Melanoma is an extremely aggressive tumor with a high metastatic rate, whose diagnosis in advanced stages was associated, until a decade ago, with minimal chances of survival [1]. It is a tumor originating in melanocyte cells which are formed during embryogenesis from the neural crest of the trunk [2]. Melanocytes come from progenitor cells with a high migration capacity. This migratory capacity explains why they are distributed and present at the level of a large number of structures: the skin—the basal layer of the epidermis (with an important role in the uniform pigmentation by forming epidermal–melanin units)—the inner ear, gastrointestinal tract and the nerve structures [3]. The incidence of melanoma has seen a spectacular increase in the last two decades, with over 300,000 new cases in 2018, the most affected countries being Australia and New Zealand (over 33 cases/100,000 inhabitants), the average age of onset of melanoma being 65 years old [4,5]. Regarding the distribution according to sex, in the case of melanomas discovered in adulthood, there is a predominance of cases in males, with a reversal of the phenomenon for melanomas diagnosed between 15–39 years, over 60% of cases appearing in the female population [6,7]. An interesting phenomenon was observed in the young population diagnosed with melanoma, the cases experiencing an alarming increase between 1999–2006, with a decrease in the next 10 years, dynamics overlapping with the increasing popularity of photoprotection methods and awareness of the danger posed by tanning beds exposure [6,8]. Due to the poorly associated prognosis determined mainly by the discovery of melanoma in advanced stages, melanoma remains the skin cancer with the highest mortality, the 5-year survival rate being less than 80% and depending on the degree of local tumor extension, lymphatic invasion, and presence of metastases [9,10]. Although dermatoscopic evaluation has become essential in pigmentary lesions screening, the diagnosis of melanoma is difficult even for experienced dermatologists, the clinical and dermatoscopic appearance presenting a great variability and sometimes even possessing misleading histological aspects that can lead to false-negative results [9,11,12].

Melanomas with genital location are included in the category of rare neoplasms, vulvar and vaginal melanomas totaling less than 1–2% of all melanomas diagnosed in females [13,14]. Vulvar melanoma represents approximately 5% of all cancers located in the vulva, being more common in adulthood (average age is 68 years) [15,16]. Between 2.5 and 4.5 patients/100,000 inhabitants are affected each year by vulvar cancer, melanoma with this location being among the top four most common vulvar cancers, the most frequent being squamous cell carcinoma (over 75% of cases versus 5.6% for melanoma) [17,18].

On the other hand, vaginal melanoma is even a rarer condition, accounting for less than 0.05–0.1% of genital neoplasms [19]. The average age of onset is lower than in the case of vulvar melanoma (57 years), and the prognosis is much worse, less than a third of patients surviving 5 years after diagnosis, despite the correct instituted treatment [20]. In a study performed on 1400 patients with vulvar melanoma and 463 with vaginal melanoma, Wohlmuth et al. highlight the occurrence of the former at a significantly younger age (*p* < 0.001) [21].

Unlike cutaneous melanoma, which is much more common in Caucasians, the frequency of vulvar and vaginal melanomas has very little variability related to race, but there is a slight increase in frequency in Whites (3.14 vs. 1.02: 1) [22]. This increase is statistically significant for vulvar melanoma, but not for those with vaginal localization (*p* < 0.001) [21].

Particular attention has been paid in the last years to the necessity of avoiding sun exposure using screen protective agents, periodic self-examination of pigmented lesions and periodic dermatoscopic surveillance of potentially evolving lesions, these being considered the most important methods to decrease the incidence and mortality of melanoma [23]. However, the genital area is often overlooked as a possible site of melanoma and other skin cancers, both by patients and doctors. Yet, melanoma with localization in the vulva and vagina is characterized by increased severity especially due to late diagnosis, most often in metastatic stages [18]. Moreover, the therapeutic means used in cutaneous melanomas are of little use in the treatment of melanomas with vulvar and vaginal localization, mortality

having high values and not changing considerably in the last three decades (5-year survival rates vary between 10% and 63%) [17,24–26]. Thus, the present review aims to highlight the main risk factors identified in the occurrence of vulvar and vaginal melanomas, clinical manifestations, molecular changes underlying these neoplasms, as well as the main therapeutic means and their effectiveness in terms of survival.

#### **2. Risk Factors**

Melanoma, like most neoplastic pathologies, is a multifactorial disease, its occurrence being related to interactions between environmental and host factors, thus appearing as a consequence of genetic and epigenetic modifications that eventually lead to alteration of regulatory processes [27]. Although a number of risk factors involved in the etiopathogenesis of the disease have been identified for cutaneous melanoma, such as intermittent exposure with increased intensity to ultraviolet (UV) radiation, history of sunburns (more common at high latitudes), the presence of atypical nevi, specific skin phenotype (lightly pigmented skin, with light eyes, blond or reddish hair, presence of freckles), family history with specific genetic changes that alter the ultraviolets repair of melanocytes subjected to UV radiation, the use of Psoralen and Ultraviolet A therapy (PUVA) for a long time (over 15 years after exposure), the etiopathogenic mechanisms for vulvar and vaginal melanomas are not yet elucidated, and no specific factors involved in their occurrence have been identified [17,28–32]. Moreover, in a study published by Heinzelmann-Schwartzet et al., the onset of vulvar melanoma was regarded as spontaneous, de novo, with changes in a single melanocyte cell being enough to trigger the process of oncogenesis and determine the formation of melanoma [17].

However, there are studies that show a greater association between mucosal melanomas in general, and vaginal and vulvar melanomas in particular, and certain factors:

Sex. The female gender appears to be a risk factor for mucosal melanomas in general, which are twice as common as in men, compared to cutaneous melanomas whose distribution is similar between the two sexes [33].

Age. Vulvar melanoma is a disease that has an average age of onset of 68 years old, the risk of onset increasing with age (the number of cases increases from 0.11/1 million inhabitants for 15–29 years range, to 3.5/1 million inhabitants for those over 60 years old [34,35]). In contrast, cutaneous melanoma has a maximum incidence around the fourth decade of life [35].

Family history of cutaneous melanoma appears to be a risk factor for vulvar melanoma [36].

Ethnicity also seems to play an important role in the evolution of melanomas with genital localization and in their prognosis. Even if the association is not as strong as in the case of cutaneous melanomas, vulvar and vaginal melanomas are three times more common in the white race (*p* < 0.001) [22]. However, the prognosis has an inverse association, the mortality being much higher among the African population [37].

Lichen sclerosus is a precursor lesion of squamous cell carcinoma of the genital area which appears in its evolution in a percentage of approximately 5%, the causal link between the two pathologies being well known. Regarding the occurrence of vulvar melanoma in patients with lichen sclerosus, while a causal link has not been clearly established, although the number of reported cases is quite small, an increased incidence of vulvar melanoma has been observed among these patients (relative risk = 341) [38,39].

UV radiation. Though vulvar melanoma does not occur in photo-exposed areas, some studies suggest the indirect involvement of UV radiation in the pathogenesis of vulvar melanoma, through alterations of the immune response that favor modifications of the pathways involved in oncogenesis. However, it is important to note that the role of radiation in the pathogenesis of the disease is significantly lower than in skin melanoma [34].

