According to the World Health Organization, infertility affects approximately 15% of all couples, and 40% to 50% of cases are caused by male infertility. The main causes of male infertility are shown in Fig. 1.
Spermatozoa are produced in the testes and mature in the epididymis. After ejaculation, spermatozoa undergo several structural and biochemical changes before fertilizing an ovum. Calcium regulates several reproductive processes. Semen quality is also influenced by fatty acid (FA) consumption. Polyunsaturated FAs (PUFAs), such as linoleic acid, a-linolenic acid (ALA), eicosapentaenoic acid, and docosahexaenoic acid (DHA) target generative tissues, and change conceptive capacity and fertility[1-4].
FAs and infertility
Descriptive diagnoses for male infertility usually include “oligozoospermia” (reduced sperm count), “asthenozoospermia” (reduced sperm motility), and “teratozoospermia” (reduced percentage of sperm with normal morphology). Oligoasthenozoospermia (OA), also known as oligoasthenospermia, is a common urological condition, observed in approximately 6.1% to 13.6% of patients undergoing fertility evaluation. In nonobstructive azoospermia (NOA), sperm count is significantly reduced due to impaired spermatogenesis, which is the most severe form of male infertility. The etiology of NOA is either intrinsic testicular impairment or inadequate gonadotropin production. NOA is mainly caused by genetic mutations, altered epigenetic (DNA methylation) regulation, chromosomal aberrations, and defects in the Y chromosome. FAs and calcium can specifically be used to treat OA. OA is a sperm disorder characterized by a low sperm count (<15 million spermatozoa/mL) and significantly reduced motility (>60% immotile spermatozoa). Relationships between male infertility and PUFA/Ca levels are shown in Table 1. PUFAs play an important role in maintaining the fluidity of the sperm plasma membrane and its sensitivity to lipid oxidation, which results in the formation of peroxides.
Approximately, 30% to 50% of the overall FA present in mammalian sperm plasma membrane consists of omega-3 and omega-6. These molecules are important for reproduction, fluidity control, and the promotion of acrosome reactions. A previous study reported that an increase in omega-3 and omega-6 PUFA levels in pig sperm plasma membranes improved sperm characteristics. Similar results were observed in bovine, goat, and ram spermatozoa.
The structure of unsaturated FAs in sperm and ovum plasma membranes influences conception[1,2]. LC-FAs (>C20) influence the seminal quality and the development of spermatozoa, maintaining membrane structural integrity by improving membrane fluidity owing to their specific structural orientation.
Lipid metabolism and male fertility
Cholesterol and lipid balance may regulate male fertility as the lipid composition of the plasma membrane influences several key characteristics of spermatozoa (Fig. 2)[17,18]. The liver is the primary organ controlling lipid metabolism in mammals, and adipose tissue acts as an endocrine organ by regulating FA metabolism and lipid storage. Dietary medium-chain triglyceride is known to improve spermatogenesis in the epididymis and enhance forward movement. Medium-chain triglyceride can be used to treat abiogenic supportive oligospermia/asthenospermia in men with low chylomicron levels and high very-low-density lipoprotein levels. The various reproductive processes that are regulated by PUFA are shown in Table 2.
Table 1 -
List of notable causes of male infertility and their relationship with polyunsaturated fatty acid/calcium (PUFA/Ca) levels
Notable causes of male infertility
Relationship with PUFA/Ca levels
Fatty acid profile and metabolism are related to human sperm parameters and are relevant to idiopathic infertility and varicocele
Testicular dysfunction and malignancy
Butylated hydroxyanisole induces testicular dysfunction in mouse testis cells by dysregulating calcium homeostasis and stimulating endoplasmic reticulum stress
Both calcium and PUFAs regulate the biosynthesis of steroid hormones that are required for sperm formation[1,7,8]
Flaxseed oil containing oleic acid and linolenic acid is an important source of cholesterol, a precursor for the synthesis of steroid hormones, such as testosterone; increased testosterone levels improve libido
Providing boars with 3% fish oil led to an increase in the levels of docosahexaenoic acid in their sperms by 33%-45% and increased the volume of sperms in their ejaculate
Reactive oxygen species (ROS)
High levels of PUFAs increase reactive oxygen species (ROS) levels, which cause lipid peroxidation. Peroxidative damage disrupts the ability of the plasma membrane of the sperm to fuse with that of the ovum as well as its capacity to assist membrane-bound enzymes such as ATPase
Role of dietary fish oil (FO) on sperm characteristics and FA configuration
Dietary fish oil (FO) significantly enhanced sperm characteristics and function in rams[23,24]. The steady body weight of the rams throughout the study implied that the enhanced effect on sperm characteristics and functions was caused by FAs present in the FO. Sperm parameters improved during the first phase after supplementation with dietary FO. The positive effect of FO on the sperm count and whole sperm yield, which was observed for 2 months following the withdrawal of FO, remained unchanged. The sperm plasma membrane of ruminants contains high levels of long-chain PUFAs, especially omega-3[1,24]. Although the mechanisms by which dietary n-3 unsaturated fats affect sperm parameters require further investigation, researchers have proposed several hypotheses for sperm assembly, anti-apoptosis, eicosanoid union, and hormone production. A potential mechanism is the regulation of gene expression. DHA is required to facilitate the development of certain mammalian spermatozoa and can increase the sperm count. Dietary FAs are known to affect the sperm count of mammals; elevated levels of dietary saturated FAs or trans unsaturated fats play important roles in improving sperm quality.
