Methods for determining human sperm quality

Abstract

The present invention relates generally to the fields of reproductive medicine. More specifically, the present invention relates to methods and kits for determining the human sperm quality.

Claims

1. A method for assessing sperm fertility rate in an infertile or hypofertile subject or a subject with difficulty to conceive for more than one year, comprising the steps of: i) liquefying a semen sample obtained from said subject at 37° C. for 30 minutes, ii) isolating spermatozoa from said semen sample through a single-step density gradient centrifugation, iii) washing said isolated spermatozoa with tris-buffered saline, iv) homogenizing said isolated spermatozoa with a lysis buffer to obtain a spermatozoa protein lysate, v) measuring the expression level of one or both of a kinase anchoring protein 4 (AKAP4) and Hexokinase 1 in said protein lysate, and vi) comparing said measured expression level with a reference value, wherein detecting a differential in the expression of the one or both of AKAP4 and Hexokinase 1 between the sample and the reference value is indicative of sperm fertility rate.

2. The method according to claim 1 wherein said subject is classified as a normozoospermic subject.

3. The method according to claim 1, wherein said tris-buffered saline comprises 0.1 mM of HCl, 100 mM of NaCl, and has a pH equal to 7.6.

4. The method according to claim 1, wherein step v) is performed by analyzing said spermatozoa protein lysate obtained in step iv) in a 1D SDS-PAGE with blue staining, providing a numerical analysis of said blue stained gel, selecting protein bands between Low molecular weights 15 to 30 kDA and protein bands between High molecular weights 80 and 110 kDa, representing each band by a densitometric curve and calculating the area under the densitometric curve, and calculating a ratio between the area under the densitometric curve from Low molecular weight and High molecular weight band intensities.

5. The method according to claim 4 wherein a ratio between 1 to 2 is indicative of good sperm quality, and a ratio above 2 is indicative of bad sperm quality.

6. The method according to claim 3, wherein step v) is performed by analyzing said spermatozoa protein lysate obtained in step iv) in a 1D SDS-PAGE with blue staining, providing a numerical analysis of said blue stained gel, selecting protein bands between Low molecular weights 15 to 30 kDA and protein bands between High molecular weights 80 and 110 kDa, representing each band by a densitometric curve and calculating the area under the densitometric curve, and calculating a ratio between the area under the densitometric curve from Low molecular weight and High molecular weight band intensities.

7. A method enabling an infertile or hypofertile subject or a subject with difficulty to conceive for more than one year to determine the most successful assisted-reproductive technology comprising the steps of: i) liquefying a semen sample obtained from said subject at 37° C. for 30 minutes, ii) isolating spermatozoa from said semen sample through a single-step density gradient centrifugation, iii) washing said isolated spermatozoa with tris-buffered saline, iv) homogenizing said isolated spermatozoa with a lysis buffer to obtain a spermatozoa protein lysate, v) measuring the expression level of one or both of AKAP4 and Hexokinase 1 in said protein lysate, and vi) comparing said measured expression level with a reference value, wherein detecting a differential in the expression of the one or both of AKAP4 and Hexokinase 1 between the sample and the reference value is indicative of a given assisted reproductive technology.

8. The method according to claim 7, wherein step v) is performed by analyzing said spermatozoa protein lysate obtained in step iv) in a 1D SDS-PAGE with blue staining, providing a numerical analysis of said blue stained gel, selecting protein bands between Low molecular weights 15 to 30 kDA and protein bands between High molecular weights 80 and 110 kDa, representing each band by a densitometric curve and calculating the area under the densitometric curve, and calculating a ratio between the area under the densitometric curve from Low molecular weight and High molecular weight band intensities, wherein when said ratio is above 2 choosing intrauterine insemination (IUI) as an assisted-reproductive technology.

9. The method according to claim 7, wherein said tris-buffered saline comprises 0.1 mM of HCl, 100 mM of NaCl and has a pH equal to 7.6.

10. The method according to claim 7, wherein said subject is classified as a normozoospermic subject.

Description

FIGURES

(1) FIG. 1: AKAP4 and Hexokinase 1 expression revealed by immunoblotting.

(2) 15 μg of spermatozoa total protein lysates were loaded. Lane T98 is corresponding of T98 glioblastoma cell lysate. Lane A is representative of sperm sample with a quality index comprised between 1 and 2. Lane B is a spermatozoa sample with a quality index upper/over than 1 and 2. AKAP4 N-ter/C-ter, Hexokinase N-ter/C-ter and Histone H3 antibodies were used. The full-length AKAP4 protein is detected at an apparent MW of 95 kDa. The apparent MW of hexokinase 1 is of 100 kDa. Histone H3 were detected at apparent MW of 17 kDa and assessed that the same quantity of proteins were loaded.

