{"paper_id":"fe578b72-5bb9-4e3c-9f0e-9c03c30aa0d2","body_text":"ORIGINAL ARTICLE\nCharacterization of Mechanical Signature of Eutopic Endometrial\nStromal Cells of Endometriosis Patients\nAhmad Altayyeb1 & Essam Othman2,3 & Maha Khashbah2 & Abdelhady Esmaeel4 & Mohamed El-Mokhtar5 &\nCornelis Lambalk3 & Velja Mijatovic3 & Mohamed Abdelgawad4,6\nReceived: 14 December 2018 / Accepted: 10 April 2019 / Published online: 6 January 2020\n# Society for Reproductive Investigation 2019\nAbstract\nEndometriosis affects 5–10% of women in reproductive age and causes pelvic pain and subfertility. Exact etiology of the disease\nis unknown. Here, we present a microfluidic platform for characterizing mechanical properties of eutopic endometrial stromal\ncells of endometriosis patients based on cellular deformability inside narrow microchannels. Primary human endometrial stromal\ncells were isolated from eutopic endometrium of endometriosis patients (4407 cells, from 7 endometriosis patients) and from\ndisease-free women (4541 cells, from 6 control women) and were pumped through microchannels (formed of polydimethylsi-\nloxane (PDMS) by standard soft lithography, with dimensions of 8 × 20 × 150μm, as width × height × length) at a constant flow\nrate of 2 μL/min. High-speed imaging was used to capture videos of cells as they flow inside microchannels, and a computer\nvision code was used to track cells, measure their area, and calculate the time each cell takes to pass through the microchannel.\nCompared with their counterparts from control women, eutopic endometrial stromal cells from endometriosis patients showed\nsignificantly increased deformation index (1.65 ± 0.2 versus 1.43 ± 0.19, respectively, P value < 0.001), and higher velocity in\ntravelling through narrow microchannels (96.530 ± 0.710 mm/s versus 57.518 ± 0.585 mm/s, respectively,P value < 0.001). The\nsame difference in velocities between the two cell types was maintained after controlling for cell area. Eutopic endometrial\nstromal cells of endometriosis patients showed a mechanical phenotype characterized by high deformability and reduced stiff-\nness. This mechanical signature can represent basis of a mechanical biomarker of endometriosis.\nKeywords Mechanical stiffness . Endometrium . Stromal cells . Endometriosis . Microfluidics\nBackground\nEndometriosis is an estrogen-dependent disorder character-\nized by the presence of endometrial glands and stroma outside\nthe uterine cavity [ 1]. It affects around 5 –10% of women in\ntheir reproductive years, and up to 50% of women with chron-\nic pelvic pain and/or infertility [2]. Treatment is either medical\nor surgical, and symptoms often recur after treatment discon-\ntinuation [ 3]. Severity of the symptoms, expenses and side\neffects of medications, and the need for multiple surgeries all\nreduce the quality of life of affected women [4]. Adding to the\nsuffering of endometriosis patients are reports of increased\nrisk of ovarian cancer in these patients [ 5].\nThe gold standard of endometriosis diagnosis is surgery\nthrough direct laparoscopic visualization of lesions [ 6].\nLaparoscopy is invasive and requires anesthesia. It is not with-\nout risks including injury to a viscus, hemorrhage, or infec-\ntion. Moreover, laparoscopy is expensive and depends on the\nlevel of training of the surgeon [ 7]. Because of these\nElectronic supplementary material The online version of this article\n(https://doi.org/10.1007/s43032-019-00042-3) contains supplementary\nmaterial, which is available to authorized users.\n* Essam Othman\nessamrash@yahoo.com\n* Mohamed Abdelgawad\nm.abdelgawad@aun.edu.eg\n1 Center for Nanotechnology, Zewail City of Science and Technology,\nGiza, Egypt\n2 Department of Obstetrics and Gynecology, Assiut University,\nAssiut, Egypt\n3 Amsterdam University Medical Center, Location VUmc, Academic\nEndometriosis Center, Amsterdam, The Netherlands\n4 Mechanical Engineering department, Assiut University,\nAssiut, Egypt\n5 Department of Medical Microbiology and Immunology, Assiut\nUniversity, Assiut, Egypt\n6 Mechanical Engineering Department, American University of\nSharjah, Sharjah, UAE\nReproductive Sciences (2020) 27:364–374\nhttps://doi.org/10.1007/s43032-019-00042-3\n\ndrawbacks, endometriosis patients suffer for 8–11 years from\nendometriosis-associated symptoms before a definitive diag-\nnosis is made [ 8].\nA great deal of the puzzling nature of endometriosis stems\nfrom the fact that its etiology is not exactly known [ 9].