1.1.

Uterine Transplantation Surgery

Absolute uterine factor infertility (AUFI) renders women unconditionally infertile due to the absence of the uterus (premenopausal hysterectomy or congenital reasons).² UTx has been proposed as a potential solution in such cases. A large body of surgical research in animal transplant models,³ in addition to experience in fertility-sparing oncological surgery,⁴ has resulted in the development of surgical techniques that recently led to the first successful UTx in humans.¹

Reperfusion of the organ, through connection of its major blood vessels to the recipient’s vasculature, is necessary to ensure long-term viability of the uterus. This is particularly important as the organ is deprived of oxygenated blood during periods of vascular clamping (“warm” ischaemia) or while preserved outside the body during surgery (“cold” ischaemia). Ischemia-reperfusion injury to the parenchyma and microcirculation may occur during these periods.²

In previous work by our group, a pulse oximeter probe was used to measure oxygen saturation and “perfusion index” (a relative measurement of blood volume) by attaching it directly to the organ.⁵ Although this method had the advantage of being readily available, it suffered from that fact that it only reflected arterial oxygenation and returned just single-point measurements.

1.2.

Optical Imaging of Hemodynamics

Recently published results from our group have shown the feasibility of intraoperative visualization of oxygen saturation using a multispectral imaging (MSI) laparoscope.⁶ This technique, validated in a porcine abdominal procedure, was demonstrated to be capable of following changes in oxygenation across a wide range (30% to 100%), as well as being sensitive to the spatial heterogeneity of the tissue’s vasculature. Studies by other researchers have also demonstrated the effectiveness of this approach for assessing perfusion of the retina,⁷ brain,⁸ and kidneys.⁹ Spectral reflectance imaging has also been used to investigate cold and warm ischaemia-reperfusion injury on the kidneys.¹⁰

In this paper, we describe the use of our laparoscopic MSI system to assess changes in uterine oxygen saturation during transplant surgery on rabbit and sheep models. The system incorporates a standard surgical white light source and a liquid crystal tuneable filter (LCTF) to provide spectral selection. Results were obtained in parallel with pulse oximeter point measurements in short- and long-term survival studies. Detailed descriptions of the surgical method and results have been published separately.^11,12 These measurements enable intraoperative quantitative measures of perfusion to be recorded along with spatial information, at different time points in addition to subjective observations. The results of this study help to identify typical trends in organ oxygen saturation that may occur during surgery and recovery, with reference to a baseline.

2.1.

Multispectral Imaging Laparoscope

A laparoscopic MSI system developed by our group, and characterized and validated in a separate publication,⁶ has been used in the previous pilot studies of uterine transplantation procedures.^13,14 The clinical imaging system comprises a 30-deg laparoscope (Karl Storz GmbH, Tuttlingen, Germany), LCTF (Varispec, Cri, Inc.), and monochrome camera (DCU223M, Thorlabs Ltd., UK). A surgical xenon light source (xenon 300, Karl Storz GmbH, Tuttlingen, Germany) was used for illumination. The laparoscope was secured to a flexible mounting arm and the whole system placed on a trolley that could be conveniently positioned at the bedside for measurements. A data cube of 13 images (500 to 620 nm) was acquired to generate reflected intensity spectra of the tissue at each spatial location. Exposure times of $\sim 200\mathrm{ms}/\text{image}$ were required, resulting in a total acquisition time of $\sim 3.2\mathrm{s}$. A preprocessing image registration step using custom-written feature-tracking software^15,16 was carried out to compensate for misalignments caused by breathing and peristalsis motion during this time. The spectral sensitivity of the optical system was corrected for using a white reflectance target (Spectralon, Labsphere, Inc.) before converting the data to absorbance spectra. Figure 1 shows the laparoscope in position during imaging of the rabbit uterus.

Fig. 1

Intraoperative MSI during rabbit UTx. The distance between the laparoscope tip and the organ is $\sim 10\mathrm{cm}$ and the field-of-view of the system is $\sim 6\mathrm{cm}$ in diameter.