(3) FIG. 2: Method to calculate SPQI

(4) 15 μg of spermatozoa proteins isolated from one ejaculate are loaded on a 1D SDS-Page and Coomassie stained (A). Picture from gel was analyzed. Background was eliminated. Windows from 15 to 30 kDa and 80 to 110 kDa were selected (B). Each bands were represented by a pic. Area under curve was calculated (C). Ratio LMW/HMW was established (D).

(5) FIG. 3: Sandwich ELISA to measure the concentration of full-length AKAP4.

(6) A. Growing concentration of recombinant human AKAP4 [AKAP4 (Human) Recombinant Protein (Q01) (Cat#: H00008852-Q01)] from 0.1 ng to 120 ng were measured using the Sandwich AKAP4 ELISA. B. Serial dilutions of sperm samples from a subject of the index A group or the index B group were assayed. Note that at a dilution of 1/2000 AKAP4 concentration is measured in sample A42 but not in the B92. According to the standard curve, in subject A, the estimated concentration of full-length AKAP4 is above 120 ng at a dilution of 1/2000 whereas in subject B, the concentration is below 1 ng.

EXAMPLES

Example 1

(7) Material & Methods

(8) Semen and Semen Quality Assessment

(9) Human semen samples were collected from anonymous men who attended the infertility department (Lille University center hospital). The semen were obtained between 3 and 5 days of sexual abstinence following masturbation and allowed to liquefy 30 min at 37° C. After liquefaction, the semen parameters (volume, pH, cell counting and motility) were evaluated according to the WHO criteria 2010. Spermatozoa were diluted ( 1/20) in Ringer medium and counted with an hemocytometer (Malassez cell counting). The progressive motility was evaluated by light microscopy on 5 fields and 100 spermatozoa were observed. The progressive motility (PR) is defined by the number of spermatozoa cells moving actively, either linearly or in a large circle, regardless of speed (World Health Organization 2010). The sperm morphology characterization (spermocytogram) was performed after Schorr's staining Only sperm above the lower references limits of all semen parameters were included in the present study. The local ethic committee on human subjects has approved the collection of semen samples. Men were given informed consent for the use of their semen for research purposes.

(10) Follow-Up of Men in Assisted Reproductive Technology (ART)

(11) Six months following the sperm analysis, couples remaining with difficulties to conceive were oriented to the Lille Hospital infertility department for reproductive assistance. Information was collected about the birth outcomes of ART pregnancies which had been performed in the infertility centre of the CHRU of Lille (Table 3).

(12) Isolation of Spermatozoa

(13) Sperm cells were separated from the semen plasma and round cells by a density gradient composed of 50% ferticult (Fertipro, Beernem, Belgium)/50% PureSperm (Nidacon, Mölndal, Sweden) in a 15 ml sterile Falcon tube (Falcon). After centrifugation (350×g, 20 min at room temperature), the sperm pellet was washed once with 1 mL of Tris buffer saline (TBS) (Tris-HCl 0.1 mM pH 7.6, 100 mM NaCl) by centrifugation (350×g, 20 min at room temperature).

(14) Sperm Protein Extracts

(15) The pellet was resuspended in 500 μl of lysis buffer containing non-buffered 20 mM Tris, 2% SDS, 1% Nuclease Mix (Life Science GE Healthcare, Piscataway N.J. USA). The lysates were sonicated on ice (40 Hz, 60 pulses) and centrifuged (14,000×g, 20 min at 4° C.). The supernatants were recovered and pellets were discarded. The protein concentrations were established according to the manufacturer's instructions (Bradford Assay, BioRad, Calif. USA). Protein lysates were frozen at −80° C. until used. Proteins from a cell lineage of human glioblastoma (T98) were extracted following the same procedure as for spermatozoa and was used in parallel of sperm protein to assess the antibody labeling by western blotting.

(16) SDS-PAGE of Sperm Proteins

(17) A total of 15 μg of sperm or T98 cell protein extracts was diluted in LDS 2× (Lithium dodecyl sulfate, Invitrogen) according to the manufacturer's instructions. Protein homogenates were heated at 100° C. during 10 min, quickly spun at 500×g using a bench centrifuge and loaded on 4-12% acrylamide gel (NuPage® Bis-Tris PreCast 12 wells, Life Technologies, Carlsbad Calif. USA). Molecular weight markers (Novex and Magic Marks, Life Technologies) were loaded in the first well. Electrophoresis was performed under a continuous potential of 200V during 1 h. Polyacrylamide gel was stained with Coomassie Blue [0.1% Blue G250 (Biorad, Calif. USA), 50% ethanol (v/v), 10% acetic acid (v/v) and H.sub.2O]. Protein staining was visualized after washing with a destaining solution containing 7% (v/v) of acetic acid, 10% ethanol in H.sub.2O. The gel was digitized with an Epson perfection V750 Pro scanner (Epson, France) and acquired using Adobe Photoshop Element 9 Software (Adobe, France). Images were analyzed using ImageJ software version 1.47c (NIH). ImageJ numerical treatment only included noise background reduction without modifying the original digitized image.