\nDescribed for the first time in 1927, the retrograde menstrua-\ntion theory of Sampson is a hypothesis that stood the test of\ntime. It states that during menstruation, some viable endome-\ntrial cells flow in a retrograde manner, against current, through\nthe Fallopian tubes to reach the peritoneal cavity [10]. It is the\nmost accepted theory by researchers to explain the develop-\nment of endometriosis. However, because retrograde menstru-\nation occurs in most cycling women with patent Fallopian\ntubes, other permissive factors must be playing in the back-\nground to determine why a particular subgroup, not all, of\nwomen develops endometriosis [11].\nDespite being a benign disorder, endometriosis has some\ncommon features with cancer [ 12, 13]. Endometriosis cells\nshow high proliferation, enhanced survival, and diminished\napoptosis [14], and the disease often recurs after surgical re-\nmoval [ 15]. Moreover, endometriosis cells may disseminate\nvia blood or lymphatic streams to initiate lesions in distant\nlocations [ 16]. In addition, deep infiltrating endometriosis\nhas been shown to harbor cancer-driver mutations [ 17]. Such\nsimilarities provoked investigators to look into some patho-\nphysiologic mechanisms in cancer to see if they are operating\nin endometriosis as well.\nAs cells change from benign to malignant nature, they un-\ndergo several changes at the level of their cytoskeleton [ 18].\nThis renders malignant cells several folds more elastic than\ntheir benign counterparts [19]. Being softer and more deform-\nable, metastatic cancer cells can penetrate through the colla-\ngen cross-linked fibers of damaged extracellular matrix at\ntheir primary site and squeeze themselves through narrow\nspaces between endothelial cells lining blood vessels to enter\ninto the circulation. In addition, the high elasticity of malig-\nnant cells enables them to tolerate mechanical shears imposed\non them by the circulation which is a main defense mechanism\nof the body against cancer metastasis [20]. Similarly, in endo-\nmetriosis, endometrial cells leave their location in the uterus,\nflow against the current in the Fallopian tubes, and penetrate\nthrough damaged extracellular matrix of the peritoneal sur-\nfaces where they implant. They can also squeeze themselves\nbetween endothelial cells lining blood or lymphatic capillaries\nto get implanted in faraway organs such as the lungs [ 16].\nTo this end, we hypothesize that eutopic endometrial stro-\nmal cells of endometriosis patients show lower mechanical\nstiffness and higher elasticity/deformability, contributing to\ntheir migratory and invasive nature, compared with their\ncounterparts from disease-free women.\nMicrofluidics emerged recently as a new high throughput\ntool for mechanical characterization of cells. Due to its\nmatching size scale, microfluidics offers complete control over\nthe ex vivo cellular environmentincluding the ability to induce\nmechanical stresses. Consequently, many microfluidics-based\ntechniques were developed in the last decade to measure me-\nchanical stiffness of cells. These include wall-induced forces\n[21], shear stress–induced forces [22], hydrodynamic stretching\n[23], and optical stretchers [24].\nHere, we propose a microfluidics-based platform for iden-\ntifying the mechanical properties of eutopic endometrial stro-\nmal cells of endometriosis patients. Our platform is based on\nwall-induced forces technique [21] in which cells are forced to\nflow inside a narrow microchannel with channel width smaller\nthan cell size. Soft cells can deform easily and squeeze\nthrough this narrow microchannel, whereas stiff cells cannot\neasily deform and take longer time to flow through the same\nmicrochannel. Our findings have shown that cells from endo-\nmetriosis patients could easily deform and flow inside the\nnarrow microchannel at much higher velocities compared\nwith cells from healthy women.\nMethods\nHuman Endometrial Sample Collection\nWe obtained endometrial tissue biopsies from 7 endometriosis\npatients and 6 control women. Endometriosis was diagnosed\nduring laparoscopy performed to diagnose the cause of pelvic\npain and/or infertility experienced by these women. The con-\ntrol group consisted of women whose laparoscopic examina-\ntion revealed no abnormalities. Endometrial biopsies were ob-\ntained via curettage during the time of laparoscopic surgery in\none session for the convenience of the patient and the research\nteam. The same endometrial biopsy can be taken as an outpa-\ntient procedure using a pipelle device in office setting, without\nanesthesia.