The Utility of Chondrocutaneous Advancement Flap in Conjunction with Postauricular SkinFlap for Reconstruction of Large Helical Rim Defects

Introduction

The auricle’s complex surface anatomy, intricate architecture, and detailed topography present unique challenges for facial plastic surgeons during reconstruction procedures. The auricle’s shape and position play a significant role in contributing to the overall facial aesthetics. During reconstruction, surgeons must carefully consider the size, position, and contour of the new auricle to achieve harmony with other facial landmarks and restore a natural appearance. Beyond its aesthetic role, the auricle serves important functional purposes. For instance, it houses the ear canal, which is vital for sound transmission and hearing. The auricle provides a platform for wearing eyeglasses and hearing aids, which are essential accessories for many individuals. Reconstructive techniques aim to create an external ear that not only looks natural but also functions as closely as possible to a normal auricle. [1] Patients with auricular deformities can experience significant physical and psychological challenges. The aesthetic and practical functions of the ear are often taken for granted, but when they are compromised due to deformities, the impact on a person’s life can be profound. [1] Indeed, the impact of auricular deformity creates a challenging yet ultimately rewarding arena for both the reconstructive surgeon and the patient. The majority of acquired auricular defects are a result of trauma or surgical extirpation of cutaneous malignancies, particularly skin cancer [2,3].

Auricular skin cancer represents approximately 6% of all cutaneous malignancies in the head and neck area [4]. The pinna, also known as the auricular cartilage or external ear, is a common site for the development of cutaneous malignancies, especially due to prolonged sun exposure. The specific region of the pinna that is often vulnerable to these malignancies is the helical rim. The most common types of cutaneous malignancies that affect the ear, especially the helical rim, are squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) [5,6]. These types of skin cancer are often associated with sun exposure and can present as non-healing sores, ulcers, or abnormal growths on the ear. The development of Mohs micrographic surgery has revolutionized the treatment of nonmelanoma skin cancers, particularly basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). This specialized surgical technique allows for high cure rates while preserving as much healthy tissue as possible, making it especially beneficial for skin cancers occurring in aesthetically and functionally sensitive areas, such as the face, including the ear.

Mohs micrographic surgery can leave significant defects after tumor removal, especially in challenging locations like the ear [7,8]. Indeed, there are several options available for reconstruction, such as primary closure, secondary healing, vascularized cutaneous flap, split- thickness skin graft, full-thickness skin graft, and using a partial or total auricular prosthesis. Choosing the appropriate reconstructive method is a decision that involves careful consideration of the individual patient’s needs, preferences, and overall health. In some cases, a combination of techniques may be used to achieve optimal results. Each reconstructive method has its advantages and is selected based on several factors, including the location and size of the defect, patient preference, and the need for ongoing tumor surveillance [9].

The Antia-Buch flap is a classic and well-known reconstructive technique used for chondrocutaneous ear defects. It was first described by Indian plastic surgeons, Dr. Surendra Kumar Antia and Dr. Rustom Jal Vakil Buch, in 1967. The Antia-Buch flap is particularly useful for reconstructing defects that involve both skin and cartilage, commonly seen in cases of ear deformities or cancer excisions [10,11]. The surgeon designs the flap by bisecting the ear into two flaps: an anterior skin-cartilage flap and a posterior skin flap. The anterior flap includes cartilage, which is essential for restoring the ear’s structural integrity. Indeed, during flap elevation in the Antia-Buch flap technique, a V-Y advancement flap is sometimes utilized to add more length to the medial flap, particularly at the base of the helical crus [11,12]. This additional length can be valuable in ensuring sufficient coverage of the chondrocutaneous defect and achieving better reconstructive outcomes.

While the Antia-Buch approach can be an effective method for providing adequate closure for defects up to 2 cm in size, it is essential to recognize that this technique may have some limitations and potential drawbacks, including the risk of a loss of ear height and unsatisfactory aesthetics [13]. The use of postauricular skin incision in combination with retroauricular advancement flaps is a modification that aims to address the potential drawbacks of the traditional Antia-Buch flap thecnique and avoid scaphal resection to prevent a decrease in ear height. In this modification, a postauricular skin incision is made behind the ear. The posterior flap of skin is then elevated from the postauricular region. The posterior flap is advanced toward the defect site, utilizing retroauricular advancement flaps [14,15]. These flaps allow for the mobilization of postauricular skin to help close the defect without resecting the scapha. The flaps are transposed and sutured into position to cover the chondrocutaneous defect, and appropriate dressings are applied. By utilizing retroauricular advancement flaps, the technique avoids the need for scaphal resection. This helps preserve the ear’s natural height and contours, minimizing the risk of ear height loss and maintaining aesthetics. As with any surgical procedure, the modified Antia-Buch flap technique requires careful patient selection and surgical expertise. The surgeon’s experience in performing this modified approach is essential to achieve optimal results while preserving the ear’s functionality and aesthetics.

Materials and Methods

The authors provide a description of a retrospective study conducted at the IRCCS – Centro di Riferimento Oncologico della Basilicata, in Rionero in Vulture (PZ), Italy. The study focused on ear reconstruction after cancer excision using the modified Antia-Buch technique in patients who underwent surgery between February 2019 and November 2022. The inclusion criteria involved auricle reconstruction after cancer excision and a minimum follow- up time of one year. This approach helps to ensure that the data collected is relevant and allows for a more comprehensive analysis of the patients’ experiences and post-reconstruction outcomes. Exclusion criteria included smaller defects treated with primary closure, larger defects requiring major ear reconstruction, multiple-stage procedures, patients who missed follow-ups, and those lacking documentation. The study was conducted following the ethical standards of the Declaration of Helsinki, and patients provided informed consent, including a photo release section. Fifteen patients participated in the study, with each undergoing the modified Antia-Buch technique for ear reconstruction after cancer excision.

The patients’ ages ranged from 54 to 89 years, with an average age of 76 years. Basal cell carcinoma was the leading reason for ear reconstruction in the study. The mean area of the defects was 3.4 cm2, with the helix being the most frequent location for these defects. The study ensured that the technical details, risks, and benefits of the procedure were thoroughly discussed with the patients during medical interviews, and they were required to provide informed consent before the surgery, which included reporting possible surgical and cosmetic risks. After a minimum follow-up period of one year, post-operative pictures were taken, and patients were reassessed for various aspects, including differences in skin pigmentation between the reconstructed site and adjacent areas, altered and depressed contour of the reconstructed site, constriction of the external auditory canal, and ear asymmetry. The procedure is performed under local anesthesia, where the posterior aspect of the ear pinna is infiltrated with a solution containing xylocaine 1% and epinephrine 1:100,000 [16,17].

This helps in numbing the area and minimizing bleeding during the surgery. Epinephrine is indeed not used on the anterior aspect of the ear pinna to avoid the risk of skin necrosis. Instead, xylocaine 1% may be infiltrated as needed to provide local anesthesia for the procedure. The initial steps are consistent with tumor resection, where the lesion is marked, and margins are determined accordingly (Figure 1). The excisional defect is then converted to a rectangular shape, and immediate reconstruction follows (Figure 2). In the Antia-Buch flap technique, incisions are made along the helical sulcus to create an anterior and posterior flaps. The anterior flap, which includes both skin and underlying cartilage, is advanced and rotated to cover the chondrocutaneous defect. Additionally, for larger defects, further mobilization of the upper segment can be achieved through a V-Y advancement flap of the helix root. The Antia-Buch flap technique can be effective for smaller defects, but for larger defects (>3 cm), a crescent chondrocutaneous scapha resection may be necessary, albeit with potential risks of auricular deformities. The modification, described in the study, involves a postauricular skin incision, which allows for the elevation and mobilization of the posterior flap of skin from the postauricular region (Figure 3).

Figure 1

Figure 2

Figure 3

The retroauricular advancement flaps are utilized to aid in the closure of the defect without the need for resecting the scapha. By utilizing these flaps, the surgeons can achieve effective coverage of the chondrocutaneous defect while preserving the natural anatomy of the ear (Figure 4). Regarding the follow-up process, patients either attended an in-person appointment or were contacted by telephone for a postoperative survey. Those who underwent the modified Antia-Buch approach were asked to complete an evaluative questionnaire, which was designed by the author in collaboration with a clinical psychologist (Table 1). This questionnaire likely aimed to assess the patients’ satisfaction and outcomes after the surgery, including their cosmetic results and overall experience with the modified Antia-Buch technique [18,19]. The evaluative questionnaire used in the study consists of several scales to assess different aspects of the patients’ experiences and outcomes after ear reconstruction using the modified Antia-Buch technique.

Figure 4

Table 1: Post-reconstructive evaluation questionnaire.

These scales include:

1. Satisfaction with the appearance of the ear: This scale is used to measure how satisfied patients are with the cosmetic outcome of the reconstructed ear, compared to their expectations.

2. Excessive asymmetry with the contralateral ear: This scale evaluates whether there is any significant asymmetry between the reconstructed ear and the ear on the opposite side (contralateral ear).

3. Psychosocial well-being: This scale aims to assess the patients’ emotional and psychological well-being following the ear reconstruction, considering any potential impacts on their self-esteem, body image, and overall quality of life.

4. Difficulty wearing glasses and ear devices: This scale focuses on any challenges or difficulties patients may experience when wearing glasses or other ear devices (e.g., hearing aids) after the reconstruction.

By incorporating these scales into the questionnaire, the authors can gain valuable insights into various aspects of the patients’ postoperative experiences, functional outcomes, and overall satisfaction with the procedure. This information is crucial for assessing the success and effectiveness of the modified Antia-Buch technique from a patient-centered perspective.

Results

Patients

Between February 2019 and November 2022, a total of 18 patients underwent auricle reconstruction. Among these patients, three were excluded from the study as they passed away during the follow-up period, unrelated to the cause of reconstruction. Out of the remaining 15 patients included in the study, 9 were males, and 6 were females, with an average age of 76 years (range: 64 to 89 years). The most common reason for ear reconstruction was basal cell carcinoma, representing 20-43.5% of cases, followed by squamous cell carcinoma (16-34.8%) and precancerous lesions (10-21.7%). The standard excision limits for the primary lesions were set at ≥0.5 mm. The mean area of the defect was 3.4 cm2, with the helix being the most frequent location of the defect. All patients underwent our modified Antia- Buch flap (Table 2).

Table 2: Patients.

Post-Operative Complications

The study achieved a 100% rate of total excision of the tumor in all cases. Moreover, it’s remarkable that no complications, such as infection, bleeding, hematoma, wound dehiscence, ear cupping, or cauliflower deformity, were observed among the patients. The absence of surgical revisions in any of the patients further highlights the success and favorable outcomes of the modified Antia-Buch technique used for ear reconstruction after cancer excision. These positive results demonstrate the effectiveness and safety of the procedure in this particular study cohort.

Post-Reconstructive Evaluation

All the enrolled patients answered the evaluative questionnaire and reported positive outcomes. The morphologic results were rated as satisfactory or very satisfactory in all patients, with no self-image distortion or social obstacles resulting from the plastic reconstruction. This indicates that the modified Antia-Buch technique, which combines a helical chondrocutaneous advancement flap and a retroauricular cutaneous transposition flap, proved to be highly successful for ear reconstruction, even in cases with large defects of the superior ear.

Furthermore, the evaluative questionnaire demonstrated significant improvements in patient satisfaction across all four categories (satisfaction with the appearance of the ear, excessive asymmetry with the contralateral ear, psychosocial well-being, and difficulty wearing glasses and ear devices). This suggests that patients perceived notable positive changes in their overall results. The study’s findings support the effectiveness and viability of the modified Antia-Buch flap for ear reconstruction, providing encouraging results for patients who undergo the procedure to address large defects of the superior ear caused by tumor excision.

Discussion

Successful repair of auricle defects requires a well-designed framework and adequate vascularized tissue coverage. The ear’s blood supply comes from various auricular branches of the posterior auricular and superficial temporal arteries, while its sensory supply comes from nerves like the auriculotemporal, great auricular, lesser occipital, vagus, and glossopharyngeal nerves [20]. As mentioned, a significant proportion of auricular malignancies occur on the rim of the helix, and external ear defects can be classified based on location (superior, middle, or inferior third), thickness (partial or full), and size (small, medium, or large) [21]. When reconstructing helical defects, achieving a perfect approximation of the wound edges is crucial to prevent notching and contour irregularities, which can be highly noticeable in this area. Choosing the appropriate reconstructive method involves considering factors such as the size and complexity of the wound, the structures exposed, and the availability of local tissues.

The choice of reconstruction technique should be tailored to each patient’s specific case to achieve optimal functional and aesthetic outcomes [22]. Small skin defects in the upper third of the helix can often be closed directly or with minimal undermining and mobilization of native tissues [23]. Alternatively, geometric patterns, as described by Tanzer, can be used to approximate skin defects without tension [24]. The Antia-Buch auricular repair technique, introduced in 1967, is suitable for moderate-sized defects. This method involves creating a chondrocutaneous advancement flap by making an anterior incision along the helical sulcus to separate the helix and the scapha [25]. A superficial dissection is performed in the posterior auricle to create flaps that will converge at the wedge cut in the antihelix. Originally designed for helical rim defects up to 20 mm in size, this flap technique has been adapted and modified to repair larger defects.

One such modification involves V-Y advancement of the helical root, which extends the application of the technique to address larger defects. By employing these different approaches, surgeons can effectively reconstruct various sizes of auricular defects, providing better cosmetic and functional outcomes for patients [26]. The selection of the appropriate method depends on the size and complexity of the defect, ensuring a successful reconstruction with minimal complications. For even larger defects that extend beyond the helical rim of the upper third into the scapha and antihelix, the use of single-stage pedicled chondrocutaneous transposition flaps based on the root of the helix or the caudal part of the helix, as described by Davis, becomes a viable option [27].

While this technique can be used for the reconstruction of the entire superior pole of the ear, it requires a skin graft to the donor site. Another option is the Orticochea composite chondrocutaneous rotation flap, which is used for the reconstruction of the entire superior pole of the ear and is based on the lateral rim of the ear [28]. However, both of these techniques often involve multiple surgical steps and may require a higher level of surgical expertise to achieve successful outcomes. In contrast, the Antia-Buch flap, even though it was described more than 50 years ago, remains a reliable and effective option among the various techniques for helix reconstruction. Its simplicity, single-stage nature, and favorable outcomes contribute to its continued relevance in addressing defects in the helical region of the ear. While the classic Antia-Buch technique is effective for defects up to 2 cm in size, one potential drawback remains the risk of experiencing a loss of ear height and unsatisfactory aesthetics [29,30].

As a result, modifications have been developed, including the addition of crescentic scaphal excision, Burrow’s triangle, and transposition flaps, with the goal of enhancing the overall aesthetic appearance of the reconstructed ear and minimizing complications like the loss of ear height [31,32]. The modification introduced in this study, combining a postauricular skin incision with retroauricular advancement flaps, was intended to avoid scaphal resection, which can lead to decreased ear height. The postauricular skin incision likely provided additional tissue mobility, while the retroauricular advancement flaps utilized skin from the retroauricular region to enhance coverage of the helical rim defect. Preserving the scapha is essential for maintaining the natural contour of the ear, and increasing flap mobility allows for better reshaping and positioning of the ear during reconstructive surgeries, contributing to improved aesthetics and symmetry [33]. The authors’ modification, involving the incision of postauricular skin and the addition of a retroauricular advancement flap, has had favorable outcomes without the need for scaphal resection. This successful result emphasizes the potential benefits of the modified technique in achieving both good cosmetic outcomes and safety for patients.

Our experience further supports the effectiveness of this modified Antia-Buch flap for helical rim defects up to 3 cm wide. Such positive feedback is valuable in the field of ear reconstruction, and it indicates that the modified approach can provide reliable results for patients with larger defects in the helical region. Patient-reported improvements in satisfaction and quality of life indicate that the modified Antia-Buch technique, with the addition of the postauricular skin incision and retroauricular advancement flap, has been successful in achieving its intended goals. These improvements may include enhanced appearance and functionality of the reconstructed ear, leading to increased confidence and improved social interactions for the patients. Such positive results are valuable in guiding future treatment decisions and advancements in the field of ear reconstruction.

Conclusion

Helical rim defects can present complex challenges in ear reconstruction. While several other methods have been described for addressing these defects, they may involve more complicated procedures compared to the modified Antia and Buch technique. Complex reconstructive methods may involve multiple stages, use of tissue flaps from distant donor sites, or involve intricate surgical maneuvers to restore the helical rim’s natural shape and contour [34-36]. These approaches can be effective in certain cases but may also carry increased risks and longer recovery times. The modified Antia and Buch technique offers a valuable single-stage option for repairing large helical rim defects, providing excellent cosmetic outcomes with technical simplicity and reduced risk of complications such as tip necrosis. This simplicity, coupled with its ability to preserve anatomical landmarks and achieve superior cosmesis, makes it a favorable choice for many surgeons and patients. The classic Antia-Buch chondrocutaneous advancement flap is well-suited for small- to medium-sized helical defects [37].

The addition of an incision in the helical root and closure as a V-Y advancement allows for obtaining additional length to close the helical rim. However, for deformities greater than 2 cm, auricular distortions may occur with this flap technique [38,39]. The modified Antia and Buch approach presented in the study provides a single-stage technique specifically designed for large (up to 3 cm) upper pole defects of the ear, resulting in a superior aesthetic appearance. By combining a postauricular skin incision with retroauricular advancement flaps, they aimed to avoid scaphal resection, which is known to lead to a decrease in ear height. The postauricular skin incision likely allowed for additional tissue mobility, while the retroauricular advancement flaps utilized skin from the retroauricular region to enhance coverage of the helical rim defect [40-43].

