Physiology and metabolism

The inactive form of vitamin D, cholecalciferol, is synthesised in the skin following the conversion of 7-dehydrocholesterol by UV B radiation from the sun^{(Reference Cheng, Levine and Bell25,Reference Prosser and Jones26)} . Cholecalciferol can also be absorbed from the diet or via supplementation. In detail, cholecalciferol must undergo two hydroxylations to form the active form of vitamin D, 1,25(OH)₂D₃ that can bind and activate the VDR^{(Reference Ohyama27–Reference Gray, Omdahl and Ghazarian29)}. These steps involve the microsomal cytochrome P450 (CYP) enzymes. The first step is 25-hydroxylation by hepatic CYP2R1 which forms the prohormone 25OHD (calcidiol) followed by 1α-hydroxylation by renal CYP27B1 to form 1,25(OH)₂D₃, whereas CYP24A1 inactivates 25OHD and 1,25(OH)₂D₃^{(Reference Bouillon, Carmeliet and Verlinden30)}. Parathyroid hormone (PTH) is the main inducer of renal CYP27B1 expression and thus the main inducer of circulating 1,25(OH)₂D₃ and a powerful mobiliser of calcium from the skeleton^{(Reference Yuan, Sitara and Sato31)}. 1,25(OH)₂D₃ binds and activates the VDR which forms a heterodimer with the retinoid X receptor. This complex recognises vitamin D response elements in the promoter region of target genes and regulates transcription^{(Reference Bouillon, Carmeliet and Verlinden30)}. The DNA-binding domain of the VDR mediates the transcriptional effects, but VDR also has an alternative ligand-binding pocket that mediates rapid non-genomic effects by regulating second messenger systems. In clinical practice, vitamin D status is determined by measuring serum levels of 25OHD and not 1,25(OH)₂D₃, as 25OHD correlates with bone mass density, serum calcium level and PTH secretion^{(Reference Lips32,Reference Holick33)} . However, cellular responsiveness to circulating vitamin D metabolites goes beyond VDR expression as the presence of activating and inactivating enzymes locally in the target organs influences the availability of substrates for VDR and thereby influences the effect of vitamin D on the target tissue^{(Reference Nagakura, Abe and Suda34,Reference Choudhary, Jansson and Stoilov35)} .

Expression of vitamin D regulating enzymes and vitamin D receptor in the male reproductive system

Studies have consistently found mRNA and protein expression of the VDR and the vitamin D activating and inactivating enzymes in various cell types of the gonads and male reproductive tract^{(Reference Blomberg Jensen9)}. In most studies, VDR is co-expressed with the activating enzyme, CYP27B1, and the deactivating enzyme, CYP24A1. This co-expression occurs throughout the reproductive tract, including in some Sertoli cells, most germ cells, spermatozoa, Leydig cells and the epithelial cells lining the tract^{(Reference Johnson, Grande and Roche36–Reference Merke, Kreusser and Bier42)}. The local presence and activity of the enzymes play a crucial role in regulating the concentration of 1,25(OH)₂D₃ within cells and consequently activating the VDR.

In adult testes, the VDR and metabolising enzymes are primarily expressed in human germ cells, with a marked expression in spermatogonia, spermatocytes and spermatids in both human subjects and rodents^{(Reference Blomberg Jensen, Nielsen and Jørgensen18,Reference Zanatta, Zamoner and Zanatta43)} . Studies have also found VDR expression in both the nucleus and cytoplasm of primary cultures of immature Sertoli cells, in fetal Sertoli cells and in the immature mouse Sertoli cell line TM4 where 1,25(OH)₂D₃ is known to mediate fast non-genomic effects^{(Reference Zanatta, Bouraïma-Lelong and Delalande44–Reference Menegaz, Barrientos-Duran and Kline50)}. The expression levels of CYP2R1, CYP27B1 and CYP24A1 in the testes indicate that the cells are equipped to respond to and regulate the effects of vitamin D metabolites^{(Reference Jensen8,Reference Blomberg Jensen, Nielsen and Jørgensen18)} . Furthermore, the effects of cholecalciferol or calcitriol treatment on gonadal function may vary depending on the specific expression pattern of these enzymes. Cholecalciferol can be activated locally, potentially eliciting a response, whereas calcitriol is already active and immediately triggers responses or undergoes inactivation. This suggests that the local regulation of vitamin D is important for spermatogenesis and sperm function. The expression of VDR in germ cells suggests that calcitriol may play a role in sperm function^{(Reference Foresta, Strapazzon and De Toni51)}. Multiple studies have reported the presence of VDR in specific regions of mature human spermatozoa, including the post-acrosomal part of the head, the neck region and the midpiece^{(Reference Blomberg Jensen, Nielsen and Jørgensen18,Reference Aquila, Guido and Middea52)} . Additionally, the vitamin D activating enzymes are also expressed in spermatozoa, and the inactivating enzyme CYP24A1 exhibits a distinct expression pattern at the annulus. Notably, the expression levels of VDR and CYP24A1 are higher in spermatozoa from healthy men compared with infertile men^{(Reference Blomberg Jensen, Jørgensen and Nielsen53)}, which supports that vitamin D may be beneficial for sperm function.

