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Histological findings of endometrial biopsies. (A-C) Group 1. (A) abundant mucopolysaccharide extracellular matrix (Alcian blue, 40x), (B) PASpositive material in the glandular lumen (PAS, 40x); (C) Interstitial lymphomonocytic infiltration in the lamina propria of endometrium (H&E, 40x); (D-F) Group 2. (D) Endometriosis of grade II characterized by 4-10 cell layers thick fibrosis (H&E, 40x); (E) focal deposit of mucopolysaccharide extracellular matrix (Alcian blue, 40x); scattered glands showing intraluminal PAS-positive material in the glandular lumen (PAS, 40x)
Source publication
Background:
Despite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that o...
Contexts in source publication
Context 1
... Fig. 1 histology findings of uterine biopsies, as representative of G1 and G2 are shown. In G1 mares ( Fig. 1 a, b, c), periglandular fibrosis was observed in 13 cases, namely 9 and 4 cases of grades I and II of fibrosis, respectively. Fibrosis of grade III was totally absent. Periglandular fibrosis of grades I and II was associated to a mild ...
Context 2
... Fig. 1 histology findings of uterine biopsies, as representative of G1 and G2 are shown. In G1 mares ( Fig. 1 a, b, c), periglandular fibrosis was observed in 13 cases, namely 9 and 4 cases of grades I and II of fibrosis, respectively. Fibrosis of grade III was totally absent. ...
Citations
... Indeed, the granules stored in the phagocytic cells contain microbicidal and digestive molecules, including AMPs. Finally, the recurrence of inflammatory stimuli, following repeated mating, foaling and veterinary interventions, leads to an evolution towards chronicity: this condition seems to be due to an imbalance in the innate mechanisms of local immunomodulation of the endometrial tissue and results in the development of fibrosis.5,7,10,16 In this context, the aim of the present study was to examine theexpression of genes coding for AMPs involved in the local innate immune response of the endometrial tissue. ...
... 21,22 Beta-defensins are expressed in numerous tissues, including the reproductive tract, of human, mouse, rat, bovine and equine species, suggesting a possible role in the defence of the reproductive tract against pathogens and/or in the regulation of fertility. 16,23,24 A greater expression of this molecule at the level of the endometrial tissue was reported by Marth et al. both following the induction of bacterial acute endometritis,9,24 and in subjects defined as susceptible to PBIE.10 Crociati et al.16 divided mares into two groups homogeneous for histopathological examination (mares with different degrees of fibrosis and/or chronic inflammation were present in both groups) but different in terms of age class. The authors reported a significantly higher expression of the gene in young mares (<10 years old) Scatter plot showing significant (two-tailed) Spearman positive correlation between endometrial LYZ expression and biopsy grade, according to Kenney and Doig classification (I, IIA, IIB, III), of 26 mares classified as reproductively normal (N = 7) or affected by subclinical endometritis (N = 19) (moderate, r = 0., >20 years), was not observed. ...
Background
Endometritis is a major cause of subfertility in mares. Multiparous old mares are more susceptible to developing endometritis given that ageing is associated with an altered immune response and with inadequate physiological uterine clearance after breeding, which can lead to degenerative changes in the endometrium. Molecules such as antimicrobial peptides (AMPs) have been proposed as endometritis markers in the equine species.
Study design
Cross‐sectional.
Objectives
To investigate the endometrial expression of defensin‐beta 4B (DEFB4B), lysozyme (LYZ) and secretory leukocyte peptidase inhibitor (SLPI) genes in mares either affected or not by subclinical endometritis, due to the role of these AMPs in the immune response to bacteria and inflammatory reactions.
Methods
Endometrial biopsy for histopathological and gene expression examinations was performed on 26 mares. The inclusion criteria for the normal mare group (NM, N = 7) were 2–4 years of age, maiden status, no clinical signs of endometritis and a uterine biopsy score of I, while for mares affected by subclinical endometritis (EM, N = 19) the inclusion criteria were 10–22 years of age, barren status for 1–3 years, no clinical signs of endometritis and a uterine biopsy score between IIA and III.
Results
A significantly higher expression of LYZ (NM: 0.76 [1.84–0.37] vs. EM: 2.78 [5.53–1.44], p = 0.0255) and DEFB4B (NM: 0.06 [0.11–0.01] vs. EM: 0.15 [0.99–0.08], p = 0.0457) genes was found in endometritis mares versus normal mares. Statistically significant moderate positive correlations were found between the level of expression of LYZ gene and both the age (r = 0.4071, p = 0.039) and the biopsy grade (r = 0.4831, p = 0.0124) of the mares.
