Maria Kazour
email: maria.kazour@univ-littoral.fr

Rachid Amara

Is blue mussel caging an efficient method for monitoring environmental microplastics pollution?

Keywords: Microplastics, Biomonitoring, Caging, Blue mussels, micro-Raman

The effectiveness of mussel caging for active microplastics (MPs) biomonitoring was investigated for the first time by comparing abundance and characteristics (shape, size, color and type of polymers) of MPs ingested by caged depurated blue mussels with those ingested by native mussels collected at the same sites and with those found in their surrounding environment (surface water and sediments). Mussels were exposed along a pollution gradient originating from a wastewater treatment plant discharge and near an abandoned coastal landfill. After 6 weeks of deployment, the majority (93%) of clean transplanted mussels had ingested MPs with a mean number of items ranging from 0.61 to 1.67 items/g. The occurrence, abundance and properties of MPs ingested by caged mussels were similar to those found in native mussels. Among the debris items detected in caged and native mussels, fragments were the most predominant type, consistent with the MPs found in their surrounding environment. MPs sizes were very similar whether in the water, sediments and both caged and native mussels, with a dominance of items < 150 µm. Although some polymers were under-represented or totally absent in the caged mussels compared to overlying seawater or surrounding sediment, there was a good overlap in polymer types proportion being found between caged mussels and sediments (Morisita's index of similarity = 0.93) or seawater (0.86). Polystyrene dominated all samples in all the different matrices.

Our study suggests that blue mussels caging may be a promising tool for MPs biomonitoring making monitoring more reliable with an accurate assessment of the biological effects of MPs over a predetermined exposure period. However, further methodological improvements should be considered to define a uniform protocol for blue mussels caging to allow spatial and temporal microplastics active biomonitoring.

Introduction

Filter-feeding bivalves such as mussels of the genus Mytilus have been widely used as sentinel organisms to monitor coastal water pollution [START_REF] Beyer | Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review[END_REF][START_REF] Marigómez | Marine ecosystem health status assessment through integrative biomarker indices: A comparative study after the Prestige oil spill "mussel Watch[END_REF][START_REF] Turja | The mussel caging approach in assessing biological effects of wastewater treatment plant discharges in the Gulf of Finland (Baltic Sea)[END_REF]. The popularity of mussels as bioindicator stems from their biological and ecological characteristics which make them virtually ideal for pollution monitoring. These sedentary organisms filter large amounts of water efficiently and use waterborne particles as food and, thus, are able to bioaccumulate high amounts of different contaminants [START_REF] Beyer | Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review[END_REF][START_REF] Farrington | Goldberg's proposal of "the Mussel Watch": Reflections after 40 years[END_REF]. Their broad geographical distribution, abundance and ability to tolerate a wide range of environmental parameters make them species of choice in large-scale and long-term marine monitoring programs [START_REF] Andral | Monitoring chemical contamination levels in the Mediterranean based on the use of mussel caging[END_REF][START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF][START_REF] Turja | The mussel caging approach in assessing biological effects of wastewater treatment plant discharges in the Gulf of Finland (Baltic Sea)[END_REF]. Most often mussel biomonitoring studies involve collection of samples from natural populations, but the adoption of an active biomonitoring alternative by using transplanted mussel (caging) has gained considerable popularity in ecotoxicology research and monitoring [START_REF] Andral | Monitoring chemical contamination levels in the Mediterranean based on the use of mussel caging[END_REF][START_REF] Brooks | An integrative biological effects assessment of a mine discharge into a Norwegian fjord using field transplanted mussels[END_REF][START_REF] Galgani | Assessment of polycyclic aromatic hydrocarbon concentrations in mussels (Mytilus galloprovincialis) from the Western basin of the Mediterranean Sea[END_REF][START_REF] Lacroix | Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France)[END_REF][START_REF] Marigómez | Marine ecosystem health status assessment through integrative biomarker indices: A comparative study after the Prestige oil spill "mussel Watch[END_REF][START_REF] Tsangaris | Active biomonitoring in Greek coastal waters: Application of the integrated biomarker response index in relation to contaminant levels in caged mussels[END_REF].

Mussel caging is particularly useful when indigenous mussels are scarce or absent at the planned study sites [START_REF] Li | Using mussel as a global bioindicator of coastal microplastic pollution[END_REF]. In addition, using caged mussels from a single population minimizes confounding factors such as the genetic variability and the physiological, age or reproductive status of the organisms that influence both contaminant bioaccumulation [START_REF] Schøyen | Comparison of caged and native blue mussels (Mytilus edulis spp.) for environmental monitoring of PAH, PCB and trace metals[END_REF][START_REF] Viarengo | The use of biomarkers in biomonitoring: A 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms[END_REF]. However, several studies have previously reported differences in response between caged and native organisms exposed to environmental pollution. Observed differences were attributed to several factors: absence of cumulative long-term effects of pollutants in caged organisms [START_REF] Nigro | Cellular biomarkers for monitoring estuarine environments: Transplanted versus native mussels[END_REF], the existence of adaptive traits or compensatory mechanisms in native mussels chronically exposed to pollution, leading for example to lower bioaccumulation levels than in short-term caged organisms [START_REF] Greenfield | The usefulness of transplantation studies in monitoring of metals in the marine environment: South African experience[END_REF][START_REF] Lacroix | Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France)[END_REF][START_REF] Marigómez | Marine ecosystem health status assessment through integrative biomarker indices: A comparative study after the Prestige oil spill "mussel Watch[END_REF][START_REF] Schøyen | Comparison of caged and native blue mussels (Mytilus edulis spp.) for environmental monitoring of PAH, PCB and trace metals[END_REF]. Others studies found that native mussels accumulated higher concentrations of chemicals [START_REF] Bolognesi | Genotoxicity biomarkers in Mytilus galloprovincialis: Wild versus caged mussels[END_REF][START_REF] Piccardo | Polycyclic aromatic hydrocarbon pollution in native and caged mussels[END_REF]. In some studies, the two monitoring approaches showed similar temporal and spatial changes in chemical contaminant concentrations [START_REF] Hunt | Long-term monitoring using resident and caged mussels in Boston Harbor yield similar spatial and temporal trends in chemical contamination[END_REF][START_REF] Piccardo | Polycyclic aromatic hydrocarbon pollution in native and caged mussels[END_REF]. The caging influence on mussels contaminants accumulation is still poorly understood [START_REF] Schøyen | Comparison of caged and native blue mussels (Mytilus edulis spp.) for environmental monitoring of PAH, PCB and trace metals[END_REF]. Many studies have suggested that an integrated use of monitoring data from both native and transplanted mussels may provide a more accurate assessment of environmental pollution [START_REF] Bebianno | Glutathione Stranferases and cytochrome P450 activities in Mytilus galloprovincialis from the South coast of Portugal: Effect of abiotic factors[END_REF][START_REF] Brooks | Integrated coastal monitoring of a gas processing plant using native and caged mussels[END_REF][START_REF] Hunt | Long-term monitoring using resident and caged mussels in Boston Harbor yield similar spatial and temporal trends in chemical contamination[END_REF][START_REF] Marigómez | Marine ecosystem health status assessment through integrative biomarker indices: A comparative study after the Prestige oil spill "mussel Watch[END_REF][START_REF] Serafim | A multi-biomarker approach in cross-transplanted mussels Mytilus galloprovincialis[END_REF].

Microscopic particles and fragments of plastics smaller than 5 mm (commonly called microplastics, MPs) represent a matter of growing concern for the marine environment. Their ingestion has been documented in several marine species worldwide [START_REF] Cole | Microplastic ingestion by zooplankton[END_REF][START_REF] Watts | Ingestion of Plastic Microfibers by the Crab Carcinus maenas and Its Effect on Food Consumption and Energy Balance[END_REF] particularly in coastal marine environments acting as recipients for plastics and MPs input sources [START_REF] Leslie | Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota[END_REF][START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]. The occurrence and sources of MPs in the marine environment and their potential biological impact are subjects of major scientific and public concern. To monitor spatial and temporal trends of MPs pollution and to understand the risk they pose to the health of marine species, it is important to obtain accurate measurements of MPs abundance in environmental compartments (such as water, sediments and biota). However, in highly variable environment as marine coastal areas, the assessment of MPs abundance in water and sediments is often imprecise and tend to be affected by a variety of environmental factors such as winds, tides, currents and bioturbation [START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF]. A key challenge for marine MPs biomonitoring is to develop an appropriate and standardized method with a sentinel organism that can be applied over large spatial and temporal scales to allow cross calibration between studies worldwide. Recently, many studies have focused on the uptake of MPs by mussels, both in laboratory experiments [START_REF] Browne | Ingested Microscopic Plastic Translocates to the Circulatory System of the Mussel, Mytilus edulis (L.)[END_REF]Van Cauwenberghe et al., 2015) and in their natural habitats [START_REF] De Witte | Quality assessment of the blue mussel (Mytilus edulis): Comparison between commercial and wild types[END_REF][START_REF] Li | Microplastics in mussels sampled from coastal waters and supermarkets in the United Kingdom[END_REF][START_REF] Phuong | Quantification and characterization of microplastics in blue mussels (Mytilus edulis): protocol setup and preliminary data on the contamination of the French Atlantic coast[END_REF][START_REF] Qu | Assessing the relationship between the abundance and properties of microplastics in water and in mussels[END_REF][START_REF] Santana | Microplastic contamination in natural mussel beds from a Brazilian urbanized coastal region: Rapid evaluation through bioassessment[END_REF][START_REF] Zhao | Field-Based Evidence for Microplastic in Marine Aggregates and Mussels: Implications for Trophic Transfer[END_REF]. The species faces a strong exposure to microplastics because of its high filtering activity [START_REF] Riisgård | Allometric equations for maximum filtration rate in blue mussels Mytilus edulis and importance of condition index[END_REF]. Microplastic abundance in field collected mussels is closely related to human activity [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF][START_REF] Li | Microplastics in mussels along the coastal waters of China[END_REF], and there is evidence for a positive and quantitative correlation between MPs in mussels and surrounding waters [START_REF] Qu | Assessing the relationship between the abundance and properties of microplastics in water and in mussels[END_REF].