\nAll laparoscopic surgeries were performed in the prolifer-\native phase of the menstrual cycle. Menstrual dates were\nassessed based on patient’s menstrual history and histological\nexamination of endometrial biopsy. Women were excluded\nfrom the study if they had irregular menstrual cycles, had\nany pelvic pathology other th an endometriosis (such as\nadenomyosis, fibroids, non-endometriotic adhesions, etc.), re-\nceived hormonal treatment, or were pregnant in the last\n3 months before surgery. All participating women provided\nwritten informed consent. Institutional Review Board at\nFaculty of Medicine, Assiut University, approved the use of\nhuman endometrial tissue samples for this study.\nEndometrial Stromal Cell Culture\nEndometrial stromal cell cultures were established from\neutopic endometrial biopsies of endometriosis patients and\ncontrol women as described before [ 25]. Briefly, endometrial\nReprod. Sci. (2020) 27:364–374 365\n\nbiopsies were sent immediately to the lab where they were\nwashed several times with phosphate buffered saline (PBS)\nto remove blood clots. Endometrial tissues were minced into\n1–2-mm pieces using small scalpel before they were enzymat-\nically digested. Digestion was done in 10 ml of PBS contain-\ning 0.1% collagenase type I and continued for 2 h in a shaking\nwater bath at 37 °C. Endometrial stromal cells were separated\nthrough filtration using 40 and 20 μms i e v e s .T h ef i l t r a t ew a s\ncentrifuged at 1200 rpm at room temperature for 10 min, and\nsupernatant discarded. The cellular pellet was dissolved in\n10 ml of DMEM-F12 supplemented with 10% fetal bovine\nserum (FBS) and cultured in cell culture incubator at 37 °C\nand 5% CO\n2 until confluence. At the time of microfluidics\nexperiment, cells were harvested with 0.05% trypsin-EDTA\nand washed with PBS, and cell suspension was loaded to the\nsystem.\nCharacterization of Endometrial Stromal Cells\nCells were stained with anti-human CD90 FITC –conjugated\nantibodies (Novus Biologicals; Centennial, CO, USA, cata-\nlogue# NBP1 –96125), anti-human Vimentin Alexa Fluor®\n488–conjugated monoclonal antibodies (R&D Systems;\nMinneapolis, MN, USA, cat alogue# IC2105G) or anti-\ncytokeratin (ThermoFisher; Waltham, MA, USA,\ncatalogue#MA5 –18158) Alexa Fluor® 488 –conjugated\nmonoclonal antibodies following fixation, permeabilization\nand intracellular staining against isotype-matched controls.\nCells were acquired using the FACSCalibur flow cytometer\nand the Cell Quest Pro software. The acquired data were an-\nalyzed using FlowJo software.\nMicrochannel Fabrication\nStandard soft lithography technique was used to fabricate the\nmicrofluidic devices [ 26]. A 20- μm-thick layer of negative\nphotoresist (SU-8-2010) was deposited on a clean, dry silicon\nwafer by spin-coating (1000 rpm, 1 min) followed by soft\nbake (65 °C for 2 min followed by 5 min at 95 °C).\nMicrochannel design was patterned using UV micro-pattern\ngenerator ( μPG101, Heidelberg Instruments, Heidelberg,\nGermany) at 60% of the power of the 70 mW UV diode.\nThe SU-8 layer was baked again (95 °C, 12 min) then devel-\noped for 6 min using diacetone alcohol (Sigma, Cairo, Egypt).\nMechanical properties of the SU-8 layer were improved by\nhard baking at 200 °C for 30 min. Polydimethylsiloxane\n(PDMS) was prepared by mixing prepolymer base and curing\nagent at 10:1 ratio by weight. Negative replica of the master\nwas created by casting in PDMS. The PDMS was cured at\n100 °C for 45 min then it was peeled off; the master and holes\nwere punched for inlets and outlets. PDMS slabs were bonded\nto clean microscope slides after treating it with a portable\ncorona treater (Electro-Technic Products, Chicago, IL) for\n2 min, as described elsewhere [ 27]. The final fabricated\nmicrochannel had constrictions of the following dimensions:\n8 μm×2 0 μm × 150 μm (width × height × length).