Correlation between very long chain (VLC)-PUFA levels and sperm quality and amount
There is a positive correlation between very long chain (VLC)-PUFA levels and sperm count. The total number of motile sperm and sperm characteristics depend on the presence of VLC-PUFAs. Low levels of VLC-PUFAs are associated with reduced sperm counts and diminished sperm motility. VLC-PUFAs carrying sphingomyelin on the sperm head undergo several modifications, such as capacitation. Further studies are required to assess the correlations among in vitro fertilization, parameters of spermatozoa, levels of VLC-PUFAs, effects of VLC-PUFAs on pregnancy and live birth, and whether they can restore fertility.
Table 2 -
Summary of the studies performed, along with the source of polyunsaturated fatty acids (PUFAs)
Name of study
Impact of dietary omega-6 PUFAs and vitamin C (VC) in rams
Sunflower oil (contains approximately 60% of linoleic acid)
Increased fertility rate, enhanced progressive mobility of sperm, increased sperm count.
Impact of PUFA on libido and characteristic of sperm in Nilli-Ravi buffalo bull
Flaxseed (Linum usitatissimum) oil
Enhancement of libido, increased semen volume and mass and sperm activity, enhanced motility, increased sperm count.
Impact of n-3: n-6 fatty acid (FA) proportions and vitamin E (vit E) on male conceptive fitness in aged roosters
Sperm volume, count, and structure was not influenced by the proportion of omega-3: omega-6 used for supplementation (possible explanation for this could be the age of the roosters [45-60 weeks], as the birds matured during the study). The mobility, sustainability, and membrane quality of male gametes; testosterone levels; and fertility were increased after the roosters were provided with omega-3: omega-6 (4:25). Vitamin E was provided as an antioxidant, which increased sperm motility.
Severely unadulterated and undiluted docosahexaenoic acid (DHA) supplementation on fertility
DHA facilitated acrosome reaction and increased progressive sperm motility; no unfavorable effects were observed.
Effect of supplementation of omega-3 fatty acids on Holstein bull to measure semen quality during heat stress
Freshly ejaculated sperm samples from bulls after omega-3 fatty acids showed enhanced total motility and progressive motility of the sperms, as well as mean path velocity; the sperms were hypo-osmotic swelling test-positive.
This table summarizes the result of the different studies performed to assess the effect of PUFAs obtained from different sources on sperm motility, maturation, and other parameters.
Fluctuations in phosphatidylcholine (PC) and FA levels in reproductive cells during testicular development
Testis development, sperm maturation, and sperm capacity are correlated with lipid configuration. Phosphatidylcholines (PCs) play a vital role in determining sperm composition and capacity in the testis. PCs are also the most abundant type of lipids in all mammalian cell membranes. Omega-3 FAs are esterified to either the triglyceride or phospholipid fraction. PC-LCFA is one of the most bioavailable forms for incorporation into cellular systems. Siangcham et al. reported the configuration and dispersion of PC and absolute FAs within 3 batches of seminiferous tubules characterized by cell affiliations within 3 morphotypes of Macrobrachium rosenbergii (small males, orange claw males, and blue claw males). They observed that the testes of orange claw males had the highest levels of these FAs, whereas the testes of blue claw males had the lowest FA levels. Eicosapentaenoic acid (20:5) levels were higher in the testes of small males and orange claw males than in the testes of blue claw males. The increased levels of FAs in the testes of small males and orange claw males indicate the significance of FAs for spermatogenesis. Thus, providing diets rich in PUFAs and MUFAs to prawn broodstocks enables testis development and male fertility enhancement.