VDR expression has also been demonstrated in Leydig cells, suggesting a potential direct impact of vitamin D on steroidogenesis and thereby also an indirect effect on spermatogenesis and male fertility. Recent studies have detected VDR at the protein level in fetal and adult Leydig cells in human subjects, roosters and mice^{(Reference Blomberg Jensen, Nielsen and Jørgensen18,Reference Oliveira, Dornas and Kalapothakis38,Reference Blomberg Jensen, Jørgensen and Nielsen54,Reference Hirai, Tsujimura and Ueda55)} . Moreover, VDR, CYP27B1 and CYP24A1 are all expressed in the epididymis, prostate and seminal vesicles^{(Reference Blomberg Jensen, Nielsen and Jørgensen18,Reference Mahmoudi, Zarnani and Jeddi-Tehrani37,Reference Oliveira, Dornas and Kalapothakis38)} , which highlights a potential direct effect throughout the male reproductive system.

Animal models: vitamin D deficiency and male fertility

Numerous animal models have been utilised to investigate the relationship between vitamin D and male fertility (defined as successful conception). An overview of the models is shown in Table 1. The first association was found in a study exploring vitamin D deficiency in rats^{(Reference Kwiecinski, Petrie and DeLuca56)}. Vitamin D deficiency in male rats led to a 45 % decrease in successful mating, defined as the presence of sperm in the vaginal tract, compared with rats with sufficient vitamin D levels. Additionally, rats with vitamin D deficiency had 71 % fewer pregnancies compared with vitamin D-sufficient rats^{(Reference Kwiecinski, Petrie and DeLuca56)}. When the rats were supplemented with calcium, the fertility potential was restored, implying an indirect effect of hypovitaminosis D leading to hypocalcaemia and secondarily reduced fertility^{(Reference Uhland, Kwiecinski and DeLuca57)}. However, after reassessment of the data by adjusting for the mating period and male:female ratio, and by determining the time to pregnancy, it was evident that the rats still had lower pregnancy rates after insemination with sperm from normocalcemic vitamin D-deficient rats than normocalcemic vitamin D-replete rats^{(Reference Uhland, Kwiecinski and DeLuca57)}. This indicates that vitamin D itself is important for semen quality and that the impaired fertility caused by vitamin D deficiency cannot be fully reversed by correcting hypocalcaemia alone^{(Reference Blomberg Jensen9)}. Newer animal studies on vitamin D deficiency have supported the negative effects of inadequate vitamin D levels on semen quality and male fertility. In general, the low fertility rates in vitamin D-deficient male rodents seem to be caused by impaired sperm count, motility and occasionally poor sperm morphology^{(Reference Audet, Laforest and Martineau58,Reference Kinuta, Tanaka and Moriwake59)} .

Table 1. Six studies investigating the relationship between vitamin D deficiency and fertility in rodents

N.D., not determined.

Animal models made vitamin D-deficient either by diet, knockout of the vitamin D receptor or the 1α-hydroxylase (Cyp27b1).

*Phenotype reported for prolonged vitamin D-deficient mice; ↓, negatively associated; →, no association.

