Anatomy of the Fetal Membranes: Insights from Spinning Disk Confocal Microscopy

Anatomy of the Fetal Membranes: Insights from Spinning Disk Confocal Microscopy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anatomy of the Fetal Membranes: Insights from Spinning Disk Confocal Microscopy Hannah Marie Eichholz, Alissa Cornelis, Benjamin Wolf, Hanna Grubitzsch, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2644986/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 May, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted 4 You are reading this latest preprint version Abstract Purpose The fetal membrane is essential for the maintenance of pregnancy, and its integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies. Methods In this proof of principle study, fetal membranes were collected from both vaginal delivery and cesarean section. Small membrane sections were then fixed, fluorescently stained for nucleic acids, actin, and collagen, and subsequently imaged in three dimensions using a spinning disk confocal microscope. Results Four fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro)fractures could be identified in several samples. Conclusion The presented method proves to be well suited to determine whether and how the occurrence of membrane (micro)fractures and cellular jamming correlates with the timing of membrane rupture and the mode of delivery. In future measurements, this method could possibly be combined with mechanical probing techniques to compare optical and mechanical sample information. Fetal membranes chorion amnion anatomy confocal microscopy premature preterm rupture of membranes preterm labor fluorescence microscopy confocal microscopy fetal membrane microfractures Figures Figure 1 What does this study add to the clinical work? Premature and/or preterm fetal membrane rupture is a disastrous tissue failure whose underlying mechanisms are not completely understood. This study demonstrates the great potential of spinning disk confocal microscopy for the investigation of fetal membrane anatomy and provides a tissue processing protocol for future studies. Introduction The fetal membranes surround the fetus and the amniotic fluid during pregnancy. They are composed of three main layers: i) the amnion, the innermost epithelial layer surrounding the amniotic fluid, ii) the chorion connecting the fetal membranes to the maternal decidua, and iii) a mesenchymal layer between the amnion and chorion containing predominantly collagen-rich extracellular matrix and interspersed amnion mesenchymal cells [ 1 , 2 ]. The membrane’s integrity until parturition is of vital importance for fetal and maternal health. Preterm birth (PTB) – i.e., delivery earlier than 37 + 0 weeks of pregnancy – is a tremendous public health concern and affects about 15 million babies each year worldwide [ 3 ]. PTB manifests initially either as preterm labor or preterm premature rupture of membranes (pPROM). In this process, the fetal membranes are generally regarded as passive bystanders while maternal structures such as the decidua, the myometrium, and the uterine cervix are considered the active players in labor initiation and therefore also the targets of possible intervention [ 4 ]. For example, the process of matrix breakdown, membrane separation from the decidua, and ultimately membrane rupture is referred to biochemically as “membrane activation” [ 4 ]. This pathway involves the switch from anti-inflammatory to pro-inflammatory signaling leading to increased oxidative stress and expression of matrix metalloproteinases (among other enzymes) resulting in membrane breakdown [ 2 , 5 ]. Important research over the past decade has shed more light on the active role of fetal membranes in the initiation of parturition, however. In brief, it has been demonstrated that fetal membrane senescence leads to focal apoptosis and tissue microfractures [ 6 ], resulting in the release of damage associated pattern (DAMP) molecules that lead to a proinflammatory senescence associated secretory phenotype (SASP) [ 7 – 9 ]. Most research has focused on dynamic biochemical changes and little attention has been paid to constitutional variations of membrane architecture. Whether such interindividual variants can itself be determinants of membrane breakdown remains to be investigated. We hypothesize that membrane integrity can be conceptualized in anatomical and functional terms (which are necessarily interrelated). While anatomical integrity refers to the structural composition of the membranes as a physical barrier preventing amniotic fluid from leakage and inhibiting the ascent of microorganisms into the amniotic cavity, functional (biochemical) integrity refers to the membranes’ ability to secrete biologically active substances such as prostaglandins, matrix degrading enzymes, and antimicrobial components. Here we report first an imaging technique suitable for detailed structural analysis of fetal membrane architecture. Materials And Methods Ethics approval, patient selection, and sample collection: This was a single center study, and all specimens were collected at the Perinatal Medicine Department of the Leipzig