Esomeprazole reduces sperm motility index by targeting the spermic cholinergic machinery: A mechanistic study for the association between use of proton pump inhibitors and reduced sperm motility index

Recent studies have linked prolonged use of the most commonly prescribed proton pump inhibitors (PPIs) with declined human sperm function and infertility. Here, we report for the first time the most plausible underlying mechanism for this unwarranted secondary mode of action. We followed up on a recent serendipitous discovery in our laboratory regarding PPIs’ off-target action and performed detailed pharmacodynamic analyses by combining in silico and in vitro studies to determine the off-target effect of one of the most commonly used PPI, esomeprazole, on the key human acetylcholine biosynthesizing enzyme, choline acetyltransferase (ChAT; EC 2.3.1.6). A pivotal enzyme in the spermic cholinergic system that governs the sperm motility, concentration and quality. Our results were conclusive and showed that both the racemic form, omeprazole and its pure S-en-antiomer, esomeprazole, acted as potent mixed-competitive inhibitor of human ChAT with a global inhibition constant ( K i ) of 88 nM (95%CI: 10–167 nM) for esomeprazole and 178 nM (95%CI: 140–230 nM) for the racemic drug omeprazole. Most importantly, esomeprazole substantially reduces both total number of motile sperm (by 36%, p < 0.001; and 21% p < 0.0001, at 10 and 100 nM, respectively) as well as the total number of sperm with progressive motility (by 42% p < 0.0016 and by 26% p < 0.0001, respectively) after 60 min relative to 20 min incubation in our ex vivo functional assay performed on ejaculated human sperm. In conclusion, this study presents a completely new perspective regarding PPIs secondary mode of action/unwarranted side effects and calls for further mechanistic and larger clinical studies to elucidate the role of PPIs in infertility.


Introduction
The cholinergic signaling system is believed to have been developed during very early evolutionary stages of life as the key classical neurotransmitter acetylcholine (ACh) has been observed in all forms of life and widely synthesized and used by neuronal non-neuronal cells [1].The expression and activity of ACh-biosynthesizing enzyme, choline acetyltransferase (ChAT) (EC: 2.3.1.6;Choline O-acetyltransferase) defines cholinergic neurons/cells.Whereas, the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which break down ACh and abolish neurotransmission, are expressed by the downstream cholinoceptive cells/neurons as well as in soluble forms in whole blood, plasma, cerebrospinal fluids and interstitial fluids [2].Acetylcholine impart its signaling through activation of two main classes of receptors.The nicotinic receptors are ion-channels while the muscarinic receptors are metabotropic G-protein coupled receptors, mediating activation of intracellular second messenger signaling.
The role of the central cholinergic system in cognition is well-established [3] and its early and selective degeneration is considered one of the hallmarks of Alzheimer's disease, Lewy body dementias (DLB, including dementia with Lewy body and Parkinson's dementia) and Down's syndrome [4][5][6][7].
In addition, extensive cholinergic neuronal projections existing throughout the body, partly originated from 12 cranial nerves (CN I-XII), which form the parasympathetic system [8], controlling diverse organs, muscles, and glands.Another major cholinergic neuronal circuitry is the enteric nervous system (ENS) responsible for controlling the motility (muscular) and secretory (mucosal) reflexes within the gastrointestinal tracts [9].It is estimated that over 60% of neurons in this autonomic neuronal system are cholinergic [10], which seems to be affected in the course of many neurodegenerative diseases [11].
However, there are also two other non-neuronal cholinergic systems.Firstly, several different immune-competent cells have all of the necessary cholinergic machinery.Substantial evidence indicates that this system cross-talks with the brain through cranial nerves, in particular Vagus nerve (or CN-X), together building up the so called cholinergic anti-inflammatory pathway [12].
The second non-neuronal system that is relatively less known concerns spermatozoa.Similar to immune cells, spermatozoa possess most of the cholinergic machinery, including ChAT.For a comprehensive review on spermic cholinergic system see [13].Briefly, reports indicate that ChAT is present both intra-and extracellularly in spermatozoa [14].In this system, the synthesized ACh acts as an autocrine chemotactic agent that regulates the sperm motility and acrosome reaction initiation process via activation of nicotinic ACh receptors [13,[15][16][17].In other words, the spermic cholinergic machinery controls and regulates the sperm motility and is crucial for biological processes of fertilization/reproduction [13].
Proton pump inhibitors (PPIs) are a widely used class of drugs against a variety of gastroesophageal reflux disorders (GERD), such as gastric and duodenal ulcer, and hyperacidity conditions, such as Zollinger-Ellison syndrome [18,19].As the class name implies, their primary pharmacodynamics mode of action, exerted through an irreversible inhibition of the H + K + -ATPase (proton) pumps in parietal cells of the stomach, resulting in long-lasting reduction in the production and secretion of gastric acid.The most commonly used PPIs are omeprazole/esomeprazole, lansoprazole, and pantoprazole.Omeprazole was the first PPI, introduced in 1989 for the clinical use.Nowadays, in its S-enantiomeric form, esomeprazole is used more extensively relative to the original racemic formulation, omeprazole, due to claimed higher oral-bioavailability and more favorable pharmacokinetic profile [20].Several recent studies have shown that esomeprazole is well tolerated [21][22][23][24], and used together with two antibiotics for the eradication of H. Pylori infection in patients with duodenal ulcers [25][26][27].
A recent serendipitous discovery in our laboratory on a secondary mode of action and target of PPIs brought our attention to a plausible mechanism that seems to link the undesirable effect of prolonged use of PPIs on the quality, motility, and concentration of sperm [34][35][36], with the crucial role of spermic cholinergic system [17].In the course of a combined in silico and in vitro approach, we found that both lansoprazole and omeprazole/esomeprazole can interact and inhibit the activity of the core ACh biosynthesizing enzyme, ChAT [37].
We hypothesized that PPIs could impair the motility of sperm by directly inhibiting the activity of ChAT, thereby affecting the de novo synthesis of ACh, a required chemotactic agent for sperm motility.To challenge this hypothesis we performed detailed in silico and in vitro enzyme kinetic analyses, followed by an ex vivo functional study using Computer-assisted sperm analysis (CASA) system [38,39], to assess motility index of sperm in human ejaculate.In this paper, we selected esomeprazole to test our hypothesis and explore the possible molecular mechanism underlying the unwanted secondary effect of PPIs on sperm motility.We chose esomeprazole because our initial in vitro studies indicated that esomeprazole was several folds more potent than lansoprazole with regard to inhibition of ChAT activity, and esomeprazole is a more used PPI.