Dietary vitamin D-deficient mice have also been shown to have lower testicular weight and fewer spermatozoa in the cauda epididymis compared with controls^{(Reference Fu, Chen and Xu60)}. Another study revealed adverse impacts on semen quality in vitamin D-deficient rats when compared with control groups. These effects included reductions in germ cell numbers, sperm count, sperm morphology, motility and viability. Furthermore, a decrease in serum testosterone levels was also observed in vitamin D-deficient rats^{(Reference Zamani, Saki and Hatami61)}. Duration and severity of the deficiency may be an important factor in reproductive function. Vitamin D deficiency through one spermatogenic cycle induced a significant increase in sperm DNA fragmentation but no significant effect on other semen parameters^{(Reference Shahreza, Hajian and Gharagozloo62)}. In contrast, prolonged vitamin D deficiency through two spermatogenic cycles resulted in a modest but significant decrease in sperm motility. Furthermore, different degrees of vitamin D deficiency have been induced in mice, and no effect was observed on sperm concentration and sperm viability, but sperm morphology was decreased in the severe deficiency group^{(Reference Sun, Chen and Zhang63)}. In another study, DNA fragmentation has also been shown to increase significantly in spermatozoa of vitamin D-deficient rats, which could explain the impaired fertility^{(Reference Merino, Sánchez and Gregorio64)}. Combined, these studies indicate that vitamin D is involved in the regulation of male fertility in rodents, suggesting a potential link between vitamin D status and reproductive health.

Human studies

Many of the cross-sectional studies investigating the link between vitamin D status and testicular function have been reviewed and discussed before, and the link critically depends on the fraction of men with low vitamin D status in the study population^{(Reference Lorenzen, Boisen and Mortensen65)}. Recent years have seen a surge in human intervention trials with vitamin D and male fertility outcomes. In this review, we have evaluated six randomised clinical intervention studies, with four of them being conducted after 2020. An overview of the trials is shown in Table 2. In the first randomised controlled trial from 2014, 86 infertile men with idiopathic oligoasthenozoospermia were included and randomised to receive either a 3 month supplementation of cholecalciferol (5 μg daily) and calcium (600 mg daily), or placebo^{(Reference Deng, Li and Yang66)}. This study found a significant increase in the number of progressive motile sperm and a higher pregnancy rate in the vitamin D v. placebo-treated men. These findings are supported by the most recent randomised clinical trial of 120 men conducted in 2022 on Indian men^{(Reference Padmapriya, Archana and Sharma67)}. They found an increased sperm volume, progressive motility, total motility and sperm count in men treated with cholecalciferol (100 μg daily for 10 weeks) when compared with the placebo group. The study does not report the serum 25OHD levels before or after treatment, so it is not possible to determine whether the effect was found in vitamin D insufficient, deficient or sufficient men.

Table 2. Interventional studies of vitamin D and semen quality in infertile men

25OHD, 25-hydroxyvitamin D; N, cohort size; N.D., not determined.

Six human randomised controlled studies investigating the relationship between serum vitamin D levels and semen quality in human subjects were evaluated.

↓, decreased with supplementation; ↑, increased with supplementation ; →, no changes.