University Medical Center. All experiments conducted were approved by the local Ethics committee (2021–12 – 20, 540/21-ek). Women eligible for inclusion were older than 18 years and their babies were delivered at term (37 + 0–41 + 3 weeks of pregnancy) either vaginally or by cesarean section (see Table 1 for patient characteristics). On admission to the labor and delivery ward, women were asked if they were willing to donate the fetal membranes directly after delivery. After the placenta and fetal membranes were inspected for completeness, the membranes were separated from the placenta and directly transferred to the laboratory at the Peter Debye Institute for Soft Matter Physics in Leipzig for further analysis. Preparation of the fetal membranes: Upon arrival at the laboratory, the specimens were rinsed with phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, Massachusetts) three times to eliminate any fetal contaminations (e.g., meconium stains). A representative part of the membranes measuring approximately 1.5 cm 2 was excised and fixed with 10% neutral buffered formalin (NBF, Sigma-Aldrich, St. Louis, Missouri) at 4°C for 24 hours. Staining: To visualize the cell nuclei, all samples were stained with SPY555-DNA (Spirochrome AG, Stein am Rhein, Switzerland) which is a non-toxic, cell permeable, and highly specific live cell DNA probe that can be excited with wavelengths around 555 nm. In addition, actin and collagen were stained using SiR-actin (Spyrochrome AG, Stein am Rhein, Switzerland) and Col-F Collagen Binding Reagent (ImmunoChemistry Technologies, Davis, California), respectively. The cell permeable SiR-actin probe is highly specific for filamentous actin (F-actin), has an excitation maximum at 652 nm and emits light in the far-red region. Col-F binds to collagen and elastin fibers, which are main components of the extracellular matrix (ECM). The fluorescent probe can be excited at wavelengths around 490 nm and emits green light. Furthermore, 1% of Triton X-100 (Sigma-Aldrich, St. Louis, Missouri) was added to the staining solution to permeabilize the cell membranes and facilitate the uptake of stain molecules. The tissues were submerged in the staining solution and incubated at 4°C for 12–24 hours. Thereafter, the stained samples were washed with PBS three times and placed in glass bottom Petri dishes (ibidi, Gräfelfing, Germany) with the amnion or chorion facing down. Depending on the size of the tissues, a few drops of ibidi Mounting Medium (ibidi, Gräfelfing, Germany) were added to the samples to reduce photobleaching effects and ensure optimal conditions for microscopic imaging. Imaging: Imaging was performed using an inverted Zeiss Axio Observer.Z1 research microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a Yokogawa CSU-X1A 5000 spinning disk confocal scanning unit (Yokogawa Denki, Musashino, Japan). This setup enables high-resolution spinning disk confocal microscopy with up to four different wavelengths. The stained tissue samples were excited with lasers corresponding to their respective stains, namely 488 nm (Col-F), 561 nm (SPY555-DNA), and 638 nm (SiR-actin). They were then imaged for different magnifications and to a depth of about 50 µm from their surface. Subsequently, the individual channels were slightly adjusted in terms of contrast and superimposed. Results For this proof of principle investigation, four fetal membranes were processed and imaged as described above. The characteristics of the donor women are described in Table 1 . Fixation, staining and imaging of the tissue was successful in all cases, irrespective of delivery mode (vaginal delivery or cesarean section). Image resolution was expectedly best in the most superficial layer (amnion), but sufficient resolution was achieved in the deeper layers of the mesenchymal membrane part as well (compare Fig. 1 ). Nuclear and cellular outline was excellent on the amniotic surface. In all cases, we could identify focal membrane (micro)fractures as described before [ 6 ]. Table 1 Fetal and maternal characteristics Case No. GA (weeks) Delivery mode Number of pregnancies Number of prev. deliveries Sex Birth weight Additional maternal morbidity 1 38 + 0 VB (induction because of IUGR) 2 1 Male 2700 g History of HELLP-Syndrome in previous pregnancy 2 40 + 3 cesarean (two previous c-sections) 15 10 Female 3290 g none 3 39 + 0 cesarean (maternal tocophobia) 1 0 Male 3610 g none 4 39 + 4 VB 6 5 Female 3520 g insulin dependent gestational diabetes Discussion We report here for the first time the utility of spinning disc confocal microscopy for imaging fetal membranes and provide a protocol for tissue processing and staining. A high-resolution, three-dimensional image data acquisition method, as we report here, is the prerequisite for an in-depth investigation of fetal membrane structure. A key advantage of our method is that tissues can be imaged after they have been used for other mechanical studies such as atomic force microscopy (AFM). This will help us to precisely correlate visual and mechanical information in the future. Richardson et al. have used a combination of multiphoton autofluorescence microscopy and