Effect of ALA on membrane stability and oxidative pressure in solidified defrosted sperms
Treatment with ALA and bovine serum albumin (BSA) or methyl-β-cyclodextrin (MBCD) not only negatively affects the acrosome, plasma membrane, mitochondrial activity, structure, and mobility of solidified defrosted boar sperms but also induces oxidative stress in the sperms. The addition of defined proportions of BSA and MBCD in the absence of ALA during cryopreservation results in morphological abnormalities and variable tail motion in the spermatozoa. Adding ALA along with BSA and MBCD has been shown to significantly enhance sperm function.
The plasma membrane and acrosome were intact and sperm motility was unaffected when sperm were treated with the ALA + MBCD-treated group; significant results were not obtained in the ALA + BSA group. During cryopreservation of boar spermatozoa, transporter proteins, such as BSA and MBCD, may enhance the effect of ALA, which improves membrane integrity and mitochondrial action by decreasing reactive oxygen species (ROS)-induced lipid peroxidation levels.
Role of calcium in male fertility
Calcium ions (Ca2+) regulate several natural cycles, such as cell proliferation, protein discharge, and muscle contractionvia calmodulin, which is an intra-cellular calcium receptor that regulates most of these processes[34,35]. The prostate gland, which acts as a source of calcium for human semen, seminal vesicles, and epididymis have a markedly high concentration of calcium. Several studies have documented the effects of intra-cellular and seminal plasma calcium on sperm function and motility. Men with hypomotility show low levels of calcium in semen than those with typical motility, indicating a positive correlation between high calcium levels and fecundity in men[37,38]. Moreover, in vitro studies have shown that high levels of calcium increase fecundity in men. Low levels of vitamin D are associated with reduced levels of intra-cellular calcium, which lead to impaired sperm motility, abnormal acrosome reaction, and increased chances of male infertility[39,40]. The effects of calcium supplementation on male fertility are shown in Fig. 3.
Role of calcium in the process of spermatogenesis
Calcium plays a significant role in the regulation of spermatogenesis and fertilization, in addition to other processes, such as the development, differentiation, proliferation, and apoptosis of male gametogonium and male gametocytes. Intra-cellular calcium levels are higher in spermatozoa than in spermatogonia (sperm > spermatids > spermatocyte > spermatogonia). This suggests that calcium levels are altered and maintained in equilibrium at specific stages of sperm production and maturation. Calmodulin is expressed in high quantities in the testes, thereby suggesting the significance of calcium in typical spermatogenesis.
Many calcium channels are present in male gametogonia and gametes, and these are important for the regulation of calcium signaling pathways. Calcium signaling is important for the development of spermatogonia to the next stage during spermatogenesis. Changes in calcium balance and homeostasis can be induced by activating different calcium channels during the formative phases of spermatogenesis. This delicate balance is crucial in maintaining spermatocyte activity; however, any increase in free calcium levels may also harm sperm function, including chromatin condensation, mitochondrial damage, and the release of degradative enzymes that promote apoptosis.
Role of calcium in testosterone secretion
Calcium is important for the synthesis of steroid hormones in the Leydig cells of the testes. The release of testosterone increases the levels of calcium, whereas calcium chelators curb steroidogenesis. Calcium also plays a role in Sertoli-Sertoli junction dynamics. Testosterone plays an important role in calcium influx, thus demonstrating that calcium channels present in the plasma membrane play a vital role in testosterone-calcium signalling. Calcium plays an important role in the modulation of testosterone to facilitate gonad cell differentiation and development and spermatogenesis.
Impact of calcium influx on male fecundity
Ca2+ are important for regulating several physiological processes in spermatozoa, such as chemotaxis, hyperactivation, capacitation, and acrosome reaction, which are all necessary for favorable fertilization. Various calcium channel proteins play a vital role in the transfer of intra-cellular pH. Several studies have indicated the existence of a potential correlation between the levels of sperm proteins and calciumpermeable channel proteins, which modulate the mechanism underlying calcium influx, thus playing a vital role in sperm motility.
Sperm capacitation and calcium levels
Capacitation is a process through which a sperm undergoes functional maturation to render it competent to fertilize an oocyte. Capacitation usually occurs after ejaculation when semen is deposited in the female reproductive tract. The uterus plays a vital role during capacitation by releasing sterol-binding albumin, lipoproteins, proteases, and glycosidases (eg, heparin). Capacitation is regulated by the organs of the female reproductive tract. Calcium is considered one of the critical components that affect capacitation. During capacitation, intra-cellular calcium levels and membrane hyperpolarization increase. Calcium controls sperm capacitation by modulating cAMP-dependent signaling and tyrosine phosphorylation pathways in a biphasic manner. The dispersion of intra-cellular calcium alters capacitation. Several proteins, such as ryanodine receptor, troponin, and sarcoplasmic calcium-binding protein, influence calcium signaling activities in spermatozoa throughout various reproductive phases.