Their capacity to integrate and amplify the MPs contamination signal [START_REF] Karlsson | Screening for microplastics in sediment, water, marine invertebrates and fish: Method development and microplastic accumulation[END_REF] led to their potential consideration as bioindicator for coastal MPs pollution [START_REF] Beyer | Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review[END_REF][START_REF] Li | Using mussel as a global bioindicator of coastal microplastic pollution[END_REF][START_REF] Wesch | Towards the suitable monitoring of ingestion of microplastics by marine biota: A review[END_REF]. The International Council for the Exploration of the Sea (ICES) has suggested blue mussels as suitable sentinels for monitoring of MPs contamination [START_REF] Ospar | Request on Development of a Common Monitoring Protocol for Plastic Particles in Fish Stomachs and Selected Shellfish on the Basis of Existing Fish Disease Surveys[END_REF][START_REF] Vandermeersch | A critical view on microplastic quantification in aquatic organisms[END_REF]. Although Mytilus spp. controlled deployment has been largely used to study diffuse chemical contaminants and biomarkers, this approach was only recently used to study MPs pollution. To our knowledge, only three studies caged blue mussels in specific areas to investigate the MPs pollution related to specific anthropogenic activity such as wastewater discharge [START_REF] Railo | Application of an enzyme digestion method reveals microlitter in Mytilus trossulus at a wastewater discharge area[END_REF], wreck removal [START_REF] Avio | Microplastics pollution after the removal of the Costa Concordia wreck: First evidences from a biomonitoring case study[END_REF] or to assess seasonal changes in plastic pollution [START_REF] Catarino | Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal[END_REF]. This latter study suggested that the use of cage deployed mussels can be an effective method to quantify and assess MPs pollution in the field whereas the study of [START_REF] Avio | Microplastics pollution after the removal of the Costa Concordia wreck: First evidences from a biomonitoring case study[END_REF] highlighted the limited capability of transplanted mussels to discriminate MPs pollution around the wreck area.

Mussels are hardy creatures tolerant to handling that are easy to keep in culture, making them suitable for translocation and caging exposure. In addition, the mussels can be depurated in the laboratory before caging and, thus, allow the use of reference individuals to be deployed in different areas. The objective of this research was to investigate the MPs ingestion in caged blue mussels exposed along a pollution gradient originating from a WWTP discharge and near an abandoned coastal landfill [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]. The effectiveness of the mussel caging approach to quantify and assess MPs pollution in the field was assessed for the first time by comparing abundance and characteristics (shape, size, color and type of polymers) of MPs ingested by caged mussels with those ingested by native mussels collected at the same site and MPs found in their surrounding environment (surface water and sediments).

Material and methods

2.1 Mussels field deployment and sampling a. Mussels depuration One week prior to field deployment, blue mussels (Mytilus edulis) were collected from farmed Bouchot mussels from Camiers near Boulogne-sur-mer, (50°35'43.1"N, 1°34'41.3"E;

French Eastern English Channel coast). Mussels were sorted in the laboratory to get 157 individuals of the same size range (5.77 ± 0.5 cm shell length, mean ± standard deviation).

Seven of these mussels were immediately frozen at -20°C (reference samples collected prior to the start of the depuration experiment) and the remaining 150 individuals were pre-cleaned to remove any adhering organisms and placed in a 100 L glass aquarium (1 m x 0.45 m x 0.3 m) meticulously pre-rinsed with Milli-Q water. The aquarium was maintained in a thermoregulated room with a constant temperature of 8 ± 1°C and a 12 h light-dark illumination regime. The aquarium was equipped with an air pump and was supplied by filtered sea water (filtered on Polycarbonate filters; 0.1 µm) and the water was changed every 24 hours for 7 consecutive days. During the experiment, the mussels were daily fed (except on Saturday and Sunday) with live microalgae, Rhodomonas sp. and Isochrysis sp. in order to enhance the mussels' filtration capacity. After 7 days of depuration, seven mussels were frozen at -20°C in order to compare the MPs concentration in both non-depurated and depurated mussels. The rest of the mussels were used for the caging experiment.

b. Transplantation and caging experiment

After the depuration period, mussels were transported to caging sites in a small aquarium (60 L, 0.6 m x 0.35 m x 0.3 m) supplied by oxygenated filtered sea water. The translocation experiment was carried out between the 3 rd and the 4 th of February 2018 and lasted 42 days (until the 4 th and 5 th of April 2018). Five marine coastal sites were chosen for the caging experiment: four sites in Le Havre harbor along a pollution gradient originating from a WWTP discharge and one site near an abandoned coastal landfill (Figure 1; see [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF] for more detailed sampling locations). At each site, a stainless-steel cage (50 cm x 50 cm x 50 cm, length x width x height) with no plastic materials to avoid contamination was deployed. The cage's mesh size was 15 mm allowing water circulation and stopping mussels from falling out of the cages. A stainless-steel grid was placed in the mid-height of the cages as a suitable substrate allowing mussels' attachment and avoiding direct contact with sediments. 17 to 23 mussels were put in each cage. The cages were deployed during low tide in the lower intertidal shore zone and attached to the bottom using four reinforced iron rods.

The study area is characterized by semi-diurnal tide with an average tidal range of about 1 m at neap tides and 6 m at spring tides.

After retrieval, each mussel was put separately inside an aluminum foil and frozen at -20°C until analysis. Water temperature, salinity and dissolved oxygen were measured using a multiparameter probe (Mutiparameter HI 982, HANNA instruments) during cages transplantation and retrieval.

Water, sediment and native mussels sampling

Water, sediments and native mussels were taken on the 3 rd and 4 th of April as described in [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]. In short, at each site, 1 to 2 m 3 of the first 10 cm of the water column was pumped and filtered on stainless steel sieves of different mesh sizes (500 µm, 200 µm, 80 µm and 20 µm). Infralittoral sediments were also collected at each site except for site 4 (due to its rocky substrate). Sea water and sediments analyses were preformed according to [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF].

Mussels from the French Eastern English Channel coast are dominated by Mytilus edulis, but some areas are also populated by hybrids between M. galloprovincialis and M. edulis. This is the case of Le Havre Harbor where 70% of the mussels are hybrids (J. Couteau, personal communication). Therefore, for native collected mussels the term Mytilus spp. is used in this study. Native mussels (Mytilus spp.) were hand collected from the lower intertidal shore zone from the same sites near the deployed cages. At each site, 10 mussels of similar size were collected and immediately conserved inside an aluminum foil and frozen at -20°C until analysis. In the Harbor (sites 1 to 4), mussels were bigger 6.44 ± 0.73 cm (mean shell length ± standard deviation) than at site 5 (3.2 ± 0.4 cm).

Microplastics analyses a. Contamination control

Microplastics contamination control is an important step during microplastics analysis.

Several precautions were followed. Cotton lab coats were worn throughout the experiments, and all steps (filtration, measurements and digestion) were realized under a laminar flow hood. Only laboratory materials (bottles, petri dishes, Erlenmeyers, filtration system) made of glass were used. All used solutions were filtered three times on glass fibers filters (GF/A) Whatman, (France) in order to remove any unwanted particles and fibers. All surfaces and equipment were cleaned using filtered ethanol 70% and MilliQ water. With every manipulation, a control was made following the same conditions as the samples in order to track the contamination.

b. Samples preparation

Water and sediment samples were prepared as described by [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]. In short, water samples were rinsed inside a separation funnel and underwent a density separation step using zinc chloride (ZnCl2; 1.8 g/cm 3 ) and the supernatant was filtered on GF/A filters.

Sediment samples were digested using hydrogen peroxide 30% (H2O2 30%) then they underwent density separation using ZnCl2.

Mussels were defrosted 4 hours prior to digestion and their total length, width (cm), total and net weight (g) were measured. The condition index (CI) was calculated as follow (AFNOR, 1985):

= ℎ ℎ × 100
This index is a good indicator for the physiological condition (tissue growth, energy reserves) of mussels (Orban et al., 2002).

Mussels were digested using a solution of potassium hydroxide (KOH) 10% (m/v, ChimiePlus, France) [START_REF] Dehaut | Microplastics in seafood: Benchmark protocol for their extraction and characterization[END_REF]. Caged mussels were thawed, dissected and put inside graduated Erlenmeyers equipped with glass stopper containing a volume of 150 mL of KOH 10% (the volume 150 mL was used because it was the necessary volume that led to an optimal digestion efficiency percentage). These Erlenmeyers were maintained on a heating magnetic stirrer at 60 °C for 24 h. After digestion, the digestat of each individual was filtered on a GF/A filter (90 mm diameter). The same procedure was done with native mussels but a volume of 250 mL was added instead (see [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]). Suspected microplastics were identified using Micro-Raman Xplora Plus (HORIBA Scientific ® , France). The machine is protected by a door so that the filter placed inside would not be affected by any airborne contamination. Due to the time consumption that takes when analyzing filters under micro-Raman, five filters of caged and native mussels containing the highest number of potential MPs from each sampling site were chosen and analyzed. All sediments items, and subsamples of water items were also taken for Raman analysis. For identification, two lasers with a wavelength of 532 nm and 785 nm and a range of 200-3400 cm -1 were used with x10 and x100 objectives (Olympus). Each particle spectrum is compared to a polymer database identification software (KnowItAll, BioRad ® ) and a personal library made with standard polymers obtained from Goodfellow (France). KnowItAll software gives a resemblance (correlation) between the obtained spectra and the one existing in the database.

If the obtained spectrum showed a high fluorescence, several acquisition parameters are changed from one sample to another: whether in the acquisition time (0.2s, 1s, 2s, or 5s), the number of accumulations (2, 5, 10, or 15), the laser wavelength and intensity (0.1%, 1%, 10%, 50% or 100%), the slit and the hole, to obtain a better identifiable spectrum. The identification is considered correct when the HQI (Hit Quality Index) was above 70 (ranging from 0 to 100). Baseline correction was realized on spectra in order to have a cleaner identifiable spectrum. Pigments spectra (Copper phthalocyanine, Hostasol Green, etc…) obtained were identified using an option called "Mixture Analysis" existing in "KnowItAll software" that is capable to decompose a mixed spectrum into two spectra consisting of the dye and its associated polymer (see Supplementary Figure 1 for an example).

Statistical and data analyses

Microplastics found in water, sediments and mussels were reported in unit volume This index ranges from 0 (when no common size or polymer types are shared between the two sample types) and 1 (when all sizes or polymer types are common between the two sample types).