second harmonic generation microscopy to generate three-dimensional images of native fetal membranes [ 6 ]. Although this technique provides excellent images, the use of autofluorescence is rather a surrogate than a precise mapping of extracellular matrix components as can be accomplished using specific staining. Furthermore, our images provide a clearer outline of cellular, and more importantly nuclear shapes by actin staining. We have demonstrated before that cell and nuclear shape are a surrogate parameter of cellular unjamming, which is an important determinant of tissue fluidity and therefore stability [ 10 ]. It will be interesting to investigate whether cellular unjamming as indicated by cellular and nuclear shapes – which can be analyzed easily – are correlated with time of membrane rupture, pPROM, and delivery mode. We hypothesize that a more fluidic cell shape is associated with pPROM and vaginal birth, while a more static tissue architecture will be observed in patients undergoing cesarean section or iatrogenic membrane rupture during vaginal birth. While a wealth of literature has been published on regional membrane weakening in the supracervical zone [ 11 – 13 ], it will be especially interesting to determine whether architectural changes occur more ubiquitously in cases of (repeated) pPROM. This could indicate a structural predisposition to pPROM, as such membranes are more susceptible to inflammatory attack. Our investigations will therefore focus on membrane sections distant from the supracervical region. Fittingly, in a recent review, Ramkumar Menon and John Moore – two of the most accomplished researchers in the area – have raised the question of how membranes might fight off small inflammatory challenges that might otherwise lead to fetal membrane rupture and labor onset. The answer might lie in architectural robustness, just as a well-built fortress will resist heavier attacks. The methods presented in this paper will help us to answer this question. Declarations Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Conflicts of interest: Financial interests: BA has received honoraria from Pfizer, Roche, Novartis, AstraZeneca, Amgen and Daichi Sankyo. None of the payments is related to any aspect of this publication. BW has received consulting fees from Roche Pharma. HS has received consulting and speaker fees from Roche Diagnostics, Alexion, GE ultrasound and Sanofi. None of the payments is related to any aspect of this publication. All other authors declare no potential conflict of interest. Author contributions: Eichholz: protocol/project development, data collection and management, data analysis, manuscript writing Cornelis: protocol/project development, data collection and management, manuscript writing Wolf: project development, manuscript writing and editing Grubitzsch: data collection Friedrich: data analysis, manuscript writing and editing Makky: data collection Aktas: project development, manuscript editing Käs: project development, manuscript editing Stepan: project development, manuscript editing All authors read and approved the final manuscript. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee at the Medical Faculty of the University of Leipzig (2021-12-20, 540/21-ek). Consent to participate: Informed consent was obtained from all individual participants included in the study. References Menon R, Richardson LS, Lappas M (2019) Fetal membrane architecture, aging and inflammation in pregnancy and parturition. Placenta 79:40–45. https://doi.org/10.1016/j.placenta.2018.11.003 Bryant-Greenwood GD (1998) The extracellular matrix of the human fetal membranes: Structure and function. Placenta 19:1–11. https://doi.org/10.1016/S0143-4004(98)90092-3 Walani SR (2020) Global burden of preterm birth. International Journal of Gynecology & Obstetrics 150:31–33. https://doi.org/10.1002/ijgo.13195 Romero R, Dey SK, Fisher SJ (2014) Preterm labor: One syndrome, many causes. Science 345:760–765. https://doi.org/10.1126/science.1251816 Strauss Jerome F (2013) Extracellular Matrix Dynamics and Fetal Membrane Rupture. Reprod Sci 20:140–153. https://doi.org/10.1177/1933719111424454 Richardson LS, Vargas G, Brown T, et al (2017) Discovery and Characterization of Human Amniochorionic Membrane Microfractures. The American Journal of Pathology 187:2821–2830. https://doi.org/10.1016/j.ajpath.2017.08.019 Dutta EH, Behnia F, Boldogh I, et al (2016) Oxidative stress damage-associated molecular signaling pathways differentiate spontaneous preterm birth and preterm premature rupture of the membranes. Molecular Human Reproduction 22:143–157. https://doi.org/10.1093/molehr/gav074 Behnia F, Taylor BD, Woodson M, et al (2015) Chorioamniotic membrane senescence: a signal for parturition? American Journal of Obstetrics and Gynecology 213:359.e1-359.e16. https://doi.org/10.1016/j.ajog.2015.05.041 Menon R, Moore JJ (2020) Fetal Membranes, Not a Mere Appendage of the Placenta, but a Critical Part of the Fetal-Maternal Interface Controlling Parturition. Obstetrics and Gynecology Clinics 47:147–162. https://doi.org/10.1016/j.ogc.2019.10.004 Grosser S, Lippoldt J, Oswald L, et al (2021) Cell and Nucleus