During capacitation, the ion channels undergo several modifications that are caused by changes in the membrane potential, which markedly influences calcium channels. The mechanism underlying this phenomenon is still not clearly understood. It is suggested that pH modulation and depolarization play important roles in calcium transfer.
Role of calcium in acrosome reaction
Calcium influx via membrane channels plays a critical role in acrosome reaction and fertility. The source of internal calcium storage is calcium itself, not the complex molecule broken down into calcium and stored. Both plasma and acrosomal membranes are calcium pumps that deliver calcium during the acrosome reaction[8,65-67]. Acrosome reaction occurs only when calcium is present in the extra-cellular region[8,68]. Treating sperm with the ionophore A23187 effectively initiates calcium influx, giving rise to acrosome reaction[8,69]. Progesterone plays a vital role in the influx of calcium, thus initiating acrosome reaction.
Role of PUFAs in calcium assimilation
Dietary fat intake is associated with increased intestinal calcium absorption. FAs seem to influence the small intestine differently. Compared with MUFAs, saturated FAs reduce lipid fluidity of the intestinal membrane and attach to calcium, thereby increasing fecal fat ejection and reducing calcium bioavailability.
Calcium bioavailability is greater in the distal jejunum than in the proximal ileum of humans who consume high-fat diets. The increase in intestinal calcium absorption with high fat-diet intake is not due to increased availability of calcium from food, thus indicating some biological significance. Calcium assimilation further impact sperm motility by several key processes during spermatogenesis and capacitation reaction[71-74].
Conjugated linoleic acid (CLA) treatment for more than 2 months has been found to enhance calcium assimilation in young developing rats that were fed high amounts of omega-3 PUFAs.
Impact of calcium on lipid metabolism
High levels of calcium are known to reduce the synthesis of FAs from carbon precursors of acetyl coenzyme-A (Co-A) (lipogenesis) and increase hydrolysis of triglycerides into glycerol and FAs (lipolysis) in fat tissue, thus leading to low muscle-to-fat ratio. Providing rats with calcium lowered the levels of parathyroid hormone (1,25 nutrient D was not estimated). The rats did not show reduced absolute fat bulk. Thus, the physiological effects of the changes in dietary calcium levels influence the synthesis of FAs from the carbon precursor of acetyl Co-A or the hydrolysis of triglycerides into FAs and glycerol within adipose (fat) tissue is insignificant. Calcium can bind to and precipitate unsaturated fats within the gastrointestinal tract, reducing its assimilation and increasing discharge into defecation.
If calcium specifically decreases the ingestion of saturated/trans FAs compared with MUFAs or PUFAs, excessive consumption of calcium might be expected to beneficially affect the serum lipid profile. In a previous study, levels of FAs in the fecal discharge increased markedly after rats were fed diets containing high levels of calcium. Rats that received diets with high levels of calcium showed a decrease in the evident edibility of FAs. Diets with excessive calcium reduce the edibility of saturated FAs more considerably than MUFAs and PUFAs.
This study highlights the changes in dietary calcium levels have a minor impact on body weight or fat mass in rats, except if extremely low levels of calcium are administered. A decrease in the edibility of total FAs with excessive dietary calcium was also noted.
Further studies are required to confirm whether calcium in dairy items reduces the antagonistic effects of TFA on these items. This is significant if natural TFA has similar antagonistic consequences for blood lipids, such as industrial trans FAs but is very difficult to eliminate from edible accommodation.
LC-PUFAs have been identified in humans and several other animal species, such as birds (eg, chickens, ducks, and turkeys) and mammals (eg, rams, bulls, and boars). PUFAs confer fluidity to the sperm plasma membrane and are required for the fusion of the sperm into the ovum. These molecules are susceptible to ROS, which cause lipid peroxidation. Peroxidative damage disrupts the ability of the plasma membrane of the sperm to fuse with that of the ovum as well as the capacity of the former to assist membrane-bound enzymes (eg, ATPase). Theoretically, changes in membrane fluidity can delay or prevent the initiation of signal transduction pathways, which are important for fertilization. Extra-cellular antioxidants play an important role in protecting spermatozoa from lipid peroxidation. High levels of dietary PUFAs decrease antioxidant levels in sperms. The significance of LPO is suggested by the action of vitamin E, an important antioxidant that reverses the adverse effects of dietary PUFAs.