Results

Mussels caging experiment

Before translocation, mussels were depurated. There was no significant difference in mussel's condition index after seven days of depuration (p = 0.264 > 0.05). However, a significant difference in the amount of MPs was observed between non-depurated and depurated mussels (p = 0.018 < 0.05). Non-depurated mussels had an average of 2.22 ± 1.35 items/g with fragments constituting 82.3% of the observed items (Figure 2). The number of items significantly decreased by 97.6% after 7 days of depuration reaching 0.05 ± 0.15 items/g consisting only of fibers. The size of observed fibers before depuration ranged from 41.54 µm to 622.04 µm with a mean size of 284 ± 156.8 µm; these fibers were of different colors: black, green, blue and red. Only two blue fibers were observed after depuration with a respective length of 304.06 and 415.73 µm.

Even though the cages were transplanted during the winter period when the temperature had been at its lowest (around 6-7°C), the mean mussels survival rate was 73%. After 42 days of caging experiment, mussels shell length (5.7 ± 0.47 cm) and condition index (54.7 ± 3.8%)

were not significantly different from the beginning of the experiment (p = 0.621> 0.05). Also, there was no significant inter-sites difference in the caged mussels condition index (p = 0.521).

Microplastics abundance, type and color

The absence of fragments, microbeads and films in our procedural background blanks indicates that contamination was only limited to airborne fibers. The average fiber contamination during caged mussels dissection and digestion had an average of 0.72 ± 0.78 fibers (representing the average found for a batch of 9 mussels representing a total one hour constant laboratory work under the laminar flow hood). Similar values were observed for the other three matrices (see [START_REF] Kazour | Sources of microplastics pollution in the marine environment: Importance of wastewater treatment plant and coastal landfill[END_REF]). The importance of following procedural blanks is that any potential contamination is then eliminated from the analyzed samples.

Microplastic contamination of seawater, sediments, and mussels was evident at all of our sampling sites. The number of suspected MPs in the water decreased with the increasing distance from the effluent of the WWTP and the highest amount was found near the landfill (site 5). Such pattern was not observed for MPs found in the sediments (Figure 3 -A).

Suspected MPs were found in mussels of all sites; 93% of native mussels and 94.7% of caged mussels had ingested suspected MPs with a mean number of items ranging from 0.41 to 2.76 items/g. The spatial pattern of MP abundances in mussels was different from that observed for water or sediment samples (Figure 3). Caged mussels ingested the same or higher amount of MPs than native mussels at all sites except for site 5; however for the same site, the amount of ingested MPs by caged and native mussels were not significantly different (p > 0.05) (Figure 3).

Both fragments and fibers were found in the water, sediment and in both caged and native mussels. In all the matrices analyzed, fragments dominated the type of MPs except at sites 1, 3 and 4 for water samples (Figure 3). Primary microplastics (polystyrene raw microbeads)

were found only at site 5 in the water but were not detected in mussels nor in sediment samples. Films were observed only in native mussels. Blue fragments dominated the water (45%) and sediment (69%) samples whereas red and blue fragments dominated in both caged (76%) and native mussels (42% each). For the fibers, black (51%) and blue and green color (71.4%) dominated the water and sediment samples respectively whereas blue and black fibers dominated in caged (85%) and native (80%) mussels. At each site, the proportion of MPs items type or color was quite similar among caged and native mussels. For fragments, the order of dominant colors was blue, red, green, white and transparent; whereas for fibers, it was blue, black, green and red.

Microplastics size and polymer types

Suspected MPs sizes were very similar whether in the water, sediments and both caged and native mussels, with a dominance of items < 150 µm (Figure 4). This was confirmed by the very high overlaps between caged mussels vs native mussels (0.98), vs sediments (0.9) or vs water samples (0.84). Smaller size classes (< 200 µm) were more abundant in native mussels compared to caged mussels (Figure 4 -A). Caged mussels showed greater number of items with a size class > 200 µm compared to native mussels. Sediments and water samples showed higher proportion of items of size class < 150 µm compared to caged mussels (Figure 4 -B-C).

Of the 503 items analyzed with the micro Raman, 422 (83.9%) were positively identified as a known polymer (examples of various parameters used to identify samples are indicated in Supplementary Table 2). Among the non-polymers items, only 2% were identified as cellulose-based fibers. The rest of the spectra were highly fluorescent and non-identifiable (for example if a sample is too thin, Raman tends to detect the underlying substrate instead of the sample [START_REF] Käppler | Analysis of environmental microplastics by vibrational microspectroscopy: FTIR, Raman or both? Anal[END_REF]).

Five polymers were identified in caged mussels, by order of dominance: polystyrene (PS), acrylonitrile butadiene styrene (ABS), polyurethane (PUR) and polyethylene (PE),. A higher number of polymers were identified in native mussels (9), water (11) and sediments (8) (Figure 5). PS dominated all samples in all the different matrices with ABS and PA the second most abundant polymers. The highest overlap in polymer types was found between caged mussels and sediments (0.93) followed with native mussels (0.88) and water samples (0.86).

Discussion

The key advantage of using blue mussels for microplastics biomonitoring, is that they can be depurated in the laboratory, reducing initial contamination bias, and thus, clean reference individuals will reflect the MPs contamination of their specific deployment sites. In mussels, MPs may be retained for extended periods of time, for example, complete clearance of MPs was not achieved after a seven-days depuration period under laboratory conditions with microbeads (2,6 µm) being retained within the digestive tracts [START_REF] Paul-Pont | Exposure of marine mussels Mytilus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation[END_REF]. Our results showed that after 7 days of depuration, 97.6% of the MPs were eliminated by the mussels. Similar depuration rate (85%) was observed in the Mediterranean mussel, M.

galloprovincialis, after seven days depuration period [START_REF] Fernández | Insights into the uptake, elimination and accumulation of microplastics in mussel[END_REF]. Lower depuration period seems however to be insufficient for total depuration; in a three days depuration experiment of farmed M. edulis, Van Cauwenberghe and Janssen (2014) found a reduction of ca. 33% in the MPs quantity. Since depuration significantly decreased the quantity of MP in blue mussels, the eliminated particles were probably located in the digestive system as suggested for the brown mussels (Perna perna) by [START_REF] Birnstiel | Depuration reduces microplastic content in wild and farmed mussels[END_REF].

The main objective of the study was to test effectiveness of the mussel caging for active microplastics biomonitoring, and this was investigated by comparing MPs ingested by caged blue mussels with those ingested by native mussels collected at the same site and MPs found in their surrounding environment (surface water and sediments). Two aspects can be compared: the quantitative aspect (MPs concentrations in the different matrices) and the qualitative aspect (shape, size, color and type of polymers) of MPs.

Quantitative MPs comparison

A key question for microplastics caging studies is how long the blue mussels should stay deployed to be equalized with native mussels and to reflect MPs concentrations of their surrounding environment. For chemical pollution, the reported caging periods lasted generally between 3 to 6 weeks but may last 18 -24 weeks or up to 2 years (see review in [START_REF] Beyer | Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review[END_REF]. Short term mussel deployments, such as one or two months, has been shown to be suitable for chemical pollutant monitoring in coastal waters, e.g. metals [START_REF] Greenfield | The usefulness of transplantation studies in monitoring of metals in the marine environment: South African experience[END_REF][START_REF] Lacroix | Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France)[END_REF][START_REF] Piccardo | Polycyclic aromatic hydrocarbon pollution in native and caged mussels[END_REF]. To date, only three studies used caged blue mussels to investigate the MPs pollution [START_REF] Avio | Microplastics pollution after the removal of the Costa Concordia wreck: First evidences from a biomonitoring case study[END_REF][START_REF] Catarino | Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal[END_REF][START_REF] Railo | Application of an enzyme digestion method reveals microlitter in Mytilus trossulus at a wastewater discharge area[END_REF]. These studies showed that mussels transplanted for 4 weeks period have accumulated MPs in their tissues. However, they have not compared the ingested MPs with those found in native mussels or in their surrounding environment. In the present study, even though mussels were not transplanted during their optimal condition (the seawater temperature has been at its lowest: around 6-9°C), mussels' condition and growth were maintained after 6 weeks, and their survival rate was high (73%). More importantly, the majority (93%) of transplanted mussels had ingested MPs. This percentage was similar to that of native mussels (94,7%) collected in the same areas. In addition, the spatial pattern in the amount of ingested MPs by native and caged was quite the same except in site 5 where the native mussels ingested higher number of MPs. This difference in ingested MPs can be due to the significantly lower body weight of native mussels from the site 5 compared to that of transplanted mussels (1.07 ± 0.35 g and 5.15 ± 1.11 g, respectively). The increase on the retention time of plastics particles with decreasing size in mussel has been reported [START_REF] Fernández | Insights into the uptake, elimination and accumulation of microplastics in mussel[END_REF][START_REF] Van Cauwenberghe | Microplastics in bivalves cultured for human consumption[END_REF]. Using caged mussels from a single population avoids bias related to the age, the size or the reproductive status of the organisms that can influence ingestion, retention or clearance rates.

Among the detected debris items in native and caged mussels, fragments were the most predominant type observed, consistent with the MPs found in mussels and cockles [START_REF] Hermabessiere | Microplastic contamination and pollutant levels in mussels and cockles collected along the channel coasts[END_REF] and juvenile fish (Kazour et al., 2018) collected along the Eastern English Channel coast. This high occurrence and abundance of fragments in mussels reflect those found in their surrounding environment (seawater and sediments). A recent study showed a positive and quantitative correlation of MPs in mussels (M. edulis and Perna viridis) and their surrounding waters [START_REF] Qu | Assessing the relationship between the abundance and properties of microplastics in water and in mussels[END_REF]. The authors suggested that the amount of MPs in mussels can reflect the real abundance of MPs in the coastal water but that mussels were more likely to ingest smaller MPs. In our study, the spatial pattern of MPs concentrations in mussels (both caged and native) was different from that observed for water or sediment samples. Similar results were observed along the coastal waters of the U.K., with no quantitative correlation between MPs in mussels and their ambient seawaters [START_REF] Li | Microplastics in mussels sampled from coastal waters and supermarkets in the United Kingdom[END_REF][START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF]. Such absence of correlation may be due to the limited sampling sites (only five in our study and seven in [START_REF] Li | Microplastics in mussels sampled from coastal waters and supermarkets in the United Kingdom[END_REF] study) which are insufficient for robust correlation analyses. In addition, it is likely that a range of factors influence mussel encounter rates with particles within their immediate environment, including hydrodynamics processes in the study area, particle behavior in the water column and the mussels or seawater sampling location in the water column [START_REF] Avio | Microplastics pollution after the removal of the Costa Concordia wreck: First evidences from a biomonitoring case study[END_REF][START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF]. In the study of Avio et al.

