Does Placenta donors’ age significantly impact on amniotic membrane quality?

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Marco Campagnaro, Roberto Pasqualini, Niccolò Stefanato, Rudy De Vettor, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7799881/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Placenta is a very versatile tissue in the field of transplantation also thanks to the fact that, since there is no upper limit to the age of placental donors, is a really useful and easy tool to manage in different tissue banks. Several tissues can be extracted from the placenta as it is made up of various types of membranes and having a heterogeneous population of cells; among these tissues, the most used and studied in the field of transplantation is the amniotic membrane, which is really useful in different kind of surgeries having a relevant role in skin wound repair, in ophthalmic surgery and in maxillofacial surgery. In this study we asked ourselves if the lack of an upper age limit to placenta donors lead to a virtuous process in which the retrieving and processing costs are justified by the quality of the AM extracted from them in an age-independent manner: we started from a database of 2002 placenta donors collected from 2018 until 2024 and we have analyzed a possible correlation between the age of placenta donors and the quality of the amniotic membrane extracted from them, highlighting a statistically significant correlation between them. Furthermore, this statistical study showed that the amniotic membranes of donors with an age from 30 to 40 years have surprisingly the same quality as donors with an age from 40 to 50 which both differ from donors with an age below 30 and that therefore they can be divided into exactly two separate groups: donors under 30 and donors over 30. Amniotic membrane statistics placenta age vs quality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Amniotic membrane (AM) is a thin membrane located in the innerside of the placenta, next to the fetus. It comprises three major histological structures: an epithelial monolayer, a thick basement membrane, and an avascular stroma in contact with the underlying chorion. The innermost epithelium layer, facing the fetus, in direct contact with the amniotic fluid, consists of a single layer of cuboidal epithelial cells with apical microvilli uniformly arranged on the basement membrane, which help in the absorption and secretion of solutes and water (Ingraldi AL, Audet RG et al. 2023). Due to specific properties (such as anti-inflammatory, anti-viral, anti-bacterial and promoter of epithelization) the AM is used in different kind of surgeries for various applications including treatment of skin burns, ocular surface reconstruction and as a biological barrier for improving the natural scarring process in wound healing (Mamede AC, Carvalho MJ et al. 2012; Jirsova K, Jones GLA 2017); moreover, the AM is the only tissue for which no age limit is currently set for its retrieval and for the processing and it has no limits dictated by ethical reasons, which makes it easy to retrieve for many tissue banks in every country. There are several variables that contribute to define the quality of the amniotic membrane in the therapeutic field, not only based on the processing method or on the properties that the amniotic membrane acquires as a result of various kinds of molecules to which it is exposed to reach the desired end product, which can be decellularized amniotic membrane, cryopreserved, lyophilized, freeze dried, injectable or a combination of at least two of them (Mamede AC, Carvalho MJ et al 2012).These variables are not all well defined and the effects on the quality and the properties of AM are not yet been fully established. Here we report a statistical analysis that correlate the quality of the amniotic membrane extracted from placentas from different donors since 2018 and the donors age. MATERIAL AND METHODS All 2002 human placentas donors since 2018 from our database were screened for age criteria and quality of the amniotic membrane. The placentas were sourced from elective cesarean sections after obtaining written informed consent in hospitals belonging to our tissue bank procurement network. Donors were selected on the basis of strict criteria that also include guidelines for harvesting, processing and distributing tissues for transplantation as approved by the National Transplant Centre. Selection criteria included the absence of any kind of malignancy, infant malformation or pathology, a gestation period of at least 35 weeks, negative family medical history for genetic diseases, and lifestyles of both parents not at risk for infectious diseases. On arrival at the bank the tissues were anonymized with a unique code number used for all processing phases. Working in sterile conditions in a laminar flow cabinet within 24 h of tissue retrieval, HAM was carefully detached from the chorion and rinsed with saline solution to remove blood clots. All the procedure were performed only by qualified personnel. From this database we removed all donors rejected for reasons not linked to the quality of the amniotic membrane (such as meconium-stained amniotic membranes or placentas not well stored after retrieval or donors anamnestic reasons) and we obtained the final database composed of 1323 different donors. We then divided all donors in two groups: the one with amniotic membranes rejected for low quality of the tissue immediately after the extraction from placentas like, for example, yellow-stained