Noninvasive electrophysiological imaging identifies 4D uterine peristalsis patterns in subjects with normal menstrual cycles and patients with endometriosis

Throughout the menstrual cycle, spontaneous mild contractions in the inner layer of the uterine smooth muscle cause uterine peristalsis, which plays a critical role in normal menstruation and fertility. Disruptions in peristalsis patterns may occur in women experiencing subfertility, abnormal uterine bleeding, ovulatory dysfunction, endometriosis, and other disorders. However, current tools to measure uterine peristalsis in humans have limitations that hamper their research or clinical utilities. Here, we describe an electrophysiological imaging system to noninvasively quantify the four-dimensional (4D) electrical activation pattern during human uterine peristalsis with high spatial and temporal resolution and coverage. We longitudinally imaged 4968 uterine peristalses in 17 participants with normal gynecologic anatomy and physiology over 34 hours and 679 peristalses in 5 participants with endometriosis over 12.5 hours throughout the menstrual cycle. Our data provide quantitative evidence that uterine peristalsis changes in frequency, direction, duration, magnitude, and power throughout the menstrual cycle and is disrupted in endometriosis patients. Moreover, our data suggest that disrupted uterine peristalsis contributes to excess retrograde menstruation and infertility in patients with endometriosis and potentially contributes to infertility in this cohort.

Human uterine activity changes dynamically across the menstrual cycle. Menses begins when serum 31 concentrations of the hormones progesterone and estrogen drop, signaling the uterus to shed blood and 32 epithelial cells through the cervix. In the proliferative phase, the uterine epithelium grows in thickness to 33 prepare for potential embryo implantation as a follicle develops on one or both ovaries to release an oocyte. 34 During the peri-ovulatory phase, an oocyte is released and travels down the fallopian tube. If unprotected 35 sexual intercourse occurs during this time, fertilization may occur. During the secretory phase, the uterine 36 epithelium continues to thicken in preparation for potential embryo implantation. 37 Most research on the menstrual cycle has focused on hormones and their effects on the epithelium. 38 However, some evidence indicates that the smooth muscle layer, the myometrium, also contributes to 39 uterine functions by generating slow, low-magnitude, spontaneous contractions, termed uterine peristalsis 40 1-10 . Unlike labor contractions, in which the entire myometrium produces faster and stronger contractions, 41 uterine peristalsis only involves the inner layer of the myometrium, the stratum subvasculare. Uterine 42 peristalsis, first observed on ultrasound 5 , has been shown to vary in direction and frequency throughout the 43 phases of the menstrual cycle 1 . During menses, peristalsis waves travel from the fundus to the cervix and 44 help expel blood and tissue. Conversely, peristalsis waves travel from the cervix toward the fundus during 45 the peri-ovulatory phase and help transport sperm toward the fallopian tubes. 46 Several studies have suggested that uterine peristalsis plays an essential role in uterine pathology. 47 6 and dominant direction ratio (the percentage of Cervix-Fundus peristalsis waves over the percentage of 122 Fundus-Cervix peristalsis waves) (Fig. 4 A-B). We also graphed the average magnitude, duration, and 123 power of peristalsis waves from each participant, with data from the Fundus-Cervix waves plotted 124 separately from the data from Cervix-Fundus waves (Fig. 4 C-H). We observed significant differences in 125 multiple uterine peristalsis indices between healthy participants and those with endometriosis ( Fig. 4 I-X). 126 During the menses phase, peristalsis waves were significantly more likely to be Fundus-Cervix in healthy 127 participants than in those with endometriosis (Fig. 4J). The Fundus-Cervix waves were longer (Fig. 4R) 128 and had a higher magnitude (Fig. 4N) in healthy participants than in those with endometriosis. Conversely, 129 the Cervix-Fundus waves were longer duration (Fig. 4Q) and higher magnitude (Fig. 4M) and power (Fig.  130 3U) in the participants with endometriosis than in the healthy patients. In the peri-ovulatory phase, 131 peristalsis waves were more likely to be Cervix-Fundus in the healthy participants than in the participants 132 with endometriosis (Fig. 4K), and the Cervix-Fundus waves were longer (Fig. 4S) and higher magnitude 133 ( Fig. 4O) and power (Fig. 4W) in the healthy participants than in those with endometriosis. Conversely, 134 the Fundus-Cervix waves in the peri-ovulatory phase were longer duration (Fig. 4T) and higher magnitude 135 ( Fig. 4P) in the participants with endometriosis than in the healthy participants. 136 Peristalsis wave direction during ovulation correlates with dominant follicle laterality 137 Finally, we found that Cervix-Fundus peristalsis waves during the peri-ovulatory phase tend to move 138 preferentially toward one fallopian tube. In nine of the healthy participants and two of the participants with 139 endometriosis, we were able to determine which ovary had a dominant follicle by clinical TVUS and then 140 observe whether the peristalsis propagated in the direction of the dominant follicle. Fig. 5A shows an 141 example of UPI from a healthy participant with a dominant follicle in the right ovary. In this patient, 5 of 8 142 Cervix-Fundus peristalsis episodes moved toward the right ovary. The other 3 waves showed no 143 preferential direction. In the eight healthy participants for whom we had TVUS imaging demonstrating the dominant follicle, 151 peristalsis waves during the ovulatory phase more often moved toward the side with the dominant follicle 152 than toward the side with no dominant follicle. In two participants with endometriosis for whom we had 153 data regarding the dominant follicle, the peristalsis waves during the ovulatory phase more often moved 154 toward the side without the dominant follicle than toward the side with the dominant follicle ( Table 1). 155