(2017) mussels were caged for 4 weeks at 2 different depths, approximately at 1.5 m from the surface and from the bottom (30-45 m). They found some differences in the occurrence of ingested MPs as a function of caging depth, with higher ingested particles (32%) in surface deployed mussels compared to those caged in proximity of the bottom (12%). In addition, some variations were observed in terms of typology and size of particles between surface and bottom-caged mussels highlighting the influence of a different distribution of MPs along the water column. The density of the plastic particles will determine their position in the water column, and, therefore, the likelihood of encounter by an organism [START_REF] Desforges | Widespread distribution of microplastics in subsurface seawater in the NE Pacific Ocean[END_REF]. In our study, we sampled surface water whereas mussels were near the bottom. When comparing different matrices or compartments, a more adapted sampling strategy should take into account numerous parameters such as the sampling time according to the tide, the time lag necessary for MPs to sink in the water column, to be ingested by mussels and to be sedimented at the bottom [START_REF] Schmidt | Occurrence of microplastics in surface waters of the Gulf of Lion (NW Mediterranean Sea)[END_REF]. As these conditions have not been implemented in the present study, it is therefore difficult to compare the MP concentrations found in mussels, water and sediments. Thus, the present data should be considered as semiquantitative until the influence of environmental factors on the measured MP concentrations found in the seawater and sediments and those ingested by blue mussels have been further investigated. In addition, an understanding of MPs bioaccumulation is a prerequisite for the use of mussel as biomonitoring organisms in the aquatic environment.

Qualitative (size, color and polymers) MPs comparison

Comparison between native and caged mussels showed that, at each sampling site, the proportion of MPs items type, size, color and to some extent their composition (polymer types) were quite the same with a high overlap index. Both native and caged mussels from all sites have mainly ingested small sized MPs (< 200 μm). This was similar to other studies that demonstrated that MPs below 300 μm were the most found in mussels [START_REF] Leslie | Microplastics en route: Field measurements in the Dutch river delta and Amsterdam canals, wastewater treatment plants, North Sea sediments and biota[END_REF][START_REF] Naji | Microplastics contamination in molluscs from the northern part of the Persian Gulf[END_REF][START_REF] Phuong | Quantification and characterization of microplastics in blue mussels (Mytilus edulis): protocol setup and preliminary data on the contamination of the French Atlantic coast[END_REF]. Blue fragments were the dominant items found in all the analyzed compartments. Although small sized MPs dominated the water and sediments samples, it is currently unknown whether their abundance in mussels are due to mussels size preferences in regards to prey, or because the majority of MPs in the environment were typically in this size range [START_REF] Bråte | Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study[END_REF][START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF].

Concerning polymer types, a good overlap was found between caged mussel and the others compartment analyzed. The highest overlap in polymer types being found between caged mussels and sediments (0.93). Polystyrene (PS) dominated all samples in all the different matrices. This polymer was among the most common plastics used worldwide whether in packaging or other household materials. We found that some polymers were underrepresented or totally absent in the caged mussels compared to overlying seawater or surrounding sediment. More polymer types (N=13) were identified in the surface water compared to sediments (N=9) or caged mussels (N=6). In the South West of England, [START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF] found significant differences in the relative abundance of polymer types and particle sizes between seawater, sediment, and mussels. However, they found that particles within mussels reflected more closely those found in the intertidal surface sediment compared to those found in the surface seawater. This is also the case in our study where particles found within caged mussels presented the highest similarity with those found in the sediments. The bioavailability of MPs to aquatic organism depends on their characteristics, such as polymer type, chemical composition, size, shape, density, etc. [START_REF] Carbery | Trophic transfer of microplastics and mixed contaminants in the marine food web and implications for human health[END_REF]. The shape of MPs may also influences their accumulation (persistence inside mussels) or elimination [START_REF] Browne | Accumulation of Microplastic on Shorelines Woldwide: Sources and Sinks -Environmental Science & Technology (ACS Publications)[END_REF][START_REF] Thompson | Lost at Sea: Where Is All the Plastic?[END_REF]. As suggested by [START_REF] Scott | Particle characteristics of microplastics contaminating the mussel Mytilus edulis and their surrounding environments[END_REF], it is likely that both environmental and biological partitioning of microplastic particles and the selective feeding ecology of this species is responsible for the under-representation of certain polymer types and particle sizes within the mussels. This should be taken into consideration when using mussels for MPs pollution biomonitoring.

Mussels biomonitoring as a standardized tool for microplastics assessment

In this work, we were able to demonstrate the potential use of mussels as a MPs biomonitoring tool. But a lot of factors should be considered to optimize and standardize this tool. The first issue is that mussels represent different filtration rates depending on the region and genotype, for example [START_REF] Lüskow | In Situ Filtration Rates of Blue Mussels (Mytilus edulis) Measured by an Open-Top Chamber Method[END_REF][START_REF] Riisgård | Effect of salinity on filtration rates of mussels Mytilus edulis with special emphasis on dwarfed mussels from the low-saline Central Baltic Sea[END_REF]. The difference in the uptake rate for analogous species needs to be further studied to appropriately adjust interspecific difference. Algal abundance, temperature and salinity of water, as well as the depth of the transplanted cages could alter mussel's filtration and clearance rates [START_REF] Comeau | Comparison of eastern oyster (Crassostrea virginica) and blue mussel (Mytilus edulis) filtration rates at low temperatures[END_REF][START_REF] Riisgård | Allometric equations for maximum filtration rate in blue mussels Mytilus edulis and importance of condition index[END_REF][START_REF] Riisgård | Effect of salinity on filtration rates of mussels Mytilus edulis with special emphasis on dwarfed mussels from the low-saline Central Baltic Sea[END_REF] and, therefore, their MPs uptake. For example, differences in MPs ingestion have been observed between caged mussel's transplanted at 1.5m and 30-45m depth [START_REF] Avio | Microplastics pollution after the removal of the Costa Concordia wreck: First evidences from a biomonitoring case study[END_REF]. In addition, seasonal variation for MPs intake has been observed in caged mussels but it has been more linked to an important water flow from a river rather than the different seasons [START_REF] Catarino | Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal[END_REF]. More progression in this area is highly needed to obtain a standardized protocol. Such tool would constitute plenty of advantages for MPs evaluation such as allowing a microplastics assessment while using similar population (genetic and physiological) in areas lacking mussel beds, controlling exposition period and to potentially study the toxicological impact of microplastics on mussels exposed in the natural environment.

Conclusion

In summary, the present study demonstrated for the first time the effectiveness of the mussel caging approach. The abundance and properties of MPs found in caged and native mussels were quite similar. If we rely on the assumption that MPs concentrations in native mussels have reached steady-state (balance between intake and defecation/egestion), the results of this study indicate that short term mussel deployments (i.e. 6 weeks) appear to be suitable to reflect native mussels MPs ingestion. Although microplastics monitoring is a complex task that it is in its initial phases [START_REF] Carbery | Trophic transfer of microplastics and mixed contaminants in the marine food web and implications for human health[END_REF], caging presents many advantages including the selection of well-characterized homogenous organisms (number, age, size, weight, sex)

and the control of exposure (location, time, season). To the best of our knowledge, mussels caging as tool to study microplastics pollution has not been investigated before. Though the abundance and properties of microplastics in mussels does not exactly match those in their environment (seawater and sediments), our study suggests that blue mussels caging may be a promising tool for MPs biomonitoring in marine coastal environments. The caging approach makes monitoring more reliable and may allow accurate assessment of the biological effects of MPs over a predetermined exposure period. However, further methodological improvements should be considered, such as the choice of the deployment time and the caging location (with respect to the shore and the depth of their implantation in the water column), to define a uniform protocol for blue mussels caging allowing an accurate spatial and temporal microplastics biomonitoring.

Funding

Authors Contributions

  c. Visual and Raman spectroscopy analysisAll filters were observed under 120x magnification using Leica M165 C Stereomicroscope and images of suspected MPs particles were taken with a Leica M170 HD camera and LAS (Leica application suite) software. All suspected MPs particles were counted, categorized by type (fragments, fibers, microbeads or films) and color. The following criteria were taken into consideration while observing the items: (1) absence of cellular or organic structures; (2) a homogenous thickness across the particles; and, (3) homogenous colors[START_REF] Hidalgo-Ruz | Microplastics in the marine environment: A review of the methods used for identification and quantification[END_REF]. Measurements were done on the suspected items at their longest dimension and they were divided into different size classes of 50 µm.

:

  (per L) and per gram of sample, respectively. Statistics were performed with SPSS software (IBM SPSS STATISTICS 20). The Wilcoxon signed-rank test was used to compare the ingested MPs between depurated and non-depurated mussels and a Student t-test was used to compare the condition index. For inter-site comparisons, if data did not comply with the parametric assumption of normality (Shapiro-Wilk tests) and homogeneity of variance (Levene tests), the non-parametric Kruskall-Wallis test and Mann-Whitney U test for post hoc pairwise comparisons were used. All results are expressed as mean ± SD, and p-value < 0.05 was considered as statistically significant. Morisita's index of similarity (or Morisita overlap index) is a measure of how similar or different two sets of data are. This index was calculated to compare size and polymer types similarity found in caged mussels and those found in native mussels, water and sediments samples. Morisita's overlap index between j and k j: Caged mussels k: Native mussels or water or sediments ̂ : Proportion of a size class or a polymer i used by j ̂ : Proportion of a size class or a polymer i l used by k 3 : Number of size class or polymer j that use i 3 : Number of size class or polymer k that use i ; : Total number of size class or polymer in sample

Maria

  Kazour is financially supported by a PhD fellowship from the National Council for Scientific Research (Lebanon) and Université du Littoral Côte d'Opale (France). This work has been partially financially supported by the European Union (ERDF), the French Government, the Région Hauts-de-France and IFREMER, in the framework of the project CPER MARCO 2015-2020.