Main limitations
The study investigated a limited number of genes and mares, and the presence/location of the proteins coded by these genes was not confirmed within the endometrium by IHC.
Conclusions
If the results of this study are confirmed, LYZ and DEFB4B genes can be used as markers to identify mares that are affected by subclinical endometritis.
... In research, sometimes only the application of special staining is mentioned, suggesting that the purpose is to facilitate fibrosis assessment [22,48,49]. Among the specific staining for connective tissue, Masson trichrome staining (MT) [43,[50][51][52][53][54][55][56][57], picrosirius red staining (PSR) [15,18,35,[58][59][60], Gomori trichrome staining (GT) [61,62], elastica van Gieson staining (EVG) [4,63,64], Azan staining [65], resorcin-fuchsin staining (RF) [65], periodic acid-Schiff staining (PAS) [4,52,65,66], and, in highly specific way, IHC [6,16,51,60,67], may be utilized for enhancing the histological visualization of connective tissue in light microscopy. Although electron microscopy is a more expensive technique, it may be employed for the evaluation of smaller structures in ECM or cellular degeneration in fibrosis studies; thus, methylene-blue-azur2-basic fuchsin staining (MBABF) on semi-thin slices for area selection [35], uranyl acetate-lead citrate staining (UALC) [4,35,64], and gold-coating [64,66] can be used. ...
... In research, sometimes only the application of special staining is mentioned, suggesting that the purpose is to facilitate fibrosis assessment [22,48,49]. Among the specific staining for connective tissue, Masson trichrome staining (MT) [43,[50][51][52][53][54][55][56][57], picrosirius red staining (PSR) [15,18,35,[58][59][60], Gomori trichrome staining (GT) [61,62], elastica van Gieson staining (EVG) [4,63,64], Azan staining [65], resorcin-fuchsin staining (RF) [65], periodic acid-Schiff staining (PAS) [4,52,65,66], and, in highly specific way, IHC [6,16,51,60,67], may be utilized for enhancing the histological visualization of connective tissue in light microscopy. Although electron microscopy is a more expensive technique, it may be employed for the evaluation of smaller structures in ECM or cellular degeneration in fibrosis studies; thus, methylene-blue-azur2-basic fuchsin staining (MBABF) on semi-thin slices for area selection [35], uranyl acetate-lead citrate staining (UALC) [4,35,64], and gold-coating [64,66] can be used. ...
... In contrast, Grüninger et al. stated that MT is widely used for detecting endometrial fibrosis [15]. Interestingly, in some scientific studies, mostly MT [16,22,43,52,56,57] and PSR [15,18,60] were utilized to stain collagen fibers, while EVG [4,64], PAS [4,52,66], Alcian blue (AB) staining [16,52], immunohistochemical (IHC) staining [6,16,60,67] (light microscopy), UALC [4,64], and gold sputter (GS) coating [66] (electron microscopy) were used for other specific assessments of other ECM components co-occurring alterations. Such a wide use of specific staining in the scientific research indicates that for diagnostic purposes, connective tissue staining would be beneficial. ...
Simple Summary
Uterine diseases are the leading cause of infertility in mares, causing increasing costs and losses in horses’ breeding. Their current diagnosis is often supported by obtaining endometrial biopsies and hematoxylin–eosin staining, which is the basic staining used in histopathology. This review aims to show the variety of uterine changes affecting fertility and highlights the usefulness of special stains for connective tissue visualization—Masson trichrome, picrosirius red, elastica van Gieson, or periodic acid–Schiff—for a more comprehensive diagnosis. The fibrosis evaluation includes connective tissue changes in the cervix, the endometrium, and around/in the wall of blood vessels. Cervical connective tissue undergoes cyclic changes impacting fertility, whereas vascular changes, especially in multiparous mares, are crucial for adapting to physiological shifts, affecting early pregnancy and hindering placental development. Special stains are valuable for the identification of structural changes in the cervix and fibrosis in uterine blood vessels. Moreover, equine endometrosis, linked to fibrotic processes in the endometrium, emphasizes the need for wider use of special stains in diagnosis. Therefore, we advocate for special staining in reproductive tract evaluation due to its simplicity, accessibility, and effectiveness. We encourage scientists and diagnosticians to adopt additional tools for clearer pathology visualization, enabling reliable fertility predictions.