In silico analyses
In silico docking analyses was performed on all FDA-approved PPI drugs identified after initial virtual screening step to elucidate their mode of binding in the active sites of hChAT protein.The crystal structure of hChAT (PDB ID: 2FY3) [40] was downloaded from PDB database and a 3D structure of hChAT was prepared by addition of hydrogens, repairing side chain, treating termini, fixing atom type, setting protonation state, fixing bond order, adding charge, and fixing side chain amide.The prepared structure was minimized in order to remove the strain produced during the earlier protein preparation steps.The defined binding pocket, i.e. a 'Protomol' was generated using the co-crystallized ligand in the active site of hChAT.The chemical structure of the drugs were sketched covering both R and S stereoisomers and converted into 3D conformation.Finally, the prepared dataset of compounds were docked into the active site of hChAT using Surflex-Dock GeomX (SFXC) module interfaced in SYBYL-X 2.1.1 [41] and the compounds were ranked using Total_Score (−logK d ).

Purification of recombinant ChAT
The recombinant ChAT protein was purified at the Protein Science Facility (PSF), Karolinska Institutet, Stockholm, Sweden, as described previously.Briefly, DYT media (16 g/l Tryptone, 10 g/l yeast extract, 5 g/l NaCl, 100 µg/ml ampicillin, 34 µg/ml chloramphenicol) was inoculated with a preculture of E. coli BL21 Rosetta2 transformed with pProEXHTa-ChAT (a gift from Dr. Brian Shilton, Department of Biochemistry, University of Western Ontario, London, Canada).The bacteria were grown in a shaking incubator at 37 °C with 200 rpm until the optical density at 600 nm reached 0.5.After which, 0.5 mM IPTG was added and His 6 -ChAT was expressed for circa 16 h at 18 °C.The bacteria were harvested and stored at −80 °C.
His 6 -ChAT was purified with "Ni-NTA fast start Kit" (Qiagen) following the manufacturer's instructions.The elution buffer was exchanged to storage buffer (10 mM Tris pH 7.4, 500 mM NaCl, 10% (v/ v) glycerol) using 30 kDa molecular weight cutoff Amicon Ultra concentrators (Merck Millipore).The protein was purified at the Protein Science Facility (PSF), Karolinska Institutet.The purified protein was aliquoted, and stored at −80 °C.The purity and molecular weight of the protein was determined using SDS-PAGE electrophoresis.The total protein concentration was measured with DC Protein Assay (BioRad).
Our ChAT assay is optimized to run as high-throughput assay and could be run in either 96-well or 384-well plates.For 96-well plates, 50 μL/well of 75 ng (final concentration) of the recombinant hChAT was incubated with 100 μM esomeprazole (50 μL/well) for 10-30 min at room temperature in dilution buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.0 mM EDTA, 0.05% (v/v) Triton X-100).Then, 50 µL of a cocktail-A [dilution buffer containing choline chloride (final concentration 150 µM), ACoA (final concentration 13.3 µM) and CPM (final concentration 15 µM)] was added to each well.Immediately after adding the cocktail-A, the changes in fluorescence was monitored kinetically at 479 nm after exciting at 390 nm at 1-2 min intervals for 15-20 min using a microplate spectrophotometer reader (Infinite M1000, Tecan).
Esomeprazole was run in triplicates.On each 96-wells plate, several enzyme wells without esomeprazole were also included during measurements as control and for estimating the inhibition level.Negative controls were wells without enzyme.The percentage inhibition was calculated based on the enzyme control value as a reference (100% activity).

