Microsurgical Dissection of Fresh Human Mastectomy Specimens for Visualization of Breast Fascial and Ligamentous Support Structures

Summary

This study describes the microsurgical dissection of fascial and ligamentous support structures in breast tissues removed during prophylactic mastectomy. The findings provide a valuable resource for surgeons and anatomists studying human female breast anatomy.

Abstract

Ligamentous and fascial support structures, such as Cooper’s ligaments and the superficial fascial system, are important structural components of the female breast, contributing to breast shape, mechanical support, and surgical plane definition during breast surgery. Although previous studies have investigated these structures through cadaveric dissection, intraoperative observation, and biomechanical analysis, there have been no reports describing their relationships using microsurgical dissection of fresh human breast tissue. In this study, fresh nonfixed breast tissue specimens were obtained from patients undergoing prophylactic mastectomy and sectioned into 2-cm-thick sagittal slices by a breast pathologist. Microsurgical dissection of each slice was performed under approximately 5×–10× magnification to visualize and examine Cooper’s ligaments and the superficial layer of the superficial fascia (SLSF) and deep layer of the superficial fascia (DLSF). Using this protocol, microsurgical dissections were completed in 24 breast tissue specimens, enabling visualization of the observed structural continuity between Cooper’s ligaments, the SLSF, the dermis, the DLSF, and the pectoral fascia. Specifically, Cooper’s ligaments were observed extending through the SLSF into the dermis and posteriorly through the DLSF toward the fascia overlying the pectoralis major muscle. This protocol enables direct visualization of the fascial-ligamentous support network of the breast and facilitates anatomical understanding of the relationships among these structures. This study and its protocol may serve as valuable educational and research resources for breast surgeons, plastic surgeons, and cross-disciplinary researchers seeking a better understanding of the fascial-ligamentous support structures of the breast.

Introduction

The anatomy of the breast has long been a subject of interest in both clinical practice and anatomical research, especially owing to its implications for breast aesthetic surgery and breast cancer treatment. One of the most important, yet underexplored, components of breast anatomy is Cooper’s ligaments, a network of fibrous connective tissue that runs through the breast, providing structural support. Initially described by Sir Astley Cooper in his 19th-century book, On the Anatomy of the Breast, Cooper’s ligaments play a critical role in maintaining the shape and position of the breast by anchoring the skin to the underlying pectoral fascia¹. Their relevance in both surgical and aesthetic procedures has increased, particularly as breast surgery continues to evolve. In the 1990s, Ted Lockwood highlighted the significance of the superficial fascial system, especially in the context of breast and body contouring surgeries. His work emphasized the importance of this fascial layer for maintaining breast shape, distributing mechanical forces during surgery, and providing a surgical plane for procedures, such as breast lifts (mastopexy) and breast reconstructions^2,3. Since then, the focus on breast anatomy has intensified, with various studies investigating Cooper’s ligaments and the superficial fascial system through cadaveric dissection^4,5,6, histological characterization⁷, imaging⁸, endoscopy⁹ intraoperative findings⁴, and biomechanical studies^7,10,11. Collectively, these investigations have broadened current understanding of the structural organization of the breast fascial system and highlighted the diversity of anatomical observations reported in the literature.

These investigations have enriched our understanding of the structural dynamics of the breast and how they change under physiological and pathological conditions. However, they lack the detailed, fine-scale observations necessary to fully understand the relationships between tissues as seen during microsurgical dissection. Despite the wealth of information from macroscopic dissections and clinical procedures, there have been no reports of microsurgical dissection of these structures in human breast tissue. Such observations are crucial for gaining a deeper understanding of how these anatomical systems work together to maintain the breast’s architecture. Therefore, there remains a significant knowledge gap regarding the features of Cooper’s ligaments and the superficial fascial system. For surgeons, especially those in breast oncology and plastic surgery, having a detailed understanding of these systems is important for surgical planning and anatomical orientation during procedures, such as breast reconstruction, mastectomy, and augmentation¹².