membranes, AM with fragile features such as large areas with massive perforations or membranes too much attached to the corion (group 0) and the ones admitted to further processing (group 1). RESULTS From the whole database we divided the total population in two groups, group 0 (discharged AMs) and group 1 (admitted to further processing AMs) respectively, and we calculate the mean and median age and the standard deviation from each one of them (Table 1 ). Table 1 characteristics of the total population, group 0 and group 1. In here we report even minimum age, maximum age and standard deviation of each group Mean age Median age Standard deviation Minimum age Maximum age Donors number Total population 34,15 34 4,852 19 50 1323 Group 0 34,602 35 4,70 21 50 453 Group 1 33,919 34 4,84 19 49 870 We then take the donors from each of the two groups and see the distribution based by age (Fig. 1 ). In Fig. 2 we can appreciate the difference in age from donors in the two groups (p < 0,05). Starting from the whole data, we divided the entire population in 3 subgroups based on the donors age: under 30, 30–40 and over 40 to find out how if there are any major differences. We also calculate the ratio between group 1 and group 0 population in each of the sub-groups created and the percentage of group 0 amniotic membrane in each sub-group on the total population (R) (Table 2 ). Table 2 characteristics of total population of placenta donors divided in 3 groups based on age. We can appreciate the difference in R rate between the under 30 group and the 30–40 and over 40 group, with the last two that have a really similar rate. Group 1 Group 0 Group1/Group0 rate R Under 30 166 60 2,77 0,265 30–40 633 348 1,82 0,354 Over 40 71 45 1,57 0,387 We then analyze the two subgroups created and the total population highlighting the distribution of each group of donors based by age (Fig. 3 ) Figure4 comparative study of R rate between under 30 and over 30 group. (***) indicates a p-value <0,001 Looking at the R rate of the 3 age groups in which the donors were grouped, we realized that the 30–40 age group and the over 40 group had very similar R rates. Based on the assumption that the two groups were equal for that parameter, we wondered if there was a statistically significant difference between the R rate of the under 30 group and that of the over 30 group. To make our analysis strong, we considered only the age groups that had at least 10 donors within them and that had a normal distribution; for this reason, the age group considered is from 23 to 42 years old. As described by Fig. 4 we see that the difference between the mean and the median of the rate R between the two groups is very high and with considerable significance (p < 0.001). The distribution of the rate R varies between the different age groups (Fig. 5 ). The complete data from both populations are listed below: Table 3 characteristics of rate R of donors between age 23–29 and age 30–42 Mean Median Standard deviation Age 23–29 0,286 0,285 0,039 Age 30–42 0,367 0,365 0,025 DISCUSSION Placenta is a really useful tissue for a lot of tissue banks. Due to the lack of an upper limit age placenta is really easy to retrieve and has no ethical issue that donation of other tissues have. With this study we demonstrate that the lack of the age limit of placenta donation makes sense in the context of tissue banks as there are small percentage differences between the discard rate of donors under 30 and donors over 30, thus making the process of donation and processing of the amniotic membrane efficient regardless the age of the donors. The amniotic membrane is a very versatile tissue, which can be subjected to various types of processing, storage and decontamination methods that has shown to not affect its beneficial properties (Paolin A, Trojan D et al. 2016 , Rodríguez-Ares MT, López-Valladares MJ et al. 2009). The comparison between the quality of the amniotic membrane and the age of placental donors is an aspect that has not yet been fully explored: for example, Valladares et al. tried to establish whether there was a correlation between the content of total proteins and growth factors and the age of the donors and the gestation time. In the article they found that the concentration of bFGF, HGF, KGF, NGF, and TGF-β1 was lower in older donors than in younger ones highlighting a possible age-related effect. The study was limited to 13 placentas so the population was not so wide. It’s also the only study, as far as we know, that analyzes the possible correlation between donor age and the quality of the amniotic membrane (López-Valladares MJ, Teresa Rodríguez-Ares M et al. 2010). This study tries to shed a light on this field of investigation that is still little known. Although the statistical analysis is affected by some biases (such as the quality of the amniotic membrane which is evaluated by each operator without the use of an instrument, like for example a microscope, and therefore not from measurable values) preliminary evaluations can be made on a possible effect of age on the quality of the amniotic membrane. First of all, the analysis showed a statistically significant difference between the age of donors in group 0 and group 1. If we then shift our attention to the percentage of discarded amniotic membranes out of the total of each subgroup created, we note that there is a marked difference between the incidence of discarded amniotic membranes in donors under the age of 30 compared to both the 30–40 group and the over 40 group, with the latter two that we