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Human amniotic fluid as a source of stem cells

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Veröffentlicht/Copyright: 4. April 2022
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Open Medicine
Aus der Zeitschrift Open Medicine Band 17 Heft 1

Abstract

Human amniotic fluid collected during amniocentesis contains a heterogeneous population of differentiated and undifferentiated cells. Properties and number of these cells vary depending on the gestational age and the presence of potential fetal pathologies. The aim of this study was to analyze the effects of maternal, fetal, and environmental factors on the success rates of amniotic fluid stem cell cultures, the number of human amniotic fluid stem cells (hAFSC), their growth rates in primary cultures, and the number of cell passages. The study included 355 patients qualified for genetic amniocentesis at the Prenatal Genetic Unit, Department of Obstetrics, Gynecology and Oncologic Gynecology, Nicolaus Copernicus University Medical College in Bydgoszcz in 2011–2017. The mean age of the study participants was 34 ± 6.2 years, and mean gravidity amounted to 2.48 ± 1.4. Amniotic fluid sample volume turned out to be a highly significant (p < 0.01) predictor of culture success, and the relationship was particularly evident in women older than 40 years. Another highly significant predictor of culture success was the presence of two cell populations in the sample (p < 0.01). The likelihood of culture success correlated significantly (p < 0.05) with the season of the year at the time of amniocentesis. The number of cell passages differed significantly depending on the maternal age (p < 0.01). The number of passages also showed a highly significant relationship with the season of the year the sample was obtained (p < 0.01). Younger maternal age was identified as a determinant of high passage number (≥3), and another highly significant determinant of high passage number was the presence of two cell populations in the amniotic fluid sample (p < 0.01). Percentage of successfully established hAFSC cultures and the number of passages depended on amniotic fluid volume, the presence of two cell populations within the sample, and the season of the year. Individual characteristics of the donors, such as age and gravidity, did not exert a significant effect on the number of isolated hAFSCs and the rate of their growth. Patients’ place of residence, fetal karyotype, transportation time, and purity of the samples did not affect the success rates for primary cultures and the number of passages.

1 Introduction

The term “stem cells” refers to the cells that are capable of self-renewal, i.e., can undergo unlimited divisions and differentiate into many various cell types [1]. Depending on their differentiation potential, stem cells can be classified as totipotent (i.e., the cells that can differentiate into all the cell types in a body, as well as into extra-embryonic cells), pluripotent (that can differentiate into cells derived from all three germ layers), multipotent (differentiating only into cells characteristic for one specific germ layer), and unipotent (that can turn into only one particular type of cell) [2,3]. Based on their origin, stem cells are classified into embryonic stem cells (ESCs), fetal stem cells (FSCs), adult stem cells (ASCs), and induced pluripotent stem cells (iPSCs) [4]. Despite their relatively lower differentiation potential and limited possibilities of their genetic modification, ASCs represent a potential source of stem cells used for clinical purposes.

Recently, the role of stem cells is extensively examined in the development of different benign and malignant gynecological pathologies. Endometrial stem progenitor cells were proposed as cells that give rise to the origin of benign and malignant endometriotic lesions. Endometrial stem cells demonstrated a high plastic capacity of differentiation by the characterization of several lines of cells with a different expression pattern of cell surface markers, endometrial localization, and clonal efficiency. The endometrial stem cells have demonstrated the ability to migration, adhesion, proliferation, and induction of angiogenesis. It is proposed that physical and biochemical injuries caused by inflammatory cytokines and reactive oxygen species trigger the activation of endometrial stem cells inducing local production of estrogen and tissue injury-repair mechanisms such as cell cycle activation. On that basis, endometrial stem progenitor cells may be involved in the etiopathogenesis of benign and malignant endometrial aberrations such as endometriosis, endometrial hyperplasia, and endometrial cancer [5,6].

Moreover, the possible role of ovarian stem cells in the initiation and progression of ovarian cancer is getting growing attention. Accumulating evidence suggest that stem cells may play pivotal role in recurrence disease. The initial clinical response is primarily due to the therapeutic efficacy of chemotherapy against differentiated cancer cells that constitute the bulk of the tumor, whereas the high rate of recurrence is thought to be due to remaining drug-resistant cells, biologically distinct, identified as cancer stem cells. Current efforts focus on the genetic and cytological definition of cancer stem cells, to guide the development of new diagnostic and therapeutic perspectives [7,8].

Human amniotic fluid collected during amniocentesis contains a heterogeneous population of differentiated and undifferentiated cells. Properties and number of these cells vary depending on gestational age and the presence of potential fetal pathologies. Periodical changes in fetal status may also contribute to changes in the population of amniotic fluid stem cells [9]. In addition, both total cell count and cell viability may differ considerably from sample to sample. For example, the total cell count in the amniotic fluid from the second trimester was shown to vary from 10 to 1,000 cells/µL [10]. The heterogeneity of the cells can also be a consequence of direct contact of the fluid with fetal tissues due to its variable flow between the growing fetus and the amniotic sac. Some cells found in the amniotic fluid were identified as fetal cells derived from the skin or amniotic membrane [10]. Therefore, the amniotic fluid is routinely used for genetic and biochemical testing for various fetal anomalies and sometimes also to establish the sex of a growing fetus [9].

Based on their morphology, cells present in the amniotic fluid can be divided into three types: amniotic fluid-specific cells (AF-type), epithelioid cells (E-type), and fibroblastic-type cells (F-type). The largest group among these types are AF-type cells that account for 60–70% of all cells found in the amniotic fluid. E-type and F-type cells constitute 20–30% and less than 10% of all cells, respectively [11]. While the published terminology used to describe the amniotic fluid cells varies from paper to paper, usually two main populations, amniotic fluid mesenchymal stem cells (AF-MSCs) and amniotic fluid-derived stem cells (AFSCs), are identified [9]. AF-MSCs are postulated to be more abundant among these two types. However, according to the available literature, we conclude that both have the similar properties, and authors describing mesenchymal stem cells isolated from the amniotic fluid use also both terms interchangeably. That is why we decided to focus on the most common term, which is AFSCs, and its population was subjected to all analysis.

MSCs are a population of multipotent stem cells that can differentiate into mesoderm-derived cells, such as adipocytes, chondrocytes, myoblasts, and osteoblasts [12]. However, these cells were also shown to turn into the cells of ectodermal and endodermal lineages [13,14]. MSCs were first identified in the bone marrow, whereby they constitute 0.001–0.01% of all nuclear cells [15]. However, the presence of these cells, with similar properties to those isolated from the bone marrow, was later demonstrated in many adults, fetal, and extra-embryonic tissue [16].

First reports on the presence of a cellular subpopulation with biological properties resembling those of MSCs in the amniotic fluid were published in 2001 [17]. In 2003, Prusa et al. demonstrated that the cells present in the amniotic fluid could express OCT4, a pluripotency marker [10]. In the same year, In’t Anker et al. showed that the amniotic fluid could be a source of multipotent stem cells [18].

The pool of amniotic fluid stem cells includes a population expressing markers of mesenchymal cells, such as CD90, CD105, CD73, and CD166 and a population lacking expression of hematopoietic cell markers, CD45, CD34, and CD14 [19]. These cells constitute 0.9–1.5% of all cells found in the amniotic fluid [19]. The results of a comprehensive analysis of these cells were published in 2007 by De Coppi et al. who isolated a population of CD117+ cells with clonogenic potential [20]. Colony-forming amniotic fluid stem cells are also capable of self-renewal and can maintain the constant telomere length in late passages. Moreover, despite their high proliferation potential, these cells have typical morphological features of MSCs and express pluripotency markers, such as OCT4, NANOG, SSEA3, SSEA4, and c-MYC even up to 25 passages [21]. Moreover, they maintain normal karyotypes in culture and do not form neoplastic tissue in vivo [22]. In vitro studies demonstrated that these cells could differentiate into the cells derived from all three germ layers, turning into adipocytes, osteocytes, myocytes, as well as into endothelial cells and neurons. Given those findings, MSCs derived from the amniotic fluid were classified into a new type of multipotent cells that combine the characteristics of embryonic and adult stem cells [23]. Amniotic fluid stem cells also have the ability to modulate immune cells. Several research groups demonstrated that these cells showed decreased expression of HLA-DR and proinflammatory molecules and enhanced the activity of anti-inflammatory molecules, e.g., interleukin 10 [24,25]. In addition, they do not express major histocompatibility complex antigens MHC class II and some clusters of differentiation markers, namely, CD40, CD80, and CD86 [21]. These findings suggest that these cells can release immunosuppressive factors in response to the activation of the immune system, which seems vitally important in the context of prevention of the rejection of transplanted amniotic stem cells by the recipient [9].

The aim of this study was to analyze the effects of maternal, fetal, and environmental factors on the success rates of amniotic fluid stem cell cultures, the number of harvested human amniotic fluid stem cells (hAFSC), their growth rates in primary cultures, and the number of cell passages.