Kinetic studies and in vitro estimation of IC 50 and mode of action of esomeprazole
For kinetic studies, a similar protocol as inhibition assay was followed; a dilution series of different concentrations ranging from 0 to 20 μM were prepared for esomeprazole.ChAT was pre-incubated with specified concentrations of esomeprazole and different substrate concentrations ranging from 10 to 320 µM at room temperature for 10-30 min.After incubation period, the activity of ChAT was measured in real-time using the ChAT fluorescence assay as described above.The effect of different concentrations of esomeprazole on ChAT at varied substrate concentrations was compared to the activity of a ChAT protein in presence of DMSO (Vehicle-control).
In these enzyme kinetic analyses, the A-CoA concentration was kept constant at 10 μM (final) but the concentration of choline chloride was varied between 10 and 320 μM.Each concentration was measured in duplicates.The rate of enzyme activity (as ΔFU/min) was calculated and processed using the GraphPad Prism 8 analysis software [48].The same method was also followed for omeprazole inhibition assay.The half-maximal inhibitory concentration (IC 50 ) values were calculated by plotting the percentage enzyme activity vs. the log of the esomeprazole and omeprazole concentrations at a single substrate concentration of 160 μM and fitting the data using the nonlinear regression Enzyme Kinetics-Inhibition function.
The Michaelis-Menten constant (K m ) and maximal velocity (V max ) values were calculated from substrate-velocity curve after fitting the data with non-linear regression Michaelis-Menten kinetic function.The kinetic data analyses and curve fitting was performed using GraphPad Prism 8 software [48].

Collection of human ejaculates
Ejaculates from men aged between 27 and 35 years attending the ANOVA Centre for infertility investigation were used after clinical routine examinations were handled.ANOVA has ethical approval for using anonymized aliquots of ejaculates after clinical routine examination for research, development, quality control, training and education (Swedish Ethical Review Authority Dnr 2015/2326-31, Dnr 2019-02466).Each man was given instructions regarding the semen collection.
All subjects were referred to ANOVA for semen analysis (usually due to couple subfertility).All the ejaculates that were used in the current study were from men who did not require clinical andrology work-up due to normal routine semen analysis outcome, i.e. normal total number of spermatozoa and motility (based on WHO 1999 four category: rapid, slow, non-progressive, and immotile) [49].
Ejaculates were collected after 2-5 days of sexual abstinence and stored at 37° C immediately after collection for liquefaction.After liquefaction, an experienced biomedical technician examined an aliquot under the phase contrast microscope and estimated the content of motile spermatozoa.Ejaculate volume was determined by weight according to the WHO guidelines (2010) [50].The samples that were deemed to contain sufficient live, motile spermatozoa were selected for experiments and further analysis with Computer-Assisted Sperm Analysis (CASA).The ex vivo experiment was designed so that every sample constituted its own control.All samples were used without patient data.

Measurement of human sperm motility using Computer-Assisted sperm analysis (CASA)
Sperm motility analysis for each semen sample was performed as soon as practically possible after liquefaction at room temperature using CASA tool [38,39].Briefly, the liquefied semen sample was mixed with equal volume of filtered sterilized phosphate buffered saline (PBS, 50 mM Na + /K + -phosphate buffer, 150 mM NaCl, pH 7.4) and quickly analyzed for the sperm motility before incubation with esomeprazole.
The in vivo concentration of esomeprazole in testes and or in ejaculate is unknown we therefore used information available for the brain since reports indicate the presence of a blood-testis barrier (or bloodsertoli cell barrier) similar to the brain.This may suggest a reduced tissue distribution of esomeprazole in testes, which in turn may limit the in vivo testes concentration of esomeprazole relative to its plasma concentration (which for PPIs is in 1-20 µM ranges).The brain concentration of PPI is expected to be about 10-15% of their plasma concentration [33].We hence chose two esomeprazole concentration in the ranges that are expected to be present in the brain, namely 10 nM and 100 nM of esomeprazole concentrations.Each semen sample was incubated with 10 and 100 nM concentrations of esomeprazole and stored at room temperature (ranging between 21 and 23 °C).
Esomeprazole is not readily water soluble, so we prepared stock solution in DMSO, which was then used to prepare working solutions in PBS buffer.To account for a possible effect of DMSO as well as the buffer, aliquots of the semen sample were also incubated with buffer alone (Buffer-Control) and buffer containing equal amount of DMSO corresponding to highest concentration of esomeprazole (100 nM) as Vehicle-Control.The sperm motility was measured at 20-and 60-min time points by loading 6 µL of sample in 20-µm Leja disposable slides (Leja Products B.V., The Netherlands) with two counting chambers and monitoring the slides at 10x magnification under Nikon microscope (Eclipse 50i with 10 × phase contrast objective and intermediate TV relay lens of × 0.5 for IDS uEye camera -UI-1540LE-M-HQ) connected to a V3.0.9.486 (https://www.akymed.com/)CASA-QualiSperm™ instrument [51].
While performing human sperm analysis, we checked and followed all the standard requirements as described by Björndahl et al. [52].When performing the analyses the fresh ejaculate were prepared and kept at room temperature with a fluctuation range between 21 and 23 °C.Nonetheless, to avoid long incubation time at room temperature which could affect the motility assessment, the whole procedure was completed within 2 h of collection of each ejaculate.These ex vivo experiments had repeated measured design so that each sample could serve as its own control when comparing the changes at 60 min relative to 20 min ex vivo incubation.Four independent experiments were performed using ejaculates collected from four different subjects on four different days.For each time point, samples were analyzed in duplicate and at least 10 microscopic fields were examined for each count.The data sheet containing the following four-category scheme defined by Barratt et. al., 2011[53] for sperm motility assessment: rapid progressive, fast progressive, slow progressive, and immotile, was exported from QualiSperm™ analysis software and was further analyzed using StatView 5.0 software.The data is presented as mean ± SEM.