The technique’s advantages, including technical simplicity, low risk of tip necrosis, patient convenience, and superior cosmesis, make it an excellent choice for repairing many defects of the helical rim. By restoring anatomical landmarks and concealing scars in the natural concavities and convexities of the ear, the technique ensures a more natural and pleasing outcome for patients. Based on the positive outcomes and advantages reported, the authors highly recommend using this modified Antia and Buch technique for the reconstruction of defects in the helical rim.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest

The authors declare no conflict of interest.

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Oncology and Cancer Medicine: Understanding the Complexities in Older Patients

Oncology, the branch of medicine that deals with the diagnosis, treatment, and prevention of cancer, presents unique challenges when it comes to caring for older patients (Komatsu [1]). As the global population continues to age (Horta Reis da Silva [2-6]), the incidence of cancer among older adults is expected to rise significantly (Pilleron, et al. [1,7]). However, older patients often face distinct physiological, psychological, and social factors that can impact cancer care and outcomes. By 2040, there would be 28.4 million cases worldwide, with 14 million additional cases among older persons (Sung, et al. [1,8]). This is a 47% increase in the cancer burden since 2020. Because of the length of carcinogenesis, the susceptibility of ageing tissues to environmental carcinogens, and other physiological changes, age is a risk factor for cancer (Balducci [1,9]). When treating older persons with cancer, healthcare practitioners must deal with comorbidities, polypharmacy, and age-related frailty (van der Poel, et al. [1,6,10-14]). While loneliness may not directly cause cancer in older adults, it is intricately linked to cancer risk, progression, and survivorship through its impact on psychological stress, immune function, health behavior, social support, healthcare utilization, and quality of life (Horta Reis da Silva [15-17]).

Recognizing and addressing loneliness as a psychosocial determinant of health is essential for promoting resilience, well-being, and optimal cancer outcomes among older adults. Interventions aimed at enhancing social connectedness, fostering supportive relationships, and addressing emotional distress may play a critical role in mitigating the adverse effects of loneliness on cancer-related outcomes in this vulnerable population (Horta Reis da Silva [15-17]). Understanding these complexities is essential for providing comprehensive and personalized care to older individuals with cancer. The use of proven clinical assessment instruments and decision-making models is advised by clinical practice guidelines (Soto Perez de Celiz, et al. [1,12]). However, during the first three months of chemotherapy, significant toxicity affects more than 50% of older patients with advanced disease (Hurria, et al. [1,18]). Making educated treatment decisions requires taking older individuals’ values and preferences into consideration. Geriatric evaluations can support shared decision-making and assist control the vulnerability of older persons with cancer (Komatsu [1]). Geriatric evaluations can support shared decision-making (SDM) between patients, carers, and oncologists as well as assist control the vulnerability of older persons with cancer (Mohile, et al. [1,19]).

Although their responsibilities in the treatment decision-making process are complicated and need flexibility, nurses play a vital role in determining and enabling patients’ choices during cancer SDM (Komatsu [1]). This article provides an overview of the intersection between oncology, cancer medicine, and older people, highlighting key considerations and approaches to improve care for this vulnerable population. Komatsu [1]’s study centers on the role that nurses play in helping older persons with cancer make decisions about their course of treatment. It emphasizes the significance of geriatric screenings, or GAs, and the function that they play in detecting geriatric syndromes, gathering patient preferences, and encouraging effective communication. According to the available research, patients’ outcomes improve when treatment choices are made with their preferences and frailty state in mind. But there are time limits associated with implementing GAs, and nurses must become proficient in performing GAs in an efficient and effective manner (Komatsu [1]).

Comprehensive Geriatric Assessment (CGA)

To address the unique needs of older patients with cancer, comprehensive geriatric assessment (CGA) has emerged as a valuable tool for oncology clinicians (Parker, et al. [20]). CGA involves a multidimensional evaluation of older adults’ physical, functional, cognitive, psychological, and social status to identify vulnerabilities, optimize care plans, and tailor treatment approaches. CGA enables oncologists to assess older patients’ overall health status, frailty, comorbidities, medication use, nutritional status, psychosocial needs, and preferences, which inform treatment decisions and supportive care interventions (2018). The American Society of Clinical Oncology (Giri, et al. [21]), National Comprehensive Cancer Network (DuMontier, et al. [22]), and International Society of Geriatric Oncology (Extermann, et al. [23]) have advised geriatric screening and assessment (GAs) for all older persons with a new cancer diagnosis (Komatsu [1]). By determining each patient’s level of comprehension and aiding in interpreting the material, nurses play a crucial role in detecting the information requirements of older patients. Many elderly cancer patients have faith in their doctors and are content with the information they receive, but they also encounter inadequate communication both before and after making treatment decisions (Komatsu [1]). In addition to providing psychological support, eliciting, and identifying the information requirements particular to each patient, and facilitating appropriate risk perception, nurses must employ the teach-back approach (Komatsu [1]).

Sometimes older cancer patients may not see nurses as experts who can provide them with vital information about their treatment. Obstacles in practice, education, institutional regulations, and administration are among the difficulties faced by oncology nurses (Komatsu [1]). To lower these obstacles and elevate nurses’ responsibilities as essential healthcare providers for elderly cancer patients, nurses must cultivate communication skills that enable them to direct patients’ information demands. Policymakers and clinical practice guidelines propose using Structural Decision Making (SDM) as a conventional strategy in the decision-making process (Maes-Carballo, et al. [1,24,25]). Putting in place a communication training program encourages SDM and patient involvement. Adult children or spouses of older cancer patients are frequently involved in treatment decision-making. Using the fundamentals of a family system approach and family health talks as a basis, nurses should create workable solutions for triadic conversations pertaining to treatment decisions (Komatsu [1]).

Epidemiology of Cancer in Older Adults

Cancer is predominantly a disease of aging, with most cancer diagnoses occurring in individuals aged 65 and older (Pilleron, et al. [1,7]). As life expectancy increases and the population ages, the burden of cancer among older adults is expected to grow (Pilleron, et al. [1,7]). Common types of cancer in older adults include breast cancer, prostate cancer, lung cancer, colorectal cancer, and hematologic malignancies such as leukemia and lymphoma (Pilleron, et al. [7]). Additionally, older adults are more likely to have multiple comorbidities and complex medical histories, which can impact cancer diagnosis, treatment decisions, and outcomes (Reis da Silva [6]).

Major Cancer Sites Among Older Adults

Prostate cancer was the most common cancer among older men worldwide (Figure 1) and in every area except in Asia, where lung cancer outnumbered all other cancers. In most areas, lung and colorectal cancer were also prevalent malignancies (Pilleron, et al. [7]). In Asia, the Middle East, North Africa, and sub-Saharan Africa, stomach cancer was the second most common cancer diagnosed in men over the age of fifty. Liver cancer was more common in these regions. In all locations, these five malignancies accounted for more than two thirds of the overall cancer burden among those 65 years of age and older (Pilleron, et al. [7]). Breast cancer was the most frequent cancer among older females worldwide (Figure 1) and in most of the world’s regions, except for Asia, which includes China, and sub-Saharan Africa, where cancers of the colon, the lung, and the cervical area were more common, respectively. In Asia, the Middle East, North Africa, and sub-Saharan Africa, liver cancer was the most common cancer site among men, while colorectal cancer was among the top five cancer sites worldwide (Pilleron, et al. [7]). The top five malignancies in this age range explained less of the overall cancer burden than did men, but they nevertheless accounted for 46% of cancer cases in the Middle East and North Africa and 66% of cancer cases in China (Pilleron, et al. [7]). Oceania, northern America, and Europe had the greatest rates of prostate and colorectal cancers in older males, and the highest rates of breast and colorectal cancers in older females, with respect to the rates of the individual primary cancer sites (Pilleron, et al. [7]).

Figure 1

Sub-Saharan Africa had the lowest rates of lung and colorectal cancers for both sexes, whereas Asia (including China and India) had the highest rates of stomach and liver cancer among older males and females. The cancer site determined how much the rates varied between areas. When comparing the rates of lung cancer in sub-Saharan Africa (24 per 100,000) with northern America (325 per 100,000 in both sexes combined), the biggest difference was seen in lung cancer (Pilleron, et al. [7]). The combined rate of colorectal cancer in Oceania (286 per 100,000) and sub-Saharan Africa (32 per 100,000) was found to be nine times higher. When it came to liver cancer, the variations were smallest, with rates varying three times between Northern America and Asia (from 29 per 100,000 in both sexes to 89 per 100,000) (Figure 2).

Figure 2

Challenges in Oncology Care for Older Patients

Caring for older adults with cancer presents several challenges due to age-related factors, including physiological changes, comorbidities, polypharmacy, cognitive impairment, functional decline, and social isolation (Seghers, et al. [1,26]). Older patients may also experience disparities in access to cancer screening, diagnosis, and treatment due to ageism, lack of awareness, and underrepresentation in clinical trials. Furthermore, older adults are more susceptible to treatment-related toxicities and adverse events, which can compromise treatment efficacy and quality of life (Seghers, et al. [1,26]). The study (Seghers, et al. [26]) examined the difficulties of treating elderly individuals with multiple comorbidities, such as cancer, in clinical practice. To create a new treatment route for these patients, five elements were found to be important: care coordination, patient support and monitoring, decision making, and patient monitoring. A limited number of patients and carers, selection bias, recollection bias, and the study’s emphasis on geriatric oncology were among its drawbacks (Seghers, et al. [26]). For older cancer patients, geriatric assessment (GA) has been shown to improve the incorporation of non-oncologic information and support decision-making (Wagner, et al. [11, 19,26-31]). For older patients with multimorbidity, including cancer, it is critical to consider GA findings when making treatment decisions and to make sure impairments are targeted with treatments (Seghers, et al. [26]).

More attention was paid by patients and their carers to the aftereffects of therapy, including managing side effects and preserving functionality and quality of life. It is necessary to place a strong emphasis on maintaining and improving functionality, quality of life, and care reliance both during and after therapy (Seghers, et al. [26]). In older patients with multimorbidity, follow-up and reevaluation of ongoing treatment are crucial; however, geriatric oncology has not yet completely perfected this follow-up. Studies have demonstrated increased quality of life, longer survival times, and reduced healthcare utilization using remote symptom monitoring. For our demographic, concurrent monitoring of other illnesses or functioning can be just as crucial (Basch, et al. [26,32-35]). Only patients and their careers, not medical experts, recognized the significance of carer burden and carer engagement (Seghers, et al. [26]). Although it may not receive enough attention in the present healthcare system, carer engagement is crucial and should be considered when developing a care plan for elderly people who have many medical conditions, including cancer. It has been demonstrated that including a nurse navigator and holding multidisciplinary team meetings enhances communication and care coordination within the multidisciplinary team as well as the patient experience (Seghers, et al. [26]). The necessity of switching from a single-disease treatment pathway to an integrated patient-centered strategy has been underlined by research on multimorbidity.

For patients with multimorbidity, each pathway will need to be customized based on a detailed assessment of all issues pertaining to multimorbidity, cancer, general health, and patient values. In summary, the care of elderly individuals with many medical conditions, such as cancer, is intricate and necessitates a departure from a singular disease-focused treatment approach. A patient-centered care route for elderly patients with multimorbidity, including cancer, may be further developed using the information provided here.

Personalized Cancer Care for Older Adults

Personalized cancer care involves tailoring treatment strategies to individual patient characteristics, preferences, and goals. In older adults, personalized cancer care emphasizes a holistic approach that considers not only cancer-related factors but also overall health status, functional status, quality of life, and treatment tolerability. Treatment decisions for older patients with cancer should be guided by evidence-based guidelines, multidisciplinary collaboration, and shared decision-making between patients, caregivers, and healthcare providers. Additionally, supportive care interventions, such as palliative care, symptom management, psychosocial support, and rehabilitation, play a crucial role in optimizing outcomes and enhancing quality of life for older adults with cancer.

Nursing Diagnosis

Jomar [36] wrote a integrative review and highlighted the ten most common nursing diagnosis in hospitalized older adults with cancer:

Nursing Interventions

Farrington [37] purposed in their scoping review to identify and describe supportive treatments designed for elderly patients receiving cancer therapy. The emphasis on geriatric evaluation, particularly about oncological decision making, is one of its features. Prior research has demonstrated the potential advantages of geriatric evaluation for cancer patients under consideration for therapy in terms of managing complexity, uncovering latent issues, enhancing functional status, and choosing suitable interventions. Given population trends, certain localities are shockingly lacking, nonetheless (Farrington [37]). The interventions found in this review downplay the complexity of the elder cancer patient’s experience. Complex concerns including frailty, multimorbidity, or the effects of other geriatric disorders in addition to cancer were seldom addressed by the therapies (Farrington [37]). If researchers and practitioners want to make health care responsive to the unique requirements of this population, they must recognize and address complexity in the design and assessment of treatments. Lastly, research (Farrington [37]). indicates that older adults place a high value on freedom, and that preserving independence is just as crucial to maintaining good health. Although several of the described therapies implied the development of independence for this patient population, the outcome measures employed do not explicitly reflect this as an objective (Table 1) [38,39].

Table 1: Domains and tools used for CGA of older adults with cancer.

Note: Source: Caillet, et al. [38]

Conclusion

Caring for older adults with cancer requires a comprehensive and multidisciplinary approach that addresses the unique needs and challenges faced by this population. By understanding the complexities of oncology care in older patients, healthcare providers can deliver personalized and compassionate care that optimizes outcomes and enhances quality of life. Moving forward, efforts to improve cancer care for older adults should focus on integrating geriatric principles into oncology practice, promoting age-inclusive research and clinical trials, enhancing access to supportive care services, and fostering collaboration among healthcare professionals, patients, families, and communities. Through a holistic approach to cancer care, we can strive to improve the health and well-being of older individuals affected by cancer.

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Role of Diet Therapy in the Treatment of Polycystic Ovary Syndrome (PCOS)

Introduction

Polycystic Ovary Condition (PCOS) is a perplexing endocrine problem in regenerative matured ladies with a pervasiveness from 4% up to 18%, is the most widely recognized reason for fruitlessness and is related to conceptive, metabolic, and hormonal brokenness and higher relationship of pregnancy complications. Women with PCOS with feminine inconsistencies, identified with ovulation, may experience issues with conceiving. 5 Most ladies with PCOS have hyperinsulinemia and Insulin Opposition (IR), which have a critical job in the pathogenesis of the disorder and over the long haul may prompt impedance of glucose digestion, androgen abundance with hyperandrogenic side effects, gonadotropin anomalies, ovulatory brokenness and polycystic ovaries, higher intra-stomach fat and fat tissue Brokenness, Freely Of Weight List (BMI) [1]. Women with PCOS generally gauge more than ladies without PCOS. Numerous examinations show that the greater part of ladies with PCOS are discovered to be hefty and will generally put on more weight longitudinally contrasted with ladies without PCOS. However, regardless of the way that most ladies with PCOS are overweight or fat, many fit ladies with PCOS are additionally viewed as an expanded danger of metabolic disturbances. Central stoutness, race, and age are emphatically connected with IR. Central heftiness and overabundance muscle to fat ratio intensify IR, dyslipidemia, and hormonal dysfunction.

Moreover, ladies with PCOS have expanded danger of metabolic disorder, gestational diabetes, type 2 diabetes, and cardiovascular illnesses Diet and its impact on metabolic results ought to be all the more completely inspected in ladies with PCOS. Ladies with PCOS appear to have a more noteworthy hunger, devour more energy-thick high Glycemic Record (GI) food sources and immersed fat, have insufficient fiber allowed, and have low scores for PCOS-related personal satisfaction, even though their general energy consumption, active work, and resting metabolic rate are like controls. A decrease in IR has been proposed as the central objective of PCOS treatment. Way of life changes (diet in addition to active work), alongside weight reduction (5–10%), is proposed as the principal line technique for improvement of IR, ovulatory work, and diminished free testosterone levels in ladies with PCOS. Trunk fat, Midriff Periphery (WC), and BMI are the best indicators of IR in PCOS [2]. Other dietary intercessions, including starch conveyance, feast recurrence, timing, sufficient admission of n-3 unsaturated fats, or potentially nutrient D supplementation, have been recommended to bring to the table some extra advantages for markers of glucose and energy digestion and regenerative hormonal guidelines.

The methodology of the eating regimen treatment in these patients should be to arrive at explicit objectives like improving insulin opposition, and metabolic and regenerative capacities that will be conceivable through the plan of a low-calorie diet to accomplish weight reduction or keep a sound weight, limit the admission of straightforward sugars and refined carbs and admission food varieties with a low glycemic list, decrease of soaked and trans unsaturated fats and consideration regarding potential inadequacies like nutrient D, chromium and omega-3 [3].

PCOS and Hormones

At the point when you have PCOS, your regenerative chemicals are out of equilibrium. This can prompt issues with your ovaries, for example, not having your period on schedule or not getting it. Your body builds hormones to keep various things going. Some influence your monthly cycle and are attached to your capacity to have a child [4]. The hormones that assume a part in PCOS include

1. Androgens: They’re frequently called male hormones, however, ladies have them, as well. Ladies with PCOS will in general have more elevated levels.

2. Insulin: This hormone deals with your glucose level. If you have PCOS, your body probably won’t respond to insulin in the manner in which it ought to.

3. Progesterone: With PCOS, your body might not have enough of this hormone. You may miss your periods for quite a while or experience difficulty foreseeing when they’ll come.

Common Basic Signs and Symptoms of PCOS are

Hair development in undesirable regions: Your primary care physician might call this “hirsutism” (articulated HUR-soo-tiz-uhm). Undesirable hair may become all over the jaw, bosoms, stomach, or thumbs and toes.

