Populations of NK Cells and Regulatory T Cells in the Endometrium of Cycling Mares-A Preliminary Study.

Endometrial immune cells are essential to support uterine functions across the estrous cycle and in preparation for pregnancy. It has been acknowledged that changes in phenotype and/or numbers of lymphocytes, such as regulatory T cells (Tregs) and NK cells, might result in lower fertility in women and mice. Little is known about equine endometrial immune cells across the estrous cycle. Here, we compared the populations of endometrial Tregs and NK cells in estrus and diestrus in mares. Endometrial biopsy and blood samples were taken in estrus and diestrus from 11 mares ages 4-12 years. Flow cytometry with anti-CD4, -CD25 and -FOXP3 and anti-NKp46 and -CD3 antibodies was used to determine the populations of Tregs and NK cells, respectively. The concentration of progesterone was measured with chemiluminescence immunoassay. The results were analyzed with paired Student t tests. The mean percentage of endometrial CD4+FOXP3+ Tregs was 13.7 ± 6.2% in diestrus and 14.5 ± 5.9% in estrus, while the mean percentage of endometrial CD4+FOXP3+CD25+ Tregs changed from 3.6 ± 2.1% in diestrus to 2 ± 2% in estrus (p = 0.0947). The mean proportion of CD3-NKp46+ lymphocytes in the endometrium was not significantly different, with 6 ± 1% in estrus and 6.5 ± 1.4% in diestrus. There was a large variation in the percentage of NK cells between mares of 2.1-12.7%. This study showed, for the first time, the presence of CD4+FOXP3+CD25+ Tregs and CD3-NKp46+ NK cells in the endometrium of non-pregnant cycling mares. The percentage of Tregs, and to a greater extent NK cells, showed large fluctuations between mares. Both Tregs and NK cells might be important for the preparation of the endometrium for semen deposition and pregnancy; however, further research is required.

Humoral Response to SARS-CoV-2 Vaccine of a Patient Receiving Methotrexate Treatment and Implications for the Need of Monitoring.

We report a case of monitoring the antibody response to the BioNTech-Pfizer vaccine of a 50-year-old female diagnosed with rheumatoid arthritis undergoing treatment with methotrexate (MTX). Antibody levels were measured 21 days after dose 1 (i.e., on the day of dose 2) and then 8, 14 and 30 days after dose 2 with Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics). Patient showed a negative result after dose 1 and had the serum sample retested using a LIAISON® SARS-CoV-2 TrimericS IgG assay (DiaSorin), which showed a positive result. Subsequent samples were tested using both assays. Antibody levels kept increasing but at a much slower rate than in patients not receiving any immunomodulatory therapies. Other research indicates that among patients with autoimmune diseases, those receiving disease-modifying antirheumatic drugs (DMARDs) have higher COVID-19 mortality than those treated with tumor necrosis factor inhibitors (TNFis). These results indicate the need for people with autoimmune diseases to be carefully observed following vaccinations, including testing of antibody levels, and treated as potentially at risk until the effect of vaccination is confirmed. The different available vaccines should also be tested to verify their usefulness in the case of people with autoimmune diseases and those who take different immunomodulatory medications.

Hypothyroidism Affects Uterine Function via the Modulation of Prostaglandin Signaling.

Thyroid hormones control the functions of almost all body systems. Reproductive dysfunctions, such as abnormal sexual development, infertility, or irregularities in the reproductive cycle, might be associated with thyroid disorders. Uterine receptivity is the period when the uterus is receptive to the implantation of an embryo. During the receptivity period (implantation window), a newly formed blastocyst is incorporated into the uterine epithelium. Prostaglandins are well-known primary mediators of pathological conditions such as inflammation and cancer but are also essential for the physiology of female reproduction. The aim of this study was to evaluate the possible relationship between hypothyroidism and changes in the prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model. The results show that hypothyroidism impaired uterine receptivity by decreasing the level of E2 as well as decreasing the expression of the uterine-receptivity factors homeobox A10 and osteopontin. Moreover, hypothyroidism caused changes in the expression of elements of the prostaglandin E2, F2α, and I2 signaling pathways and changed the levels of those prostaglandins in the uterine tissue. The results suggest that the mechanisms by which hypothyroidism affects female reproductive abnormalities might involve the prostaglandin signaling pathway, resulting in a subsequent reduction in uterine receptivity.

