{"paper_id":"cff0dc4c-a2fe-45a2-b39e-0aafc28f327d","body_text":"The coronavirus disease 2019 (COVID-19) starts by the entrance of SARS-CoV-2 into\nhuman cells, by binding to its cell surface receptor, the membrane bound form of a\nprotein also known as type-2 Angiotensin Converting Enzyme (ACE2). 1  We have\npreviously demonstrated that ACE2 is present in a variety of reproductive tissues,\nsuch as testis, 2  ovary, 3 – 5  and endometrium,\nwith predominance in the endometrial epithelium and during the secretory phase of\nmenstrual cycle. 6  Therefore, the human reproductive system is a potential target\nfor SARS-CoV-2, but whether ACE2 is present in other uterine tissues like the\nmyometrium remains unknown. 7\nIf the myometrium and/or uterine leiomyomas express the protein ACE2, there is a\nplausible mechanism by which SARS-CoV-2 might infect these tissues, where it might\ntrigger a local inflammatory response and might disrupt functional mechanisms of\ntissue repair. 8 , 9  Inflammation in\nthe myometrium may predispose to adenomyosis 10  and also boost the growth of\nuterine fibroids, through paracrine actions of transforming growth factor β3 and\nproinflammatory cytokines released in the extracellular matrix. 11  Therefore, the\npresent study evaluated whether uterine fibroids and normal myometrium express ACE2\nand, if so, at which tissue compartments.\n\nThis cross-sectional study was approved by the Research Ethics Committee of\nUniversidade Federal de Minas Gerais under protocol number CAAE\n60375616.5.0000.5149, version 3. The participants signed an informed consent before\nbeing enrolled.\nWe included 13 premenopausal women (age range 33–50 years, median 40 years) with\nuterine fibroids undergoing elective hysterectomy or myomectomy for abnormal uterine\nbleeding between January and May 2019. Exclusion criteria were uterine atrophy,\nmalformation or malignancy. Fibroid classification according to the FIGO\ncriteria 12  ranged from 2 to 4 and 54% of the participants were using\nprogestins at the time of surgery. Samples of leiomyoma ( n  = 12)\nand normal myometrial ( n  = 8) tissue were obtained in the operating\nroom. Half of the tissue fragments (six samples of fibroids and four of myometrium)\nwere fixed in buffered formaldehyde and embedded in paraffin for subsequent use in\nimmunohistochemistry, and the remaining samples were immediately immersed in RNA\nstabilization solution (RNAlater, ThermoFisher, São Paulo, Brazil).\nTissue sections of 5-μm thickness were mounted in silanized slides, deparaffinized in\nxylene, and rehydrated in serially diluted ethanol baths. Antigen retrieval was\nperformed by microwave heating in EDTA buffer pH 8.0 for 5 min followed by cooling\nat room temperature. Using the Novolink™ non-biotin polymer detection system kit\n(Novocastra ® , Newcastle Upon Tyne, UK), endogenous peroxidase\nactivity was blocked with peroxidase block for 5 min, followed by 5 min incubation\nwith protein block to reduce background staining. Next, sections were incubated with\nrabbit polyclonal antibody anti-human ACE2 (Abcam, Cambridge, UK, catalog number\nab15348) 1:10 overnight at 4°C. Afterwards, post-primary block was added for 5 min\nto enhance penetration of the polymer reagent. Reactions were developed using\n3,3′-diaminobenzidine and sections were counterstained with hematoxylin. Negative\ncontrols were processed with PBS instead of the primary antibody. High resolution\nimages of the stained sections were acquired through a Pannoramic Digital Slide\nScanner (3DHistech, Budapest, Hungary) and analyzed in full using CaseViewer 2.4\nsoftware, then representative areas were chosen to illustrate the findings.\nTotal RNA was isolated from samples using homogenization in Trizol reagent\n(Invitrogen, Carlsbad, CA, USA) and reverse transcription was performed on 1 μg of\nDNAse I treated RNA using SuperScript III reverse transcriptase kit (Invitrogen,\nCarlsbad, CA, USA). The cDNA was subsequently subjected to real time polymerase\nchain reaction using SYBR Green Master Mix kit (Life Technologies, Invitrogen,\nCarlsbad, CA, USA) as detailed elsewhere. 3  The oligonucleotide primers used\nfor human ACE2 (Accession number  NM_021804.2 ) were 5′-GGATGGAGTACCGACT-3′ (forward)\nand 5′-TCCATTTACAGGCCCTTCTTCC-3′ (reverse). ACE2 gene expression was normalized by\nthe reference gene  S26  (Accession number  NM_001029.3 , primer\nsequences 5′-CCGCCATCCGGCTAAATAGT-3′ and 5′-GGGTGGAAATGCGTTCCTAGT-3′) and the\nresults were expressed as fold change (2 −ΔΔCT ).\n\nFigure 1  shows representative\nexamples of ACE2 localization in normal myometrial tissue dissected out of\nhysterectomy specimens. There was diffuse immunostaining in smooth muscle fibers\n( Figure 1(a)  and ( b )), particularly around the\nnuclei, but also in the interstitial space. Of note, ACE2 immunostaining was evident\nin the circular layer of arteriolar smooth muscle ( Figure 1(c) ) as well as in the endothelium of\nsmall myometrial veins ( Figure\n1(d) ).