**3. Effect of omega-3 fatty acids on endometrial function: in vivo and in vitro studies**

Several studies have suggested that supplementation of omega-3 fatty acids during pregnancy is beneficial for establishment and maintenance of pregnancy, maintains gestation length and fetal growth, prevents preterm birth, and decreases the rate of gestational diabetes [43]. These effects of omega-3 fatty acids have been mainly studied in animals such as bovine and ovine; however, some recent studies have begun to be performed to demonstrate the beneficial effect of these fatty acids in humans and mice. Consumption of omega-3 fatty acids has been associated with a reduction of the symptoms and lower risk of developing endometriosis in women, a hormone-dependent chronic inflammatory condition [44, 45]. In wild-type mice, the administration of EPA reduced the number of endometriotic lesions, similarly as was observed in a transgenic mouse model with high levels of omega-3 fatty acids [46]. In a rat model of endometriosis, the EPA supplementation reduced the endometriotic lesions and expression of pro-inflammatory gene, suggesting that the EPA supplementation might be a strategy for the treatment of endometriosis [47].

*Apolipoproteins, Triglycerides and Cholesterol*

inflammation [9].

nancy [7, 8].

fertilization, and implantation. PGs are synthesized from arachidonic acid by different cells, and five types of PGs have been described, with specific roles and mechanism in the female reproductive system [5, 6]. Prostaglandin F2α (PGF2α) has a luteolytic effect, whereas prostaglandin E2 (PGE2) is central in ovulation, fertilization, embryo development, and implantation [7, 8]. In addition, PGE2 plays important roles in inflammatory processes, being increased at first phases of

Inflammation is a complex process with two differentiated steps or conditions: acute and chronic inflammation. A number of lipid mediators act as pro-inflammatory (i.e., leukotriene and prostaglandins) or anti-inflammatory and pro-resolving (lipoxins, resolvins, maresins, and protectins) mediators. Lipid mediators derived from polyunsaturated fatty acids (PUFA) have potent anti-inflammatory effect and promote the resolution of inflammation, through specialized pro-resolving lipid mediators (SPM) [10]. Omega-3 fatty acids are a type of PUFA with known beneficial effect, which, in addition to its pro-resolving mechanism, have shown two additional anti-inflammatory mechanisms: activation of the free fatty acid (FFA)-4 receptor and inflammasome inhibition. Recent evidences have suggested an anti-inflammatory effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in the endometrium. Furthermore, FFA4 receptor was detected in the human, mouse, and bovine uterus [11, 12]. The following sections describe the effect and potential mechanisms of omega-3 fatty acids in the immune cells and

endometrium and perspectives of these fatty acids in health and disease.

**2. Eicosanoids in fertility and immune function in the uterus**

icity and may reduce the risk of uterine infections in cows [22].

Eicosanoids, which include prostaglandins and leukotrienes, are members of a large family of compounds that are synthesized from arachidonic acid through the cyclooxygenase and lipoxygenase pathways [13]. PGF2α and PGE2 exert opposite actions on the corpus luteum (CL); therefore, control over their synthesis and secretion is critical either for the initiation of luteolysis or maintenance of preg-

PGF2α is considered a pro-inflammatory molecule, and it may stimulate the synthesis of pro-inflammatory cytokines that enhance phagocytosis and lymphocyte functions [14]. PGF2α can increase IL1β, IL6, CCL2, and CXCL8 via ERK1/ERK2, PI3K, NFAT, and NF-κB pathways in the myometrial cells from term pregnant women, suggesting that PGF2α induces an inflammatory environment during the late stage of human pregnancy [15]. PGF2α in vitro enhanced neutrophil chemotaxis and the ability of neutrophils to ingest bacteria, and anti-PGF2α antibody blocked the chemotactic effects of PGF2α [16, 17]. Exogenous PGF2α increases uterine secretion of PGF2α through the activation of phospholipase A2 (PLA2) and cyclooxygenase 2 [18–20]. Also, it has been proposed that exogenous PGF2α increases luteal leukotriene B4 (LTB4) production [21]. LTB4 can stimulate chemotaxis, random migration, and antibody-independent cell-mediated cytotox-

Most studies about PGF2α and fertility have been performed in production animals. PGF2α and its analogs have been used to resolve uterine infections in livestock; however, its mechanism of action is not known. Moreover, it is unclear if modulation of sexual steroids levels induced by PGF2α directly alters the immune response in postpartum. Cattle are resistant to uterine infections when progesterone concentrations are basal, and they are susceptible to uterine infections when progesterone concentrations are increased [23, 24]. It has been proposed that exogenous PGF2α is an effective luteolytic factor to reduce progesterone levels

**214**

Some studies in vitro have evidenced that the supplementation of mice with omega-3 fatty acids increased implantation markers such as laminin and leukemia inhibitory factor in endometrial epithelium and stroma, which would encourage the endometrium for a favorable environment of implantation [48]. Through an abortion mouse model and human stromal cells, it was suggested that omega-3 fatty acids activate the signaling pathways ERK1/ERK2 and AMPK, which increase FOXO1 and GLUT 1 expression, and the increased glucose uptake would be important for the maintenance of pregnancy [11].