Expression profile of proinflammatory mediators in the placenta of mares during physiological detachment and retention of fetal membranes.

Physiological parturition is characterized by sterile, inflammatory-like processes. During parturition, the placenta expresses various proinflammatory mediators, such as chemokines and IL-17. Nevertheless, inflammatory processes present in the parturient mare are poorly characterized. The aim of this study was to investigate the expression of selected chemokines and IL-17 in the allantochorion and the endometrium of mares that retained fetal membranes (RFM) and expelled them physiologically. We hypothesized that the expression of these mediators may be altered in the placenta of mares with RFM and result in RFM occurrence. Differences in mRNA expression in the placenta of investigated groups of mares were detected for CCL2, CCL3, CCL4, CCL8, CXCL1, CXCL8, CXCL10, CX3CL1 and IL-17. There were no differences in mRNA expression of CCL5 and CXCL6. Gene ontology network analysis showed enrichment in genes related to leukocyte migration, cell chemotaxis and response to chemokine in tissues of RFM mares. Analysis of association network suggested denotations between CXCL6, CXCL8, CXCL1, CCL5, CCL4, CX3CL1 and CXCL10. Moreover, possible inhibition of CXCL10 by IL-17A and prostaglandin peroxide synthase 2 (PTGS2) by CXCL1 was detected. Our results suggest that, based on differences in chemokines and IL-17 expression, recruited subsets of leukocytes might differ between the analyzed groups of mares, which in turn may impair the separation of fetal membranes in the group of RFM mares. In addition, the results of the expression analysis suggest that macrophages might be one of the most abundant cells infiltrating the equine placenta during the expulsion of fetal membranes. Furthermore, we suspect that the synthesis of PTGS2 might be inhibited in mares with RFM.

Prostaglandin E2 affects in vitro maturation of bovine oocytes.

The role of prostaglandin E2 (PGE2) in the successful resumption of oocyte meiosis and cumulus expansion has been well-documented. However, there remains very little information available on the influence of PGE2 on other processes that occur during oocyte maturation. In this study, we supplemented a maturation medium with PGE2 and monitored oocyte quality markers, glucose metabolism, mitochondrial status, oxidative stress, and apoptosis in the cumulus-oocyte complexes (COCs), using a well-established in vitro model of embryo production in cattle. We found that this increased availability of PGE2 during maturation led to an increase in the expression of genes associated with oocyte competence and improved the quality of blastocysts produced. Prostaglandin E2 also appeared to stimulate glucose uptake and lactate production in the COCs, both influencing the expression of enzymes involved in glycolysis and the hexosamine biosynthetic pathway. We found that PGE2 reduced intracellular reactive oxygen species levels, and simultaneously increased glutathione concentration and stimulated antioxidant gene expression in the oocyte. These results indicate that PGE2 has an important role in the protection of oocytes against oxidative stress. Mitochondrial membrane potential was also improved in PGE2-treated oocytes, and there was a reduction in the occurrence of apoptosis in the COCs. Promotion of an anti-apoptotic balance in transcription of genes involved in apoptosis was present in both oocytes and the cumulus cells. In summary, PGE2 could represent a novel autocrine/paracrine player in the mechanisms that can facilitate successful oocyte maturation and oocyte survival in the cow.

LPAR2 and LPAR4 are the Main Receptors Responsible for LPA Actions in Ovarian Endometriotic Cysts.