The present study aims to fill this gap by providing detailed microsurgical observations of Cooper’s ligaments and the superficial fascial system in breast tissue obtained from prophylactic mastectomy specimens. Using tissue slices, we performed dissections of these structures using the microsurgical dissection techniques described in the protocol below. Unlike previous cadaveric or macroscopic studies, this protocol enables microsurgical visualization of Cooper’s ligaments and the superficial fascial system in fresh human breast tissue.

This study provides clear, visually documented evidence of the relationships between Cooper’s ligaments, the superficial fascial system, and surrounding breast tissues at the microsurgical level. These anatomical observations may facilitate future anatomical and surgical investigations. The potential applications of these findings in oncologic breast surgery require further validation using specimens from breast cancer patients. For junior breast and plastic surgeons, as well as cross-disciplinary researchers, the findings presented here will serve as an educational tool, enhancing their understanding of the intricate details of the breast’s fascial and ligamentous anatomy.

Protocol

This protocol was approved by The University of Texas MD Anderson Cancer Center’s Institutional Review Board (IRB) on 05/06/2016 with the record number PA16-0364. As per institutional guidelines, written informed consent was obtained from all patients who provided prophylactic mastectomy breast tissue samples.

NOTE: This protocol provides detailed steps for microsurgical dissection of breast tissue to visualize and study the ligaments and fascia of the breast structural support system. The breast surgeons at the institution where this study was conducted routinely use electrocautery to dissect the pectoralis major fascia (pectoral fascia) to remove the mastectomy specimen from the chest wall during a total skin-sparing or nipple-sparing mastectomy. Specimen preparation, orientation, and microsurgical dissection are described in detail in the following sections.

1. Preparation of tissue slices from mastectomy specimens

1. Collect mastectomy specimens.
   1. Obtain the breast tissue from patients undergoing prophylactic mastectomy following approval of the protocol by the institutional IRB. Obtain informed consent and collect tissues in accordance with ethical and clinical guidelines.
2. Section the mastectomy specimens.
   1. Immediately after the mastectomy, examine grossly and begin processing the mastectomy specimen. This is done by a breast pathologist. A schematic illustrating the specimen sectioning strategy is shown in Figure 1.
   2. Before sectioning, orient and ink the intact mastectomy specimen according to the standardized pathology color scheme: red (superior), orange (inferior), green (anterior), and black (posterior).
   3. Use a #20 pathology scalpel blade to section the specimen into approximately 2-cm-thick sagittal slices while maintaining its native anatomical orientation. Subsequently, use the retained ink markings to preserve the anatomical orientation of the tissue slice during subsequent handling and microdissection.
   4. Use a single representative peripheral tissue slice provided by the breast pathologist for microsurgical dissection, as the central slices containing the nipple or a portion of the nipple are usually retained for routine clinical pathologic evaluation.
3. Transfer and store the breast tissue.
   1. Wrap each tissue slice in a saline-moistened paper towel and seal the tissue slice in a plastic biohazard bag. Transfer the wrapped slice to the institutional tissue bank (ITB), where they are refrigerated at 4 °C until microsurgical dissection.
      NOTE: Specimens are not frozen or rewarmed prior to dissection. The time from mastectomy to refrigerated storage typically ranges from 2–3 h. Microsurgical dissection is typically performed within 6–24 h after mastectomy. The effect of storage duration on tissue quality was not formally evaluated in this study.

2. Microsurgical setup

1. Position the microscope.
   1. Position the surgical microscope and set it to 5×–10× magnification to ensure optimal visualization of tissue structures for precise microsurgical dissection. Use an adapter to connect a smartphone to the microscope (Figure 2) and ensure that the field of view is properly aligned.
      NOTE: The smartphone setup provides sufficient image quality for visualization, documentation, and video recording of the dissection. Alternatively, an operating microscope equipped with an integrated camera may be used when available.
2. Prepare dissection tools.
   1. Arrange all dissection tools, including forceps and scissors, on the dissection platform for easy access during the procedure.
3. Set up the vacuum and solution.
   1. Use a vacuum system equipped with a 3-mm Frazier suction tip to assist with tissue removal during dissection. Set the vacuum pressure between 180 mmHg and 200 mmHg.
   2. Use normal saline to keep the tissue slices hydrated and prevent tissue desiccation throughout the procedure.
      NOTE: When greater suction is needed, temporarily cover the vent hole of the Frazier suction tip with the thumb to increase suction. Apply suction gently to the adipose tissue on either side of the ligament, removing only enough adipose tissue to expose the ligament. Avoid direct suction of the ligament or fascial structures. Keep additional saline available to flush the suction tip if it becomes blocked or the suction strength weakens. This ensures continuous suction and prevents interruptions during dissection.

