Penicillin-Streptomycin

Spontaneous healing of human amnion in the premature rupture of membrane model

Ah-young Lee a,1, Ki-Jin Ryu b,1, Ki Hoon Ahn b, Dahyeon Kang a, Dong Ho Geum a, Byung-Soo Kim c,d, Geum Joon Cho b, Min-Jeong Oh b, Hai-Joong Kim b, Soon-Cheol Hong b,*

Abstract

Introduction: This study aimed to explore the spontaneous healing of ruptured fetal membranes experimentally in the prelabor rupture of membrane model using the amnion pore culture technique.
Methods: The human amniotic membrane was separated from the post-delivery term placenta in women with normal pregnancies who delivered by scheduled unlabored cesarean section and stained immunohistochemically with primary antibodies against SSEA-4, OCT-3/4, and TRA-1-60. The characteristics of the cultured amniotic epithelial cells were examined by fluorescence-activated cell sorting analysis. Amniotic membranes with perforations that were 1, 2, and 3 mm in diameter were cultured in αMEM containing 10% heat-inactivated FBS, 1% penicillin-streptomycin, and 10 ng/mL EGF at 37 C in a humidified incubator with 5% CO2. Next, the pore sizes were calculated to evaluate the healing process.
Results: The amniotic membrane stained positive for CD49d and pluripotent stem cell markers such as SSEA-4, TRA 1-60, and OCT-4 in the stromal and epithelial cell layers. In the flow cytometry analyses, the extracted amniotic epithelial stem cells were observed to express indicator markers for stem cells such as SSEA-4, OCT-4, SOX-2, and Nanog. In the amnion pore culture technique model, the 1-mm pores healed completely, whereas the 2- and 3-mm pores did not heal substantially.
Discussion: The amnion pore culture technique was useful for demonstrating the natural healing process of the human amniotic membrane. Stem cells in the human amnion might facilitate the resealing of small pores in the amniotic membrane, as observed in this model.

Keywords:
Amnion pore culture technique model
Premature rupture of membrane (PROM)
PROM model
Tissue regeneration

1. Introduction

Prelabor rupture of membrane (PROM) in pregnant women should be managed on the basis of the gestational week and the level of complications. While labor induction and prompt delivery are generally recommended for at- or near-term pregnancies with PROM, expectant management may yield better outcomes in pregnancies with preterm PROM (PPROM) at 24–34 weeks of gestation without any complications compared to those in immediate delivery [1]. With expectant management, 2.8–13% of women can anticipate the cessation of amniotic fluid leakage and the possible restoration of normal amniotic fluid volume such that preterm labor is not induced or it subsides [2]. Spontaneous resealing of the fetal membranes has been observed in up to 5% of PROM cases [3], and a small iatrogenic puncture in fetal membranes caused by amniocentesis generally heals spontaneously [4]. However, the exact probability of healing of the ruptured membranes in various clinical conditions and the underlying mechanisms remain unclear.
The amnion plays a major role in load bearing and in maintaining the integrity of the fetal membranes owing to its tensile strength, even though it is thinner than the chorion and accounts for only 20% of the thickness of fetal membranes [5]. Therefore, resealing of the damaged amnion may play a crucial role in spontaneous healing of ruptured fetal membranes, and the regenerative potential of the amnion has been considered to serve as one of the key factors in the resealing phenomena [6,7]. The amnion might possibly retain pluripotent properties that could be attributed to the amniotic epithelial stem cells (AESCs) derived from the amnion, which were shown to promote cell regeneration in previous studies [8]. However, the role of AESCs in the regeneration of the amnion and the mechanisms underlying spontaneous healing of the fetal membranes remain unknown; the lack of an efficient experimental model of PROM may be one of the major limitations in this context.
In this study, we designed a novel in vitro human PROM experimental model with human amnion using the amniotic pore culture technique (APCT). The study primarily aimed to investigate the process of spontaneous healing of the punctured amnion (with different pore sizes) in the PROM model and to determine the role of AESCs in the process.