2 Materials and methods

2.1 Patients

This study included 355 patients qualified for genetic amniocentesis at the Prenatal Genetic Unit, Department of Obstetrics, Gynecology and Oncologic Gynecology, Nicolaus Copernicus University Medical College in Bydgoszcz in 2011–2017. Only women with singleton pregnancy were included in our study. Women with multiple pregnancies and after in vitro fertilization were excluded from the study. The mean age of the study participants was 34 ± 6.2 years, and mean gravidity amounted to 2.48 ± 1.4. Amniocentesis was carried out at a mean gestational age of 16.4 ± 1.6 weeks. All patients were qualified for amniocentesis after noninvasive testing. Women who were referred to the prenatal genetic unit between 11 and 13 + 6 weeks of gestation underwent the integrated test in which the risk of fetal abnormalities was estimated based on the results of the ultrasound and biochemical tests. The latter included the determination of serum concentration of free beta-hCG and PAPP-A. The results expressed as multiples of the median (MoM) were 2.4 ± 7.4 for beta-hCG and 0.8 ± 1.4 for PAPP-A (the mean values were as follows: 44.18 ± 23.4 IU/l for free beta-hCG and 3.36 ± 2.27 IU/l for PAPP-A). Comprehensive ultrasonographic examination (GE Voluson E8 apparatus with a volumetric probe) was conducted by one of the experienced operators trained according to the FMF and PTG (Polish Society of Gynecologists and Obstetricians) standards. The list of parameters determined during the ultrasonographic examination included crown-rump length (CRL, mm) to confirm gestational age (range 45–84 mm), the presence of nasal bone (NB), nuchal translucency (NT, mm), and fetal heart rate (FHR).

Patients who were qualified for invasive prenatal testing due to medical indications were first consulted by a physician. During the consultation with a specialist in clinical genetics, they were familiarized with detailed indications for amniocentesis and potential complications of this procedure and asked about their preferences. On a scheduled date of the procedure, each patient signed the informed consent form prepared according to the Polish Society of Gynecologists and Obstetricians recommendations. During the consultation, the patients were also informed about the option to culture stem cells isolated from the first collected volume of amniotic fluid, which in line with the current recommendations should be discarded as it cannot be used to determine fetal karyotype. After expressing their consent to participate in the study and signing the informed consent form, 355 enrolled women were qualified for the amniotic fluid analysis.

Amniocentesis was carried out in an outpatient setting under ultrasonographic guidance. After initial ultrasonographic examination, including determination of the fetal heart rate, the position of the fetus, and chorion, an optimal puncture site was selected. Routinely, amniocentesis was carried out without anesthesia and antibiotic prophylaxis. Aseptic preparation included disinfection of the puncture site and ultrasonographic probe, inserting the latter into a sterile cuff, and the use of sterile syringes and ultrasonographic gel. The amniotic fluid at a volume of 15–18 mL was collected with a 20-gauge Brown needle, using a free-hand technique or a guidewire. The needle was inserted in such a way that it did not puncture the chorion, or if not feasible, it punctured the placenta at its thinnest part, avoiding the placental margin and insertion of the umbilical cord. The entire puncture procedure was carried out under ultrasonographic guidance. After the assistant connected the syringe to the needle, the first volume of the amniotic fluid, which is not used routinely to determine the fetal karyotype, was collected and sent to the Department of Tissue Engineering to establish the cell culture. Then, the syringe was replaced by the new one, and another volume of amniotic fluid was collected for the determination of the fetal karyotype. After the amniocentesis was completed and the needle was removed, the fetal heart rate was reassessed. After securing amniotic fluid samples under sterile conditions and labeling the syringes appropriately, they were sent to the laboratory. The patients were instructed to self-control for potential amniotic fluid leakage, bleeding, and uterine contractions. Anti-D prophylaxis was recommended to pregnant women who were Rh negative.

Fetal karyotype was determined at the Department of Clinical Genetics, Nicolaus Copernicus University Medical College in Bydgoszcz (Prof. Jurasz Memorial University Hospital No. 1), based on chromosome banding. The most commonly found abnormalities included trisomy 21, trisomy 18, and trisomy 13.

2.2 Methodology of laboratory tests

2.2.1 Isolation and in vitro culture of human amniotic fluid mesenchymal stem cells

Collected amniotic fluid, at a 0.1–5 mL volume, was transferred from syringes to tubes under sterile conditions of a laminar chamber. The content of the syringes was inspected macroscopically for contamination, such as residual blood. Then, the tubes were centrifuged at 350×g for 10 min, the supernatant was discarded, and the precipitate was suspended in Dulbecco’s Modified Essential Medium (DMEM/Ham’s F12, Sigma, Germany) supplemented with 20% fetal bovine serum (FBS, Sigma, Germany), 10 ng bFGF (Sigma, Germany), and 1% antibiotic solution (penicillin/streptomycin, amphotericin B, Sigma, Germany). The viability of the cells was determined by the trypan blue test. Then, the cells were transferred to 35 mm Petri dishes. The dishes were incubated at 37°C with 5% CO2 and constant humidity. The culture medium was replaced every 48 h. The cells were cultured until they reached confluence and then transferred to new Petri dishes.

2.2.2 Passaging of amniotic fluid mesenchymal stem cells

All procedures related to maintenance of in vitro cell cultures and cell passaging were carried out under sterile conditions of class II laminar chamber (Jouan, France). The first stage of passaging was the removal of the culture medium; then, the growth surface was rinsed with phosphate-buffered saline (PBS) without calcium and magnesium (Sigma, Germany). Subsequently, the cells were treated with 0.05% trypsin solution (Sigma, Germany) mixed with 0.5 mM EDTA (POCh, Poland) in 1:1 ratio. The cells were incubated at 37°C for 5 min, while the degree of their detachment was controlled under an inverted microscope (Nikon, Japan). The trypsinization was inhibited by adding an equal volume of culture medium; then, the material was transferred to a sterile tube, centrifuged at 350×g for 10 min, and the sediment was suspended in 1 mL of the culture medium. The number of viable cells was determined in a hemocytometer based on the result of the trypan blue test. Cell morphology was examined under an inverted phase-contrast microscope (CKX53 Olympus); the cells were classified as epithelioid or fibroblastic type.

2.2.3 Determination of amniotic fluid mesenchymal cell viability

To determine cell viability, an equal volume of 0.4% trypan blue solution (Sigma, Germany) was added to the cell suspension. The mixture was applied onto the Neubauer chamber, and the number of viable cells was determined under an inverted microscope. Viable cells were counted in four squares of the Neubauer chamber, and the cell viability was calculated as follows: L = A/4 × 2 × 104 × B, where L is the total number of viable amniotic fluid mesenchymal stem cells and A is the number of viable cells in four squares of the chamber.

Assays confirming the mesenchymal nature of isolated stem cells, in accordance with the recommendations of the International Society for Cytotherapy, were performed at the Department of Tissue Engineering, Nicolaus Copernicus University Medical College in Bydgoszcz and published previously [26].

2.3 Statistical analysis

Statistical analyses were carried out with PQStat package, version 1.6.4.110. A relationship between maternal age and cell culture success rate was analyzed using a logistic regression model, and by the chi-squared test when the age was converted into a categorical variable. Cell counts at the beginning of culture and following subsequent passages were compared with Mann–Whitney U test. A relationship between amniotic sample volume and cell culture success rate was analyzed using a logistic regression model. Effects of grouping variables on cell culture success rate were analyzed with the chi-squared test. Relationships among maternal age, cell count at the beginning of the culture (P0), or the number of cell passages were analyzed by the Kruskal–Wallis test, Dunn–Bonferroni post-hoc test, and Jonckheere–Terpstra trend test; also, Spearman’s coefficients of correlation between maternal age and the numbers of cells/passages were calculated. Relationships among various dichotomous grouping variables, cell count at the beginning of the culture (P0), and the number of cell passages were analyzed by the Mann–Whitney U test. Relationships among the season of the year at the time of amniocentesis, cell count at the beginning of the culture (P0), or the number of cell passages were analyzed with the Kruskal–Wallis test and Dunn–Bonferroni post hoc test. Predictors of high passage number (≥3) were identified through the logistic regression analysis, and predictors of cell count at the beginning of the culture (P0) were determined through the multiple regression analysis. The results of all tests were considered significant at p < 0.05 and highly significant at p < 0.01.

  1. Ethics statement: The protocol of the study was approved by the Local Bioethics Committee at Ludwik. Rydygier Collegium Medicum in Bydgoszcz (decision no. KB 239/2011), and written informed consent was sought from all participants.

3 Results

As shown in Table 1, culture success did not depend on maternal age (p > 0.05). No statistically significant differences in culture success rates were observed (p > 0.05) on comparative analysis of the results for women from various age groups (Table 2).