Shape as an Indicator of Tissue Fluidity in Carcinoma. Phys Rev X 11:011033. https://doi.org/10.1103/PhysRevX.11.011033 Malak TM, Bell SC (1994) Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site. Br J Obstet Gynaecol 101:375–386. https://doi.org/10.1111/j.1471-0528.1994.tb11908.x Reti NG, Lappas M, Riley C, et al (2007) Why do membranes rupture at term? Evidence of increased cellular apoptosis in the supracervical fetal membranes. American Journal of Obstetrics and Gynecology 196:484.e1-484.e10. https://doi.org/10.1016/j.ajog.2007.01.021 El Khwad M, Stetzer B, Moore RM, et al (2005) Term Human Fetal Membranes Have a Weak Zone Overlying the Lower Uterine Pole and Cervix Before Onset of Labor1. Biology of Reproduction 72:720–726. https://doi.org/10.1095/biolreprod.104.033647 Cite Share Download PDF Status: Published Journal Publication published 15 May, 2023 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted Reviewers agreed at journal 31 Mar, 2023 Editor invited by journal 07 Mar, 2023 Editor assigned by journal 03 Mar, 2023 First submitted to journal 01 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2644986","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":187990489,"identity":"9aade5f7-3658-4505-bf7f-d62c84b06a10","order_by":0,"name":"Hannah Marie Eichholz","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"Marie","lastName":"Eichholz","suffix":""},{"id":187990490,"identity":"fd97e8d6-a197-440d-86e0-b459c2250036","order_by":1,"name":"Alissa Cornelis","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alissa","middleName":"","lastName":"Cornelis","suffix":""},{"id":187990491,"identity":"47ad64e8-f607-4728-bb99-45b2f96b16b2","order_by":2,"name":"Benjamin Wolf","email":"","orcid":"https://orcid.org/0000-0002-1931-5198","institution":"University Hopsital Leipzig","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Benjamin","middleName":"","lastName":"Wolf","suffix":""},{"id":187990492,"identity":"2e47bb95-a41a-4958-a46d-a10e8579fec3","order_by":3,"name":"Hanna Grubitzsch","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanna","middleName":"","lastName":"Grubitzsch","suffix":""},{"id":187990493,"identity":"7c728497-0849-4675-b511-fe5905e3a154","order_by":4,"name":"Philip Friedrich","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Friedrich","suffix":""},{"id":187990494,"identity":"f5b37e37-d8e3-4c35-9cbf-782e84ba5ebe","order_by":5,"name":"Ahmad Makky","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Makky","suffix":""},{"id":187990495,"identity":"3b0d7c51-433e-49d3-95db-21d493f605e6","order_by":6,"name":"Bahriye Aktas","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bahriye","middleName":"","lastName":"Aktas","suffix":""},{"id":187990496,"identity":"5fd4d92d-67bf-450d-9b2b-24f794a6c06c","order_by":7,"name":"Josef A. Käs","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Josef","middleName":"A.","lastName":"Käs","suffix":""},{"id":187990497,"identity":"27bf9008-f503-489b-a9b4-d15f59018a62","order_by":8,"name":"Holger Stepan","email":"data:image/png;base64,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","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Holger","middleName":"","lastName":"Stepan","suffix":""}],"badges":[],"createdAt":"2023-03-01 23:31:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2644986/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2644986/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00404-023-07070-0","type":"published","date":"2023-05-15T20:52:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":35207131,"identity":"bb626fed-f985-4ebb-9b8e-243ecaf44766","added_by":"auto","created_at":"2023-04-03 14:09:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":954019,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic fetal membrane anatomy visualized by spinning disc microscopy. On the left, a schematic drawing indicates the relevant anatomical structures. In the middle column, confocal microscopy images with nuclear staining for DNA (red) and collagen (green) is shown. In the right column, corresponding images with staining for actin (red) and collagen (green) is shown.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2644986/v1/133a7e4f4112832ed2745b66.png"},{"id":44729082,"identity":"7dcade9f-197c-4d9f-8d25-8403465373c1","added_by":"auto","created_at":"2023-10-16 21:11:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1243472,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2644986/v1/b60b4c81-41f7-4c4d-af46-29572d8fe49f.pdf"}],"financialInterests":"","formattedTitle":"Anatomy of the Fetal Membranes: Insights from Spinning Disk Confocal Microscopy","fulltext":[{"header":"What does this study add to the clinical work?","content":"\u003cp\u003ePremature and/or preterm fetal membrane rupture is a disastrous tissue failure whose underlying mechanisms are not completely understood. This study demonstrates the great potential of spinning disk confocal microscopy for the investigation of fetal membrane anatomy and provides a tissue processing protocol for future studies.