Author Correction: Alterations in seminal plasma proteomic profile in men with primary and secondary infertility

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Infertility globally affects 15% of couples and is now classified as a disease of the reproductive system by the World Health Organization (WHO) 1 . Based on the presence or absence of previous successful pregnancies, infertility can be divided as primary and secondary. Couples who were unable to become pregnant after at least 1 year of sexual intercourse without contraceptive methods are referred as primary infertility. On the other hand, couples who were able to get pregnant at least once, but not subsequently are referred as secondary infertility. Prevalence of primary infertility (1.5 to 2.6%) is reported to be lower than secondary infertility (7.2 to 18%) 2 . Approximately, 50% of all reported couple infertility cases can be attributed to the male factor 3,4 though the reasons remain unknown. Basic semen analysis is one of the first steps in the evaluation of male infertility. This analysis provides both macroscopic (volume, pH, color, viscosity) and microscopic characteristics (sperm concentration, total motility, progressive motility, sperm morphology) of semen 5 . The semen analysis remains the cornerstone of male fertility evaluation. However, it does not provide a systematic explanation for the subcellular changes that occur in the spermatozoa of infertile men, which necessitates a more in-depth analysis and understanding at molecular level 6,7 .
Spermatozoa acquires fertilizing potential during their epididymal maturation phase before ejaculation 8 . The ejaculated semen contains both cellular (spermatozoa) and non-cellular (seminal plasma) components. The seminal plasma is composed of secretions from testis, epididymis, prostate, seminal vesicles and bulbo-urethral glands; 9,10 it provides nourishment and protection to spermatozoa 11,12 . It also plays a crucial role in sperm maturation, capacitation, acrosome reaction and fertilization 11,12 . Composition of the seminal plasma protein and their interaction with sperm surface influence the fertilizing capacity of spermatozoa 12 .
In recent years, there is an increased number of reports on seminal plasma proteome to identify potential biomarkers for different pathologies and conditions related to infertility. This includes varicocele [13][14][15][16] , oxidative stress mediated male infertility [17][18][19][20] , nonobstructive azoospermia [21][22][23] , asthenozoospermia 24,25 , Semen analysis. Semen samples were collected at the Andrology Laboratory by masturbation after sexual abstinence of least 2-7 days. Samples were allowed to liquefy completely for 20 minutes at 37 °C, and semen analysis was performed according to the WHO (2010) guidelines 31 using a disposable Leja sperm counting chamber (Spectrum Technologies, Healdsburg, CA) to evaluate sperm count, motility and round cells. After routine semen analysis, the left-over samples were centrifuged for 7 minutes at 1000 × g. The clear seminal plasma was aspirated from the samples and stored at −80 °C for proteomic studies.
Sample preparation for proteomic analysis. The samples used for proteomic analysis were in compliance with the Minimum Information about a Proteomics Experiment (MIAPE) guidelines of the Human Proteome Organization's Proteomics Standards Initiative (HUPO-PSI) for reporting proteomics studies 32 . Seminal plasma samples were thawed at room temperature and centrifuged at 3000 × g for 30 minutes to remove any contamination with spermatozoa or other cellular debris. The samples were mixed (1:1 ratio) with the proteinase inhibitor cocktail (Roche, Indianapolis, IN) prepared in phosphate buffer saline (PBS) to prevent proteolysis during sample handling. The protein concentration was determined using a commercial kit, bicinchoninic acid (BCA) kit (Thermo, Rockford, IL), following the manufacturer instructions.