3. Dissection process

NOTE: Handle fresh human breast tissue in accordance with institutional biosafety procedures. Wear disposable gloves, a laboratory coat, and a surgical mask throughout specimen handling and microsurgical dissection. Collect suctioned material through the institutional wall suction system using a disposable suction collection canister equipped with an overflow protection mechanism to prevent accidental entry of fluids and suction debris into the wall vacuum system. Dispose of sharps, tissue specimens, suctioned material, and other biohazardous materials according to institutional biosafety procedures.

1. Confirm the tissue orientation.
   1. Follow the orientation, labeled by the pathologist (Figure 3).
   2. Before microscopic dissection, routinely orient the tissue slice with the posterior (black-inked) surface facing the dissector and the skin surface facing the assistant.
      NOTE: This standardized orientation places the pectoral fascia closest to the dissector and allows systematic identification of the DLSF, Cooper’s ligaments, and SLSF in a consistent anatomical sequence during dissection.
   3. Double-check the orientation under low power magnification by identifying key anatomical landmarks, such as the epidermis, dermis, subcutaneous tissue, and pectoral fascia.
2. Identify the superficial fascial system.
   1. Locate and observe the superficial fascial system, paying attention to how it connects with the skin and integrates with subcutaneous adipose tissue and deeper layers.
   2. Locate the superficial layer of the superficial fascia (SLSF):
      1. Identify the SLSF layer, which typically presents as a distinct, thin, somewhat translucent fascial layer beneath the skin and subcutaneous adipose tissue.
      2. Under 5×–10× magnification, it appears as a somewhat continuous linear membrane running parallel to the dermis, distinguishing it from the surrounding adipose tissue. The fascia often has an adipose lobule “traversing” the fascia and creating a “hole” in the fascia. Follow this plane carefully during dissection to confirm the fascia's identity and visualize its attachments to surrounding structures (Figure 4A,A’).
   3. Locate the deep layer of the superficial fascia (DLSF):
      1. Identify the DLSF as a somewhat translucent fascial layer located immediately superficial to the pectoral fascia and extending transversely across the chest wall. It runs parallel to the pectoral fascia and forms a continuous fascial plane during gentle dissection.
      2. Trace this structure carefully during dissection to confirm the fascia’s identity and visualize the adjacent anatomical structures (Figure 5A,A’).
         NOTE: The DLSF lies close to the pectoral fascia and may not be easily distinguishable in all specimens. Compared to the superficial layer, the deep layer is more challenging to identify and usually requires additional time and careful dissection to locate and confirm. If the expected continuous fascial plane cannot be confidently identified, return to low magnification to relocate the fascial plane before continuing the dissection.
3. Trace Cooper's ligaments and dissect adipose pockets.
   1. Recognize Cooper's ligaments as smooth, translucent fiber bundles, typically less than 1 mm thick, extending approximately perpendicular to the dermis, SLSF, DLSF, and pectoral fascia. They remain intimately adherent to the surrounding adipose lobules and breast ducts. Follow the ligaments continuously during gentle traction.
   2. (Exception) Dissection of Cooper's ligaments may not be feasible in specimens with dense glandular tissue. Carefully identify a candidate ligamentous structure and trace it in both the superficial and deep directions while preserving its relationships with the surrounding tissues.
      1. Document its observed anatomical course, including whether it terminates, branches, merges with adjacent fascial structures, or becomes disrupted during dissection.
      2. Manipulate ligamentous tissues gently with micro-forceps to prevent damage to the fascia. Cooper's ligaments are composed of collagen fibers that are predominantly oriented in a uniform direction, whereas ordinary fibrous septa contain collagen fiber bundles arranged in multiple directions.
   3. As dissection proceeds, incise the ligamentous cover of adjacent adipose pockets to expose the adipose tissue for dissection.
      1. For cut specimens with exposed adipose tissue, apply suction gently to the adipose tissue on both sides of the target ligament to remove only enough adipose tissue to expose the edge of the ligament to facilitate its microsurgical dissection (by scissor dissection). Avoid direct suction of the ligament or fascial structures. The adipose tissue is organized into lobules that remain adherent to the surrounding Cooper's ligaments.
      2. Carefully remove the adipose tissue to expose the ligamentous framework and facilitate visualization of the anatomical relationships between Cooper's ligaments, the superficial fascial system, and the surrounding adipose tissue (Figure 6).
   4. Maintain hydration with saline irrigation.
      1. Use normal saline irrigation throughout the procedure to prevent tissue desiccation.
      2. Perform irrigation as needed, guided by the tissue’s appearance rather than a predetermined flow rate or total volume.
      3. Adjust the suction evacuation as needed to keep the dissection field clear of excess fluid and debris, ensuring uninterrupted observation and precise dissection of each ligament.