Hair Loss: Ladies with PCOS may see diminishing hair on their heads, which could deteriorate in middle age.

Acne and Oily Skin: Chemical changes because of PCOS can cause slick skin and pimples. (You can have these skin issues without PCOS, obviously).

Darkening of Skin: You might see thick, dim, smooth patches of skin under your arms or bosoms, on the rear of your neck, and in your crotch region. This condition is called acanthosis nigricans.

Sleeping Issues or Feeling Tired Constantly: You could experience difficulty nodding off. On the other hand, you may have an issue known as rest apnea. This implies that in any event, when you do rest, you don’t feel all around rested after you awaken.

Migraines: The flooding chemicals that cause PCOS can give you migraines, as well.

Heavy Periods: PCOS can cause huge swings in the feminine cycle, including exceptionally substantial draining and continuous periods.

Irregular Periods: You might not have a period, or it might skirt a couple of months.

Inconvenience getting pregnant: Not having customary periods can make it hard to get pregnant. PCOS is one of the main sources of fruitlessness.

Weight Acquisition: About a portion of ladies with PCOS battle with weight acquisition or struggle to lose pounds. PCOS can make you acquire a great deal of weight. Furthermore, being overweight can make PCOS side effects more genuine. Shedding even only a couple of pounds might work on the circumstances of your periods. Shedding pounds can be a solid method to keep your cholesterol and glucose levels within proper limits, the two of which are significant if you have PCOS.

Causes of PCOs

The specific reason for PCOS isn’t known. Components that may assume a part include:

Excess Insulin: Insulin is the hormone created in the pancreas that permits cells to utilize sugar, your body’s essential energy supply. If your cells become impervious to the activity of insulin, your glucose levels can rise and your body may create more insulin. An overabundance of insulin may build androgen creation, causing trouble with ovulation.

Low-Grade Aggravation: This term is utilized to depict white platelets’ creation of substances to battle contamination. Exploration has shown that ladies with PCOS have a kind of poor-quality aggravation that animates polycystic ovaries to create androgens, which can prompt heart and vein issues.

Heredity: Exploration recommends that specific qualities may be connected to PCOS.

Excess Androgen: The ovaries produce unusually undeniable degrees of androgen, bringing about hirsutism and skin breakout.

Health Complications Linked to PCOS Include

1. Infertility

2. Gestational diabetes or pregnancy—high blood pressure

3. Non-Alcoholic steatohepatitis–liver inflammation

4. Metabolic syndrome—a cluster condition including high blood pressure, high sugar level, and an abnormal cholesterol level that increases the risk of cardiovascular diseases.

5. Type 2 diabetes

6. Sleep apnea.

7. Depression, anxiety, and eating disorders.

8. Abnormal uterine bleeding

9. Cancer of the uterine lining

PCOS Will in General Altercation Families, Yet the Specific Reason isn’t Known. Side Effects May Include

1. Rare feminine periods, no feminine periods as well as sporadic dying

2. Barrenness because of the absence of ovulation

3. Expanded hair development on the face, chest, stomach, back, thumbs or toes.

4. Skin breakout, slick skin, and dandruff

5. Weight acquires, particularly around the midriff.

6. Diminishing hair on head

7. Pelvic torment

Dietary Strategies During PCOS

Pick High Quality, High Fiber Carbohydrates

Ladies with PCOS are bound to be more determined to have type 2 diabetes than ladies who don’t have PCOS. Like a diabetic eating routine, it is significant for ladies with PCOS to devour top-caliber, high-fiber starches. This will help in settling your glucose levels.

Eat a Balanced Diet

Burning through an even PCOS Diet will assist with keeping your body in an impartial, homeostatic state. A reasonable PCOS Diet permits insulin to work appropriately by carrying glucose to your cells for energy. This cycle brings about less insulin in your circulatory system, at last diminishing androgen creation and lightening your PCOS indications.

A Low Glycemic Record (GI) Diet

The body digests food varieties with a low GI all the more leisurely, which means they don’t cause insulin levels to ascend much or as fast as different food sources, like a few sugars. Food varieties in a low GI eating regimen incorporate entire grains, vegetables, nuts, seeds, organic products, boring vegetables, and other natural, low-sugar food sources [5].

An Anti-Inflammatory Diet

Anti-fiery food varieties, like berries, greasy fish, mixed greens, and additional virgin olive oil, may diminish aggravation-related indications, like weariness.

The Dash Diet

Doctors regularly prescribe the Dietary Approaches to Stop Hypertension (DASH) diet to decrease the danger or effect of coronary illness. It might likewise assist with overseeing PCOS side effects. A DASH diet is rich in fish, poultry, organic products, vegetables entire grain, and low-fat dairy produce. The eating regimen debilitates food varieties high in soaked fat and sugar TYA [6-12].

Follow a Consistent Routine and Regular Mealtimes

Try not to skip suppers. Skipping suppers can crash your glucose levels, prompting food desires and overindulgence. Keeping a standard will permit your glucose levels to settle. Stable glucose helps in legitimate androgen creation in your body. Legitimate androgen creation = less extreme PCOS side effects. A few specialists suggest eating more modest, more incessant dinners to manage glucose and set up better propensities more readily. PCOS patients are not in every case especially overweight but rather PCOS is firmly connected with stomach heftiness and insulin obstruction. Successful ways to deal with nourishment and exercise work on endocrine highlights, regenerative capacity, and cardiometabolic hazard profile- – even without stamped weight reduction. Late investigations permit us to make proposals on macronutrient admission. Fat ought to be limited to < or =30% of all calories with a low extent of soaked fat. High admission of low GI sugar adds to dyslipidemia and weight acquisition and invigorates appetite and carb needs. Diet and exercise should be custom-made to the person’s necessities and inclinations. Calorie admission should be conveyed between a few dinners each day with low admission from bites and beverages. The utilization of medications to either further develop insulin affectability or to advance weight reduction is defended as a momentary measure and is destined to be gainful when utilized right off the bat in the mix with diet and exercise.

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Problems with Bekesy’s Traveling Wave Theory

Introduction

The theory of hearing under the name of traveling wave theory was announced in 1928 by Georg Bekesy, a 29-year-old engineer from Budapest. In 1961, it was awarded the Nobel Prize [1]. It has been revised and supplemented many times. The progress of science over the century has been much faster than the evolution of this theory. Many years of analysis and consideration of the logic of what is assumed to be a mechanistic theory of hearing indicate the need for a new discussion on a seemingly already closed issue. New studies and experiments not previously known have emerged [2]. Processes at the molecular and electron levels are coming to the fore [3]. Less important is the mechanics and hydrodynamics, which so far was the center stage. Some fundamental assumptions of the theory need to be verified. In effect of the analysis of countless works originating from numerous specialties and consultations with specialists in many fields of science, a picture of hearing has emerged that is significantly different from that presented in textbooks and publications related to hearing. For 20 years now, there have been voices of criticism of the current philosophical system of our hearing [4]. Today, despite the censorship of orthodox reviewers, increasingly more is heard about the frailty of the traveling wave theory. But it is still impossible to think about the possibility of making a mistake in the assumptions of the theory of hearing almost 100 years ago.

Thanks to advances in science, this is becoming more and more apparent. A consequence of this is the need to introduce new information related to hearing theory into textbooks. This is met with some significant resistance from potent decision makers, accustomed to the existing state of affairs, despite contradictions with current knowledge and the logic of Nature. There have been signals about the problems of the traveling wave theory for a long time, but they are certainly too weak because they do not stimulate even the slightest discussion on this topic. It seems to be a forbidden topic; it is forbidden to challenge a Nobel Prize-winning theory. As evidence pointing to the need for discussions and analysis, I present some of the most important issues related to hearing theory.

Problems of Hearing Theory for Discussion

1. Human threshold hearing needs to be clarified, where the hearing threshold of 0 dB means a sound wave in EAC with an amplitude of 0.008 nm. This is a pressure of 2.0 x 10^-5Pa – the amplitude of this wave = 8 x 10^-12 m [5]. This wave, approx. 0.01 nm, fades several hundred times on its way to the cap. It does not have enough energy to induce a wave traveling on the basilar membrane, to move the fluid mass according to the amplitude and frequency of the sound wave. It has no energy to tilt or bend the hairs of the hair cells. Thus, it has no ability to induce OHC depolarization. Despite this, such a signal is detected along the auditory nerve. According to the logic, there is another simple signal path to the receptor.

2. The main pillar of Bekesy’s theory is the resonance of the sound wave with the basilar membrane. The problem arises: how does the longitudinal wave resonate with the transverse wave of the basilar membrane? Bekesy assumed, based on simple studies, that the natural vibrations of the basilar membrane are between 16 Hz and 20,000 Hz. Studies of natural vibrations of human tissues have shown that the results range from 8-100 Hz [6]. In addition, Bekesy assumed false dimensions of the basilar membrane for his calculations. The width of the vestibular duct at the oval window is 4.3 mm. In contrast, the width of the basilar membrane at this location taken for calculation is 0.1 mm. In a narrowing cochlea, the width of the vestibular canal near the cap is 1.7 mm, while the width of the basilar membrane increases to 0.5 mm. The thickness of the basilar membrane increases from 0.025 mm at the oval window to 0.075 mm at the cap region. According to Bekesy, this membrane, occupying only 1/42 of the width of the septum between the canals, with an average thickness of 5 micrometers, houses the entire organ of Corti with Deiters cells, Hensen cells, Claudius cells, phalanx cells, external and internal hair cells, Nuel’s space, internal tunnel of Corti, reticular membrane, nerves and vessels, and a layer of connective tissue on the lower surface of the basilar membrane. By itself, the anatomical basilar membrane is only a small part of the entire vibrating mass. It should be added that these vibrations take place in the fluid of the two cochlear ducts, which have high vibration damping capabilities. The transmission of accurate auditory information through this route is very questionable, even impossible. Consider that small mammals and birds have basilar membranes 2-5 mm long and can hear sounds up to 100 kHz [7]. There is no explanation of how resonance is created for a 100 Hz wave when this wavelength in the cochlear fluid is 1450 cm, and the basilar membrane is 5-32 mm. There is no explanation of how the resonance of a wave lasts a tenth of a ms. is formed when the wave has only 1 or 2 wave periods? [8]. The significance of the difference in the speed of the longitudinal wave in the cochlear fluid – 1450 m/s – and that on the basilar membrane – a traveling wave of 8-100 m/s depending on the frequency and location on the basilar membrane – has not been explained. The wave traveling on the basilar membrane grows from the oval window toward the cap. On what principle, since the energy of the sound wave decreases rapidly and, besides, low frequencies cannot resonate in the initial section of the basilar membrane due to the incompatibility of the forcing vibrations with the forced ones. How does resonance arise in small mammals and birds that have a basilar membrane of 2-5 mm, hearing sounds with a frequency of 10 Hz (a pigeon hears sounds of 5 Hz), when the wavelength in the cochlear fluid is 145 m?

3. The sound wave transmits not only energy, which encodes auditory information. How are polytones with aliquots, phase shifts, and accent transmitted? The same is true for cochlear fluid moved by a traveling wave. The same information is supposed to be conveyed by the tilting of auditory cell hairs and the tightening of cadherin filaments, connecting neighboring hairs and the gating mechanism of the potassium mechanosensitive channel. The energy of the sound wave encoding the information is quantized [3]. The mechanisms described above do not have the ability to quantize the energy transferred.

4. Signal amplification, according to theory, is typically mechanical amplification by contraction of the OHC and pulling up the basilar membrane in the appropriate place. Quiet sounds are amplified by 40 dB, i.e. their amplitude increases 10,000 times. It is difficult to understand that we still hear them as quiet sounds. In addition, for loud sounds, OHC contraction after depolarization and basilar membrane pull-up occur, all the same. Doesn’t it interfere with the wave at that time traveling along the basilar membrane? Tones that are below the auditory threshold cannot be amplified because they do not cause depolarization and contraction of the OHC. There is a problem of amplifying polytones, containing quiet and loud tones with harmonic tones. Mechanical amplification is time-consuming. Loud tone information is sent to the brain, while quiet tones are separated and amplified. Information cannot be transmitted along with loud tones. Besides, amplification of quiet tones interferes with extraneous new waves existing on the basilar membrane. Such mechanical amplification could only exist for a continuous harmonic tone. The sound wave does not meet such conditions. Intracellular amplification has no such problems.

5. To simplify calculations, Bekesy assumed that the cochlea is a straight pipe narrowed in half. This changes the mechanics of the cochlea. In the coiled cochlea, wave reflections from the wall surfaces of the double concave ducts play an important role, resulting in a concentration of reflected rays. There is absorption attenuation, reflection attenuation and interference attenuation. Additionally, the dispersion of the wave on the fluid contents of the cochlea and the increasing distance from the oval window cause a dramatic decrease in the energy of the sound wave, which makes it difficult to transmit information to the brain.

6. The fading of wave energy on its way to the receptor: Laser Doppler vibrometry studies have shown that the amplitude of the 90 dB (500 nm) and 10 kHz wave in the EAC, examined on oval window, has an amplitude of 0.5 nm [2]. The path to the oval window is not the path to the receptor, but the greatest energy loss occurs on the way to the cap. Please note that the input is 90 dB. A human hears a tone at an input of 0 dB = 0.01 nm. If such a wave amplitude fades on its way to the cap several hundred times, how is the wave traveling on the basilar membrane formed? This is a size 10 times smaller than the diameter of the atoms that make up the basilar membrane. Is such a wave capable of bending the hairs of hearing cells 10,000 times thicker? And if it is assumed that these hairs are connected to the covering membrane, they must be bent to change the tension of the cadherin filaments. If this is impossible, then inducing OHC depolarization is impossible, mechanical amplification is impossible. We hear it! So there is another signal pathway to the receptor.

7. Cochlear Implant Surgery for Partial Deafness: The electrodes inserted into the tympanic canal immobilize the basilar membrane, but this does not change the hearing of tones heard before the surgery. The signal must go to the receptor by another route.

8. Stapedotomy surgery improves hearing only of low and medium frequencies [9]. The piston prosthesis mimics only the movements of the piston. It does not imitate the physiological movements of the stapes plate in the transverse axis at high frequencies, or movements in the longitudinal axis of the stapes plate during the highest frequencies. The absence of these rocking (oscillating) movements is the reason for the lack of improvement in high frequencies after surgery.

9. The incudostapedial joint is a spherical joint. It allows the stapes plate to move in various planes, which allows you to hear high frequencies by transmitting them from the middle ear bones to the cochlear bone casing. In the case of rocking movements, half of the staples generate fluid movement in the direction of the cap, while the other half of the plate generates fluid movement in the opposite direction. Adjacent fluid and wave streams with opposite directions are formed. The transmission of information through this route is disturbed. And precise information reaches the receptor. It is believed that it takes a different route – through the bone housing of the cochlea.

10. The signal travel time from the EAC to the auditory nerve according to electrophysiological studies is 1.5 – 1.9 ms. On the other hand, the signal travel time, including all sections of the path through the basilar membrane, cochlear fluids and the tip-links mechanism, is approximately 5 ms. This indicates that there is a path twice as fast. The bone conducts sound waves at a speed of 4,000 m/s.

11. Bekesy incorrectly assumed that the sound wave resonating with the basilar membrane travels on both sides of the membrane. For this purpose, for calculation purposes, he removed Reissner’s membrane from the ear and connected the vestibular duct with the cochlear duct, regardless of the difference in electrolyte concentrations of these fluids. In this way, he obtained a different (artificial) path of sound wave along the basilar membrane. However, he neglected the important facts that along the way the sound wave encounters the tegmental membrane with very low natural vibrations, then crosses the subtegmental endolymph layer to pass through the organ of Corti with receptors in the form of hair cells. It passes through these cells, without transmitting information, and heads for the basilar membrane, where it is supposed to induce a traveling wave, which is supposed to activate the cochlear fluids to tilt the hairs of the hair cells. This resulting movement of the cochlear fluid due to the traveling wave has a direction opposite to the direction of the wave heading to the basilar membrane. Nature could not accept such an illogical solution, incompatible with anatomy. The auditory receptor receives a relevant stimulus, which is the energy of the sound wave. So the hair cell = receptor receives information from the sound wave that reaches it. In Bekesy’s concept, the sound wave passed through the auditory cells without passing information to the receptor. This goes against the logic of Nature.

12. A sound wave has no mass and is not subject to the law of inertia, possessing motion and acceleration. In contrast, the vibrating elements of the middle ear (ossicles) and the vibrating elements of the inner ear – the basilar membrane, inner ear fluids, OHCs and hairs of hair cells – have mass and are subject to positive or negative motion and acceleration in wave motion. There is a formula for acceleration in wave motion. (2π x frequency)2 x amplitude. Acceleration times mass = inertia g/mm/s2. The higher the frequency and amplitude of vibrating element, the higher the inertia. This issue is not analyzed in the traveling wave theory, because it indicates the difficulty of transmitting high frequencies through the basilar membrane and cochlear fluids. On the other hand, there is no problem with signal transmission through the bone housing of the cochlea.

13. The tip-links mechanism, with the pulling of cadherin links acting as the molecular mechanism of channel gates, is supposed to be responsible for the opening of mechanosensitive potassium channels in the hair cell membrane. J. Hudspeth announced that myosin is responsible for closing potassium channels. None of the entire range of myosins are able to operate this mechanism during high frequencies. They are too slow to act.