Abstract
Uterine diseases stand as the primary cause of infertility in mares; however, the diagnostic process often relies on obtaining endometrial biopsies and their hematoxylin–eosin staining. This review seeks to present the variability of uterine changes and their impact on fertility and underscore the utility of special stains, such as Masson trichrome, picrosirius red, elastica van Gieson, or periodic acid–Schiff, in enhancing diagnostic breadth. Connective tissue evaluation in the cervix is discussed, as it is subjected to cyclic changes and the impact on overall fertility. Vascular changes, particularly prevalent in multiparous mares, play a crucial role in adapting to physiological and pathological alterations, affecting early gestation and impeding placental development. Given that uterine vascular pathologies often involve fibrotic changes, connective tissue stains emerge as a valuable tool in this context. Moreover, equine endometriosis, predominantly associated with endometrial fibrosis, further highlights the relevance of special stains, suggesting their underutilization in the diagnostic process. Recognizing the subjective nature of diagnosing uterine pathologies and the need for additional diagnostic tools, we advocate for using dedicated stains in the histopathological evaluation of uterine samples. In conclusion, we encourage scientists and diagnosticians to embrace additional tools that enhance pathology visualization, enabling more reliable diagnoses concerning expected fertility.
... Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases (10) that are important factors in the process of fibrosis. Data concerning MMP expression in equine endometrial fibrosis are limited but MMP-2 and MMP-9 (now called 72 kDa gelatinase and 92 kDa gelatinase, in the horse), seem to be involved in this process (11)(12)(13)(14). MMP-2 and MMP-9 are gelatinases that denature collagens (gelatins) and other ECM substrates (15,16). ...
Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.
... Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases (10) that are important factors in the process of fibrosis. Data concerning MMP expression in equine endometrial fibrosis are limited but MMP-2 and MMP-9 (now called 72 kDa gelatinase and 92 kDa gelatinase, in the horse), seem to be involved in this process (11)(12)(13)(14). MMP-2 and MMP-9 are gelatinases that denature collagens (gelatins) and other ECM substrates (15,16). ...
Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.
... Mare aging, but not so much parity, is associated with the severity of endometrosis [36,76,79]. It appears that as mares age, dysfunction of modulators of the immune system or of tissue remodeling, such as defensin β, clusterin, uterine serpin, complement C3, neutrophil gelatinase-associated lipocalin (NGAL), or connective tissue growth factor (CTGF), among others, indirectly impair the extracellular matrix (ECM) homeostasis [73,80], which might predispose to fibrogenesis. In addition, mare's aging has been associated to increased COL deposition in the equine endometrium and in the oviduct [81], and to deficient development of placental microcotyledons [82]. ...
... Since equine conceptus development relies initially on the nutrients that derive from exocrine secretions of endometrial glands (histotroph), and later on the placenta, endometrium and placenta's Mare aging, but not so much parity, is associated with the severity of endometrosis [36,76,79]. It appears that as mares age, dysfunction of modulators of the immune system or of tissue remodeling, such as defensin β, clusterin, uterine serpin, complement C3, neutrophil gelatinase-associated lipocalin (NGAL), or connective tissue growth factor (CTGF), among others, indirectly impair the extracellular matrix (ECM) homeostasis [73,80], which might predispose to fibrogenesis. In addition, mare's aging has been associated to increased COL deposition in the equine endometrium and in the oviduct [81], and to deficient development of placental microcotyledons [82]. ...
In this paper, the evolution of our understanding about post breeding endometritis (PBE), the susceptibility of mares, and events leading to endometrosis are reviewed. When sperm arrive in the uterus, pro-inflammatory cytokines and chemokines are released. They attract neutrophils and induce modulatory cytokines which control inflammation. In susceptible mares, this physiological defense can be prolonged since the pattern of cytokine release differs from that of resistant mares being delayed and weaker for anti-inflammatory cytokines. Delayed uterine clearance due to conformational defects, deficient myometrial contractions, and failure of the cervix to relax is detected by intrauterine fluid accumulation and is an important reason for susceptibility to endometritis. Multiparous aged mares are more likely to be susceptible. Untreated prolonged PBE can lead to bacterial or fungal endometritis called persistent or chronic endometritis. Exuberant or prolonged neutrophilia and cytokine release can have deleterious and permanent effects in inducing endometrosis. Interactions of neutrophils, cytokines, and prostaglandins in the formation of collagen and extracellular matrix in the pathogenesis of fibrosis are discussed. Endometritis and endometrosis are interconnected, influencing each other. It is suggested that they represent epigenetic changes induced by age and hostile uterine environment.