Relative concentrations of oxygenated (${\mathrm{HbO}}_2$) and deoxygenated hemoglobin (Hb) were determined using linear least squares regression⁶ of the experimental data to the known pure component spectra.¹⁷ The sum of the concentrations (total hemoglobin; Hbt) was calculated, along with the concentration of ${\mathrm{HbO}}_2$ expressed as a percentage of Hbt (oxygen saturation; ${\mathrm{StO}}_2$). This approach is subject to the assumption that oxy- and deoxyhemoglobin are the only optical absorbers in the field-of-view, and that scattering and penetration depth are approximately constant across the spectrum. Pixel locations with a poor match ($r^2<0.9$) between experimental and model absorbance curves were discarded. All data processing was conducted offline using MATLAB (The Math Works, Inc.).

2.2.

Intraoperative Imaging

Uterine oxygenation was measured during short-term (sheep) and long-term (rabbit) animal UTx viability studies. The sheep ($\sim 60\mathrm{kg}$) study was conducted as an autotransplant under terminal anaesthesia, with the uterus removed, stored externally temporarily, and replaced into the same animal. New Zealand white rabbits ($\sim 4\mathrm{kg}$) were used in a series of allotransplant procedures, where the uterus of a donor animal was transplanted to a recipient animal. The recipient subsequently recovered following surgery.

The transplant procedure involved removal of the uterus and its blood supply en bloc, in a microvascular patch, followed by flushing of the blood vessels with saline and a preservation solution (Custodiol HTK, Dr. Franz Köhler Chemie GmbH, Germany). The organ was stored on ice in a bath of the same preservation solution while the recipient was prepared. During implantation, the cornua and vagina were also attached to provide mechanical stability for the organ. Reconnection of the blood supply (anastomosis) was achieved either through an aorta–aorta/inferior vena cava (IVC)–IVC anastomosis (rabbit) or internal/external iliac anastomosis (sheep), as illustrated in Fig. 2.

Fig. 2

Schematic showing the harvested uterus, its major supplying blood vessels, and the points of anastomosis for the rabbit and sheep transplants. The numbered regions of interest indicate measurement points for oxygen saturation on the medial and lateral parts of the uterine cornua. The photograph on the right shows a rabbit uterus immediately prior to removal from the donor animal. The aorta and inferior vena cava appear below the organ following dissection of the microvascular patch.

Oxygenation measurements were made of the uterus in its native condition, during organ retrieval surgery in the donor, and immediately after implantation and reperfusion in the recipient. In the case of the allotransplant study, the recipient’s own uterus was also measured before a hysterectomy was performed. A pulse oximeter (Datex-Ohmeda 3600P; Datex-Ohmeda, Colorado) was used to measure oxygen saturation and perfusion index (PI) at the medial and lateral aspects of the right and left cornua (Fig. 2). These measurements were followed by MSI acquisitions of the entire uterus. Three MSI measurements were made at each site to reduce the risk of random error. Mean ${\mathrm{StO}}_2$ was calculated in the regions-of-interest within the images that corresponded to the pulse oximeter measurement sites.

3.1.

Sheep Study (Autotransplantation)

Color and processed MSI data from the sheep transplants are shown in Fig. 3. On appearance alone, the uterus, in its native state, appears bright red/pink as it is well-perfused with oxygenated blood. The transplanted uterus (“graft”), while also red, is not as saturated as in the native state. The processed MSI data also support this, with the bright red and yellow shades indicating relatively high oxygenation in both cases. Areas that could not be adequately described by the absorbance model are seen as “gaps” in the processed images. This is particularly evident in regions where specular highlights are present, such as those visible in Fig. 3.

Fig. 3

Complete sheep imaging results showing the uterus in the donor, prior to harvesting, and the transplanted graft. UTx 1 and 4 were abandoned during implantation.

The quantitative results are shown in Fig. 4. Transplant numbers 1 and 4 had to be abandoned due to intraoperative complications.¹¹ The rest were completed as planned.