Although the Human Papilloma Virus (HPV) is known for its roles in the development of benign (condyloma acuminata) and malignant tumors (invasive squamous cell carcinoma, anorectal cancers, penile cancers, squamous vaginal cancers) with genital localization, there is yet no mechanism to demonstrate its involvement in melanoma. There is no evidence that the HPV infection elevates the risk of vulvar or vaginal melanomas [32,40–42].

#### **3. Clinical Manifestations**

Unlike cutaneous melanoma which is most frequently distributed on photo-exposed areas (face, trunk, lower limbs) making it possible to diagnose it from early stages, vulvar and vaginal melanomas have origins that are generally overlooked by frequent inspection, the anatomical position of the lesion being the main reason for its late diagnosis and poor prognosis [43]. Melanomas of the vulvar region can occur in a variety of sites starting from the hairy skin of the labium majus to the introitus (Figure 1). The most common sites are the clitoris, the labia majora, and the labia minora, and in most cases, multiple tumors can be detected [34,44,45].

Vulvar and vaginal melanomas are pigmented lesions with variable diameters (generally over 7 mm), with macular (most frequently), papular or nodular appearance, most often asymmetrical and with an inhomogeneous appearance in terms of pigmentation [44,46,47]. There are also reports in the literature of cases with amelanotic manifestations of genital melanomas, especially at the vaginal level [20,48]. The first case of genital melanoma was described in 1861 in a 35-year-old patient who presented with multiple pigmentary lesions and evolved with neurological and digestive symptoms in the context of multiple secondary determinations and, subsequently, exitus [49]. Since then, multiple cases of vulvar and vaginal melanomas with various clinical, dermatoscopic aspects, and symptoms have been depicted in the literature (for additional details see [16,20]).

As it is a rare pathology, most of the clinical data presented in the literature are obtained from isolated case presentations or case series, most of which highlight the presence of genital bleeding, a palpable, white-gray color mass, itching, dyspareunia, yellow genital secretions, and local pain [50–54]. Cases that mention the presence of painless masses are also reported [55]. Most of the cases presented depict the tumor in the labia minora. The diagnosis is generally late, with half of the patients being diagnosed in advanced stages, with the invasion of deep structures [56].

**Figure 1.** Most common sites for vulvar melanomas [15,36,47,57–59].

Although not so numerous and generally on small patient samples, there are prospective or retrospective clinical studies and meta-analyses that have highlighted the main clinical manifestations and the most common sites of vulvar and vaginal melanomas, which are further illustrated in Table 1.

Regarding the dermatoscopic examination, it is characterized by asymmetrical, irregular aspects, both in terms of contour and pigmentation, lacking a clear structure. In addition, multiple cases report the presence of blue-gray or white-gray areas and globular punctiform black-brown structures [45,58].

#### **Table 1.** Clinical findings in melanoma of the vulva and vagina [15,26,36,47,57–60].

#### **4. Histological Aspects**

Melanoma may arise de novo or within an existing benign or dysplastic nevus. Lesions usually measure between 2–4 mm in thickness and are frequently ulcerated. In addition to this clinical aspect that raises the suspicion of a malignant lesion, histopathological examination is absolutely necessary to establish the diagnosis.

#### *4.1. Macroscopic Examination*

The distribution of melanomas on the skin can be at any level, therefore areas not exposed to the sun should not be neglected, which is practically the case of vulvar melanomas. Considering that vulvar melanomas often appear in older women, they should be advised to self-examine with a hand-held mirror [13]. The first step in examining pigmented nevi, moles, and brown spots and growths is to apply the ABCDEs rule and the Ugly Duckling sign (although not applicable for vulvar melanomas) [61]. As for the rest of the skin, vulvar melanomas are black or dark brown, but may also vary from white, pink, red, to other colors [13,62,63].

The ABCDE rule refers to the main macroscopical aspects taken into consideration when assessing cutaneous lesions, namely Asymmetry (the two halves of the mole are not identical), Border irregularity (edges are ragged or notched), Color variation (shades of tan, brown, or black and sometimes patches of red, blue, or white), Diameter (>6 mm or >1/4 inch) or Dermoscopic structure, and Evolution (Figure 2) [64]. The same rule can be regarded from a histological point of view, thus becoming the ABCDE(FG) rule, which refers to Asymmetry (silhouette and color imbalance), Buckshot scatter (pagetoid distribution), Cytological atypia, Deep mitosis, Enclosing lymphocytes, Fibrosis, and Gainsaying (=no) maturation [65].

Another step in the diagnosis of vulvar melanoma is dermoscopy, which facilitates early detection based on the identification of a lesion with irregular dots, multiple colors (black, blue, brown, pink, gray, and white), a blue-white veil, and atypical vessels [66]. When the lesion is assessed and determined as suspicious, the next step is to determine the most important factor for the future evolution and prognosis—the vertical depth of invasion [67]. Therefore, the most effective way to assess the local invasion is the sampling of the lesion. The evolution of melanoma depends on the correctness and accuracy of the biopsy technique. Thus, two types of biopsies of suspicious formations are described: an excisional biopsy that removes the entire lesion, and an incisional biopsy, which removes only a portion from the suspicious cutaneous lesion [68]. Excision biopsy is considered the

gold standard in the correct diagnosis of melanomas, and has the advantage of providing accurate microstaging [69].

**Figure 2.** ABCDE evaluation of a pigmentary skin lesion [64].

The 7th edition of The American Joint Committee on Cancer (AJCC) Cancer Staging Manual published in 2009 was the first to give up the Clark score, which was largely based on the level of anatomical invasion in the skin layers [70]. Further on, the 8th AJCC edition on melanoma staging and classification relies on the Breslow thickness in order to establish the depth of invasion, so as to accurately assign a stage based on the classical tumor, node, metastasis (TNM) scores [71]. Additionally, this latest edition specifies that tumor depth should be measured to the nearest 0.1 mm instead of 0.01 mm, in order to improve precision [72]. Although there is not yet a standardized and unanimously accepted staging of vuvar melanomas, the Gynecologic Oncology Group (GOG) recommends the use of the AJCC staging manual instead of the International Federation of Gynecology and Obstetrics (FIGO) system, in spite of the several uncertainties that still persist [18]. Specifically, while generally regarded as mucosal tumors, the molecular characteristics of vulvar melanomas differ from those of both cutaneous and mucosal melanomas in terms of mutational signatures, thus prompting some authors to consider them a unique subclass [21,73].

#### *4.2. Microscopic Examination*

From a cytological point of view, melanoma cells are of two types: epithelioid and spindle cells. The epithelioid type is characterized by large, round cells with abundant eosinophilic cytoplasm. They have vesicular nuclei with coarse irregular chromatin with peripheral condensation (pattern known as "peppered moth" nuclei). This cell type is most commonly found in nodular and superficial spreading melanomas. The stromal compartment is accompanied by a variable inflammatory infiltrate (brisk, non-brisk, absent), irregular distribution of the pigment, and dermal fibrosis.

#### *4.3. Histological Subtypes of Melanoma*

The class of tumors known as melanoma contains numerous histological subtypes that are not often considered of the utmost importance to clinicians or patients, especially in terms of prognosis [74]. Histopathological characteristics of the gynecological melanomas should be interpreted in the context of other clinical diagnostic criteria and macroscopic data.