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe fetal membranes surround the fetus and the amniotic fluid during pregnancy. They are composed of three main layers: i) the amnion, the innermost epithelial layer surrounding the amniotic fluid, ii) the chorion connecting the fetal membranes to the maternal decidua, and iii) a mesenchymal layer between the amnion and chorion containing predominantly collagen-rich extracellular matrix and interspersed amnion mesenchymal cells [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe membrane\u0026rsquo;s integrity until parturition is of vital importance for fetal and maternal health. Preterm birth (PTB) \u0026ndash; i.e., delivery earlier than 37\u0026thinsp;+\u0026thinsp;0 weeks of pregnancy \u0026ndash; is a tremendous public health concern and affects about 15\u0026nbsp;million babies each year worldwide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. PTB manifests initially either as preterm labor or preterm premature rupture of membranes (pPROM). In this process, the fetal membranes are generally regarded as passive bystanders while maternal structures such as the decidua, the myometrium, and the uterine cervix are considered the active players in labor initiation and therefore also the targets of possible intervention [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For example, the process of matrix breakdown, membrane separation from the decidua, and ultimately membrane rupture is referred to biochemically as \u0026ldquo;membrane activation\u0026rdquo; [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This pathway involves the switch from anti-inflammatory to pro-inflammatory signaling leading to increased oxidative stress and expression of matrix metalloproteinases (among other enzymes) resulting in membrane breakdown [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Important research over the past decade has shed more light on the active role of fetal membranes in the initiation of parturition, however. In brief, it has been demonstrated that fetal membrane senescence leads to focal apoptosis and tissue microfractures [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], resulting in the release of damage associated pattern (DAMP) molecules that lead to a proinflammatory senescence associated secretory phenotype (SASP) [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost research has focused on dynamic biochemical changes and little attention has been paid to constitutional variations of membrane architecture. Whether such interindividual variants can itself be determinants of membrane breakdown remains to be investigated. We hypothesize that membrane integrity can be conceptualized in anatomical and functional terms (which are necessarily interrelated). While anatomical integrity refers to the structural composition of the membranes as a physical barrier preventing amniotic fluid from leakage and inhibiting the ascent of microorganisms into the amniotic cavity, functional (biochemical) integrity refers to the membranes\u0026rsquo; ability to secrete biologically active substances such as prostaglandins, matrix degrading enzymes, and antimicrobial components. Here we report first an imaging technique suitable for detailed structural analysis of fetal membrane architecture.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval, patient selection, and sample collection:\u003c/h2\u003e \u003cp\u003eThis was a single center study, and all specimens were collected at the Perinatal Medicine Department of the Leipzig University Medical Center. All experiments conducted were approved by the local Ethics committee (2021\u0026ndash;12 \u0026ndash; 20, 540/21-ek). Women eligible for inclusion were older than 18 years and their babies were delivered at term (37\u0026thinsp;+\u0026thinsp;0\u0026ndash;41\u0026thinsp;+\u0026thinsp;3 weeks of pregnancy) either vaginally or by cesarean section (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for patient characteristics). On admission to the labor and delivery ward, women were asked if they were willing to donate the fetal membranes directly after delivery. After the placenta and fetal membranes were inspected for completeness, the membranes were separated from the placenta and directly transferred to the laboratory at the Peter Debye Institute for Soft Matter Physics in Leipzig for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the fetal membranes:\u003c/h2\u003e \u003cp\u003eUpon arrival at the laboratory, the specimens were rinsed with phosphate buffered saline (PBS, Thermo Fisher Scientific, Waltham, Massachusetts) three times to eliminate any fetal contaminations (e.g., meconium stains). A representative part of the membranes measuring approximately 1.5 cm\u003csup\u003e2\u003c/sup\u003e was excised and fixed with 10% neutral buffered formalin (NBF, Sigma-Aldrich, St. Louis, Missouri) at 4\u0026deg;C for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStaining:\u003c/h2\u003e \u003cp\u003eTo visualize the cell nuclei, all samples were stained with SPY555-DNA (Spirochrome AG, Stein am Rhein, Switzerland) which is a non-toxic, cell permeable, and highly specific live cell DNA probe that can be excited with wavelengths around 555 nm. In addition, actin and collagen were stained using SiR-actin (Spyrochrome AG, Stein am Rhein, Switzerland) and Col-F Collagen Binding Reagent (ImmunoChemistry Technologies, Davis, California), respectively. The cell permeable SiR-actin probe is highly specific for filamentous actin (F-actin), has an excitation maximum at 652 nm and emits light in the far-red region. Col-F binds to collagen and elastin fibers, which are main components of the extracellular matrix (ECM). The fluorescent probe can be excited at wavelengths around 490 nm and emits green light. Furthermore, 1% of Triton X-100 (Sigma-Aldrich, St. Louis, Missouri) was added to the staining solution to permeabilize the cell membranes and facilitate the uptake of stain molecules.