Endometriosis has been considered as an estrogen (E2)-dependent and progesterone (P4)-resistant disease. On the other hand, lysophosphatidic acid (LPA) has been suggested as a significant modulator of ovarian pathology, acting via both LPA levels and LPA receptor (LPAR) upregulation. Therefore, the objective of the present study was to evaluate LPA concentration as well as LPARs, autotaxin (ATX), and phospholipase A2 (PLA2) expression in ovarian endometriotic cysts and normal endometrium with correlation of the expression of E2 and P4 receptors in endometriotic cysts. The analyses were carried out using the tissues derived from 37 patients with ovarian endometriosis and 20 endometrial samples collected from women without endometriosis were used as a control. We found that ovarian endometriotic cysts are a site of LPA synthesis due to the presence of enzymes involved in LPA synthesis in the tissue. Additionally, when we compared endometriotic cysts versus normal endometrium, we were able to show overexpression of 3 from 6 examined LPARs and both enzymes responsible for LPA synthesis in endometriotic cysts. Finally, we found the correlations between LPARs, ATX, and PLA2 and the expression of E2 and P4 receptors in endometriotic cysts. Owing to the high LPAR2 and LPAR4 transcript and protein expression in endometriotic ovarian cysts and positive correlations of both these receptors with the PR-B and ERß, respectively, those receptors seem to be the most promising predictors of the endometriotic cysts as well as the main receptors responsible for LPA action in the ovarian endometriosis.

Altered expression of lysophosphatidic acid receptors, in association with the synthesis of estrogens and androgens in type 1 endometrial cancer biology.

To establish association between two main lysophosphatidic acid (LPA) receptors (LPAR2 and LPAR1) with the synthesis of estrogens and androgens in type-1 endometrial carcinoma (EC), we evaluated correlation of LPARs expression with expression of steroid 5 alpha-reductase 2 - aromatase (SRD5A2), or cytochrome P450 family 19 subfamily A member 1-5α-reductase (CYP19A1) in EC. Moreover, we aimed to investigate SRD5A2 and CYP19A1 expression in type 1 endometrial cancer and normal endometrium with its correlation to selected clinicopathological features. The studied cancerous samples showed higher CYP19A1 and SRD5A2 expression comparing to normal endometria. We also documented positive correlations between LPAR1 and LPAR2 with responsible for proliferation SRD5A2 in EC tissue which suggests that intratumoral estrogen metabolism and synthesis are pivotal in endometrial carcinoma progression, with the involvement of LPA in this process. However, positive correlation between CYP19A1 and LPAR1 accounts for supporting role of LPA acting via LPAR1 in intratumoral DHT concentration and the ethiology of endometrial cancer progression. Also, owing to the highest expression of LPARs, CYP19A1 and SRD5A2 as well as their association with depth of myoinvasion and FIGO stage LPAR2 and LPAR1 seem to be the efficient candidate prognostic markers in the individual, targeted therapies for EC.

Expression of factors involved in apoptosis and cell survival is correlated with enzymes synthesizing lysophosphatidic acid and its receptors in granulosa cells originating from different types of bovine ovarian follicles.

BACKGROUND: Lysophosphatidic acid (LPA) regulates reproductive processes in the cow. Ovarian granulosa cells play a pivotal role in follicle growth and development. Nevertheless, the role of LPA in the local regulation of granulosa cell function in different follicle categories in the bovine ovary has not been investigated. METHODS: Ovarian follicles were divided into healthy, transitional and atretic categories. The expression levels of AX, PLA2, LPARs and factors involved in apoptosis and cell survival processes in granulosa cells in different types of follicles were measured by real-time PCR. The correlations between the expression levels of AX, PLA2, LPARs and the examined factors were measured. The immunolocalization of AX, PLA2 and LPARs in different ovarian follicles was examined by immunohistochemistry. Statistical analyses were conducted in GraphPad using a one-way ANOVA followed by the Kruskal-Wallis multiple comparison test or a correlation analysis followed by Pearson's test. RESULTS: The expression levels of AX, PLA2 and LPARs, with the major role of LPAR2 and PLA2, were found in the granulosa cells originating from different follicle types. The expression levels of the factors involved in cell apoptosis (TNFα and its receptors, FAS, FASL, CASP3, CASP8, ß-glycan, and DRAK2) were significantly higher in the granulosa cells of the atretic follicles compared to the healthy follicles. A number of correlations between LPARs, AX, PLA2 and factors associated with apoptosis were observed in the atretic but not in the healthy follicles. A greater expression of the factors involved in differentiation and proliferation in the granulosa cells (DICE1 and SOX2) was found in the healthy follicles in comparison with the atretic. A number of correlations between LPARs, AX, PLA2 and the factors associated with cell survival were observed in the healthy but not in the atretic follicles. CONCLUSIONS: Granulosa cells are the target of LPA action and the source of LPA synthesis in the bovine ovarian follicle. We suggest that the participation of LPA in apoptosis in the atretic follicles mainly occurs through the regulation of TNF-α-dependent and caspase-induced pathways. In the transitional follicles, LPA might influence the inhibins to shift the balance between the number of healthy and atretic follicles. In the healthy follicle type, LPA, acting via LPAR1, might regulate MCL1 and estradiol-stimulating ERß mRNA expression, leading to the stimulation of anti-apoptotic processes in the granulosa cells and their differentiation and proliferation.