(18) NanoLC-MALDI-TOF-MS/MS Experiments

(19) Following tryptic digestion of Coomassie blue stained protein bands, nanoseparation was performed on an U3000 nano-HPLC system (Dionex-LC-Packings, Sunnyvale, Calif., USA). After a pre-concentration step (C18 cartridge, 300 μm, 1 mm), peptide samples were separated using a Pepmap C18 column (75 μm, 15 cm) by applying an acetonitrile/0.1% TFA gradient from 0% acetonitrile over 3 minutes, 0% to 15% over 7 minutes, 15% to 65% over 42 minutes, 65% to 90% over 5 minutes and, lastly, 6 minutes in 90% of acetonitrile. The flow rate was set to 300 nL/min and 110 fractions were automatically collected every 30 seconds on an AnchorChip™ MALDI target using a Proteineer™ FC fraction collector (Bruker Daltonics, Germany). Two μl of α-cyano-4-hydroxycinnamic acid matrix (0.3 mg/mL in acetone:ethanol:0.1% TFA-acidified water, 3:6:1 v/v/v) were added during the collection process. The MS and MS/MS mass measurements were performed off-line using the Ultraflex™ II TOF/TOF mass spectrometer (Brucker Daltonics). The apparatus parameters were set to values given below. Peptide fragmentation was driven by Warp LC™ software (Bruker Daltonics) according to the following parameters: signal-to-noise ratio>15, more than 3 MS/MS by fraction if the MS signal was available, 0.15 Da of MS tolerance for peak merge and elimination of peaks which appears in more than 35% of the fractions. The protein identification was performed as described below.

(20) Mass Spectrometry

(21) The molecular mass measurements were performed in automatic mode using the FlexControl™ 3.0 software on an Ultraflex™ II TOF/TOF instrument and in reflectron mode for MALDI-TOF peptide mass fingerprinting (PMF) or “LIFT” mode for MALDI-TOF/TOF peptide fragmentation fingerprinting (PFF). External calibration over a 1000-3200 mass range was performed using the [M+H]+ monoisotopic ions of bradykinin 1-7, angiotensin I, angiotensin II, substance P, bombesin and adrenocorticotropic hormone (clip 1-17 and clip 18-39) using a peptide calibration standard kit (Bruker Daltonics). Briefly, an accelerating voltage of 25 kV, a reflector voltage of 26.3 kV and a pulsed ion extraction of 160 ns were used to obtain the MS spectrum. Each spectrum was produced by accumulating data from 800 laser shots. A maximum of five precursor ions per sample were chosen for LIFT-TOF/TOF MS/MS analysis. Precursor ions were accelerated to 8 kV and selected in a timed ion gate. Metastable ions generated by laser-induced decomposition were further accelerated by applying 19 kV in the LIFT cell and their masses were measured in reflectron mode. Peak lists were generated from MS and MS/MS spectra using Flexanalysis™ 3.3 software (Bruker Daltonics). Database searches with Mascot 2.3.02 (Matrix Science Ltd, London, UK) using combined PMF and PFF datasets were performed in the UnitProt 2012_06_database via ProteinScape™ 2.1 (Bruker Daltonics). Taxonomy was restricted to Human in raison of the protein source nature. A mass tolerance of 75 ppm and 1 missing cleavage site for PMF and an MS/MS tolerance of 0.5 Da and 1 missing cleavage site for MS/MS searching were allowed. Carbamidomethylation of cysteine and oxidation of methionine residues were considered as fixed and variable modifications, respectively. The relevance of protein identities was judged according to the probability-based MOlecular Weight SEarch (MOWSE) score calculated with a p value equal or lower than 0.05 (p<0.05).

(22) Western Blotting

(23) After electrophoresis, proteins were transferred to 0.45 μm nitrocellulose membrane (Amersham, Life Science GE Healthcare, Piscataway N.J. USA) using the NuPage Liquid Transfer System (Life Technologies) and according to the manufacturer's instructions. Membranes were blocked with a solution containing 5% skimmed milk-TNT (Tris-HCl15 mM pH 8.0-NaCl 140 mM, 0.05% (w/v) Tween-20). Membranes were incubated with the primary antibodies overnight at 4° C. (complete antibody references and dilutions are listed in Table 1). Immuno-complexes were revealed after incubation with secondary antibodies coupled to horseradish peroxidase (horse anti-mouse: 1/50000 in TNT or goat anti-rabbit: 1/5000 in TNT, Vector Laboratories, Burlingham USA) and detected using the ECL™ luminescence kit (Amersham, Life Science GE Healthcare, Piscataway N.J. USA). The luminescence of immune-complexes was acquired with a LAS3000 imaging system (Fujifilm).