Statistical analysis
The relative effect of different concentrations of esomeprazole, DMSO and dilution buffer on human sperm motility was statistically examined by using One-way ANOVA analysis.The changes in sperm motility/progressive parameters caused by esomeprazole or DMSO were compared with buffered control sample (no esomeprazole or DMSO).The changes in sperm motility index as a function of incubation time was assessed by Repeated measured ANOVA.An ANOVA p-value of < 0.05 was considered significant, and followed by Fisher's least significant difference (LSD) post-hoc test to compare each esomeprazole concentrations (10 nM and 100 nM) with DMSO sample (Vehicle-control) or buffered sample (Buffer-control), as well as with incubation times, T0 (within five minutes after addition of additive to the samples), and after 20 (T20) and 60 min (T60) of incubation..

In silico analyses revealed the possible binding site of esomeprazole on human ChAT
Virtual in silico screening analyses of PPIs estimated a high total binding score (-log Kd) of 9.37 for esomeprazole, which was slightly lower than the estimated value for the R-enantiomer of rabeprazole (a -log Kd value of 9.902 that was the highest binding score among all PPIs) [33].Given that esomeprazole is used more commonly than rabeprazole, we selected esomeprazole and performed advanced in silico docking analyses to determine its possible binding site/pocket and interacting residues on ChAT enzyme (Fig. 1).Human ChAT catalyzes both forward and backward reactions involved in biosynthesis of ACh.In other words, ChAT is an enzyme capable of both synthesizing and degrading ACh.The synthesis of ACh mainly takes place inside the catalytic tunnel, which have two putative entrances, one for the cofactor, acetyl-CoA (A-CoA) or CoA (represented by yellow circle; Fig. 1) and another for the ACh/choline.Our analyses indicated that the binding pocket for esomeprazole (highlighted by red color; Fig. 1

Esomeprazole significantly inhibited human ChAT activity in vitro
As the in silico docking analyses confirmed a direct interaction between esomeprazole and ChAT, we further explored the implication of this direct interaction in vitro using an in-house developed real-time kinetic ChAT fluorescence assay.First, we studied the effect of esomeprazole at a single concentration of 100 µM to determine whether the binding of this drug alters the activity of ChAT.Surprisingly, this single concentration analysis revealed that esomeprazole was able to completely (> 99%) inhibit the activity of ChAT.Next, we performed detailed dose-response analyses at several different concentrations of esomeprazole (0-20 µM) and the substrate choline (10-320 µM, Fig. 2).The dose-response analyses demonstrated that esomeprazole reduced ChAT activity by ~78 and 92% at concentrations of 4 and 20 µM, respectively (Fig. 2A).The analyses also indicated the inhibition of ChAT at a given esomeprazole concentration (in particular in 0.8-0.05µM ranges) was significantly affected by the substrate (choline) concentration (Fig. 2B).For instance, 0.8 µM of esomeprazole inhibited ChAT by ~20% at choline concentration of 160-320 µM, while the same esomeprazole concentration inhibited ChAT by ~70% at 10-20 µM choline concentrations (Fig. 2B).These findings indicated that esomeprazole might behave as a competitive inhibitor of human ChAT with respect to the substrate, choline.This is an important finding as it indicates that choline and esomeprazole might compete with each other for the binding site in the enzyme, thereby suggesting that high endogenous concentrations of choline may be able to counteract the inhibition of ChAT by esomeprazole as well as that the binding of esomeprazole to ChAT is reversible.

Determination of kinetic parameters (K m and V max ), mode of inhibition and half-maximal inhibitory concentration value (IC 50 )
To fully evaluate and confirm the type of binding of esomeprazole to ChAT enzyme and the nature of its inhibitory mode of action we performed enzyme kinetic analyses.These analyses were done by non- linear regression analysis of GraphPad Prism 8.0 software, which determined the kinetic parameters such as K m and V max values (Fig. 3A and Table 1).The K m and V max values define enzyme catalytic behavior as a function of substrate concentration (here choline).Esomeprazole significantly affected both V max and K m values with increasing concentrations (Table 1).V max was reduced from 58.7 µmol/min (in the absence of esomeprazole) to 15.3 µmol/min at 4 µM esomeprazole, whereas K m value was increased from 16.8 µM (at 0.0 µM esomeprazole) to 98.0 µM in the presence of 4 µM esomeprazole, further reinforcing our observation that esomeprazole is a competitive inhibitor of human ChAT.
Next, we estimated the half-maximal inhibitory concentration value (IC 50 ) for the hChAT-esomeprazole interaction.IC 50 values indicate the potency of an enzyme inhibitor by quantifying inhibitor concentration required to reduce the rate of an enzyme-catalyzed reaction by 50%.