The effect of lysophosphatidic acid together with interferon tau on the global transcriptomic profile in bovine endometrial cells.

In cows, lysophosphatidic acid (LPA), which acts in an auto/paracrine manner, serves as a luteotropic factor during early pregnancy by stimulating progesterone and prostaglandin E2 secretion, thus protecting the bovine corpus luteum and early embryo development. Our hypothesis was that LPA exerted some local effects on the bovine endometrium prior to early embryo-maternal interactions and that interferon tau (IFNτ), the pregnancy recognition signal, modulated this action. In the present study, we applied an in vitro model involving whole-transcriptomic profiling to examine the effects of LPA on gene expression in bovine endometrial cells. Microarray analyses revealed 36, 269 and 284 differentially expressed transcripts in bovine endometrial cells in the control vs. LPA, control vs. LPA + IFNτ and LPA vs. LPA + IFNτ groups, respectively. The expression of matrix metalloproteinase 13 (MMP13) and radical S-adenosyl methionine domain containing 2 (RSAD2) was increased in the LPA-treated endometrial cells. Among the transcripts differentially regulated by LPA together with IFNτ, many of the genes were classical- or novel-type I IFN-stimulated genes (ISGs). The results indicated that 10 of the 16 analyzed genes showed a positive correlation with their corresponding microarray data upon real-time PCR validation, indicating a considerable consistency between both techniques. In summary, these transcriptional profiling studies identified a number of genes that were regulated by LPA alone and LPA together with IFNτ in endometrial cells from the bovine uterus. Available studies support the idea that LPA, which acts in an auto/paracrine manner on the endometrium, alters the expression of genes that are probably important for uterine receptivity, maternal immune tolerance to the embryo and conceptus growth and development during early pregnancy. Moreover, the differentially expressed genes (DEGs) that increased in the LPA + IFNτ-treated endometrial cells are largely in response to IFNτ actions and are possibly associated with crucial biological processes during the peri-implantation period of pregnancy.

The role of vitamin D in reproductive dysfunction in women - a systematic review.

Vitamin D is essential for the proper functioning of the human body. There is also evidence of its strong association with fertility problems in women. This review aims to evaluate the relationship between vitamin D and diseases affecting women's fertility (polycystic ovarian syndrome (PCOS), uterine leiomyomas and endometriosis), and in vitro fertilization (IVF) outcome. A systematic review of the literature was conducted in Scopus and PubMed for relevant English language publications since 1989. Vitamin D influences the functioning of the reproductive system in women and has been associated with PCOS, uterine leiomyomas, endometriosis and in vitro fertilization (IVF) outcome. However, further studies on larger groups of patients are needed to establish what role vitamin D plays in the treatment of female infertility.

[First in the world application of next generation sequencing in preimplantation genetic diagnostics in clinical practice - a case report]. / Pierwsze w swiecie zastosowanie sekwencjonowania nastepnej generacji w diagnostyce przedimplantacyjnej w praktyce klinicznej - opis przypadku.