(24) TABLE-US-00001 TABLE 1 Proteins, manufacturers, species and dilutions of the primary antibodies used in western-blotting and immunocytochemistry. Dilution Dilution Immunocyto- Antibody Manufacturer Epitope Species Immunoblotting chemistry Proacrosin mAb 4D4 C-ter Mouse 1/1000 - TNT — (Gallo et al., 1991) Histone H3 05 - 928 C-ter of total H3 Rabbit 1/2000 - TNT — (Millipore, independant of Bellerica, post- USA) translational modifications HSP 90 Sc 13119 - Amino acids Mouse 1/500 - TNT — A1510 (Santa 610-723 Cruz, California, USA) Cytochrome C 89515 (BD — Mouse 1/500 - TNT — Pharmingen, San José, USA) GAPDH Sc25778 Amino acids Rabbit 1/10000 - TNT — FL335 (Santa 1-335 Cruz, California, USA) Tubulin β MAb 3408 - — Mouse 1/10000 - TNT — LV 1475931 (Millipore, Bellerica, USA) AKAP4 N-ter Ab 56551 Amino acids Mouse 1/10000 - TNT 1/500 - TBS 1X (Abcam, UK) 1-101 AKAP4 C-ter Sc-66308 C-ter Goat 1/5000 - TNT 1/400 - TBS 1X (Santa Cruz, California, USA) Hexokinase 1 Ab 55144 Amino acids Mouse 1/5000 - TNT 1/200 - TBS 1X N-ter (Abcam, UK) 101-201 Hexokinase 1 Sc-6518 C-ter Goat 1/1000 - TNT 1/100 - TBS 1X C-ter (Santa Cruz, California, USA) TNT = Tris-NaCl-Tween. TBS = Tris Buffer Saline

(25) Immunochemistry

(26) After sperm isolation, 100 μL of homogenate were fixed with 2% paraformaldehyde and incubated 15 min at room temperature. Spermatozoa were isolated by centrifugation (10 min, 350×g) and supernatant was removed. Sperm pellet was suspended in TBS 1× and washed by centrifugation two times (10 min, 350×g). Sperm pellet was suspended in 100 μL of TBS 1×. Thick drop was dropped-off on Superfrost slide (Menzel-Glazer, Germany) and conserved at 4° C.

(27) Slides were placed in acetone bath during 5 min at ambient temperature and washed twice in TBS 1× (5 min). 100 μL of TBS 1×-BSA 0.2% were deposed on thick drop and slides were incubated 1 h at room temperature. 100 μL of primary antibody diluted in TBS 1× (Table 1) covered thick drop and slides were incubated at 4° C. overnight. Slides were washed two times 5 min in TBS 1× at room temperature. Secondary antibodies (Vector Laboratories, Clinisciences, SAS France) were diluted in TBS 1× and slides were incubated during 1 h at room temperature. Slides were washed twice 5 min in TBS 1×. ABC mixture (Vectastain ABC Kit, Vector Laboratories) covered slides according to manufacturer's instructions. Immunolocalization was revealed by DAB (Vectastain ABC Kit) and reaction was stopped with water. Sperm head was stained with hematoxylin Mayer's solution (SIGMA-Aldrich, France) during 1 min. Slides were placed in successive bath of ethanol 70/95/100% during 1 min/bath and toluene bath (10 min). Slides were mounted with the VectaMount™ AQ Mounting Medium (Vector Laboratories). Images from slides were obtained with a Leica DM 2000 LED microscope (Leica, France).

