Prolonged Cold Ischemia Did Not Impair Mitochondrial Oxygen Consumption or Reactive Oxygen Species Production in Human Uterine Fundus and Horn Myometrium

Uterine transplantation may be a solution for infertility of uterine origin. Nevertheless, only three pregnancies with a live birth have so far been possible involving a uterine transplant from a brain-dead donor. Particularly, the impact of ischemia needs a better understanding. Analysis of mitochondrial respiration and production of reactive oxygen species (ROS) in muscle are of interest since they are pertinent markers of the harmful effects of ischemia. We therefore studied both uterine fundus and horn muscle mitochondrial use of oxygen and ROS production in eight women needing hysterectomy. High resolution respirometry and electron paramagnetic resonance allowed the determination of, respectively, myometrium oxidative capacity, hydrogen peroxide, mitochondrial free radical leak and superoxide anion production early (2 and 7 h) and late (24 h) following surgery. Mitochondrial oxygen consumption of the uterine fundus and horn tended to decrease with time but this was not statistically significant. Concerning ROS production, globally, we observed no significant change for H2O2, superoxide anion and free radical leak. In conclusion, a long period of cold ischemia did not impair myometrium mitochondrial respiration, only generating a transient H2O2 increase in uterine fundus. These data support that cold ischemia, even when prolonged, does not significantly alter uterine muscle oxidative capacity.


Introduction
Almost 200 women of reproductive age per million are suffering from uterine infertility that need uterine transplantation to allow pregnancy [1].
This clearly raises the issue uterine viability when subjected to long-lasting ischemia.
Although they are placed in a hypothermic preservation medium, all organs can be significantly impaired by cold ischemia depending on its duration. In fact, organ characteristics modulate the tolerance to cold ischemia. Experimental and human studies investigating the uterine tolerance to cold ischemia demonstrated a lack of significant change in markers of necrosis and apoptosis till 6 and 24 h after cold ischemia [21][22][23].
Interestingly, studies of ischemia reperfusion performed upon skeletal muscle showed an early mitochondrial dysfunction suggesting that such a parameter might also demonstrate an eventual ischemia-induced uterine alteration [24,25].
Accordingly, mitochondrion is an important organelle producing energy and its dysfunction causes depletion in ATP and increased reactive oxygen species (ROS) production, particularly in the case of ischemia reperfusion [26]. Oxygen consumption by mitochondria is therefore widely used as a biological marker of its functionality.
Currently, little is known about the fate of mitochondria in the human uterus during transplantation and the purpose of this study was to determine the kinetics of mitochondrial respiration and of ROS production (2, 7 and 24 h after surgery) in two different segments of uterine myometrium subjected to cold ischemia as potentially observed during the clinical setting. Indeed, in skeletal muscles, the sensitivity to ischemia depend on muscles characteristics [27] and it appears to interestingly challenge the hypothesis of a differential effect of ischemia on either uterine fundus or horn, localizations often involved in embryo implantation.

Population and Methods
Eight women needing hysterectomy gave their informed consent and participated in the study carried out in the university teaching hospital of Strasbourg CHU and approved by the French Committee for the Protection of Persons (CPP) and registered on clinicaltrials. gov (RIN 2018-HUS N 7222).

Study Design
Inclusion criteria were patient >18 years old, affiliated to a social security regime and undergoing scheduled hysterectomy for an ostensibly benign pathology.
Tissue samples were provided by the surgeon immediately following surgical removal of the uterus during a total hysterectomy procedure conducted vaginally, laparoscopically or by laparotomy in the gynecological surgical service at the Strasbourg University Hospitals, France. The exact time at which the second uterine artery was ligated was recorded (ischemia T0). The surgeon cut the tissue directly post-operatively, with a cold scalpel to remove two samples of one cm 3 myometrium per uterus, one at the fundal midline and the other at the right uterine horn (Figure 1). transplantation and the purpose of this study was to dete chondrial respiration and of ROS production (2, 7 and 24 h different segments of uterine myometrium subjected to cold served during the clinical setting. Indeed, in skeletal muscle depend on muscles characteristics [27] and it appears to int pothesis of a differential effect of ischemia on either uterine f often involved in embryo implantation.