Superficial spreading melanoma (SSM) is the most common subtype, characterized by asymmetrical proliferation of atypical melanocytes. Individual melanocyte units are distributed at the dermo-epidermal junction and are characterized by the presence of large pagetoid areas (>0.5 mm2) [75].

Lentigo maligna melanoma (LMM) is characteristic for elderly patients repeatedly exposed to chronic sun damage. Melanocytes arranged in solitary units along the dermoepidermal junction are organized in small nests (lentiginous pattern), sometimes the nests being horizontally confluent and variable in size and shape (nevoid/dysplastic-like pattern). Dermal invasion of atypical melanocytes is frequent, as well as the extension into the hair follicles [76].

Acral lentiginous melanoma (ALM) is more common in the Afro-Asian population, on palms and soles, and involves the eccrine glands excretory ducts [77].

Nodular melanoma (NM) is considered the most aggressive form, and grows in depth rather than in diameter, so that the ABCDE rule does not apply [78]. It is characterized by a nodular dermal proliferation of atypical melanocytes.

Regarding vulvar melanomas, a retrospective study based on 43 patients, performed by DeMatos et al., showed that most of the mucosal melanoma lesions (vulvar, vaginal, and cervical) were not classified (33%), while the rest were acral lentiginous (30%), or superficial spreading melanoma (26%). Unclassified tumors were either nodular or polypoid melanoma [79].

The most common vulvar melanomas subtypes are mucosal lentiginous and nodular melanoma. The characteristic microscopic aspects are represented by impairment of the epidermis, pagetoid spread of melanocytes, or melanocytes organized in nests (Figures 3 and 4) [80]. Usually, melanocytes are variable in size and shape, and are sometimes localized within lymphovascular spaces. Furthermore, they can frequently become confluent and lack maturation, showing atypical mitoses and increased apoptotic activity (Figures 5 and 6) [81]. The presence of an ulcerated tumor is associated with a poor prognosis.

**Figure 3.** Vaginal melanoma. Vaginal squamous epithelium with areas of ulceration. In lamina propria one can observe tumoral cells organized in nests. Hematoxylin-eosin stain, original magnification ×4 (histological image courtesy of dr. Ileana Popa).

**Figure 4.** (**A**) Vaginal melanoma. Tumor cells organized in nests, some fusiform-looking cells (Hematoxylin-eosin stain, original magnification ×10.) (**B**) Vaginal melanoma. Epithelioid-looking malignant cells in lamina propria outlooking a nest disposition, abundant eosinophilic cytoplasm. Hematoxylin-eosin stain, original magnification ×20 (histological image courtesy of dr. Ileana Popa).

**Figure 5.** (**A**) Vaginal melanoma. Tumoral cells filled with melanin organized in nests at the epithelium-lamina propria junction pathognomonic for the diagnosis, original magnification ×20. (**B**) Detail showing haphazardly distributed atypical melanocytes in lamina propria original magnification ×40 Hematoxylin-eosin stain (histological image courtesy of dr. Ileana Popa).

**Figure 6.** (**A**,**B**) Vaginal melanoma. Arrows—Nuclear mitoses—original magnification ×40.Hematoxylin-eosin stain (histological image courtesy of dr. Ileana Popa).

#### **5. Immuno(cyto)histochemistry in the Diagnosis of Melanoma**

The criteria described above are imperiled by subjective interpretations, lacking objectivity, and reproducibility. Moreover, melanomas are able to mimic a lot of other malignancies with epithelial, hematologic, mesenchymal, and neural origin, such as lymphomas, carcinomas, neuroendocrine tumors, and sarcomas [82]. Therefore, immunocytochemistry (IHC) is a step most often necessary in the correct evaluation of a melanocytic suspect lesion in patients with underlying hematological pathologies or with solid cancers with frequent skin metastasis. Immunohistochemistry can show its utility in the differential diagnosis of vulvar soft tissue lesions, i.e., Paget disease, which can mimic melanoma [83]. In addition, IHC is less expensive by comparison with electron microscopy, which can bring additional information for melanoma cells containing melanosomes and other ultrastructural particularities which are not present in Paget disease.

Melanomas are usually immunoreactive for Melanoma antigen recognized by T-cells-1 (MART-1) or melan A, S-100 protein, melanoma-specific antigen (HMB-45), tyrosinase, and Microphthalmia transcription factor (MITF) [84].

S-100 protein, a sensitive marker for melanocytic differentiation, is considered to have a sensitivity of 97–100%, but the specificity for melanoma is low since is also expressed on glial cells, Schwann cells, chondrocytes, lipocytes, dendritic cells, histiocytes [85].

One of the first specific markers that was discovered is HMB45, a marker of the cytoplasmic pre melanosomal glycoprotein gp100 [86].

Melan A is the most widely used technique for identifying basal melanocytic proliferations, having a more intense and diffuse staining than HMB45 and a specificity of 95–100% [87].

Further on, the Sry-related HMg-Box gene 10 (SOX10) nuclear transcription factor is a key player in the differentiation of pluripotent neural crest cells into melanocytes. In this regard, its use as a metastatic melanoma marker has been investigated and found to be highly specific and sensitive [88].

#### **6. Molecular Characterization**

The pathogenesis of genital tract melanomas can mimic that of cutaneous melanoma developed in areas that are sun-protected, as in the case of vulvar melanomas. Moreover, other types of mucosal melanomas (e.g., the respiratory and the gastrointestinal tract) can serve as a model for the development of vaginal or cervical melanomas. Still, there are gaps in understanding the pathogenesis of vulvar or vaginal melanomas. However, elucidating the pathways involved in melanoma genesis is a very important step towards future advanced precision treatment.

Mucosal melanomas represent a challenge in relation to treatment, since, as opposed to cutaneous melanomas, not only are they detected in more advanced stages, but they are also less responsive to immunotherapy and lack activating mutations involving dominant MAP kinases [89,90]. Mutations in KIT, NF1 and SF3B1 genes are more frequently seen in mucosal melanomas, while alterations to NRAS and BRAF genes are more common in cutaneous melanomas [91,92]. Female genital tract melanomas can sometimes contradict the pattern described above. A study involving the whole-genome analysis of 284 patients diagnosed with mucosal melanoma localized in different sites revealed 10 mutated genes (Table 2) [93].

A retrospective study performed by Rouzbachman et al. on 33 vulvar and 11 vaginal malignant melanomas identified by next-generation sequencing analysis, found that the most frequent mutations were in C-KIT and NRAS genes, while BRAF mutations were infrequent. The study did not establish a correlation with the prognosis or outcome for these patients [94].

A more recent study performed by Cai et al. on 19 melanomas of the female genital tract and paired with 25 cutaneous melanomas, 18 acral melanomas and 11 melanomas of the nasal cavity, concluded that malignant melanoma of the female genital tract harbors distinct mutation rates in the KIT, BRAF, SF3B1, KRAS, and NRAS genes. Moreover, this location of the melanoma is an entirely distinct entity from skin melanoma and melanomas of the nasal cavity, while cervix melanomas also have some particularities, and do not suffer recurrent KIT mutations, or mutations of the NRAS and SF3B1 genes [95].

Because BRAF and NRAS mutations are uncommon in non-cutaneous melanomas, we can consider that each melanoma subtype depends on a different oncogenetic pathway for its development. This leads to the idea that it will not benefit from anti-RAF treatment, although large studies are needed before considering this as a rule. However, in the era of precision medicine, the importance of driver mutations cannot be neglected, and should become part of mucosal melanoma routine clinical testing [96].