(28) AKAP4 Sandwich ELISA

(29) Nunc F8 Maxisorp 96-well plates (Ref. 469949) are coated overnight at 4° C. with a 1/500 (volume/volume) dilution of AKAP4 C-terminus antibody (Santa Cruz Calif., Sc-66308 Goat polyclonal antibody). Wells are rinsed twice with PBS (Phosphate Buffer Saline pH 7.4, P3813 Sigma-Aldrich) Tween-20 (0.05%) and saturated with 200 μl of PBS added with 0.1% casein (weight/volume) during 1 hour at 37° C. Before incubation with the sperm sample, the plate is rinsed twice with PBS Tween-20 (0.05%). Sperm sample is diluted at 1/2000 in PBS and 100 μl is incubated in the plate overnight at 4° C. The plate is rinsed twice in PBS and incubated 2 hours at room temperature with the AKAP4 monoclonal antibody (Abcam, UK, Ab 55144, mouse monoclonal antibody) diluted at 1/500 in PBS added with 0.2% BSA (weight/volume). The plate is rinsed twice with PBS Tween-20 (0.05%) and incubated with horseradish peroxidase coupled anti-mouse IgG gamma-chain (Sigma-Aldrich, Ref A3673) at 1/4000 in PBS BSA (0.2% weight/volume) during one hour at 37° C. The immune-complexes are rinsed twice with PBS Tween-20 (0.05%) and the reaction is revealed with TMB (Sigma-Aldrich ref. T3405) in citrate/phosphate buffer pH 5 added 2 μl of H.sub.2O.sub.2 at 30%. After 30 minutes at room temperature, the reaction is stop with 50 μl of H.sub.2SO.sub.4 solution (10.32 mL, of H.sub.2SO.sub.4 in 89.78 mL of H.sub.2O). The optical density is measured at 450 nm. A standard curve of recombinant AKAP4 is performed with a serial dilution from 1.2 ng to 0.36 ng in PBS.

(30) Biostatistics Analysis

(31) Quantitative data are presented as mean with standard deviation. Quantitative data were analyzed and compared using the SAS software release 9.01 (SAS Institute INC, Cary, N.C.). SPQI Group A and B were compared in terms of age, sperm volume, cell count and motility. Pearson correlation coefficient was calculated to evaluate relationship between sperm parameters and age of men. Anova (variance analysis) and Student t-test (average analysis) were used to analyze the relationship between SPQI and semen parameters. Statistical significance was set at p<0.05 for all tests.

(32) Results

(33) Sperm Isolation and Analysis of Sub-Cellular Markers

(34) For the present study, 161 human semen samples were freshly collected. Men were aged of 34.7±5.8 years old (22-56) on average. Individuals had no infectious disease or treatment that could interfere with semen parameters. For all samples, the semen volume was over the lower limits of 1.5 mL (WHO 2010). The initial number of spermatozoa counted was of 101 10.sup.6±65.5 10.sup.6 spermatozoa/mL on average (385 10.sup.6±295 10.sup.6 spermatozoa/ejaculate) and therefore above the lower reference average limit of 15 10.sup.6 spermatozoa/mL. The PR motility was of 51.27%±12.27% over the lower reference limit of 32%. Normal spermatozoa morphology was of 33.8%±12.5% above lower reference limit of 4%. After selection, no epithelial or round cells were detected as established by light microscopy assessment of spermatozoa from crude and gradient isolation.

(35) Although the protocol for the isolation of spermatozoa was routinely used in clinic, molecular markers of the sperm compartments were evaluated. Thus, the microscopic observation of spermatozoa was associated to the analysis of protein markers of spermatozoa sub-compartments: proacrosin for the acrosome, histone H3 for the nucleus, HSP90 for the cytoplasm, cytochrome C for mitochondria and tubulin-β for the flagellum. This analysis was performed in five individual sperm protein samples. Proacrosin was resolved as a single band at 50 kDa. Proacrosin was not detected in T98 protein lysate. Histone H3 (17 kDa), HSP90 (90 kDa), Cytochrome C (15 kDa) and Tubulin-β (50 kDa) were detected both in protein lysate from sperm and T98 cells. Histone H3 and Tubulin-β were proportionally more abundant in spermatozoa than in T98 glioblastoma-derived cells. Together, the microscopic and molecular markers analyses did not show obvious alteration of spermatozoa following our isolation protocol.

(36) Spermatozoa 1D Proteome Pattern

(37) Whereas equal amounts of proteins were loaded, two major protein profiles were observed in Coomassie blue stained of 1D SDS-PAGE gels. The inventors observed that 49.6% of sperm samples (80/161) presented many and several well-individualized intense bands comprised between 15 to 160 kDa and this group of electrophoretic protein pattern was defined as group A. In contrast, 50.4% of sperm samples (81/161) were included in the group B characterized principally by the lower level or absence of the strong band at 110 kDa apparent molecular weight. Higher molecular weights polypeptides were also less intensively stained in this group B 1D protein pattern. To further analyze the profiles of human sperm protein expression, proteins with apparent molecular weights were selected such as histone H3 (17 kDa), GAPDH (37 kDa), tubulin-β (50 kDa) and HSP90 (90 kDa). Strong signals were detected for these proteins in sperm protein lysate from group A as well as in T98 glioblastoma cells. Histone H3 staining was comparable in both group A and group B. A decreased signal of GAPDH, tubulin-β and HSP90 were observed in sperm protein lysates from group B.