In 2018, the biggest study to date was conducted and contradicted these findings^{(Reference Blomberg Jensen, Lawaetz and Petersen17)}. In the Copenhagen bone-gonadal study, 330 Danish infertile men with vitamin D insufficiency (defined as serum 25OHD <50 nm/l) were randomised to receive either a high single-dose cholecalciferol supplementation (7 500 μg) followed by daily supplementation of cholecalciferol (35 μg) and calcium (500 mg daily), or placebo. The study showed that supplementation did not affect semen parameters or live birth rates when comparing the groups. The spontaneous pregnancy rate tended to be higher in couples in which the men were treated with vitamin D and calcium compared with couples in which the men were in the placebo group, but the results were not statistically significant. However, subgroup analysis of oligospermic men did demonstrate an increased live birth rate in the treatment group (36 v. 18 %). One criticism of the study is the use of a high-loading dosage that could lead to high CYP24A1 activity locally in many organs and thus inactivate vitamin D activity in the gonads. The overall negative findings of the study are consistent with three other recent randomised clinical trials conducted on Iranian men^{(Reference Gheflati, Mirjalili and Kaviani68–Reference Amini, Mohammadbeigi and Vafa70)}. One trial with results published in 2020, investigated the effects of vitamin D supplementation in sixty-two infertile men with impaired semen quality and vitamin D insufficiency (defined as serum 25OHD <50 nm/l). The men were randomised to receive either cholecalciferol (1 250 μg once weekly) for 8 weeks, followed by a maintenance dose (1 250 μg once) lasting the final 4 weeks, or placebo treatment. The same study design was used by another Iranian research group from 2021, conducted on forty-four men with asthenozoospermia^{(Reference Gheflati, Mirjalili and Kaviani68)}. Both studies used vitamin D-deficient men (defined as serum levels <75 nm/l) and found no effect of cholecalciferol supplementation on semen parameters. A third Iranian study published in 2021 evaluated the effects of cholecalciferol on both endocrine markers and semen parameters in eighty-six infertile men with asthenozoospermia^{(Reference Maghsoumi-Norouzabad, Zare Javid and Mansoori69)}. Supplementation with 100 μg cholecalciferol daily for 12 weeks had no significant effects on semen volume, sperm count or sperm morphology compared with the placebo group. However, they found a positive effect on total and progressive sperm motility, to some extent supporting the results found in the Chinese and Indian study populations.

Overall, most of the interventional human data available indicate that vitamin D supplementation does not affect sperm count, volume, morphology or pregnancies. However, it must be noted that there are some contradictory reports on the presumed effect on total and progressive sperm motility, but positive data have only been generated in a few relatively small studies. Furthermore, with the diverse study populations of the mentioned studies (co-morbidities, age and BMI), design, inclusion criteria and baseline vitamin D status, it is hard to find consensus on the effect. Observational human studies and case–control studies investigating the association between vitamin D status and reproductive functions have also been conducted. These results are conflicting; however, most studies find that low vitamin D status is linked to impaired semen quality and fertility^{(Reference Heller and Clermont20,Reference Prosser and Jones71–Reference Kidroni, Har-Nir and Menczel76)} . Therefore, it seems likely that the possible effect of vitamin D supplementation on semen quality and male fertility is present under conditions with vitamin D deficiency or prolonged vitamin D insufficiency. These data are in line with animal studies showing impaired fertility and poor semen quality in males with vitamin D deficiency, which by combining suggest that vitamin D deficiency should be avoided to secure optimal reproductive function.

Systemic calcium homoeostasis

Serum calcium is maintained within a narrow physiological range to secure normal function of several organs as multiple cellular processes are influenced by calcium. Virtually all cells in the body have a 20 000-fold gradient of calcium ions (Ca²⁺) from the extracellular to the intracellular environment (about 100 nm/l-Ca²⁺ intracellularly), making them capable of using Ca²⁺ as a second messenger when entry is permitted into the cell by Ca²⁺ channels^{(Reference Clapham77)}. The total calcium concentration in serum is 2⋅20–2⋅55 mm/l and the non-protein-bound ionised calcium level is 1⋅18–1⋅32 mm/l although the exact reference levels vary among laboratories. A large fraction of calcium is protein-bound or in complex with anions such as citrate, lactate, phosphate or bicarbonate and the free and active fraction is therefore dependent on pH and availability of binding partners^{(Reference Baird78)}. To maintain serum calcium within this narrow range several potent regulators influence systemic calcium homoeostasis by modifying intestinal absorption, renal reabsorption and excretion and release from the skeletal reservoir. The calcium-sensing receptor (CaSR) in the parathyroid cells senses the Ca²⁺ concentration in serum and transduces an intracellular signal that inhibits the production and release of PTH into the bloodstream^{(Reference Brown, Gamba and Riccardi79)}. PTH binds primarily to the PTH receptor 1 and mobilises calcium to the circulation. In bone, PTH mediates calcium release by promoting bone resorption, and in the kidney, it increases calcium reabsorption and up-regulates CYP27B1 activity which ensures high vitamin D activity. In mice, the CaSR is indispensable as Casr knockout mice die shortly after birth and suffer from hypercalcaemia and hyperparathyroidism^{(Reference Ho, Conner and Pollak80)}. In human subjects, homozygous loss-of-function mutations in CASR cause neonatal severe hyperparathyroidism, a potentially life-threatening condition with severe hypercalcaemia, bone demineralisation and respiratory distress, whereas a heterozygous loss-of-function mutation in CASR causes familial hypocalciuric hypercalcaemia 1^{(Reference Boisen, Mos and Lerche-Black81)}, which shows that full compensation by other factors does not occur.