\nExperimental Setup\nThe experimental setup used for characterizing the mechanical\nproperties of endometrial ce lls is shown schematically in\nFig. 1. The platform was placed on the stage of an inverted\nmicroscope (Olympus CKX53, Shinjuku, Tokyo, Japan),\nwhich was working in phase contrast mode with × 40 objec-\ntive. We typically started each experiment with 0.5 ml of cell\nsuspension at a density of 7 × 10 5 cells/ml. A syringe pump\n(NE 4000, New Era Pump Systems, Farmingdale, NY , USA)\nwas used to pump the cell suspension into the microchannel at\naf l o wr a t eo f2 μL/min. A high-speed camera (Basler\nACA2000-340 km, Basler, Ahrensburg, Germany) was used\nto capture images of the flowing cells inside the constriction\nmicrochannel. Images were acquired using software built on\nLabVIEW (National Instruments, Austin, TX, USA). The\nframe rate of all recorded videos was around 3000~4000\nframe per second. These images were transferred to a comput-\ner to be analyzed offline using computer vision.\nImage Analysis\nWe used an in-house built computer vision code based on\nLabVIEW software [ 28], to analyze the recorded videos of\ncells flowing inside the microchannel. The computer vision\ncode detects and tracks the cell once it enters the constriction\nand calculates its projected area which was used as an indica-\ntion of cell size. The real-time velocity of cells inside the\nconstriction was calculated by measuring the distance be-\ntween the center of mass of the cell in two successive frames\nas in Eq. 1.\nVelocity ¼\ntraveled distance in two successive frames\nframe time ð1Þ\nThe average cell velocity inside the microchannel was cal-\nculated by averaging the real-time velocities all over the con-\nstriction length. Visual inspection was used to confirm that\neach cell flowed nonstop inside the microchannel and kept\ncontinuous contact with microchannel walls. Any cell that\ndid not satisfy these two conditions, due to channel blockage\nor being small in size, was excluded from data analysis.\nChoice of the proper width of the microchannel was important\nto enable testing the maximum number of cells in light of the\nabove two criteria. We tested different channel widths (7, 8,\n10, and 12 μm) before we decided to use the reported width of\n8 μm. Channels with smaller width got blocked frequently,\nwhich affected the throughput of the experiments. Whereas\nfor 10 and 12 μm channels, some cells passed the channel\n366 Reprod. Sci. (2020) 27:364–374\n\nwithout touching channel walls or without deforming (diam-\neter of endometrial stromal cells ranges between 8 and\n30 μm). Also, data of multiple cells flowing through the chan-\nnel together were excluded and not used in data analysis.\nStatistical Analysis\nStatistical analysis was done using Statistical Package of\nSocial Scientists (SPSS), version 20 (Chicago, IL, USA).\nData analysis was done using parametric statistics. Groups\nwere compared using the independent sample Student’s t test.\nV elocity of eutopic endometrial stromal cells from endometri-\nosis patients and control women was expressed as mean ±\nstandard error of the mean (SEM). Statistical significance\nwas reached if P <0 . 0 5 .\nResults\nClinical Characteristics of Study Subjects\nThese are summarized in Table 1.\nCharacterization of Endometrial Stromal Cells\nOur endometrial stromal cells were more than 95% positive\nfor anti-human Vimentin and anti-human CD90 and were\nnegative for anti-cytokeratin (Fig. 2), verifying their identity\nas endometrial stromal cells.\nPhases of Endometrial Stromal Cellular Passage\nThrough Microchannels\nWhile flowing inside the microchannel, eutopic endometrial\nstromal cells from endometriosis patients showed a different\nbehavior compared with their counterparts from healthy wom-\nen. Cell passage through the microchannel can be divided into\ntwo phases: transient and equilibrium. In the transient phase,\nthe cell deforms gradually from spherical shape to a plug-like\nshape to fit inside the microchannel. In the equilibrium phase,\nthe cell stops deforming and flows inside the microchannel\nwith almost a constant velocity (i.e., linear profile of distance\nversus time). Eutopic endometrial stromal cells from endome-\ntriosis patients took less time to deform in the transient phase\nand also passed through the microchannel in a shorter total\ntime (as shown Fig. 3), indicating that they may be less stiff\nthan cells from healthy women.\nDeformability of Eutopic Endometrial Stromal Cells\nas They Travel Through Microchannels\nDeformation index of cells, which is defined as the ratio\nbetween cell length inside the microchannel divided by its\noriginal diameter, was found to be higher in cells from\nendometriosis patients than that from control women\n(Fig. 4). Analysis of 234 cells from patients and controls\nshowed that endometrial cells f rom endometriosis patients\nhad a significantly higher deformation index of 1.65 ± 0.2\n(mean ± SD) compared with a deformation index of 1.43 ±\n0.19 (mean ± SD) for cells from healthy women ( P value <\n0.001, Table 2).