... In addition to the impaired uterine clearance, old mares have also a longer vulva (Pascoe 1979) with an overall worse conformation, which represents a larger entrance for bacteria and could explain why bacteria counts are increased in old compared to younger mare uteri (Evans et al. 1987). A recent study showed an impairment of the expression of genes involved in local uterine immune response in old mares (Crociati et al. 2019). Studying endometrial tissue cultures from mares affected by subclinical endometritis, an increased production of prostaglandin E2 (PGE2), 6-oxoprostaglandin F1alpha (6-keto-PGF1a) and leukotriene C4 (LTC4) is observed in old mares in comparison to younger mares (Siemieniuch et al. 2017). ...
Although puberty can occur as early as 14–15months of age, depending on breed and use, the reproductive career of mares may continue to advanced ages. Once mares are used as broodmares, they will usually produce foals once a year until they become unfertile, and their productivity can be enhanced and/or prolonged through embryo technologies. There is a general consensus that old mares are less fertile, but maternal age and parity are confounding factors because nulliparous mares are usually younger and older mares are multiparous in most studies. This review shows that age critically affects cyclicity, folliculogenesis, oocyte and embryo quality as well as presence of oviductal masses and uterine tract function. Maternal parity has a non-linear effect. Primiparity has a major influence on placental and foal development, with smaller foals at the first gestation that remain smaller postnatally. After the first gestation, endometrial quality and uterine clearance capacities decline progressively with increasing parity and age, whilst placental and foal birthweight and milk production increase. These combined effects should be carefully balanced when breeding mares, in particular when choosing and caring for recipients and their foals.
... In this study, we selected interferon-gamma (IFNG), interleukins (IL)-2, IL5, IL8, IL10, IL12/p35, IL12/p40, and Transforming growth factor beta 1 (TGFB1) to investigate their gene expression. Tests were assessed by RT-qPCR using previously primers tested [28][29][30][31] and summarized in Table 3. Sybr Green Real-Time PCR amplification were performed in a CFX96™ Real-Time System with 5 µL of 1:10 diluted cDNA added to 15 µL PCR mixture at final concentration of 1× master mix (Power SYBR™ Green PCR Master Mix, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and 200 nM of each primer combination. The following thermal profile was applied: 95 • C for 10 min., then 50 cycles of 95 • C for 15 s and 60 • C for 30 s. ...
... Samples with normal (+++) or high viral load (++++) showed the expression of L1 and IL12/p40 (9 out of 11; Table 6). The 65% of samples were positive for TGFB1 gene expression; in this group, a significant decrease of the IFNG (Mdn= RT-PCR data for B2M are expressed as + (amplified) or -(not amplified); Indication of the viral load was give indicating the Cq at which the positivity for L1 was detected: − (>48 Cq), + (32-37 Cq), ++ (26)(27)(28)(29)(30)(31), +++ (20)(21)(22)(23)(24)(25), and ++++ (14)(15)(16)(17)(18)(19). All samples were classified into EcPV2 high when viral load was +++ or ++++ and into EcPV2 low when viral load was ++ or +. ...
Penile squamous cell carcinomas (SCCs) are common tumors in older horses, with poor prognosis mostly due to local invasion and recurrence. These tumors are thought to be mainly caused by Equus caballus papillomavirus type 2 (EcPV-2). The aim of this study is to characterize the tumor immune environment (TIME) in equine penile tumors. Equine penile epithelial tumors (17 epSCCs; 2 carcinomas in situ, CIS; 1 papilloma, P) were retrospectively selected; immune infiltrate was assessed by histology and immunohistochemistry; RT-qPCR tested the expression of selected chemokines and EcPV-2 DNA and RNA. The results confirmed EcPV-2-L1 DNA in 18/20 (90%) samples. L1 expression was instead retrieved in 13/20 cases (65%). The samples showed an increased infiltration of CD3+lymphocytes, macrophages (MAC387; IBA1), plasma cells (MUM1), and FoxP3+lymphocytes in the intra/peritumoral stroma when compared to extratumoral tissues (p < 0.05). Only MAC387+neutrophils were increased in EcPV-2high viral load samples (p < 0.05). IL12/p35 was differentially expressed in EcPVhigh and EcPVlow groups (p = 0.007). A significant decrease of IFNG and IL2 expression was highlighted in TGFB1-positive samples (p < 0.05). IBA1 and CD20 were intratumorally increased in cases where IL-10 was expressed (p < 0.005). EpSCCs may represent a good spontaneous model for the human counterpart. Further prospective studies are needed in order to confirm these preliminary results.