2. Materials and methods

2.1. Amnion collection

Informed consent was obtained from women with normal pregnancies who delivered by scheduled unlabored cesarean section, under the approval of the Institutional Review Board of Korea University Anam Hospital (No.2009AN0036). A grossly intact amniotic tissues were collected aseptically from the membranes covering or from an area adjacent to the placentas immediately after cesarean section in cases of full-term pregnancy without any complications. All manipulations were performed under sterile conditions.

2.2. Isolation and culture of AESCs

Human AESCs were isolated as previously described [9]. Briefly, the amniotic membrane was separated from the chorion membrane and washed with PBS (Invitrogen, Carlsbad, CA, USA) several times to remove the residual blood. Thereafter, the amnion was diced with sterile scissors in a 50 mL conical tube, and 0.05% trypsin-EDTA (Life Technologies Corporation, NY, USA) was added to it. This mixture was incubated at 37 C for 10 min and centrifuged at 1800g for 8 min. The supernatant was discarded, and the pellet was suspended in 0.05% trypsin-EDTA and was further incubated at 37 C for 40 min for digestion. Next, serum-containing media was added to terminate the digestive process. The mixture was then filtered through a 100 μm nylon mesh and centrifuged at 1800g for 5 min. The cell pellet was resuspended in Eagle’s α-minimum essential medium (αMEM; WELGENE, Gyeongsangbuk-do, Republic of Korea) containing 10% heat-inactivated fetal bovine serum (FBS; Welgene), 1% penicillin-streptomycin (Welgene), and 10 ng/mL epithelial growth factor (EGF; R&D Systems, Minneapolis, MN, USA). The cells were incubated at 37 C in a humidified incubator supplied with 5% CO2. The media was replaced every 2–3 days until the cells achieved 90% confluence.

2.3. Flow cytometry (fluorescence-activated cell sorting)

The characteristics of the cultured amniotic epithelial cells were assessed by fluorescence-activated cell sorting (BD FACSCalibur, BD Biosciences). The cells were harvested and fixed with 4% paraformaldehyde (PFA; Biosesang) for 10 min and then blocked with 1% bovine serum albumin (BSA, Bovogen, Australia) solution for 20 min. The cells were then incubated with fluorescence-conjugated monoclonal antibodies (phycoerythrin (PE)-conjugated mouse anti- CD19, CD34, CD73, CD90, CD105, CD324, SSEA-4, OCT-3/4, SOX-2, Nanog, and Alexa 647-conjugated anti- CD349, Biolegend, 1:50, San Diego, CA, USA) for 30 min at 4 C in the dark. After washing the cells several times with PBS, the cells were examined under FACSCalibur (BD Bioscience). At least 10,000 cell events were recorded for each sample for further quantification.

2.4. Immunofluorescence

The amniotic membrane and AESCs were fixed with 4% PFA for 10 min and blocked with 3% BSA (Bovogen, Australia) in PBS. After washing with PBS, the amniotic membrane and AESCs were probed with the following primary antibodies: mouse anti-TRA-1-60 (1:200, Cell Signaling Technology, Danvers, MA, USA), mouse anti-SSEA4 (1:200, R&D systems), rat anti-Oct-3/4 rabbit (1:200, R&D systems) overnight at 4 C. The cells were then probed with Alexa Fluor 488 anti-mouse immuno-globulin G (IgG), Alexa Fluor 594 anti-rabbit IgG, or rat Alexa Fluor 594 anti-IgG (Life Technologies, CA, USA) (1:1000 in blocking solution) for 60 min. For counter staining, the cells were incubated with 4ʹ,6-diamidino-2-phenylinodole (1:1000, DAPI, Life Technologies) for 5 min in dark. The cells were preserved in the fluorescence mounting medium and imaged under a fluorescence microscope (Olympus, Japan).