Population and Methods
Eight women needing hysterectomy gave their informed the study carried out in the university teaching hospital o proved by the French Committee for the Protection of Perso clinicaltrials.gov (RIN 2018-HUS N 7222).

Study Design
Inclusion criteria were patient >18 years old, affiliated to undergoing scheduled hysterectomy for an ostensibly benign Tissue samples were provided by the surgeon immed moval of the uterus during a total hysterectomy procedure c scopically or by laparotomy in the gynecological surgical se versity Hospitals, France. The exact time at which the secon was recorded (ischemia T0). The surgeon cut the tissue dire cold scalpel to remove two samples of one cm 3 myometrium midline and the other at the right uterine horn ( Figure 1). These sites were readily identifiable and therefore repr to myometrium-rich areas where embryo implantation ch These sites were readily identifiable and therefore reproducible, and corresponded to myometrium-rich areas where embryo implantation characteristically takes place. Samples were dissected and consisted only of myometrium, without mucosa or serosa, and any macroscopically pathological myometrium (myoma, adenomyosis) was avoided.
Three samples per tissue were obtained at three different times: 2, 7, and 24 h after cold ischemia.
Thus, glutamate (10 mM) and malate (2.5 mM) were added to support electron flow through complex I (CI) of the electron transport system (ETS). Then, the addition of ADP (2 mM) stimulated respiration and oxidative phosphorylation (OXPHOS by CI). Finally, succinate was added (25 mM) to activate the complex II (OXPHOS by CI&II). DatLab software 4.3 was used to determine the oxygen flow (Oroboros Instruments, Innsbruck, Austria). Data are expressed in pmol O 2 /s/mg wet weight.

Reactive Oxygen Species Production
Reactive Oxygen Species (ROS) comprise several moieties such as the superoxide anion (O 2 •− ) or hydrogen peroxide (H 2 O 2 ). Hydrogen peroxide production was measured using combined AmplexRed and horseradish peroxidase (HRP). AmplexRed reacts with H 2 O 2 (1:1) and is catalyzed by HRP into resorufin, a fluorescent molecule. Resorufin is detected at 563/587nm. Fluorescence was measured simultaneously with mitochondrial respiration on the high-resolution oxygraph. AmplexRed (20 µM) and HRP (1 U/mL) were thus added into the Oroboros chambers at the beginning of the experiment. Results are expressed in pmol/min/mg wet weight.

Free Radical Leak
Hydrogen peroxide and O 2 consumption were measured simultaneously. This allowed the calculation of the fraction of out-of-sequence electrons serving to oxidize O 2 into ROS in the respiratory chain. The Free Radical Leak (FRL) is therefore the rate of H 2 O 2 production divided by twice the rate of O 2 consumption, expressed as a percentage.

Statistical Analysis
All data are expressed as mean ± standard error of the mean (SEM) and were analyzed using Prism software (GraphPad Prism 5, Graph Pad Software, San Diego, CA, USA).We tested normality for all groups, based on the Shapiro-Wilk test. When normality was not observed, the non-parametric test for repeated values (Friedman test) was applied followed by a posttest (Dunn's multiple tests). A p value < 0.05 was considered significant.

Clinical Characteristics of the Patients
The clinical characteristics of the eight women participating in the study are summarized in Table 1. The mean patient age was 47.6 years, and mean gravidity 2.4. Two women were treated for hypertension and one was smoker. The indications for hysterectomy were myomatous uterus causing symptoms, symptomatic adenomyosis, and treatment of a prolapse in, respectively, four, three and one patients. A sarcoma was discovered on histological examination in one patient. No patient was menopaused.