Several studies in bovines have proposed that omega-3-rich diet improve the reproductive performance. It has been described that incorporation of fatty acids of omega-3, and also omega-6, in the bovine diet influences some of the reproductive process involved in the follicular development [49] and progesterone and PGF2α production [50, 51] regulating embryo survival and implantation.

It has been shown that high intake of omega-6 fatty acid induces a change in membrane phospholipids, increasing the proportion of arachidonic acid, which would favor the synthesis of PG of the series 2, and eicosanoid, so it would turn into a pro-inflammatory environment [52]. By contrast, the dietary increase of the omega-3 fatty acid especially EPA and DHA would increase the proportion of these phospholipids in the cell membranes, which would ultimately result in the decrease of the synthesis of the PG series 2, whereby they would act as an anti-inflammatory mechanism [52]. Based on the effect of fatty acids on PG secretion, several studies conducted in dairy cattle have been addressed to attenuate the endometrium secretion of PG at the time of the embryo-maternal recognition of pregnancy in order to improve embryo survival and pregnancy rate [53–55]. Dairy cows supplemented with conjugated linoleic acid (CLA) have higher pregnancy rates than their nonsupplemented control group, and the probability of pregnancy increases by up to 26% and that the interval of first postpartum ovulation was reduced by 8 days [56].

Also, the supplementation of dairy cows with polyunsaturated omega-3 fatty acids as EPA can inhibit the synthesis of PGF2α through competition with arachidonic acid by COX-1 and COX-2 enzymes or in the case of DHA competing with arachidonic acid with the phospholipase A2 enzymes [57]. For this reason, fish meal included in the bovine diet could reduce PGF2α and delay regression of the CL, improving embryonic survival and female fertility [51]. The supplementation of cows with omega-3 fatty acid from the fish meal not only reduces the endometrial concentration of arachidonic acid but also increases the concentration of both EPA and omega-3 fatty acids in the endometrium [58]. When fish meal was included in the diet in a study conducted with beef cows (Angus), an increase in EPA and DHA in luteal tissue and a reduction of arachidonic acid in the endometrium resulting in an increase in the fertility of cows were observed [59]. However, in addition to its effect on PG secretion, some studies have concluded that diets rich in EPA and DHA can have a direct effect on the growth of the conceptus per se [60]. Others speculate that the delay on CL regression would allow not well-developed embryos to reach their competent size to initiate a maternal dialog before the luteolytic secretion of PG [50, 61].

The roles of omega-3 and omega-6 fatty acids on prostaglandin secretion have been well documented in in vitro and in vivo studies. The production of PGF2 α was suppressed in an endometrial cell culture when the culture medium was supplemented with omega-3 fatty acids [62]. However, when the medium was supplemented with omega-6 fatty acids, the increase in the ratio of omega-6 to omega-3 produced an increase of PGF2α [63]. Similarly, in studies conducted with dairy cows, the supplementation with different ratio of fatty acids from omega-6 to omega-3 altered the secretion on PGF2 α induced by either oxytocin [64] or spontaneous [49]. The production of PGE2 induced by LPS also was inhibited in the cellular line of bovine endometrium BEND treated with DHA [12].

**217**

unpublished data).

**Figure 1.**

about formation of SPM.

*Beneficial Effect of Omega-3 Fatty Acids on Immune and Reproductive Endometrial Function*

Additionally, recent evidences show an inhibition of the translocation of the transcription factor NF-κB induced by LPS in BEND cells treated with DHA (**Figure 1**;

*Localization of NF-κB in BEND cells treated with DHA and stimulated with LPS. BEND cells were treated with 50 μM DHA for 15 min, and then 1 μg/ml LPS was added and incubated for 30 min. NF-κB was detected* 

*by immunocytochemistry and epifluorescence microscopy. Magnification 40X [65].*

**4. Mechanisms of action of omega-3 fatty acids in endometrial cells**

Two more recent mechanisms have been described in macrophages and endothelial cells: (1) binding of DHA to FFA4 receptor/β-arrestin and inhibition of TAK1/NF-κB, thus reducing synthesis of pro-inflammatory factors, and (2) inhibition of NLRP3 inflammasome. FFA4 receptor is a G-protein-coupled receptor with high affinity by DHA described first in the intestine and macrophages. Recent studies evidenced the presence of FFA4 receptor in the human, mouse, and bovine endometrium [11, 12]. After ligand binding, FFA4 receptor couples to β-arrestin2, which is followed by receptor endocytosis and inhibition of TAB1-mediated activation of TAK1, a protein activated after inflammatory stimuli such as LPS, which