2.5. Observation of spontaneous healing using APCT

The amniotic membranes were separated from the term placentas and washed several times with PBS to remove the residual blood. The amniotic membranes were perforated with an 8 mm diameter disposable biopsy punch (Kai Medical, Japan) on a glass plate containing 1X PBS. Amniotic membrane tissues (8 mm in diameter) were then re-punctured with a disposable biopsy punch (1, 2, and 3 mm in diameter). Amniotic membrane tissues isolated by this repeated procedure were placed and dried in 24-well plates for attachment to the wells. The tissues were cultured in αMEM with 10% heat-inactivated FBS, 1% penicillin- streptomycin (Welgene), and 10 ng/mL EGF at 37 C in a humidified incubator supplied with 5% CO2. The media was replaced every 2 days during the culture period. For observation of spontaneous healing using the APCT model, a pore was imaged under an optical microscope (Olympus) using the TOMORO AcquCAM3 program. The captured images were analyzed using Image J (Broken Symmetry software). (Fig. 1).

2.6. Statistical analysis

All data are expressed in terms of mean standard error (SE). ANOVA was performed to compare the differences between the groups. Sphericity was tested using Mauchly’s test. We performed a multivariate ANOVA when sphericity was violated. Kruskal Wallis multiple comparison tests were performed using SPSS v21.0. p < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of the amniotic membrane During amniotic tissue culture, epithelial-like cells proliferated and shifted from the bottom layers of the tissue and attached to the plate (Fig. 2a). These cells had an epithelial-like morphology and proliferated in the form of a colony. These cells expressed CD49d (marker of integrin alpha subunit), which was also found to express in the amniotic mesenchymal cells (Fig. 2c). When the cells were collected and subcultured, the morphology gradually changed to that of mesenchymal- like cells (Fig. 2b). The cells that proliferated from the amnion were thought to possess characteristics of both epithelial and mesenchymal cells. 3.2. Characterization of isolated amniotic epithelial stem cells We experimentally verified that the stem cell markers TRA-1-60, SSEA-4, and OCT-3/4 were expressed in the freshly isolated amniotic membrane (Fig. 3). These results indicate that the amniotic membrane has an abundance of stem cells. 3.3. Spontaneous healing of pores in the APCT model We further studied the healing of amniotic tissue with different pore sizes in the APCT model. To evaluate the natural healing process of amniotic tissues, we used four independent placentas. After the isolation of the amniotic membrane from the placenta, a pore of 8 mm diameter was punched using a disposable biopsy punch, in which pores of 1, 2, and 3 mm diameter were punched further. The punched tissues were cultured for 22 days in an incubator (Fig. 1). We observed cellular regrowth in the punched amniotic membrane tissue that covered the pore area within 10 days of incubation in all three groups (Fig. 4a). Yellow dotted lines indicate the sites of tissue regeneration. The area of pores created with a 1-mm punch reduced significantly in size in a time-dependent manner (p < 0.05). Although the areas of 2- and 3-mm pores also reduced during the culture period, the reduction was not significant. The pore size reduction differed significantly in each group (Fig. 4b). To analyze the characteristics of the cells that proliferated within the pore, staining was performed using TRA-1-60, SSEA-4, and OCT-3/4. The cells were observed to express stem cell markers, which indicated that these cells were associated with the regeneration of amniotic tissues (Fig. 5). In the APCT model, we observed that the 1-mm pore in the amniotic tissue was spontaneously covered with grown cells within 10 days and its area reduced significantly by day 22. Conversely, although the medium- (2-mm) and large-sized (3-mm) pores in the amniotic tissue were (A) Macroscopic images of amniotic tissues with 1-, 2-, and 3-mm pores. The punched area (yellow dotted line) was measured