Pooled samples from control (n = 3); primary infertility (n = 3) and secondary infertility (n = 3) were used for proteomic analysis. Equal concentration of proteins from each individual sample was used to normalize the Quantitative proteomics. The relative quantification of the proteins was performed by comparing the number of spectra, termed spectral counts (SpCs) in control vs primary infertility group and control vs. secondary infertility group. To achieve the false detection rate (FDR) < 1%, protein identification criteria was established at >99% probability as explained in our previous study 37 . The abundance of the proteins was determined by matching the SpCs and classified as high (H), medium (M), low (L), or very low (VL). To overcome the sample-to-sample variation, normalization of spectral counts was done using the normalized spectral abundance factor (NSAF). In general, longer proteins have more peptide identifications than shorter proteins. NSAF ratio determines the actual expression of the protein in the samples, proteins with ratio <1 and >1 are considered underexpressed and overexpressed, respectively. Different constraints for fold-change cut-offs and significance tests (P value) based on the average SpCs from 3 replicate runs were applied to obtain the DEPs 36 . Appropriate filters were applied to minimize the errors due to the presence of low abundance proteins. Abundance and the expression of DEPs are based on the following criteria: (i) VL -SpC range, 1.7-7; NSAF ratio (≥2.5 for upregulated and ≤0.4 for downregulated proteins); and P ≤ 0.001, (ii) L -SpC range, [8][9][10][11][12][13][14][15][16][17][18][19]; NSAF ratio (≥2.5 for upregulated and ≤0.4 for downregulated proteins); and P ≤ 0.01, (iii) M -SpC range, between 20 and 79; NSAF ratio (≥2.0 for upregulated and ≤0.5 for downregulated proteins); and P ≤ 0.05, (iv) H -SpC, >80; NSAF ratio (≥1.5 for upregulated and ≤0.67 for downregulated proteins); and P ≤ 0.05.
Bioinformatic analysis. DEPs identified in the control vs primary infertility group and control vs secondary infertility group were subjected to functional annotation and enrichment analysis using both, publicly available bioinformatic annotation tools and databases such as GO Term Finder, GO Term Mapper, UniProt, and Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.niaid.nih.gov). Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis was performed using the online tool to identify protein-protein interaction networks (https://string-db.org/). Proprietary software package Metacore (GeneGo Inc.) was also used to identify the upstream regulators involved in the enriched pathways.

Group
Volume ± SD (ml) Concentration ± SD (x10 6 /ml) Motility ± SD (%) www.nature.com/scientificreports www.nature.com/scientificreports/ Protein validation by western blot and total protein staining. The key proteins involved in reproductive functions and fertilization process were selected for validation. The functions of these proteins are well described in the literature. Based on function and role of proteins related to fertility potential, six DEPs from primary infertility and five DEPs from secondary infertility were selected for validation. The DEPs were validated by western blot (WB) in individual samples from the control group (n = 6) and primary (n = 6) or secondary (n = 6) infertility group. A total of 20 µg of protein per sample was mixed with equal volume of loading buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and 1% β-mercaptoethanol). The sample mixture was boiled for 10 minutes and kept on the ice for 5 minutes. 30 µL of each sample was loaded into a 4%-15% SDS-polyacrylamide gel and electrophoresed for 2 h at 90 V along with a set of molecular weight marker (Sigma Chemical Co., St. Louis, MO, USA). The resolved protein bands were then transferred onto polyvinylidene difluoride (PVDF) membranes at 20 V for 30 minutes using a transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol). PVDF membranes were blocked with Tris-buffered saline-Tween-20 (TBST) with 5% bovine serum albumin (BSA) and used for immunodetection of seminal plasma proteins 38 . For each protein analysis, specific primary antibodies were incubated overnight at 4 °C overnight (Table 1). Subsequently, the membranes were washed four times with TBST for 10 minutes and incubated with the secondary antibodies at  www.nature.com/scientificreports www.nature.com/scientificreports/ room temperature for 1 h ( Table 1). The same membranes were washed four times with TBST (10 minutes) and finally treated with enhanced chemiluminescence (ECL) reagent (GE Healthcare, Marlborough, USA) for 5 minutes. ECL reacted blots were exposed to Chemi-Doc (ChemiDoc MP Imaging System, Bio-Rad, Hercules, USA) to detect the chemiluminescence signals 38 .
The total amount of protein present in the membranes were quantified using a Colloidal Gold Total Protein Stain (Bio-Rad, Hercules, USA). The protocol was performed according to manufacturer instructions. Briefly, the membranes were washed twice for 10 minutes in distilled water and stained with total colloidal gold protein stain by gentle shaking for 2 h at room temperature. Stained membranes were washed twice with distilled water for 10 minutes, and the densitometry image was captured using colorimetric mode on Chemi-Doc (ChemiDoc MP Imaging System, Bio-Rad, Hercules, USA) 38 .
Statistical analysis. MedCalc Statistical Software (V. 17.8; MedCalc Software, Ostend, Belgium) (https:// www.medcalc.org/) was used for data analysis. Mann-Whitney test was performed to compare (control vs. primary infertility group and control vs. secondary infertility group) the semen parameters and the expression levels of the proteins validated using WB technique. The results were considered significant with P < 0.05.