14. The basilar membrane, according to the traveling wave theory, is supposed to be responsible for frequency discrimination. The length of the human’s basilar membrane = 32 mm. A trained musician recognizes 3,000 frequencies. That is, for 1 frequency with the maximum wave deflection on the basilar membrane, there is 0.0106 mm of the basilar membrane. What do these waves look like in the case of polytones with harmonic components? How do these waves traveling on the basilar membrane determine frequency resolution in mammals or birds having basilar membranes 2 – 5 mm long? Birds are very musical.

15. The hair cell is an excitable cell [10]. A supra-threshold stimulus in the form of a sound wave knocks the cell out of a state of dynamic equilibrium, starting its depolarization, followed by repolarization. During these phases, the hair cell is insensitive to new stimulation. This is an absolute refraction, lasting about 1-2 ms. Assuming that all ion channels work at the same time, the cell cannot be depolarized and thus contract more often than 1000 /s. It is not possible to transmit high frequencies while depolarizing the entire cell at the same time. Only limited depolarization gives the possibility of high-frequency transmission. A problem arises with the mechanical amplification of low intensities of high frequencies if depolarization and contraction of the OHC cannot occur.

16. Directional hearing is determined by the difference in the time it takes for the signal to reach both ears. The difference in the interaural distance in humans results in a difference in the received signal time of 0.6 ms in air and 0.5 ms in water. This 0.1 ms difference means that in water, directional hearing does not work. The interaural distance in birds is much smaller, yet they have excellent directional recognition, even in the case of quiet sounds, which, according to the traveling wave theory, require time-consuming amplification and travel to the receptor in a roundabout way through time-consuming resonance, basilar membrane, cochlear fluids and the inclination of hairs of hair cells. The survival of many animal species on Earth depends on the speed of auditory reactions, the ability to recognize directions and judge distances.

17. The traveling wave theory does not clearly describe the conversion of the quantized mechanical energy of a sound wave into electrochemical energy of the auditory cell membrane [11]. There is no description of the biochemical processes inside the hair cell, intracellular amplification, or the importance of calcium in the transmission of information. These processes are described in the paper: “Processing and Transmission of Auditory Information” from 2004 and in the paper: “Submolecular Theory of Hearing” from 2022.

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Role of High-Resolution Manometry in Diagnosing Esophageal Motility Disorders – A Literature Review in Line with Chicago Classification V4.0

Introduction

The human esophagus is divided into the cervical esophagus, composed of striated muscles, and the thoracic esophagus, made of smooth muscles. Its main function is to transfer swallowed food from the throat to the gut through coordinated contractions known as peristalsis [1]. Disruption of this process can lead to esophageal motility disorders (EMDs), also considered functional disorders affecting the esophageal body, or sphincters due to neuromuscular dysfunction in smooth muscle [2]. EMDs can have a wide range of presentations, from asymptomatic to difficulty swallowing, noncardiac chest pain, or heart pain [3]. In severe cases, they may lead to aspiration, regurgitation, respiratory problems, and weight loss [4]. EMDs can occur as primary disorders or as part of systemic or secondary diseases, such as systemic sclerosis, diabetes mellitus, Chagas disease, viral infections, and malignancies [2,3]. Despite being rare and having an elusive cause, EMDs significantly impact individuals› quality of life and society. Factors like genetics and environmental factors have been evaluated as potential reasons. Some studies have shown that esophageal motility may correlate with the patient›s age [5]. The diagnosis of EMDs is challenging, requiring a combination of tests like high-resolution manometry (HRM), endoscopy, and barium swallow imaging to support the diagnosis. Advancements in manometry sensor technology and data display have improved the accuracy of EMD diagnosis [2]. HRM, with its higher spatial resolution and more sensors (36 sensors, one cm apart) covering all segments of the esophagus, including sphincters, pharynx, and stomach, has become the standard gold test for diagnosing, classifying, and managing EMDs [6,7]. The data collected is transformed into a dynamic color-coded esophageal pressure topography (EPT) using advanced software algorithms.

The Chicago classification, developed in 2007 by Ray E. Clouse, is an evolving hierarchical system that continues to be refined. The latest version, CCv4.0, was published in 2020 after two years of work by 52 international HRM experts from 20 countries. It includes updates and recommendations for a solid-state HRM catheter with less than 2 cm sensor spacing and a combined impedance catheter [8,9]. CCv4.0 encompasses seven subgroups, covering various aspects such as standard HRM protocol, achalasia, esophagogastric junction outflow obstruction (EGJOO), distal esophageal spasm (DES), hypercontractile esophagus, ineffective esophageal motility (IEM), and esophagogastric junction (EGJ) metrics [10]. These modifications make CCv4.0 more rigorous and accurate compared to the previous CCv3.0. This review aims to determine the role of HRM in diagnosing EMDs in line with CCv4.0 and to find the difference between CCv3.0 and CCv4.0.

Methodology

Search Strategy

The authors conducted a thorough literature review to update the role of HRM in CCv4.0 & the classification of EMDs. They searched multiple databases, including PubMed, ResearchGate, the Cochrane Library, and Google Scholar, using specific keywords related to CCv4.0 and EMDs. Only full-text articles were included, while unpublished data and grey literature were excluded.

Study Screening and Selection

The author conducted a comprehensive literature search and identified a total of 71 citations. After removing duplicates and screening abstracts and titles, 46 articles remained. Four articles were not accessible in full text and were excluded. Thus, 42 articles were considered potentially relevant and reviewed in full. Ultimately, ten articles were included in this study. The research was approved by the Faculty of Life Science and Education, University of South Wales Ethics Subgroup, and Faculty Research Ethics Committee. Details of the excluded articles can be found in Appendix Table 1.

Appendix Table 1.

Assessment of Methodological Quality

Each selected article was assessed based on the method of the study, literature search strategy, data extraction, risk of bias, and all parts of methodological qualities using the A Measurement Tool to Assess Systematic Reviews (AMSTAR-2) tool and presented in Appendix Table 2.

Appendix Table 2. Q1: did the research questions and inclusion criteria for the review include the components of PICO?
Q2: did the report of the review contain an explicit statement that the review methods were established prior to the conduct of the review and did the report justify any significant deviations from the protocol?
Q3: did the review authors explain their selection of the study designs for inclusion in the review?
Q4: did the review authors use a comprehensive literature search strategy?
Q5: did the review authors perform study selection in duplicate?
Q6: did the review authors perform data extraction in duplicate?
Q7: did the review authors provide a list of excluded studies and justify the exclusions?
Q8: did the review authors describe the included studies in adequate detail?
Q9: did the review authors use a satisfactory technique for assessing the risk of bias (RoB) in individual studies that were included in the review?
Q10: did the review authors report on the sources of funding for the studies included in the review. Q11: if meta-analysis was performed, did the review authors use appropriate methods for statistical combination of results?
Q12: if meta-analysis was performed, did the review authors assess the potential impact of RoB in individual studies on the results of the metaanalysis or other evidence synthesis?
Q13: did the review authors account for RoB in individual studies when interpreting/ discussing the results of the review?
Q14: did the review authors provide a satisfactory explanation for, and discussion of, any heterogeneity observed in the results of the review?
Q15: if they performed quantitative synthesis, did the review authors carry out an adequate investigation of publication bias (small study bias) and discuss its likely impact on the results of the review?
Q16: did the review authors report any potential sources of conflicts of interest, including any funding they received for conducting the review?

Quality of Evidence

The grading of the recommendation, assessment, development, and evaluation (GRADE) tool was used to assess and evaluate the quality of evidence and risk of bias for CCv4.0 recommendations in the main findings of this review as presented in Appendix Table 3.

Appendix Table 3.

Findings of HRM Protocol in the CCv4.0

CCv4.0 introduces significant changes to enhance the HRM protocol, incorporating position changes, and adding provocation tests. Standardizing the HRM protocol in CC4.0 is crucial for improving procedure reliability and consistency, enabling collaborative research among different centers [11]. CCv4.0 records HRM findings in both supine and upright positions, facilitating the diagnosis of various motility conditions. The inclusion of provocative tests in CC4.0 has led to increased sensitivity and specificity of HRM studies.

Patient›s Positions in line with CCV4.0

After HRM catheter insertion through the nostril to the esophagus and stomach, the patient rests for 60 seconds (Adaptation period) and takes three deep inspirations to confirm the catheter›s position. The procedure can begin in either an upright/supine position, with a preference for starting in the supine position according to CCv4.0 protocol recommendations. Clinicians may modify the protocol based on available resources if they adhere to the normative values [11]. Supine Position: Patients start with ten wet swallows, and if the results are inconclusive, they switch to the upright position and perform at least five more swallows. Changing positions helps eliminate conditions specific to the supine position, like false-positive EGJOO identification.

Upright Position: Patients begin with five wet swallows to determine the conditions of the upright position, such as false-positive IEM diagnosis. The upright position affects bolus transportation velocity and the distal contractile interval (DCI) in the esophagus due to the gravity effect.

The CCv4.0 working group suggests obtaining swallows in both positions, particularly if unexpected EMD is found. While single wet swallows in upright and supine positions, together with provocative tests, can be time-consuming, in certain cases, if a conclusive diagnosis of achalasia type I or II is achieved from the primary position, the full protocol can be avoided. Nevertheless, if the CCv4.0 protocol is not fully completed, applying position-appropriate normative values is recommended [11].

HRM Diagnostic Threshold

The CCv4.0 working group determines the cut-off thresholds of HRM metrics when evaluating deglutition relaxation through lower esophageal sphincter (LES)/EGJ with the use of integrated relaxation pressure (IRP). Assess vigorous esophageal body contraction using DCI. The latency of deglutition inhibition by using distal latency (DL) [10]. Table 1 illustrates the HRM metrics and thresholds according to CCv4.0 (the author inspired it from (Yadlapati, et al. [9]).

Table 1: HRM metrics and thresholds according to CCv4.

Additional HRM Maneuvers

The CCv4.0 working group recommends incorporating provocation tests in the HRM protocol to assess esophageal motility. This is essential because a limited number of wet swallows during supine or upright positions may not always be sufficient, especially in symptomatic patients [11]. These additional maneuvers include multiple rapid swallows (MRS), rapid drink challenge (RDC), ingestion of more viscous material, single solid swallows, and test meals (using either the patient›s food or pre-prepared meals). The CCv4.0 working group has reached a consensus on how to analyze, interpret, and report the results of these provocation tests [10]. Figure 1 demonstrates using of provocation tests during CCv4.0 protocol in supine and upright positions (the author inspired it from Fox, et al. [11]).

Figure 1

Multiple Rapid Swallows (MRS): The CCv4.0 working group recommends the use of a syringe to deliver 2 ml of fluid, repeated five times with 2-3-second intervals, as part of the MRS maneuver. This maneuver is performed in the supine position and may be repeated up to three times to confirm the presence of peristalsis reserve, as postcontraction augmentation varies. However, MRS lacks normative values and specific diagnostic criteria, requiring further research. The expected response during MRS is the absence of esophageal contraction (DCI < 100 mm Hg.s.cm) with complete deglutition inhibition of the LES, along with post-MRS contraction augmentation. This response would suggest the presence of peristalsis reserve, especially if a diagnosis of IEM is made during single wet swallows [11].

Rapid Drink Challenge (RDC): The CCv4.0 working group recommends performing RDC in an upright position minimizing the risk of aspiration. The RDC involves swallowing 100-200 ml of water (preferably 200 ml) with a straw. During RDC, assess the contraction inhibition of the esophageal body (DCI) & IRP. The expected response is a DCI < 100 mm Hg.s.cm, total inhibition of the lower esophageal sphincter, with no evidence of significant motility disorders post-RDC [10]. RDC is a common and easy-to-perform additional provocation test that increases the sensitivity of HRM. Studies have established normative values with a sensitivity of 80% and specificity of 93%. Furthermore, patients with moderate HRM throughout single wet swallows can receive a conclusive diagnosis by performing the RDC maneuver [11].

Three patterns can be detected during RDC:

• Hyperparasite pattern (standard).
• A brief hyperparasite pattern (weakness of deglutition inhibition) occurs in the non-obstructive hypercontractility.
• prolonged hyperparasite pattern (Impairment of IRP).

Single Solid Swallow Maneuvers: Including single solid swallows in the HRM protocol improves the diagnosis of EMDs, especially EGJOO. Studies reveal that single swallows increase DL & DCI while reducing significant breaks in the contractile front [10]. The CCv4.0 working group suggests swallowing a 1-2 cm cube of soft biscuit, buttered bread, cake, or dumpling, after chewing it. At least five (preferably 10 swallows) during HRM protocol are recommended, and the esophageal pressure is measured using Medtronic software. The diagnostic cut points for EGJ disorder are IRP > 25 mm Hg and DL > 4.5 seconds (indicating effective contraction) with fewer than a small break in the contractile front (<3 cm) & DCI > 1000 mmHg.cm. s (vigorous contraction). If a minimum of 20% of single swallows lead to effective contractions, it indicates EGJOO; otherwise, it suggests IEM for solid swallows [11].

Solid Test Meal: CCv4.0 recommends this test for patients with esophageal symptoms when other maneuvers are inconclusive or fail to identify the underlying cause. It can also help detect peristalsis reserve in patients suspected of having IEM during water swallows [12]. CCv4.0 recommends patients undergo a standard test meal (200 g) in 8 minutes, producing 20-30 pharyngeal swallows and abnormal EGJ function is considered when having two or more swallows with IRP>25 mm Hg (in the Medtronic system). The addition of the test meal has increased the diagnostic yield to nearly 50% for EGJOO, and HRM with meal test shows higher sensitivity (85%) compared to single water test (54%) and barium esophagogram. The HRM protocol with solid meal test and impedance can be extended to post-prandial periods for identifying other functional disorders, for example, rumination syndrome, volume reflux, and supra-gastric belching [11].

Findings of EMD Classification in the CCv4.0

CCv4.0 classifies EMDs based on peristalsis and EGJOO, building on CCv3.0 (Figures 2A & 2B). However, CCv4.0 extends the diagnosis beyond HRM findings, incorporating additional tests (Provocation tests, timed barium esophagogram, and FLIP) and considering clinical relevance to support HRM findings [13]. CCv4.0 has updated all EMDs, requiring additional tests and a history of obstructive symptoms for a definitive diagnosis of EGJOO. The IEM definition is now more precise, including fragmented peristalsis. However, treatment progress for achalasia and absent contractility remains limited. DES and hypercontractile esophagus diagnoses remain unchanged due to insufficient data. The categorization of major and minor disorders has been eliminated as minor disorders like IEM are now considered major disorders under the new definition [12]. The differences between the diagnosis of EMDs by CCv4.0 and CCv3.0 are summarized in Table 2.

Figure 2

Table 2: The difference between CCv4.0 and CCv3.0.

Esophagogastric Junction Outflow Obstruction (EGJOO) Disorders: The CCv4.0 working group has divided EGJOO disorders into achalasia (type I, II, III) and EGJOO with the recommended criteria of abnormal IRP in the first position [12].

Achalasia: The subclassification of achalasia in CCv4.0 remains unchanged as was in the previous classification. CCv4.0 defined achalasia as abnormal median IRP in 10 wet swallows during primary position (upright or supine) and 100% absent peristalsis. The definition of Absent peristalsis is either no peristalsis at all or premature contractility with DL < 4.5 sec. Pan esophageal pressurization (PEP) can differentiate between type I and II achalasia. CCv4.0 refined the definition of type III achalasia from the previous CCv3.0 [14].

Conclusive Diagnoses of Achalasia and Subtypes: Although HRM protocol in Chicago classification version 4 consists of primary and secondary positions for wet swallows, a conclusive diagnosis of achalasia requires a primary position only [14].

Type I Achalasia (Classic): CCv4.0 defines type I achalasia as a late stage of the disease, with median IRP raised above the upper limit of normal and 100% absent peristalsis (Figure 3A) [14]. The most common subtype is the same as in the previous Chicago classification (Figure 3B). CCv4.0 defines it with an abnormal median IRP compared to the upper limit of normal, 100% absent peristalsis, & 20% or more of swallows showing pan esophageal pressurization [12].

Type III Achalasia: It is considered a rare subtype whose definition has been changed by the CCv4.0 working group [14]. CCv4.0 defined type III achalasia as an elevated median IRP compared to the upper limit of normal with 20% or more swallows showing premature contractions (DL < 4.5 seconds and DCI ≥ 450 mmHg.s.cm) and no evidence of peristalsis [12]. However, the cutoff of 20% swallows with premature contractions is considered arbitrary, and higher numbers of premature spasms may increase confidence in diagnosing type III achalasia (Figure 3C). It is worth noting that chronic daily use of opioids has been associated with premature contractions, so CCv4.0 recommends discontinuing opioids before HRM study if possible [14].

Figure 3

Inconclusive Diagnosis of Achalasia

CCv4.0 classified type me and II achalasia as inconclusive diagnoses if median IRP in both the primary and the secondary positions fell within the upper limit of normal, in addition to failed peristalsis in less than 20% of swallows. This is regardless of signs of pan esophageal pressurization. Additionally, an inconclusive diagnosis can be made if there is evidence of peristalsis with changing position in type I or type II achalasia in the primary position, requiring a supportive test (accepted clinical observation). For type III, an inconclusive diagnosis is made if there is an abnormal median IRP and premature contractions with evidence of peristalsis. If the patient fulfills the criteria of EGJOO, the diagnosis of EGJOO with spasms can be considered [14].