In the epithelia lining the intestine and kidney, calcium is transported from the lumen to the blood through the cell cytoplasm by the transcellular pathway or between intercellular spaces by the paracellular pathway. Expression of transcellular calcium transporters in the kidney and intestine is regulated mainly by 1,25(OH)₂D₃^{(Reference Hoenderop, Nilius and Bindels82)}. Transcellular calcium transport involves three steps: first, Ca²⁺ is transported into the cytosol by transient receptor potential vanilloid −5 or −6 (TRPV −5/−6) Ca²⁺ channels. Secondly, Ca²⁺ binds intracellularly to calbindin-D_9k (or -D_28k) to maintain a low intracellular Ca²⁺ concentration. Thirdly, Ca²⁺ is removed from the cytosol by the plasma-membrane calcium ATPase (PMCA) proteins or the Na⁺/Ca²⁺ exchanger 1^{(Reference VanHouten and Wysolmerski83)}. CaSR is expressed in the thick ascending limb of Henle in the kidney, and acute inhibition of CaSR increases the permeability of the paracellular Ca²⁺ pathway^{(Reference Loupy, Ramakrishnan and Wootla84)}. More recently, it has been shown that CaSR inhibits the paracellular Ca²⁺ transport by increasing the expression of renal claudin-14 responsible for the inhibition of reabsorption in a PTH-independent manner^{(Reference Dimke, Desai and Borovac85)}. In addition, PTH directly increases renal Ca²⁺ reabsorption by inhibiting claudin-14^{(Reference Sato, Courbebaisse and Ide86)}.

Calcium balance in the male reproductive tract

Calcium and phosphate concentrations are very different in the proximal and the distal parts of the epididymis. The concentration of calcium decreases, whereas the concentration of phosphate increases^{(Reference Clulow, Jones and Hansen87)}. These variations in calcium and phosphate levels are believed to play a crucial role in sperm maturation and the initiation of sperm motility and in keeping the sperm quiescent during storage in the distal epididymis. The high calcium concentration in the seminal fluid may be of great importance as spermatozoa are transcriptionally silent and heavily rely on intracellular calcium as a signalling system^{(Reference Blomberg Jensen, Bjerrum and Jessen88)}. A list of observational studies exploring total and ionised calcium levels in the human seminal fluid/plasma is summarised in Table 3. Based on the weighted average of the studies included in Table 3, the concentration of total calcium in the seminal fluid is about 7⋅48 mm/l, and the concentration of ionised calcium is about 0⋅23 mm/l. Citrate and phosphate concentrations are also high in the seminal fluid, ensuring competent buffer systems and making the ionised calcium concentration lower than the corresponding serum level^{(Reference Owen and Katz89–Reference N'Guessan, Yaye and Coulibaly93)}. Studies exploring the potential impact of the total calcium concentration in seminal fluid on sperm parameters are inconclusive – some suggest it is associated with increased motility^{(Reference Sørensen, Bergdahl and Hjøllund94,Reference Prien, Lox and Messer95)} , another study found an inverse relationship with sperm morphology^{(Reference Salsabili, Mehrsai and Jalaie96)} and other studies found no impact on sperm function^{(Reference Kılıç, Sarıca and Yaman97–Reference Abou-Shakra, Ward and Everard99)}. One study showed that low total calcium content in the seminal fluid (<5 mm/l) was associated with fewer progressive and total motile sperm and fewer morphologically normal sperm compared with men with calcium levels between 5 and 10 mm/l^{(Reference Boisen, Nielsen and Verlinden19)}. Regarding studies that investigated the link between ionised calcium and semen quality variables, two studies found a positive association with motility^{(Reference Prien, Lox and Messer95,Reference Kılıç, Sarıca and Yaman97)} , one showed a negative association^{(Reference Arver and Sjöberg100)} and one study found no effect^{(Reference Magnus, Åbyholm and Kofstad91)}. The possible link between total calcium/Ca²⁺ levels and sperm motility/morphology/concentration is interesting because these semen variables are predictors of male fertility potential. However, the spermatozoa are only briefly exposed to the seminal fluid before it is mixed with female fluids, which questions a direct effect of calcium content on the fertility potential. The total calcium concentration in seminal fluid is more than 2-fold higher than the level found in serum, and the levels are not associated (Fig. 1A)^{(Reference Boisen, Nielsen and Verlinden19,Reference Arver and Sjöberg100)} , hence there must be an active transport of calcium from serum into the seminal fluid. The calcium concentration in the different epididymal compartments (studies have only been conducted in rodents) varies largely from low to several-fold higher than the corresponding serum concentration (Fig. 1B)^{(Reference Jenkins, Lechene and Howards101–Reference Boisen, Hansen and Mortensen103)}. To maintain this steep gradient the mechanism and proteins underlying calcium homoeostasis must be tightly regulated. Seminal fluid comprises fluid from the epididymis (about 10 %), prostate (about 20 %) and seminal vesicles (about 70 %), which indicates that the calcium content in the ejaculate is highly dependent on secretions occurring after the transit and storage of the spermatozoa in the epididymis. At the time of ejaculation, spermatozoa escape the millimolar concentrations of citrate and phosphate in seminal fluid and encounter about 2⋅2 mm/l-total calcium and about 1⋅23 mm/l-Ca²⁺ in the follicular fluid in the female reproductive tract^{(Reference Magnus, Åbyholm and Kofstad91,Reference Boisen, Rehfeld and Mos104)} . This influences sperm function and facilitates sperm capacitation and hyperactivation that help the sperm swim up in the vicinity of the oocyte.