**Table 2.** The main mutations that occur in melanomas and their frequency of occurrence [93,97–102].

#### **7. Melanoma Stage Description**

Detection of a melanoma involving the vulva or vagina associates an essential step: the exclusion of any kind of metastatic dissemination in places such as mucosal membranes, skin, or the eyes [102]. Since staging approved for melanomas, in general, cannot be extended to female genital tract melanomas, there is a great need for dedicated staging.

Clark classification that stages melanomas regarding their level of invasiveness and Breslow classification regarding the vertical depth of the lesion are dedicated for cutaneous melanomas [103–105].

A new and modified Clark classification, named Chung classification, is considered specific for micro-staging of mucosal melanomas in general (Table 3) [106].

**Table 3.** Clark and Chung classifications comparative aspects [104–106].

A 25-year study that both encompasses clinical and pathological features of vulvar melanoma revealed that American Joint Committee on Cancer (AJCC) staging system is the unique prognostic factor for vulvar melanoma (Table 4) [57]. The AJCC system was revised in 2017, associating new prognostic factors [72].

**Table 4.** American Joint Committee—prognostic stage group for melanoma 2017 [57,72].

#### **8. Therapeutic Approach**

Melanomas with vulvar and vaginal localization represent real challenges in terms of treatment, not only due to late diagnosis in locally advanced or metastatic stages, but also due to the absence of specific therapeutic guidelines due to the small number of cases. Thus, due to the impossibility of gathering a significant number of cases to establish dedicated guidelines, the guidelines for cutaneous melanoma are often used.

The main treatment for both vulvar and vaginal melanoma is surgical excision of the tumor. Clinical experience in the last few decades has shown no significant differences in survival rates between radical excision and limited excisions with safety margins (Table 5), so limited resections are currently recommended [32,103]. Pelvic exenteration may be helpful, but only in carefully selected cases [107].

**Table 5.** Safety surgical margins in limited resection of vulvar and vaginal melanomas [15,103,107].

Surgical excision may be completed in some cases by lymphadenectomy. Literature data do not provide specific information on the positive impact of lymphadenectomy in terms of survival, so it is preferred to perform the sentinel node technique in the absence of clinical involvement of regional lymph nodes, followed by lymphadenectomy in the case of a positive result [103]. In the case of clinical invasion of the regional lymph nodes, tumor extension in the adjacent structures, and/or ulceration, lymphadenectomy is recommended, not preceded by the sentinel node technique, which may or may not be followed by adjuvant treatment [108,109]. Lymphadenectomy, although a radical intervention, has no impact on long-term survival, only enabling better local control of the disease [107].

Regarding the utility of radiotherapy, chemotherapy, and immunotherapy, data presented in the literature are limited, but randomized controlled trials show minimal changes in survival [107]. Radiotherapy is generally used as neoadjuvant treatment, in case of impossibility of surgical resection, lymphatic invasion, regional extension, or as palliative treatment [103,110]. Doses of 45–55 Gy are generally used in daily sessions of 1.8–2 Gy/session [103]. The use of radiotherapy in localized forms has been associated with a decrease in survival [110,111].

Chemotherapy is mainly used as a neoadjuvant treatment or unique therapy, utilizing the same agents as those used in skin melanoma: dacarbazine and temozolomide, with dacarbazine being the first Food and Drug Administration-approved drug for melanoma treatment [103,112]. In addition, chemotherapy represents, together with surgery, the last therapeutic resource for the treatment of vulvar melanoma diagnosed in advanced stages [107].

Although on small groups of patients, there are studies showing the effectiveness of chemotherapy compared to the use of high-dose interferon for stage II vulvar melanoma [107]. Moreover, the use of temozolomide-cisplatin combination as adjuvant therapy in stages II-III showed statistically significant benefits over the use of interferon-alpha or surgery in terms of survival (*p* < 0.01) [109]. The carboplatin–paclitaxel–bevacizumab combination has been shown to be effective in decreasing tumor volume, thus allowing surgical resection without the need for further grafting [113]. Chemotherapy is used mainly in regionally advanced forms or with distant metastases, but the benefits are minimal, the use of chemotherapy is generally associated with a decrease in overall survival and recurrencefree survival [110,113]. Up until 2011, only four drugs had been approved for the treatment of melanoma; currently, their number has reached 30, and this increase is based mainly on newly emerged molecules from the class of immunotherapeutics or biological drugs [112].

The class of immunotherapeutics includes molecules that interfere with the immune cascade of carcinogenesis, such as interferon α-2b (IFN α-2b), peginterferon α-2b (PegIFN α-2b), interleukin 2 (IL-2), inhibitor of death protein 1 (PD-L1 blockade)–nivolumab, regulatory T lymphocyte inhibitors, monoclonal antibodies against cytotoxic T cell antigen 4 (CTLA-4)–ipilimumab [112]. These are especially used in advanced stages (stage III-IV) of cutaneous melanoma, but for vulvar melanoma, there are not enough clinical data to demonstrate efficacy in terms of overall survival or disease-free survival. However, their use doubled in the period 2012–2015 compared to the corresponding previous period (*p* < 0.01), most frequently for regionally advanced forms and those with distant metastases [110]. IFN α-2b appeared to lead to an increase in disease-free survival in a randomized clinical trial, while no effects on survival after the use of IL-2 alone or in combination with peptide vaccines were observed [107]. Albert et al. observed in a study on 1917 patients with vulvar melanoma (3/4 with localized disease) that the combination of immunotherapy with local surgery was associated with an increase in overall survival, although statistically insignificant [110]. On the other hand, the use of immunotherapy in combination with chemotherapy and biological therapy was associated with a decrease in overall survival and recurrence-free survival in a study on 48 patients with vulvar and vaginal melanomas [113]. The same data were supported by Tcheung et al. who did not find significant differences in overall survival in patients treated with immunotherapy and chemotherapy [114].

Although targeted therapy has evolved in the last years and significantly improved the prognosis of patients with cutaneous melanoma, its use in vulvar and vaginal melanomas is still limited by the different mutagenic profiles involving fewer BRAF mutations and more c-KIT, NF1 and SF3B1 mutations [91,112]. Thus, monoclonal antibodies such as imatinib, sunitinib, dasatinib, nilotinib (for c-kit mutations) could have been a promising option due to a large number of c-kit mutations, but the results are not as good as in the case of cutaneous melanoma. Imatinib has been shown to reduce tumor size and PET uptake, as well as the disappearance of metastases in a patient with recurrent vulvar melanoma and c-kit mutation in exon 13 [115]. There are phase II studies with promising results for the use of imatinib and dasatinib in patients with mucosal melanomas with c-kit mutations [107,116]. Cocorocchio et al. also mention a positive response to avapritinib therapy in a patient with advanced vulvar melanoma with a c-kit mutation in exon 17, who had a previous negative response to the ipilimumab–nivolumab combination [117].

The prognosis is generally poor, characterized by frequent recurrences (up to 70%), with a low overall survival (average survival between 15–78 months), and with a recurrence disease-free interval of up to 64 months [113,114,118–121] (Table 6). The main prognostic factors are tumor size, lymphatic invasion, Breslow depth, and the presence of ulcerated lesions [114,122].