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The UPI imaging data presented herein suggest that UPI can provide objective and quantitative measures 157 of uterine peristalsis throughout the human menstrual cycle. Additionally, we developed novel indices to 158 quantitatively characterize uterine peristalsis patterns automatically. Finally, we used UPI to provide 159 evidence that uterine peristalsis patterns differ in women with normal anatomy and menstrual cycles and in 160 women with endometriosis. 161 In the normal participants, the predominant peristalsis pattern in menses was Fundus-Cervix. This pattern 162 has been seen by others and postulated to facilitate the expulsion of blood and endometrial tissue while 163 protecting against ascending pathogens 36 . In the peri-ovulatory phase, the predominant peristalsis pattern 164 Endometriosis has long been hypothesized to be caused by retrograde menstruation 13,41-46 . However, as all 173 reproductive-age women have some amount of retrograde menstruation, it is unclear why only 10-15% of 174 females would develop endometriosis 42,45,47-49 . We found that all healthy participants had at least some 175 Cervix-Fundus peristalses, which could cause retrograde menstruation. Our data suggested that Cervix-176 Fundus peristalsis waves were less frequent and weaker than the Fundus-Cervix waves in subjects without 177 endometriosis. Therefore, the strong and frequent Fundus-Cervix waves may have effectively expelled 178 blood vaginally and left a small amount of blood in the uterine cavity. Although part of the blood could still 179 be transported retrogradely to the peritoneal space by the weak Cervix-Fundus waves, the level may not be 180 sufficient to cause endometriosis in healthy people. On the contrary, in participants with endometriosis, a 181 higher percentage of waves were Cervix-Fundus, and these were stronger and had longer durations than 182 the Cervix-Fundus waves in normal patients. More importantly, in healthy subjects, the Fundus-Cervix 183 peristalsis waves were less frequent and weaker in endometriosis patients than the Fundus-Cervix peristalsis 184 waves, which impair normal expulsion and leave more blood in the uterine cavity. Therefore, retrograde 185 menstruation is more likely to push much more blood and tissue into the peritoneal space in women with 186 endometriosis than in women without endometriosis 8,12,50,51 . Our work suggests that a comprehensive 187 evaluation of 4D uterine peristalsis direction distribution, frequency, magnitude, duration, and power during 188 the menses phase could be used to stratify the risk of developing endometriosis and assess the severity of 189

endometriosis. 190
Our data may also provide clues to infertility in women with endometriosis. In healthy participants during 191 the peri-ovulatory phase, uterine peristalsis waves most frequently traveled Cervix-Fundus, with most 192 peristalsis waves traveling toward the dominant follicle. These patterns could assist sperm in transit to 193 ensure interaction with an oocyte. Conversely, in participants with endometriosis during the peri-ovulatory 194 phase, uterine peristalsis waves most frequently traveled Fundus-Cervix, and those that traveled Cervix-195 9 Fundus traveled toward the ovary without a dominant follicle more often than toward the ovary with a 196 dominant follicle. These patterns could limit the number of spermatozoa that reach the oocyte 20,21,52,53 . 197 The UPI system potentially has a wide range of possible clinical research and therapeutic applications. UPI has several advantages over other modalities used to image uterine peristalsis. First, UPI is noninvasive, 210 which is optimal for long-duration uterine monitoring. Additionally, modalities using invasive monitoring 211 may iatrogenically cause non-physiologic perturbations of peristalsis. Second, UPI provides high spatial-212 temporal resolution information, including the initiation sites, direction, frequency, and duration of uterine 213 peristalsis waves. Third, UPI provides 4D data that considers the individual's unique uterine anatomy in 214 both space and time domains. Fourth, UPI software allows automatic, objective, and real-time 215 electrophysiological quantification of uterine peristalsis. Future work will focus on developing a portable, 216 low-cost, wearable UPI system to enable larger UPI studies. To make UPI more accessible to patients, we 217 will replace the current short anatomical MRI scan with a low-cost ultrasound measurement to generate the 218 patient-specific body-uterus geometry. Wearable, low-cost, printed electrodes 54,55 will also be integrated 219 into the UPI system to minimize the costs. 220 system four times during one menstrual cycle, once during menses, early proliferative, late proliferative 238 (peri-ovulatory), and secretory phases. Blood was collected at each visit to measure concentrations of the 239 hormones estradiol, progesterone, and testosterone to confirm the menstrual phase. 240

