Second harmonic generation microscopy of fetal membranes under deformation: Normal and altered morphology
Introduction
The integrity of the fetal membrane (FM) is essential during pregnancy in order to avoid preterm delivery. The preterm premature rupture of membrane affects about 3% of pregnancies and causes around 25–30% of all preterm deliveries [1]. A better understanding of the microstructure of FM, its alterations and its behaviour under deformation broaden the understanding of the mechanisms leading to premature mechanical rupture [2]. In addition, the increasing interest in the amniotic membrane as scaffold for tissue-engineered implants [3], [4], [5], [6] and as in vitro biological models [7], [8] further motivates the present investigation.
The microstructure of amnion and chorion was previously studied by using confocal light microscopy [9], [10], [11], [12], [15] and electron microscopy [11], [13], [14], [15] to identify membrane's sub-layers, collagen types, cellular and extracellular components. A zone of altered morphology (ZAM) was identified over the cervix in term FM both pre labour [16] and post labour [17], [18]. The ZAM tissue shows swelling of the amnion and reduced thickness of the choriodecidua compared to the reflected tissue (between the placental border and ZAM) [16], [21]. These microstructural alterations were considered to be a consequence of biochemical factors, such as a local increase of matrix metalloproteinases [19], [20] and poly(ADP-ribose) polymerase cleavage [21], which influence collagen remodelling and cellular apoptosis, respectively.
The microstructure of FM could not be visualized under deformation due to limitations of the used methods. Nonlinear microscopy techniques, such as second harmonic generation (SHG) and multiphoton microscopy, allow the visualization of collagen and elastin structures without fixation and staining of the tissue [22]. Moreover, the two photon excitation can penetrate deeper into the tissue allowing imaging of relatively thick samples. Thus, this new technique lends itself to investigate the response of extracellular components under deformation, e.g. for porcine arterial tissue [23] or rat-tail tendons [24].
Acute in-vitro stretching of amniotic epithelial cells and FM has been shown to enhance expression of cytokine (interleukin8 and Visfatin) that are usually up-regulated in association with labour [25], [26], [27]. Repeated physiological mechanical stimuli affect mechanical properties of FM [28], [29], but their effect on the microstructure has not been investigated yet.
In this study, for the first time SHG microscopy was used to investigate the normal and altered morphology of FM. This technique was combined with an in-situ stretching device to visualize and quantify collagen alignment under uniaxial tension. Comparison of reflected, ZAM and cyclically inflated tissue addressed the hypothesis that repeated stretching of FM over the cervix might contribute to the morphological alterations observed in the ZAM.
Section snippets
Samples procurement and preparation
Fetal membranes (n = 8) were collected from patients who underwent elective caesarean sections at 38 gestational weeks. Patients were recruited with informed written consent using a protocol approved by the Ethical Committee of the District of Zürich (study Stv22/2006). The selected pregnancies had no labour contractions prior delivery, no preterm rupture of the membrane, no diabetes mellitus and were negative for streptococcus B, HIV, hepatitis A and B, chlamydia and cytomegaly. Membranes were
Membrane morphology
Nonlinear laser scanning microscopy revealed the microstructure of FM. Fig. 2 shows layers of one representative sample of reflected FM. Under the amniotic epithelium, collagenous structures are visible and different layers can be identified by the presence (fibroblast layer, reticular layer) or absence (compact layer, spongy layer) of cellular nuclei. The amnion is characterized by a homogeneous compact layer of collagen on top of the fibroblast layer. The chorion, mainly composed by
Discussion
Second harmonic generation microscopy was used for the first time to visualize the microstructure of FM and its changes under deformation. Second harmonic generation in combination with our custom-built stretching device allows the investigation of biological membranes in their physiological hydration and without chemical fixation, which is the great advantage of this technique compared to electron microscopy. Nonlinear laser scanning microscopy allows the simultaneous investigation of collagen
Acknowledgement
The authors are grateful to the Swiss National Science Foundation (SNSF) for financial support (Project number: 205321-134803/1 and 32003B-124925/1) and to the team of Prof. Stahel (Seminar for Statistic, ETH Zurich) for statistical advice. The authors would like to thank Charlotte Burger and Theresa Lehmann (Institute of Anatomy, University of Zurich) for providing the histological sections and Raoul Hopf for the development of the stretching device.
References (43)
Preterm premature rupture of the membranes
Am Col Obstet Gynecol
(2003)- et al.
In vitro simulation of placental transport: part I. Biological model of the placental barrier
Placenta
(2013) - et al.
Confocal immunofluorescence localization of collagen types I, III, IV, V and VI and their ultrastructural organization in term human fetal membranes
Placenta
(1993) - et al.
Laser scanning confocal examination and comparison of nidogen (entactin) with laminin in term human amniochorion
Placenta
(1994) - et al.
Type VII collagen associated with the basement membrane of amniotic epithelium forms giant anchoring rivets which penetrate a massive lamina reticularis
Placenta
(2013) - et al.
Increased concentration of pro-matrix metalloproteinase 9 in term fetal membranes overlying the cervix before labor: implications for membrane remodelling and rupture
Am J Obstet Gynecol
(2000) - et al.
Fetal membrane distention: I. Differentially expressed genes regulated by acute distention in amniotic epithelial (WISH) cells
Am J Obstet Gynecol
(2000) - et al.
Fetal membrane distention: II. Differentially expressed genes regulated by acute distention in vitro
Am J Obstet Gynecol
(2000) - et al.
Contractions, a risk for premature rupture of fetal membranes: a new protocol with cyclic biaxial tension
Med Eng Phys
(2013) - et al.
Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity
Placenta
(1997)
Human fetal membranes: a source of stem cells for tissue regeneration and repair?
Placenta
Fetal membrane distention: determination of the intrauterine surface area and distention of the fetal membranes preterm and at term
Am J Obstet Gynecol
Separation of amnion from choriodecidua is an integral event to the rupture of normal term fetal membranes and constitutes a significant component of the work required
Am J Obstet Gynecol
A comparative biomechanical analysis of term fetal membranes in human and domestic species
Am J Obstet Gynecol
Myofibroblast differentiation in the connective tissues of the amnion and chorion of term human fetal membranes – implications for fetal membrane rupture and labour
Placenta
On the deformation behavior of human amnion
J Biomech
Branching toughens fibrous networks
J Mech Behav Biomed
Microstructure and mechanics of the chorioamnion membrane with an emphasis on fracture properties
Ann N Y Acad Sci
In vitro and in vivo characterization of iris pigment epithelial cells cultured on amniotic membranes
Mol Vis
Amniotic membrane use in dermatology
Int J Dermatol
Human amniotic membrane as a delivery matrix for articular cartilage repair
Tissue Eng
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These two authors contributed equally.