Esophagogastric Junction Outflow Obstruction (EGJOO)

Approximately 10% of HRM patients exhibit EGJOO motility disorder, making HRM the gold standard for diagnosing EMDs. However, around 30% of these cases may not require clinical action, leading to potentially inappropriate treatments due to factors like opioids, benign mechanical obstruction, or artifacts [10]. To enhance the specificity and reduce over-diagnosis of EGJOO, CCv4.0 reviewed previous literature and identified different overlapping patterns of peristalsis, including EGJOO with IM, EGJOO with spasm, EGJOO with hypercontractility, and EGJOO with intact peristalsis. Thus CCv4.0 working group has made a more stringent criterion for the diagnosis of EGJOO that requires all the following [12]:

• Raised IRP in both supine and upright positions.
• 20% or more of supine swallows should have elevated intrabolus pressure.
• The presence of clinical symptoms including dysphagia and/or non-cardiac chest pain.
• TBE and FLIP, two tests that are not supportive of HRM, show signs of outflow obstruction.

The CCv4.0 working group provides these recommendations to reach a «clinically relevant conclusive diagnosis» of EGJOO as summarized in Table 3.

Table 3: CCv4.0 update on esophageal motility disorders.

Inconclusive Diagnosis of EGJOO

The CCv4.0 working group defined the inconclusive diagnosis of EGJOO [10] as an isolated increase in IRP in primary or secondary positions or an isolated increase in intrabolus pressures during the supine position (Low GRADE, Conditional recommendation).

Additional points for EGJOO

The CCv4.0 working group considers the following points quite supportive but not necessary for the diagnosis of EGJOO [10]:
• Evidence of outflow obstruction and esophageal pressurization during RDC.
• If the patient›s symptoms are temporally related to an outflow obstruction through a solid test meal, this information is important.
• Abnormal EGJ function after pharmacological provocation.

Disorder of Peristalsis

Disorders of peristalsis disorders in the CCv4.0 scheme remain the same as in CCv3.0. including DES, absent contractility, IEM, and hypercontractile esophagus [13]. Furthermore, fragmented peristalsis is still not considered a separate disorder but rather a diagnostic component of IEM. The definition of Peristaltic disorders requires a normal median IRP with the exclusion of conclusive EGJOO [10].

Absent Contractility

The diagnosis of absent contractility remains unchanged according to CCv3.0. It is defined as a normal median IRP on the primary and secondary sides, together with 100% failed peristalsis (DCI <100 mmHg.s.cm) (shown in Figure 3D). An inconclusive diagnosis of absent contractility is considered when the median IRP falls within the upper limit, especially in the supine position between 10 mmHg to 15 mmHg (Medtronic system), in symptomatic patients with dysphagia. This requires excluding type I achalasia through provocation tests and supportive tests [10]. Absent contractility is idiopathic and has a prevalence of 0.4% in 469 healthy volunteers and 3.2% in 1081 patients assessed for anti-reflux surgery [15]. It can also be seen in patients having mixed connective tissue diseases and gastroesophageal reflux diseases [16].

Distal Esophageal Spasm (DES)

The CCv4.0 working group recommends considering together HRM findings and clinical presentation to define clinically relevant DES (Figure 3E). The definition of conclusive DES requires consideration of the following points [17]:

• At least 20% of premature contraction (DL less than 4.5 seconds)
• DCI more than 450mmHg.s.cm.
• Presence of dysphagia and/ or non-cardiac chest pain.
• Normal EGJ relaxation.

The finding of DES can be challenging due to difficulties in localizing contractile deceleration points (CDP). CCv4.0 recommends considering various techniques for diagnosing DES and distinguishing intrabolus pressure from esophageal contraction artifacts. Although DES is a concern, there is insufficient evidence to support these concerns [17]. Inconclusive DES is defined as having at least 20% of premature contractions with DL > 4.5 seconds & DCI < 450 mm Hg.s.cm. Supportive tests like barium esophagogram, FLIP, and MRS during HRM can aid in confirming the diagnosis. It is important to distinguish primary DES from secondary DES, where type III achalasia and factors like opioid use or GERD may contribute to secondary DES [16].

Hypercontractile Esophagus (HE)

The CCv4.0 working group suggests using manometric findings and relevant symptoms (non-cardiac chest pain and/or dysphagia) for diagnosing clinically relevant HE [10]. The manometric findings for a conclusive diagnosis remain unchanged from CCv3.0, which includes 20% or more hypercontractile supine swallows with DCI >8000 mmHg.s.cm with normal IRP (Figure 3F). Before making the HE diagnosis, the CCv4.0 working group suggests excluding distal esophageal obstruction or achalasia, as HE can be associated with other abnormalities like GERD and EGJOO. The introduction of three manometric subtypes (single peaked, multipeaked/Jackhammer, vigor LES after contraction) of HE is not recommended by the CCv4.0 working group. The following statements did not meet the criteria of agreement in CCv4.0 as they were unable to meet 85% agreement [18]:

• HE should be retained as a major condition of peristalsis and not as a minor one (65%appropriate).
• HRM should be annexed with an impedance study for the best possible detection of intrabolus pressure, flow time, and bolus clearance via esophagus and EGJ (67% appropriate).
• The hypercontractile esophagus is not synonymous with the Jackhammer esophagus and should be considered a subtype of HE (76%appropriate).
• The diagnosis of HE should be supported by the manometric findings of the elevated intrabolus pressure (60% appropriate)
• The diagnosis of HE should be supported by an abnormal RDC test (53% appropriate).
• HRM diagnosis of HE should be supported by the absence of contraction reserve on MRS (56% appropriate).
• Response to medications (nitrate, calcium channel blockers, phosphodiesterase inhibitors, etc.) should be considered as a support to HE diagnosis (51% appropriate).

Ineffective Esophageal Motility (IEM)

The CCv4.0 working group modified the previous CCv3.0 classification of the esophageal hypomotility disorder into two groups in the context of normal LES relaxation.

• Absent peristalsis
• Ineffective motility disorder

The CCv4.0 working group included fragmented peristalsis as part of the Ineffective Esophageal Motility (IEM) definition [10]. The criteria for a conclusive IEM diagnosis were refined, requiring more than 70% ineffective swallows or at least 50% failed peristalsis. These ineffective swallows are defined by a DCI of 100 to 450 mmHg.s.cm or more than 5 cm transition zone fragmentation in peristalsis, while failed peristalsis is defined as a DCI of less than 100 mmHg.s.cm. Patients with ineffective swallows between 50-70% are given an inconclusive IEM diagnosis, and the CCv4.0 working group recommends additional supportive tests like barium esophagogram or HRM with impedance to strengthen the IEM diagnosis, particularly by showing poor bolus transit during MRS and lack of contraction reserve [15]. To sum up the disorders of peristalsis by CCv4.0, Table 3 gives a complete picture in this regard.

Esophagogastric Junction Barrier Metrics

The CCv.4.0 working group guides understanding EGJ metrics, anatomy, integrity, and contractile vigor during baseline position, which was lacking in the previous Chicago classification [10]. The EGJ is a complex sphincter composed of the crural diaphragm (CD) and LES with different physiological control processes and pathophysiology. The working group identified four changes with EGJ assessment, including EGJ contractile integral (EGJ-CI), LES-CD separation, intragastric pressure, and respiratory inversion point (RIP). CCv4.0 stated the following recommendations [19]:

1. The EGJ complex should be measured during quiet respiration during the baseline recording in a segment that is comparatively free of swallowing and/or recording artifacts.
2. Intragastric pressure should be measured below the CD over three complete respiration cycles and if possible on the same segment being used to measure EGJ-CI.
3. LES-CD separation is the separation between the CD and LES signals at the time of inspiration. On exhalation, the precise location of the LES can be determined in blocked cases.
4. The RIP is the point along the axial axis where the inspiratory change in pressure changes from an inspiratory increase (characteristic of intra-abdominal recordings) to an inspiratory decrease (characteristic of intrathoracic recordings). 5. There are three different subtypes of EGJ pressure topography.
• No hiatal hernia: LES-CD separation 1 cm
• Hiatal hernia, distal RIP: LES-CD separation of more than 1 cm and RIP between the LES and CD
• Hiatal hernia, proximal RIP: LES-CD separation of more than 1 cm and RIP close to the LES
6. In the normal state (EGJ subtype 1), the RIP identifies the proximal margin of the LES-CD (EGJ) complex.
7. The RIP with hiatus hernia can localize either between the LES and CD or close to the LES.
8. The RIP location and relationship to LES may not be accurate for LES-CDs larger than 3 cm. But typically, the EGJ in these patients is dysfunctional.
9. The EGJ-CI should be expressed in mmHg.cm and referenced to intragastric pressure.

Conclusion

The Chicago classification, known as CCv4.0, is an evolving system aimed at improving the diagnosis and management of EMDs. It divides these disorders into two main categories: EGJ outflow disorders (achalasia and EGJOO) and peristalsis disorders, including esophageal spasm (DES and HE) and esophageal hypomotility (absent contractile and IEM). CCv4.0 introduces standardized HRM protocols to enhance reliability and research collaboration. It incorporates single wet swallows and provocation tests to increase sensitivity and specificity. Achieving a conclusive diagnosis involves both manometric and non-manometric evaluations. The definition of IEM now includes fragmented peristalsis. CCv4.0 no longer uses major and minor classifications but distinguishes EGJ outflow disorders from peristalsis disorders. Baseline metrics for EGJ are proposed including LES-CD, EGJ-CI, intragastric pressure, and respiratory inversion point. Although CCv4.0 is more accurate, ongoing research is needed to address certain areas like EGJ barrier function and inconclusive categorization of motility disorders and improve diagnosis and management strategies.

Author Confirmation

It is hereby confirmed that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. I further confirm that the order of authors listed in the manuscript has been approved by all of us.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethics Statement

The research was deemed as low risk and as such was reviewed by the Low-Risk Ethical procedure at the Faculty of Life Science and Education, University of South Wales, and granted approval.

Acknowledgments

The authors acknowledge all colleagues who had given consent to be a part of this project.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

No specific grant was received from any public, commercial, or not-for-profit sector funding agency.

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Examination of the Optimal Depth of Sunspaces in the South Facade of Residential Buildings with the Aim of Greater Efficiency

Introduction

Energy is one of the elements for the development of countries. Energy sources in the world can be divided into three main groups, fossil fuels (oil, gas, coal, and so on), nuclear energy, and renewable energy (solar, wind, geothermal, biomass). Nowadays, with the increase in energy consumption, using renewable energy, especially solar energy, has become particularly important. The building sector is responsible for more than 40% of the total final energy consumption and more than 30% of the greenhouse gas emissions in developed countries, more than the industry and transportation sectors (Yang, et al. [1]). From this share of energy use, the residential sector makes up more than 60% (Balaras, et al. 2007). Improving the energy performance of buildings is one of the particular ways in new construction and existing buildings. There is a growing need to implement energy conservation measures in existing buildings due to their low replacement rate which is only 0.07% annually (Poel, Van Cruchten, & Balaras, 2007). Therefore, most of these buildings will still be functional until 2025 or even 2050 (Ürge Vorsatz, Danny Harvey, Mirasgedis, & Levine, 2007). To minimize energy inefficiency and waste in residential buildings, different performance strategies have been constantly promoted. The challenge is the fact that the surging innovations in the building sector should be examined to ensure that the targeted energy efficiency is acquired.

This needs attention to a combination of influencing parameters related to climate and configuration of the building envelope. In this term, the sun is the best energy source for natural, and artificial activity and different human technologies. The current environmental crisis, like global warming, climate pollution, and so on, is due to the overuse of fossil fuels. Thus, the best solution is to use natural energy such as the sun. The term solar thermal energy includes all cases of using solar heat, which is possible using various technologies. Solar energy is of the key renewable energy sources. This type of energy could be used in the building as active and passive ways. One of the most popular inactive solar systems is “sunspace.” Recent studies show that if sunspace is added to the southern wall of the building, one can reduce its heating load (J. Schoenau, et al. [2]). Among the renewable energy resources, Iran has a great potential to use solar systems such as sunspaces. However, only a few studies have been carried out in Iran. Sadeghi et al. examined the various geometry and the shapes of solar shapes in the Tehran climate to find an optimal shape for the sunspace (Saghafi [3]). Safgafi & Yazarlou examined the effects of using the sunspace on heating load and only in Yaz hot area (Monge Barrio [4]).

Balilan examined the effect of using sunspace on the energy consumption in a building in London to emphasize the efficiency of these spaces in buildings (Balilan, et al. [5]). Gerick et al. examined the heating efficiency of four sunspaces with various shapes and dimensions in Portugal to present an optimal sample (Sadeghi, et al. [6]). Another study has been carried out to prove the efficiency of sunspaces that has led to the use of proper air conditioning in sunspace climate (Kalogirou [7]). A need is felt for an overall and holistic view and comparison of the effects of the stated systems on heating and cooling loads and in various climate types of the country. Thus, the study is an effort to examine and analyze the changes in heating and cooling loads affected by using sunspace in summer and winter, considering the various climate types. Attached sunspaces or conservatories are often referred to as sunrooms and function similar to Trombe walls. The only difference between the two systems is the availability of more space between the wall and the glass in the latter case, which could create a comfortable living space while affording energy efficiency advantages (Kesik [8]) (Figure 1).

Figure 1

Most of the studies on sunspaces try to find solutions to maximizing the benefits of heating load in winter and avoiding overheating in the summertime. Aside from overheating in summer, one drawback of passive heating is that it can only happen through the south façade, although this issue can be addressed by facilitating heating energy distribution across the space (Konstantinou [9]). Schoenau, Lumbis, and Besant examined the thermal performance of four sunspaces in Saskatoon, Canada. In order to validate an analytical model, performance was monitored hourly while a simulation was conducted to account for the annual energy performance estimations Aelenei (Schoenau, et al. [10]). De Azevedo Leal, and Aelenei used a numerical approach to investigate the thermal performance of a sunspace in a residential building in Portugal. Orientation, sunspace configuration, natural ventilation of the sunspace and position and radiative properties of the shading devices were considered as design variables and their influence on thermal behaviour and the possible amount of energy saving were analyzed (Leal [11]). Bataineh and Fayez investigated the thermal performance of an attached sunspace to a building in Amman, Jordan.

Furthermore, they evaluated the impact of the orientation of the sunspace, opaque wall and floor absorption coefficients and the number of glass layers on thermal performance. Based on their results, sunspace can decrease heating load considerably in winter. However, it causes serious overheating in summer (Bataineh [12]). Bakos and Tsagas explored the thermal and economic aspects of an attached sunspace in Greece. Thermal load was calculated by the degree-day method and, for economic performance, the LCC method was used (Bakos [13]). Oliveti, Arcuri, De Simone, and Bruno calculated the solar gains of the sunspace and the adjacent spaces in different regions of Italy based on several geometric configurations including a system of windows made up of clear double-glazing. They considered and analyzed the impact of factors such as different levels of exposure, optical properties and thermal aptitude of the opaque areas, the ventilation capacity and the shading mechanism (Oliveti, et al. [14]). Sánchez Ostiz, et al. [15] investigated thermal performance and design of two passive solar systems including attached sunspace with horizontal heat storage and an attached sunspace with vertical thermal storage. These two sunspaces were tested under the real conditions in two residential buildings in Spain. (Sánchez Ostiz, et al. [15]).

Monge Barrio and Sánchez Ostiz studied the behaviour of sunspaces as passive elements in summer for different climatic regions in Spain. The results show that sunspaces can be configured to perform efficiently in summer, even in extremely hot conditions (Monge Barrio [16]). Zhu, Liu, Yang, and Hu evaluated the thermal performance of new Yaodong dwellings by adding an attached sunspace to the old building located in the Zaoyuan village in Yanan City, China. By using EnergyPlus software, they conducted numerical simulations of heating and cooling energy consumption. Fernández-González assessed the thermal performance of five passive solar testcells including Direct Gain, Trombe wall, Water wall, Sunspace, and Roof pond by considering a control test-cell in Muncie, Indiana in order to identify the limitations of these passive solar heating systems (Fernández González [17]). Rempel, Rempel, Gates, and Shaw modelled a series of field-validated sunspaces in Pacific Northwest to quantify their thermal mass design issues and to investigate the impact of factors such as the sizing and ground configuration of floor-based thermal mass (Rempel, et al. [18]).

Lucas, Hoese, and Pontoriero analyzed and compared the thermal performance of three passive systems including Trombe wall, direct gain and sunspace, in a region with a continental Mediterranean climate. The results show that all mentioned passive systems gained solar radiation throughout all the seasons of the year. Among them, the Trombe wall joined to a sunspace provided the best results, with small energy gain in summer and high energy contribution in winter (Lucas, et al. [19]). Mottard and Fissore proposed a new thermal simulation model for an attached sunspace by paying attention to the internal long-wave radiation exchanges and solar radiation distribution within the sunspace. For validation, the calculated results were compared to the empirical data. Furthermore, a sensitivity analysis was used to determine the parameters of the model with the strongest impacts on energy performance (Mottard [20]). Babaee, Fayaz, and Sarshar proposed a design modification for sunspaces to enhance the thermal performance of dwellings in Tabriz, Iran which has a cold climate. Six sunspace configurations with different ratios of glazed to opaque surfaces were modelled and simulated to identify an optimum dimension of the sunspace. The orientation, the number of glazed surfaces, the direction and inclination angle of the surfaces, the glazing material, and the common wall material of the sunspace as design criteria were also assessed (Babaee, et al. [21]).

In this research, we seek to Examination of the Optimal Depth of Sunspace in the Residential Building climate of Iran (Kashan city), which, despite the reduction of the heating load in the cold days of the year, does not impose an excess cooling load on the building in the summer. In this regard, Common and diverse types of sunspaces with different depths and native and new materials were investigated. For this purpose, the residential houses of Kashan city were selected and the studies were done on the south facade of these settlements.