Table 1

Culture success rates depending on maternal age

Beta p-Value Odds ratio −95% CI +95% CI
Intercept 1.7194 0.0093 5.5810 1.5286 20.3769
Age −0.0360 0.0530 0.9646 0.9301 1.0005
Table 2

Culture success rates in various age groups

Culture success Age
Up to 25 26–30 31–35 36–40 41 and above
N % N % N % N % N %
No 13 32.5 13 27.66 28 34.15 59 45.38 24 42.86
Yes 27 67.5 34 72.34 54 65.85 71 54.62 32 57.14
Chi-square 6.6415
Df 4
p-value 0.1561

The amniotic sample volume turned out to be a highly significant (p < 0.01) predictor of the culture success. The larger the sample volume, the more likely successful the culture (Table 3). While the relationship was particularly evident in women older than 40 years, it was not observed in patients younger than 25 years (Tables 4 and 5). No significant relationship between the sample volume and culture success was observed in younger women (p > 0.05) (Table 4). Among older women, the sample volume was a significant (p < 0.05) determinant of culture success. In this age group, the likelihood of culture success increased proportionally to the sample volume (Table 5).

Table 3

Culture success rates depending on sample volume (overall)

Beta p-value Odds ratio −95% CI +95% CI
Intercept −0.3471 0.0986 0.7068 0.4682 1.0669
Sample volume (mL) 0.4189 <0.0001 1.5203 1.2574 1.8380
Table 4

Culture success rates depending on sample volume in women aged 25 years and younger

Beta p-Value Odds ratio −95% CI +95% CI
Intercept 0.2642 0.7188 1.3024 0.3091 5.4879
Sample volume (mL) 0.2749 0.4844 1.3164 0.6091 2.8452
Table 5

Culture success rates depending on sample volume in women older than 40 years

Beta p-Value Odds ratio −95% CI +95% CI
Intercept −0.8663 0.0863 0.4205 0.1563 1.1315
Sample volume (mL) 0.6406 0.0111 1.8976 1.1577 3.1102

Another highly significant predictor of culture success was the presence of two cell populations in the amniotic fluid sample (p < 0.01); the cultures prepared from the amniotic fluid containing two populations of cells were more likely to be successful (Table 6). While this relationship was not observed among younger women (p > 0.05), it was statistically significant in the group of older patients (p < 0.05).

Table 6

Culture success rates depending on the presence of two cell populations within the sample

Culture success Overall
Presence of two cell populations
No Yes
N % N %
No 121 52.84 13 10.66
Yes 108 47.16 109 89.34
Chi-square 60.0076
Df 1
p-Value <0.0001
Culture success Younger (<25 years)
Presence of two cell populations
No Yes
N % N %
No 12 40% 1 10
Yes 18 60% 9 90
Chi-square 3.0769
Df 1
p-Value 0.0794
Culture success Older (>40 years)
Presence of two cell populations
No Yes
N % N %
No 21 56.76 2 11.11
Yes 16 43.24 16 88.89
Chi-square 10.3695
Df 1
p-value 0.0013

The likelihood of culture success correlated significantly (p < 0.05) with the season of the year at the time of amniocentesis, with the lowest success rates for the samples collected in summer and the highest for the stem cells harvested in winter. The relationship between culture success and the season of the year was not observed in the group of younger women (p > 0.05) but was statistically significant among older patients (p < 0.05) in whom the highest success rates were obtained for the stem cells harvested in winter (Table 7).

Table 7

Culture success rates depending on the season of the year

Culture success Overall
Season
Spring Summer Autumn Winter
N % N % N % N %
No 38 40.43 30 50.85 30 42.86 39 29.55
Yes 56 59.57 29 49.15 40 57.14 93 70.45
Chi-square 8.9685
Df 3
p-Value 0.0297
Culture success Younger (<25 years)
Season
Spring Summer Autumn Winter
N % N % N % N %
No 5 41.67 2 25 3 42.86 3 23.08
Yes 7 58.33 6 75 4 57.14 10 76.92
Chi-square 1.5332
Df 3
p-Value 0.6746
Culture success Older (>40 years)
Season
Spring Summer Autumn Winter
N % N % N % N %
No 7 41.18 2 28.57 10 76.92 5 26.32
Yes 10 58.82 5 71.43 3 23.08 14 73.68
Chi-square 8.8860
Df 3
p-Value 0.0308

No statistically significant relationship was found between the success rates at the time elapsed since amniocentesis to the establishment of the culture (p > 0.05). The success rates for cultures established up to 10 h from harvesting and later did not differ significantly (Table 8).

Table 8

Culture success rates depending on the time elapsed since amniotic fluid sampling to the establishment of the culture

Culture success Overall
Time elapsed since the sampling
<10 h >10 h
N % N %
No 108 38.85 29 37.66
Yes 170 61.15 48 62.34
Chi-square 0.0358
Df 1
p-Value 0.8499
Culture success Younger (<25 years)
Time elapsed since the sampling
<10 h >10 h
N % N %
No 10 29.41 3 50
Yes 24 70.59 3 50
Chi-square 0.9854
Df 1
p-Value 0.3209
Culture success Older (>40 years)
Time elapsed since the sampling
<10 h >10 h
N % N %
No 18 42.86 6 42.86
Yes 24 57.14 8 57.14
Chi-square 0
Df 1
p-Value 1

The culture success rate was also not associated with contamination of the sample (p > 0.05).

Cell count at the beginning of the culture (P0) did not correlate significantly with maternal age, sample volume, the presence of two cell populations in the sample, and the season of the year at the time of amniocentesis.

However, multiple regression analysis demonstrated that P0 correlated strongly (p < 0.01) with the sample volume (Table 9).

Table 9

Predictors of high passage number at the beginning of the culture (P0)

Beta p-value Standardized beta Standard error for beta
Intercept 327077 0.4912
Age 12,842 0.3010 0.0538 0.0519
Sample volume (mL) −205,548 0.0005 −0.1835 0.0520
Season 46,518 0.4442 0.0397 0.0519
Time since the sampling −163,851 0.3597 −0.0477 0.0520

The number of cell passages differed significantly depending on maternal age; a strong inverse correlation was observed between these two parameters (p < 0.01; Table 10). It also differed in morphology and cell density in following passages. In early one, cells were smaller and proliferated very intensively. However, changes in cells’ morphology, their size, and growth were observed in later passages. Human AFSCs were larger and proliferated slower, which can also indicate the undergoing aging process (Figure 1).

Table 10

Number of passages in various age groups

Overall Up to 25 26–30 31–35 36–40 41 and more
Median 2 1.5 2 2 1 1
Minimum 0 0 0 0 0 0
Maximum 9 7 8 9 9 8
Lower quartile 0 0 0.5 0 0 0
Upper quartile 3 3 2 3 2 2
Kruskal–Wallis test H 7.6830
p 0.1039
Jonckheere–Terpstra test Z 2.4620
p 0.0138
Up to 25 26–30 31–35 36–40 41 and more
Dunn–Bonferroni test Up to 25 1 1 1 1
26–30 1 1 1 1
31–35 1 1 0.3933 0.3274
36–40 1 1 0.3933 1
41 and more 1 1 0.3274 1
Spearman correlation R −0.1361
p 0.0095
Figure 1 Isolation and in vitro culture of hAFSCs. (a) hAFSCs after 2nd passage, (b) hAFSCs after 6th passage.
Figure 1

Isolation and in vitro culture of hAFSCs. (a) hAFSCs after 2nd passage, (b) hAFSCs after 6th passage.

Moreover, the number of passages was significantly higher when the culture was prepared from a larger volume of the amniotic fluid (p < 0.05; Table 11).

Table 11

Number of passages depending on sample volume

Up to 1 mL More than 1 mL
Median 1 2
Minimum 0 0
Maximum 9 9
Lower quartile 0 0
Upper quartile 2.25 3
Z 2.2114
p 0.0270

A highly significant difference was also found in the number of passages prepared from the amniotic fluid containing one and two populations of cells (p < 0.01); in the latter case, the number of passages turned out to be significantly higher (Table 12).

Table 12

Number of passages depending on the presence of two cell populations within the sample

No Yes
Median 1 2
Minimum 0 0
Maximum 8 9
Lower quartile 0 2
Upper quartile 2 3
Z 6.9950
p <0.0001

The number of passages also showed a highly significant relationship with the season of the year the sample was obtained (p < 0.01). The number of passages for cultures established in winter was significantly higher than for those established in spring and summer (Table 13).

Table 13

Number of passages depending on the season of the year

Overall Spring Summer Autumn Winter
Median 2 1 1 1 2
Minimum 0 0 0 0 0
Maximum 9 5 5 9 9
Lower quartile 0 0 0 0 0
Upper quartile 3 2 2 2 3
Kruskal–Wallis test H 12.9948
p 0.0046
Spring Summer Autumn Winter
Dunn–Bonferroni test Spring 1 1 0.0290
Summer 1 1 0.0191
Autumn 1 1 0.1372
Winter 0.0290 0.01914 0.1372

No statistically significant relationships were observed between the number of passages and sample contamination, fetal karyotype, and indications for amniocentesis (p > 0.05).