\nFig. 1 Schematic of the time of\nflight principle for differentiating\nbetween cells based on its\ndeformability. A high-speed\ncamera (HSC) was used to\ncapture images of cells as they\nflow through the region of interest\n(ROI) and the velocity of each cell\ninside the microchannel was\ncalculated\nReprod. Sci. (2020) 27:364–374 367\n\nVelocity of Eutopic Endometrial Stromal Cells Derived\nfrom Endometriosis Patients and Control Women\nas a Surrogate of Their Cellular Stiffness\nWe assessed the velocity of 4407 individual eutopic endome-\ntrial stromal cells derived from seven endometriosis patients\nand 4541 cells derived from six control women inside our\nmicrochannel system. As shown in Fig. 5, the mean velocity\nof eutopic endometrial stromal cells derived from all endome-\ntriosis patients (96.530 ± 0.710 mm/s) is significantly higher\nthan that of their counterparts derived from control women\n(57.518 ± 0.585 mm/s); P value is < 0.001. Supplementary\ntable 1 shows velocities of eutopic endometrial stromal cells\nderived from individual endometriosis cases and control\nwomen. Supplementary video S1 shows flow of eutopic en-\ndometrial stromal cells from a control woman inside the\nmicrochannel constriction. Supplementary video S2 is the\nsame as video S1 after being processed using the machine\nvision program which shows velocity of each cell while\nflowing inside the microchannel. Supplementary videos S3\nand S4 are similar videos but for eutopic endometrial stromal\ncells from an endometriosis patient.\nTable 1 Clinical characteristics\nof study subject Case/control Age Indication of surgery Operative findings\nEndometriosis case# 1 30 years Secondary infertility Peritoneal endometriotic spots\nEndometriosis case# 2 31 years Secondary infertility Ovarian endometriotic cyst\nEndometriosis case# 3 29 years Primary infertility Ovarian endometriotic cyst\nEndometriosis case# 4 26 years Primary infertility Ovarian endometriotic cyst\nEndometriosis case# 5 25 years Chronic pelvic pain Ovarian endometriotic cyst\nEndometriosis case# 6 30 years Primary infertility Bilateral ovarian endometriotic cysts\nEndometriosis case# 7 32 years Chronic pelvic pain Peritoneal endometriotic spots\nControl# 1 30 years Primary infertility Normal laparoscopic findings\nControl# 2 36 years Secondary infertility Normal laparoscopic findings\nControl# 3 34 years Primary infertility Normal laparoscopic findings\nControl# 4 32 years Secondary infertility Normal laparoscopic findings\nControl# 5 32 years Primary infertility Normal laparoscopic findings\nControl# 6 25 years Secondary infertility Normal laparoscopic findings\nFig. 2 Characterization of eutopic endometrial stromal cells. Flow\ncytometry analysis after staining with a Vimentin Alexa Fluor® 488 –\nconjugated monoclonal antibodies, b CD90 FITC –conjugated\nantibodies, and c anti-cytokeratin Alexa Fluor® 488 –conjugated\nmonoclonal antibodies . Shaded histograms were stained with\nrepresentative isotype-matched control antibodies\n368 Reprod. Sci. (2020) 27:364–374\n\nDistribution of Velocities of Eutopic Endometrial\nStromal Cells Inside Microchannels\nWhen a histogram is plotted for the distribution of velocities\nof eutopic endometrial stromal cells from endometriosis pa-\ntients and control women inside the microchannels, cells from\nendometriosis patients show more or less a normal distribution\ncurve (skewness is 0.35), whereas cells from control women\nFig. 3 I Series of pictures\nshowing an endometrial cell\nwhile deforming to squeeze\nthrough the narrow microchannel.\nII Series of pictures of a cell\npassing through a microchannel\n(8 μmW×2 0 μmH×\n150 μm L). The machine vision\ncode we developed captured the\ncell once it enters the\nmicrochannel and calculated its\narea and the time it took to pass\nthrough the microchannel. III\nDisplacement vs. time curve for\ntwo cells imposed on the same\nfigure, one cell from a healthy\nwoman (cell area = 254μm\n2), and\none cell from an endometriosis\npatient (cell area = 242 μm2). The\ncell from the endometriosis\npatient passed through the\nchannel in a much shorter time\n(i.e., with higher velocity)\nFig. 4 S c a t t e rp l o to fd e f o r m a t i o nindex (cell length inside the\nmicrochannel divided by its original diameter) of cells from healthy\nwomen and endometriosis patients. Figure is based on analysis of 125\ncells from endometriosis patients and 109 cells from healthy women.