Vaginal melanoma is characterized by higher recurrence rates and lower average survival, requiring wider excisions with complete lymphadenectomy and even pelvic exenteration [113,121,123,124]. Along with the surgical excision, adjuvant chemotherapy (dacarbazine) or the combination of targeted therapy (ipilimumab)–radiotherapy have been mentioned in the literature in isolated case presentations or case series, with favorable results [113,121]. In the case of the combination of ipilimumab–radiotherapy, it was found that the administration of radiotherapy in doses of 3000 cGy in 5 fractions was accompanied by the absence of recurrences compared to radiotherapy in doses of 6020 cGy in 28 fractions (for isolated cases, no statistical significance was demonstrated in randomized clinical trials) [113].

**Table 6.** Survival rates according to type of treatment [110,114–116,118].

#### **9. Conclusions**

Vulvar and vaginal melanomas are rare neoplasms, with aggressive evolution, most often diagnosed in advanced stages. Gynecological melanomas currently represent a discouraging diagnosis due to the absence of specific therapeutic guidelines and management measures with satisfactory results. Despite the significant development of targeted therapy that has brought major benefits in the prognosis of cutaneous melanoma, vulvar and vaginal melanoma have a low response to these therapies due to the presence of different mutagenic profiles. Thus, despite the use in various clinical trials of many types of targeted therapies, or combinations of chemotherapy and immunotherapy, surgical excision within safe limits remains the therapeutic approach with the best survival rate. The molecular mechanisms of the vulvar and vaginal melanomas are not yet fully understood, these types of melanomas being considered an independent subcategory of melanoma. As new mechanisms are discovered, new targeted therapies may appear to improve the prognosis and subsequently, the survival rates.

**Author Contributions:** Conceptualization, E.-C.D., C.E.C., B.S.G. and S.M.C.; methodology, E.-C.D.; software, E.-C.D., C.V., S.M.C. and D.C.; validation, E.-C.D., C.V., S.M.C. and D.C.; formal analysis, E.-C.D., C.V., S.M.C., N.S. and D.C.; investigation E.-C.D., C.E.C., S.M.C. and V.N.V.; resources, E.-C.D., C.V., S.M.C., I.P. and D.C.; data curation, E.-C.D., C.V., N.S. and S.M.C.; writing—original draft preparation, E.-C.D., C.V., I.P. and S.M.C.; writing—review and editing, E.-C.D., B.S.G., C.E.C., S.M.C. and V.N.V.; visualization, E.-C.D., C.E.C. and S.M.C.; supervision, B.S.G.; S.M.C.; project administration, E.-C.D., C.V. and S.M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant of the Romanian Ministry of Research and Innovation, PCCDI-UEFISCDI, projects number PN-III-P1-1.2-PCCDI-2017-0833/68/2018, and PN-III-P1-1.2- PCCDI-2017-0820/67/2018 within PNCDI III.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

MDPI St. Alban-Anlage 66 4052 Basel Switzerland Tel. +41 61 683 77 34 Fax +41 61 302 89 18 www.mdpi.com

*Biomedicines* Editorial Office E-mail<EMAIL_ADDRESS>www.mdpi.com/journal/biomedicines

MDPI St. Alban-Anlage 66 4052 Basel Switzerland

Tel: +41 61 683 77 34 Fax: +41 61 302 89 18

www.mdpi.com

ISBN 978-3-0365-4384-0


**1.1 Addressing the need for disinfectants**

A dichotomy of virology work is the need for both viral presence within the confines of research and the equally consistent need to establish pathogen-free research spaces. Throughout the world, contagious disease through viral contamination is an ever-present concern, and SARS-CoV-2 has brought the need to decontaminate to the forefront of virtually every industry. Scientific industries performing research, manufacturing pharmaceuticals, or providing healthcare services, all employ protocols for the disinfection of their environments in order for safe, successful, timely work to take place. These industries depend upon disinfection chemicals, and perhaps just as importantly the chemical delivery systems, that ensure the integrity of their work, personnel safety, and efficient transition from one research project or product type to the next.

### **1.2 Classification of antimicrobial effectiveness**

Today, a number of distinct categories are used to classify and understand disinfection methods. Disinfection chemicals are tested with established protocols and classified according to their relative success at eliminating specific pathogens. The *Biosafety in Microbiological and Biomedical Laboratorie*s (BMBL) 6th edition makes a distinction between the inactivation of pathogens (rendering them nonviable) and the destruction of pathogens and their infectious particles (decontamination) [1]. This distinction is highly relevant to industries where establishing a sterile surface can be a critical determinant of success or failure [2]. The United States Environmental Protection Agency (EPA) classifies disinfectants by their ability to inactivate certain challenging pathogens, such as *Clostridioides difficile* (*C. diff*) and *Bacillus anthracis* (Anthrax), which delineates if the disinfectant is classified a sterilant, decontaminant, or sporicide [3] (**Box 1**). This delineation is based on the Spaulding classification, the microbiological hierarchy model standard, which classifies pathogens based on their environmental hardiness and relative resistance to disinfection [8, 9]. In this hierarchy, small non-enveloped viruses are considered moderately resistant, whereas spores are most resistant to disinfection methods. Beginning in 2016, the EPA developed its Emerging Viral Pathogen category to fast-track products proven against bacterial spores for use against newly appearing viral threats [9–12]. Beyond this classification testing, commercially available spore-based biological indicators can be used with certain solutions as an ongoing measurement and verification of sterilization results [2].

#### **Box 1.**

*Definitions. Definitions relating to achieving and evaluating levels of antimicrobial effectiveness on environmental surfaces [1]. Definitions of substance phase or classification [4–7]. Depending on device design, the chemical being dispersed throughout the treatment space may be delivered in a variety of forms, phases, or states of matter. These definitions are provided for the sake of our understanding the differences in technologies and delivery methods described within this chapter.*


**4. Measures for management of ravine land**

The agricultural activity with land leveling is preferred for slope stabilization and its management in shallow ravines due to presence of deep and alluvial soil. The increased cost of land leveling operation is a major constraint and key factor in making decision for management of medium and deep ravines. The appropriate ways for management of ravines is reducing runoff intensity and safe disposal of runoff in ravines. The runoff generated from the adjacent marginal land is managed

**183**

*Ecological Engineering Measures for Ravine Slope Stabilization and Its Sustainable Productive…*

by constructing peripheral bund, checking of runoff/sediment in the interconnected gullies by earthen gully plugs/bori-bund/composite check dams at proper horizontal interval. The terracing is recommended in shallow and medium ravines slopes along with agroforestry measures for sustainable land use. The deep and very deep ravines are recommended to be kept under permanent vegetative cover along with site specific soil and water conversation measures. The medium and deep gullies having steeper slopes and economically not suitable for cultivation can be put under silvi-pasture permanent vegetation cover with protection from grazing for

**4.1 Engineering measures for management of shallow ravine land**

increases the water and nutrient use efficiency of the cropping systems.