Fig. 2. Esomeprazole significantly inhibited ChAT activity in vitro.
A shows the activity inhibition curves of ChAT at different concentrations of esomeprazole and the substrate choline.Esomeprazole inhibited ChAT activity by ~92% inhibition at 20 µM concentration as compared to vehicle-control (no inhibitor).B is bar chart graph illustration of inhibition of ChAT activity by different concentrations of esomeprazole at various concentration ranges of the substrate, choline.This analysis reveals an inverse relationship between the degree of ChAT inhibition and the substrate concentration at a given concentration of esomeprazole.For example, at 0.8 µM esomeprazole much higher ChAT inhibition is seen at 10-20 µM range of choline concentrations than at higher substrate concentrations, in particular 160-320.This suggest that the inhibitor and the substrate may compete with each other.The ChAT activity inhibition curves were fitted using the non-linear regression analysis function of GraphPad Prism 8 software.The values are shown as mean of one representative independent experiment out of the several performed in duplicate.Fig. 3. Esomeprazole is a much potent reversible mixed-competitive inhibitor of hChAT than parent, omeprazole.(A) The ChAT activity dose-response curve for estimation of IC 50 value for omeprazole (racemic mixture) and its s-enantiomer, esomeprazole at a single substrate concentration of 160 μM.The IC 50 value was estimated after fitting the curves using nonlinear regression function of GraphPad Prism 8. (B) Enzyme-inhibition kinetic analysis for estimation of the global inhibition constant (K i ) for esomeprazole as ChAT inhibitor at concentration range of 0-320 µM of the substrate, choline, using nonlinear regression analyses in GraphPad.Mixed model equation analyses was used to determine the mode of activity of esomeprazole as ChAT inhibitor.The model indicated with 99% probability that esomeprazole behaved as a mixed-competitive inhibitor as was compared to competitive, noncompetitive and uncompetitive modes of activities.The values are shown as mean of one representative independent experiment out of the several performed in duplicate.

Table 1
The K m and V max values for Esomeprazole at different concentrations.The IC 50 values were determined for both omeprazole and esomeprazole at a single choline concentration of 160 µM.Esomeprazole showed an IC 50 value of ~53 nM, which was almost 3 times lower than the IC 50 value of omeprazole (~153 nM; Fig. 3B).Unlike IC 50 value, K i is independent on substrate concentrations and hence provide a universal measure of potency of an inhibitor.Non-linear regression analyses suggested a global K i of 88 nM (95%CI: 9.6-167 nM) for esomeprazole but a K i of 178 nM (95%CI: 140-230 nM) for the racemic drug omeprazole.Enzyme-inhibition kinetic analyses using a mixed-model equation (in GraphPad software) in addition established (with 99% probability) that both omeprazole and esomeprazole behaved as mixedcompetitive ChAT inhibitor.In simple terms, a mixed-competitive behavior indicates that esomeprazole shows some preference to bind to the free enzyme (like a competitive ligand) but also binds to the enzyme-substrate complex like a noncompetitive ligand.Overall, the results indicated that esomeprazole is about twice as potent inhibitor of human ChAT than the parent racemic mixture formulation of omeprazole.

