inflammatory effects of androgens. Indeed, hypogonadism increases inflammatory markers (TNFα and IL-6). Conversely, testosterone replacement therapy in patients with hypogonadism decreases inflammatory markers (C-reactive protein, TNFα, and IL-1). 31 From the US point of view, we found a correlation between low serum total testosterone levels and higher frequency of complicated forms of MAGI (bilateral prostate-vesiculo-epididymitis and prostate-vesiculitis), confirming the protective role of androgens against a greater extension of the inflammatory process. This suggests using TRUS in predicting patients who should undergo blood sampling for testosterone measurements. 32 Moreover, eugonadal men have a lower prevalence of the FSUF variant of MAGI that, as above mentioned, represents a US form associated with persistent low-quality sperm parameters after pharmacological treatment 11,32 ( Figure 5).
Another endocrinological condition often related to MAGI is type 2 diabetes mellitus (DM2). We have previously reported that MAGI is present in 43% of patients with DM2 33 with a higher prevalence in DM2 patients with autonomic neuropathy compared to those without this complication. 9 Indeed, these patients present pe- On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35,37 while the pathological characteristics in survivors remain unknown. In this review, we summarize the current research focusing on the effects of COVID-19 on male reproduction from the following 3 aspects: detection of SARS-CoV-2 in the male reproductive tract, determination of the impact of COVID-19 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.  before and after ejaculation and ejaculation fraction of the seminal vesicles. US changes also correlate with increased seminal fluid concentration of fructose and sperm parameter improvement. These findings suggest the use of PDE5i as a valid therapeutic strategy to treat the consequences of diabetic neuropathy on male reproductive health. 37

| SYS TEMI C A S PEC TS
Prostatitis is often associated with irritable bowel disease. In a previous study, we found a prevalence of this disease in 30.3% of patients with prostatitis and 31.9% of prostatitis in patients with irritable bowel disease. 38 In particular, patients with chronic bacterial prostatitis associated with irritable bowel disease have a higher frequency of MAGI compared with patients with chronic bacterial prostatitis without irritable bowel disease. 39 Furthermore, the contemporary presence of both conditions is associated with greater severity of prostatic symptoms. 38 Probably, the same mechanism involved in the pathogenesis of irritable bowel disease symptoms, such as an imbalance between commensal and pathogen bacteria of the intestinal microflora, local low-grade inflammation associated with abnormal immune function, altered intestinal motility, and the intraluminal environment may play a role in the chronicization of bacterial prostatitis. 38 In these patients, the main US characteristic indicative of MAGI is represented by the dilation of the periprostatic venous plexus. 40 This finding could represent another mechanism by which rectal inflammation can affect the male accessory glands, given also the anatomical proximity of these structures. 40

| UROLOG IC AL A S PEC TS
Patients with varicocele, frequently have a concomitant dilation of the periprostatic venous plexus. 41 In a previous study, we reported that patients with concomitant presence of these two venous alterations, maintain seminal fluid hyperviscosity and consequently decreased sperm motility after surgical varicocele repair compared with patients without dilation of the periprostatic venous plexus.
This suggests lower effectiveness of varicocele correction on sperm parameters in these patients. 42 In this study, we excluded MAGI as a confounding factor; however, in clinical practice, MAGI may associate with the presence of varicocele. In these cases, an adequate diagnostic path, also based on a US approach, allows the clinician to be guided toward the most appropriate therapeutic decision.
A recent study also showed an increased risk of prostate cancer in patients with MAGI and a positive correlation of this condition with the Gleason score; thus suggesting that the presence of chronic inflammation may be associated with more aggressive forms of cancer. In particular, the prevalence of prostate cancer was significantly higher in patients with US signs of prostatitis alone and prostate-vesiculitis. 43 However, this aspect still needs to be further investigated.

| S E XUAL A S PEC TS
We have previously shown a higher prevalence of sexual dysfunction in patients with MAGI. In particular, this was more frequent in patients who, in addition to the diagnosis of MAGI made according to the WHO criteria, present also typical US signs, suggesting once again On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35,37 while the pathological characteristics in survivors remain unknown. In this review, we summarize the current research focusing on the effects of COVID-19 on male reproduction from the following 3 aspects: detection of SARS-CoV-2 in the male reproductive tract, determination of the impact of COVID-19 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| RE SULTS AND D ISCUSS I ON
After reviewing the studies retrieved from the database, citations and references were added based on a review of the title or abstract ( Figure 1). Fourteen studies were eligible and were included in this study, with 12 studies detecting SARS-CoV-2 in the male reproductive tract, 3 determining the impact of COVID-19 on sperm quality and 3 exploring pathological changes in the testes of COVID-19 patients.

| Detection of COVID-19 in the male reproductive tract
Twelve studies investigated the presence of SARS-CoV-2 in the male reproductive tract (eg semen, prostatic secretion or testicular tissue) and are shown in Table 1. Most studies were cross-sectional in design and included mainly Chinese subjects. In brief, ten of 12 studies reported that none of the participants had SARS-CoV-2 RNA | 7 LA VIGNERA Et AL. the importance of US for a better characterization of MAGI. 44 The presence of sexual dysfunctions in these patients is probably to be associated with the neuropathy resulting from chronic prostatic and periprostatic inflammation given the proximity of these structures to the nerve pathways responsible for the erectile and ejaculatory mechanisms. 44 According to our findings, Screponi and colleagues found a prevalence of 62.1% of premature ejaculation in patients with CP. 45 Similarly, a Chinese study found a higher prevalence of premature ejaculation and erectile dysfunction in patients with CP and the duration of the chronic inflammatory process. 46 Finally, a meta-analysis showed an overall prevalence of 62% of sexual dysfunctions in patients with CP/CPPS. In particular, the prevalence of erectile dysfunction and premature ejaculation was 29% and 40%, respectively. 47

CO N FLI C T O F I NTE R E S T
All authors declare no competing interests.  On the other hand, many viruses, such as mumps virus, HIV and HSV, 7,14 have been found to impair semen quality, and they may directly interact with spermatozoa or affect spermatogenesis by inducing local inflammation. [15][16][17] Previous studies found that SARS, 1 of the 3 epidemic coronaviruses to emerge in the past 20 years and that shows similar clinical presentations to COVID-19, 18 could cause orchitis 19 and focal testicular atrophy. 20 Considering the tens of millions of COVID-19 cases and that men are more vulnerable to COVID-19 than women, [21][22][23] it is imperative to determine the effect of COVID-19 on male reproduction. 24 Several studies have been performed on this topic. However, the results are controversial. For example, some researchers have reported that SARS-CoV-2 was not detected in the male reproductive tract, [25][26][27][28][29][30][31][32][33][34] while others reported that SARS-CoV-2 RNA was found in the semen or testes of COVID-19 patients. 35,36 There are also unknown factors regarding COVID-19 and male reproduction.

Conceptualization
Orchitis and broad destruction of the testes were found in deceased COVID-19 patients, 35 on sperm quality and exploration of pathological changes in the testes of COVID-19 patients. We further discuss the discrepancies and summarize the unknown topics, which we believe will be helpful for future research.

| reprodu
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