Semen parameters of men with primary infertility and men with secondary infertility. Semen
analysis showed significant decrease (P < 0.05) in sperm concentration, motility, and total count, and total motile sperm count in both primary infertility and secondary infertility group compared to control group (Table 2).

Seminal plasma proteome of primary and secondary infertility. LC-MS/ MS analysis identified a
total of 515 different peptides in primary infertility and control group. Out of these, 392 peptides were common to primary infertility and control group, 8 were unique to control group and 115 were unique to primary infertility group (Fig. 1A). Relative abundance of identified peptides revealed 265 peptides with very low, 126 had low, 91 had medium and 25 had high relatively abundance (Fig. 1E). Only 48 seminal plasma proteins were identified as DEPs between primary infertility and control group (Table 3). Of the 48 identified DEPs, 40 were overexpressed, 4 were underexpressed and 4 were unique to primary infertility group compared with control group (Fig. 1C).
The analysis identified a total of 523 different peptides in secondary infertility and control group. Of these, 415 peptides were common to secondary infertility and control group, 6 were unique to control group and 102 were unique to secondary infertility group (Fig. 1B). Relative abundance of the identified proteins revealed 245 proteins with very low abundance, 143 with low abundance, 110 with medium abundance and 15 proteins with high relative abundance (Fig. 1F). A total of 53 seminal plasma proteins were found to be differentially expressed between secondary infertility and control group (Table 4). Of these 53 DEPs, 2 were underexpressed, 38 were

Bioinformatic analysis of seminal plasma DEPs in men with primary and secondary infertility.
STRING analysis was performed to clarify the protein-protein interaction networks of DEPs identified in the seminal plasma of men with primary infertility (Fig. 2A). This online tool also enabled the identification of biological processes more relevant in the network based on Gene Ontology (GO). In the seminal plasma of men with primary infertility, we identified regulated exocytosis (GO:0045055) (False discovery rate (FDR) = 1.09e −6 ) and secretion by the cell (GO:0032940) (FDR = 1.09e −6 ) as the two most important biological processes. Other biological processes identified are listed in Fig. 2A. Using DAVID we were able to identify functional annotations for DEPs identified in seminal plasma of men with primary infertility. Key processes and functions involved in overexpressed, underexpressed and DEPs unique to seminal plasma of men with primary infertility are presented in Table 5. Finally, the diseases related to DEPs from seminal plasma of men with primary infertility were identified using MetaCore enrichment (Fig. 3).
String analysis was performed with the DEPs identified in men with secondary infertility (Fig. 2B). In the seminal plasma of men with secondary infertility, regulated exocytosis (GO:0045055) (FDR = 1.09e −12 ) and cell activation involved in immune response (GO:0002263) (FDR = 2.81e −12 ) were the two most important biological processes identified. Other recognized biological processes are presented in Fig. 2B. Using DAVID, we identified key processes and functions involved in overexpressed, underexpressed and DEPs unique to seminal plasma of men with secondary infertility (Table 5). Using MetaCore enrichment, the diseases related with DEPs from seminal plasma of men with secondary infertility were identified and presented in Fig. 4.

Western blot validation of seminal plasma proteins.
Based on selection criteria and the biological role, six proteins (ANXA2, PRDX2, CDC42, CD63, SEMG1 and SEMG2) from men with primary infertility and five proteins (ANXA2, C4, APP, SEMG1 and SEMG2) from men with secondary infertility were validated by WB.
Of the proteins analyzed in men with primary infertility, ANXA2 showed an increased expression when compared to fertile donors (p < 0.05) (Fig. 5A). Other three proteins (PRDX2, CD63 and SEMG1) were detected by WB, but no differences were observed in their expression levels compared to control group (Fig. 5C,D,F). The WB validation of the protein CDC42 showed an increase in protein expression (p < 0.05) (Fig. 5B) that was in concordance with the proteomic results. The other protein SEMG2 selected for validation by western blot was underexpressed in seminal plasma of men with primary infertility when compared to fertile donors (p < 0.05) (Fig. 5E). The protein C4, SEMG1 and SEMG2 selected for validation using western blot in secondary infertility Scientific RepoRtS | (2020) 10:7539 | https://doi.org/10.1038/s41598-020-64434-1 www.nature.com/scientificreports www.nature.com/scientificreports/ group did not show any change in the expression (Fig. 6B,D,E). Other key proteins such as ANXA2 and APP were overexpressed in secondary infertility group compared with control group (p < 0.05) (Fig. 6A,C).