can surprisingly consider as one group based on these data. The R rate between under 30 and over 30 donors show a statistically significant difference, although the analysis cover the age range between 23 and 42 due to lack of consistent data from age below 23 and above 42. Although further verification from a molecular point of view seems to be very useful, all together the data presented in this study show that there is a strong correlation between the age of placenta donors and the quality of the amniotic membrane extracted from them in order to be able to subject it to further processing. Declarations Funding No funds, grant or other support was received Confict of interests The authors have no competing interests to declare that are relevant to the content of this article. Ethical approval The retrieval of human tissues are authorized by national and regional competent authorities. This study does not contain any sensitive donor information. The consent for tissue retrieval was obtained from the tissue donor according with the Italian national regulation on tissue banking. Consent to Publish declaration All authors agreed with the content and all gave explicit consent to submit. Informed consent all participants signed an informed consent agreement. The study has been approved by the ethical territorial committee of Area Nord Veneto. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modifed the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by-nc-nd/4.0/ Author Contribution MC wrote the manuscript and analyze the dataRP prepared the dataNS collected and prepared the dataRDV collected dataGM revise the manuscript and support ethical issueDT revise the manuscript and correct the data References Ingraldi AL, Audet RG, et al. The Preparation and Clinical Efficacy of Amnion-Derived Membranes: A Review. J Funct Biomater. 2023;14(10):531. doi: 10.3390/jfb14100531 . Mamede AC, Carvalho MJ et al. Amniotic membrane: from structure and functions to clinical applications. Cell Tissue Res. 2012;349(2):447–58. doi: 10.1007/s00441-012-1424-6 . Jirsova K, Jones GLA. Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review. Cell Tissue Bank. 2017;18(2):193–204. doi: 10.1007/s10561-017-9618-5 . López-Valladares MJ, Teresa Rodríguez-Ares M et al. Donor age and gestational age influence on growth factor levels in human amniotic membrane. Acta Ophthalmol. 2010;88(6):e211-6. doi: 10.1111/j.1755-3768.2010.01908.x Paolin A, Trojan D et al. Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and γ-irradiated human amniotic membranes. Cell Tissue Bank. 2016;17(3):399–406. doi: 10.1007/s10561-016-9553-x . Rodríguez-Ares MT, López-Valladares MJ et al. Effects of lyophilization on human amniotic membrane. Acta Ophthalmol. 2009;87(4):396–403. doi: 10.1111/j.1755-3768.2008.01261.x . Additional Declarations No competing interests reported. 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08:47:14","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42710,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/90ce20f161f2d0d2d31f5248.html"},{"id":95818992,"identity":"170fe1c9-60b5-452f-b587-4cf7a4a73793","added_by":"auto","created_at":"2025-11-13 10:37:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":84652,"visible":true,"origin":"","legend":"\u003cp\u003ea) distribution of amniotic membrane in group 0 (yellow) and group 1 (blue) based on age of donors; b) distribution of group 0 (red) and group 1 (green) per age; c) distribution of group 0 (red) and group 1 (green) per age range.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/709a0614b4b496c73e0fe7bf.png"},{"id":95805984,"identity":"017fa64d-abfb-46ea-892c-9ede55699018","added_by":"auto","created_at":"2025-11-13 08:47:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15112,"visible":true,"origin":"","legend":"\u003cp\u003emean age, median age and standard deviation of data from both groups. (*) indicates a p-value \u0026lt;0,05\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/12dee036d31137f7e5ef3222.png"},{"id":95819054,"identity":"9ddb7557-db54-47e3-a1c5-56b635818f0a","added_by":"auto","created_at":"2025-11-13 10:37:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":169938,"visible":true,"origin":"","legend":"\u003cp\u003epie charts from group 0 (a) group 1 (b) and the total population (c).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/d12ea315fd0efabfeb3dab63.png"},{"id":95805910,"identity":"f3ea4bb7-d6a2-4176-af97-7297a34c3597","added_by":"auto","created_at":"2025-11-13 08:47:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13190,"visible":true,"origin":"","legend":"\u003cp\u003ecomparative study of R rate between under 30 and over 30 group. (***) indicates a p-value \u0026lt;0,001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/dc79e14acbf4a0869d4bedf3.png"},{"id":95806285,"identity":"ff7548d1-18bc-4aff-bb19-3a74ac3d9263","added_by":"auto","created_at":"2025-11-13 08:47:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":23031,"visible":true,"origin":"","legend":"\u003cp\u003edistribution R rate vs age in age range 23-42\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/39c216f4f8f2d9ec472bfd11.png"},{"id":100583628,"identity":"443608ee-770d-4f88-abdc-c58a8a970dad","added_by":"auto","created_at":"2026-01-19 11:33:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":689933,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7799881/v1/dbf08e16-8924-4e56-8836-9ea861178d27.