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Patients were determined to be in one of four menstrual phases (menses, early proliferative, late 242 proliferative, and secretory) by using a combination of patient-reported bleeding, cycle length, ultrasound 243 findings, ovulation predictor kit (Clearblue, Geneva, Switzerland) results, and hormonal measurements. 244 Serum blood (5-10 ml) was collected and sent to the Core Laboratory for Clinical Studies at Washington 245 University in St. Louis to measure concentrations of the hormones (estradiol, progesterone, and 246 testosterone). The menses phase was assigned when a patient-reported bleeding. The early proliferative 247 phase was assigned after the patient had stopped bleeding, ultrasound demonstrated early follicular activity 248 (largest follicle size <16 mm), serum estradiol <200 pg/ml, and serum progesterone <3 ng/ml. The late 249 proliferative (peri-ovulatory) phase was defined by a positive result on an ovulation predictor kit, serum 250 estradiol >200 pg/ml, serum progesterone <3 ng/ml, and/or a dominant follicle on ultrasound (≥16mm). 251 The secretory phase was assigned when serum progesterone was >3 ng/ml. 252

Uterine peristalsis imaging (UPI) procedure
253 First, a woman underwent a one-time, fast, anatomical (T2W sequence) 3T Siemens Prisma MRI scan (~10 254 mins) to acquire the patient-specific uterus-body surface geometry while wearing up to 8 patches containing 255 up to 128 MRI-compatible fiducial markers around the abdomen and lower back (Fig. 1A). Uterus and 256 body geometry were generated (Fig. 1 B&C). Second, after the MRI scan, customized BioSemi pin-type 257 electrode patches were applied to the same locations on the body surface as the MRI fiducial markers. An 258 ADC box was used to record the body surface electrical signals (Fig. 1D&E) for 20 minutes. The body 259 surface electrical signals were processed with a band-pass filter (0.01-0.1 Hz) 25,34,35 to generate wave 260 electrical signals (peristalsis waves) over the entire abdomen surface (Fig. 1F)

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With the electro-quasi-static assumption of the bioelectric field, the inverse computation combines the 269 patient-specific uterus-abdomen surface and electrical potentials measured on the abdominal surface to 270 reconstruct the potential distribution over the entire 3D uterine surface. We assume that the medium is 271 homogeneous between the uterine surface and abdominal surface without any primary electrical source. UPI data processing 293 The inverse computation described above was employed to compute the uterine surface electrical signals 294 ( Fig. 1 G&H) on the three-dimensional uterine surface. The times when the uterine surface electrical 295 signals at various uterine surface areas reached the steepest negative slope 57-61 were extracted and defined 296 as electrical activation times at those uterine areas during peristalsis waves (red dots in Fig. 1 G&H). 297 During each peristalsis wave, sequential time frames were generated as the activation sequences (Fig. 1I) 298 to reflect the detailed 4D spatial-temporal activation patterns of the uterine peristalsis. Within each time 299 frame, the red region indicated the electrically activated myometrium areas currently experiencing 300 peristalsis, and the blue region indicated the inactive areas of the uterus. The isochrone map was generated 301 as a color-coded 3D map to summarize the electrical activation sequence (Fig. 1J). In the isochrone map, 302 warm and cool colors denote regions of the uterus that activated early and late, respectively, during the 303 peristalsis wave. The UPI isochrone maps contained rich spatial-temporal information of uterine activation, 304 including the activation and termination sites, propagation direction, and duration. In addition, uterine 305 potential maps were generated to reflect the 4D electrical potential distribution during peristalsis waves: 306 1D electrical signals (Fig. 1 G&H) over the entire 3D uterine surface (Fig. 1K). The distributions of uterine 307 peristalsis propagation direction, initiation, and termination sites (Fig. 1L)  (waves/min); (G,H,I) Boxplots of uterine peristalsis duration (downsampled to 1 Hz, seconds), magnitude (mV), and power (mV*sec) for all peristalsis waves in each phase (each dot represents one uterine peristalsis wave). In the UPI activation sequences and isochrone maps, the white asterisks indicate the peristalsis wave initiation sites, and the white arrows indicate the propagation directions. *P <0.05 Figure 3 Uterine peristalsis imaging in one participant with surgically con rmed endometriosis during four phases of the menstrual cycle. (A) Dominant Cervix-Fundus uterine peristalsis pattern during the menses phase; (B) Cervix-Fundus uterine peristalsis pattern during the proliferative phase; (C, D) Fundus-Cervix uterine peristalsis pattern during the (C) peri-ovulatory and (D) secretory phases; (E) Pie charts showing the uterine peristalsis direction distribution in each phase; (F) Bar plot of uterine peristalsis frequency (waves/min); (G,H,I) Boxplots of uterine peristalsis duration (downsampled to 1 Hz, seconds), magnitude (mV) and power (mV*sec) for all peristalsis waves in each phase (each dot represents one uterine peristalsis wave). In the UPI activation sequences and isochrone maps, the white asterisks indicate the peristalsis wave initiation sites, and the white arrows indicate the propagation directions. *P <0.05, **P<0.01  Table 1 Supplementary Files This is a list of supplementary les associated with this preprint. Click to download.