Predicted COVID-19 molecular effects on endometrium reveal key dysregulated genes and functions.

COVID-19 exerts systemic effects that can compromise various organs and systems. Although retrospective and in silico studies and prospective preliminary analysis have assessed the possibility of direct infection of the endometrium, there is a lack of in-depth and prospective studies on the impact of systemic disease on key endometrial genes and functions across the menstrual cycle and window of implantation. Gene expression data have been obtained from (i) healthy secretory endometrium collected from 42 women without endometrial pathologies and (ii) nasopharyngeal swabs from 231 women with COVID-19 and 30 negative controls. To predict how COVID-19-related gene expression changes impact key endometrial genes and functions, an in silico model was developed by integrating the endometrial and COVID-19 datasets in an affected mid-secretory endometrium gene co-expression network. An endometrial validation set comprising 16 women (8 confirmed to have COVID-19 and 8 negative test controls) was prospectively collected to validate the expression of key genes. We predicted that five genes important for embryo implantation were affected by COVID-19 (downregulation of COBL, GPX3 and SOCS3, and upregulation of DOCK2 and SLC2A3). We experimentally validated these genes in COVID-19 patients using endometrial biopsies during the secretory phase of the menstrual cycle. The results generally support the in silico model predictions, suggesting that the transcriptomic landscape changes mediated by COVID-19 affect endometrial receptivity genes and key processes necessary for fertility, such as immune system function, protection against oxidative damage and development vital for embryo implantation and early development.

Stem cell paracrine actions in tissue regeneration and potential therapeutic effect in human endometrium: a retrospective study.

OBJECTIVE: Determining genetic and paracrine mechanisms behind endometrial regeneration in Asherman's syndrome and endometrial atrophy (AS/EA) patients after autologous CD133+ bone marrow-derived stem cell (CD133+ BMDSC) transplantation. DESIGN: Retrospective study using human endometrial biopsies and mouse models. SETTING: Fundación-IVI, IIS-La Fe, Valencia, Spain. SAMPLES: Endometrial biopsies collected before and after CD133+ BMDSC therapy, from eight women with AS/EA (NCT02144987) from the uterus of five mice with only left horns receiving CD133+ BMDSC therapy. METHODS: In human samples, haematoxylin and eosin (H&E) staining, RNA arrays, PCR validation, and neutrophil elastase (NE) immunohistochemistry (IHQ). In mouse samples, PCR validation and protein immunoarrays. MAIN OUTCOME MEASURES: H&E microscopic evaluation, RNA expression levels, PCR, and growth/angiogenic factors quantification, NE IHQ signal. RESULTS: Treatment improved endometrial morphology and thickness for all patients. In human samples, Jun, Serpine1, and Il4 were up-regulated whereas Ccnd1 and Cxcl8 were down-regulated after treatment. The significant decrease of NE signal corroborated Cxcl8 expression. Animal model analysis confirmed human results and revealed a higher expression of pro-angiogenic cytokines (IL18, HGF, MCP-1, MIP2) in treated uterine horns. CONCLUSIONS: CD133+ BMDSC seems to activate several factors through a paracrine mechanism to help tissue regeneration, modifying endometrial behaviour through an immunomodulatory milieu that precedes proliferation and angiogenic processes. Insight into these processes could bring us one step closer to a non-invasive treatment for AS/EA patients. TWEETABLE ABSTRACT: CD133+ BMDSC therapy regenerates endometrium, modifying the immunological milieu that precedes proliferation and angiogenesis.

Bioengineering strategies of the uterus towards improving current investigative models and female reproductive health.

Ever since the inception of artificial reproductive technologies (ART), new advances have been developed in the lab and translated to the clinic to improve the reproductive outcome of patients. Tissue engineering (TE) adopts ideas and concepts from biology, bioengineering and material science amongst others, resulting in a promising and burgeoning multidisciplinary field of investigation within regenerative medicine. The main objective of the work presented in this thesis was to use TE based approaches to create different types of natural biomaterials obtained from decellularized porcine or rabbit uteri. We investigated if these different bioscaffolds could improve current investigative in vitro models while also showing potential to be used as therapeutic solutions. Decellularized whole organs are acellular vascularized scaffolds that could be used to create tissue-engineered, transplantable organs. However, they can also be processed further into thin sections, ECM hydrogels and coatings, and were used as biocompatible tissue-specific substrates for cell and embryo culture. Two animal models were used, the pig model demonstrated that our perfusion-based protocol (with or without a freeze/thaw step) successfully decellularizes large uteri, yielding a biocompatible material. Secondly, we adapted this protocol for the rabbit uterus and converted the acellular endometrium into tissue-specific ECM hydrogels and coatings. After characterization of these substrates their effect on in vitro embryo development was also examined. While DC organs could one day be used to resolve the main issues plaguing transplantations, endometrial ECM sections, hydrogels and coatings have shown the potential to become a platform used in the culture of stem/progenitor cells and primary culture cells to better maintain their tissue-specific phenotype, improving in vitro models. Furthermore, ECM hydrogels could possibly be used in the future in vivo, as part of a treatment of Asherman's syndrome and endometrial atrophy.