using Image J ( 40). Scale bars: 20 μm 1 mm (n ¼ 16), 2 mm (n ¼ 16), 3 mm (n ¼ 14). (B) The pore sizes were measured for 22 days using ImageJ. Area of pores in amnion with 1-, 2-, and 3-mm punch at 1, 10, and 22 days of culture. Each symbol represents the area of the pore in a single rupture. The area 1-mm (white dots), 2-mm (gray dots), and 3-mm (black dots) pores were reduced significantly during the culture period. The blue bar represents mean SEM. *: P < 0.05, repeat ANOVA and paired t-test. Error bars represent SEM. Stem cell-like characters of cells that were proliferating in amniotic tissues with pores (3 mm) at 22 days. a) TRA-1-60 (green), b) SSEA-4 (green), and c) OCT-3/4 (red) were expressed in cells derived from amniotic tissues with pores. Scale bars: 100 μm. The nucleus was counterstained with DAPI (blue). covered within 10 days, the pore sizes did not decrease significantly (Fig. 4b). During the culture period, the cells that originated from the amniotic tissue consisted of both epithelial and mesenchymal cells. Once the pores were covered, the cells formed multiple layers within the pore. After the healing process, the cells present in the pores were found to test positive for the stem cell markers TRA-1-60, SSEA-4, and OCT-3/4 (Fig. 5). 4. Discussion In this study, we developed a novel in vitro APCT system using human amnion samples and observed a significant reduction in the size of a small pore (1 mm in diameter) without any treatment, which suggests the possibility of spontaneous healing of ruptured fetal membranes. Both epithelial and mesenchymal cells were observed to grow in the pore in the APCT model, and we also detected stem cells among other cell types by immunofluorescence analysis using human embryonic stem cell markers, including TRA 1-60, SSEA-4, and OCT-4 [10]. Our results indicate that stem cells in the human amnion might facilitate the spontaneous resealing of small pores in the fetal membranes, although the exact mechanisms remain unclear. In this study, we demonstrated that the spontaneous healing of a sterile ruptured amniotic membrane is dependent on the size of the pore. There was no significant change in the size of the large pores (2 and 3 mm in diameter), although the diameter of the small-sized pore (1 mm) was reduced significantly in the APCT model. A previous study conducted on an in vivo PROM animal model also revealed the regenerative potential of the amnion, which was also restricted to the small pores [6]. A mouse model of sterile rupture of fetal membranes was developed by puncturing amniotic membranes with 26 or 20 G needles (diameter: 0.47 or 0.91 mm), and it was shown that almost 100% of the small ruptures healed, whereas the majority of the large ruptures did not [6]. These findings are consistent with the clinical findings, which suggests that damage to the fetal membranes after amniocentesis does not lead to amniorrhexis in most cases, whereas spontaneous resolution rarely occurs in case of a larger rupture caused by fetoscopy [11]. The authors of the aforementioned animal study suggested that the mechanism of the healing of the fetal membranes includes recruitment of macrophages from the amniotic fluid and epithelial-mesenchymal transition, which is also observed in wound healing and tissue regeneration [12]. A large pore size would limit the healing of the membrane primarily because of the challenges in mesenchymal cell transition and migration. Therefore, resealing may be more difficult in actual cases of PROM in which the tears in fetal membranes are often ragged and pulled apart in three dimensions, and are not neatly pricked as in this study. The possibility of cell migration might remain if the ruptured membranes slide over each other, which would reduce the distance between them. The presence of stem cells among other proliferative cells in the pore of the APCT model indicates that these cells might play a vital role in the regeneration of amnion and spontaneous healing of PROM. Stem cells that originate from the amniotic membrane include AESCs and amniotic mesenchymal stem cells, both of which