About one third of the ravine lands in the country are shallow ravines (<1 m deep) and can be easily reclaimed with simple earth moving machinery for cultivation of crops. The management of shallow ravines instigates with designing and instituting a peripheral bund along the gully head to check the runoff generated from the adjoining marginal lands. The land leveling operations across the slope and smoothening on the upstream side of the peripheral bund increases the infiltration opportunity time for the runoff generated from the crop field. The in-situ soil moisture conservation

Vegetative barriers are used either for supplementing or substituting earthen

The Napier, Guinea and Para fodder grasses strips of 1 m and 2 m were grown as vegetative barrier in 2% slope in shallow ravines. The lowest sediment yield was observed in 2 m width of Napier grass strip (1.43 t/ha) followed by 2 m width of Guinea and Para grass strips as compared to control (4.07 t/ha), respectively. The 2 m width of Napier grass strips has lowest nutrient loss of (N-5.64 kg/ha, P-3.1 kg/ ha, K-5.4 kg/ha) followed by Guinea and Para were found almost equally efficient in reducing nutrient losses from crop field which is 28 to 30% of nutrient losses from control (N-20.01 kg/ha, P-8.3 kg/ha, K-17.00 kg/ha). The equivalent yield of 2 m width of Napier grass strip and cotton crop (1293.5 kg/ha) has highest yield followed by others. The economic analysis shows that Napier grass strips of 2 m width was found best in reducing runoff, soil loss, nutrient losses and net return from crop field [10].

Grassed waterways are important for preventing the scouring of channel bed in shallow ravines. The *Para* grass strips in waterways were optimized for grass cover to check runoff velocity and reduce sediment concentration in downstream water bodies. The different *Para* grass cover (0–100%) in waterways was studied at 2% slope. The different grass covers are 100%, 75%, 50%, 25% and no grass cover. These *Para* grass strips were able to produce green grass yield of 14.5 kg/m2

*para* grass strips in waterways is able to reduce the outflow up to 22% with 100%

. The

bunds. The *Dichanthium annulatum, Cenchrus ciliaris, Vetiveria zizanioides, Eulaliopsis binata, Saccharum munja* and *Aloe barbadensis* vegetative barriers were evaluated for their effectiveness in reducing runoff, soil and nutrients losses from 2% slope of marginal shallow ravines. Vegetative barriers were grown across the slope at 45 m horizontal interval, in paired rows of 10 cm slip to slip spacing. The cultivation of pigeon pea (BDN-2) was done at 120 × 30 cm spacing in all plots. These vegetative barriers reduced the annual runoff by 19.7 to 50.1% and soil loss by

*DOI: http://dx.doi.org/10.5772/intechopen.94136*

proper management of these lands.

*4.1.1 Vegetative barrier*

*4.1.2 Grassed waterway*

51.1 to 80.3% over the control plot [10].

#### *Ecological Engineering Measures for Ravine Slope Stabilization and Its Sustainable Productive… DOI: http://dx.doi.org/10.5772/intechopen.94136*

by constructing peripheral bund, checking of runoff/sediment in the interconnected gullies by earthen gully plugs/bori-bund/composite check dams at proper horizontal interval. The terracing is recommended in shallow and medium ravines slopes along with agroforestry measures for sustainable land use. The deep and very deep ravines are recommended to be kept under permanent vegetative cover along with site specific soil and water conversation measures. The medium and deep gullies having steeper slopes and economically not suitable for cultivation can be put under silvi-pasture permanent vegetation cover with protection from grazing for proper management of these lands.


*3.5.4 Magnetic tracer method*

The application of magnetic tracers has two aspects: (i) to trace sediment sources using magnetic minerals in the environment, (ii) to indicate environmental change in basins. Hence Li *et al.* [34] synthesized that the magnetic tracer method can reflect the history of land use pattern, vegetation succession, and soil erosion in a watershed. It can also identify the soil distribution and the erosion rate for certain period. Therefore, this method can be used to provide a theoretical basis for soil erosion prediction and monitoring, and a history of the development of small watersheds. The advantages of this method are the transportability of the equipment, the methods simplicity; meeting the need of large samples and nondestructive nature. The method is however constrained by inability to trace magnetic properties and depth of soil erosion or deposition. Presently, magnetic tracers have been used to study soil formation, classification of soils, and the quantitative description of evolution, occurrence, and development of erosion.

## **3.6 Soil erosion models**

Soil erosion models are quantitative approaches in study of soil erosion. Based on literature searches, the application of models in Southeastern Nigeria is still minimal. Models can be classified into three groups viz. Empirical, Physically-based and Conceptual (partly empirical/mixed) [41].

#### *3.6.1 Empirical statistical model*

Empirical models are based primarily on observation and inductive logic from the environment. Empirical models such as the Universal Soil Loss Equation (USLE), Revised Universal Soil Loss Equation (RUSLE), the Unit Stream Power Based Erosion/Deposition model (USPED), Erosion Productivity Impact Calculator (EPIC). Empirical models are mainly based on the USLE and remain widely used till date even in regions with limited data. Li *et al.* [34] documented the advantage and disadvantage of these models to include: (1) the formula is concise and the meaning of each factor is clear. (2) The calculation method of the factor has been basically mature and the parameters are easy to obtain for the continuous improvement and perfection of the model. (3) After several years of verification and testing, the accuracy of the model meets the needs of the application. In the tropics, the incompetence of the USLE in its rainfall erosivity component has been overcome by the incorporation of rainfall erosivity values in the RUSLE [27]. Applying the equation in the Anambra area, they observed that about 1804.39 km2 (39.49%) of the area had slight erosion rate of 0–10 t/ha/yr., while rates of erosion in 746.60 km2 (16.34%), 1025.38 km2 (6.28%) and 45.59 km2 (1.02%) of the area are 10.6–85.3, 85.4–235.2, 235.3–608, 608.1–2200 and > 2200.1 t/ha/yr. respectively. They noted that high rainfall erosivity, moderate to high slope and decreasing vegetal cover were the major factors driving soil loss in the area. In an earlier study, Igwe *et al.* [24] compared the USLE and the Soil Loss Estimation Model for South Africa (SLESMA) in producing soil erosion working maps in Anambra and Enugu States, South-East Nigeria. They found out that the USLE model reflected better the actual field situations except for its high values, absolute values compared to the global scale. The values were categorized into very slight (<50 Mg/ha/yr); slight (50–150 Mg/ha/yr); moderate (151–500 Mg/ha/yr); severe (501–1500 Mg/ ha/yr) and very severe (>1500 Mg/ha/yr). A similar high value of above 200 t/ ha/yr. was reported in Uyo metropolis, Nigeria by Fashae *et al.* [42] who observed that the values corresponded with areas with active gullies and altered vegetation cover. Obinna *et al.* [43] applied RUSLE model on the entire Southeastern Nigeria and observed that the results corresponded with known areas of gully menace in the region. Most of the erosion hotspots were located around the north-eastern part of the region covering most parts of Ebonyi State, some parts of Enugu State (Northwest axis), Anambra State (South East and Central axis), and most parts of Abia State. It could, therefore, be concluded that the high number of active gully occurrence may translate into the likelihood of other forms of erosion in the tropics.