Table 3. Studies investigating ionised and total calcium levels in human seminal fluid/plasma

CASA, computer-assisted semen analysis; CaSR, calcium-sensing receptor; n, cohort size; N.A., not applicable (the studies did not contain a conclusion on calcium levels and sperm function).

The ionised calcium and/or total calcium concentration measured in the seminal fluid or seminal plasma. The conclusions drawn by the authors of the papers regarding the calcium content and semen parameters are mentioned in the third column. All studies are mean(sd) except for Sørensen et al.^{(Reference Sørensen, Bergdahl and Hjøllund94)}, which is given as the median. The weighted averages are calculated based on the study sizes.

Fig. 1. Calcium and vitamin D in the male reproductive tract. (A) Seminal fluid concentration relative to serum concentration of calcium, 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) and 24,25-dihydroxyvitamin D3 (24,25(OH)₂D₃) in men. Dotted lines represent the detection limit in seminal fluid, solid lines represent the constant concentration in seminal fluid and grey solid lines represent the undetectable levels in seminal fluid. The figure is based on data presented in^{(Reference Boisen, Nielsen and Verlinden19,Reference Hansen, Rehfeld and De Neergaard138)} . (B) The concentration of calcium and vitamin D in the intraluminal fluid of rodent epididymis relative to the serum concentration. In caput, the 25OHD concentration is 15× lower and 11× lower in cauda compared with serum levels. The calcium concentration is 2× higher in caput and 4× lower in cauda. The figure is based on data presented in^{(Reference Kidroni, Har-Nir and Menczel76,Reference Jenkins, Lechene and Howards101,Reference Jenkins, Lechene and Howards102)} .