can differentiate into multiple cell lineages [13,14]. Human AESCs offer several advantages over other stem cells and have drawn considerable attention from researchers because these are easily available (can be collected non-invasively after delivery), their collection poses no ethical concerns, these have limited risks of tumorigenicity and immunogenicity, and accumulating evidences have revealed that AESCs can also promote cell survival and regeneration [8]. These cells have already been utilized in various stem cell-based therapies for skin-regeneration, chronic wound healing, and corneal regeneration therapy [15–17]. Based on the promising applications of AESCs and the results of our study, we hypothesized that the stem cell-like activity of AESCs induces amnion regeneration and could be the mechanism underlying membrane resealing. Conversely, amniotic connective tissue contains a large number of fibroblasts/myofibroblasts that exhibit mesenchymal stem cell-like characteristics in vitro, and we detected mesenchymal cells in the pore in the APCT model. Amniotic fibroblasts/myofibroblasts may play an important role in the natural healing process of fetal membranes, as these cells are commonly known to play critical roles in wound healing and are involved in creating new extracellular matrix and collagen structures to support other cells associated with effective wound healing, as well as in contracting the wound area [18]. Further investigation is warranted to determine the exact relationship between amniotic stem cells, fibroblasts, and membrane healing. The physiological rupture of the fetal membranes occurs as a consequence of different factors, including mechanical modifications, remodeling of extracellular matrix components, and apoptosis of amniotic cells; however, the exact mechanism remains unclear [19]. Because the amnion has poor vascularization, agglutination of blood in clots and epithelial wound healing, which occurs in other connective tissues (e.g., skin), would not be applicable to the resealing of the damaged amnion [20]. Instead, the rupture in the amnion might be concealed by the attachment of the membrane to the decidua (by one sliding over another) or by contraction and scarring in the myometrial and decidual layers of the uterus, rather than due to active healing of the amnion itself [11]. However, the spontaneous healing capacity of the ruptured fetal membranes has been demonstrated in several studies [21], and the phenomenon of spontaneous regeneration of the amnion was also confirmed in our APCT model. The possible mechanisms underlying amnion regeneration may include the activity of aforementioned stem cells, macrophage recruitment, and epithelial-mesenchymal transition [6]. However, it is unclear whether the regeneration of the amniotic membrane observed in the small-sized pore of the APCT model in this study involved complete healing facilitated by regenerated amniotic epithelial cells or simply sealing by other cells originating from amniotic stem cells for healing the rupture. Further study involving transmission electron microscopic observation of the recovered fetal membranes may be helpful for addressing these issues. The use of various materials have been proposed for the treatment of PROM, including collagen plugs, fibrin glue, amniopatches, cryoprecipitates, and polytetrafluoroethylene; however, none of these have been proven to be effective thus far [22]. Although several animal models of PROM, including rodent or rabbit models, have been developed and studies on these have yielded valuable results [23,24], the proposed APCT model using human amnion might be useful for investigating the mechanism of spontaneous healing of fetal membranes as well as for identifying a suitable treatment for PROM. The resealing of a small pore in the human amniotic membrane was observed in this novel PROM model developed using APCT, which may partly replicate the process of spontaneous healing in PROM or PPROM cases. In this model, we also demonstrated that stem cell recruitment may contribute to the spontaneous healing of human