Ex vivo treatment of human spermatozoa by esomeprazole significantly affected sperm motility
Finally, to provide more direct biological evidence and challenge our hypothesis, we studied ex vivo the effect of esomeprazole on human sperm motility using CASA system.Therefore, we used two esomeprazole concentrations (namely 10 nM and 100 nM) that are < 10% of the expected concentration of PPIs in plasma.These concentrations can be safely considered as expected physiologically concentrations of esomeprazole reaching testes tissue.
The ex vivo effect of esomeprazole on human sperm motility was monitored immediately (within five minutes after addition of esomeprazole to the sample) as well as after 20 and 60 min of incubation.In this experiment, the measured average number of motile cells were 15.4 and 17.6 mil/ml at 20 and 60 mins, respectively.The results following 20 and 60 min ex vivo incubation are shown in Figs.4-6.
Repeated-measured ANOVA analyses showed statistically significant difference between groups (Buffer-control, DMSO-control and 10 nM and 100 nM esomeprazole samples; p < 0.014), incubation times (20 vs 60 min; p < 0.0001) as well as groups-incubation time interaction (p < 0.0015) for the total number of motile sperm.
DMSO-treated controls did not show any significant changes on the total motile sperm (Fig. 4A, p > 0.5) and number of sperm with progressive motility after 60 mins (Fig. 4B, p > 0.6) when compared with 20 min ex vivo incubation.Moreover, there was no statistically significant differences between the buffer-treated and DMSO-treated control samples (Fig. 4A and B; p = 0.20 and p = 0.14) with regards to the total number of motile sperm and the number of sperm with progressive motility, respectively.
Nonetheless, both 10 nM and 100 nM ex vivo concentrations of esomeprazole substantially reduced the total number of motile sperm (by 36%, p < 0.0011; and 21% p < 0.0001, respectively), as well as the total number of sperm with progressive motility (by 42% p < 0.0016 and by 26% p < 0.0001, respectively) after 60 min ex vivo incubation with sperm samples as compared to 20 min incubation (Fig. 4A and B).
We further analyzed the data to observe the effect of esomeprazole on sperm progressive motility parameters.These parameters represent the swimming behavior/movement of individual sperm and indicate whether they are moving in straight line slowly or fast (i.e.Slow-and Fast-progressive, respectively).The motile but non-moving sperm (e.g.circle movement) are termed as non-progressive.In these respects, repeated-measure ANOVA analyses showed a statistically significant difference with regards to incubation times (20 and 60 min; p-values 0.0017 and 0.0007 for slow-and fast-progressive motile sperm, respectively), and the interaction between group and incubation time (pvalues 0.021 and 0.024 for slow-and fast-progressive motile sperm, respectively).
Further analyses indicated that esomeprazole at 10 nM concentration considerably reduced the number of slow-and fast-progressive motile sperm after 60 min as compared to 20 min ex vivo incubation time (52% p < 0.0076 and 67% p < 0.0051, respectively, Fig. 5A and  B).Similarly, esomeprazole at 100 nM concentration reduced the number of slow-and fast-progressive motile cells after 60 min as compared to 20 min (32% p < 0.0001 and 34% p < 0.0047, respectively, Fig. 5A and B).
The reduction was also significant when compared with buffertreated control samples, for 10 nM esomeprazole concentration (by 42% p < 0.0026, and by 39% p < 0.0017) and the 100 nM concentration (by 44%, p < 0.0016 and 35% p < 0.012) on the number of slow-and fast-progressive sperm, respectively.Most importantly, DMSO-treated samples did not show any statistically significant difference with the buffer-treated samples, with regards to number of slow-and fast-progressive motile sperm (p > 0.2 and p > 0.15, respectively).
Esomeprazole at 10 nM and 100 nM, and DMSO-and buffer-treated control samples did not show any significant effect on the number of non-progressive motile sperm (Fig. 6).Fig. 4. Ex vivo incubation of human sperm with esomeprazole at expected in vivo physiological concentrations in testes decreases the total number of motile spermatozoa.A illustrates the changes in the total number of motile sperm in presence of 10 and 100 nM esomeprazole as well as DMSO (vehicle control) and dilution buffer (Control) as function of incubation time with the additives.B illustrates the corresponding changes in the total number of progressive sperm in the ejaculates.The ex vivo effect of the additives was monitored with CASA system after 20 and 60 min on fresh human sperm.For each time point, samples were analyzed in duplicate and at least 10 microscopic fields were examined for each count.*, ** and *** represents p-value < 0.05, 0.01 and 0.001, respectively.The lines with arrowheads defines the comparisons.