Gene expression is silenced during spermatogenesis as histones are replaced by protamines that further condense DNA and prevent transcription within the small spermatozoa (about 4⋅4 × 2⋅8 μm)^{(Reference Miller and Ostermeier105,Reference Bellastella, Cooper and Battaglia106)} . Protein synthesis and processing are also hampered and therefore, spermatozoa critically depend on second messenger systems to transmit extracellular signals, and they mainly rely on changes in the intracellular calcium concentration ([Ca²⁺]_i). An increase in [Ca²⁺]_i occurs by opening Ca²⁺ channels across the cell membrane or from intracellular stores – constituting a Ca²⁺ signal. [Ca²⁺]_i regulates capacitation, hyperactivation and the acrosome reaction^{(Reference Publicover, Harper C and Barratt107)}. Regulation of the [Ca²⁺]_i ensures the activation of spermatozoa at the appropriate time. The importance is indicated by studies showing that loss-of-function mutations in the gene coding for the main Ca²⁺ channel in sperm, CatSper, lead to infertility, and spermatozoa from men in fertility treatment have lower Ca²⁺ influx in response to progesterone compared with spermatozoa from healthy men^{(Reference Kelly, Brown and Costello108,Reference Williams, Mansell and Alasmari109)} . Studies have shown that men with severely impaired semen quality and men who had unsuccessful fertility treatment have significantly lower Ca²⁺ influx upon stimulation with female secreted factors compared with spermatozoa from healthy men indicating that these Ca²⁺ signals are clinically relevant^{(Reference Kelly, Brown and Costello108,Reference Alasmari, Barratt and Publicover110)} .

Animal models with ablation of genes related to calcium homoeostasis in the testes or epididymis

Calcium homoeostasis in the male reproductive tract and its impact on male fertility has been studied in various animal models. Table 4 presents rodent models where a gene ablation has led to changes in local calcium homoeostasis in the testes or epididymis. The Cyp27b1 ^−/− mice exhibit hypocalcaemia, decreased sperm count and motility resulting in impaired fertility. Cyp27b1 ^−/− mice also have lower expression of calcium transport proteins including CaSR, CaV3⋅1 and TRPV5 in testes compared with wild-type mice^{(Reference Sun, Chen and Zhang63)}. Interestingly, a rescue diet (containing high calcium, phosphate and lactose) reestablished both the fertility and the expression of CaSR, CaV3⋅1 and TRPV5 in the testes of Cyp27b1^−/− mice^{(Reference Sun, Chen and Zhang63)}. A study in Vdr ^−/− mice also found reduced Casr expression in the testes compared with wild-type mice^{(Reference Boisen, Nielsen and Verlinden19)}, which is in accordance with the role of VDR as a regulator of calcium transporters in various organs. Previous studies have shown CaSR expression in the testes and spermatozoa from different species^{(Reference Sun, Chen and Zhang63,Reference Mendoza, Perez-Marin and Garcia-Marin111–Reference Ellinger117)} , and a proteomic study in bulls showed that the CaSR was the most differentially expressed protein when comparing good v. bad quality spermatozoa^{(Reference Singh, Sengar and Singh118)}. Moreover, treatment with different CaSR agonists increased sperm motility in rodents^{(Reference Mendoza, Perez-Marin and Garcia-Marin111)}. However, a germ cell-specific Casr knockdown model in male mice had no major reproductive phenotype compared with tamoxifen-treated controls irrespectively of the timing of Casr knockdown as conducted both pre- and post-pubertally (70 and 84 % reduction, respectively)^{(Reference Boisen, Nielsen and Verlinden19)}, which questions the importance of CaSR in spermatozoa, at least in mice. In a recent paper investigating CaSR function in human sperm, three patients with loss-of-function mutations in CASR, and one patient with a gain-of-function mutation were included. Spermatozoa from all patients had aberrant Ca²⁺ signalling. Moreover, two of the patients with loss-of-function mutations in CASR had either low sperm motility and few morphologically normal spermatozoa, or calcifications in the efferent ducts^{(Reference Boisen, Rehfeld and Mos104)}.

Table 4. Mouse knockout models that impact local calcium homoeostasis in the male reproductive tract

CaSR, calcium-sensing receptor; Ip3r1, intracellular ion channel inositol 1,4,5-triphosphate receptor 1; Itpr1/2, inositol 1,4,5-trisphosphate receptor type 1/2; KO, knockout; Mtmr14, myotubularin related protein 14; N.A., not applicable; N.D., not determined; Pmca1/4, plasma membrane Ca²⁺ ATPase 1/4; Ryr, ryanodine receptor; SERCA2, sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase; Sppl2c, signal peptide peptidase-like 2C; Trpm5/8, transient receptor potential cation channel subfamily M 5/8; Trpv5/6, transient receptor potential vanilloid sub-type 5/6; Vdr, vitamin D receptor.