#### *3.6.2 Physical process model*

The physical process model is based on the study of the processes and mechanisms of soil erosion e.g. stream flow or sediment transport. Examples of physical models include Water Erosion Prediction Project (WEPP), European Soil Erosion Model (EUROSEM). The WEPP model can simulate soil erosion, non-regular steep slope, and soil, tillage, and management measures by calculating the temporal and

#### *Erosion Quantification and Management: Southeastern Nigeria Case Study DOI: http://dx.doi.org/10.5772/intechopen.99551*

spatial distribution of soil erosion and predicting the movement of sediment in the slope and basin [34]. The WEPP model reflects the applicability and ductility of the temporal and spatial distribution of erosion and sediment; thus, numerous scholars still use this method. Although the physical model greatly compensates for the defects of the empirical model, this approach also has some shortcomings. (1) The physical mechanism of soil erosion is relatively complex and unclear. Some parameters in the physical process model are still dependent on the empirical model. (2) The large range of the study area is the major obstacle that hinders the use of the model because of the exacting demand of the model parameters. (3) The structure of the physical process model is complex and may change because the form has not been unified.


**Acknowledgements**

The project was supported by grants from the National Basic Research Program of China (No. 2015CB351905), the National Key Research and Development Program of China (No. 2016YFA 0302300, No. 2016YFA0200400), the National Natural Science Foundation of China (No. 61306105).


**2. Post-transplant liver biopsy techniques**

Liver biopsies can be performed with various techniques, including a percutaneous ap‐ proach (with marking by percussion/palpation, marking by ultrasound (US), or under realtime US or computed tomography (CT) guidance), a transjugular approach, or a surgical/ laparoscopic approach. Although percutaneous liver biopsies on non-transplant patients can be done without the use of imaging, it is recommended that patients who have undergone any abdominal surgery (including liver transplantation) undergo biopsies aided by the use of US to avoid vascular or other structures [1]. While US marking followed by biopsy is suf‐ ficient in most post-transplant patients, in certain situations (such as split-liver recipients), biopsy under real-time US or CT guidance is preferred to avoid encountering intervening bowel loops. While specimens at least 1.5cm in length and containing at least 6-8 portal tri‐ ads are considered adequate for the diagnosis of chronic liver disease [2,3], some advocate a minimum length of 2.0 cm and at least 11 complete portal tracts for accurate grading and staging of liver disease [4].

© 2012 Limaye et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Percutaneous liver biopsy can be performed rapidly and safely in an outpatient setting with the appropriate monitoring equipment and staff availability [5]. After discharge, patients are typically instructed to avoid strenuous physical activity or driving for 24-48 hours, and are asked to contact the clinical provider in the event of concerning symptoms. In our institu‐ tion, a review of over 3,000 liver biopsies (including liver transplant patients) demonstrated that the majority of complications were discovered within the first hour after percutaneous liver biopsy, and that shortening the recovery time to 1-2 hours did not impact the frequen‐ cy of complications [6].

plications between 0.2% and 1.8% [13]. While early studies suggested an increased risk of post-biopsy sepsis in patients with Roux-en-Y choledochojejunostomy, subsequent studies

Liver Biopsy After Liver Transplantation http://dx.doi.org/10.5772/52617 191

Abnormalities in liver enzyme levels are often encountered in liver transplant patients, and can represent hepatocellular injury (reflected by the transaminases), biliary injury [reflected by alkaline phosphatase or gamma-glutamyl transferase (GGT)], or hepatic synthetic dys‐ function (reflected by the albumin or by coagulation abnormalities). While the use of serum blood tests (such as viral or autoimmune serologies) and imaging techniques (such as ultra‐ sound with Doppler, angiography, and magnetic resonance cholangiography) can be useful to determine the etiology of abnormal liver enzymes, liver biopsy is often necessary for a

The differential diagnosis of liver enzyme abnormalities varies with the amount of time which has passed since liver transplant. In the normal post-transplant course, liver enzymes typically rise immediately following transplant and become normal or near-normal within 3-5 days. If the enzymes fail to improve or normalize but soon rise again, it is likely that an early complication has occurred (Table 1). In the very early post-transplant period (within the first week), liver enzyme abnormalities can be related to primary graft nonfunction (PNF) or dysfunction, hepatic arterial insufficiency, small for size syndrome (SFSS), or portal

PNF and primary graft dysfunction are associated with prolonged ischemia time [15], and it is likely that preservation and reperfusion injury play a role. PNF is heralded by a precipi‐ tous rise in hepatic transaminases in the second or third postoperative day accompanied by signs of hepatic failure (encephalopathy and coagulopathy). Once hepatic artery thrombosis (HAT) has been ruled out by imaging, a liver biopsy confirms the diagnosis. Biopsies in this setting typically show centrilobular hepatocyte dropout due to hepatocellular necrosis, with

The clinical presentation of HAT is quite similar to that of PNF, with a dramatic increase in hepatic transaminases and bilirubin in the very early post-transplant period. A liver biopsy is typically not required, as the diagnosis can usually be confirmed with angiography. When

compensatory zone 2 hepatocyte proliferation and bile ductular proliferation [16].

show that the risk is similar to patients with a duct-to-duct anastomosis [14].

**4. Post-transplant liver enzyme abnormalities**

**5. Early post-transplant liver enzyme abnormalities**

definitive diagnosis.

venous thrombosis (PVT).

**5.1. Primary graft dysfunction**

**5.2. Hepatic artery thrombosis**

Percutaneous liver biopsy can be performed with suction needles (such as Jamshidi needle or Menghini needle), cutting needles (such as the Tru-Cut needle), or spring-loaded needle "guns". Specimens adequate for diagnosis, grading, and staging can usually be obtained by all of the biopsy needles used in current practice.

In patients with severe/uncorrectable coagulopathy, thrombocytopenia (typically platelet count < 50,000/mm3 ), large ascites, morbid obesity, or an inability to cooperate, a transjugu‐ lar liver biopsy (TJLB) is typically recommended [7]. In addition, TJLB is useful in patients for whom wedged hepatic venous pressure gradient (HVPG) measurement would be clini‐ cally useful. Miraglia et al reported on the safety of TJLB in liver transplant patients, with only one complication in 183 biopsies (0.5%) [8].

TJLB is typically performed with the use of automated needle systems, such as the Quick-Core needle and the Flexcore needle. It has been established that these automated needle systems often require multiple passes, and usually collect smaller core samples than those obtained by percutaneous liver biopsy [9]. Despite this fact, specimens ob‐ tained via TJLB are adequate for diagnosis, staging, and grading liver disease in greater than 90% of cases [10,11].

Surgical liver biopsies (either open liver biopsy or laparoscopic liver biopsy) are typically performed when patients require a surgical procedure for another indication. In liver trans‐ plant patients, this often involves repair of postoperative hernias. Biopsies in this setting can be performed with either automated needle systems or with a wedge resection, and the pro‐ cedure provides the advantage of direct visualization of the liver and the ability to immedi‐ ately diagnose and treat any bleeding which occurs.


*4.6. Vitamin C*

Vitamin C is classified as a natural antioxidant compound and has a potential role in reducing the risk of cancer by scavenging ROS in the human body [50]. However, vitamin C is rapidly degraded in fresh fruit and vegetables by several factors including storage [17]. Therefore, there is a need to minimize the decrease in vitamin C during refrigerated storage. Our results showed that the applied treatments of essential oil, ethanol and AsA presented a higher vitamin C value only towards the end of the storage period (9 and 12 d) The same results have been observed in previous studies for treated loquat fruit [18] with ethanol and litchi fruit with AsA [40].