(38) Mass Spectrometry Identification of Proteins Contained in 110 kDa Bands in Coomassie Blue Strained 1D SDS-PAGE Gel

(39) The protein electrophoretic pattern of group B was suggestive of a proteolysis of polypeptides during spermatozoa isolation. Proteolysis of sperm proteins during protein extraction or due to a defective quantification was ruled out. Thus addition or protease inhibitors following sperm liquefaction or the loading of increasing volumes of protein samples did not modified the electrophoretic protein pattern. High molecular weights polypeptides and especially the intensively Coomassie blue stained band at 110 kDa was poorly stained and therefore, the Coomassie Blue-stained band was isolated and polypeptides present in this band were analysed by mass spectrometry. Two proteins were identified by Nano-LC MS/MS in the 110 kDa Coomassie-stained band of group A sample. These proteins corresponded to the A-kinase anchor protein 4 (AKAP4) and hexokinase-1 (HK1) (Table 2).

(40) TABLE-US-00002 TABLE 2 Identification of proteins contained in 110 kDa bands following in gel trypsic digestion of the polypeptides and mass spectrometry identification. Accession MW [kDa] pI Score #Pept. SC [%] Protein AKAP4_HUMAN 94.4 6.6 377.5 5 8.7 A-kinase anchor protein 4 OS = Homo sapiens GN = AKAP4 HXK1_HUMAN 102.4 6.4 118.6 2 3.2 Hexokinase-1 OS = Homo sapiens GN = HK1 MW = Theoretical molecular weight; pI = isoelectric point; Score = identification score: #Pept. = number of peptide sequenced; SC = overlap with the protein sequence (%).

(41) AKAP4 and HK1 Profile Explored by Immunoblotting

(42) AKAP4 and HK1 expression was analyzed by immunoblotting in more than 30 samples from group A and from group B (FIG. 1) with antibodies directing against N-ter and C-ter parts of both proteins. A single band at 85 kDa and 110 kDa corresponding to AKAP4 and HK1 respectively was detected in all human sperm samples from group A with antibody directing against N-ter part of both proteins. Two bands at 85 and 95 kDa corresponding to pro-AKAP4 and AKAP4 were observed in sperm samples from group A with a C-ter antibody. In sharp contrast, AKAP4 and HK1 were not or poorly detected in samples from group B. Instead of the 110 kDa band, several bands with molecular weights lower than 110 kDa were visualized for both proteins in group B samples whatever antibodies used but were different (FIG. 1). No signal corresponding to AKAP4 was detected in T98 cells lysate suggesting that the band detected in sperm protein is not artefactual. Our results strongly suggest that the 110 kDa bands observed by Coomassie Blue staining contain the AKAP4 and HK1 proteins and the disappearance of staining is likely related to change in AKAP4 and HK1 expression in group B samples when compared to group A.

(43) Sub-Cellular Localization of AKAP4 and HK1 by Immunochemistry

(44) AKAP4 and HK1 sub-cellular localization were analyzed by immunochemistry. As ever described in literature (Eddy, Toshimori, and O'Brien 2003), both proteins were located in fibrous sheath of sperm sperm flagellum. Any differences were observed between group A and B.

(45) Sperm Protein Quality Index (SPQI)

(46) After numerical analyses of Coomassie blue stained gels with Image J (FIG. 2A), bands with the strongest intensities were used to compare the 161 human sperm protein samples. Two windows of molecular weight were defined. Thus, protein bands between 15 to 30 kDa (Low molecular weights LMW) and secondly bands between 80 to 110 kDa (high molecular weights HMW) were selected. These two windows were reproducibly defined using molecular weight markers to calibrate the gels. Each band visualized on gel was defined as a pic by the software (FIG. 2B). For each window, area under curve was calculated (FIG. 2C). To establish a sperm proteome quality index (SPQI), the ratio between the area under curve from HMW and LMW band intensities (FIG. 2D) was calculated. When the 1D proteome was resolved as numerous intense protein bands over the whole 1D SDS-PAGE lane, the ratio was comprised between 1 and 2, named SPQI A and represented 80/161 samples. A second category of proteome profiles with a SPQI B below 1 or above 2 included 81/161 samples was defined.

(47) SPQI and Sperm Parameters

(48) Any correlation between sperm parameters and the age of men was revealed in whole population. With regards to the SPQI, the sperm parameters were reported. Group A were classified with a SPQI A (Table 3) whereas group B had an SPQI B (Table 3). SPQI B group (n=80, 35.6 years old) was 1.9 years older on average than SPQI A group (n=81, 33.7 years old). The number of spermatozoa per mL of semen (SPQI A=111.8 10.sup.6±72 spz/mL vs SPQI B 90.5 10.sup.6±56 spz/mL, p=0.041) as well as the PR motility (SPQI A=53.9±9% vs SPQI B 48.6±14%, p=0.006) were significantly higher for SPQI A than for SPQI B (Table 3). The PR motility showed the strongest statistical difference between SPQI A and B. Sperm morphology was not different between the two groups.