\u003c/p\u003e \u003cp\u003eThe tissues were submerged in the staining solution and incubated at 4\u0026deg;C for 12\u0026ndash;24 hours. Thereafter, the stained samples were washed with PBS three times and placed in glass bottom Petri dishes (ibidi, Gr\u0026auml;felfing, Germany) with the amnion or chorion facing down. Depending on the size of the tissues, a few drops of ibidi Mounting Medium (ibidi, Gr\u0026auml;felfing, Germany) were added to the samples to reduce photobleaching effects and ensure optimal conditions for microscopic imaging.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImaging:\u003c/h2\u003e \u003cp\u003eImaging was performed using an inverted Zeiss Axio Observer.Z1 research microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a Yokogawa CSU-X1A 5000 spinning disk confocal scanning unit (Yokogawa Denki, Musashino, Japan). This setup enables high-resolution spinning disk confocal microscopy with up to four different wavelengths.\u003c/p\u003e \u003cp\u003eThe stained tissue samples were excited with lasers corresponding to their respective stains, namely 488 nm (Col-F), 561 nm (SPY555-DNA), and 638 nm (SiR-actin). They were then imaged for different magnifications and to a depth of about 50 \u0026micro;m from their surface. Subsequently, the individual channels were slightly adjusted in terms of contrast and superimposed.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eFor this proof of principle investigation, four fetal membranes were processed and imaged as described above. The characteristics of the donor women are described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Fixation, staining and imaging of the tissue was successful in all cases, irrespective of delivery mode (vaginal delivery or cesarean section). Image resolution was expectedly best in the most superficial layer (amnion), but sufficient resolution was achieved in the deeper layers of the mesenchymal membrane part as well (compare Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Nuclear and cellular outline was excellent on the amniotic surface. In all cases, we could identify focal membrane (micro)fractures as described before [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFetal and maternal characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"+\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGA (weeks)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDelivery mode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of pregnancies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNumber of prev. deliveries\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBirth weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAdditional maternal morbidity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e38\u0026thinsp;+\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVB (induction because of IUGR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2700 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHistory of HELLP-Syndrome in previous pregnancy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e40\u0026thinsp;+\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecesarean (two previous c-sections)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3290 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e39\u0026thinsp;+\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecesarean (maternal tocophobia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3610 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"+\" colname=\"c2\"\u003e \u003cp\u003e39\u0026thinsp;+\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3520 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003einsulin dependent gestational diabetes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe report here for the first time the utility of spinning disc confocal microscopy for imaging fetal membranes and provide a protocol for tissue processing and staining. A high-resolution, three-dimensional image data acquisition method, as we report here, is the prerequisite for an in-depth investigation of fetal membrane structure. A key advantage of our method is that tissues can be imaged after they have been used for other mechanical studies such as atomic force microscopy (AFM). This will help us to precisely correlate visual and mechanical information in the future.