Discussion
The global proteomic approach is currently being used to identify the molecular factors associated with the cause of male infertility. As spermatozoa are transcriptionally and translationally silent they depend on the sperm and the seminal plasma proteins for their normal biological functions. Seminal plasma harbors factors that are essential for protection of spermatozoa during its transit through female reproductive tract and assist in the fertilization process. Semen analysis of men with primary or secondary infertility revealed that all semen parameters were above the WHO 2010 reference values 39 (Table 2). Since semen quality seems not to be the cause for infertility in these men, we used proteomic approach to identify the dysregulated seminal plasma proteins. Intasqui et al. carried out proteomic profiling of sperm in men with primary and secondary infertility, and proposed BAG6 and HIST1H2BA as potential biomarker of male infertility 40 . As a follow up, the current study focused on the proteomic analysis of seminal plasma and its bioinformatic analysis that provides an extensive information about distribution, molecular and functional analysis of the identified proteins 9,11,38,41 . Furthermore, the proteins selected based on reproductive functions and fertilization process such as regulation of exocytosis 42,43 , regulation of cell motility 44,45 or vesicle mediated-transport 46 were validated using WB technique. These biological processes are crucial for fertilization and our analysis revealed dysregulation in both men with primary and secondary infertility. Role of key proteins associated with the pathophysiology of primary and secondary male infertility are discussed in detail.
Annexin 2 (ANXA2) is a Ca 2+ -dependent phospholipid-binding protein, which is associated with plasma membrane of cells and endosomes 47 . ANXA2 plays an important role in cellular processes, such as, membrane trafficking events 48 , lipid reorganization in the membrane and endocytosis 49 . During abnormal ubiquitination process, an aberrant expression of ANXA2 was observed 50,51 . With respect to reproductive functions, ANXA2 is involved in maintaining the integrity of blood-testis-barrier and in the release of spermatozoa 52 . Presence of ANXA2 in sperm is essential for the binding of the sperm in the female tract that is crucial for fertilization 53 . www.nature.com/scientificreports www.nature.com/scientificreports/ Earlier studies have reported abnormal expression of ANXA2 in seminal plasma and prostasomes of subfertile or infertile men [54][55][56] . In the current study, ANXA2 was found to be involved in several biological function such as exocytosis and secretion by cell, both processes essential in the binding to sperm. Proteomic analysis revealed overexpression of ANXA2 in the seminal plasma of men with primary and secondary infertility. Furthermore, WB findings also confirmed the overexpression of ANXA2 in both primary and secondary infertile men compared to control group. Hence, we suggest that aberrant expression of ANXA2 in the seminal plasma affects the maturation process of the spermatozoa 55 , resulting in production of the immature sperm in both primary and secondary male infertility conditions. Peroxiredoxins (PRDXs) are the key proteins that regulate ROS levels and plays an important role in male fertility 57 . Bioinformatic analysis of DEPs present in the seminal plasma of primary infertility men confirmed that PRDX2 was involved in the oxidation-reduction process. In spermatozoa, the presence of PRDX2 was detected in plasma membrane, acrosome, nucleus, midpiece and flagellum 58 . PRDX2 reduces the availability of iron which in turn decreases oxidative stress 59 . Alteration in the expression of this protein has been reported in men with immunological infertility 60 . Proteomic analysis of seminal plasma of men with primary infertility revealed overexpression PRDX2, whereas WB validation did not reveal difference in the expression levels of PRDX2 compared to the control group. The discrepancies in the results of LC-MS/MS and WB could be mainly due to the differences in the specificity and sensitivity of both techniques. Our seminal plasma proteomic results are concurrent with the findings from earlier reports on male infertility conditions related to varicocele and in men with Hodgkin's disease 61,62 . Increased levels of PRDX2 in the seminal plasma of men with primary infertility indicates oxidative stress mediated damage to the spermatozoa leading to impairment of physiological functions related to fertilization process such as hyperactivation, capacitation, acrosome reaction and sperm-oocyte fusion. www.nature.com/scientificreports www.nature.com/scientificreports/ The protein CD63 antigen (CD63) is a cellular trafficking molecule 63 and is considered an exosomal marker 64 . Dysregulation in the expression of CD63 affects the exosome-sperm fusion, which is responsible for the production of immature spermatozoa 65 . In the current study, CD63 was overexpressed in primary infertility group and was involved in vesicle-mediated transport. Alteration in the expression of CD63 protein was reported in spermatozoa of men with testicular cancer 66 and seminal plasma of infertile varicocele men 55 . Furthermore, our bioinformatic analysis revealed that the overexpressed seminal plasma protein CDC42 was also involved in vesicle-mediated transport. CDC42 proteins are expressed in the head of elongated spermatids 67 and are involved in the formation of sperm tail and head 68 . CDC42 are identified as key controllers of capacitation-dependent actin dynamics 69 and regulators of the acrosome reaction 70 . Dube et al. reported decreased levels of CDC42 transcripts in obstructive azoospermia 71 . Increase levels of CDC42 and CD63 in seminal plasma may play a major role in the pathology associated with vesicle-mediated transport in men with primary infertility. Further, CD63 has a direct impact on the transport of exosomal molecules/ factors to the spermatozoa required for the maturation process whereas CDC42 protein dysfunction may serve as factor for compromised acrosome reaction in men with primary infertility. We were also able to demonstrate the presence of both the proteins (CDC42 and CD63) using WB in the seminal plasma of men with primary infertility.
Immune response and inflammation have negative impact on the male reproductive system and male fertility 72 . LC-MS/MS analysis and further confirmation by WB, revealed that C4 protein, which is involved in body's immune response 73 , was overexpressed in secondary infertile men. Further, bioinformatics analysis also predicted infection as the most relevant dysregulated pathway in secondary infertility. Likewise, the multifunctional protein APP, which was detected in the seminal plasma of secondary infertility men, is involved in many biological processes such as exocytosis, cell activation to response of immune system, proteolysis regulation and regulation of iron homeostasis. Earlier studies demonstrated the localization of APP in tail and head of spermatozoa 74,75 . In oxidative stress mediated male infertility aberrant expression of APP results in acrosome dysfunction 76 . In the current study APP was overexpressed and described to have a role in sperm motility and interaction of the sperm with the oocyte 75 . Moreover, the validation of proteomic results using WB suggests that APP can serve as a seminal plasma marker in diagnosis of secondary infertility. Hence, from the global proteomic and WB results it is clear that proteins associated with immune response are overexpressed in the seminal plasma of men with secondary infertility and thus can a predisposing cause for this condition.
The most abundant proteins in seminal plasma are the semenogelins (1 and 2) 77 . These proteins are secreted by the seminal vesicles and form the gel-like coagulum with the fibronectin in the ejaculated semen 78 . The coagulum is liquefied through the action of prostate specific antigen protein, responsible for semenogelins cleavage, inducing motility and releasing spermatozoa. de Lamirande et al. reported that SEMGs are involved in the inhibition of capacitation process of human spermatozoa 79 . Other global proteomic studies also demonstrated the abnormal expression of SEMGs in seminal plasma of men with abnormal semen parameters 80 , seminal oxidative stress 81 and varicocele 82,83 . In the present study, proteomic profile of seminal plasma showed both SEMG1 and SEMG2 were underexpressed in primary and secondary infertile men compared to control group. Dysregulation of the semenogelins in both primary and secondary infertility condition may have a severe impact on the capacitation process of the spermatozoa. Furthermore, validation of SEMG2 using WB in the seminal plasma of men with primary infertility suggests that SEMG2 levels can serve as candidate marker to differentiate primary from secondary infertility.

Conclusion
The proteomic data from this pilot study shows an altered seminal plasma proteome in primary and secondary infertility compared with fertile men. Our preliminary results have identified maturation failure and immune reaction response as the causes for infertility in men with primary and secondary infertility. Validation of key proteins suggests that ANXA2 can be a screening biomarker for both primary and secondary infertility, whereas CDC42 and SEMG2 can be useful candidate biomarkers for primary infertility and APP for secondary infertility. This is the first report showing DEPs in seminal plasma of men with primary and secondary infertility, however further studies are warranted to confirm and validate these biomarkers.