Acknowledgments

The authors would like to thank the administration of Le Havre harbor (especially Miss.

Magali HAUTOT who helped obtaining permissions to enter the harbor and was our companion during sampling), Khalef Rabhi and Sarah Terki for field assistance. The author would like also to thank Sami Souissi and Capucine Bialais for providing use live microalgae cultures.

Declaration of interests: None


Dr Stephanie Nougaret
email: stephanienougaret@free.fr.

PhD Martina Sbarra

Jessica Robbins

Imaging Spectrum of Benign Uterine Disease and Treatment Options

Keywords: Leiomyoma, Leiomyosarcoma, Adenomyosis, MRI, Uterus

Benign uterine disease is mainly comprised of adenomyosis and leiomyomas, common gynecologic conditions affecting women of all ages.

♦ Magnetic resonance imaging (MRI) is the most accurate imaging modality for detection and localization of benign uterine disease and their mimics.

♦ MRI is the diagnostic tool of choice for pretreatment evaluation, assessing potential procedural risk and predicting treatment response, and monitoring treatment outcomes.

Introduction:

Benign uterine disease, such as adenomyosis and leiomyomas, are common gynecologic conditions affecting women of all ages [START_REF] Association | Advancing Minimally Invasive Gynecology W. AAGL practice report: practice guidelines for the diagnosis and management of submucous leiomyomas[END_REF][START_REF] Brosens | Uterine junctional zone: function and disease[END_REF][START_REF] Habiba | Adenomyosis and Endometrial Cancer: Literature Review[END_REF][START_REF] Krentel | From Clinical Symptoms to MR Imaging: Diagnostic Steps in Adenomyosis[END_REF][START_REF] Munro | Uterine polyps, adenomyosis, leiomyomas, and endometrial receptivity[END_REF][START_REF] Vannuccini | Recent advances in understanding and managing adenomyosis[END_REF] . Although ultrasound (US) is the first line imaging technique in the examination of the uterus, magnetic resonance imaging (MRI) has become very useful and is the most accurate tool in lesion diagnosis and patient management [START_REF] Agostinho | MRI for adenomyosis: a pictorial review[END_REF][START_REF] Bolan | MR imaging of atypical fibroids[END_REF][START_REF] Brown | Uterine junctional zone: correlation between histologic findings and MR imaging[END_REF][START_REF] Demulder | Uterine Leiomyosarcoma: Can MRI Differentiate Leiomyosarcoma From Benign Leiomyoma Before Treatment?[END_REF][START_REF] Deshmukh | Role of MR imaging of uterine leiomyomas before and after embolization[END_REF][START_REF] Dudiak | Uterine leiomyomas in the infertile patient: preoperative localization with MR imaging versus US and hysterosalpingography[END_REF][START_REF] Kubik-Huch | European Society of Urogenital Radiology (ESUR) Guidelines: MR Imaging of Leiomyomas[END_REF] . Indeed, benign uterine disease may manifest with atypical features and can mimic malignancy, making the correct diagnosis a challenge [START_REF] Demulder | Uterine Leiomyosarcoma: Can MRI Differentiate Leiomyosarcoma From Benign Leiomyoma Before Treatment?[END_REF][START_REF] Lakhman | Differentiation of Uterine Leiomyosarcoma from Atypical Leiomyoma: Diagnostic Accuracy of Qualitative MR Imaging Features and Feasibility of Texture Analysis[END_REF][START_REF] Sato | Clinical application of diffusion-weighted imaging for preoperative differentiation between uterine leiomyoma and leiomyosarcoma[END_REF] . MRI also assists in the triage of symptomatic patients to the most appropriate treatment modality, including surgery (hysterectomy or myomectomy), interventional procedures (uterine artery embolization) and medical therapy [START_REF] Dubuisson | The current place of mini-invasive surgery in uterine leiomyoma management[END_REF][START_REF] Lewis | A Comprehensive Review of the Pharmacologic Management of Uterine Leiomyoma[END_REF][START_REF] Owen | Clinical management of leiomyoma[END_REF] . Hysterectomy is curative, but uterus-sparing therapies are a valid alternative for eligible women.

In this article we highlight the utility of MRI in the diagnosis of the most common benign uterine diseases, such as leiomyomas and adenomyosis, discuss their typical and atypical MRI findings, describe the therapeutic options, and delineate the role of MRI in treatment planning.

MRI Protocol:

Clinical guidelines for diagnosis and management of patients with leiomyomas are extensive [START_REF] Association | Advancing Minimally Invasive Gynecology W. AAGL practice report: practice guidelines for the diagnosis and management of submucous leiomyomas[END_REF][START_REF] Stokes | Quality improvement guidelines for uterine artery embolization for symptomatic leiomyomas[END_REF][START_REF] Burke | ACR Appropriateness Criteria(R) on treatment of uterine leiomyomas[END_REF] .

Recently, recommendations for MRI were proposed by the European Society of Urogenital Radiology [START_REF] Kubik-Huch | European Society of Urogenital Radiology (ESUR) Guidelines: MR Imaging of Leiomyomas[END_REF] .

Patient Preparation:

Fasting and the use of an antiperistaltic agent such as Buscopan or glucagon is useful to minimize motion artifacts related to small bowel peristalsis. A moderately full bladder is recommended to reduce artifacts related to bladder filling [START_REF] Kubik-Huch | European Society of Urogenital Radiology (ESUR) Guidelines: MR Imaging of Leiomyomas[END_REF] .

Imaging Protocol

High resolution thin-section images acquired at 1.5 T or 3.0 T are recommended.

The optimal protocol, according to recent ESUR guidelines is summarized in table 1 [START_REF] Kubik-Huch | European Society of Urogenital Radiology (ESUR) Guidelines: MR Imaging of Leiomyomas[END_REF] .

Leiomyomas Epidemiology, pathophysiology:

Leiomyomas are the most common benign uterine tumor affecting up to 20-30% of reproductive-aged women [START_REF] Deshmukh | Role of MR imaging of uterine leiomyomas before and after embolization[END_REF] . They are benign neoplasms of unknown cause composed of multiple layers of smooth muscle fascicles and fibrous connective tissue anchored in the muscular wall of the uterus. These tumors may be solitary or, most frequently, multiple [START_REF] Murase | Uterine leiomyomas: histopathologic features, MR imaging findings, differential diagnosis, and treatment[END_REF] . The size of leiomyomas is variable and influenced by estrogen and progesterone; they often grow during pregnancy and with oral contraceptive use and regress after menopause [START_REF] Murase | Uterine leiomyomas: histopathologic features, MR imaging findings, differential diagnosis, and treatment[END_REF] . They may be asymptomatic but 20-50% of women with leiomyomas present with symptoms such as menorrhagia, dysmenorrhea, pressure, urinary frequency, pelvic and back pain, and dyspareunia [START_REF] Munro | Uterine polyps, adenomyosis, leiomyomas, and endometrial receptivity[END_REF] .

Location:

Leiomyomas generally involve the myometrium of the uterine corpus and are classified by their location as submucosal, intramural and subserosal. In accordance with the FIGO classification system, they can be further subdivided in 8 categories (Figure 1) [START_REF] Munro | FIGO classification system (PALM-COEIN) for causes of abnormal uterine bleeding in nongravid women of reproductive age[END_REF] . Pedunculated lesions can become detached from the uterus, receiving blood supply from other adjacent structures, and are called "parasitic" leiomyomas [START_REF] Araki | MR Imaging of Parasitic Leiomyoma with Red Degeneration[END_REF][START_REF] Garrido Oyarzun | Anterior abdominal wall parasitic leiomyoma: case report[END_REF] . Leiomyomas not related with the myometrium may be located in the cervix and in the round or broad ligaments.

Leiomyomas may occur in unusual locations such as diffuse peritoneal leiomyomatosis, intravenous leiomyomatosis or benign metastasizing leiomyoma. Peritoneal leiomyomatosis is characterized by multiple lesions along the peritoneal surfaces probably due to the iatrogenic dissemination throughout the peritoneal cavity after surgery [START_REF] Fasih | Leiomyomas beyond the uterus: unusual locations, rare manifestations[END_REF] . A previous history of hysterectomy for leiomyomas or a diagnosis of uterine leiomyomas may point to the correct diagnosis. Intravenous leiomyomatosis has an aggressive intravascular growth pattern within intrauterine and systemic veins [START_REF] Fasih | Leiomyomas beyond the uterus: unusual locations, rare manifestations[END_REF] .

Imaging features

Ultrasound:

Ultrasound (US) is usually the initial imaging study.

On US, a typical leiomyoma is a well-defined mass, often shadowing at the margin and/or producing internal linear shadowing. The echogenicity varies from hypo to hyperechoic in relationship to the myometrium. Dystrophic calcifications can be seen particularly in postmenopausal women. On color -Doppler US, circumferential blood flow around the lesion is often seen [START_REF] Van Den Bosch | Terms, definitions and measurements to describe sonographic features of myometrium and uterine masses: a consensus opinion from the Morphological Uterus Sonographic Assessment (MUSA) group[END_REF] .

MRI:

US can be limited by co-existing pelvic diseases, uterine anomalies, unusually small or large tumors, and tumor location [START_REF] Hricak | Uterine leiomyomas: correlation of MR, histopathologic findings, and symptoms[END_REF] . Thus, MRI is considered the most accurate imaging modality to detect, locate and characterize myometrial lesions prior to patient management [START_REF] Lakhman | Differentiation of Uterine Leiomyosarcoma from Atypical Leiomyoma: Diagnostic Accuracy of Qualitative MR Imaging Features and Feasibility of Texture Analysis[END_REF][START_REF] Dueholm | Accuracy of magnetic resonance imaging and transvaginal ultrasonography in the diagnosis, mapping, and measurement of uterine myomas[END_REF] . MRI has reported sensitivities and specificities of 94.1% and 68.7%, respectively, for uterine leiomyomas [START_REF] Murase | Uterine leiomyomas: histopathologic features, MR imaging findings, differential diagnosis, and treatment[END_REF][START_REF] Hricak | Uterine leiomyomas: correlation of MR, histopathologic findings, and symptoms[END_REF][START_REF] Mittl | High-signal-intensity rim surrounding uterine leiomyomas on MR images: pathologic correlation[END_REF] .