\u003c/p\u003e \u003cp\u003eRichardson et al. have used a combination of multiphoton autofluorescence microscopy and second harmonic generation microscopy to generate three-dimensional images of native fetal membranes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although this technique provides excellent images, the use of autofluorescence is rather a surrogate than a precise mapping of extracellular matrix components as can be accomplished using specific staining. Furthermore, our images provide a clearer outline of cellular, and more importantly nuclear shapes by actin staining. We have demonstrated before that cell and nuclear shape are a surrogate parameter of cellular unjamming, which is an important determinant of tissue fluidity and therefore stability [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It will be interesting to investigate whether cellular unjamming as indicated by cellular and nuclear shapes \u0026ndash; which can be analyzed easily \u0026ndash; are correlated with time of membrane rupture, pPROM, and delivery mode. We hypothesize that a more fluidic cell shape is associated with pPROM and vaginal birth, while a more static tissue architecture will be observed in patients undergoing cesarean section or iatrogenic membrane rupture during vaginal birth. While a wealth of literature has been published on regional membrane weakening in the supracervical zone [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], it will be especially interesting to determine whether architectural changes occur more ubiquitously in cases of (repeated) pPROM. This could indicate a structural predisposition to pPROM, as such membranes are more susceptible to inflammatory attack. Our investigations will therefore focus on membrane sections distant from the supracervical region. Fittingly, in a recent review, Ramkumar Menon and John Moore \u0026ndash; two of the most accomplished researchers in the area \u0026ndash; have raised the question of how membranes might fight off small inflammatory challenges that might otherwise lead to fetal membrane rupture and labor onset. The answer might lie in architectural robustness, just as a well-built fortress will resist heavier attacks. The methods presented in this paper will help us to answer this question.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial interests: BA has received honoraria from Pfizer, Roche, Novartis, AstraZeneca, Amgen and Daichi Sankyo. None of the payments is related to any aspect of this publication. BW has received consulting fees from Roche Pharma. HS has received consulting and speaker fees from Roche Diagnostics, Alexion, GE ultrasound and Sanofi. None of the payments is related to any aspect of this publication.\u003c/p\u003e\n\u003cp\u003eAll other authors declare no potential conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEichholz: \u0026nbsp;protocol/project development, data collection and management, data analysis, manuscript writing\u003c/p\u003e\n\u003cp\u003eCornelis: \u0026nbsp;protocol/project development, data collection and management, manuscript writing\u003c/p\u003e\n\u003cp\u003eWolf: \u0026nbsp; project development, manuscript writing and editing\u003c/p\u003e\n\u003cp\u003eGrubitzsch: \u0026nbsp;data collection\u003c/p\u003e\n\u003cp\u003eFriedrich: \u0026nbsp;data analysis, manuscript writing and editing\u003c/p\u003e\n\u003cp\u003eMakky: \u0026nbsp;data collection\u003c/p\u003e\n\u003cp\u003eAktas: \u0026nbsp;project development, manuscript editing\u003c/p\u003e\n\u003cp\u003eK\u0026auml;s: project development, manuscript editing\u003c/p\u003e\n\u003cp\u003eStepan: \u0026nbsp;project development, manuscript editing\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee at the Medical Faculty of the University of Leipzig (2021-12-20, 540/21-ek).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMenon R, Richardson LS, Lappas M (2019) Fetal membrane architecture, aging and inflammation in pregnancy and parturition. Placenta 79:40\u0026ndash;45. https://doi.org/10.1016/j.placenta.2018.11.003\u003c/li\u003e\n\u003cli\u003eBryant-Greenwood GD (1998) The extracellular matrix of the human fetal membranes: Structure and function. Placenta 19:1\u0026ndash;11. https://doi.org/10.1016/S0143-4004(98)90092-3\u003c/li\u003e\n\u003cli\u003eWalani SR (2020) Global burden of preterm birth. International Journal of Gynecology \u0026amp; Obstetrics 150:31\u0026ndash;33. https://doi.org/10.1002/ijgo.13195\u003c/li\u003e\n\u003cli\u003eRomero R, Dey SK, Fisher SJ (2014) Preterm labor: One syndrome, many causes. Science 345:760\u0026ndash;765. https://doi.org/10.1126/science.1251816\u003c/li\u003e\n\u003cli\u003eStrauss Jerome F (2013) Extracellular Matrix Dynamics and Fetal Membrane Rupture. Reprod Sci 20:140\u0026ndash;153. https://doi.org/10.1177/1933719111424454\u003c/li\u003e\n\u003cli\u003eRichardson LS, Vargas G, Brown T, et al (2017) Discovery and Characterization of Human Amniochorionic Membrane Microfractures. The American Journal of Pathology 187:2821\u0026ndash;2830. https://doi.org/10.1016/j.ajpath.2017.08.019\u003c/li\u003e\n\u003cli\u003eDutta EH, Behnia F, Boldogh I, et al (2016) Oxidative stress damage-associated molecular signaling pathways differentiate spontaneous preterm birth and preterm premature