Younger maternal age was identified as a determinant of high passage number (three and more passages); the older the maternal age, the lower the number of obtained passages. Another highly significant determinant of high passage number was the presence of two cell populations in the amniotic fluid sample (p < 0.01; Table 14).

Table 14

Predictors of high passage number (at least three passages)

Beta p-Value Odds ratio −95% CI +95% CI
Intercept 0.2415 0.7329 1.2732 0.3181 5.0968
Age −0.0478 0.0178 0.9534 0.9164 0.9918
Sample volume (mL) −0.0093 0.9270 0.9907 0.8120 1.2088
P0 (cell count) 0.0000 0.1074 1.0000 1.0000 1.0000
Presence of two cell populations 1.0671 0.0001 2.9068 1.7295 4.8857

4 Discussion

The applicability of stem cells for regeneration of tissues and organs is a subject of many currently ongoing studies in the field of tissue engineering and regenerative medicine. In recent years, amniotic fluid has emerged as an alternative source of cells for therapeutic purposes. The uniqueness of this source of stem cells is also associated with the fact that the latter can be used even before birth, in fetal life [27]. Aspiration of the amniotic fluid does not raise controversies of ethical nature as it is a part of a routine amniocentesis procedure. Amniotic fluid stem cells constitute not only a diagnostic instrument but also a potential treatment of many congenital defects [28]. Hence, amniotic fluid stem cells are an interesting research model from a perspective of their applicability in preclinical studies. The volume of amniotic fluid collected for diagnostic purposes is small, approximately 20 mL. If the total volume of amniotic fluid, expressed as an amniotic fluid index (AFI), is normal, amniocentesis does not pose a threat for a growing fetus. Another argument, vitally important from a perspective of the clinical application, is the possibility of storing the sample frozen, even if the pregnancy was terminated by the cesarean section [29].

An interesting problem, albeit rarely addressed in the available literature, is a relationship between the maternal age and the possibility to isolate amniotic fluid stem cells and to culture them in vitro. In this study, we did not find a significant difference in culture success rates for amniotic fluid stem cells obtained from pregnant women of different ages. In the study conducted by Romani et al., the culture success rate for amniotic fluid stem cells obtained from a group of 35- to 40-year-old women was 40% [30]. In other studies, which analyzed gestational rather than maternal ages, culture success rates reached up to 93% [31]. We also did not find statistically significant differences in the number of amniotic fluid stem cells obtained from women of various ages. Bielec-Berek et al. and Azouna et al. also did not observe a significant relationship between maternal age and the number and viability of fetal stem cells isolated from another source, i.e., umbilical cord blood [32,33]. Similar findings were also reported by Ballen et al. who did not find an association between maternal age and biological properties of stem cells [34].

Our present study demonstrated that the culture success rate for hAFSCs obtained from women older than 40 years depended on the volume of the amniotic fluid sample, whereas no such relationship was observed for the stem cell cultures from younger patients, younger than 25 years. According to the study by Sessarego et al., the culture success rate for stem cells obtained from 2 mL of amniotic fluid was 91%. However, the authors of that study did not analyze the association between the success rate and the maternal age [35]. Further analyses did not show a significant effect of amniotic fluid volume, less than 1 mL or >1 mL, on the number of isolated cells. In the most previous studies, stem cells were obtained from amniotic fluid volumes greater than 1 mL, and also the results of those experiments suggest that the sample volume does not affect the number of isolated cells [31,36].

In our study, the presence of two cell populations within the sample exerted a significant effect on the success of amniotic fluid stem cell culture, especially if the cells were originated from women older than 40 years. The success rates were higher if the sample contained two cell populations, epithelial and fibroblast-like cells. However, no such relationship was observed in the case of stem cells obtained from younger women younger than 25 years. The presence of two cell populations is a common finding at early stages of in vitro culture. After a certain number of passages, the culture contains solely the cells with a fibroblast-like morphology [9], i.e., the proper MSCs [37]. Additional cell population present within the sample might promote proliferation or migration of stem cells, acting in a paracrine manner. However, regardless of the maternal age, we did not observe a significant effect of the additional cell population on the number of isolated amniotic fluid stem cells.

The success rate of hAFSCs cultures depended on the season of the year when amniocentesis was conducted. Irrespective of the maternal age, the highest success rates, more than 70%, were observed for the cultures established in winter. The relationship between the season of the year and culture success rate was statistically significant in older women, older than 40 years, but not in the younger group, younger than 25 years. Moreover, no statistically significant differences were observed in the number of amniotic fluid stem cells isolated in various seasons of the year. While the published data about the isolation of stem cells and their biology are sparse, seasonal and circadian physiological fluctuations are a common phenomenon. Seasonality, e.g., seasonal changes in air temperature, might exert an effect on body weight and reproductive and immune functions; these fluctuations are controlled primarily by the endocrine system. Also, concentrations of hormones in body fluids frequently show circadian or seasonal rhythms. Garde et al. observed seasonal variability in concentrations of total cholesterol, dehydroepiandrosterone sulfate (DHEA-S), prolactin, and free testosterone in healthy women [38]. According to the study by van Anders et al., the highest concentrations of testosterone in women were found in samples collected in autumn and winter [39]. It cannot be excluded that those seasonal fluctuations might also exert an effect on the success rates of amniotic fluid stem cell cultures.

Regardless of the maternal age, sample transportation time, i.e. the time elapsed since the collection of amniotic fluid to the establishment of hAFSC culture, did not exert a significant effect on the culture success rate. Culture success rates exceeded 60% whether the transportation time was below 10 h or above. Whenever the time elapsed since amniocentesis to the isolation of hAFSCs was longer than 10 h, the samples were always stored at 4°C, which probably contributed to the good quality of the amniotic fluid. Pamphilon et al., who analyzed the effect of transportation time on progenitor cells from umbilical cord blood, also concluded that a primary factor determining culture success and influencing the number of isolated cells was storage temperature, rather than the time elapsed since the sampling to the establishment of the culture [40]. Similarly, Matsumoto et al. did not find significant differences in the number of adipose tissue-derived stem cells isolated after various storage times [41]. Published data about optimal storage time, transportation time, and conditions are sparse not only for amniotic fluid stem cells but also for the stem cells from other sources. A comprehensive analysis-based recommendation regarding both storage and transport of the stem cells seems to be of utmost importance, especially in the context of potential clinical applications of this material. Our findings suggest that 4°C is an optimal storage temperature for amniotic fluid, but due to safety concerns, hAFSCs should be isolated immediately after amniocentesis and amniotic fluid collection.

Our study did not reveal a statistically significant relationship between hAFSC culture success rate and purity of the amniotic fluid sample. We observed a tendency to higher success rates of cultures prepared from a noncontaminated amniotic fluid, especially in the case of older women. The number of isolated cells turned out to be significantly higher for contaminated amniotic fluid samples. Contamination of amniotic fluid with the blood affects the initial number of isolated cells. In this study, we used the isolation method based on adherent properties of hAFSCs, which after multiple passages enabled us to select a clone of fibroblastic-type cells and, hence, to minimize the effect of initial sample contamination.

Further analyses did not show an effect of fetal karyotype on hAFSC culture success rates. Without stratification according to maternal age, the success rates for cultures prepared from maternal amniotic fluid from pregnancies with normal and abnormal fetal karyotype were similar. However, the success rates in the group of older women (over 40 years of age) were higher whenever an abnormal karyotype was found in the fetus. Nevertheless, this observation should be interpreted with caution as pregnancies with abnormal karyotype constituted only 11% of the study material. Furthermore, abnormal karyotype exerted no effect on the number of isolated amniotic fluid stem cells. These findings imply that amniotic fluid stem cells with the same karyotype as fetal one can be successfully cultured in vitro. This observation seems particularly important from a perspective of research on chromosomal aberrations or mutations accounting for monogenic disorders. According to the study by Rosner et al., mutation-harboring amniotic fluid-derived stem cells can serve as a model for research on many various disease entities [42].

hAFSCs culture success rates for women living in smaller towns, up 50 000, and those from cities over 50 000 were similar. However, it needs to be stressed that a statistically insignificant tendency to higher success rates among younger women living in larger cities was observed. Nevertheless, also this result should be interpreted with caution considering the small size of this group. The lack of statistically significant association between the place of residence and culture success rate should be regarded as a promising finding, especially given published data on the influence of environmental pollution on human health [43]. Stock and Clemens demonstrated that exposure to environmental pollutants might contribute to low birthweight, preterm labor, and respiratory diseases in a growing fetus [44]. While we did not analyze birth-related parameters and postnatal data in this study, the place of residence had no significant effect on the number of isolated cells, their morphology, and the proliferation potential.