4. Video documentation

1. Record the entire dissection.
   1. Use the microscope’s built-in camera system to capture the complete dissection process in real time for reference and review, ensuring no steps are missed. Perform the dissection under 5×–10× optical magnification using the operating microscope.
2. Focus on key anatomical structures.
   1. During dissection and recording, zoom in from low to high power on critical structures, including Cooper’s ligaments and their structural relationships with the superficial fascial system, to provide detailed views that highlight their relationships with surrounding tissues.
3. Annotate findings.
   1. During the dissection, record verbal narration. After acquiring the video, review the recorded video and audio to add text annotations during postproduction.
      NOTE: These annotations highlight key anatomical structures, procedural steps, and important observations, thereby improving the educational value and clarity of the final video.

Results

Over the course of 1 year, microsurgical dissections were performed on 24 unilateral prophylactic mastectomy specimens obtained from 24 patients. The visibility of individual fascial and ligamentous structures varied among specimens; therefore, the representative images presented here were selected to illustrate the protocol, the anatomical appearance of the fascial and ligamentous structures, and the observed anatomical relationships among Cooper's ligaments, the DLSF, SLSF, and the surrounding adipose tissue. A summary...

Discussion

The dissection process outlined in this study emphasizes several critical steps necessary to achieve optimal visualization of Cooper’s ligaments and the superficial fascial system. Proper sagittal sectioning of the breast tissue into 2-cm-thick slices under the guidance of a pathologist is fundamental to preserving the anatomical orientation and integrity of the structures of interest. Additionally, the use of a surgical microscope with 5×–10× magnification ensures precise dissection and observation...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% NaCl solution	General use	N/A	Used for tissue irrigation and periodic flushing of the Frazier suction tip during dissection.
Adapter (Smartphone and Microscope)	GOSKY	Standard	Smartphone adapter used to attach the camera to the operating microscope.
Camera	Apple	iPhone SE (2nd generation)	Used for video recording through the operating microscope.
CDI tissue marking dye	Cancer Diagnostics, Inc.	#0727-1, #0727-3, #0727-4, #0727-6	Used to ink the intact mastectomy specimen before sectioning for orientation (Red = superior, Orange = inferior, Green = anterior, Black = posterior).
Forceps	Precision Medical Devices	N/A	Used for microsurgical tissue manipulation.
Frazier suction tip	Integra Miltex	Model not specified	3-mm Frazier suction tip instrument used for tissue suction during dissection.
Hospital wall suction	N/A	N/A	Hospital wall suction system connected to the operating room vacuum source.
Microscissors	Precision Medical Devices	S1059 1575	Used for fine microsurgical tissue dissection.
No.20 scalpel blade	Swann-Morton	Example	Used for gross specimen trimming.
Non-absorbable suture (Silk)	Ethicon	Mersilk 4-0	Used to mark anatomical structures for photography and video documentation.
Scalpel handle	General surgical supply	N/A	Compatible with No.20 scalpel blade.
Surgical microscope	Med Link	Z880	Operating microscope used for microsurgical dissection
Tissue culture dishes	Corning	430167	Used to hold saline for periodic flushing of the Frazier suction tip.