... Protocols for interleukin-1β (IL-1B), IL-4, and IL-6 genes had been previously set up [21,49], while primers for IL-2, IL-3, IL-5, IL-8, transforming growth factor β1 (TGFB1), tumour necrosis factor-α (TNFA), and interferon-γ (IFNG) were designed using the Primer-BLAST free software available on line on https://www.ncbi.nlm.nih.gov/tools/primer-blast/; these were placed at exon-exon junctions or at different exons to avoid biases due to genomic DNA amplification. ...
Simple Summary: Stressful stimuli, both infectious and non-infectious, can modify and trigger an innate immune response and inflammation, via an attempt to restore a homeostatic state. Coping with stressors can be measured by different procedures, including the evaluation of immunological parameters. These are also modulated by exercise, which can be considered stress prototypic in the Thoroughbred racehorse. To evaluate the complex of physiological regulations during the training period, twenty-nine clinically healthy, two-year-old Thoroughbred racehorses were followed during their first 3 months of sprint training. Blood collection was performed at rest, three times until 90 days of training, for testing immunological parameters during incremental sprint training to evaluate its effect on the immunological status of the animals. During the training period, we observed the following: (A) an increase in red blood cell parameters that are crucial for exercise performance adaptation, improving O 2 transport and muscle cell respiration; (B) variations of blood granulocytes; and (C) changes in inflammatory cytokine gene expression. On the basis of clinical and laboratory findings, training exercise probably played a major role in the modulation of the above parameters. These latter changes could be seen as a preparation of the innate immune system to respond quickly and adequately to environmental conditions. Abstract: Training has a great impact on the physiology of an athlete and, like all stressful stimuli, can trigger an innate immune response and inflammation, which is part of a wider coping strategy of the host to restore homeostasis. The Thoroughbred racehorse is a valid animal model to investigate these changes thanks to its homogeneous training and highly selected genetic background. The aim of this study was to investigate modifications of the innate immune response and inflammation in young untrained Thoroughbred racehorses during the first training season through haematological and molecular investigations. Twenty-nine Thoroughbred racehorses were followed during their incremental 3-month sprint exercise schedule. Blood collection was performed at time 0 (T0; before starting the intense training period), 30 days after T0 (T30), and 90 days after T0 (T90). Haematological parameters (red and white blood cells, haemoglobin, and platelets) were evaluated and haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), and red cells width distribution + standard deviation (RDW-SD) were calculated. Moreover, via RT-qPCR, we investigated the expression of, Interleukin 1β (IL-1β),Interleukin 4 (IL-4) Interleukin 6 (IL-6), Interleukin 2 (IL-2), Interleukin 3 (IL-3), Interleukin 5 (IL-5) Interleukin 8 (IL-8), Trasformig Growth Factor βand α (TGF-β), Tumor necrosis factor α(TNF-α), and Interferon γ(IFN-γ)genes. Main corpuscular volume (MCV) showed a significant (p = 0.008) increase at T90. Main corpuscular haemoglobin (MCH) and haemoglobin concentration (MCHC) values were significantly augmented at both T30 (p < 0.001) and T90 (p < 0.001). Basophils were significant increased at T30 (p = 0.02) and eosinophils were significantly increased at T90 (p = 0.03). Significant differences in gene expression were found for all the genes under study, with the exception of IFN-γand TNF-α. In particular, IL-2 (T30, p = 0.011; T90, p = 0.015), IL-4 (T30, p = 0.009; T90, p < 0.001), and IL-8 (T30, p < 0.001; T90, p < 0.001) genes were significantly upregulated at both T30 and T90 with respect to T0, TGF-βwas intensely downregulated at T30 (p < 0.001), IL-5 gene expression was significantly decreased at T90 (p = 0.001), while IL-1β(p = 0.005) and IL-3 (p = 0.001) expression was strongly augmented at the same time. This study highlighted long-term adjustments of O 2 transport capability that can be reasonably traced back to exercise adaptation. Moreover, the observed changes of granulocyte numbers and functions and inflammatory cytokine gene expression confirm a major role of the innate immune system in the response to the complex of stressful stimuli experienced during the training period.