(49) TABLE-US-00003 TABLE 3 Sperm parameters related to the 1-D sperm proteome index. SPQI A (n = 80) SPQI B (n = 81) Sperm parameters Mean ± SD (min-max) Mean ± SD (min-max) P (t-test) Age (years) 33.7 ± 5.1 (22-46)  35.6 ± 6.3 (23-56) 0.043 Volume (mL)  3.7 ± 2.2 (1.5-5.9)    4.2 ± 1.1 (1.9-5.7) NS Sperm count (10.sup.6/mL)  111.8 ± 72.4 (23.9-380)   90.5 ± 56.3 (15.5-300) 0.041 Progressive motility (%) 53.9 ± 9.4 (40-70) 48.6 ± 14.1 (35-70) 0.006 Normal sperm morphology (%) 33.6 ± 11.8 (17-65)  33.9 ± 13.1 (16-58) NS SPQI = Sperm Protein Quality Index; SD = Standard Deviation; NS = No Significant.

(50) Full-Length AKAP4 Sandwich ELISA

(51) In order to discriminate between group A and group B, the inventors have developed an ELISA with a C-terminus goat anti-AKAP4 antibody and a N-terminus mouse monoclonal anti-AKAP4 to reveal. Accordingly to our observations, AKAP4 proteolysis occur at the C-terminus of the protein. The ELISA would therefore enable to quantify the amount of full-length AKAP4 whereas, the proteolysis of AKAP4 would lead to a reduce amount of the full-length and therefore a lower level of detection. Thus, the inventors expected to detect subject from the group A and a reduced amount of AKAP4 should be detect in group B. The inventors first determined the functionality of the assay using incremental quantity of the human recombinant AKAP4 protein. The sandwich ELISA enables the detection of 10 ng of recombinant protein and reach a plateau over 100 of ng (FIG. 3A). Using the assay, the quantity of AKAP4 was measured in serial dilution of sperm sample from a subject of the group A and one subject of the group B. In the sperm sample of the group A, the DO was increasing from dilution of 1/10000 to 1/1000. In sharp contrast, the DO only slightly increases in sperm sample from the subject of group B. Noticeably, at a dilution of 1/2000 the DO was of 0.7 for the sample of group A whereas the DO was of 0.47 for the sample of the group B subject, knowing that the basal background signal is of 0.35. This results therefore shows that a sandwich ELISA using a C-terminus anti-AKAP4 antibody to capture the protein enable to discriminate between the two groups.

(52) Distribution of SPQI Group A and B in ART and Success of Birth

(53) To date, among the 161 couples that were followed at the Lille Hospital Fertility department, ART has been offered to 150 (93.1%) of them. Intrauterine insemination (IUI) has been performed in 76 couples, current in vitro fecondation (IVFc) was performed in 60 couples and 14 of them were proposed intracytoplasmic sperm injection (ICSI). Independently of the ART proposed to patients, frequency of birth success between group A (34%) and group B (22%) was slightly higher for group A. However, differences were more pronounced depending of the ART performed. Thus, among couples with IUI 19 birth were observed. The frequency of success was not significantly differently between categorized in group A (8/35; 23%) or group B (11/41; 27%). Among couples with IVFc, 42.8% (15/35) of success was observed with men belonging to group A whereas a lower frequency of success (28%, 7/25) for men of the group B. Interestingly, among couples with ICSI, 6 births were obtained 2/3 (66.6%) for men included in the group A and 4/11 (36.3%) for men in the group B.

(54) Discussion

(55) In order to further investigate the potential modification of the sperm proteome that could be associated to the sperm quality index, a simple biochemical method enabling to define a sperm proteome quality index, herein named SPQI, based on analysis of the whole 1D sperm proteome profile and/or proteolysis of two major sperm proteins AKAP4 and Hexokinase 1 have been designed. Over the analysis of sperm proteomes from 161 ejaculates with normal sperm parameters widely above lower references limits given by WHO (World Health Organization 2010), two categories of proteome profiles were established and a modification of this proteome was identified. SPQI is significantly correlated with lower progressive motility of spermatozoa. To the best our knowledge, it is the first study to report a modified 1D proteome profile in normozoospermic samples significantly associated with a change in the progressive motility of spermatozoa in men with normozoospermia.