\nLocalization of ACE2 in normal human myometrium by immunohistochemistry.\nWhite arrows: smooth muscle cells; black arrows: venous endothelial cells;\nasterisk: arteriolar wall.\nNC: negative control.\nUterine fibroids also had abundant expression of ACE2 ( Figure 2 ). The protein was detected in\nleiomyoma cells characterized by elongated spindle shape nuclei, with indistinct\ncell borders and no evidence of mitotic activity, cellular and nuclear pleomorphisms\n( Figure 2(a) ). In areas\nrich in extracellular matrix, ACE2 expression was much stronger in the leiomyoma\nsmooth muscle fibers than in the surrounding collagen deposits ( Figure 2(b)  and ( c )). Venous endothelia were also positive for\nACE2 ( Figure 2(c)  and ( d )).\nLocalization of ACE2 in uterine leiomyoma by immunohistochemistry. White\narrows: smooth muscle cells; black arrows: venous endothelial cells.\nLSM: leiomyoma smooth muscle cells; ECM: extracellular matrix – collagen\nfiber components between the cells; NC: negative control.\nACE2 mRNA expression was quantified in myometrium and in uterine leiomyoma samples,\nalthough no mRNA quantitative difference between myometrium and leiomyoma was\ndetected for ACE2 ( Figure\n3 ).\nRelative expression of ACE2 mRNA in human myometrium ( n  = 4)\nand leiomyoma ( n  = 6).\n\nThe present study investigated the mRNA expression and protein immunolocalization of\nACE2 in uterine leiomyoma and normal myometrial tissue from premenopausal women. The\nresults obtained clearly indicate the presence of ACE2 mRNA in both tissue types and\nshow evidence of ACE2 localization in myometrium smooth muscle and blood vessels, as\nwell as in leiomyoma cells.\nWhile ACE2 had been detected in human endometrial cells, 6  myometrial expression of ACE2\nhad been reported only in pregnant guinea-pig, particularly in the vascular smooth\nmuscle of spiral, myometrial, and mesometrial arteries. 13  In human, a previous study\ndetected ACE2 mRNA in term gestation myometrial strips, but without protein\nassessment in this specific tissue. 14  Thus, to the best of our\nknowledge, the present study is the first to show ACE2 expression and localization\nin non-pregnant human myometrium.\nThe potential implications of this finding reside, firstly, on the characteristic of\nACE2 of being the main cell surface receptor for SARS-CoV-2, meaning that the\npresence of ACE2 is a precondition for a cell be susceptible to this type of\ncoronavirus. 1  However, another critical molecule to allow SARS-CoV-2 infection\nis the serine protease TMPRSS2, which is necessary for priming of the viral spike\nproteins. 15  The presence of TMPRSS2 in human myometrium and uterine\nfibroids remains uncertain and therefore should be further assessed in conjunction\nwith ACE2, as only cells co-expressing both proteins are potentially susceptible to\nSARS-CoV-2 entry. 15  A second potential implication of the presence of ACE2 in human\nmyometrium and uterine leiomyoma is the enzymatic activity after which ACE2 is\nnamed, that is, the cleavage of peptides from the renin-angiotensin system. ACE2\nconverts angiotensin II into angiotensin-(1–7), an heptapeptide that has\nanti-fibrotic properties in extra-reproductive tissues, 16  a protective mechanism\npotentially relevant to stall the development of uterine fibroids 17  and\nadenomyosis. 18\nThis study has several limitations. We did not perform a quantitative comparison of\nACE2 levels between normal myometrium and leiomyoma, but only a descriptive analysis\nof the protein distribution in the tissue compartments. The similar levels of ACE2\nmRNA cannot be taken as conclusive evidence that myometrium and fibroids have the\nsame levels of protein nor the same functional activity of ACE2 either as a\nSARS-CoV-2 receptor or as an angiotensin-(1–7) generating enzyme. The sample size\ndid not confer statistical power to rule out a difference between myometrium and\nleiomyoma as regards ACE2 mRNA levels. In addition, the small sample size and the\nlack of quantitative protein measurements did not allow us to investigate whether\nACE2 associates with clinical symptoms, fibroid topography, uterine volume, or\npreoperative medical treatments, which should be evaluated in the future.\nNevertheless, as a proof of concept our results provide evidence that both normal\nmyometrium and uterine fibroid possess ACE2 mRNA and protein and therefore fulfill\none of the molecular conditions to be a SARS-CoV-2 target tissue.\nIn conclusion, human myometrium and uterine leiomyoma express ACE2 mRNA and have\nabundant distribution of ACE2 protein in their smooth muscle cells and\nmicrovasculature. It is still early to know whether women infected by SARS-CoV-2\nwill have any damage in their reproductive organs leading to a worse fertility\nprognosis 19 , 20  or to severer fibroid symptoms. Nevertheless, prospective\nstudies should assess these hypotheses since the virus is able to elicit intense\ninflammatory response in target tissues 9  with still unknown sequelae.","source_license":"CC0","license_restricted":false}