Research Method

As already stated, this study aims to examine the shape of sunspace in the heating load of hot and dry climates to reach the optimal shape to increase the efficiency of this element. Thus, considering the physical nature of this study, in the first step, using the average annual climatic data of Kashan and considering the materials listed in “Table 1 & 2”, and then using modeling and analysis in Energy Design Builder and Energy Plus, the average internal temperature of the dwelling adjacent to the sunspace, the heat absorption through solar energy (Solar Gains Interior Windows) and heat output via the glazing are determined and compared to these models and determine the model with optimal performance.

Table 1: Specifications of opaque walls for models 1 to 4.

Table 2: Specifications of opaque walls for models 1 to 4.

Examining Heating Load Reduction Solutions Using Sunspace

Recently, using solar greenhouses has become a popular solution to enhance the heating performance of buildings in winter. Solar greenhouses are a passive solar system usually consisting of a south-facing exterior room made mainly of transparent walls greenhouse. The greenhouse is a retaining space between the building and the external environment that allows a large amount of solar radiation to enter (Asdrubali, et al. [22]). Although using a greenhouse is very common in temperate climates, it has not found its status in hot and dry climates. Due to its hot and dry climate, the central regions of Iran have always been looking for solutions to deal with summer heat and have paid less attention to heating loads in winter. The solution for traditional Iranian houses is to use materials with a delay time [23,24]. However, due to the design movement of low-consumption and green buildings, this method does not have much place and effect in reducing heat exchange and energy consumption. Thus, it is necessary to comprehensively study the composition of indigenous buildings and other systems of passive solar design. Residential buildings in this climate have two sections, winter and summer quarters, whose inhabitants are settled due to seasonal changes. This section follows the combination of sunspace with the winter section of a native house. The hot and dry climate of Iran (Kashan), despite the reduction of heating load on cold days of the year, does not impose excess cooling load on the building in summer. Thus, the new Boroujerdi house, among the residential houses in Kashan, was selected, and studies were conducted based on its winter front.

Examining the Effect of Greenhouse Shape

This section has considered four models of sunspace designed and assumed with the same volumes, the depth of the greenhouse is 1.73, and its height is 3.00 meters, to study the heating, and cooling loads in the residential space joined to them at the height of 3.00, length of 5.27 and the width of 6.00 meters. The cases examined are: Sunspace, rectangular cube, Sunspace, a rectangular cube with an angle of 30 degrees, Sunspace, a rectangular cube with an angle of 45 degrees, Sunspace, a rectangular cube with an angle of 60 degrees (Figure 2).

Figure 2

Climate Chart of Kashan

Kashan is in a climatic zone with relatively cold winters and hot and dry summers. It is one of the factors affecting the climatic components of air temperature. The following table is based on data obtained from the Meteorological Department in the 19-year period of Kashan (1999-2017). Concerning Kashan, it shows average monthly temperature, average minimum monthly temperature, average maximum monthly temperature, the number of sunshine hours, humidity and rainfall in Kashan synoptic station. The feeling of comfort is exposed to the radiant heat of the sun or any other source by examining these data in January, February, March, December, and November. It is relatively comfortable in about two months of the year, equal to April and October, and the five months of May, June, July, August, and September are in a state where it is not possible for people to feel comfortable without airflow and coldness due to evaporation (Figures 3 & 4).

Figure 3

Figure 4

Materials Used in the Model Simulation

The materials used for all models “Figure 2” are the specifications of the base state and the structures in Tables 1 & 2.

Simulation of Thermal Performance of Sunspace Models

In this section, four sunspaces “Figure 1” are designed and assumed that using software (Energy Builder Design and Energy Plus), the average internal temperature of the dwelling adjacent to the sunspace, the heat absorption through solar energy (Solar Gains Interior Windows) and heat dissipation was examined through the transparent wall (glazing). In all cases, the same climatic conditions based on climatic data of Kashan “Figure 2”, “Figure 3”, the dimensions of the settlement joining the sunspace in all models with a length of 5.27 m and a width of 6.00 m and a height of 3.00 m are considered. The wall, ceiling, floor, and glass materials specifications are considered based on the specifications listed in “Tables 1 & 2”. Since our goal has been to reach a range of optimal heating and cooling load in the sunspace we intend to check the types of suitable depth of the sunspace assuming the materials listed in Tables 1 & 2, i.e. the use of building and insulating materials and double-glazed glass, so We divided the different depths of this space, and since the sunspace with a depth of fewer than 20 centimeters and a depth of more than 2 meters is not meaningful, therefore, the depth range of 0.2 meters, 0.5 meters, 1 meter, and more than 1 meter is also considered as the depth between Two intervals were selected. The direction of the said sunspaces facing south and the common wall of the room and the sunspace have been considered for direct heat transfer and exchange in a transparent manner (no thermal barrier).

The height of the sunspace is 3.00 meters, and in two cases, assuming the presence of a shade, 0.8 meters and without a shade, we have investigated the heating and cooling loads in this space. In all models, the common wall between the sunspace and the building material room, the volume of the room under investigation is 103.61 m3. The structure of other windows is according to Table 1. All the glasses are double-glazed according to Table 2 and the only common glass between the room and the sunspace is simple single-glazed. In the above table, in case 1, the model has an optimal canopy, summer ventilation, and heat transfer with the ground through the floor. In the case of case 2, only the summer performance of the model (without canopy, summer ventilation, and heat exchange) is assumed.

Discussing the Influence of the Depth of the Sunspace

By examining Tables 3-6, the following results are obtained. According to the results of the calculations related to the heating loads in the optimal winter conditions, the studied models, the priority of use are sunspace with an angle of 60 degrees, sunspace with an angle of 45 degrees, sunspace with a 30-degree angle, rectangular cube sunspace. In the rectangular cube sunspace, we examine two modes; First, the sunspace has an optimal canopy, summer ventilation, winter night insulation, and heat exchange with the soil in contact with it. In this model, by reducing the width of the sunspace to 1 meter, the heating requirement of the room increases slightly. While the cooling load has been significantly reduced. In widths of less than 1 meter, the heating load is reduced, which in the best winter mode (2-4 V) has improved by about 21% compared to the base mode of its model (V0). The model has a variable behavior by reducing the width of the sunspace, and heating and cooling loads. In this optimal state (2-4 V), the heating and cooling load requirements have been reduced by 89.1% and 34.5%, respectively, compared to the existing state. In the second case, the sunspace without a canopy is summer ventilation and heat exchange with the soil.

Table 3: Sunspace, rectangular cube (energy plus).

Table 4: Sunspace, a rectangular cube with 30 degrees of angel (energy plus).

Table 5: Sunspace, a rectangular cube with 45 degrees of angel (energy plus).

Table 6: Sunspace, a rectangular cube with 60 degrees of angel (energy plus).

In this model, the heating load of all cases is lower than in model 1, and there is a direct relationship between the width and the heating requirement of the space. also decreases, but they impose a huge cooling load on the building. In fact, for the second case where there is no canopy, it can be said: “The smaller width of the sunspace causes its better heating performance (Table 3). In the sunspace of 30 degrees, V0 mode with the largest width has almost the same heating requirement as V4 mode with It has the smallest width, but its cooling load is about 120kwh more than V4. The optimality of this model (V4) has about 89.7% reduction in heating load and 45.3% cooling load compared to the existing state (Table 4). V0 state is the optimal state of the rectangular cube sunspace with an angle of 45 degrees, which requires. The heating and cooling loads have be reduced by 93% and 27.2%, respectively. The V4 mode of this model, with a width of 0.2 meters, is only 10kwh more than the optimal mode, but it has a lower cooling load of around 106kwh (Table 5) The 60-degree model has the most optimal mode among these seven models. In this case, the heating requirement is zero and the cooling load is reduced by about 5% compared to the current state. As the width of the sunspace decreases, the heating loads increase irregularly, but the cooling loads decrease continuously. In this model. With less than half of the width of the optimal state, the V1 mode has a favorable heating and cooling load. and its cooling requirement has irregular changes and decreases for widths below 1 meter.

Its optimal mode has an improvement of 99.5% and 22% for heating and cooling loads, which considering the cooling load, this mode is more efficient than the optimal mode of 60 degrees (Table 6). In general, two ranges can be defined according to the width for the sunspaces studied in this research: first, widths of more than 1 meter that can add living space to the building, and second, widths of less than 1 meter. which act similar to Trombe’s wall and do not add livable space to the building. For the studied models, the following numerical relationship can be defined between the volume of the existing room and the appropriate volume of its sunspace: (if the volume of the room in contact with the sunspace is VR and the volume of the sunspace is VG)

1. The volume of the sunspace for the rectangular cube model is the same for each room; VG = 0.04 VR

2. The volume of sunspace for 60 and 45-degree models per room is equal to; VG = 0.42 VR

3. The volume of sunspace for 30 models per room is equal to: VG = 0.05 VR

Conclusion

The purpose of sunspaces in hot and dry climates is to reduce the heating load during the cold days of the year and to minimize the excess cooling load in the summer. A depth of more than 1 meter can add living space to the building and secondly, a depth of less than 1 meter that acts like a Trombe wall and does not add a living space to the building. In each of the studied models, if the sunspace is used, due to the importance of the cooling load in the city of Kashan on the hottest summer day, the model that has a lower excess cooling load and creates a suitable heating load in the winter season is determined as the optimal model. According to the results of the calculations related to the heating loads in optimal winter conditions, the studied models of priority use are sunspace with an angle of 60 degrees, sunspace with an angle of 45 degrees, sunspace with an angle of 30 degrees, rectangular cube sunspace. The 60-degree model has the most optimal mode among these seven models. The heating requirement in this mode is zero and the cooling load is reduced by about 5% compared to the existing mode. As the width of the sunspace decreases, the heating loads increase irregularly. but the cooling loads decrease continuously. In this model, the V1 mode having less than half of the width of the optimal mode, has a favorable heating and cooling load, so it can be concluded that the winterized spaces use the optimal V0 model and in the spaces for summer residents, it is optimal to use the V4 model. In addition, considering the small difference between the above two modes, if you move the sunspace and the shade in such a way that the shade is created in the summer and the shade is removed in the winter, it is possible to benefit from the optimal It is the most possible state.

Author Contributions

Karbasforoushha performed the literature review and model design, analyzed and interpreted the data, and prepared the manuscript text and edition. prepared the manuscript text and manuscript edition. Compiled the data and manuscript preparation.

Acknowledgement

I want to thank the personnel of the Sciences Library of the Tehran Branch. Department of architecture, Tehran-west Branch, Islamic Azad University, for helping us to gather the data and articles.

Conflict of Interest

The authors declare no potential conflict of interest regarding the publication of this work. In addition, the ethical issues, including plagiarism, informed consent, misconduct, data fabrication, falsification, double publication and, or submission, and redundancy, have been completely witnessed by the authors.

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Trends in Plasma Kisspeptin Level and Cellular Experiment Insights During Progestin-Primed Ovarian Stimulation in Assisted Reproductive Technology

The global prevalence of infertility is 8%–12%, with Assisted Reproductive Technology (ART) as an effective treatment method [1]. ART consists of three principal components, including oocyte retrieval, fertilization and embryo culture, and embryo transfer. Among these, the achievement of high-quality oocyte retrieval is of utmost importance [2]. The appropriate choice of controlled ovarian stimulation (COS) is crucial for high-quality oocyte collection. Recently, progestin-primed ovarian stimulation (PPOS) has become popular, although the exact mechanism by which it suppresses ovulation remains unclear [3]. Kisspeptin, a hypothalamic neuropeptide hormone encoded by the Kiss1 gene, acts through the hypothalamic kisspeptin 1 receptor (Kiss1R) to induce the release of endogenous gonadotropin-releasing hormone (GnRH), which in turn increases Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) secretion from the pituitary gland, thereby inducing ovulation [4]. Kisspeptin plays a pivotal role in reproductive function by regulating the hypothalamic–pituitary–gonadal axis. One of the functions of progesterone (P4) in humans is to suppress GnRH and LH secretion, and thus ovulation, but the detailed mechanism of action remains unclear [5]. Estradiol (E₂) in rodents acts on kisspeptin neurons in the Anteroventral Periventricular Nucleus (AVPV) and Arcuate Nucleus (ARC) regions, thereby adjusting GnRH secretion through positive and negative feedback, respectively [6].

Additionally, a rodent study revealed that P4 administration to the AVPV region suppressed ovulation [7], indicating that progestin may suppress ovulation in humans by inhibiting kisspeptin gene expression. In this study, we continuously monitored plasma kisspeptin levels during PPOS to investigate their changes. Further, we measured changes in kisspeptin expression with progestin administration in mHypoA-50 cells, which were derived from mouse Kiss-1-expressing neurons in the AVPV region.

Materials and Methods

Ethical Approval

The Ethical Review Board of Osaka University Hospital (No. 21113-2) approved this study. Samples were obtained from patients who signed written informed consent before inclusion and who underwent PPOS at the Reproduction Clinic Osaka, Osaka, Japan, from November 2021 to January 2022.

Serum and Plasma Hormone Profiles During PPOS

Table 1 shows the baseline characteristics of five patients during PPOS. Kisspeptin-54 was measured in plasma from days 2–5 of the menstrual cycle to the oocyte pick-up day using a commercial enzyme-linked immunosorbent assay kit (Peninsula Laboratories International, Inc.; San Carlos, CA) [8]. Blood samples were immediately transferred into a polypropylene tube that contains ethylenediaminetetraacetic acid on ice and then centrifuged for 15 min at 1,600 g at 4 ℃. Plasma layer samples were collected and stored at −80 °C. The concentrations of FSH, LH, E₂, P₄, and kisspeptin-54 are presented in Figure 1.

Table 1: Baseline characteristics of patients undergoing the PPOS protocol: serum and plasma hormone profiles.

Note: AMH: anti-müllerian hormone; E₂: estradiol; FSH: follicle-stimulating hormone; IQR: interquartile range; LH: luteinizing hormone; OPU: oocyte pick-up; P₄: progesterone; PPOS: progestin-primed ovarian stimulation.

Figure 1

Materials

The following chemicals and reagents were obtained from the indicated sources: GIBCO fetal bovine serum (FBS) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA); Dulbecco’s modified Eagle’s medium (DMEM) at 4.5 mg/mL of glucose (Invitrogen); phenol red-free DMEM (FUJIFILM Wako Chemicals, Osaka, Japan); charcoal-stripped FBS (Hyclone, Logan, UT); penicillin–streptomycin and water-soluble β-estradiol (Sigma-Aldrich Co., St. Louis, MO); and GnRH and Chlormadinone Acetate (CMA) (Fuji Pharmaceutical Co., Tokyo, Japan).

Cell Culture and Stimulation

CEDAR-LANE (Ontario, Canada) supplied mHypoA-50 cells. Cells were plated in 60-mm tissue culture dishes and incubated with high-glucose DMEM containing 10% heat-inactivated FBS and 1% penicillin–streptomycin at 37 ℃ under a humidified atmosphere of 5% CO₂. The cell culture medium was changed to phenol red-free high-glucose DMEM with 10% charcoal-stripped FBS and 1% penicillin–streptomycin for a minimum of 24 h before the reagent and vehicle treatments in the mHypoA-50 cells. Each experiment used cells grown in culture plates to 80% confluence. Cells were incubated without (vehicle) or with the test reagents for the indicated concentrations and periods when stimulated with the test reagents. RNA preparation, reverse transcription (RT), and quantitative real-time polymerase chain reaction (PCR) A Nucleo Spin RNA Plus Kit (Takara Bio Inc., Shiga, Japan) was used to extract total RNA, and Super Script IV VILO Master Mix (Invitrogen) was used to synthesize cDNA from 2.5 μg of total RNA, following the manufacturer’s instructions. The following RT-PCR protocol was used for the initial identification of mPRα, mPRβ, and PgRMC1 mRNAs in mHypoA-50 cells. The forward primer for mPRα was 5ʹ-CAGAAGCCTCCGCAACCAGAAC-3ʹ, and the reverse primer was 5ʹ-GAGCCACAGCACTGAACGAGAG-3ʹ, whereas the forward primer for mPRβ was 5ʹ-TGACGACTGCCATCCTAGAGCG-3ʹ, and the reverse primer was 5ʹ-CAATGCCCCTGCCTCCACAAAG-3ʹ. The forward primer for PgRMC1 was 5ʹ-AGGGCAGGAACAGGTATGTG-3ʹ, and the reverse primer was 5ʹ-CCAAAGGAGTATTACCCAAGACC-3ʹ.

This primer sets generated products of 310, 305, and 205 bp for mPRα, mPRβ, and PgRMC1, respectively. Thermal cycling conditions were as follows: one cycle at 94 °C for 5 min; 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 min; one cycle at 72 °C for 5 min. Amplified PCR products were run on a 2.0% agarose gel and investigated for appropriate size band production. Quantitative real-time PCR with Taqman™ Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the mRNA expression of kisspeptin (Kiss1, Mm03058560_m1). Samples were run in triplicate using optical 96-well plates, and relative gene expression levels were evaluated using the 2−ΔΔCT method. Gene expression was normalized to histone 3a (Mm00517632_s1) mRNA levels, which were used as internal controls for the gene expression assay [9]. Each measurement is based on three biological replicates, and the values are presented as the means ± Standard Error of the Mean (SEM).

Statistical Analyses

One-way analysis of variance with the post hoc Tukey–Kramer test and GraphPad Prism version 8.0 were used for statistical analyses.

Results

Plasma Kisspeptin Levels During PPOS

Table 1 shows the baseline characteristics of patients undergoing PPOS. The median age and anti-müllerian hormone level were 32.0 years (interquartile range [IQR]: 30.0–37.0) and 4.88 (ng/mL) (IQR: 4.12–5.42), respectively. While E₂ serum levels continuously increased, no significant differences in plasma kisspeptin-54 and serum LH values were found during PPOS. No cases of ovulation were observed before oocyte retrieval.