High levels of PUFAs enhance fecundity, as reported in different studies showing the effects of this dietary supplementation on domesticated animals. Providing boars with 3% FO showed increased levels of DHA in sperm by 33% to 45% and increased the volume of sperms in the ejaculate. Shark oil enhanced sperm motility and velocity parameters, but other examinations revealed no enhancement in fertilizing capacity in sperm used for artificial insemination obtained boars that were administered cod liver oil. Therefore, it is evident that PUFAs play a critical role in regulating several key mechanisms that influence sperm maturation, motility, and acrosome reaction. Furthermore, FA profiles in men with different seminal characteristics due to reproductive pathologies, such as varicocele and infections, are altered. Basically, PUFAs modulate oxidative stress, ROS production, and inflammatory processes during spermatogenesis.
Calcium plays a vital role in male fertility by regulating several cellular and molecular mechanisms. Calcium is responsible for regulating steroid hormone synthesis and sperm mobility, chemotaxis, capacitation, and acrosome reactions. Dietary calcium is known to improve infertility. Hence, the role of calcium and its levels must be evaluated in human semen exhibiting idiopathic defects. PUFAs regulate the fluidity of the sperm plasma membrane, which is important for gamete fusion. PUFAs are components of fucosylated lipids and glycosphingolipids, which are essential for fertility. PUFAs regulate several biochemical and physiological mechanisms differently in the different sections of the male reproductive system, such as the synthesis of steroid hormones in Leydig cells.
This review mainly focuses on ruminants; these animals are exposed to a wide range of temperatures throughout the year, unlike humans. Seminal quality largely varies throughout the year because of exposure to seasonal variations. Unlike humans, the metabolic fate of n-3 FAs in ruminants primarily differs because of the biohydrogenation system present in these animals.
PUFA consumption, particularly CLA, enhances calcium absorption. Dietary calcium controls body configuration and lipid metabolism in rats. Thus, dietary PUFA and calcium levels may significantly affect sperm function.
PUFAs affect various reproductive processes (Table 2). Flaxseed oil containing oleic acid and linolenic acid is an important source of cholesterol, a precursor for the synthesis of steroid hormones such as testosterone. Increased testosterone levels improve libido. PUFAs have a positive effect on the integrity of cell membranes. A normal sperm plasma membrane is important for successful acrosome reaction and binding to the ovum, thus increasing the ability to fertilize. A PUFA-rich diet leads to enhanced biosynthesis of prostaglandin and steroid hormones, influencing the hypothalamic-pituitary axis and acting as hormone regulators for spermatogenesis[1,7]. During heat pressure, dietary supplementation with unsaturated FAs improves semen quality. N-3 LC-PUFAs influence the sperm plasma membrane structure, movement of spermatozoa, and sustainability. N-3 PUFAs enhance semen quality by modulating thermogenesis, which is important for the maintenance of testicular function. Intra-cellular calcium levels influence male fertility, and it is involved in capacitation, spermatogenesis, and acrosome reaction. FAs esterified into phospholipids help maintain the structural integrity and composition of the cell membrane. FAs are an important source of phospholipids in the membrane and act as precursors for bioactive lipid mediators, thus regulating cellular processes. Phospholipases enable the release of long chain-n-3 PUFAs into the target tissue. These phospholipases are regulated by calcium. DHA inhibits an increase in intra-cellular Ca2+ concentration through a mechanism mainly involving voltage-dependent Ca2+ channels. CLA treatment enhances calcium assimilation in young developing rats that are fed omega-3 PUFA in large amounts. Therefore, dietary supplementation with FAs and calcium may be an option for regulating male fertility.
The authors would like to thank the Amity University Uttar Pradesh, Noida, India, University Grants Commission, New Delhi, India, Council of Scientific and Industrial Research, New Delhi, India and the Science and Engineering Research Board (SERB), New Delhi, India.
T.N. and S.C. have written the paper under the guidance of S.S. All authors have contributed in conceiving and designing the manuscript. All authors contributed to the final manuscript and approved the submitted version.
The first author Taniya Naz is funded by University Grants Commission Junior Research Fellowship (UGC-JRF) and the lab expenses including second author's fellowship (JRF) is funded by the Science and Engineering Research Board - Start up Research Grant (SERB-SRG) (Grant No.: SRG/2019/000501) of Department of Science and Technology, New Delhi, India.
Conflicts of interest
All authors declare no conflict of interest.
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