Preimplantation genetic diagnosis (PGD) is a well established method for detecting genetic abnormalities during the course of infertility treatment, resulting in thousands of healthy newborns delivered worldwide. PGD with next generation sequencing (NGS) provides new possibilities for diagnosis and new parameters for evaluation. The use of next-generation DNA sequencing technique has lead to great progress in the human genome analysis. The aim of this study was molecular analysis using next generation sequencing technique of embryos from a couple suffering from recurrent pregnancy losses. As a result of in vitro fertilization procedure, seven embryos were created. Seven blastomeres, one from each embryo, were analyzed. Transfer of two blastocysts in a fresh cycle resulted in the singleton pregnancy. Healthy baby girl was delivered via caesarean section after 28 weeks of gestation (weight: 1250g, Apgar score: 8/9). The reason for the premature labor was likely caused by mother's pneumonia. This is the first case of clinical use of the NGS in PGD in fresh cycle after blastomere biopsy.

Lysophosphatidic acid modulates prostaglandin signalling in bovine steroidogenic luteal cells.

We examined whether lysophosphatidic acid affects prostaglandin biosynthesis, transport, and signalling in bovine steroidogenic luteal cells. The aim of the present study was to determine the influence of LPA on PGE2 and PGF2α synthesis and on the expression of enzymes involved in PG biosynthesis (PTGS2, mPGES-1, cPGES, mPGES-2, PGFS and 9-KPR), prostaglandin transporter (PGT), and prostaglandin receptors (EP1, EP2, EP3, EP4 and FP) in bovine steroidogenic luteal cells. We found that LPA inhibited PGF2α synthesis in steroidogenic luteal cells. Moreover, LPA increased mPGES1 and cPGES and decreased PGFS expression in cultured bovine steroidogenic luteal cells. Additionally, LPA stimulated EP2 and EP4 receptor and PGT expression. This study suggests that LPA activity in the bovine CL directs the physiological intraluteal balance between the two main prostanoids towards luteotropic PGE2.

The significance of the altered expression of lysophosphatidic acid receptors, autotaxin and phospholipase A2 as the potential biomarkers in type 1 endometrial cancer biology.

In order to study lysophosphatidic acid (LPA) signaling associated with type 1 endometrial carcinoma (EC), we evaluated the LPA receptors (LPARs), autotaxin (ATX) and phospholipase A2 (PLA2) expression in EC and normal endometrium with correlation to clinicopathological features. We investigated LPAR1, LPAR2, LPAR3, LPAR4, ATX and PLA2 expression at mRNA and protein levels using quantitative real-time PCR and western blot analyses in 37 ECs and 10 normal endometria. All the examined LPARs (except for LPAR3 protein), ATX and PLA2 were overexpressed in cancerous compared to healthy endometrium. The studied ECs showed the highest LPAR2 and LPAR1 expression. Statistically positive correlations were found between depth of myoinvasion and levels of LPAR1, LPAR2 and PLA2 transcripts and proteins. We also found positive correlations between LPAR1, LPAR2, LPAR4 and PLA2 expression with the International Federation of Gynecology and Obstetrics (FIGO) stage. The expression of LPAR1, LPAR2 and PLA2 was positively associated with the age of patients. Positive correlations were found between the expression of LPAR1 mRNA, LPAR2 mRNA and protein and LPAR3 mRNA and body mass index (BMI) of the examined patients. We found no association between the expression levels of the studied factors and diabetes or hypertension among the examined patients. Owing to the highest LPAR2 and LPAR1 expression in EC and positive correlations of these two receptors with the depth of myoinvasion and the FIGO stage, we believe that LPAR2 and LPAR1 show promise as predictors of the EC progression as well as the main receptors responsible for LPA action in the EC tissue.

Lysophosphatidic acid (LPA) signaling in human and ruminant reproductive tract.

Lysophosphatidic acid (LPA) through activating its G protein-coupled receptors (LPAR 1-6) exerts diverse cellular effects that in turn influence several physiological processes including reproductive function of the female. Studies in various species of animals and also in humans have identified important roles for the receptor-mediated LPA signaling in multiple aspects of human and animal reproductive tract function. These aspects range from ovarian and uterine function, estrous cycle regulation, early embryo development, embryo implantation, decidualization to pregnancy maintenance and parturition. LPA signaling can also have pathological consequences, influencing aspects of endometriosis and reproductive tissue associated tumors. The review describes recent progress in LPA signaling research relevant to human and ruminant reproduction, pointing at the cow as a relevant model to study LPA influence on the human reproductive performance.