Discussion
In the present study, we followed up a recent serendipitous discovery in our laboratory concerning an unprecedented secondary mode of action of PPIs, namely inhibition of the key cholinergic enzyme ChAT.A prolonged use of PPIs has been shown to affect male fertility and semen quality [34][35][36].Sperm have cholinergic machinery and use ACh as a chemotactic agent [13].We hypothesized that PPIs affect male fertility by inhibiting the spermic ACh-biosynthesizing enzyme, ChAT.We first performed detail ChAT kinetic analyses on esomeprazole as our preliminary analyses had indicated that esomeprazole was about 15fold more potent ChAT inhibitor than lansoprazole.These in silico and in vitro enzyme kinetic analyses conclusively confirmed that indeed PPIs, in particular esomeprazole, inhibit ChAT activity with high potencies [33].Here, we showed that esomeprazole inhibited ChAT with a K i value ranging between 10 and 166 nM.The analyses also revealed that this drug behaves as a mixed-competitive reversible inhibitor of human ChAT, with regard to choline as one of the substrates of the enzyme.
We then challenged our hypothesis by setting up an ex vivo functional cholinergic assay using computer-assisted sperm motility analysis (CASA) system.The principle of the assay is to use freshly ejaculated spermatozoa and assess the motility of the sperm using the CASA system [54].The rationale is based on the understanding that human spermatozoa use ACh-signaling as a chemotactic agent for flagellar movement of the tail (swimming) [14,17,55,56].Thus inhibition of sperm ChAT by esomeprazole is expected to cause a net reduction in production of ACh, thereby affecting the motility of sperm.We found that esomeprazole at low concentrations of 10-100 nM significantly reduced the overall number of motile sperm as well as the total number of progressive (both slow and fast) sperm.The results of this ex vivo experiment strongly supported our mechanistic hypothesis that inhibition of ChAT spermatozoa may account for the link between PPIs and male infertility.
Thereby our findings further reinforce the recent reports of Huijgen et al. [35,36] and Banihani et al. [34] indicating an association between long-term PPIs usage and male infertility due to poor sperm quality and motility.Huijgen and colleagues performed a patient-based study in Dutch men and observed two-to three-fold higher risk of lower total motile sperm count in patient using PPIs between 6 and 12 months prior to semen analysis [35,36].Surprisingly, they didn't observe any significant effect between 0 and 6 months or when they combined the two time periods in a window of 1 year prior to semen analysis.They suggested that the effect of PPIs could be due to increased gastric pH resulting in poor absorption and depletion of vitamin B 12 and other micronutrients resources needed for sperm function over time (4-6 months) [36].In contrast, a large patient based retrospective study by Keihani et al. contradicted these findings and reported that PPIs didn't affect semen quality in subfertile males [57].Banihani et al. specifically focused on lansoprazole and studied its effect in vitro on human sperm motility, viability, nitric oxide production as well as on semen calcium chelation capacity.They showed that lansoprazole decreased both total and progressive motility, but not viability of human sperm.The authors suggested lansoprazole may exerted this effect by altering calcium chelating capacity of semen or by reducing Na + /K + -ATPase activity [34] but they didn't observe any changes in nitric oxide production.
Up-to-date, none of these studies suggested any possible link between PPIs and sperm cholinergic machinery, which based on the hindsight provided by the current results, is one of the most plausible possible underlying mechanisms.At the time of the previous studies [35,36], the authors had no knowledge of PPI acting as inhibitor of sperm ChAT, as we are reporting this for the first time.Nonetheless, the effect of various cholinergic agents on human spermatozoa motility has been studied [13,16].Reports indicate that ACh, pilocarpine as well as physostigmine and neostigmine (inhibitors of ACh-degrading enzyme, AChE) increase the motility of human spermatozoa [16].Studies by Fig. 5. Ex vivo incubation of human sperm with esomeprazole at expected in vivo physiological concentrations in testes decreases the number of Slow-and Fast-progressive spermatozoa.The ex vivo effect of esomeprazole (0.01 and 0.1 µM), DMSO (vehicle control) and dilution buffer (Control) on the total number of sperm with Slow (A) and Fast (B) progressive motility was monitored with CASA system after 20 and 60 min on fresh human ejaculates.For each time point, samples were analyzed in duplicate and at least 10 microscopic fields were examined for each count.*, ** and *** represents p-value < 0.05, 0.01 and 0.001, respectively.The lines with arrowheads defines the comparisons.Fig. 6.Ex vivo incubation of human sperm with esomeprazole or vehicle controls do not affect the number of non-progressive spermatozoa.The ex vivo effect of esomeprazole (0.01 and 0.1 µM), DMSO (vehicle control) and dilution buffer (Control) on the total number of sperm with non-progressive motility was monitored with CASA system after 20 and 60 min on fresh human sperm.For each time point, samples were analyzed in duplicate and at least 10 microscopic fields were examined for each count.
ACh receptors antagonists have shown that muscarinic antagonists (atropine and hyoscine) do not greatly influence spermatozoa motility [13,16].In contrast, nicotinic antagonists (hexamethonium, dtubocurarine, and succinylcholine) significantly inhibit the motility of spermatozoa [13,16].Thus, one or more subtypes of nicotinic ACh receptors are the key mediators in these contexts.Intriguingly, it has been shown that ACh affect the levels of calcium in sperm [15], which may indicate that α7nAChR subtype may be involved.
The effect of ChAT inhibitors has also been examined.Studies in rats and rabbits have shown that ChAT is present in various segments of epididymis, with the highest levels found in distal cauda [13], indicating that high ChAT is crucial for fully matured sperm.In addition, both ChAT and AChE can be extracted from various part of spermatozoa (i.e.head, midpiece and tail), where the specific activities of these two enzymes in the tail seem to be five times higher than those measured in the head or midpiece sections [13].Nonetheless, the earliest and strongest evidence for the crucial role of spermic cholinergic system in motility of sperm comes from the direct effect of one of the classic inhibitors of ChAT, known as BETA (2-benzoylethyltrimetylammonium).BETA exhibits an IC 50 of 1.0 µM for ChAT extracted from rat spermatozoa.It is shown that BETA at this concentration decreases the motility index of sperm by 95% after 1 h [13].Given that our current and previous in silico and in vitro enzyme kinetic analyses on both omeprazole, lansoprazole and esomeprazole conclusively show that these PPIs are more potent ChAT inhibitors than BETA [33], a direct inhibition of ChAT by PPIs is indeed the most plausible underlying mechanism for the PPIs depression of sperm motility.
