Sequence variation at KLK and WFDC clusters and its association to semen hyperviscosity and other male infertility phenotypes.
Are kallikreins (KLKs), the whey-acidic-protein four-disulfide core domain (WFDCs) and their neighbors, semenogelins (SEMGs), known to play a role in the cascade of semen coagulation and liquefaction, associated with male infertility?



Several KLK and SEMG variants are overrepresented among hyperviscosity, asthenozoospermia and oligozoospermia, supporting an effect of abnormal semen liquefaction on the loss of semen quality and in lowering male reproductive fitness.



In the cascade of semen coagulation and liquefaction the spermatozoa coated by EPPIN (a protease inhibitor of the WFDC family) are entrapped in a cross-linked matrix established by SEMGs. After ejaculation, the SEMG matrix is hydrolyzed by KLK3/2 in a fine-tuned process regulated by other KLKs that allows the spermatozoa to increase motility.



This study includes a cohort of 238 infertility-related cases and 91 controls with normal spermiogram analysis. The remaining 126 controls are healthy males with unknown semen parameters. Sample collection was carried out from June 2011 to January 2015 and variant screening from May 2013 to August 2015.



We performed a screening by massive parallel sequencing in a pooled sample (N = 222) covering approximately 93 kb of KLK (19q13.3-13.4) and WFDC (20q13) clusters, followed by the genotyping of most promising variants in the full cohort.



Overall, 160 common and 296 low-frequency variants passed the quality control filtering. Statistical tests disclosed an association with hyperviscosity of a KLK7 regulatory variant (P = 0.0035), and unveiled a higher burden of deleterious mutations in KLKs than expected by chance (P = 0.0106). KLK variants found to be overrepresented in cases included two substitutions likely affecting the substrate binding pocket, two nonsynonymous variants overlapping in the three-dimensional structure and two mutations mapping in consecutive N-terminal residues. Other variants identified in SEMGs possibly contributing to hyperviscosity and asthenozoospermia consisted of three replacements predicted to modify targets of proteolysis (P = 0.0442 for SEMG1 p.Gly400Asp) and a copy number variation associated with a reduced risk of oligozoospermia (P = 0.0293).



Not applicable.



The sampling of a few hundred individuals has limited power to detected associations with low-frequency variants and only a small set of variants was prioritized for genotyping. Other susceptibility variants for male infertility may remain unidentified.



We provide important evidence for an effect of KLKs and SEMGs variability on semen quality and for modifications in the process of semen liquefaction as a possible cause for male infertility.



This work was funded through the Portuguese Foundation for Science and Technology (FCT) and FEDER through COMPETE and QREN. The authors have no conflict of interest to declare.
Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal.|Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-135 Porto, Portugal.|Department of Biochemistry and Molecular Biology-IUOPA, University of Oviedo, 33006 Oviedo, Spain.|Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal.
Patrícia Isabel
PI
Marques
Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal.|Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-135 Porto, Portugal.
Filipa
F
Fonseca
Human Genetics Department, National Institute of Health Dr Ricardo Jorge (INSA), 1649-016 Lisboa, Portugal.
Ana Sofia
AS
Carvalho
Department of Biochemistry and Molecular Biology-IUOPA, University of Oviedo, 33006 Oviedo, Spain.
Diana A
DA
Puente
Center of Infertility and Sterility Studies (CEIE), 4050-345 Porto, Portugal.
Isabel
I
Damião
Center of Infertility and Sterility Studies (CEIE), 4050-345 Porto, Portugal.|Department of Biology, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal.
Vasco
V
Almeida
Center for Reproductive Genetics Alberto Barros, 4100-009 Porto, Portugal.
Nuno
N
Barros
Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal.|Center for Reproductive Genetics Alberto Barros, 4100-009 Porto, Portugal.|Department of Genetics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal.
Alberto
A
Barros
Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal.|Department of Genetics, Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal.
Filipa
F
Carvalho
Proteomics Platform CICbioGUNE, CIBERehd, ProteoRed-ISCIII, Parque Tecnologico de Bizkaia, Derio, Spain.
Mikel
M
Azkargorta
Proteomics Platform CICbioGUNE, CIBERehd, ProteoRed-ISCIII, Parque Tecnologico de Bizkaia, Derio, Spain.
Felix
F
Elortza
Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal.|Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-135 Porto, Portugal.|Faculty of Medicine of the University of Porto, 4200-319 Porto, Portugal.
Hugo
H
Osório
Human Genetics Department, National Institute of Health Dr Ricardo Jorge (INSA), 1649-016 Lisboa, Portugal.
Rune
R
Matthiesen
Department of Biochemistry and Molecular Biology-IUOPA, University of Oviedo, 33006 Oviedo, Spain.
Victor
V
Quesada
Instituto de Investigação e Inovação em Saúde, Universidade do Porto (i3S), 4200-135 Porto, Portugal sseixas@ipatimup.pt.|Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-135 Porto, Portugal.
Susana
S
Seixas
D005838:Genotype; D006801:Humans; D007248:Infertility, Male / Q000235:genetics*; D007610:Kallikreins / Q000235:genetics*; D008297:Male; D010641:Phenotype; D011506:Proteins / Q000235:genetics*; D012661:Semen; D055101:Semen Analysis; D029607:Seminal Vesicle Secretory Proteins / Q000235:genetics; D013081:Sperm Motility / Q000235:genetics; D013091:Spermatogenesis / Q000235:genetics; D014783:Viscosity
D011506:Proteins; D029607:Seminal Vesicle Secretory Proteins; C484310:WFDC1 protein, human; C424482:seminal vesicle-specific antigen; D007610:Kallikreins
genetic susceptibility; prostate-specific antigen; rare variants; semen coagulation and liquefaction; semenogelins