We also did not find a significant association between maternal gravidity and culture success rates. Primiparas and multiparas did not differ significantly in terms of hAFSCs culture success rates. Since the amniotic fluid contained also fetal cells, among them exfoliated amniotic cells, one can assume that the sample cellularity, and hence, culture success might depend on the fetal growth and well-being, rather than gravidity [28].

Amniocentesis is used as a prenatal test in clinical practice since the 1950–60s. Initially, the primary indication for this procedure was advanced maternal age defined as more than 35 years [45]. This age limit was specified based on the observed association between older maternal age and increased likelihood of fetal chromosomal aberrations and some diseases, e.g., Down syndrome. In this study, we verified if the stem cell culture success was associated with any of the established indications for amniocentesis. The first analyzed indication was maternal age. Culture success rates in women referred to amniocentesis because of advanced age, and those without this indication amounted to 57% and 66%, respectively, and did not differ significantly. Another analyzed indication was an abnormal result of the integrated test and triple test. In the case of an abnormal result of the integrated test, culture success rates were higher, approximately 68%. However, the success of amniotic fluid stem cell culture did not depend on the result of the triple test. We also did not find statistically significant relationships between culture success rate and another two indications for amniocentesis, evidence of ultrasonographic abnormalities, and fetal hydrops. The last analyzed indication was remarkable obstetrical history. This indication turned out to be a highly significant determinant of culture success rate in the study group. The success rates in women referred to amniocentesis because of remarkable obstetrical history were significantly lower than in other participants of the study. However, this finding should be interpreted with caution as women with the remarkable obstetrical history accounted for only 6% of the study group.

5 Conclusion

  1. Percentage of successfully established hAFSC cultures and the number of passages depended on amniotic fluid volume, the presence of two cell populations within the sample, and the season of the year.

  2. Individual characteristics of the donors, such as age and gravidity, did not exert a significant effect on the number of isolated hAFSCs and the rate of their growth.

  3. Patients’ place of residence, fetal karyotype, transportation time, and purity of the samples did not affect the success rates for primary cultures and the number of passages.

  1. Funding information: The authors received no financial support for the research, authorship, and/or publication of this article.

  2. Conflict of interest: The authors declare that they have no competing interests.

  3. Data availability statement: The data used to support the findings of this study are available from the corresponding author upon request.

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Received: 2021-07-02
Revised: 2022-02-28
Accepted: 2022-03-10
Published Online: 2022-04-04