Our hypothesis and the current findings have important clinical implications.Over the last few years, PPIs have been overly used throughout the world, most likely because of easy availability, acceptable cost and a perceived high safety-index [58][59][60].For example, in US alone the use of PPIs was increased from 4% in 2002 to 9.2% in 2009 [60].Shockingly, a study shows that ~63% of the patients, who used PPIs had no gastrointestinal complaints, gastrointestinal diagnoses, or other indicated reason for their use [60].In 2010, FDA issued an advisory warning against long-term use of PPIs as several experimental and clinical studies implicated the use of PPIs with increased risk of hip fracture, vitamin B12 deficiency, hypergastrinemia, clostridium difficile infections, and dementia [28][29][30][31][32]. Intriguingly, cholinergic dysfunction is also associated with an increased risk of fall and dementia [61][62][63][64][65][66][67][68], indicating the implication of this anti-ChAT activity of PPIs, goes beyond their effect on male fertility [34][35][36].
We selected one of the most popular and commonly used PPIs, esomeprazole, based on our initial in silico virtual screening studies.The results of our detailed enzyme kinetic analyses were conclusive in terms that esomeprazole acted as a mixed-competitive ChAT inhibitor.This pure S-enantiomer exhibited about 2-fold higher potency than the racemic mixture omeprazole, indicating that the R-enantiomer may lack anti-ChAT activity.The inhibition constants for esomeprazole and omeprazole were in the current study estimated in low nM ranges.The concentration of PPIs in human plasma varies between 1 and 23 µM [69].For instance, plasma concentration of esomeprazole ranges from 2.1 to 2.4 µM following a dose of 20 mg to 4.7-5.1 µM of a dose of 40 mg [69].These could be even higher in elderlies and patients with hepatic dysfunctions as PPIs mainly undergo metabolism in the liver.The concentrations that we used in our ex vivo experiment safely represent the physiological levels of the drug in vivo even in the testes assuming that the blood-testis barrier is as effective as the BBB, where the expected esomeprazole concentration is 100-2300 nM [33].Nonetheless, it should be pointed out that esomeprazole as supported by the current ex vivo findings is a rapidly acting inhibitor of ChAT, and thereby does not require prolonged incubation to inhibit sperm ChAT.Thus esomeprazole may originate from prostate and/or seminal vesicles fluid during ejaculation.Sperm might also be exposed to PPIs in the in vivo microenvironment in female reproductive tracts.In all these instances, esomeprazole concentration may be as high as its plasma concentration (2.1-5.1 µM) [69], since neither prostate, seminal vesicles nor uterus are subjected to any particular blood barrier to substantially restrict the distribution of the drug.
The effect of esomeprazole on the motility of the sperm seems to be time dependent.This is somewhat expected for two reasons, one is the presence of an ACh reservoir and the other is the partial displacement of esomeprazole (competition) by free choline as the substrate of ChAT.Indeed, both esomeprazole and omeprazole behaved as reversible mixed-competitive inhibitors with regards to choline which has been determined between 0.9 and 1.4 mg/ml (i.e.9-13 mM) in human semen [70].However, it should be pointed out that the current analyses were run in whole liquefied ejaculate which was merely diluted 50% with buffer rather than on completely washed-out sperm.Thereby we may conclude that the ex vivo design of the experiment suggests that the observed effect of esomeprazole on sperm motility parameters includes the possible interference of endogenous choline concentration which is expected to be the range of 4.5-6.5 mM in the 50% diluted ejaculates.Furthermore, the findings are in agreement with esomeprazole K i value of 88 nM (10-166 nM), a parameter that in contrast to IC 50 is independent of the substrate (i.e.choline) concentration.Nonetheless, future studies are required to assess the possible interference of full range choline concentration of 9-13 mM [70].
A limitation of our study was the small sample size used for the ex vivo sperm motility analysis.However, we were using sperm samples as an ex vivo model to test PPI-ChAT system on sperm motility rather than investigating the clinical effects PPIs on fertility.The ex vivo part of this study was hence designed as pilot experiment to provide a proof-ofconcept for our hypothesis.We are now planning to test all other commonly prescribed PPIs and to expand our studies to include large number of healthy volunteers, subfertile males and patients with other sexual disorders.
In conclusion, we report here that esomeprazole behaves as a potent mixed-competitive inhibitor of key cholinergic enzyme human ChAT, and thereby significantly exhibited ex vivo depression of the motility index of fresh sperm.The study as a whole was a proof-of-concept favoring our hypothesis that PPIs affect the motility of sperm by targeting spermic cholinergic machinery.Given that this is a novel and unexplored hypothesis, our study provides a completely new perspective regarding PPIs secondary mode of action/unwarranted side effects and calls for further mechanistic and larger patient based clinical studies to elucidate the role of PPIs in infertility and other hypo-cholinergic disorders such as Alzheimer's and ALS.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
) lies inside the catalytic tunnel of ChAT and almost overlaps the A-CoA/CoA binding site.The data indicate that esomeprazole mainly interacts with ChAT via π-stacking (non-covalent aromatic-aromatic interaction) and hydrogen bond formation with tyrosine residue at position 552.The 3D and 2D representation of esomeprazole binding pocket and active site interacting residues on human ChAT are shown in Fig. 1.

Fig. 1 .
Fig. 1.Binding pocket of esomeprazole lies within the ChAT catalytic tunnel.The in silico docking analyses was performed as described in the Materials and Method section to identify the potential binding site of esomeprazole on ChAT.The figure shows the 3D representation of hChAT protein (PDB id: 2FY5), where the ACoA/ CoA entrance site of the hChAT catalytic tunnel is highlighted by dashed yellow circle.The in silico analyses indicated that the esomeprazole binding pocket (highlighted by red color) lies within the catalytic domain and overlap's with the ACoA/CoA entrance site.The 2D and 3D representation of esomeprazole binding pocket on hChAT are further presented here in detail, to reveal the binding orientation within the catalytic tunnel and key interacting residues.Esomeprazole is shown as ball and stick and mainly interacts with the active site amino acid residue tyrosine (552) by forming non-covalent aromatic and hydrogen bonds as shown by dark green and dotted pink lines, respectively.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)