Effect of CMA on Kiss-1 Gene Expression in mHypoA-50 Cells

We investigated the effect of CMA on Kiss-1 gene expression, which was increased with E₂ and GnRH treatment, in mHypoA-50 cells. Expression of estrogen and GnRH receptors was confirmed in mHypoA-50 cells [9,10]. RT-PCR analysis revealed that the cells expressed mRNA for mPRα, mPRβ, and PgRMC1 (Figure 2a). E₂ stimulation significantly increased Kiss-1 mRNA expression in mHypoA-50 cells by 1.47 ± 0.13-fold at 100 nM of E₂ ([vehicle vs. E₂], P < 0.05) and 1.06 ± 0.13- fold at 100 nM of E₂ + 1.5 ng/mL of CMA ([E₂ vs. E₂ + CMA], P < 0.05) (Figure 2b). GnRH stimulation significantly increased Kiss-1 mRNA expression in mHypoA-50 cells by 1.41 ± 0.01-fold at 100 nM of GnRH ([vehicle vs. GnRH], P < 0.001) and 1.05 ± 0.07-fold at 100 nM of GnRH + 1.5 ng/mL of CMA ([GnRH vs. GnRH + CMA], P < 0.001) (Figure 2c).

Figure 2

Discussion

COS enables the collection of a large number of oocytes in a single retrieval and improves cumulative pregnancy rates as the number of retrieved oocytes increases, thereby demonstrating their clinical efficacy [11]. However, ovulation suppressant administration is essential during COS to prevent breakthrough ovulation. The PPOS method uses progestin as an ovulation Inhibitors and is reported to be cost-effective and simple compared with other methods and yields reproductive outcomes, including ovulation suppression rates, comparable with those of other methods [3,12]. However, recent reports revealed a possible deterioration in the quality of embryos and a decrease in pregnancy rates [13,14]. Furthermore, slight increases in P₄ levels before natural ovulation have potentially induced an LH surge, indicating that the exact mechanism by which P₄/progestins suppress ovulation remains unclear [15]. The patients in our study were undergoing COS, and no cases of ovulation before oocyte retrieval were observed, confirming the ovulation-suppressing effect of progestin. Kisspeptin, a neuropeptide hormone that originates in the hypothalamus, is encoded by the Kiss1 gene and exerts its effect via Kiss1R located in the hypothalamus, thereby facilitating the release of endogenous GnRH [4]. Pubertal development was absent in families with Kiss1R mutations, thereby establishing an association between kisspeptin and reproduction [16]. Kisspeptin acts on various tissues, including the hypothalamus, ovaries, uterus, and placenta, thereby regulating their functions [6].

Kisspeptin in humans and many other species increases FSH and LH secretion from the pituitary gland, thereby triggering ovulation [4]. A single-blinded placebo-controlled physiological study [17] revealed that GnRH administration did not increase kisspeptin levels, indicating that kisspeptin operates upstream of GnRH. Two randomized, placebo-controlled, parallel-group, dose-finding trials (Phase I and Phase IIa) revealed that kisspeptin receptor agonist (MVT-602) administration induced an LH surge of similar amplitude to the physiological LH surge, indicating its use as a trigger for oocyte maturation [4]. Furthermore, the administered dose of MVT-602 induced a dose-dependent increase in LH levels [4]. Plasma kisspeptin concentrations increase before natural ovulation during the menstrual cycle and correlate with an increase in LH levels [18]. This study revealed that kisspeptin and LH concentrations did not increase before oocyte retrieval, indicating that LH surge suppression by progestin could be due to kisspeptin level reduction. The ovulation control mechanism via kisspeptin in rodents is different from humans, but rodents serve as an excellent model for investigating aspects that are difficult to analyze in humans. GnRH neurons in the hypothalamus do not express estrogen or P4 receptors in both humans and rodents [19]. There are two types of kisspeptin-producing neurons located in the hypothalamus: the Arcuate Nucleus (ARC) and the Anteroventral Periventricular Nucleus (AVPV) [19].

Estrogen suppresses Kiss1 gene expression in the former (negative feedback) and promotes it in the latter (positive feedback). Additionally, the former regulates pulsatile GnRH secretion (promoting follicular development), whereas the latter regulates surge-like GnRH secretion (inducing an LH surge and ovulation) [6]. In rats, kisspeptin neurons express estrogen and P4 receptors, which act on kisspeptin secretion from these neurons to regulate ovulation [20]. Studies have shown that P4 administration suppresses the LH surge, and RU486 injection (a P₄ receptor antagonist) into the AVPV region increases LH secretion, indicating that suppression of the LH surge through P₄ in rats involves AVPV neurons [20]. Estrogen administration in mice increases kisspeptin neurons in AVPV, whereas it decreases in ARC [21]. Furthermore, P₄ administration suppressed the LH surge induced by estrogen administration, indicating the ovulation-suppressing effect of P₄ [7]. However, the LH surge was not suppressed in groups administered both P₄ and kisspeptin, indicating that P₄’s ovulation-suppressing function may act upstream of kisspeptin, thereby controlling its secretion. mHypoA-50 cells, derived from mouse Kiss-1-expressing neurons in the AVPV region, have confirmed Kiss-1 and estrogen receptor expression, as well as GnRH/GnRH receptors [9,10]. Furthermore, we confirmed P₄ receptor expression in mHypoA-50 cells and revealed that progestin (CMA) suppresses kisspeptin gene expression induced by E₂ or GnRH.

The mechanisms of ovulation control differ between rodents and humans, but progestin in humans may suppress kisspeptin secretion through upstream mechanisms, thereby inhibiting LH surge occurrence. Plasma kisspeptin levels in nonpregnant adult women vary according to previous reports. Previous studies have reported plasma kisspeptin concentrations to be 1.65 ± 0.1 ng/mL [mean ± SEM], consistent with our findings [18]. Other studies have documented both lower [22] and higher concentrations [23] than those observed in our examination. The half-life of kisspeptin is short [4,24], and it is easily degraded, making the sample collection and preservation methods crucial. Studies retrospectively investigated the relationship between plasma kisspeptin levels and miscarriage [8,25] have also been published, proving the feasibility of measuring serum kisspeptin concentrations. Our study used the same assay system. Furthermore, our research did not establish a control group without the use of ovulation suppressants in COS, thereby not directly proving the suppression of plasma kisspeptin by progestin. However, conducting COS without ovulation suppressants is ethically untenable. An increase in estrogen levels can induce an LH surge, while plasma kisspeptin concentrations increase before ovulation even in natural menstrual cycles with lower estrogen levels [18]. Therefore, kisspeptin levels are expected to increase in this COS if progestin had not been used. Further research with an increased sample size is essential to confirm our study results. In summary, our human plasma analysis during PPOS and the additional experiments with mHypoA-50 cells indicate that PPOS with progestin significantly suppressed the LH surge rate, which may be due to the suppression of Kiss-1 gene expression in the hypothalamus.

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Cluster Analysis of RNA m⁶A Methylation Regulators Based on TCGA and GEO Database in Hepatocellular Carcinoma

Introduction

In recent years, as one of the most common and fatal malignant tumors in the world, liver cancer has become the focus of medical and public attention. According to the statistics of the World Health Organization, the incidence of liver cancer ranks fifth and the mortality ranks third, causing more than 800,000 deaths each year, and the number is still increasing [1-4]. As the main pathological type, more than 90% of liver cancer is hepatocellular carcinoma. Unfortunately, because the onset of HCC is hidden and there are no obvious symptoms in its early stage, most patients are found in the middle and late stage, which causes poor therapeutic effect [5]. Although great progress has been made in targeting, immunity, intervention, radiotherapy and other fields in recent years, the prognosis of patients with HCC is still not ideal enough [6]. Therefore, the study of HCC is still very urgent and of great significance. m⁶A RNA refers to the methylation of the sixth nitrogen atom of adenine in RNA. Since it was first discovered in the 1970s, m⁶A methylation has gradually become a research hotspot in life science [7]. M⁶A RNA can regulate many biological processes such as RNA degradation, transcription, splicing and translation, thus affecting gene expression and protein synthesis, and plays an important regulatory role in the occurrence and development of HCC. In HCC, the increase of m⁶A RNA expression is mainly regulated by three key proteins: «writer», «eraser» and «reader». More and more studies have shown that the abnormal expression and regulatory mechanism of m⁶A RNA are closely related to the occurrence, progression and prognosis of HCC [8-10].

As potential targets and candidate molecules for the treatment of liver cancer, m⁶A RNA and its related genes have important application prospects in individualized therapy and immunotherapy of HCC. The purpose of this study is to use bioinformatics technology to analyze the gene chips of patients with HCC in public database, comprehensively and systematically sort out and analyze the mutation, expression and survival differences of m⁶A RNA and its related genes in HCC. Through the study of the expression level, function, regulatory mechanism, related proteins and signal pathways of m⁶A RNA, we can better understand the key role of m⁶A RNA in the occurrence, progression and prognosis of HCC. Through a comprehensive and in-depth exploration of the study of m⁶A RNA in HCC, we are expected to provide new targets and methods for early diagnosis, treatment and prognosis evaluation, provide more effective and individual treatment for patients, and contribute to the development and progress of liver cancer research.

Materials and Methods

Data Selection and Processing

The RNA-Seq gene expression profiles of patients with HCC were downloaded from The Cancer Genome Atlas(TCGA) portal (https://cancergenome.nih.gov/) and International Cancer Genome Consortium (ICGC) portal (https://dcc.icgc.org/). We downloaded gene chip GSE10143 from NCBI Gene Expression Omnibus(GEO) database (http://www.ncbi.nlm.nih.gov/geo/). All the samples used by GSE10143 were resected from patients with HCC, including 80 tumor tissue samples and 82 normal liver tissue samples. The platform is GPL5474, Human 6k Transcriptionally Informative Gene Panel for DASL.

Differential Expression Gene Analysis

Use the “Limma” package of R software (version 4.1.2) to analyze the differential expression of mRNA in the dataset. The data is selected as adjusted «P < 0.05 and |logFC|> 2» (logFC is defined as threshold mRNA differential expression screening) as Differential Expression Genes(DEGs). The heat map was drawn by R software package “pheat” thermal map.

Functional Enrichment Analysis

In order to further screen and confirm the potential function of DEGs, the functional enrichment analysis of the data was carried out by means of Gene Ontology (GO). GO is a widely used tool to annotate genes with potential functions through Molecular Function (MF), Biological Pathway (BP), and Cellular Component (CC). Kyoto gene and genome database (Kyoto Encyclopedia of Genes and Genomes, KEGG) enrichment analysis is a method used to annotate gene function and related signal pathways. In this study, the Cluster function enrichment and KEGG pathway of DEGs were analyzed by using the “Cluster bubble Profiler” package in R language software, and the bubble diagram was drawn by R software package “ggord”.

Establishment of m⁶A Subgroups

All genes were used to conduct a univariate Cox survival regression. P<0.1 was used to create a Least Absolute Shrinkage and Selection Operator (LASSO) regression model. R package “glmnet”, “forest” and “survival” were used during this process. Unsupervised clustering was performed to identify subgroups based on the m⁶A regulators. Package “Consensus Cluster Plus” in R software were used.

Statistical Analysis

Categorical data were analyzed by chi-square test or Fisher exact test. Normal data were analyzed using the t-test, and non-normal data were analyzed using the Wilcox on rank sum test. The Kaplan-Meier method was used for survival analysis. The COX model was used for univariate and multifactorial analyses. Differences were considered statistically significant at P < 0.05. All results were double counted three times.

Results

Variation of m⁶A Regulators in HCC

By downloading data from TCGA and GEO databases, we collected 595 liver cancer patients, including 480 HCC samples and 115 normal samples. Copy number variations including both gain and loss of copies were found in 23 m⁶A regulated genes (Figure 1A). The position of the CNV change of the 23 m⁶A regulated genes on the chromosome is shown in the (Figure 1B). Considered as DEGs by adjusted P<0.05, differences in expression were found in 21 of all 23 m⁶A regulated genes (Figure 1C). The gene mutation data are also downloaded from the TCGA database, among which 364 samples have complete mutation data. Single Nucleotide Variation (SNV) were found in 31 samples including synonymous mutation, missense mutation, shift mutation and nonsense mutation , accounting for 8.52% of the total (Figure 1D).

Figure 1

Survival Analysis of m⁶A Regulators in HCC

The survival data of HCC patients were downloaded from TCGA database and analyzed by Kaplan-Meier survival analysis. The statistical difference of Overall Survival (OS) was distinguished by P < 0.05. A total of 6 of the 23 m⁶A regulated genes can affect the prognosis of patients with HCC (Figures 2A-2F). High expression level all of these 6 genes promote the development of HCC and lead to a worse prognosis.

Figure 2

Cluster Analysis of m⁶A RNA in HCC

According to the difference of m⁶A regulated gene expression, we clustered all the HCC samples obtained from TCGA database. This process is accomplished through machine learning. We tried to divide all the samples into 2-9 categories, and there was the most obvious difference when they were divided into 3 categories (Figures 3A-3C). We named these three clusters as m⁶A cluster A, B and C. Gene Set Variation Analysis (GSVA) was used to find out pathways that genes in different m⁶A clusters enriched in. The results show that genes are enriched in cell homeostasis, cellular micro-environment, metabolism and a variety of tumor-related signaling pathways (Figures 4A-4C).

Figure 3

Figure 4

Cluster Analysis of Differential Genes by m6A Clusters in HCC

There are also differentially expressed genes in different m6A clusters. In order to find out these genes, we found the gene expression data of individuals in different m6A clusters from the TCGA database. Then, we clustered these genes using the method mentioned above. After we divided all the samples into 2-9 categories, there was the most obvious difference when they were divided into 3 categories (Figure 5A). We named these three clusters as gene cluster A, B and C. A total of 1046 genes show difference between gene cluster A and B. The number of genes between cluster A and C is 94 while the number between cluster B and C is 2109 (Figure 5B). To explore the biological functions of these genes, they were categorized into BP, CC and MF. Under stringent threshold conditions by adjusted P value less than 0.05, top 10 terms of each part are in (Figure 5C). Through Kaplan-Meier survival analysis, we found that there were differences in individual prognosis among different gene clusters. The overall survival time of patients in gene cluster A was the longest while in cluster C was the shortest (Figure 5D). The basic information, general situation, tumor stage and other data of these individuals are shown in the (Figure 5E). It is worth mentioning that we analyzed the differentially expressed genes that affect the prognosis of HCC patients in different gene clusters and found that m6A regulated genes were all differentially expressed in different gene clusters (Figure 6). However, it needs further research.

Figure 5

Figure 6

Discussion

In recent years, with the deepening of research, the important role of m6A modification in HCC has been widely recognized. More and more studies have gradually revealed the process of m6A modification and the effect of m6A regulators on the biological behavior of HCC [11,12]. For example, Chen, et al. [13]) found that METTL3 in human HCC was significantly u up regulated compared with non-tumor liver control, and the overall m6A modification level in human HCC was also higher than that in normal individuals. At the same time, the team also proved that METTL3-mediated m6A hypermethylation is a new mechanism of epigenetic silencing of tumor suppressor gene expression in human cancer. Another study also proved that m6A modification is very important for the regulation of EMT process of HCC. The level of m6A modification increases significantly when EMT occurs, which can affect the invasion, metastasis and EMT process of HCC both in vivo and in vitro [14]. (Lin, et al. [15]) further identified Snail as an important transcription factor involved in EMT, has become the target of METTL3-mediated m6A modification. METTL3 works with YTHDF1 to promote protein translation of Snail, explaining how overexpression of METTL3 promotes HCC transfer. More and more m6A regulated genes have been found. For example, Chen, et al. [16,17]) found that the other two components of m6A complex, WTAP and KIAA1429, were significantly up regulated in HCC, which was related to the low overall survival rate of patients (Ma, et al. [18]).

Proved that METTL14 interacts with microprocessor protein DGC8 to promote the maturation of microRNA-126, and the downregulation of METT14 weakens the expression of microRNA-126, thus promoting HCC transfer (Li, et al.[19]). Observed the overexpression of FTO in HCC tissues. At the same time, with the increase of m6A level, the knockdown of FTO induced cell cycle arrest and inhibited the colony formation ability of HCC cells. In addition, it has been reported that the expression of YTHDF2 is down-regulated in human HCC, and the loss of YTHDF2 destroys the m6A-dependent mRNA decay of IL11 and SERPINE2, resulting in increased invasiveness of HCC [20]. It can be seen that m6A RNA is closely related to HCC. It has been confirmed that different gene mutations may be the key factors leading to the change of m6A RNA expression level. In addition, some m6A regulator genes have also been confirmed to play a cancer-promoting role in a variety of malignant tumors [21-23], such as non-small cell lung cancer [24], gastric cancer [25], bladder cancer [26], colon cancer [27] and so on. More significantly, it has also been confirmed to be associated with drug resistance of cancer, which indirectly leads to a poor prognosis of tumor patients [28]. For this reason, we analyzed the large sample size data in TCGA and GEO database to find the differentially expressed m6A regulator gene which may affect the prognosis of HCC patients. Through LASSO regression and other machine learning algorithms, we carried out cluster analysis of these differentially expressed genes, and enriched and analyzed the biological pathways that each type of genes may affect.