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Does Placenta donors’ age significantly impact on amniotic membrane quality?","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAmniotic membrane (AM) is a thin membrane located in the innerside of the placenta, next to the fetus. It comprises three major histological structures: an epithelial monolayer, a thick basement membrane, and an avascular stroma in contact with the underlying chorion. The innermost epithelium layer, facing the fetus, in direct contact with the amniotic fluid, consists of a single layer of cuboidal epithelial cells with apical microvilli uniformly arranged on the basement membrane, which help in the absorption and secretion of solutes and water (Ingraldi AL, Audet RG et al. 2023). Due to specific properties (such as anti-inflammatory, anti-viral, anti-bacterial and promoter of epithelization) the AM is used in different kind of surgeries for various applications including treatment of skin burns, ocular surface reconstruction and as a biological barrier for improving the natural scarring process in wound healing (Mamede AC, Carvalho MJ et al. 2012; Jirsova K, Jones GLA 2017); moreover, the AM is the only tissue for which no age limit is currently set for its retrieval and for the processing and it has no limits dictated by ethical reasons, which makes it easy to retrieve for many tissue banks in every country.\u003c/p\u003e\u003cp\u003eThere are several variables that contribute to define the quality of the amniotic membrane in the therapeutic field, not only based on the processing method or on the properties that the amniotic membrane acquires as a result of various kinds of molecules to which it is exposed to reach the desired end product, which can be decellularized amniotic membrane, cryopreserved, lyophilized, freeze dried, injectable or a combination of at least two of them (Mamede AC, Carvalho MJ et al 2012).These variables are not all well defined and the effects on the quality and the properties of AM are not yet been fully established. Here we report a statistical analysis that correlate the quality of the amniotic membrane extracted from placentas from different donors since 2018 and the donors age.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cp\u003eAll 2002 human placentas donors since 2018 from our database were screened for age criteria and quality of the amniotic membrane. The placentas were sourced from elective cesarean sections after obtaining written informed consent in hospitals belonging to our tissue bank procurement network. Donors were selected on the basis of strict criteria that also include guidelines for harvesting, processing and distributing tissues for transplantation as approved by the National Transplant Centre. Selection criteria included the absence of any kind of malignancy, infant malformation or pathology, a gestation period of at least 35 weeks, negative family medical history for genetic diseases, and lifestyles of both parents not at risk for infectious diseases. On arrival at the bank the tissues were anonymized with a unique code number used for all processing phases. Working in sterile conditions in a laminar flow cabinet within 24 h of tissue retrieval, HAM was carefully detached from the chorion and rinsed with saline solution to remove blood clots. All the procedure were performed only by qualified personnel. From this database we removed all donors rejected for reasons not linked to the quality of the amniotic membrane (such as meconium-stained amniotic membranes or placentas not well stored after retrieval or donors anamnestic reasons) and we obtained the final database composed of 1323 different donors. We then divided all donors in two groups: the one with amniotic membranes rejected for low quality of the tissue immediately after the extraction from placentas like, for example, yellow-stained membranes, AM with fragile features such as large areas with massive perforations or membranes too much attached to the corion (group 0) and the ones admitted to further processing (group 1).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eFrom the whole database we divided the total population in two groups, group 0 (discharged AMs) and group 1 (admitted to further processing AMs) respectively, and we calculate the mean and median age and the standard deviation from each one of them (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003echaracteristics of the total population, group 0 and group 1. In here we report even minimum age, maximum age and standard deviation of each group\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean age\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian age\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStandard deviation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMinimum\u003c/p\u003e\n \u003cp\u003eage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaximum age\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDonors number\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal population\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34,15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4,852\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1323\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGroup 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34,602\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4,70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e453\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33,919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4,84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e870\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe then take the donors from each of the two groups and see the distribution based by age (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e we can appreciate the difference in age from donors in the two groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,05).