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) as a putative human endometrial stem cell marker.

The endometrium is recognized for its remarkable regenerative and remodeling capacity. Every month this hormonally regulated organ undergoes cycles of growth (from 0.5-2 to 7 mm), regression and shedding of two-third of the tissue, leading to its monthly renewal that occurs ∼400 times in a woman's reproductive lifetime. Several groups have suggested the existence of a human endometrial somatic stem cell (SSC) population located around the spiral arterioles of the basalis. Different groups have isolated, identified and characterized putative endometrial SSC populations in human endometrium based on the general features of undifferentiated cells, such as slow cycling detected using the 5-bromo-2-deoxyuridine technique or identification of a side population using the Hoechst efflux dye technique. Nevertheless, specific markers to isolate these endometrial SSC have not yet been consistently elucidated. Accumulated evidence based on lineage tracing studies indicates that a surface protein named Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is a marker that can identify SSC in several tissues such as small intestine mucosa (endodermal origin), hair follicles (ectodermal origin) or mature kidney nephrons (mesodermal origin). This protein plays a crucial role in the Wnt/ß-catenin signaling system by acting on the self-renewal and maintenance of the SSC population. In this work, we present novel data suggestive of Lgr5 as a putative human endometrial SSC marker, and since this is a mesoderm-derived tissue, these findings reinforce the concept that Lgr5 can be considered a universal SSC marker.

Identification, characterization and co-localization of label-retaining cell population in mouse endometrium with typical undifferentiated markers.

BACKGROUND: The endometrium, lining of the uterus, is a highly active organ that is remodelled periodically during the lifespan. Different studies suggest the presence of an adult or progenitor stem cell (PSC) population in this tissue because of its cyclic regenerative capacity. METHODS: In this study, we aim at identifying and localizing the putative PSC population in the murine uterus using the 5-bromo-2'-deoxyuridine (BrdU) labelling method to detect label-retaining cells (LRCs) that cycle slowly. Uteri from BrdU-treated mice were analysed via single and double immunohistochemistry to co-localize them with the markers of undifferentiation already described such as c-KIT and POU5F1 (also known as OCT-4). Finally, we confirmed the presence of the indicated markers at mRNA level. RESULTS: We observed the presence and gradual decrease of LRCs in the endometrium during the lifespan of the mice. In adulthood, the LRC population decreased notably and remained in the lower region of the stroma in the murine endometrium. Some of the endometrial LRCs co-localized with c-KIT and POU5F1. PCR and nested-PCR confirmed the presence of these undifferentiated markers. CONCLUSIONS: We demonstrated that the murine endometrium possesses LRCs with the features of a putative PSC population localized at the lower region of the stroma.

Glutathione, glutathione S-transferase and reactive oxygen species of human scalp sebaceous glands in male pattern baldness.

We investigated the contribution of reactive oxygen species to the development of sebaceous gland hyperplasia and the characteristics of the glutathione S-transferase/glutathione system in male pattern baldness. Glutathione S-transferase, glutathione, and thiobarbituric acid-reactive substances were determined in sebaceous gland-enriched scalp skin of men affected by male pattern baldness and were subjected to hair autotransplantation. In comparison with the hairy occipital-donor areas, the following results were obtained in alopecic frontoparietal samples: glutathione S-transferase-specific activity increased 7-fold (p < 0.001); enzyme affinity towards 1-chloro-2,4-dinitrobenzene decreased 2-fold (p = 0.009); glutathione content decreased 2.5-fold (p = 0.017); and thiobarbituric acid reactive substances increased 2-fold (p = 0.006). Chromatofocusing analysis, bromosulfophthalein IC50 values, enzyme-linked immunosorbent assay, and immunohistochemistry with polyclonal antibodies raised against glutathione S-transferases alpha, mu, and pi demonstrated the presence of alpha, pi, and probably the 5.8 alpha isoenzymes in the sebaceous gland. These results support the hypothesis that reactive oxygen species are involved in the pathogenesis of sebaceous gland hyperplasia in male pattern baldness.

Inefficacy of collagen shields in the rabbit corneal wound-healing process.