The first known anti-inflammatory mechanism of omega-3 fatty acids was the formation of specialized pro-resolving mediators (SPMs) derived from DHA. The enzymatic oxygenation of DHA via 12−/15-lipoxygenase (LOX) and 5-LOX leads to the formation of the D-series resolvins (RvD1, RvD2, RvD3, RvD4, RvD5, and RvD6), neuroprotectins/protectins, and maresins in different cells [10, 66], and resolvins have a potent effect on leukocyte migration and also reduce production of pro-inflammatory cytokines [67]. All those evidences have been obtained in different cellular types, but in the uterus or endometrial cells, there are not yet studies

*DOI: http://dx.doi.org/10.5772/intechopen.89351*

*Beneficial Effect of Omega-3 Fatty Acids on Immune and Reproductive Endometrial Function DOI: http://dx.doi.org/10.5772/intechopen.89351*

**Figure 1.**

*Apolipoproteins, Triglycerides and Cholesterol*

tant for the maintenance of pregnancy [11].

Some studies in vitro have evidenced that the supplementation of mice with omega-3 fatty acids increased implantation markers such as laminin and leukemia inhibitory factor in endometrial epithelium and stroma, which would encourage the endometrium for a favorable environment of implantation [48]. Through an abortion mouse model and human stromal cells, it was suggested that omega-3 fatty acids activate the signaling pathways ERK1/ERK2 and AMPK, which increase FOXO1 and GLUT 1 expression, and the increased glucose uptake would be impor-

Several studies in bovines have proposed that omega-3-rich diet improve the reproductive performance. It has been described that incorporation of fatty acids of omega-3, and also omega-6, in the bovine diet influences some of the reproductive process involved in the follicular development [49] and progesterone and PGF2α

It has been shown that high intake of omega-6 fatty acid induces a change in membrane phospholipids, increasing the proportion of arachidonic acid, which would favor the synthesis of PG of the series 2, and eicosanoid, so it would turn into a pro-inflammatory environment [52]. By contrast, the dietary increase of the omega-3 fatty acid especially EPA and DHA would increase the proportion of these phospholipids in the cell membranes, which would ultimately result in the decrease of the synthesis of the PG series 2, whereby they would act as an anti-inflammatory mechanism [52]. Based on the effect of fatty acids on PG secretion, several studies conducted in dairy cattle have been addressed to attenuate the endometrium secretion of PG at the time of the embryo-maternal recognition of pregnancy in order to improve embryo survival and pregnancy rate [53–55]. Dairy cows supplemented with conjugated linoleic acid (CLA) have higher pregnancy rates than their nonsupplemented control group, and the probability of pregnancy increases by up to 26% and that the interval of first postpartum ovulation was reduced by 8 days [56]. Also, the supplementation of dairy cows with polyunsaturated omega-3 fatty acids as EPA can inhibit the synthesis of PGF2α through competition with arachidonic acid by COX-1 and COX-2 enzymes or in the case of DHA competing with arachidonic acid with the phospholipase A2 enzymes [57]. For this reason, fish meal included in the bovine diet could reduce PGF2α and delay regression of the CL, improving embryonic survival and female fertility [51]. The supplementation of cows with omega-3 fatty acid from the fish meal not only reduces the endometrial concentration of arachidonic acid but also increases the concentration of both EPA and omega-3 fatty acids in the endometrium [58]. When fish meal was included in the diet in a study conducted with beef cows (Angus), an increase in EPA and DHA in luteal tissue and a reduction of arachidonic acid in the endometrium resulting in an increase in the fertility of cows were observed [59]. However, in addition to its effect on PG secretion, some studies have concluded that diets rich in EPA and DHA can have a direct effect on the growth of the conceptus per se [60]. Others speculate that the delay on CL regression would allow not well-developed embryos to reach their competent size

production [50, 51] regulating embryo survival and implantation.

to initiate a maternal dialog before the luteolytic secretion of PG [50, 61].

The roles of omega-3 and omega-6 fatty acids on prostaglandin secretion have been well documented in in vitro and in vivo studies. The production of PGF2 α was suppressed in an endometrial cell culture when the culture medium was supplemented with omega-3 fatty acids [62]. However, when the medium was supplemented with omega-6 fatty acids, the increase in the ratio of omega-6 to omega-3 produced an increase of PGF2α [63]. Similarly, in studies conducted with dairy cows, the supplementation with different ratio of fatty acids from omega-6 to omega-3 altered the secretion on PGF2 α induced by either oxytocin [64] or spontaneous [49]. The production of PGE2 induced by LPS also was

inhibited in the cellular line of bovine endometrium BEND treated with DHA [12].

**216**

*Localization of NF-κB in BEND cells treated with DHA and stimulated with LPS. BEND cells were treated with 50 μM DHA for 15 min, and then 1 μg/ml LPS was added and incubated for 30 min. NF-κB was detected by immunocytochemistry and epifluorescence microscopy. Magnification 40X [65].*

Additionally, recent evidences show an inhibition of the translocation of the transcription factor NF-κB induced by LPS in BEND cells treated with DHA (**Figure 1**; unpublished data).