↓, negatively associated; →, no association; *the low caudal sperm count after swim-out may be explained by impaired motility rather than impaired sperm production or storage.

Myotubularin-related protein 14 (MTMR14) is a phosphoinositide phosphatase and knockout of the gene results in impaired male fertility. In the Mtmr14 ^−/− mice, the mRNA expression of the calcium channels inositol 1,4,5-trisphosphate receptor type 1 and 2 (Itpr −1/−2), and ryanodine receptor 3 (Ryr3) was decreased and [Ca²⁺]_i in spermatozoa derived from epididymis was lower compared with spermatozoa from wild-type mice^{(Reference Wen, Yu and Liu119)}. Additionally, Tmem203-deficient male mice are sterile and exhibit a profound defect in spermatogenesis. mRNA expression profiling revealed down-regulation of the calcium channels Trpv6 and transient receptor potential cation channel subfamily M members 5 and 8 (Trpm −5/−8), and an increase in Pmca1 and intracellular ion channel inositol 1,4,5-triphosphate receptor (Ip3r1) in testes. Testicular cells from Tmem203 mice also exhibited altered calcium mobilisation^{(Reference Shambharkar, Bittinger and Latario120)}. Signal peptide peptidase-like 2C (Sppl2c) deficiency in male mice leads to dysregulation of the calcium pump sarcoendoplasmic reticulum calcium ATPase (SERCA2) involved in intracellular Ca²⁺ handling in male germ cells. The disturbed calcium homoeostasis resulted in impaired motility of spermatozoa but preserved fertility^{(Reference Niemeyer, Mentrup and Heidasch121)}. Efflux of intracellular Ca²⁺ can be mediated by the PMCA pump, which is essential for spermatozoa as Pmca4 ^−/− mice are infertile due to Ca²⁺ overload and inability of spermatozoa to undergo capacitation^{(Reference Okunade, Miller and Pyne122,Reference Prasad, Okunade and Miller123)} .

The epididymis requires active calcium transport to maintain the calcium concentration gradient (Fig. 1B), which is necessary to ensure normal sperm function. In cauda epididymis in global Trpv6 knockout mice a 10-fold higher calcium concentration impaired sperm motility^{(Reference Weissgerber, Kriebs and Tsvilovskyy124)}. Ma et al.^{(Reference Ma, Zhang and Liu125)} found an excessive calcium accumulation in the epididymis of vitamin K2-deficiency rats due to dysregulation of γ-glutamyl carboxylase (GGCX) and matrix gla protein (MGP). The vitamin K2-deficiency rats had lower sperm count and sperm motility. In agreement with the results in rats, the study also identified an SNP mutation in GGCX in an infertile man with asthenozoospermia, indicating that the influence of GGCX and MGP on fertility is conserved between species.

Human relevance of calcium in male fertility

The link between calcium, vitamin D, bone and gonadal function^{(Reference Oury, Ferron and Huizhen126–Reference Bøllehuus Hansen, Kaludjerovic and Nielsen129)} complicates interpretations of human and animal studies as many of the effects of calcium may be indirect. In most human studies on vitamin D and male fertility, potential calcium-mediated effects have been neglected. In a study of infertile men, serum Ca²⁺ levels were negatively associated with sperm motility indicating that Ca²⁺ may influence semen quality in infertile men^{(Reference Blomberg Jensen, Gerner Lawaetz and Andersson10)}. This association suggests that systemic regulators of calcium homoeostasis may also influence local calcium transport in the male reproductive tract, as calcium levels in serum and calcium levels in the epididymis and seminal fluid are not associated (Fig. 1A).

Significant interspecies differences exist in fertility and sperm function. Hence, extrapolating results from mice to human subjects in the fertility field is problematic and should be performed with caution^{(Reference Kaupp and Strünker130)}. Depending on the species, fertilisation occurs under entirely different aqueous conditions with different calcium and vitamin D levels, which remains to be thoroughly studied in vivo. Most data have been generated by in vitro studies and the true levels of calcium surrounding the spermatozoa as it moves through the female reproductive tract to the oocyte and in the moment of fertilisation are largely unknown.