A controlled study in the rabbit eye was performed to clarify the suitability of collagen shields in accelerating the wound healing process in damaged cornea. After a standardized bilateral keratectomy, the spontaneous evolution of the healing process in the right eye (control eye) was compared with the evolution in the left eye, treated with a collagen shield (a shield of the 12- or 24-h type). The healing area was measured by planimetry after fluorescein staining at 0, 24, 48, and 72 h after keratectomy. Histological and immunohistochemical analysis of the healing process was also performed. Reepithelization of the damaged cornea was almost complete at 72 h, and there were no differences in the time course of the healing process between control and treated eyes. There was an important polymorphonuclear infiltration in treated eyes, mainly composed of eosinophiles, which was not present in control eyes. This indicates a subacute inflammatory immunological reaction. It is concluded that the usefulness of collagen shields should be reappraised, especially in injured corneas.

Corneal wound healing in the rabbit eye: activity of a fibronectin-splitting product.

The corneal wound-healing properties of fibronectin (FN) and a fibronectin hydrolysate (FNH) have been evaluated in comparison with commonly used drugs. Nonpenetrating bilateral surgical keratectomy was performed in male albino rabbits. The left eye was treated with the active product, whereas the right eye served as a control (vehicle). The healing area was measured by planimetry after fluorescein staining at 0, 24, 48, and 72 h after keratectomy. Histological and immunohistochemical analysis of the healing process was also performed. Results were as follows: (a) Nandrolone (p < 0.005) and asiaticoside (p < 0.001), both at 10 mg/ml, in eyedrops delayed the healing process. (b) An ointment containing vitamin A and amino acids also delayed the process but at the limit of statistical significance (p = 0.055). (c) FNH (20-80 mg prot/ml) significantly improved the quality and shortened the time of the healing process at 60 mg prot/ml and above. (d) Human FN (100-800 micrograms/ml) did not affect the healing process. Immunohistochemical analysis demonstrated that FNH accelerated the appearance of endogenous FN in the damaged cornea earlier than in the control eyes. It is concluded that FNH may be useful in the management of corneal wounds, whereas the effectiveness of FN is doubtful.

[The kinetic characteristics of the erythrocyte glutathione S-transferase system as a function of sex and the tobacco habit]. / Características cinéticas del sistema glutatión S-transferasa eritrocitario en función del sexo y del hábito tabáquico.

BACKGROUND: Erythrocytic glutathion S-transferase (GST) plays an important role as a protective mechanism against oxidative stress. The present study was conducted to evaluate the influence of both smoking habit and sex upon the kinetic characteristics of the enzyme. SUBJECTS AND METHODS: 176 healthy subjects (100 men and 76 women), smokers and nonsmokers, were included. Enzyme parameters were calculated in erythrocytic haemolysates using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathion (GSH) as substrates. Haemoglobin (Hb) was removed by affinity chromatography. In samples coming from 51 men and 42 women the native haemolysate was subjected to thermal shock (52 degrees C) and the enzyme parameters were compared with those obtained in the non-denatured samples. RESULTS: In non-denatured samples, Km (mM) and Vmax (mumol/min/g Hb) values for CDNB were significantly higher (p < 0.001) in smokers than in non smokers, especially in women. Thus, respectively for Km and Vmax (mean +/- standard deviation): for men non smokers, 1.43 +/- 0.54, 1.63 +/- 0.42 and smokers, 1.74 +/- 0.5, 1.8 +/- 0.69; for women, non smokers 1.42 +/- 0.56, 1.57 +/- 0.46 and smokers, 2.05 +/- 0.59, 2.51 +/- 0.6. Thermal denaturation diminished the enzyme activity in all cases and modified the Km values, these results were opposite to those obtained in the non-denatured samples. Thus, for Km and Vmax respectively: for men, smokers, 1.6 +/- 0.71 and 0.9 +/- 0.32 and non smokers, 1.4 +/- 0.66 and 0.53 +/- 0.29; for women, non smokers, 2.00 +/- 0.58, 1.13 +/- 0.29 and smokers 1.22 +/- 0.77, 0.52 +/- 0.23. The GSt content was similar in the four groups studied (3.75 +/- 1.15 mumol SH/g Hb). CONCLUSIONS: The greater thermolability of GST activity and the increase in the Km values observed in smokers, especially in women, should be considered as indicative of an increased risk for the erythrocytes against oxidative stress.

Glutathione S-transferase in normal human anagen hair follicles.