Then, we analyze the differences of gene expression in different m6A clusters. We clustered the different genes by machine learning method and analyzed the clinical manifestations of individuals in different clusters. By detecting the expression of these genes, we can establish a prognostic risk model for patients with HCC, which may be helpful for the diagnosis and treatment of HCC patients. However, it needs to be verified by further experiments and clinical cohort studies. Our study had some limitations. First, although we found that m6A regulators play different roles in the alternative pathways, the potential molecular mechanisms were not evaluated, it warrants further investigation. Second, m6A clusters and their risk gene clusters might improve prognosis of HCC patients. Further research is necessary to explore whether these genes could be used as diagnostic markers or therapeutic targets in HCC and guiding more effective treating strategies.

Conclusion

In summary, m6A regulators have CNV and SNV mutations in HCC patients, which may lead to poor prognosis of HCC. Different clusters of m6A regulators play different roles in multiple biological pathways. Different gene clusters can affect the prognosis of patients with HCC. There is a differential expression of m6A regulators in different gene clusters. The comprehensive evaluation of m6A modification pattern in HCC will contribute to enhancing our understanding of tumor characterization and may guide more effective therapy of HCC patients.

Author Contributions

Xiaoshi Zhang designed this study, Zhuo Yu collected the data, Xiaoyi Zhang and Zhuo Yu analysised the data, Xiaoshi Zhang wrote this manuscript. Jianqiang Cai revised the manuscript. All authors read and approved the final manuscript.

Conflict of Interest Statement

There was no potential conflicts of interest to declare among the authors.

Funding Sources

This work was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS) (grant number 2021-I2M-1-066) and the Sanming Project of Medicine in Shenzhen (grant number SZSM202011010).

Data Availability Statement

The datasets generated from this research can be disclosed only in specific circumstances. Further inquiries can be directed to the corresponding author.

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Prevalence of Hard Tick Infestation Load on Cattle in and Around Kore zone, Southern Ethiopia

Introduction

Ethiopia represent varioin theatic zones and livestock production system in tropical Africa (Solomon, et al. [1]) it has the largest number of livestock in Africa approximately 53.99 million cattle 25.5 million sheep and 24.06 million goats 6.91 million horses 0.92 million camels 6.75 million donkeys, 0.35 million mules 50.38 million poultry and 5.21 million bee hives central statistic authority (CSA, [2]) among livestock population cattle play a significant role in the socio economic aspects of the life of people of Ethiopia. In additional to the product like meat and milk cattle provide drought for cultivation of the agricultural land of many peasants, skin and hides are also important components of the livestock sector in generating foreign export earnings (Tamiru and Abebaw [3]). Even though they are important components of the Ethiopia farming system, their contribution to food production rural income and export earnings are for below the expected potential. This is because cattle promotion in Ethiopia is constrained by the compound effect on animal disease poor feeding and poor management (Getachew [4]). Now a day parasitism represents a major obstacle to development and utilization of animal resources. In Ethiopia ecto parasites in ruminant cause serious economic loss to small holder farmers, the training industry and the country as a whole through morality as animals decreased production down grading and rejection of skin and hide (Tike and Addis [5]) as a result of their activity ecto parasites may have a variety of direct and indirect effects on their hosts.

Ecto parasite commonly tick, mite and the in injection they inflict on the skin (Taylor, et al. [6]) and by their effect on the physiology of the animal as well as through transmission of deferent diseases (Wall and Sherear, 2001) infection by ecto parasites significantly effects the quality of hide they’re by effecting the economy on Ethiopia farmers as well as international market (Bekele [7]). Ticks are the most important ecto parasites of livestock in tropical area and sub-tropical areas and are responsible for secure economic loss in livestock. The major losses however, caused by ticks are due to their ability to transmit protozoan Rickettsia and viral disease of livestock which are great economic importance in worldwide. Tick born protozoan dieses (example Babesiosis and Theileriosis, Anaplasmosis and Cawdrosis and tick associated dermatophilosis are a major healthy and management problem in many developing countries. The economically most important Ixodid ticks of livestock in tropical region belongs to the general of Hyalomma, Boophilus, Rhipicephalus and Ambyelomma (Faris, 2000). In Ethiopia there are 47 species of ticks are found on livestock and most of them have importance as vector for disease causing agents and also have damaging effect on skin and hide production (Bayu [8]).

Ticks besides being important vectors for diseases of ecto parasites are organisms that spend all or part of their life cycle on the external of another organism the host and in the process extract nutriment from it for survival. They could live on puncture, burrow or attach on the surface of their host causing discomfort, weight loss, loss of body condition, reduction in milk production and irritation of the skin, which subsequently leads to ulceration and secondary infection these result negative effect on animal welfare, animal husbandry and generally quality of production (Colebrook and Wall [9]).The pathogens transmitted by major ticks’ genera can cause high morbidity and mortality in livestock. These diseases generally affect the blood and lymphatic system and cause fever, anemia, jaundice, anorexia, weight loss, milk drop, swelling of lymph node, dyspnea, nervous disorder and even death these factors also contribute to losses in milk production calving interval and weaning performance (Jonson 2006).The distribution of ticks is determined by a complex interaction of factors such as climate, host density, host susceptibility and grazing habits (Minijauw and Decastro, 2000) one of Ethiopia belongs a tropical country. Tick belong to the phylum arthropod class Arachind and order Acari the families of ticks parasiting livestock are categorized in to two the first one is Ixodidae (hard tick) and the second Argasidae/soft ticks/ these are shearing certain basic properties they differed in many structures’ behavior, physiological, feeding and reproduction pattern (Kassa [10]) According to the number of hosts Ixodidae ticks are classified as one host ticks two host ticks and three host ticks in one host ticks all the parasitic stages (larva, nymph and adult) feed on the same hosts. In two host tick’s larvae attach to one host feed and mount on the ground to adult and three host ticks the larva, nymph and adult attach to different hosts and all detach from the host after engorging and moult on the ground (Kettle [11]).

Although species of ticks and ticks born disease offer among ecological regions their impact on animal production in important wherever they occur. Ticks are wide spread in Ethiopia (Pegram, et al. [12]) ticks apart from transmitting protozoal rickettsia and viral disease.A complex of problem related to ticks and tick born disease of cattle created a demand for methods to control ticks and losses of cattle production and productivity (George, et al. [13]). Control of tick infestation and transmission of tick-borne diseases remain a challenge for the cattle industry in tropical and sub-tropical area of the world. Tick control is a priority for many countries in tropical and sub- tropical regions (Faris, 2000). In the study area there is no enough information that determine the prevalence of hard tick infestation on cattle even though, tick infestation in cattle was prevalent in selected zonal administrative. Therefore, objective of this study was to determine the prevalence and associated risk factors of hard tick genera on cattle in and around Kore zone Sarmale district, Southwestern Ethiopia.

Material and Methods

The study was conducted in and around Kore zone, Sarmale district in selective kebeles such as, Derba, Buniti, Abulo and Alefacho. Kore zone is one of the zonal administrations of Southern Ethiopian region with a total land coverage area 179980 hectare of land. The altitude of the zone 1200-3601m above sea level with annual temperature of 12-25oc. The rain fall distribution various from year to years and rainy periods June to November. The population of zone is 183056. Of whom 89095 are females and the rest 93961 are males and about 76% of their livelihood depend on traditional pastoralist and 24% depend on agro pastoral (AWAO, 2016). Kore zone is located of the bordered with to the North lake Abaya North East Oromia region, South Burji zone South-West Konso zone and to the West lake Chamo. The land scape of Kore zone is characterized by steeply sloping maintains 30% hills 20% undulating 25% and gently to plan land features 25% and the population of livestock Bovine 242211, Caprine 259032, Equine 23705 (AWLFDO, [14]).

Study Design

A cross-sectional study was conducted from December 2022 to May 2023 in selective area of Kore zone. Active data was generated from randomly selected cattle. In this study simple random sampling method was employed. Then the collected ticks were carefully examined to different groups them in to the genera using the guide indicated in (Walker et al., 2003).

Study Animal

The study animal was cattle of different breed, origin, age, sex and body condition around Kore zone. A total of 384 cattle was randomly selected from total number of cattle selected by numbering and examined which are managed under extensive farming system. The age, origin, sex, breeds and body condition score of each cattle was recorded.

Sample Size Determination and Sampling Technique

The sample size was determined by assuming the expected prevalence of 50%, 95% confidence interval and 5% absolute precision according to (Thrusfield, 2005). The desired sample for the study was calculated by the formula of by substituting this formula the sample size was taken 384.

where n= required sample size

pexp= minimum expected prevalence 50%

d= desired accuracy level at 0.05 %

Data Collection Procedures

Before tick collection animals were casting by using physical restraining. The body surfaces of animals were inspected for tick infestations alternative one sides were made by using damage to the tick. Adult ticks were collected from different parts of body regions from the ear, neck, dewlap, abdomen, anus, vulva, hip, udder, scrotum, base of tail prepuce, hind leg, flank, belly, and data of collected, address, site of attachment, breed, age, sex and body condition scores of animals were labeled. The collected ticks were put in to universal bottle, which are labeled according to the site of collection in to the 70% alcohol. Before the transport of all tick collected from different animals’ body were separately examined under stereo microscope.

Data Management and Data Analysis

The collected data was entered into Microsoft excel and was transferred to statistical package for social science (spss version20). The prevalence of ticks was determined by dividing the number of positive samples by total sample size and expressed as percentage. Chi square test was used to assess statistical significance tick infestation rate with different origins, sex, breed, age groups as well as body condition scores. In this study a total of 384 animals were examined and overall 52.33% prevalence for hard tick infestation was recorded in the study area. The prevalence of tick in study area 55(14.32%), 39(10.15%), 55(14.32%) and 52(13.54%) across the selected kebele Abulo, Derba, Bunit, Alfacho respectively below (Table 1). Out of 384 cattle Variation in breed also occurs in that local breeds were affected as compared with cross breeds 38.28% and 14.06% respectively (Table 2). Based on sex 16.66% female and 35.67% male animals were found to had more than one genus of tick and statistically significant association was recorded indicated below (Table 3). Based on body condition scoring poor 23.43% medium 18.48% and good 10.41% poor body condition animals were found severely affect with ticks than medium and good body condition animals respectively. Body condition was significant associated with tick infestation at which engorging the diseases in poor body condition were higher than cattle that have good body condition (Table 4). From total of 384 cattle the prevalence of hard tick infestation was found 11.45% in young, 20.57% in adult and 20.31% in old animals (Table 5).

Table 1: Prevalence of hard tick infestation based on origin at Sarmale district.

Table 2: Prevalence of hard tick infestation based on breed.

Table 3: Prevalence of hard tick infestation based on sex.

Table 4: Prevalence of hard tick infestation based on body condition scores.

Table 5: Prevalence of hard tick infestation based on age.

Discussion

Different tick genera are widely distributed in Ethiopia and number of researchers reported the distribution and abundance of sticks in different parts of the country (Solomon, et al. [1]). In the present study the total tick infestation prevalence was found 52.33% this found were greater than the reports of (Kassa and Talew, 2012) with a prevalence of 33.21% in Haramaya district and (Tesfahewet and Simeon [15]) a prevalence of 16% Benchi Maji Zone of the South Western Ethiopia those study contrast to the presence study (Nigatu and Teshome [16]) were reported a higher prevalence of ticks (89.4%) from Western Amhara region. The presence study had high result of tick infestation recorded due to poor management system of pasture, in adequate control of tick and care of his cattle.Risk factor (sex, origins, breed and body condition score) were also involved in the variation of the prevalence of ticks in the study area. The prevalence of tick was 23.43%, 18.48% and10.41% in poor, medium and good body condition score respectively. These was lower than indicated in (Bossena and Andu [17]) were poor body condition 62.9%, medium body condition 59.4% and good body condition scoring 41.2%. In this less than one year it was 66.91% while in one three years and greater than three years were 101% and 49.45% respectively. In general, the prevalence of ticks in all the researchers’ indicated that very young animals are affected less than adult animals. This could be due to the less exposure to field grazing with other animal in the field and adults are exposed due to the communal grazing habit (Gedilu et al., 2014) local breed 38.28% was affected higher than that of cross breed 14.06%. This result was agreed with the finding of Kassa and yalew, [18] who reported the prevalence of tick infestation was significantly higher in local cattle (58.18%) than cross breed (10.55%).

Conclusion and Recommendations

The important and abundant tick genera investigated in this study were Boophilus, Ambyelomma, Hyalomma and lastly Rhipicephalus. The study indicated that was high prevalence of ticks in the study area. However, the attention given to controlling the infestation had not been sufficient the control methods necessary were selection of tick resistance cattle, acaricide treatment, appropriate livestock management evaluation and incorporation of traditional practices. Generally, the distribution of ticks is not fixed but determined by a complex interaction of factors such as climate, host density, host susceptibility, grazing habit and pasture herd management. Therefore, an effective tick control program should be formulated and implemented based on the distribution pattern of ticks and factors responsible for their distribution. Based on the above conclusion the following recommendations are forwarded:

• Awareness creation on regular deworming and topical acaricide application should be implemented.

• A proper posture management through rotational grazing

• Further detailed epidemiological study should be conducted at the study area.

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Urinary Retention After Haloperidol Decanoate and Fluoxetine Combination Reversed After Discontinuation of Fluoxetine

Introduction

Defined as the inability to completely empty the bladder [1], urinary retention is a side effect not commonly observed during the use of psychotropics, although it has been reported that 2% of acute urinary retention episodes are induced by drugs [2]. A variety of classes of drugs can interfere with the physiology of the lower urinary tract, contributing to the development of urinary retention, including some psychotropic agents [3]. A previous review of the literature observed that there were several case reports of selective serotonin reuptake inhibitors (SSRI) causing urinary retention in combination. They hypothesized that these combinations may have affected the hepatic metabolism of the other drugs, like the case of fluoxetine impairing the cytochrome CYP2D6 enzyme metabolism of risperidone when administered in combination, increasing the plasma levels of risperidone and therefore contributing to its side effects [4]. Overall, there is good evidence that urinary retention is very rarely if at all associated with SSRI used in monotherapy [3]. Concerning the effects of typical antipsychotics, haloperidol promoted urinary retention in 5% of patients, but the lack of homogeneous definitions of urinary retention among these studies make it hard to conclude the real role of haloperidol for the development of urinary retention, with a possibility that the typical antipsychotics only caused mild voiding dysfunction instead [3]. This report describes a case of a young adult with a premorbid schizoid personality disorder, who evolved with urinary retention after using combined therapy with haloperidol decanoate and fluoxetine.

Case Report

A man, 24 years old, only child, Body Mass Index 15.16 kg/ m², with pre-morbid schizoid personality disorder and probable intellectual disability, admitted to a psychiatric ward due to a severe depressive episode with loss of self-care and food refusal. About nine days before admission to the psychiatric ward, he had used 01 intramuscular ampoule of haloperidol decanoate, and started biperiden 2 mg/d orally for three days, after an outpatient consultation with a psychiatrist. There was no report of previous use of psychotropic drugs. On admission, olanzapine 5 mg/d was prescribed (in the presence of psychotic symptoms: probable audiovisual hallucinations), fluoxetine 20 mg/d and Clonazepam 0.5 mg/d at night. On the sixth day of hospitalization, olanzapine was suspended to maintain the use of haloperidol decanoate for greater therapeutic adherence after hospital discharge, with 1 ampoule administered intramuscularly, maintaining an interval of fifteen days from the applied dose prior to admission. On the tenth day of hospitalization, he developed dysuria, voiding effort and reduced urinary output, without an increase in nitrogen slag, signs of infection or other changes in the urine summary. After about two days without maintenance of satisfactory debt and with the exclusion of other causes, the hypothesis was raised that such an occurrence could be related to adverse effects due to the combination of psychotropics, and fluoxetine was withdrawn, considering the impossibility of suspension of the depot antipsychotic. Around two to three days after the suspension of fluoxetine, the patient evolved with resolution of the urinary retention, a time interval that coincides with the half-life of fluoxetine (2 to 4 days)1(Figure 1).

Figure 1

Discussion

It is already known that both fluoxetine and haloperidol are metabolized by the hepatic enzyme CYP450 2D6 [5,6], and that fluoxetine is an important inhibitor of this same enzyme, so that their concomitant use, due to these pharmacokinetic interactions, predisposes to increased serum levels of both, augmenting adverse effects, especially in patients with a low Body Mass Index like the one of this report. These knowledge derives from studies with oral formulations, but the interaction between oral fluoxetine and haloperidol decanoate was already described in the literature, with one study showing the 14 days after the addition of fluoxetine, a very significant increase in haloperidol concentrations (100%) was observed, reinforcing the knowledge that the pharmacokinetic interactions with haloperidol of fluoxetine is clinically significant, either by inhibiting its hepatic metabolism or and by displacing it from protein binding sites [7]. Some cases with similarities were previously reported in the literature [8,9], including one that reported urinary retention also in a young patient, but due to fluoxetine and oral haloperidol combination, that was reverted after haloperidol discontinuation, emphasizing that this is the first report of a case of urinary retention with fluoxetine/haloperidol combination reverted with the maintenance of haloperidol use. The recovery of spontaneous diuresis after only two days of fluoxetine suspension in our case report emphasizes the hypothesis that the major mechanism underlying this interaction was the probable fluoxetine property of displacing haloperidol from protein binding sites, knowing that as stated before, both medications are substrates of the same cytochrome P450 enzyme, CYP 2D6 [5,6].

Conclusion

To our knowledge, this is the first report of urinary retention with haloperidol decanoate associated with fluoxetine, reverted after discontinuation of fluoxetine and maintenance of haloperidol, considering that haloperidol couldn’t be suspended because of its long-acting presentation. This report emphasizes the importance of the knowledge of pharmacokinetic interactions in the management of psychiatric patients, by bringing an infrequent side effect, managed with the help of the concepts of drug interactions.

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