© 2022 Pawel Walentowicz et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  81. Comprehensive analysis of molecular mechanism and a novel prognostic signature based on small nuclear RNA biomarkers in gastric cancer patients
  82. Smog and risk of maternal and fetal birth outcomes: A retrospective study in Baoding, China
  83. Let-7i-3p inhibits the cell cycle, proliferation, invasion, and migration of colorectal cancer cells via downregulating CCND1
  84. β2-Adrenergic receptor expression in subchondral bone of patients with varus knee osteoarthritis
  85. Possible impact of COVID-19 pandemic and lockdown on suicide behavior among patients in Southeast Serbia
  86. In vitro antimicrobial activity of ozonated oil in liposome eyedrop against multidrug-resistant bacteria
  87. Potential biomarkers for inflammatory response in acute lung injury
  88. A low serum uric acid concentration predicts a poor prognosis in adult patients with candidemia
  89. Antitumor activity of recombinant oncolytic vaccinia virus with human IL2
  90. ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway
  91. Risk prediction of cardiovascular disease using machine learning classifiers
  92. Value of ultrasonography parameters in diagnosing polycystic ovary syndrome
  93. Bioinformatics analysis reveals three key genes and four survival genes associated with youth-onset NSCLC
  94. Identification of autophagy-related biomarkers in patients with pulmonary arterial hypertension based on bioinformatics analysis
  95. Protective effects of glaucocalyxin A on the airway of asthmatic mice
  96. Overexpression of miR-100-5p inhibits papillary thyroid cancer progression via targeting FZD8
  97. Bioinformatics-based analysis of SUMOylation-related genes in hepatocellular carcinoma reveals a role of upregulated SAE1 in promoting cell proliferation
  98. Effectiveness and clinical benefits of new anti-diabetic drugs: A real life experience
  99. Identification of osteoporosis based on gene biomarkers using support vector machine
  100. Tanshinone IIA reverses oxaliplatin resistance in colorectal cancer through microRNA-30b-5p/AVEN axis
  101. miR-212-5p inhibits nasopharyngeal carcinoma progression by targeting METTL3
  102. Association of ST-T changes with all-cause mortality among patients with peripheral T-cell lymphomas
  103. LINC00665/miRNAs axis-mediated collagen type XI alpha 1 correlates with immune infiltration and malignant phenotypes in lung adenocarcinoma
  104. The perinatal factors that influence the excretion of fecal calprotectin in premature-born children
  105. Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study
  106. Does the use of 3D-printed cones give a chance to postpone the use of megaprostheses in patients with large bone defects in the knee joint?
  107. lncRNA HAGLR modulates myocardial ischemia–reperfusion injury in mice through regulating miR-133a-3p/MAPK1 axis
  108. Protective effect of ghrelin on intestinal I/R injury in rats
  109. In vivo knee kinematics of an innovative prosthesis design
  110. Relationship between the height of fibular head and the incidence and severity of knee osteoarthritis
  111. lncRNA WT1-AS attenuates hypoxia/ischemia-induced neuronal injury during cerebral ischemic stroke via miR-186-5p/XIAP axis
  112. Correlation of cardiac troponin T and APACHE III score with all-cause in-hospital mortality in critically ill patients with acute pulmonary embolism
  113. LncRNA LINC01857 reduces metastasis and angiogenesis in breast cancer cells via regulating miR-2052/CENPQ axis
  114. Endothelial cell-specific molecule 1 (ESM1) promoted by transcription factor SPI1 acts as an oncogene to modulate the malignant phenotype of endometrial cancer
  115. SELENBP1 inhibits progression of colorectal cancer by suppressing epithelial–mesenchymal transition
  116. Visfatin is negatively associated with coronary artery lesions in subjects with impaired fasting glucose
  117. Treatment and outcomes of mechanical complications of acute myocardial infarction during the Covid-19 era: A comparison with the pre-Covid-19 period. A systematic review and meta-analysis
  118. Neonatal stroke surveillance study protocol in the United Kingdom and Republic of Ireland
  119. Oncogenic role of TWF2 in human tumors: A pan-cancer analysis
  120. Mean corpuscular hemoglobin predicts the length of hospital stay independent of severity classification in patients with acute pancreatitis
  121. Association of gallstone and polymorphisms of UGT1A1*27 and UGT1A1*28 in patients with hepatitis B virus-related liver failure
  122. TGF-β1 upregulates Sar1a expression and induces procollagen-I secretion in hypertrophic scarring fibroblasts
  123. Antisense lncRNA PCNA-AS1 promotes esophageal squamous cell carcinoma progression through the miR-2467-3p/PCNA axis
  124. NK-cell dysfunction of acute myeloid leukemia in relation to the renin–angiotensin system and neurotransmitter genes
  125. The effect of dilution with glucose and prolonged injection time on dexamethasone-induced perineal irritation – A randomized controlled trial
  126. miR-146-5p restrains calcification of vascular smooth muscle cells by suppressing TRAF6
  127. Role of lncRNA MIAT/miR-361-3p/CCAR2 in prostate cancer cells
  128. lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2
  129. Noninvasive diagnosis of AIH/PBC overlap syndrome based on prediction models
  130. lncRNA FAM230B is highly expressed in colorectal cancer and suppresses the maturation of miR-1182 to increase cell proliferation
  131. circ-LIMK1 regulates cisplatin resistance in lung adenocarcinoma by targeting miR-512-5p/HMGA1 axis
  132. LncRNA SNHG3 promoted cell proliferation, migration, and metastasis of esophageal squamous cell carcinoma via regulating miR-151a-3p/PFN2 axis
  133. Risk perception and affective state on work exhaustion in obstetrics during the COVID-19 pandemic
  134. lncRNA-AC130710/miR-129-5p/mGluR1 axis promote migration and invasion by activating PKCα-MAPK signal pathway in melanoma
  135. SNRPB promotes cell cycle progression in thyroid carcinoma via inhibiting p53
  136. Xylooligosaccharides and aerobic training regulate metabolism and behavior in rats with streptozotocin-induced type 1 diabetes
  137. Serpin family A member 1 is an oncogene in glioma and its translation is enhanced by NAD(P)H quinone dehydrogenase 1 through RNA-binding activity
  138. Silencing of CPSF7 inhibits the proliferation, migration, and invasion of lung adenocarcinoma cells by blocking the AKT/mTOR signaling pathway
  139. Ultrasound-guided lumbar plexus block versus transversus abdominis plane block for analgesia in children with hip dislocation: A double-blind, randomized trial
  140. Relationship of plasma MBP and 8-oxo-dG with brain damage in preterm
  141. Identification of a novel necroptosis-associated miRNA signature for predicting the prognosis in head and neck squamous cell carcinoma
  142. Delayed femoral vein ligation reduces operative time and blood loss during hip disarticulation in patients with extremity tumors
  143. The expression of ASAP3 and NOTCH3 and the clinicopathological characteristics of adult glioma patients
  144. Longitudinal analysis of factors related to Helicobacter pylori infection in Chinese adults
  145. HOXA10 enhances cell proliferation and suppresses apoptosis in esophageal cancer via activating p38/ERK signaling pathway
  146. Meta-analysis of early-life antibiotic use and allergic rhinitis
  147. Marital status and its correlation with age, race, and gender in prognosis of tonsil squamous cell carcinomas
  148. HPV16 E6E7 up-regulates KIF2A expression by activating JNK/c-Jun signal, is beneficial to migration and invasion of cervical cancer cells
  149. Amino acid profiles in the tissue and serum of patients with liver cancer
  150. Pain in critically ill COVID-19 patients: An Italian retrospective study
  151. Immunohistochemical distribution of Bcl-2 and p53 apoptotic markers in acetamiprid-induced nephrotoxicity
  152. Estradiol pretreatment in GnRH antagonist protocol for IVF/ICSI treatment
  153. Long non-coding RNAs LINC00689 inhibits the apoptosis of human nucleus pulposus cells via miR-3127-5p/ATG7 axis-mediated autophagy
  154. The relationship between oxygen therapy, drug therapy, and COVID-19 mortality
  155. Monitoring hypertensive disorders in pregnancy to prevent preeclampsia in pregnant women of advanced maternal age: Trial mimicking with retrospective data
  156. SETD1A promotes the proliferation and glycolysis of nasopharyngeal carcinoma cells by activating the PI3K/Akt pathway
  157. The role of Shunaoxin pills in the treatment of chronic cerebral hypoperfusion and its main pharmacodynamic components
  158. TET3 governs malignant behaviors and unfavorable prognosis of esophageal squamous cell carcinoma by activating the PI3K/AKT/GSK3β/β-catenin pathway
  159. Associations between morphokinetic parameters of temporary-arrest embryos and the clinical prognosis in FET cycles
  160. Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis
  161. The incidence of bronchiectasis in chronic obstructive pulmonary disease
  162. Integrated bioinformatics analysis shows integrin alpha 3 is a prognostic biomarker for pancreatic cancer
  163. Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway
  164. Comparison of hospitalized patients with severe pneumonia caused by COVID-19 and influenza A (H7N9 and H1N1): A retrospective study from a designated hospital
  165. lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1
  166. Pathological characteristics of liver injury induced by N,N-dimethylformamide: From humans to animal models
  167. lncRNA ELFN1-AS1 enhances the progression of colon cancer by targeting miR-4270 to upregulate AURKB
  168. DARS-AS1 modulates cell proliferation and migration of gastric cancer cells by regulating miR-330-3p/NAT10 axis
  169. Dezocine inhibits cell proliferation, migration, and invasion by targeting CRABP2 in ovarian cancer
  170. MGST1 alleviates the oxidative stress of trophoblast cells induced by hypoxia/reoxygenation and promotes cell proliferation, migration, and invasion by activating the PI3K/AKT/mTOR pathway
  171. Bifidobacterium lactis Probio-M8 ameliorated the symptoms of type 2 diabetes mellitus mice by changing ileum FXR-CYP7A1
  172. circRNA DENND1B inhibits tumorigenicity of clear cell renal cell carcinoma via miR-122-5p/TIMP2 axis
  173. EphA3 targeted by miR-3666 contributes to melanoma malignancy via activating ERK1/2 and p38 MAPK pathways
  174. Pacemakers and methylprednisolone pulse therapy in immune-related myocarditis concomitant with complete heart block
  175. miRNA-130a-3p targets sphingosine-1-phosphate receptor 1 to activate the microglial and astrocytes and to promote neural injury under the high glucose condition
  176. Review Articles
  177. Current management of cancer pain in Italy: Expert opinion paper
  178. Hearing loss and brain disorders: A review of multiple pathologies
  179. The rationale for using low-molecular weight heparin in the therapy of symptomatic COVID-19 patients
  180. Amyotrophic lateral sclerosis and delayed onset muscle soreness in light of the impaired blink and stretch reflexes – watch out for Piezo2
  181. Interleukin-35 in autoimmune dermatoses: Current concepts
  182. Recent discoveries in microbiota dysbiosis, cholangiocytic factors, and models for studying the pathogenesis of primary sclerosing cholangitis
  183. Advantages of ketamine in pediatric anesthesia
  184. Congenital adrenal hyperplasia. Role of dentist in early diagnosis
  185. Migraine management: Non-pharmacological points for patients and health care professionals
  186. Atherogenic index of plasma and coronary artery disease: A systematic review
  187. Physiological and modulatory role of thioredoxins in the cellular function
  188. Case Reports
  189. Intrauterine Bakri balloon tamponade plus cervical cerclage for the prevention and treatment of postpartum haemorrhage in late pregnancy complicated with acute aortic dissection: Case series
  190. A case of successful pembrolizumab monotherapy in a patient with advanced lung adenocarcinoma: Use of multiple biomarkers in combination for clinical practice
  191. Unusual neurological manifestations of bilateral medial medullary infarction: A case report
  192. Atypical symptoms of malignant hyperthermia: A rare causative mutation in the RYR1 gene
  193. A case report of dermatomyositis with the missed diagnosis of non-small cell lung cancer and concurrence of pulmonary tuberculosis
  194. A rare case of endometrial polyp complicated with uterine inversion: A case report and clinical management
  195. Spontaneous rupturing of splenic artery aneurysm: Another reason for fatal syncope and shock (Case report and literature review)
  196. Fungal infection mimicking COVID-19 infection – A case report
  197. Concurrent aspergillosis and cystic pulmonary metastases in a patient with tongue squamous cell carcinoma
  198. Paraganglioma-induced inverted takotsubo-like cardiomyopathy leading to cardiogenic shock successfully treated with extracorporeal membrane oxygenation
  199. Lineage switch from lymphoma to myeloid neoplasms: First case series from a single institution
  200. Trismus during tracheal extubation as a complication of general anaesthesia – A case report
  201. Simultaneous treatment of a pubovesical fistula and lymph node metastasis secondary to multimodal treatment for prostate cancer: Case report and review of the literature
  202. Two case reports of skin vasculitis following the COVID-19 immunization
  203. Ureteroiliac fistula after oncological surgery: Case report and review of the literature
  204. Synchronous triple primary malignant tumours in the bladder, prostate, and lung harbouring TP53 and MEK1 mutations accompanied with severe cardiovascular diseases: A case report
  205. Huge mucinous cystic neoplasms with adhesion to the left colon: A case report and literature review
  206. Commentary
  207. Commentary on “Clinicopathological features of programmed cell death-ligand 1 expression in patients with oral squamous cell carcinoma”
  208. Rapid Communication
  209. COVID-19 fear, post-traumatic stress, growth, and the role of resilience
  210. Erratum
  211. Erratum to “Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway”
  212. Erratum to “Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study”
  213. Erratum to “lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2”
  214. Retraction
  215. Expression and role of ABIN1 in sepsis: In vitro and in vivo studies
  216. Retraction to “miR-519d downregulates LEP expression to inhibit preeclampsia development”
  217. Special Issue Computational Intelligence Methodologies Meets Recurrent Cancers - Part II
  218. Usefulness of close surveillance for rectal cancer patients after neoadjuvant chemoradiotherapy
https://doi.org/10.1515/med-2022-0468
Creative Commons
BY 4.0