On MRI, most leiomyomas, without any degeneration, are easily recognized as well-delineated round or ovoid lesions homogeneously hypointense on T2WI related to the outer myometrium and isointense on T1WI, with heterogeneous and variable enhancement. The presence of a T2hyperintense rim indicates a pseudocapsule of edema due to dilated lymphatic vessels and veins [START_REF] Mittl | High-signal-intensity rim surrounding uterine leiomyomas on MR images: pathologic correlation[END_REF] (Figure 2). Typical leiomyomas do not have restricted diffusion, showing a low signal intensity both on diffusion weighted images (DWI) and on the corresponding apparent diffusion coefficient (ADC) map, as a "blackout phenomenon" 10 (Figure 2). Two particular subtypes of leiomyoma have to be mentioned: Cellular leiomyoma is a histological subtype characterized by more compact smooth muscle cells with little or no collagen; on MRI it shows higher signal intensity on T2WI and avid enhancement [START_REF] Deshmukh | Role of MR imaging of uterine leiomyomas before and after embolization[END_REF] (Figure 3). The intermediate T2 signal and avid enhancement of cellular leiomyomas can make it difficult to differentiate them from leiomyosarcoma [START_REF] Nougaret | Endometrial Cancer MRI staging: Updated Guidelines of the European Society of Urogenital Radiology[END_REF] (Figure 3). In a retrospective study of 51 patients with a single myometrial lesion at MRI, Thomassin-Naggara et al suggested that the evaluation of DWI and ADC (value cut-off 1.23 x 10 -3 mm 2 /s) may limit the misdiagnosis of uterine sarcoma as leiomyoma with diagnostic accuracy of 92.4% 31 (Figure 3). Lipoleiomyoma occurs in 0.03-0.2% of women, generally in the post-menopausal population.

Liopleiomyomas are comprised of smooth muscle, adipose and fibrous tissue. The signal intensity of the fat components is typical high signal on T1WI and T2WI and loss of signal intensity on fatsaturated sequences [START_REF] Demulder | Uterine Leiomyosarcoma: Can MRI Differentiate Leiomyosarcoma From Benign Leiomyoma Before Treatment?[END_REF] .

When leiomyomas enlarge and outgrow their blood supply (≥ 5 cm in size), they may degenerate Hyaline degeneration is the most common type (60%). Leiomyomas with hyaline degeneration have low signal intensity on T2WI, an appearance similar to that of non-degenerated leiomyomas; however, they enhance to a lesser degree than standard leiomyomas [START_REF] Arleo | Review of Leiomyoma Variants[END_REF] (Figure 4a,b).

Cystic degeneration (4%) is characterized by the presence of cystic areas with high signal intensity on T2-WI that do not enhance [START_REF] Murase | Uterine leiomyomas: histopathologic features, MR imaging findings, differential diagnosis, and treatment[END_REF] (Figure 4c,d).

Myxoid degeneration is relatively rare and depends on the presence of gelatinous intralesional foci at gross examination that contain hyaluronic acid-rich mucopolysaccharides [START_REF] Prayson | Pathologic considerations of uterine smooth muscle tumors[END_REF] . Leiomyomas with myxoid degeneration have an extremely high signal intensity on T2WI and enhance well except for foci of mucinous lakes or clefts. Delayed and prolonged contrast enhancement is due to the presence of myxoid stroma [START_REF] Ueda | Unusual appearances of uterine leiomyomas: MR imaging findings and their histopathologic backgrounds[END_REF] (Figure 4e,f).

Hemorrhagic degeneration (red degeneration) is due to hemorrhagic infarction resulting in coagulative necrosis and is associated with pregnancy and oral contraceptives [START_REF] Kawakami | Red degeneration of uterine leiomyoma: MR appearance[END_REF] . Red degeneration may also occur after uterine artery embolization (UAE). On MRI, the signal intensity is variable: peripheral or diffuse hyperintensity on T1WI and inhomogeneous signal intensity on T2WI with or without the hypointense rim. The T1-hyperintensity is due to the proteinaceous content of the blood or to the T1-shortening effect of methemoglobin [START_REF] Dudiak | Uterine leiomyomas in the infertile patient: preoperative localization with MR imaging versus US and hysterosalpingography[END_REF] . When the hyperintensity is peripheral, it may be caused by thrombosis of the vessels that surround the lesion [START_REF] Kawakami | Red degeneration of uterine leiomyoma: MR appearance[END_REF] . (Figure 4g,h).

Calcific degeneration is associated with end-stage hyaline degeneration and post-UAE treatment changes. On MRI the calcific components produce signal voids on all sequences [START_REF] Demulder | Uterine Leiomyosarcoma: Can MRI Differentiate Leiomyosarcoma From Benign Leiomyoma Before Treatment?[END_REF] .

Smooth muscle tumor of uncertain malignant potential (STUMP) is a rare heterogeneous tumor

that cannot be definitively classified histologically as leiomyoma or leiomyosarcoma [START_REF] Bolan | MR imaging of atypical fibroids[END_REF] . Multiple subtypes have been identified according to nuclear atypia, mitotic rate and necrosis. On MRI, there are no specific features, because STUMP mimics both typical leiomyoma and leiomyosarcoma. After removal they have a high rate of recurrence (7.3-12.5%) and may recur as low-grade leiomyosarcoma; therefore, long-term follow-up is needed [START_REF] Bolan | MR imaging of atypical fibroids[END_REF][START_REF] Demulder | Uterine Leiomyosarcoma: Can MRI Differentiate Leiomyosarcoma From Benign Leiomyoma Before Treatment?[END_REF] .

Pitfalls to avoid on MRI:

Distinction between an ovarian mass and a uterine mass: It might be hard to distinguish uterine leiomyoma from a fibrous ovarian mass. In the instance of an ovarian mass, sharp angles between the ovary and the lesion are known as the "beak sign". In contrast, in the case of a uterine mass, a normal ovary will be seen separate from the mass. The "claw sign" (uterine tissue draped around the mass) (Figure 4c) or the "bridging vessels" sign (enlarged and tortuous vessels extending from the uterus to the lesion) suggest the diagnosis of a uterine mass (Figure 4h,5).

Focal myometrial contractions:

Contractions may appear as low T2 signal myometrial masses and may simulate uterine leiomyomas. Since they are transient and usually do not persist during the entire examination they can be easily differentiated from leiomyomas (Figure 6).

Endometrial polyp: Submucosal leiomyoma may be mistaken for an endometrial polyp. Polyps have heterogeneous or high signal on T2WI in contrast to pedunculated submucosal leiomyomas which are low signal on T2WI and have a stalk that arises from the myometrium.

Adenomyomas: (see adenomyosis section below).

Leiomyosarcomas: It is critical to differentiate a benign leiomyoma from leiomyosarcoma. While leiomyosarcomas arise de novo and have no biologic link to leiomyomas [START_REF] Ricci | Uterine leiomyosarcoma: Epidemiology, contemporary treatment strategies and the impact of uterine morcellation[END_REF] , they may present with symptoms and imaging features similar to leiomyomas. Large size and rapid growth are unreliable signs of malignancy [START_REF] Ricci | Uterine leiomyosarcoma: Epidemiology, contemporary treatment strategies and the impact of uterine morcellation[END_REF] . Growth of a uterine mass after menopause and elevated LDH, particularly LDH isozyme type 3, is suspicious for leiomyosarcoma [START_REF] Ricci | Uterine leiomyosarcoma: Epidemiology, contemporary treatment strategies and the impact of uterine morcellation[END_REF] . Endometrial sampling may aid diagnosis of uterine sarcoma but sensitivity is limited due to the myometrial origin of the tumor [START_REF] Ricci | Uterine leiomyosarcoma: Epidemiology, contemporary treatment strategies and the impact of uterine morcellation[END_REF][START_REF] Rieber | MRI in the diagnosis of small bowel disease: use of positive and negative oral contrast media in combination with enteroclysis[END_REF] . While no single MRI feature can reliably distinguish leiomyosarcomas from atypical leiomyomas, a combination of MRI features may improve the diagnostic performance of MRI for the correct diagnosis of leiomyosarcoma. In a study from Lakhman et al, the combination of 3 or more of 4 discriminative features including nodular borders, hemorrhage (high-SI on T1WI), T2W dark areas and central areas of nonenhancement were associated with an improved sensitivity and specificity for the diagnosis of leiomyosarcoma 14 (Figure 7). Uterine sarcoma also demonstrates rapid early enhancement of the solid components and restricted diffusion [START_REF] Bolan | MR imaging of atypical fibroids[END_REF] . Diffusion restriction alone is insufficient for diagnosis as there is considerable overlap in ADC values between leiomyomas and leiomyosarcoma; leiomyomas may demonstrate restricted diffusion especially cellular leiomyomas [START_REF] Namimoto | Combined use of T2-weighted and diffusionweighted 3-T MR imaging for differentiating uterine sarcomas from benign leiomyomas[END_REF] (Figure 7). Notably, the presence of restricted diffusion and a T2 blackout effect is highly specific for a leiomyoma [START_REF] Sato | Clinical application of diffusion-weighted imaging for preoperative differentiation between uterine leiomyoma and leiomyosarcoma[END_REF] (Figure 2). More recently, Thomassin-Naggara et al. reported that using a recursive model combining T2 signal intensity, b1000 images and ADC map with a cut off value 1.23, MRI achieved 92.4% accuracy in distinguishing benign and uncertain or malignant myometrial tumors. The authors concluded that DWI may be of interest to distinguish between uterine sarcomas and benign leiomyomas [START_REF] Thomassin-Naggara | How to differentiate benign from malignant myometrial tumours using MR imaging[END_REF] . Figure 8 is an algorithm to help differentiate leiomyoma and leiomyosarcoma.