rupture of the membranes. Molecular Human Reproduction 22:143\u0026ndash;157. https://doi.org/10.1093/molehr/gav074\u003c/li\u003e\n\u003cli\u003eBehnia F, Taylor BD, Woodson M, et al (2015) Chorioamniotic membrane senescence: a signal for parturition? American Journal of Obstetrics and Gynecology 213:359.e1-359.e16. https://doi.org/10.1016/j.ajog.2015.05.041\u003c/li\u003e\n\u003cli\u003eMenon R, Moore JJ (2020) Fetal Membranes, Not a Mere Appendage of the Placenta, but a Critical Part of the Fetal-Maternal Interface Controlling Parturition. Obstetrics and Gynecology Clinics 47:147\u0026ndash;162. https://doi.org/10.1016/j.ogc.2019.10.004\u003c/li\u003e\n\u003cli\u003eGrosser S, Lippoldt J, Oswald L, et al (2021) Cell and Nucleus Shape as an Indicator of Tissue Fluidity in Carcinoma. Phys Rev X 11:011033. https://doi.org/10.1103/PhysRevX.11.011033\u003c/li\u003e\n\u003cli\u003eMalak TM, Bell SC (1994) Structural characteristics of term human fetal membranes: a novel zone of extreme morphological alteration within the rupture site. Br J Obstet Gynaecol 101:375\u0026ndash;386. https://doi.org/10.1111/j.1471-0528.1994.tb11908.x\u003c/li\u003e\n\u003cli\u003eReti NG, Lappas M, Riley C, et al (2007) Why do membranes rupture at term? Evidence of increased cellular apoptosis in the supracervical fetal membranes. American Journal of Obstetrics and Gynecology 196:484.e1-484.e10. https://doi.org/10.1016/j.ajog.2007.01.021\u003c/li\u003e\n\u003cli\u003eEl Khwad M, Stetzer B, Moore RM, et al (2005) Term Human Fetal Membranes Have a Weak Zone Overlying the Lower Uterine Pole and Cervix Before Onset of Labor1. Biology of Reproduction 72:720\u0026ndash;726. https://doi.org/10.1095/biolreprod.104.033647\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fetal membranes, chorion, amnion, anatomy, confocal microscopy, premature preterm rupture of membranes, preterm labor, fluorescence microscopy, confocal microscopy, fetal membrane microfractures","lastPublishedDoi":"10.21203/rs.3.rs-2644986/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2644986/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe fetal membrane is essential for the maintenance of pregnancy, and its integrity until parturition is critical for both fetal and maternal health. Preterm premature rupture of the membranes (pPROM) is known to be an indicator of preterm birth, but the underlying architectural and mechanical changes that lead to fetal membrane failure are not yet fully understood. The aim of this study was to gain new insights into the anatomy of the fetal membrane and to establish a tissue processing and staining protocol suitable for future prospective cohort studies.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this proof of principle study, fetal membranes were collected from both vaginal delivery and cesarean section. Small membrane sections were then fixed, fluorescently stained for nucleic acids, actin, and collagen, and subsequently imaged in three dimensions using a spinning disk confocal microscope.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFour fetal membranes of different types were successfully processed and imaged after establishing a suitable protocol. Cellular and nuclear outlines are clearly visible in all cases, especially in the uppermost membrane layer. Focal membrane (micro)fractures could be identified in several samples.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe presented method proves to be well suited to determine whether and how the occurrence of membrane (micro)fractures and cellular jamming correlates with the timing of membrane rupture and the mode of delivery. In future measurements, this method could possibly be combined with mechanical probing techniques to compare optical and mechanical sample information.\u003c/p\u003e","manuscriptTitle":"Anatomy of the Fetal Membranes: Insights from Spinning Disk Confocal Microscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-03 14:09:09","doi":"10.21203/rs.3.rs-2644986/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-03-31T04:49:17+00:00","index":0,"fulltext":""},{"type":"editorInvited","content":"Archives of Gynecology and Obstetrics","date":"2023-03-07T14:05:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-04T03:23:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2023-03-01T18:31:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b69e8cc-bc67-4216-b269-425d83a3527e","owner":[],"postedDate":"April 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:59:52+00:00","versionOfRecord":{"articleIdentity":"rs-2644986","link":"https://doi.org/10.1007/s00404-023-07070-0","journal":{"identity":"archives-of-gynecology-and-obstetrics","isVorOnly":false,"title":"Archives of Gynecology and Obstetrics"},"publishedOn":"2023-05-15 20:52:26","publishedOnDateReadable":"May 15th, 2023"},"versionCreatedAt":"2023-04-03 14:09:09","video":"","vorDoi":"10.1007/s00404-023-07070-0","vorDoiUrl":"https://doi.org/10.1007/s00404-023-07070-0","workflowStages":[]},"version":"v1","identity":"rs-2644986","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2644986","identity":"rs-2644986","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}