Artikel in diesem Heft

  1. Research Articles
  2. AMBRA1 attenuates the proliferation of uveal melanoma cells
  3. A ceRNA network mediated by LINC00475 in papillary thyroid carcinoma
  4. Differences in complications between hepatitis B-related cirrhosis and alcohol-related cirrhosis
  5. Effect of gestational diabetes mellitus on lipid profile: A systematic review and meta-analysis
  6. Long noncoding RNA NR2F1-AS1 stimulates the tumorigenic behavior of non-small cell lung cancer cells by sponging miR-363-3p to increase SOX4
  7. Promising novel biomarkers and candidate small-molecule drugs for lung adenocarcinoma: Evidence from bioinformatics analysis of high-throughput data
  8. Plasmapheresis: Is it a potential alternative treatment for chronic urticaria?
  9. The biomarkers of key miRNAs and gene targets associated with extranodal NK/T-cell lymphoma
  10. Gene signature to predict prognostic survival of hepatocellular carcinoma
  11. Effects of miRNA-199a-5p on cell proliferation and apoptosis of uterine leiomyoma by targeting MED12
  12. Does diabetes affect paraneoplastic thrombocytosis in colorectal cancer?
  13. Is there any effect on imprinted genes H19, PEG3, and SNRPN during AOA?
  14. Leptin and PCSK9 concentrations are associated with vascular endothelial cytokines in patients with stable coronary heart disease
  15. Pericentric inversion of chromosome 6 and male fertility problems
  16. Staple line reinforcement with nebulized cyanoacrylate glue in laparoscopic sleeve gastrectomy: A propensity score-matched study
  17. Retrospective analysis of crescent score in clinical prognosis of IgA nephropathy
  18. Expression of DNM3 is associated with good outcome in colorectal cancer
  19. Activation of SphK2 contributes to adipocyte-induced EOC cell proliferation
  20. CRRT influences PICCO measurements in febrile critically ill patients
  21. SLCO4A1-AS1 mediates pancreatic cancer development via miR-4673/KIF21B axis
  22. lncRNA ACTA2-AS1 inhibits malignant phenotypes of gastric cancer cells
  23. circ_AKT3 knockdown suppresses cisplatin resistance in gastric cancer
  24. Prognostic value of nicotinamide N-methyltransferase in human cancers: Evidence from a meta-analysis and database validation
  25. GPC2 deficiency inhibits cell growth and metastasis in colon adenocarcinoma
  26. A pan-cancer analysis of the oncogenic role of Holliday junction recognition protein in human tumors
  27. Radiation increases COL1A1, COL3A1, and COL1A2 expression in breast cancer
  28. Association between preventable risk factors and metabolic syndrome
  29. miR-29c-5p knockdown reduces inflammation and blood–brain barrier disruption by upregulating LRP6
  30. Cardiac contractility modulation ameliorates myocardial metabolic remodeling in a rabbit model of chronic heart failure through activation of AMPK and PPAR-α pathway
  31. Quercitrin protects human bronchial epithelial cells from oxidative damage
  32. Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2
  33. circRNA_0001679/miR-338-3p/DUSP16 axis aggravates acute lung injury
  34. Sonoclot’s usefulness in prediction of cardiopulmonary arrest prognosis: A proof of concept study
  35. Four drug metabolism-related subgroups of pancreatic adenocarcinoma in prognosis, immune infiltration, and gene mutation
  36. Decreased expression of miR-195 mediated by hypermethylation promotes osteosarcoma
  37. LMO3 promotes proliferation and metastasis of papillary thyroid carcinoma cells by regulating LIMK1-mediated cofilin and the β-catenin pathway
  38. Cx43 upregulation in HUVECs under stretch via TGF-β1 and cytoskeletal network
  39. Evaluation of menstrual irregularities after COVID-19 vaccination: Results of the MECOVAC survey
  40. Histopathologic findings on removed stomach after sleeve gastrectomy. Do they influence the outcome?
  41. Analysis of the expression and prognostic value of MT1-MMP, β1-integrin and YAP1 in glioma
  42. Optimal diagnosis of the skin cancer using a hybrid deep neural network and grasshopper optimization algorithm
  43. miR-223-3p alleviates TGF-β-induced epithelial-mesenchymal transition and extracellular matrix deposition by targeting SP3 in endometrial epithelial cells
  44. Clinical value of SIRT1 as a prognostic biomarker in esophageal squamous cell carcinoma, a systematic meta-analysis
  45. circ_0020123 promotes cell proliferation and migration in lung adenocarcinoma via PDZD8
  46. miR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2
  47. hsa-miR-340-5p inhibits epithelial–mesenchymal transition in endometriosis by targeting MAP3K2 and inactivating MAPK/ERK signaling
  48. circ_0085296 inhibits the biological functions of trophoblast cells to promote the progression of preeclampsia via the miR-942-5p/THBS2 network
  49. TCD hemodynamics findings in the subacute phase of anterior circulation stroke patients treated with mechanical thrombectomy
  50. Development of a risk-stratification scoring system for predicting risk of breast cancer based on non-alcoholic fatty liver disease, non-alcoholic fatty pancreas disease, and uric acid
  51. Tollip promotes hepatocellular carcinoma progression via PI3K/AKT pathway
  52. circ_0062491 alleviates periodontitis via the miR-142-5p/IGF1 axis
  53. Human amniotic fluid as a source of stem cells
  54. lncRNA NONRATT013819.2 promotes transforming growth factor-β1-induced myofibroblastic transition of hepatic stellate cells by miR24-3p/lox
  55. NORAD modulates miR-30c-5p-LDHA to protect lung endothelial cells damage
  56. Idiopathic pulmonary fibrosis telemedicine management during COVID-19 outbreak
  57. Risk factors for adverse drug reactions associated with clopidogrel therapy
  58. Serum zinc associated with immunity and inflammatory markers in Covid-19
  59. The relationship between night shift work and breast cancer incidence: A systematic review and meta-analysis of observational studies
  60. LncRNA expression in idiopathic achalasia: New insight and preliminary exploration into pathogenesis
  61. Notoginsenoside R1 alleviates spinal cord injury through the miR-301a/KLF7 axis to activate Wnt/β-catenin pathway
  62. Moscatilin suppresses the inflammation from macrophages and T cells
  63. Zoledronate promotes ECM degradation and apoptosis via Wnt/β-catenin
  64. Epithelial-mesenchymal transition-related genes in coronary artery disease
  65. The effect evaluation of traditional vaginal surgery and transvaginal mesh surgery for severe pelvic organ prolapse: 5 years follow-up
  66. Repeated partial splenic artery embolization for hypersplenism improves platelet count
  67. Low expression of miR-27b in serum exosomes of non-small cell lung cancer facilitates its progression by affecting EGFR
  68. Exosomal hsa_circ_0000519 modulates the NSCLC cell growth and metastasis via miR-1258/RHOV axis
  69. miR-455-5p enhances 5-fluorouracil sensitivity in colorectal cancer cells by targeting PIK3R1 and DEPDC1
  70. The effect of tranexamic acid on the reduction of intraoperative and postoperative blood loss and thromboembolic risk in patients with hip fracture
  71. Isocitrate dehydrogenase 1 mutation in cholangiocarcinoma impairs tumor progression by sensitizing cells to ferroptosis
  72. Artemisinin protects against cerebral ischemia and reperfusion injury via inhibiting the NF-κB pathway
  73. A 16-gene signature associated with homologous recombination deficiency for prognosis prediction in patients with triple-negative breast cancer
  74. Lidocaine ameliorates chronic constriction injury-induced neuropathic pain through regulating M1/M2 microglia polarization
  75. MicroRNA 322-5p reduced neuronal inflammation via the TLR4/TRAF6/NF-κB axis in a rat epilepsy model
  76. miR-1273h-5p suppresses CXCL12 expression and inhibits gastric cancer cell invasion and metastasis
  77. Clinical characteristics of pneumonia patients of long course of illness infected with SARS-CoV-2
  78. circRNF20 aggravates the malignancy of retinoblastoma depending on the regulation of miR-132-3p/PAX6 axis
  79. Linezolid for resistant Gram-positive bacterial infections in children under 12 years: A meta-analysis
  80. Rack1 regulates pro-inflammatory cytokines by NF-κB in diabetic nephropathy
  81. Comprehensive analysis of molecular mechanism and a novel prognostic signature based on small nuclear RNA biomarkers in gastric cancer patients
  82. Smog and risk of maternal and fetal birth outcomes: A retrospective study in Baoding, China
  83. Let-7i-3p inhibits the cell cycle, proliferation, invasion, and migration of colorectal cancer cells via downregulating CCND1
  84. β2-Adrenergic receptor expression in subchondral bone of patients with varus knee osteoarthritis
  85. Possible impact of COVID-19 pandemic and lockdown on suicide behavior among patients in Southeast Serbia
  86. In vitro antimicrobial activity of ozonated oil in liposome eyedrop against multidrug-resistant bacteria
  87. Potential biomarkers for inflammatory response in acute lung injury
  88. A low serum uric acid concentration predicts a poor prognosis in adult patients with candidemia
  89. Antitumor activity of recombinant oncolytic vaccinia virus with human IL2
  90. ALKBH5 inhibits TNF-α-induced apoptosis of HUVECs through Bcl-2 pathway
  91. Risk prediction of cardiovascular disease using machine learning classifiers
  92. Value of ultrasonography parameters in diagnosing polycystic ovary syndrome
  93. Bioinformatics analysis reveals three key genes and four survival genes associated with youth-onset NSCLC