\u003c/p\u003e\n\u003cp\u003eStarting from the whole data, we divided the entire population in 3 subgroups based on the donors age: under 30, 30\u0026ndash;40 and over 40 to find out how if there are any major differences. We also calculate the ratio between group 1 and group 0 population in each of the sub-groups created and the percentage of group 0 amniotic membrane in each sub-group on the total population (R) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003echaracteristics of total population of placenta donors divided in 3 groups based on age. We can appreciate the difference in R rate between the under 30 group and the 30\u0026ndash;40 and over 40 group, with the last two that have a really similar rate.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup 0\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup1/Group0\u003c/p\u003e\n \u003cp\u003erate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnder 30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2,77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u0026ndash;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e348\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,354\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOver 40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,387\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe then analyze the two subgroups created and the total population highlighting the distribution of each group of donors based by age (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eFigure4 comparative study of R rate between under 30 and over 30 group. (***) indicates a p-value \u0026lt;0,001\u003c/p\u003e\n\u003cp\u003eLooking at the R rate of the 3 age groups in which the donors were grouped, we realized that the 30\u0026ndash;40 age group and the over 40 group had very similar R rates. Based on the assumption that the two groups were equal for that parameter, we wondered if there was a statistically significant difference between the R rate of the under 30 group and that of the over 30 group. To make our analysis strong, we considered only the age groups that had at least 10 donors within them and that had a normal distribution; for this reason, the age group considered is from 23 to 42 years old.\u003c/p\u003e\n\u003cp\u003eAs described by Fig. 4 we see that the difference between the mean and the median of the rate R between the two groups is very high and with considerable significance (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The distribution of the rate R varies between the different age groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The complete data from both populations are listed below:\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003echaracteristics of rate R of donors between age 23\u0026ndash;29 and age 30\u0026ndash;42\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStandard deviation\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge 23\u0026ndash;29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,285\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,039\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge 30\u0026ndash;42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePlacenta is a really useful tissue for a lot of tissue banks. Due to the lack of an upper limit age placenta is really easy to retrieve and has no ethical issue that donation of other tissues have. With this study we demonstrate that the lack of the age limit of placenta donation makes sense in the context of tissue banks as there are small percentage differences between the discard rate of donors under 30 and donors over 30, thus making the process of donation and processing of the amniotic membrane efficient regardless the age of the donors. The amniotic membrane is a very versatile tissue, which can be subjected to various types of processing, storage and decontamination methods that has shown to not affect its beneficial properties (Paolin A, Trojan D et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Rodr\u0026iacute;guez-Ares MT, L\u0026oacute;pez-Valladares MJ et al. 2009). The comparison between the quality of the amniotic membrane and the age of placental donors is an aspect that has not yet been fully explored: for example, Valladares et al. tried to establish whether there was a correlation between the content of total proteins and growth factors and the age of the donors and the gestation time. In the article they found that the concentration of bFGF, HGF, KGF, NGF, and TGF-β1 was lower in older donors than in younger ones highlighting a possible age-related effect. The study was limited to 13 placentas so the population was not so wide. It\u0026rsquo;s also the only study, as far as we know, that analyzes the possible correlation between donor age and the quality of the amniotic membrane (L\u0026oacute;pez-Valladares MJ, Teresa Rodr\u0026iacute;guez-Ares M et al. 2010). This study tries to shed a light on this field of investigation that is still little known. Although the statistical analysis is affected by some biases (such as the quality of the amniotic membrane which is evaluated by each operator without the use of an instrument, like for example a microscope, and therefore not from measurable values) preliminary evaluations can