amnion. However, the present study also has several limitations. First, this is an in vitro experimental model designed to investigate the regeneration of human amniotic membrane and it does not completely replicate the characteristics of PROM. We used a grossly intact amniotic tissue that was collected aseptically from the membrane covering or from an area adjacent to the placenta after cesarean section in cases of full-term pregnancy without any complications to avoid the effects of any possible infection. However, spontaneous PROM is known to occur predominantly in the cervical region of the uterus [25], possibly because the part of the fetal membranes overlying the cervix is weaker at term, as indicated by evidences of extracellular matrix degradation and/or inflammatory changes [26,27]. Further investigations with applications of these factors are required to confirm our findings. Second, membrane resealing was observed only in the small-sized (1 mm in diameter) pores after 22 days, and this finding may not exactly correspond to the healing of fetal membranes in all cases of PROM, because some cases could be characterized by large, irregular-shaped tears or infections. Despite this limitation, our study provides an evidence of spontaneous healing of the amniotic membrane, and it also can used for advanced research in this field. Third, our study did not consider the effects of the amniotic fluid. As the amnion does not contain blood vessels, the required nutrients and oxygen diffuse from the amniotic fluid [5]. In addition, the pressure of the amniotic fluid on the membrane in the amniotic sac might also affect the healing process. Further modifications can be introduced to our APCT model to verify the effects of the amniotic fluid on the healing of PROM. Fourth, although infections (e.g., chorioamnionitis) are one of the major causes of PROM, this was not recreated in this APCT model [28]. The exact mechanisms underlying PROM and PPROM remain unclear; thus, additional research should be conducted along with investigations of the healing mechanism of ruptured fetal membranes in future. 5. Conclusion APCT using human amnion is an efficient in vitro system for demonstrating the healing of amniotic membrane, and this study depicted the spontaneous regeneration and resealing of the pore in the amnion using the PROM model with APCT. The presence of stem cells among other proliferative cells in the pore of the amnion, as observed in this model, suggests that stem cell recruitment and activation may contribute to the spontaneous healing of ruptured fetal membranes in cases of PROM. Various cell culture and tissue regeneration experiments can be performed in the fetal membranes using APCT. Further studies are warranted on the treatment of PROM using candidate medications or materials, and APCT would serve as a helpful tool in such investigations. Funding This research was supported by a grant of Korea University Anam Hospital, Seoul, Republic of Korea [grant number K1809791] and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology [grant number 2011-0009551]. References [1] D.M. Bond, P. Middleton, K.M. Levett, D.P. van der Ham, C.A. Crowther, S. L. Buchanan, et al., Planned early birth versus expectant management for women with preterm prelabour rupture of membranes prior to 37 weeks’ gestation for improving pregnancy outcome, Cochrane Database Syst. Rev. 3 (2017), CD004735. [2] J.W. Johnson, R.S. Egerman, J. Moorhead, Cases with ruptured membranes that "reseal, Am. J. Obstet. Gynecol. 163 (1990) 1024–1030. ; discussion 30–32. [3] J.L. Schucker, B.M. Mercer, Midtrimester premature rupture of the membranes, Semin. Perinatol. 20 (1996) 389–400. [4] A.F. Borgida, A.A. Mills, D.M. Feldman, J.F. Rodis, J.F. Egan, Outcome of pregnancies complicated by ruptured membranes after genetic amniocentesis, Am. J. Obstet. Gynecol. 