Glutathione S-transferase (GST) has been quantified and characterized in healthy human anagen hair follicles obtained from 36 men and 36 women (26 +/- 7 years of age). GST activity was determined using 1-chloro-2,4-dinitrobenzene as a substrate, and the values in men were: 0.5 +/- 0.2 mU/follicle, significantly different from women (0.36 +/- 0.2 mU/follicle); 196 +/- 98 mU/mg protein and 309 +/- 158 mU/mg DNA without significant differences from women. Enzyme activity showed a high degree of inter-individual variability (23.5-fold when expressed per follicle, 18.29-fold expressed per mg of protein and 22.75-fold per mg of DNA) in the whole population and this variability was higher in women. Ion-exchange chromatography by KCl and enzyme immunoassay suggest that the GST present in hair follicles corresponds with the acidic form. The percentage of anagen hairs in each subject showed a positive correlation with the following parameters: GST/hair, GST/DNA and DNA/hair. It is concluded that GST may contribute to the maintenance of the hair growth cycle.

Modifications of glutathione S-transferase (GST) activity in the last period of pregnancy in rats treated with benzo(a)pyrene (BP).

Pregnant rats were treated with benzo(a)pyrene (BP) (50 mg/kg every 2 days) from day 7 of pregnancy and killed at day 16 or day 19. Km of erythrocyte glutathione S-transferase (GST) decreased during pregnancy in control rats (1.29 x 10(-3) M at day 16; 1.02 x 10(-3) M at day 19) and even more in treated rats at day 19 (0.71 x 10(-3) M). Vmax was lower in treated rats at day 19 (0.56 mumol/min/g haemoglobin) than in control rats (0.88 mumol/min/g haemoglobin) suggesting inhibition of the enzyme. Placental weight diminished in treated rats at day 19 but was not affected at day 16. Chromatofocusing of placental GST showed a single peak (pH 8.3-8.6) in control and treated rats on day 16 and an additional peak (pH 7.0-7.4) in treated rats on day 19. An increase in Km (2.84 x 10(-3) M) and Vmax (69 nmol/min/mg protein) in placental GST was observed in treated rats at day 16 (Km = 1.61 x 10(-3) M; Vmax = 43.3 nmol/min/mg protein, in control rats) followed by a decrease in these parameters in rats treated until day 19 (Km = 1.63 x 10(-3) M; Vmax = 48.7 nmol/min/mg protein). These results suggest that BP, initially, stimulates GST synthesis in placenta, followed by an inhibition of the enzyme at day 19. Fetal weight was also affected by BP treatment, especially at day 16. Km and Vmax values of fetal GST were higher in treated rats at day 16 than in control rats but these differences were not detectable at day 19. This may be explained by the more protective role of the placenta at day 19 than at day 16. Glutathione content in erythrocytes, placenta and fetus was not affected by BP.

Enhanced glutathione S-transferase (GST) activity in pregnant rats treated with benzo(a)pyrene.

Administration of Benzo(a)pyrene (BP, 50 mg/kg/d) to pregnant rats significantly increased Glutathione S-transferase (GST) activity in placental tissue-extract (Vmax = 40 nmol/min/mg protein and 69 nmol/min/mg protein in controls versus treated animals respectively; P less than 0.01) and total fetal tissue-extract (Vmax = 51 nmol/min/mg protein and 82 nmol/min/mg protein in controls versus treated animals respectively; P less than 0.01) indicating an induction effect of BP on the GST system. An increase in the Km values was also observed: 1.61 x 10(-3) M and 2.84 x 10(-3) M in control versus treated placentae; 1.38 x 10(-3) M and 2.05 x 10(-3) M in control versus treated fetuses. A competitive effect on the enzyme by the BP present in the sample may also be involved. The glutathione content in both tissues did not show any changes after the treatment with BP. This increase in the GST system was not sufficient to protect the fetus. BP affected the reproductive performance of pregnant rats by significantly increasing the number of resorptions and fetal wastage, and, also, by decreasing the fetal weight.

Glutathione-S-transferase activity in human superficial transitional cell carcinoma of the bladder. Comparison with healthy controls.

Glutathione-S-transferase (GST) activity and glutathione (GSH) content have been studied in human urinary bladder (UB) specimens obtained from healthy controls (HC) (n = 8) and from patients with superficial transitional cell carcinoma (TCC) (n = 9), either in TCC and in adjacent normal (ANE) tissues of the same patient. The GST activity was significantly higher in TCC in comparison with ANE (ten fold) and with HC (five fold). This activity was also significantly higher in HC than in ANE (two fold). The Km values obtained in the whole population (1.26 +/- 0.3 X 10(-3) mol/l) suggest that a unique form of isoenzyme is present in the UB epithelium and that it is the same acidic form "rho" described in erythrocytes. The GSH content was significantly higher in TCC than in ANE (2.5 fold) and also that in HC (three fold). A good correlation between GST activity and GSH content was observed in HC but not in TCC or ANE. These results demonstrate the relation between the activity of the GST system and the development of the TCC as well as its role in the cellular resistance to chemotherapy. A possible decrease of the GST activity before the development of the tumor is also discussed.