\nEndometrial cells from endometriosis patients had a significantly higher\ndeformation index of 1.65 ± 0.2 (mean ± SD) compared with a\ndeformation index of 1.43 ± 0.19 (mean ± SD) for cells from control\nwomen\nTable 2 Average deformation index of eutopic endometrial stromal\ncells of endometriosis patients and control cells. Data expressed as\nmean ± standard deviation\nEndometriosis\ncells (n =1 2 4 )\nControl women cells\n(n = 110)\nP value\nDeformation\nindex (DI)\n1.65 ± 0.2 1.43 ± 0.19 P value\n<0 . 0 0 1\nReprod. Sci. (2020) 27:364–374 369\n\nshow tendency of skewness to the right (skewness of 1.06), as\nseen in Fig. 6.\nCellular Velocity Versus Cell Size Inside Microchannels\nCell velocity inside the microchannel depends on cell size in\naddition to cellular stiffness. A stiffer cell will impose larger\nforces on channel walls when it deforms inside it resulting in\nhigher friction and longer passage time. Same applies for larger\ncells that may be less stiff but can still apply high forces on\nchannel walls because of the higher deformation it experiences\nto pass through the narrow microchannel. Consequently, cell size\nhad to be included as another parameter when comparing cell\nvelocity from patients and control women. Therefore, we\npresented data in the form of a heat map where cell size is plotted\non the x-axis and cell velocity is plotted on the y-axis (Fig. 7).\nEach point in the heat map represents data from one cell. When\nmany points coincide on top of each other, they are assigned a\ndifferent color. As can be seen from Fig.7, larger cells from both\npatients and controls do take longer time to pass through the\nchannel. Moreover, it is also clear that eutopic endometrial stro-\nmal cells from endometriosis patients exhibit higher velocities\nwhen passing through the microchannel compared with cells of\nthe same size from control women which is a reflection of the\nlower stiffness of cells from endometriosis patients. The data\ns h o w ni nF i g .7 is the result of analyzing 8948 cells from 7\nendometriosis patients and 6 control cases. The heat map of cells\nfrom each individual endometriosis patient and control woman is\nincluded insupplementary information.\nDiscussion\nIn the present study, we developed a high throughput\nmicrofluidics platform to characterize the mechanical signa-\nture of eutopic endometrial stromal cells of endometriosis pa-\ntients based on cellular deformability. Our results have shown\nthat eutopic endometrial stromal cells of endometriosis pa-\ntients are less stiff, more deformable, and exhibit higher ve-\nlocities in traversing narrow microchannels than their counter-\nparts from endometriosis-free women.\nTo the best of our knowledge, this is the first study to\ninvestigate the mechanical stiffness of eutopic endometrial\nstromal cells of endometriosis patients. An important advan-\ntage of the current study is the high throughput nature of our\nmicrofluidics platform in which we tested large number of\nindividual endometrial stromal cells from endometriosis\nFig. 5 V elocity of eutopic\nendometrial stromal cells of\nendometriosis patients and\ncontrol women inside\nmicrochannels. Error bars\nrepresent standard error of the\nmean (SEM). *P value < 0.001\nFig. 6 A histogram showing distribution of eutopic endometrial stromal\ncells from endometriosis patients and control women according to their\nvelocity inside the microchannel system\n370 Reprod. Sci. (2020) 27:364–374\n\npatients and control women. To confirm the accuracy of our\nresults, and as a check on the validity of the image processing\nsoftware for measuring the velocity of cells inside\nmicrochannels [28], we measured the velocity of a group of\ncells manually and compared it with the velocity automatical-\nly calculated by the software. Both values were identical.