Treatment options:

Depending on the spectrum of symptoms a patient is experiencing, they may benefit from conservative management. Asymptomatic leiomyomas do not require treatment. Nonsteroidal anti-inflammatory medications may diminish symptoms of dysmenorrhea, but it is unclear if these effects extend to women with dysmenorrhea because of leiomyomas [START_REF] College Of | ACOG practice bulletin. Alternatives to hysterectomy in the management of leiomyomas[END_REF] . Oral contraceptives or gonadotropin-releasing hormone (GnRH) agonists may be used to reduce menstrual bleeding associated with leiomyomas [START_REF] College Of | ACOG practice bulletin. Alternatives to hysterectomy in the management of leiomyomas[END_REF][START_REF] Marret | Therapeutic management of uterine fibroid tumors: updated French guidelines[END_REF] . GnRH agonists may also reduce the size of leiomyomas [START_REF] College Of | ACOG practice bulletin. Alternatives to hysterectomy in the management of leiomyomas[END_REF] . While some advocate the use of levonorgestrel-releasing intrauterine device (IUD) as a means to reduce menstrual bleeding with leiomyomas, the data to support its effectiveness are limited [START_REF] College Of | ACOG practice bulletin. Alternatives to hysterectomy in the management of leiomyomas[END_REF][START_REF] Sangkomkamhang | Progestogens or progestogen-releasing intrauterine systems for uterine fibroids[END_REF] . Since submucosal leiomyomas increase the risk of IUD expulsion and bleeding complications, they are a relative contraindication for the use of an IUD [START_REF] Marret | Therapeutic management of uterine fibroid tumors: updated French guidelines[END_REF] .

Women with refractory abnormal bleeding or those experiencing bulk symptoms, such as pelvic fullness, constipation or urinary symptoms, may be eligible for surgical management. Uterinesparing myomectomy can be considered for patients with three or fewer dominant symptomatic leiomyomas of less than 8 cm in diameter [START_REF] Marret | Therapeutic management of uterine fibroid tumors: updated French guidelines[END_REF] . Hysteroscopic myomectomy is optimal for the treatment of submucosal leiomyomas that are predominantly intracavitary, are less than 4 cm in size, and have at least 5 mm of intact myometrium overlying the leiomyoma while laparoscopic or open myomectomy is preferred for subserosal or intramural leiomyomas [START_REF] Marret | Therapeutic management of uterine fibroid tumors: updated French guidelines[END_REF] . If the patient no longer desires childbearing, hysterectomy, via abdominal, laparoscopic, or vaginal approach, is a consideration. While there is risk inherent with surgical intervention, hysterectomy yields the highest degree of symptom improvement [START_REF] Spies | Outcomes from leiomyoma therapies: comparison with normal controls[END_REF] .

Uterine artery embolization (UAE) is a minimally-invasive uterine-sparing treatment option for leiomyomas resulting in abnormal bleeding, anemia, and/or bulk symptoms. UAE is an alternative to myomectomy, but, unlike myomectomy, it is also useful in patients with multiple (>3) and large (> 10 cm) leiomyomas [START_REF] Smeets | Uterine artery embolization in patients with a large fibroid burden: long-term clinical and MR follow-up[END_REF] . UAE offers the opportunity to preserve fertility, improves or eliminates bulk symptoms and bleeding, is durable, and has a low complication rate [START_REF] Lohle | Long-term outcome of uterine artery embolization for symptomatic uterine leiomyomas[END_REF][START_REF] Goodwin | Uterine artery embolization for treatment of leiomyomata: long-term outcomes from the FIBROID Registry[END_REF] . There is a roughly 10-15% treatment failure rate in which patients will require additional treatment with hysterectomy, myomectomy, or repeat UAE [START_REF] Goodwin | Uterine artery embolization for treatment of leiomyomata: long-term outcomes from the FIBROID Registry[END_REF][START_REF] Pelage | Uterine fibroid vascularization and clinical relevance to uterine fibroid embolization[END_REF] . Some centers utilize UAE prior to myomectomy. In this scenario, UAE is performed 24-48 hours before standard myomectomy. Compared to myomectomy alone, this combined technique decreases the risk of bleeding requiring transfusion, minimizes the risk of conversion to hysterectomy, and results in a shorter postoperative hospital stay [START_REF] Mclucas | The effectiveness of combined abdominal myomectomy and uterine artery embolization[END_REF] .

Adenomyosis: Epidemiology, clinical symptoms and pathophysiology:

Adenomyosis is a common benign gynecological condition. It is defined as ectopic endometrial glands and stroma in the myometrium more than 2.5 mm from the endometrium-myometrium interface. [START_REF] Munro | Uterine polyps, adenomyosis, leiomyomas, and endometrial receptivity[END_REF][START_REF] Tetikkurt | Coexistence of adenomyosis, adenocarcinoma, endometrial and myometrial lesions in resected uterine specimens[END_REF] . While it is estimated that up to one-third of patients with adenomyosis are asymptomatic, the symptoms of adenomyosis are common but are nonspecific including dysmenorrhea, menorrhagia, and abnormal vaginal bleeding. Adenomyosis is associated with female infertility possibly in part due to the overlapping pathophysiology and association with endometriosis [START_REF] Tskhay | Diffuse massive adenomyosis and infertility. Is it possible to treat this condition?[END_REF] . The etiology of adenomyosis is still unclear [START_REF] Krentel | From Clinical Symptoms to MR Imaging: Diagnostic Steps in Adenomyosis[END_REF] . Exposure to estrogen, prior uterine surgery and parity are known risk factors.

Ultrasound Imaging Features:

Multiple ultrasound features have been associated with adenomyosis including myometrial heterogeneity with thin linear shadowing ("venetian blinds") alternating with increased echogenicity, globular uterine enlargement, asymmetric thickening of the myometrium ("pseudowidening sign") and isolated or clustered small anechoic cysts 53-62 63 .The ultrasound appearance of adenomyomas may be similar to uterine leiomyoma. Leiomyomas usually have a well-defined border and peripheral color flow, whereas adenomyomas tend to be ill-defined, demonstrate less mass effect and have diffuse and central color flow [START_REF] Sharma | Role of 3D Ultrasound and Doppler in Differentiating Clinically Suspected Cases of Leiomyoma and Adenomyosis of Uterus[END_REF] .

MRI imaging features:

MRI is highly accurate for the diagnosis of adenomyosis. In a prospective cohort, Stamatopoulos et al. described a sensitivity of 46.1%, specificity of 99.2%, and positive predictive value of 92.3% of MRI in the diagnosis of adenomyosis [START_REF] Stamatopoulos | Value of magnetic resonance imaging in diagnosis of adenomyosis and myomas of the uterus[END_REF] . An MRI classification of adenomyosis was recently proposed by Bazot et al including defining different forms of adenomyosis [START_REF] Bazot | Role of transvaginal sonography and magnetic resonance imaging in the diagnosis of uterine adenomyosis[END_REF] .

Classic MRI appearance of adenomyosis:

-Subendometrial cysts is a direct sign of adenomyosis correlated with the presence of endometrial glands within the myometrium. These microcysts typically demonstrate water signal on T1WI and T2WI. Hemorrhagic content may accumulate within the cysts and demonstrate T1 hyperintense signal [START_REF] Krentel | From Clinical Symptoms to MR Imaging: Diagnostic Steps in Adenomyosis[END_REF] . They are mainly located in the superficial myometrium and are highly specific (98%). However, they are only detected in 50% of cases [START_REF] Reinhold | Diffuse uterine adenomyosis: morphologic criteria and diagnostic accuracy of endovaginal sonography[END_REF][START_REF] Reinhold | Diffuse adenomyosis: comparison of endovaginal US and MR imaging with histopathologic correlation[END_REF][START_REF] Reinhold | Uterine adenomyosis: endovaginal US and MR imaging features with histopathologic correlation[END_REF][START_REF] Reinhold | Imaging features of adenomyosis[END_REF][START_REF] Tafazoli | Uterine adenomyosis: current concepts in imaging[END_REF] (Figure 9).

-Thickening of the junctional zone (JZ) is an indirect sign related to myometrial hypertrophy, secondary to the presence of ectopic endometrial glands within the myometrium. A JZ thickness of greater than 12 mm has a diagnostic accuracy of 85%, specificity of 96%, and sensitivity of 63% to predict adenomyosis [START_REF] Reinhold | Diffuse adenomyosis: comparison of endovaginal US and MR imaging with histopathologic correlation[END_REF] . The JZ differential sign was described by Dueholm and al as the difference between maximal and minimal thicknesses in the anterior and posterior uterine JZ; a differential of greater than 5 mm may be a more reliable marker than a JZ thickness of greater than 12 mm 71 (Figure 9).

-Because adenomyosis may demonstrate variable degrees of enhancement, intravenous contrast does not enhance the diagnostic capacity of MRI.

Subtypes of adenomyosis:

-Adenomyoma is a mass-like confluence of ectopic endometrial glands within the myometrium, distinct from the JZ. The distinction from leiomyoma may be challenging; adenomyomas are usually ill-defined low signal T2W mass which may contain punctate foci of high T2 signal [START_REF] Agostinho | MRI for adenomyosis: a pictorial review[END_REF][START_REF] Reinhold | Imaging features of adenomyosis[END_REF][START_REF] Tamai | MR imaging findings of adenomyosis: correlation with histopathologic features and diagnostic pitfalls[END_REF][START_REF] Novellas | MRI characteristics of the uterine junctional zone: from normal to the diagnosis of adenomyosis[END_REF] (Figure 10).

-Hemorrhagic cystic adenomyosis (adenomyotic cyst) is a rare subtype of adenomyosis and is frequently symptomatic with dysmenorrhea and menorrhagia. This lesion develops following spontaneous hemorrhage of ectopic endometrial glands [START_REF] Troiano | Cystic adenomyosis of the uterus: MRI[END_REF] . The hemorrhage is contained by a partial or complete rim of myometrial tissue, resulting in a cyst-like appearance with high signal on T1WI and a low signal rim on T2WI. The cyst may be submucosal, intramural, or subserosal [START_REF] Troiano | Cystic adenomyosis of the uterus: MRI[END_REF][START_REF] Takeuchi | Adenomyosis: usual and unusual imaging manifestations, pitfalls, and problem-solving MR imaging techniques[END_REF] (Figure 11).

-External adenomyosis arises in the outer part of the uterus, most likely in the posterior myometrium disrupting the serosa but not affecting the JZ. It is usually associated with deep endometriosis. On MRI, it appears as an ill-defined subserosal posterior T2 hypointense mass/pseudomass and may contains T2 hyperintense small cystic areas.

-Adenomyomatous polyp or polypoid adenomyoma presents as a polypoid mass in the lower uterine endometrium or endocervix, and accounts for about 2% of all endometrial polyps. On MRI, it is a hypointense polypoid mass associated with T2WI hyperintense foci [START_REF] Takeuchi | MR manifestations of uterine polypoid adenomyoma[END_REF][START_REF] Patel | MRI of prolapsed polypoid adenomyoma: Expanding the differential diagnosis for the broccoli sign[END_REF][START_REF] Sajjad | Polypoid Adenomyoma of the Uterus[END_REF] .