  94. Identification of autophagy-related biomarkers in patients with pulmonary arterial hypertension based on bioinformatics analysis
  95. Protective effects of glaucocalyxin A on the airway of asthmatic mice
  96. Overexpression of miR-100-5p inhibits papillary thyroid cancer progression via targeting FZD8
  97. Bioinformatics-based analysis of SUMOylation-related genes in hepatocellular carcinoma reveals a role of upregulated SAE1 in promoting cell proliferation
  98. Effectiveness and clinical benefits of new anti-diabetic drugs: A real life experience
  99. Identification of osteoporosis based on gene biomarkers using support vector machine
  100. Tanshinone IIA reverses oxaliplatin resistance in colorectal cancer through microRNA-30b-5p/AVEN axis
  101. miR-212-5p inhibits nasopharyngeal carcinoma progression by targeting METTL3
  102. Association of ST-T changes with all-cause mortality among patients with peripheral T-cell lymphomas
  103. LINC00665/miRNAs axis-mediated collagen type XI alpha 1 correlates with immune infiltration and malignant phenotypes in lung adenocarcinoma
  104. The perinatal factors that influence the excretion of fecal calprotectin in premature-born children
  105. Effect of femoral head necrosis cystic area on femoral head collapse and stress distribution in femoral head: A clinical and finite element study
  106. Does the use of 3D-printed cones give a chance to postpone the use of megaprostheses in patients with large bone defects in the knee joint?
  107. lncRNA HAGLR modulates myocardial ischemia–reperfusion injury in mice through regulating miR-133a-3p/MAPK1 axis
  108. Protective effect of ghrelin on intestinal I/R injury in rats
  109. In vivo knee kinematics of an innovative prosthesis design
  110. Relationship between the height of fibular head and the incidence and severity of knee osteoarthritis
  111. lncRNA WT1-AS attenuates hypoxia/ischemia-induced neuronal injury during cerebral ischemic stroke via miR-186-5p/XIAP axis
  112. Correlation of cardiac troponin T and APACHE III score with all-cause in-hospital mortality in critically ill patients with acute pulmonary embolism
  113. LncRNA LINC01857 reduces metastasis and angiogenesis in breast cancer cells via regulating miR-2052/CENPQ axis
  114. Endothelial cell-specific molecule 1 (ESM1) promoted by transcription factor SPI1 acts as an oncogene to modulate the malignant phenotype of endometrial cancer
  115. SELENBP1 inhibits progression of colorectal cancer by suppressing epithelial–mesenchymal transition
  116. Visfatin is negatively associated with coronary artery lesions in subjects with impaired fasting glucose
  117. Treatment and outcomes of mechanical complications of acute myocardial infarction during the Covid-19 era: A comparison with the pre-Covid-19 period. A systematic review and meta-analysis
  118. Neonatal stroke surveillance study protocol in the United Kingdom and Republic of Ireland
  119. Oncogenic role of TWF2 in human tumors: A pan-cancer analysis
  120. Mean corpuscular hemoglobin predicts the length of hospital stay independent of severity classification in patients with acute pancreatitis
  121. Association of gallstone and polymorphisms of UGT1A1*27 and UGT1A1*28 in patients with hepatitis B virus-related liver failure
  122. TGF-β1 upregulates Sar1a expression and induces procollagen-I secretion in hypertrophic scarring fibroblasts
  123. Antisense lncRNA PCNA-AS1 promotes esophageal squamous cell carcinoma progression through the miR-2467-3p/PCNA axis
  124. NK-cell dysfunction of acute myeloid leukemia in relation to the renin–angiotensin system and neurotransmitter genes
  125. The effect of dilution with glucose and prolonged injection time on dexamethasone-induced perineal irritation – A randomized controlled trial
  126. miR-146-5p restrains calcification of vascular smooth muscle cells by suppressing TRAF6
  127. Role of lncRNA MIAT/miR-361-3p/CCAR2 in prostate cancer cells
  128. lncRNA NORAD promotes lung cancer progression by competitively binding to miR-28-3p with E2F2
  129. Noninvasive diagnosis of AIH/PBC overlap syndrome based on prediction models
  130. lncRNA FAM230B is highly expressed in colorectal cancer and suppresses the maturation of miR-1182 to increase cell proliferation
  131. circ-LIMK1 regulates cisplatin resistance in lung adenocarcinoma by targeting miR-512-5p/HMGA1 axis
  132. LncRNA SNHG3 promoted cell proliferation, migration, and metastasis of esophageal squamous cell carcinoma via regulating miR-151a-3p/PFN2 axis
  133. Risk perception and affective state on work exhaustion in obstetrics during the COVID-19 pandemic
  134. lncRNA-AC130710/miR-129-5p/mGluR1 axis promote migration and invasion by activating PKCα-MAPK signal pathway in melanoma
  135. SNRPB promotes cell cycle progression in thyroid carcinoma via inhibiting p53
  136. Xylooligosaccharides and aerobic training regulate metabolism and behavior in rats with streptozotocin-induced type 1 diabetes
  137. Serpin family A member 1 is an oncogene in glioma and its translation is enhanced by NAD(P)H quinone dehydrogenase 1 through RNA-binding activity
  138. Silencing of CPSF7 inhibits the proliferation, migration, and invasion of lung adenocarcinoma cells by blocking the AKT/mTOR signaling pathway
  139. Ultrasound-guided lumbar plexus block versus transversus abdominis plane block for analgesia in children with hip dislocation: A double-blind, randomized trial
  140. Relationship of plasma MBP and 8-oxo-dG with brain damage in preterm
  141. Identification of a novel necroptosis-associated miRNA signature for predicting the prognosis in head and neck squamous cell carcinoma
  142. Delayed femoral vein ligation reduces operative time and blood loss during hip disarticulation in patients with extremity tumors
  143. The expression of ASAP3 and NOTCH3 and the clinicopathological characteristics of adult glioma patients
  144. Longitudinal analysis of factors related to Helicobacter pylori infection in Chinese adults
  145. HOXA10 enhances cell proliferation and suppresses apoptosis in esophageal cancer via activating p38/ERK signaling pathway
  146. Meta-analysis of early-life antibiotic use and allergic rhinitis
  147. Marital status and its correlation with age, race, and gender in prognosis of tonsil squamous cell carcinomas
  148. HPV16 E6E7 up-regulates KIF2A expression by activating JNK/c-Jun signal, is beneficial to migration and invasion of cervical cancer cells
  149. Amino acid profiles in the tissue and serum of patients with liver cancer
  150. Pain in critically ill COVID-19 patients: An Italian retrospective study
  151. Immunohistochemical distribution of Bcl-2 and p53 apoptotic markers in acetamiprid-induced nephrotoxicity
  152. Estradiol pretreatment in GnRH antagonist protocol for IVF/ICSI treatment
  153. Long non-coding RNAs LINC00689 inhibits the apoptosis of human nucleus pulposus cells via miR-3127-5p/ATG7 axis-mediated autophagy
  154. The relationship between oxygen therapy, drug therapy, and COVID-19 mortality
  155. Monitoring hypertensive disorders in pregnancy to prevent preeclampsia in pregnant women of advanced maternal age: Trial mimicking with retrospective data
  156. SETD1A promotes the proliferation and glycolysis of nasopharyngeal carcinoma cells by activating the PI3K/Akt pathway
  157. The role of Shunaoxin pills in the treatment of chronic cerebral hypoperfusion and its main pharmacodynamic components
  158. TET3 governs malignant behaviors and unfavorable prognosis of esophageal squamous cell carcinoma by activating the PI3K/AKT/GSK3β/β-catenin pathway
  159. Associations between morphokinetic parameters of temporary-arrest embryos and the clinical prognosis in FET cycles
  160. Long noncoding RNA WT1-AS regulates trophoblast proliferation, migration, and invasion via the microRNA-186-5p/CADM2 axis
  161. The incidence of bronchiectasis in chronic obstructive pulmonary disease
  162. Integrated bioinformatics analysis shows integrin alpha 3 is a prognostic biomarker for pancreatic cancer
  163. Inhibition of miR-21 improves pulmonary vascular responses in bronchopulmonary dysplasia by targeting the DDAH1/ADMA/NO pathway
  164. Comparison of hospitalized patients with severe pneumonia caused by COVID-19 and influenza A (H7N9 and H1N1): A retrospective study from a designated hospital
  165. lncRNA ZFAS1 promotes intervertebral disc degeneration by upregulating AAK1
  166. Pathological characteristics of liver injury induced by N,N-dimethylformamide: From humans to animal models
  167. lncRNA ELFN1-AS1 enhances the progression of colon cancer by targeting miR-4270 to upregulate AURKB
  168. DARS-AS1 modulates cell proliferation and migration of gastric cancer cells by regulating miR-330-3p/NAT10 axis
  169. Dezocine inhibits cell proliferation, migration, and invasion by targeting CRABP2 in ovarian cancer
  170. MGST1 alleviates the oxidative stress of trophoblast cells induced by hypoxia/reoxygenation and promotes cell proliferation, migration, and invasion by activating the PI3K/AKT/mTOR pathway
  171. Bifidobacterium lactis Probio-M8 ameliorated the symptoms of type 2 diabetes mellitus mice by changing ileum FXR-CYP7A1
  172. circRNA DENND1B inhibits tumorigenicity of clear cell renal cell carcinoma via miR-122-5p/TIMP2 axis
  173. EphA3 targeted by miR-3666 contributes to melanoma malignancy via activating ERK1/2 and p38 MAPK pathways
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Heruntergeladen am 5.5.2026 von https://www.degruyterbrill.com/document/doi/10.1515/med-2022-0468/html?lang=de
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