\nPrior research has confirmed the invasiveness of eutopic\nendometrial stromal cells [29]. When co-cultured with perito-\nneal explants, endometrial stromal cells breached the intact\nmesothelial cell layer in 24 h of co-culture [ 30]. Similarly,\nwhen plated with dispersed peritoneal mesothelial cells, endo-\nmetrial stromal cells extended pseudopodia under the meso-\nthelial cell layer [ 31]. In addition, endometrial stromal cells\nwere found to invade peritoneal mesothelial cells plated on\nmatrigel-coated chambers. This effect was enhanced by\nactivin A and associated with production of MMP-2 and\nMMP-9 [ 32]. Moreover, ectopic endometrial stromal cells\nwere more invasive than their eutopic counterparts, which\nwere more invasive than endometrial stromal cells of\ndisease-free women. The difference in invasiveness is related\nto increased expression of ezrin, a member of ezrin/radixin/\nmeosin family of proteins which act as linkers between actin\nfilaments and plasma membrane proteins [33]. Because cellu-\nlar motility and invasiveness are inversely proportional to their\nmechanical stiffness [34], it is not surprising that the motile\nand invasive eutopic endometrial stromal cells of endometri-\nosis patients are less mechanically stiff, as our results have\nshown.\nThe vesico-elastic properties of cells are determined by\ncharacteristics of their cytoskeleton [ 35]. F-actin, vimentin\nintermediate filaments, and microtubules represent the main\nconstituents of cellular cytoskeleton structures. F-actin fila-\nments provide high degree of resistance to deformation.\nThey polymerize to form tertiary structures known as actin\nstress fibers with the help of different actin-binding proteins\nto provide the cell with high mechanical integrity. The inter-\nmediate vimentin filaments allow moderate degree deforma-\ntion and tolerate high degrees of mechanical stress, even at\nlevels at which F-actin are unable to keep their mechanical\nintegrity. Microtubules do not have enough tensile stiffness to\nprovide mechanical support to the cytoskeleton, but they work\ntogether with other elements to stabilize the cellular cytoskel-\neton. The degree of mechanical stiffness and cellular\ndeformability exhibited by a particular cell type depends on\nthe concentration and molecular composition/regulation of the\ncytoskeleton proteins contained in these cells [36].\nKey regulators of the cytoskeleton protein dynamics are\nmembers of the small Rho- GTPase family of proteins (Rho,\nRac, and Cds42) [37]. ROCKII, which is a downstream effector\nof Rho, activates myosin light chain and regulates ezrin/radixin/\nmoesin family of actin remodeling proteins in the cell [ 38].\nEutopic endometrial stromal cells of endometriosis patients\nshowed more enhanced migratory phenotype than their coun-\nterparts from control women as a result of higher activation of\nthe Raf-1/Rho/ROCKII pathway [39]. In addition, Ectopic en-\ndometrial stromal cells containedsignificantly higher levels of\nFig. 7 V elocity of eutopic endometrial stromal cells versus cell size\n(projected cell area). a Eutopic endometrial stromal cells from control\nwomen. Cellular velocity = 57.518 ± 0.585 mm/s (mean ± SEM). Cells\ncount = 4541. b Eutopic endometrial stromal cells from endometriosis\npatients. Cellular velocity = 96.530 ± 0.710, mm/s (mean ± SEM). Cells\ncount = 4407\nReprod. Sci. (2020) 27:364–374 371\n\nphosphorylated ezrin/radixin/moesin cytoskeletal protiens than\ncells of eutopic endometrium, or control women [40]. Vinculin\nis another actin remodeling proteins that was shown to be dys-\nregulated in endometriosis [ 41]. In addition, ovarian steroid\nhormone treatment of eutopic endometrial stromal cells from\nendometriosis patients induced a promigratory phenotype char-\nacterized by cytoskeleton alteration including loss of stress fi-\nbers, progressive localization of actin toward the edge of the\ncell membrane, and simultaneous presence of numerous stress\nfiber arcs [42]. Focal adhesion kinase is an estrogen-regulated\nmolecule residing at points of contact with extracellular matrix\nforming a signaling complex to mediate important cellular\nfunctions including cytoskeleton remodeling. In endometriosis,\nfocal adhesion kinase was shown to be dysregulated in the\neutopic endometrium, and its levels correlate with the disease\nstage and pain symptoms during the secretory phase of the\nmenstrual cycle [43]. The research evidence for dysregulation\nof cytoskeleton elements and their regulatory pathways in\neutopic endometrial cells of endometriosis patients can provide\nsome explanation of our findings of altered mechanical proper-\nties and reduced stiffness of these cells.