183 (2000) 937–939. [5] S. Parry, J.F. Strauss 3rd, Premature rupture of the fetal membranes, N. Engl. J. Med. 338 (1998) 663–670. [6] H. Mogami, A. Hari Kishore, Y. Akgul, R.A. Word, Healing of preterm ruptured fetal membranes, Sci. Rep. 7 (2017), 13139. [7] S. Maxson, E.A. Lopez, D. Yoo, A. Danilkovitch-Miagkova, M.A. Leroux, Concise review: role of mesenchymal stem cells in wound repair, Stem. Cells. Transl. Med. 1 (2012) 142–149. [8] H. Xu, J. Zhang, K.S. Tsang, H. Yang, W.Q. Gao, Therapeutic potential of human amniotic epithelial cells on injuries and disorders in the central nervous system, Stem Cell. Int. (2019), 5432301. [9] T. Miki, F. Marongiu, K. Dorko, E.C. Ellis, S.C. Strom, Isolation of amniotic epithelial stem cells, Curr. Protoc. Stem. Cell. Biol. 12 (2010) 1E.3.1–1E.3.10. [10] J. Cai, W. Li, H. Su, D. Qin, J. Yang, F. Zhu, et al., Generation of human induced pluripotent stem cells from umbilical cord matrix and amniotic membrane mesenchymal cells, J. Biol. Chem. 285 (2010) 11227–11234. [11] E. Gratacos, J. Sanin-Blair, L. Lewi, N. Toran, G. Verbist, L. Cabero, et al., A histological study of fetoscopic membrane defects to document membrane healing, Placenta 27 (2006) 452–456. [12] R. Kalluri, R.A. Weinberg, The basics of epithelial-mesenchymal transition, J. Clin. Invest. 119 (2009) 1420–1428. [13] P.H. Wang, B.S. Huang, H.C. Horng, C.C. Yeh, Y.J. Chen, Wound healing, J. Chin. Med. Assoc. 81 (2018) 94–101. [14] G. Castellanos, A. Bernabe-Garcia, J.M. Moraleda, F.J. Nicolas, Amniotic membrane application for the healing of chronic wounds and ulcers, Placenta 59 (2017) 146–153. [15] T.J. Koob, R. Rennert, N. Zabek, M. Massee, J.J. Lim, J.S. Temenoff, et al., Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing, Int. Wound J. 10 (2013) 493–500. [16] T. Celik, Y.A. Katircioglu, E. Singar, M. Kosker, K. Budak, R. Kasim, et al., Clinical outcomes of amniotic membrane transplantation in patients with corneal and conjunctival disorders, Penicillin-Streptomycin Semin. Ophthalmol. 28 (2013) 41–45.
[17] N. Arai, H. Tsuno, M. Okabe, T. Yoshida, C. Koike, M. Noguchi, et al., Clinical application of a hyperdry amniotic membrane on surgical defects of the oral mucosa, J. Oral Maxillofac. Surg. 70 (2012) 2221–2228.
[18] P. Bainbridge, Wound healing and the role of fibroblasts, J. Wound Care 22 (2013) 407–412.
[19] I. Osman, A. Young, F. Jordan, I.A. Greer, J.E. Norman, Leukocyte density and proinflammatory mediator expression in regional human fetal membranes and decidua before and during labor at term, J. Soc. Gynecol. Invest. 13 (2006) 97–103.
[20] G. Bourne, The foetal membranes. A review of the anatomy of normal amnion and chorion and some aspects of their function, Postgrad. Med. 38 (1962) 193–201.
[21] V. Phupong, P. Ultchaswadi, Spontaneous reseal of ruptured membranes after genetic amniocentesis, J. Med. Assoc. Thai. 89 (2006) 1033–1035.
[22] C.M. Haller, W. Buerzle, A. Kivelio, M. Perrini, C.E. Brubaker, R.J. Gubeli, et al., Mussel-mimetic tissue adhesive for fetal membrane repair: an ex vivo evaluation, Acta Biomater. 8 (2012) 4365–4370.
[23] D. Sopher, The response of rat fetal membranes to injury, Ann. R. Coll. Surg. Engl. 51 (1972) 240–249.
[24] A.S. Mallik, M.A. Fichter, S. Rieder, G. Bilic, S. Stergioula, J. Henke, et al., Fetoscopic closure of punctured fetal membranes with acellular human amnion plugs in a rabbit model, Obstet. Gynecol. 110 (2007) 1121–1129.
[25] R. Devlieger, L.K. Millar, G. Bryant-Greenwood, L. Lewi, J.A. Deprest, Fetal membrane healing after spontaneous and iatrogenic membrane rupture: a review of current evidence, Am. J. Obstet. Gynecol. 195 (2006) 1512–1520.
[26] J. McLaren, D.J. Taylor, S.C. Bell, Increased concentration of pro-matrix metalloproteinase 9 in term fetal membranes overlying the cervix before labor: implications for membrane remodeling and rupture, Am. J. Obstet. Gynecol. 182 [28] H. Wang, S. Parry, G. Macones, M.D. Sammel, P.E. Ferrand, H. Kuivaniemi, et al., (2000) 409–416. Functionally significant SNP MMP8 promoter haplotypes and preterm premature
[27] D. Kumar, R.M. Moore, B.M. Mercer, J.M. Mansour, R.W. Redline, J.J. Moore, The rupture of membranes (PPROM), Hum. Mol. Genet. 13 (2004) 2659–2669. physiology of fetal membrane weakening and rupture: insights gained from the determination of physical properties revisited, Placenta 42 (2016) 59–73.