Fig. 4

Mean cornual ${\mathrm{StO}}_2$, plotted against sheep autotransplant number, for the four regions-of-interest in the donor uterus and transplanted graft. The error bars represent the standard deviation of repeated measures of the same organ. Pulse oximeter measurements (${\mathrm{SpO}}_2$) for the same regions of the organ are plotted alongside the MSI results.

Baseline (donor) arterial oxygen saturation values recorded by the pulse oximeter are consistently high in all animals, varying from 88% to 100% ($\text{mean}=94\%$). Tissue oxygen saturation measured by the MSI device is lower, varying from 41% to 77% ($\text{mean}=66\%$). Intraorgan variability is visible in the mean values in the four ROIs of each case, whereas interorgan variability in ${\mathrm{StO}}_2$ is evident through comparison of the donor and recipient values across all procedures. In the completed procedures (UTx 2, 3, 5), lower oxygen saturation values were recorded in the transplanted graft by both methods, with the exception of the MSI results for UTx 3.

3.2.

Rabbit Study (Allotransplant)

Macroscopically, the appearance of the rabbit uteri, shown in Fig. 5 immediately pre- and posttransplant, is similar to that of the sheep. The native organ in the donor and recipient (prehysterectomy) appears bright and well-perfused, while appearing less-so in the transplanted organ. The quantitative MSI results follow the same trend. The resolution achieved with the MSI laparoscope is $\sim 0.3\mathrm{mm}/\text{pixel}$ at a working distance of 10 cm.

Fig. 5

Complete rabbit MSI results showing images for the donor, the recipient’s native uterus and the transplanted graft. Pixels with $r^2<0.9$ are grayed out.

Quantitative results for the native (donor and recipient) and transplanted uterus are shown in Fig. 6. There were also intraoperative complications in some of the test subjects, and the rabbits in UTx 3, 4, and 6 died during anastomosis of the donor vasculature to that of the recipient.¹² In these cases, no oximetry measurements of the graft are presented.

Fig. 6

Mean cornual oxygen saturation, plotted against transplant number, in the regions-of-interest in the donor animal, the recipient (native uterus), and the transplanted graft. Identification of the ROIs at embryo transfer (UTx 5) was not possible, due to adhesions (see Fig. 7), so mean ${\mathrm{StO}}_2$ for the entire organ is shown instead. Error bars represent the standard deviation of repeated measurements on the same organ. Pulse oximeter measurements for the corresponding regions are included on the same plot.

Baseline oxygen saturation levels in the uterus (both the donors’ and recipients’ native organs) are high in all animals, varying from 87% to 100% ($\text{mean}=98\%$). Similar to the sheep results, inter- and intraorgan variability can be seen in the scatter of ${\mathrm{StO}}_2$ values in each ROI and between different animals. For a particular organ, the transplanted uterus shows a consistently lower ${\mathrm{StO}}_2$ immediately posttransplantation than the native organ across the entire dataset. This is also borne out by the pulse oximeter readings for UTx 5, 7, 8, and 9. The MSI results are lower than the pulse oximeter in every case.

The long-term survivor (UTx 5) was imaged 90 days posttransplant during the surgical transfer of embryos to the transplant recipient’s uterus. Mean ${\mathrm{StO}}_2$ in the organ recovered to $86\%\pm 1\%$ from the immediate postoperative value of 47%, which is also higher than the original baseline reading of $80\%\pm 12\%$ (medial aspect). The color and MSI images of the organ, shown in Fig. 7, indicate the changes that the organ has undergone in the intervening period. A significant number of postoperative adhesions have hidden the uterus’s distinctive shape, but it is clear that perfusion has been fully re-established.

Fig. 7

Long-term survival and embryo transfer. Rabbit test subject imaged 90 days after transplantation, during the embryo transfer procedure. The uterus, highlighted, has lost its distinctive shape due to adhesions. Background tissue has been manually masked out for clarity.