게잡이원숭이를 활용한 생의학 연구 탐구 = Exploring biomedical research on cynomolgus monkeys (Macaca fascicularis)

다국어 초록 (Multilingual Abstract)

Cynomolgus monkeys (Macaca fascicularis) are widely used in biomedical research due to their genetic, physiological, and anatomical similarities to humans, which make them valuable models in this field. However, their use as laboratory animals presents specific challenges, such as the occurrence of spontaneous tumors, the need for optimized assisted reproductive technologies, and the requirement for non-invasive prenatal monitoring.
Among the health challenges observed in cynomolgus monkeys, subcutaneous leiomyosarcoma, though rare, poses significant diagnostic and treatment challenges. Understanding the incidence, detection, and characteristics of these tumors is essential for maintaining the health of these animals and ensuring the validity of research results.
When developing gene-edited models using cynomolgus monkeys, selecting appropriate recipients for embryo transfer is a critical step. Traditional methods, which involve using uterine-embryo synchronized recipients, demand considerable time, cost, and the use of many animals.
Accurate fetal weight estimation is vital for monitoring the health and development of fetuses in both animals and humans. However, in research involving cynomolgus monkeys, fetal weight measurement often relies on invasive techniques, such as performing Caesarean sections.
The aim of this study was to advance the use of cynomolgus monkeys in biomedical research by developing refined methodologies to address the challenges associated with their use as laboratory animals. This included confirming the presence of subcutaneous leiomyosarcoma, a condition not previously documented in this species; proposing a new recipient selection strategy for generating gene-edited monkeys; and developing methods for estimating fetal weight to improve prenatal care and enhance research outcomes. Therefore, the investigations of this study were divided into the following three parts:

STUDY 1: Confirmation and characterization of subcutaneous leiomyosarcoma in a cynomolgus monkey
Leiomyosarcoma, a malignant tumor originating from smooth muscle cells, has rarely been documented in non-human primates. In this study, a 7-year-old female cynomolgus monkey presented with a rapidly growing mass overlying the left elbow joint. Radiographs indicated the presence of a soft tissue neoplasm without any associated bone involvement. The mass was surgically resected. Histological and immunohistochemical analyses revealed spindle-shaped cells with eosinophilic cytoplasm that resembled smooth muscle cells, exhibiting positive immunoreactions for vimentin, desmin, and smooth muscle actin and a negative reaction for pan-cytokeratin. This is the first reported case of subcutaneous leiomyosarcoma in a cynomolgus monkey and provides important insights into the incidence and characteristics of this condition in this species.

STUDY 2: Assessing the potential of superovulated cynomolgus monkeys used as oocyte donors to serve as surrogate recipients in embryo transfer
Assisted reproductive technologies (ARTs), such as intracytoplasmic sperm injection and embryo transfer, are crucial for generating genetically edited monkeys. Despite their importance, ARTs face challenges in recipient selection, particularly regarding time, cost, and the number of animals required. The potential of superovulated monkeys, commonly used as oocyte donors, to serve as surrogate mothers, remains underexplored. The study aimed to compare the efficacy of superovulated and uterine-embryo synchronized recipients of embryo transfer in cynomolgus monkeys. This study involved 25 cynomolgus monkeys divided into two groups—13 superovulated recipients and 12 synchronized recipients. The evaluation criteria included measuring endometrial thickness on the day of embryo transfer and calculating pregnancy and implantation rates to compare outcomes between groups. The study found no statistically significant differences in endometrial thickness (superovulated: 4.48±1.36 mm, synchronized: 5.15±1.58 mm), pregnancy rates (superovulated: 30.8%, synchronized: 41.7%), and implantation rates (superovulated: 14.3%, synchronized: 21.9%) between the groups (p > 0.05). The observations indicate that superovulated recipients are as effective as synchronized recipients for embryo transfer in cynomolgus monkeys. This suggests that superovulated recipients can serve as viable options, offering an efficient and practical approach to facilitate the generation of gene-edited models in this species.

STUDY 3: Developing ultrasonographic fetal weight estimation methods in cynomolgus monkeys at 129–132 days of gestation: Applying human fetal weight formulas and comparing with actual birth weights
The estimated fetal weight (EFW) using ultrasonography (USG) is a common practice in human prenatal management. However, its application in non-human primates, such as cynomolgus monkeys, has not been well established. This study aims to evaluate the applicability of human EFW formulas for estimating fetal weight in cynomolgus monkeys at approximately 130 days of gestation. The study involved nine pregnant cynomolgus monkeys. Fetal parameters, including biparietal diameter, head circumference, abdominal circumference, and femur length, were measured using USG. The EFW was calculated using 11 human EFW formulas. The actual birth weight (ABW) was recorded following Cesarean section, the day after the EFW calculation. To compare EFW and ABW, statistical methods such as mean absolute percentage error (APE) and Bland-Altman analysis were employed. The ABW ranged between 200.36 and 291.33 g. Among the 11 formulas, the Combs formula showed the lowest APE (4.3%) and highest correlation with ABW (p < 0.001). Notably, EFW and ABW differences for the Combs formula were ≤5% in 66.7% and ≤10% in 100% of cases. The Bland-Altman analysis supported these results, showing that all cases fell within the limits of agreement. In conclusion, the Combs formula is applicable for estimating the weight of cynomolgus monkey fetuses with USG at approximately 130 days of gestation. Observations suggest that the Combs formula can be applied in the prenatal care and biomedical research of this species.

목차 (Table of Contents)

• Abstract 1
• General introduction 6
• References 10
• Chapter 1. Confirmation and characterization of subcutaneous leiomyosarcoma in a cynomolgus monkey 14
• 1-1. Introduction 15
• 1-2. Materials and methods 17
• 1-3. Results 19
• 1-4. Discussion 21
• 1-5. References 25
• 1-6. Figures 32
• Chapter 2. Assessing the potential of superovulated cynomolgus monkeys used as oocyte donors to serve as surrogate recipients in embryo transfer 36
• 2-1. Introduction 37
• 2-2. Materials and methods 39
• 2-3. Results 45
• 2-4. Discussion 47
• 2-5. References 55
• 2-6. Tables 63
• 2-7. Figures 67
• Chapter 3. Developing ultrasonographic fetal weight estimation methods in cynomolgus monkeys at 129132 days of gestation: Applying human fetal weight formulas and comparing with actual birth weights 72
• 3-1. Introduction 73
• 3-2. Materials and methods 75
• 3-3. Results 80
• 3-4. Discussion 82
• 3-5. References 88
• 3-6. Tables 97
• 3-7. Figures 106
• General conclusion 113
• Korean abstract 115