be made on a possible effect of age on the quality of the amniotic membrane. First of all, the analysis showed a statistically significant difference between the age of donors in group 0 and group 1. If we then shift our attention to the percentage of discarded amniotic membranes out of the total of each subgroup created, we note that there is a marked difference between the incidence of discarded amniotic membranes in donors under the age of 30 compared to both the 30\u0026ndash;40 group and the over 40 group, with the latter two that we can surprisingly consider as one group based on these data. The R rate between under 30 and over 30 donors show a statistically significant difference, although the analysis cover the age range between 23 and 42 due to lack of consistent data from age below 23 and above 42. Although further verification from a molecular point of view seems to be very useful, all together the data presented in this study show that there is a strong correlation between the age of placenta donors and the quality of the amniotic membrane extracted from them in order to be able to subject it to further processing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e No funds, grant or other support was received\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfict of interests\u003c/strong\u003e The authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e The retrieval of human tissues are authorized by national and regional competent authorities. This study does not contain any sensitive donor information. The consent for tissue retrieval was obtained from the tissue donor according with the Italian national regulation on tissue banking.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration\u003c/strong\u003e All authors agreed with the content and all gave explicit consent to submit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003eall participants signed an informed consent agreement.\u0026nbsp;The study has been approved by the ethical territorial committee of Area Nord Veneto.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access\u003c/strong\u003e This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modifed the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco mmons.org/licenses/by-nc-nd/4.0/\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMC wrote the manuscript and analyze the dataRP prepared the dataNS collected and prepared the dataRDV collected dataGM revise the manuscript and support ethical issueDT revise the manuscript and correct the data\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIngraldi AL, Audet RG, et al. The Preparation and Clinical Efficacy of Amnion-Derived Membranes: A Review. J Funct Biomater. 2023;14(10):531. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/jfb14100531\u003c/span\u003e\u003cspan address=\"10.3390/jfb14100531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMamede AC, Carvalho MJ et al. 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Acta Ophthalmol. 2009;87(4):396\u0026ndash;403. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1755-3768.2008.01261.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1755-3768.2008.01261.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amniotic membrane, statistics, placenta, age vs quality","lastPublishedDoi":"10.21203/rs.3.rs-7799881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7799881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlacenta is a very versatile tissue in the field of transplantation also thanks to the fact that, since there is no upper limit to the age of placental donors, is a really useful and easy tool to manage in different tissue banks. Several tissues can be extracted from the placenta as it is made up of various types of membranes and having a heterogeneous population of cells; among these tissues, the most used and studied in the field of transplantation is the amniotic membrane, which is really useful in different kind of surgeries having a relevant role in skin wound repair, in ophthalmic surgery and in maxillofacial surgery. In this study we asked ourselves if the lack of an upper age limit to placenta donors lead to a virtuous process in which the retrieving and processing costs are justified by the quality of the AM extracted from them in an age-independent manner: we started from a database of 2002 placenta donors collected from 2018 until 2024 and we have analyzed a possible correlation between the age of placenta donors and the quality of the amniotic membrane extracted from them, highlighting a statistically significant correlation between them. Furthermore, this statistical study showed that the amniotic membranes of donors with an age from 30 to 40 years have surprisingly the same quality as donors with an age from 40 to 50 which both differ from donors with an age below 30 and that therefore they can be divided into exactly two separate groups: donors under 30 and donors over 30.\u003c/p\u003e","manuscriptTitle":"Does Placenta donors’ age significantly impact on amniotic membrane quality?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 07:52:32","doi":"10.21203/rs.3.rs-7799881/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"668fced0-63b3-4c82-8f71-2ba321211eb8","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T11:30:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 07:52:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7799881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7799881","identity":"rs-7799881","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}