The effect of postharvest treatment of TTO on vitamin C content of fruit and vegetable is not identified well due to limited research on this application. For example, a higher concentration of vitamin C was observed in lettuce plants treated before harvest with TTO

and stored for 20 d at −2 ◦C [51]; however, it was mentioned that no significant difference was observed after 3 d of storage. The difference in our study could be due to the difference in time of application and different concentration of TTO, also due to the difference in plant spices.

Our results shown in Figure 2A are in concurrence with previous work by Naeem et al. [48], who found that application of PMO acting as a semi-permeable coating around the samples' surfaces resulting in a reduction of vitamin C loss. The reduction in vitamin C loss by EOs application could be explained by the antioxidant properties of EOs resulting in a reduction in oxygen diffusion and respiration rate [52].


**Acknowledgements**

Thanks to Mr. L. Perman, Selby, SD; Mr. D. Seick, Selby, SD; and Mr. C. Totton, Chamberlain, SD for providing access to land and cattle. Funding support for this study was provided by the South Dakota Agricultural Experiment Station and USDA, NRCS Conservation Innovation Grant (CIG) 3FH560 "Demonstrating Mob Grazing Impacts in the Northern Great Plains on Grazing Land Efficiency, Botanical Composition, Soil Quality, and Ranch Economics."


*3.1. Slide Surface and Ripples*

No significant movement of the soil mass occurred within 5 min upon the wave application action, while the water body began to become turbid, and the 15-cm-thick sediment started to exhibit a weak waveform oscillation motion with a frequency and phase consistent with those of the water wave when

**3. Results and Discussion**

*3.1. Slide Surface and Ripples*

**3. Results and Discussion**

*3.1. Slide Surface and Ripples*

the wave acted for approximately 3 min. The soil mass showed distinct arc-shaped sliding surface in 20 min. The sliding surfaces located in the middle of the flume and near the wall of the flume were higher, and those at 30 cm and 90 cm approximately from the left wall of the flume were lower. The sediment on the sliding surface reciprocated the wave, and the lower part of the sliding surface was almost stationary. As the duration of the wave action increased, the sliding surface constantly expanded deeper, and the thickness of the moving soil mass increased continuously, forming a distinct "W" shaped sliding surface. When the wave application was stopped, the concentration of suspended sediments in the water gradually decreased. In addition, fine-grained sedimentary formation occurred on the surface layer (Figure 5a). a weak waveform oscillation motion with a frequency and phase consistent with those of the water wave when the wave acted for approximately 3 minutes. The soil mass showed distinct arc-shaped sliding surface in 20 minutes. The sliding surfaces located in the middle of the flume and near the wall of the flume were higher, and those at 30 cm and 90 cm approximately from the left wall of the flume were lower. The sediment on the sliding surface reciprocated the wave, and the lower part of the sliding surface was almost stationary. As the duration of the wave action increased, the sliding surface constantly expanded deeper, and the thickness of the moving soil mass increased continuously, forming a distinct "W" shaped sliding surface. When the wave application was stopped, the concentration of suspended sediments in the water gradually decreased. In addition, fine-grained sedimentary formation occurred on the surface layer (Figure 5a). sliding surface in 20 minutes. The sliding surfaces located in the middle of the flume and near the wall of the flume were higher, and those at 30 cm and 90 cm approximately from the left wall of the flume were lower. The sediment on the sliding surface reciprocated the wave, and the lower part of the sliding surface was almost stationary. As the duration of the wave action increased, the sliding surface constantly expanded deeper, and the thickness of the moving soil mass increased continuously, forming a distinct "W" shaped sliding surface. When the wave application was stopped, the concentration of suspended sediments in the water gradually decreased. In addition, fine-grained sedimentary formation occurred on the surface layer (Figure 5a).

*J. Mar. Sci. Eng.* **2019**, *7*, x FOR PEER REVIEW 5 of 10

*J. Mar. Sci. Eng.* **2019**, *7*, x FOR PEER REVIEW 5 of 10

action, while the water body began to become turbid, and the 15-cm-thick sediment started to exhibit

wave when the wave acted for approximately 3 minutes. The soil mass showed distinct arc-shaped

No significant movement of the soil mass occurred within 5 minutes upon the wave application

**Figure 5.** Experimental photographs showing the slide surface. (**a**) First day; (**b**) Seventh day. The **Figure 5.** Experimental photographs showing the slide surface. (**a**) First day; (**b**) Seventh day. The dotted lines are used to show the slide surface. dotted lines are used to show the slide surface.

dotted lines are used to show the slide surface. Similar phenomenon still occurred in the course of the wave application in subsequent days, while the maximum depth of the sliding surface gradually decreased, and the sliding surface gradually moved upwards; the surface of the soil mass started to undergo local erosion and sedimentation, and ripples appeared. Similar experimental phenomena were also observed in other experiments [22,23], but "V"-shaped sliding surfaces occurred, rather than "W"-shaped. The maximum expansion depth of the sliding surface was close to the interface of water and the soil when the last wave application was made, and the soil mass did not further oscillate. Finally, a continuous array of ripples with the wavelength of 5–10 cm formed on the surface of the soil mass (Figure 6). Additionally, sedimentation occurred in the middle position of the flume and the position near the wall of the flume, and the erosion occurred at sites approximately 30 cm and 90 cm away from the Similar phenomenon still occurred in the course of the wave application in subsequent days, while the maximum depth of the sliding surface gradually decreased, and the sliding surface gradually moved upwards; the surface of the soil mass started to undergo local erosion and sedimentation, and ripples appeared. Similar experimental phenomena were also observed in other experiments [22,23], but "V"-shaped sliding surfaces occurred, rather than "W"-shaped. The maximum expansion depth of the sliding surface was close to the interface of water and the soil when the last wave application was made, and the soil mass did not further oscillate. Finally, a continuous array of ripples with the wavelength of 5–10 cm formed on the surface of the soil mass (Figure 6). Additionally, sedimentation occurred in the middle position of the flume and the position near the wall of the flume, and the erosion occurred at sites approximately 30 cm and 90 cm away from the left wall. The surface shape of the soil mass tended to be consistent with the sliding surface, and W-shaped forms appeared. Similar phenomenon still occurred in the course of the wave application in subsequent days, while the maximum depth of the sliding surface gradually decreased, and the sliding surface gradually moved upwards; the surface of the soil mass started to undergo local erosion and sedimentation, and ripples appeared. Similar experimental phenomena were also observed in other experiments [22,23], but "V"-shaped sliding surfaces occurred, rather than "W"-shaped. The maximum expansion depth of the sliding surface was close to the interface of water and the soil when the last wave application was made, and the soil mass did not further oscillate. Finally, a continuous array of ripples with the wavelength of 5–10 cm formed on the surface of the soil mass (Figure 6). Additionally, sedimentation occurred in the middle position of the flume and the position near the wall of the flume, and the erosion occurred at sites approximately 30 cm and 90 cm away from the left wall. The surface shape of the soil mass tended to be consistent with the sliding surface, and Wshaped forms appeared.

left wall. The surface shape of the soil mass tended to be consistent with the sliding surface, and W-

**Figure 6.** Experimental photographs showing the ripples. (**a**) Top view; (**b**) Side view. **Figure 6.** Experimental photographs showing the ripples. (**a**) Top view; (**b**) Side view.