\nMechanical properties of living cells have emerged as a pos-\nsible biomarker for predicting health state of cells [ 44]. For\nexample, many types of cancers were reported to have de-\ncreased cellular stiffness. These include breast [45], lung [46],\npancreatic [47], ovarian [ 48], and bladder cancers [ 49]. This\nchange in mechanical properties between healthy and diseased\ncells leads to the emergence of mechanical properties as a bio-\nmarker for diagnostics, eliminating the need for conventional\nbiomarkers, thus reducing examination time and cost [35]. The\ndifference in cellular deformability/mechanical stiffness be-\ntween eutopic endometrial stromal cells of endometriosis pa-\ntients and those of disease-free women expressed as the velocity\nof cells as they travel through microchannels, under the condi-\ntions described in our system, can establish the basis for a non-\nsurgical test to differentiatebetween women with and without\nendometriosis.\nCompared with the current standard diagnostic modality of\nendometriosis, i.e., laparoscopy, our proposed mechanical\nbiomarker of the disease is much less invasive, as it only\nrequires an endometrial biopsy that can be taken as an office\nprocedure. Considering costs, our technique requires one dis-\nposable microfluidic chip carrying 10 microchannels and cost-\ning only $3 which is nothing compared with the expenses of\nlaparoscopy which could amount to 4289 ± $3313 [ 50].\nAlthough dissociation of the endometrial biopsy followed by\ncell culture for few days is required to obtain sufficient num-\nber of cells to perform our test, still this is cheaper, more\naccessible, and more convenient than scheduling a laparosco-\npy. It has to be noted here that cost of capital equipment for\nboth techniques (laparoscopic equipment for laparoscopy and\nan inverted microscope, high-speed camera, and a syringe\npump for our microfluidic technique) is similar.\nOur study is not without limitations. As we were trying to\nprove the concept of a difference in the mechanical properties\nin endometrial cells between women with and without endo-\nmetriosis, we used a complex platform involving cell culture\nequipment, inverted microscope, a microchip, high-speed\ncamera, and computer vision software. In order to be able to\nconfirm our results in a larger cohort of patients, our setup\nshould be simplified. The envisioned platform will comprise\na chip containing the required narrow microchannel integrated\nwith on-chip pumping [51]. Eutopic endometrial stromal cell\ncan be loaded on the inlet reservoir and pumped through the\nnarrow microchannel. Built-in optics, CMOS sensor [52], and\na field-programmable gate array (FPGA) module can be in-\ncorporated and programmed to perform computer vision steps\nto find average cell velocity inside the microchannel. To re-\nplace the cell culture step and increase the level of automation,\nthe microfluidic platform can be further developed to perform\ntissue dissociation and debris filtration on chip [ 53]. To pre-\nvent clogging of microchannels, a cross-flow filter [54]c a nb e\nbuilt at the microchannel entrance to hold large cells that may\nclog the microchannel. Cell sorting based on size prior to\npassage into the microchannel constriction is also possible.\nIn conclusion, we characterized the mechanical signa-\nture of eutopic endometrial st romal cells of endometriosis\npatients using a high throughput microfluidics platform\nimplying velocity of cells inside microchannel constriction\nas surrogate for cellular stiffness. We found that eutopic\nendometrial stromal cells of endometriosis patients have\nincreased deformation index and exhibit higher velocity\ninside our microchannel system (i.e., lower stiffness and\nhigher deformability) compared with their counterparts of\ncontrol women. These particular biomechanical features of\neutopic endometrial stromal cells of endometriosis can lay\nthe foundation for identifying a mechanical biomarker of\nthe disease.\nAcknowledgments The authors would like to acknowledge Professor\nFelice Petraglia, University of Florence, Italy, and Dr. Felice Arcuri,\nSiena University, Italy, for providing the protocol for endometrial stromal\ncell isolation and culture.\nFunding Information The study was funded by a grant from Science and\nTechnology Development Fund of Egypt (STDF) to E.O. (grant ID #\n5525). Microchannels used in this study were fabricated at the clean room\nof the Faculty of Engineering which was established through a grant from\nthe Science and Technology Development Fund of Egypt (STDF) to\nM.A. (grant ID # 4918).\nCompliance with Ethical Standards\nAll participating women provided written informed consent. 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