Discussion
This study is the first to examine the kinetic of myometrial mitochondrial respiration and reactive oxygen production at both uterine fundus and horn, two, seven and twenty-four h after cold ischemia (CI). The main results are that mitochondrial oxidative capacity remains preserved over a long period, together with a lack of major rise in ROS production. These data support that cold ischemia is relatively well tolerated by human uterine myometrium.
Ischemia is a complex pathophysiological process leading progressively to impairments in the mitochondrial respiratory chain, intracellular edema, acidosis, oxidative stress, and finally cell death. Often studied in many organs such as the heart, brain, kidney, lungs, skeletal muscle etc. [27,[30][31][32][33], there are relatively few data investigating uterine muscle ischemia-induced change in mitochondrial function [34]. Accordingly, mainly other methods were used to analyze uterine ischemia effects [35]. However, since to date, no transplant has produced a viable pregnancy after more than 6 h of cold ischemia, there is still an urgent need to better understand CI effect on uterine muscle, and particularly on mitochondria that are largely involved in many key cellular mechanisms.
Interestingly, our results demonstrate that cold ischemia (better corresponding to the clinical setting than warm ischemia) did not significantly alter uterine myometrium mitochondrial respiration, whatever the site analyzed. Thus, although porcine uterine regions might have different sensitivities to hypoxia [36], both human uterine fundus and horn remained unaltered by short term and prolonged CI supporting that if present, an eventual mitochondrial dysfunction should have been minimal. In agreement with these data, Del Priore et al. did not observe any histological modification as assessed by electron microscopy after 12 h of cold ischemia [37]. Similarly, no histological modification or increased apoptosis, as assessed by TUNEL assay, was shown after 24 h of CI [21]. In 2005, Wranning et al. studied changes in energy metabolism (ATP) and in the capacity of human uterus to contract after various lengths of CI [38]. They observed a decrease in the spontaneous contractility, irrespective of the duration of ischemia. No histological modification was observed on electron microscopy, but degeneration of the vesicular cytoplasm and chromatin condensation were observed after 24 h of CI mainly when a protective solution was not used. The authors concluded that that human myometrial tissue can tolerate cold ischaemia for at least 6 h, but mitochondrial respiration determination was not performed. Further, the fact that swollen mitochondria were observed after 8 h of warm uterine ischemia [34] suggests that warm ischemia is likely to be much more harmful than cold ischemia which reduces tissue metabolism.
Oxidative stress is also a key mechanism in ischemia pathophysiology [38]. When investigating glutathione pathway in uterine myometrium in humans or ewes, respectively, an increased oxidative stress was observed mainly after 24 h of CI [39,40]. On the other hand, in our study, despite using three complementary approaches as often suggested, we observed no significant increase in hydrogen peroxide, superoxide anion productions or mitochondrial free radical leak during this experiment except at 7 h in uterine fundus for H 2 O 2 . However, such an increase was transient and concerned only one marker of oxidative stress. Therefore, the general picture is that no major change in ROS production occurred in this experiment, although further studies will be useful to determine the antioxidant status in such a setting. This is in agreement with the lack of mitochondrial dysfunction observed since mitochondria are known to be also largely involved in ROS production, particularly in the ones we studied here. Globally, these findings confirm that uterine muscle has a marked ability to tolerate cold ischemia well, corresponding to its physiological exposure to ischemia, particularly during menstrual cycles and labor.

Conclusions
In conclusion, although mitochondrial respiration tended to decrease over time, this was not statistically significant and this study shows that uterus mitochondria are relatively resistant to long periods of ischemia. We nevertheless cannot totally exclude evidence of other mitochondrial dysfunction potentially involving mitochondrial dynamic, calcium handling etc. Further, no major increase in ROS production was observed until 24h after surgery. Thus, other predictive markers of uterine function should be investigated, probably including studies on metabolomics or progenitor stem cells of the endometrium which play an essential role in physiological remodeling and regeneration of the human uterus and might be impaired by prolonged ischemia. Additionally, reperfusion studies will be useful since it is a critical time, allowing increased oxidative stress related to oxygen reflow. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.