MRI Differential diagnosis:

-Cyclic Physiologic Changes of the Uterus (Pseudo-thickening of the JZ): thickness of the junctional zone is hormone-dependent and changes according to the menstrual cycle.

Preferably, MRI should be not performed during menstruation to avoid this pitfall [START_REF] Tamai | MR imaging findings of adenomyosis: correlation with histopathologic features and diagnostic pitfalls[END_REF][START_REF] Takeuchi | Adenomyosis: usual and unusual imaging manifestations, pitfalls, and problem-solving MR imaging techniques[END_REF] .

-Non-measurable junctional zone: the JZ may not be measurable in postmenopausal patients and in women using contraceptive drugs 2,9,79-84 .

-Myometrial contractions: transient uterine contractions are hypointense T2W bands perpendicular to the JZ or focal thickening of the JZ and can mimic focal adenomyosis [START_REF] Ozsarlak | Transient uterine contraction mimicking adenomyosis on MRI[END_REF] .

Repeating the acquisition of images within a few minutes may demonstrate their transient nature; Cine MRI may also help (Figure 6).

-Endometrial cancer: adenomyosis can be seen in 20% of patients with endometrial cancer [START_REF] Habiba | Adenomyosis and Endometrial Cancer: Literature Review[END_REF] .

Evaluation of myometrial invasion may become difficult with the coexistence of adenomyosis

as it may be responsible for a pseudo widening of the endometrium confused as myometrial invasion 3,86-88 (Figure 12). Since endometrial cancer can frequently extend into the ectopic endometrial tissue in adenomyosis, the true degree of myometrial invasion may be difficult to evaluate in the setting of concomitant adenomyosis. DWI may help to define the depth of myometrial invasion; adenomyosis does not restrict diffusion whereas endometrial cancer does [START_REF] Jha | Diffusion-weighted imaging (DWI) of adenomyosis and fibroids of the uterus[END_REF] (Figure 13).

-Leiomyoma: traditionally, adenomyomas present as a T2 hypointense mass with ill-defined borders, minimal mass effect and with multiple bright foci. In contrast, leiomyoma, besides being also T2 hypointense, also have a well-defined border, adjacent mass effect, and large vessels surrounding the lesion [START_REF] Mark | Adenomyosis and leiomyoma: differential diagnosis with MR imaging[END_REF] (Figure 10).

Treatment options:

Medical therapy for adenomyosis relies upon hormonal suppression. Oral contraceptives and GnRH agonists both induce amenorrhea and thus reduce symptoms of pain and bleeding.

Symptoms tend to recur with cessation of therapy91. Levonorgestrel-releasing IUDs may be slightly more efficacious than oral contraceptives in reducing pain and uterine bleeding92 and appear to improve quality of life measures similarly to slightly more than hysterectomy93.

Traditionally, the standard treatment for symptomatic adenomyosis had been hysterectomy91.

However, uterine-sparing techniques, including either complete or partial adenomyomectomy via

open or laparoscopic approaches, can be considered in women wishing to maintain fertility, those who cannot tolerate a large operation, or in those wishing to consider a more conservative approach. Complete adenomyomectomy is effective in the setting of a focal adenomyoma. Partial adenomyomectomy, removal of a portion of the clinically recognizable adenomyosis, is employed in the setting of diffuse adenomyosis when complete resection would effectively result in a "functional hysterectomy." All of the uterine-sparing operative techniques yield improvements in pain in 82-85% of patients, reduction in abnormal uterine bleeding in 50-69% of patients and preserve fertility in 43-47% [START_REF] Grimbizis | Uterus-sparing operative treatment for adenomyosis[END_REF] .

In select patients, UAE can be used to treat adenomyosis with or without leiomyomas. Symptom relief may not be as great or as durable as expected in the setting of isolated leiomyomas [START_REF] Froeling | Uterine artery embolization to treat uterine adenomyosis with or without uterine leiomyomata: results of symptom control and health-related quality of life 40 months after treatment[END_REF] and patients with combined adenomyosis and leiomyomas tend to experience greater symptom relief from UAE than patients with pure adenomyosis [START_REF] De Bruijn | Uterine Artery Embolization for Symptomatic Adenomyosis: 7-Year Clinical Follow-up Using UFS-Qol Questionnaire[END_REF][START_REF] De Bruijn | Uterine Artery Embolization for the Treatment of Adenomyosis: A Systematic Review and Meta-Analysis[END_REF] . Ultimately, with UAE, hysterectomy can be avoided in up to 85% of patients with symptomatic adenomyosis [START_REF] De Bruijn | Uterine Artery Embolization for Symptomatic Adenomyosis: 7-Year Clinical Follow-up Using UFS-Qol Questionnaire[END_REF][START_REF] De Bruijn | Uterine Artery Embolization for the Treatment of Adenomyosis: A Systematic Review and Meta-Analysis[END_REF] .

Conclusion

Benign uterine diseases, including adenomyosis and leiomyomas, are common conditions affecting women of all ages. Ultrasound is often the initial imaging study obtained; however, MRI is the preferred modality for additional lesion characterization and provides critical information to assist in selecting the appropriate therapies for symptomatic patients. Treatment options for adenomyosis and leiomyomas include medical, surgical, and minimally invasive techniques such as uterine artery embolization.

TABLE 1 Protocol

T2 WI

High-resolution T2 sequences in the axial, oblique, sagittal, and coronal planes. The axial oblique T2W sequence perpendicular to the corpus of the uterus is particularly useful to evaluate the location of the lesion relative to the endometrial cavity.

T1 WI

Axial T1W sequence of the pelvis with and without fat suppression. Axial T1W sequence is useful to evaluate the presence of fat or blood contents and can be used to detect the presence of lymph nodes and bone marrow abnormalities. Large FOV T1/T2WI Large FOV T1 or T2W sequence of the upper abdomen. It allows visualization of secondary signs of pelvic mass effect such as hydronephrosis and malignant disease such as lymph nodes or peritoneal carcinomatosis.

Contrast-enhanced T1WI

Contrast-enhanced axial T1-weighted images (T1WI) of the pelvis with fat saturation. It allows further lesion characterization, vascularization and its differentiation from an adnexal mass.

Dynamic contrast injection/MR angiography

Dynamic contrast injection/MR angiography is recommended if uterine artery embolization may be a possibility in order to evaluate uterine artery anatomy and collateral gonadal arterial supply.

DWI

DWI is not mandatory but has shown added value for lesion characterization and distinction between leiomyoma and leiomyosarcoma. The "blackout phenomenon" is illustrated with no signs of restricted diffusion with low signal intensity on DW image (b=1000) (d) and corresponding ADC map (e.

Figure 2 :

 2 Figure 2: Typical leiomyoma. (a) Sagittal and (b) axial T2-weighted images, (c) axial T1-weighted

Figure 3 :

 3 Figure 3: Cellular leiomyoma. (a) Sagittal and (b) axial T2-weighted images show a well-delineated

Figure 4 :

 4 Figure 4: Different types of leiomyoma degeneration. Hyaline degeneration. (a) Axial T2-weighted

Figure 5 :

 5 Figure 5: Indeterminate origin of a mass: ovarian versus uterine. Axial T2-weighted image shows a

Figure 6 :

 6 Figure 6: Transient myometrial contraction. (a) Sagittal and (b) axial T2-weighted fat saturated

Figure 7 :

 7 Figure 7: Leiomyosarcoma. (a) axial T2-weighted images, (b) axial T1-weighted image, (c) axial

Figure 8 :

 8 Figure 8: Flow chart describing the role of MRI in differentiating leiomyoma and uterine sarcoma.

Figure 9 :

 9 Figure 9: Adenomyosis. (a) Drawing shows classic features of adenomyosis: subendometrial cysts

Figure 10 :

 10 Figure 10: Adenomyoma. (a) Drawing shows the different features of adenomyoma and

Figure 11 :

 11 Figure 11: Hemorrhagic cystic adenomyosis. (a) Sagittal T2-weighted fat saturated image shows

Figure 12 :

 12 Figure 12: Pseudowidening of the JZ with coexisting endometrial cancer. (a) Drawing showing the

Figure 13 :

 13 Figure 13: Endometrial cancer with coexisting adenomyosis in 3 differents patients (a-b,c-d,e-f).

TABLE 2 MRI features of leiomyomas and leiomyosarcomas.

 2

		T2WI Signal	T2WI Border	T1WI	C+	DWI/ADC
	Type of Leiomyoma
	Non-degenerated	Hypointense	defined Well-	Isointense	Variable	Hypointense
	Cellular subtype	Hyperintense	Well-	Isointense	Vivid	Hyperintense
			defined		Enhancement
	Lipoleiomyoma	Variable -	Well-	Variable -	Variable	Hypointense
	subtype	Fat component	defined	Fat
		(hyperintense)		component
				(hyperintense)
	Degeneration Type
	Hyaline	Hypointense	defined Well-	Isointense	enhancement Moderate	Hypointense
	Cystic	Hyperintense	defined Well-	Hypointense	enhancement No	Hypointense
	Myxoid	hyperintense Very	defined Well-	Hypointense	enhancement Delayed	Hypointense
	Hemorrhagic	Variable	defined Well-	Hyperintense Variable	Hypointense
	Calcific	HypoT2	defined Well-	Hypointense	enhancement No	Hypointense
		T2 Dark areas	Nodular	Presence of	Enhancement	High DWI/Low ADC
	Leiomyosarcoma		border	High T1-SI related to	with central necrosis
				blood
	Legends
	Figure 1: Leiomyoma FIGO classification system according to location. (a) Drawing depicts the
	leiomyoma FIGO classification, as FIGO 0: pedunculated intracavitary; FIGO 1: submucosal <50%
	intramural; FIGO 2: submucosal ≥ 50% intramural; FIGO 3: contacts endometrium, 100%

[START_REF] Murase | Uterine leiomyomas: histopathologic features, MR imaging findings, differential diagnosis, and treatment[END_REF] . The types of degeneration are hyaline, cystic, myxoid, hemorrhagic, and calcific (Table2).