Evaluation of granulocyte colony-stimulating factor effects on treatment-resistant thin endometrium in women undergoing in vitro fertilization.

The aim of the study was to assess the granulocyte colony-stimulating factor (G-CSF) effects on unresponsive thin (<7 mm) endometrium in women undergoing in vitro fertilization (IVF). We included thirty-seven subjects who had thin unresponsive endometrium on the day of triggering ovulation. These patients also failed to achieve an adequate endometrial thickness in at least one of their previous IVF cycles. In all the subjects at the time of infusion of G-CSF, endometrial thickness was 6,74 ± 1,75 mm, and, after infusion, it increased significantly to 8,42 ± 1,73 mm. When we divided the group into two subgroups according to whether the examined women conceived, we showed that the endometrium expanded significantly from 6,86 ± 1,65 to 8,80 ± 1,14 mm in the first group (who conceived) and from 6,71 ± 1,80 to 8,33 ± 1,85 mm in the second, respectively. There were no significant differences between the two subgroups in respect to the endometrial thickness both before and after G-CSF infusion. The clinical pregnancy rate was 18,9%. We concluded that the infusion of G-CSF leads to the improvement of endometrium thickness after 72 hours.

Influence of lysophosphatidic acid on estradiol production and follicle stimulating hormone action in bovine granulosa cells.

The objective of the study was to examine the effect of lysophosphatidic acid (LPA) on 17ß-estradiol (E2) synthesis and follicle stimulating hormone (FSH) action in bovine granulosa cells. We found that granulosa cells in the bovine antral follicle, in addition to the uterus and the CL, are also the site of LPA synthesis and the target for LPA action in the bovine reproductive tract. Our findings suggest that LPA stimulates E2 synthesis, probably via increased expression of FSHR and 17ß-HSD genes.

Effects of lysophopatidic acid on tumor necrosis factor α and interferon γ action in the bovine corpus luteum.

We examined the effects of LPA on TNFα and IFNγ - induced decrease of P4 synthesis and on the cytokine - induced apoptosis of the cultured luteal cells. In the steroidogenic luteal cells LPA reversed the inhibitory effect of TNFα and IFNγ on P4 synthesis and also inhibited the stimulatory effects of TNFα and IFNγ on the expression of Bax, TNFR1, Fas and FasL as well as caspase 3 activity. These results suggest that TNFα and IFNγ cannot induce apoptosis in the presence of LPA, which orientates the steroidogenic luteal cells towards the survival state. In conclusion our results indicate that LPA supports P4 synthesis and action in the bovine CL.

Diverse effects of phytoestrogens on the reproductive performance: cow as a model.

Phytoestrogens, polyphenolic compounds derived from plants, are more and more common constituents of human and animal diets. In most of the cases, these chemicals are much less potent than endogenous estrogens but exert their biological effects via similar mechanisms of action. The most common source of phytoestrogen exposure to humans as well as ruminants is soybean-derived foods that are rich in the isoflavones genistein and daidzein being metabolized in the digestive tract to even more potent metabolites-para-ethyl-phenol and equol. Phytoestrogens have recently come into considerable interest due to the increasing information on their adverse effects in human and animal reproduction, increasing the number of people substituting animal proteins with plant-derived proteins. Finally, the soybean becomes the main source of protein in animal fodder because of an absolute prohibition of bone meal use for animal feeding in 1995 in Europe. The review describes how exposure of soybean-derived phytoestrogens can have adverse effects on reproductive performance in female adults.

Which bovine endometrial cells are the source of and target for lysophosphatidic acid?

The objective of the study was to examine which cultured endometrial cells are the source and which are the target for lysophosphatidic acid (LPA) in the bovine uterus. LPA concentration as well as mRNA and protein expressions of the enzymes responsible for LPA synthesis (phospholipase A2: PLA2, autotaxin: AX) were greater in epithelial than in stromal cells (P<0.05). In turn, mRNA and protein expression of LPA receptor (LPAR1) was lower in epithelial than in stromal cells (P<0.05). We suggest that LPA in bovine endometrium is produced mainly by epithelial cells and affects mostly stromal cells acting via LPAR1.