Fertility and Sterility Time series analysis of transmesothelial invasion by endometrial stromal and epithelial cells using three-dimensional confocal microscopy  Craig A Witz, M.D., Sook Cho, M.D., Ph.D., Victoria E Centonze, Ph.D., Iris A Montoya-Rodriguez, B.S., Robert S Schenken, M.D.  Fertility and Sterility  Volume 79, Pages 770-778 (March 2003) DOI: 10.1016/S0015-0282(02)04834-3

FIGURE 1 Method of confocal microscopy. (A) Images were collected using a Z-stage motor. The stage was moved up and down at 0.5- to 0.75-μm intervals and optical sections were recorded in the X–Y plane. (B) The optical sections were used to create orthogonal images in the X–Z and Y–Z planes. Witz. Endometrial invasion of mesothelium. Fertil Steril 2003. Fertility and Sterility 2003 79, 770-778DOI: (10.1016/S0015-0282(02)04834-3)

FIGURE 2 Culture of EECs with PMCs interrupted at 1 hour. (A) Single view in the X–Y plane taken at the point of attachment of a labeled EEC (round, white). The PMCs are shown with DIC (original magnification, ×450; bar = 10 μm). (B–E) serial orthogonal sections in the Y–Z plane taken through the EEC (white) at the point of attachment to the PMC monolayer (gray). The glass coverslip is on the left of each image. Attachment of the EEC to an intact layer of PMCs is demonstrated. (F–I) Three-dimensional reconstruction demonstrating an intact layer of PMCs below the attached EEC. The perspective of the initial image is from the top of the culture dish looking toward the glass coverslip. The image is rotated clockwise along the Y axis at 60-degree intervals. Witz. Endometrial invasion of mesothelium. Fertil Steril 2003. Fertility and Sterility 2003 79, 770-778DOI: (10.1016/S0015-0282(02)04834-3)

FIGURE 3 Z-series of ESCs taken at 6 hours of culture. Optical images were collected every 0.7 μm beginning at the bottom of the coverslip and moving upward. The level of the optical section is shown in the lower left of each image. An attached, spreading ESC is seen. Many unattached ESCs are also seen (white arrowheads). At 6 hours, processes of the attached ESCs can be seen extending between mesothelial cells (black arrowheads). In addition, the Z-series demonstrates an ESC passing beneath a PMC (white arrow). (Original magnification, ×250; bar = 20 μm.) Witz. Endometrial invasion of mesothelium. Fertil Steril 2003. Fertility and Sterility 2003 79, 770-778DOI: (10.1016/S0015-0282(02)04834-3)

FIGURE 4 Z-series and orthogonal view of EECs taken at 12 hours of culture. (A–D) Serial images taken in the X–Y plane beginning at the bottom of the coverslip and moving upward. The level of the optical section is shown in the lower left corner. (Original magnification, ×300; bars = 20 μm). (E–H) Orthogonal images in the Y–Z plane demonstrate the extension of an EEC process (white arrowhead) extending under mesothelial cells. Rounding of undermined mesothelial cells is seen (white arrows). Witz. Endometrial invasion of mesothelium. Fertil Steril 2003. Fertility and Sterility 2003 79, 770-778DOI: (10.1016/S0015-0282(02)04834-3)

FIGURE 5 A single attached ESC is followed over time. (A) ESCs are identified attached at the periphery of PMCs. Low-intensity laser exposure and rapid scan times were used in the live cell cultures. A single cell is followed for 24 hours (white circle). The image is a single fluorescence X–Y optical section at the level of the ESC attachment. (Original magnification, ×100; bar = 50 μm). (B) The same cell as seen in (A) is shown at 6 hours (white arrowheads). (Original magnification, ×450; bar = 20 μm). (C) The cell is seen at 24 hours. Spreading of cell processes between PMCs is evident (original magnification, ×300; bar = 20 μm). (D–G) Three-dimensional reconstruction of the Z series at 24 hours. The perspective of the initial image is from the top of the culture dish looking toward the glass coverslip. The image is rotated clockwise along the Y axis at 60-degree intervals. A rounded PMC is seen in the process of lifting from an invaded ESC (white arrow). (G) The ESC can be seen attached to the coverslip. Witz. Endometrial invasion of mesothelium. Fertil Steril 2003. Fertility and Sterility 2003 79, 770-778DOI: (10.1016/S0015-0282(02)04834-3)

FIGURE 6 Summary of the process and time course of endometrial adherence and invasion through PMCs. At 1 hour, there is attachment of ESCs and EECs (white) to the periphery of PMCs (gray). By 3 hours, ESCs and EECs extend processes to PMC junctions. These cell processes pass through PMC junctions by 6 hours. In vivo, it is postulated that the cell processes pass through the collagen IV containing basement membrane (gray) and into the underlying extracellular matrix of the peritoneum (gray strands). Between 12 and 24 hours, PMCs are undermined by ESCs and EECs. The undermined PMCs then lift from the invading ESCs and EECs. At 24–27 hours there is evidence that PMCs begin to spread over invaded ESCs and EECs. Witz. Endometrial invasion of mesothelium. Fertil Steril 2003. Fertility and Sterility 2003 79, 770-778DOI: (10.1016/S0015-0282(02)04834-3)