(56) A method was set up to select most sperm cells and extract sperm protein. Currently, they are two methods to isolate spermatozoa, the swim-up method and the density gradient. The swim-up method selects the most motile spermatozoa. However, to study the whole sperm proteome from ejaculate the lower selection of spermatozoa was necessary. A single-step density gradient centrifugation method was used to isolate spermatozoa from seminal plasma and round cells. Moreover, culture medium for spermatozoa used for sperm washing contains protein such as albumin and was replaced by a Tris buffer. In sperm pellet, round cells were rarely or often not observed and the morphology of spermatozoa was preserved. To evaluate protein extraction and protein integrity, markers from every sperm cell compartment were explored. For example, proacrosin belongs to acrosome and is cleaved in active form as β-acrosin during acrosomal reaction or consequently to sperm manipulation (Gallo et al. 1991; Zahn et al. 2002). After sperm selection proacrosin was visualized as well by immunoflurorescence (not shown) and detected by immunoblotting in sperm lysates. Together, these proteins were detected in sperm protein lysates, these data suggest that method is not selective from one cell-compartment and molecular integrity was preserved.

(57) After lysis and protein extraction, the protein quantity loaded for 1D SDS-PAGE was equal for all samples. However, the 1D SDS-PAGE profile is different. To exclude the possibility that the differences between 1D protein profiles could not result from an artifact from the method used to determine the protein concentration, BCA or the Bradford methods for protein quantification were compared. Both were giving similar sperm proteome profile. However, protein concentration determined with a kit does not indicate on the integrity of the polypeptide. Moreover, some sperm protein profile were characterized by the loss of Coomassie blue stained polypeptides of molecular weights over 50 kDa, an explanation could be that sperm proteins might be degraded during liquefaction or sperm selection. Therefore, all samples were freshly prepared after recovering ejaculates and the liquefaction time was systematically of 30 minutes. Protease inhibitors were added or not after semen liquefaction but addition or these protease inhibitors did neither improve the protein profile nor increase the number of polypeptides bands. Taken together, these observations suggest that is the protein profile observed in group B is resulting from the partial proteolysis of sperm proteome, it may possibly occur during liquefaction.

(58) Despite having normal sperm parameters, sperm is a heterogeneous complex fluid with a variable number of abnormal spermatozoa. Without having an experimental explanation to explain the difference between 1D SDS-PAGE profile, two groups were defined according to the 1D proteome quality. The group A included samples with many intense and homogeneous bands visualized between 15 to 110 kDa. Group B represented samples that showed a decrease or a disappearance of high molecular weight proteins, with regards to the sensitivity of Coomassie blue staining Bands from 110 kDa of 1D-SDS PAGE coomassie blue staining gel was analyzed and characterized by mass spectrometry. AKAP4 and Hexokinase 1 were significantly identified and there expressions were explored by western blotting. Group A presented a single band for both proteins at about 100 kDa corresponding to protein expression. Group B showed many bands from 20 to 50 kDa and 25 to 50 kDa for AKAP4 and Hexokinase 1 respectively suggesting a modification of these protein expressions. Histone H3 expression was not disturbed regardless of profile. This can be explained by biochemical proprieties of this basic protein (pI>11). Indeed, proteases implied in this elimination are probably different from the other proteins of spermatozoa, that is why we could observe no differences in its expression in our samples. Hexokinase 1 is a predominant glycolytic enzyme presents in spermatozoa (Eddy, Toshimori, and O'Brien 2003; Nakamura et al. 2008). It is mainly localized in fibrous sheath of spermatozoa and uses ATP to phosphorylate glucose to glucose-6-phosphate necessary for sperm motility. AKAP4 is a structural protein identified in fibrous sheath and that binds cAMP-dependant-kinase (PKA) to initiate and mediate sperm motility (Brown 2002; Moretti et al. 2007). Its protein is encoded by a X-linked genes into a precursor: pro-AKAP4 (97 kDa) that cleaves in AKAP4 (82 kDa) (Turner et al. 1998; Eddy, Toshimori, and O'Brien 2003). Men with dysplasia of fibrous sheath presents a gene sperm defect resulting in short or irregular flagella with disorganized axonemes (Baccetti 2005a; Baccetti 2005b; Baccetti et al. 2005). Immunostaining of sperm samples with AKAP4 antibody showed an incomplete assembled fibrous sheath scaffold (Baccetti et al. 2005). Molecular analysis demonstrated a mutation in gene that encoded AKAP4 (Turner et al. 2001; Baccetti 2005b). Recently, Xu et al. showed an down-regulation of AKAP4 in infertile normozoospermic men (Xu et al. 2012). According to literature, bands observed in our immunoblotting in Group B did not result from maturation of pro-AKAP4 to AKAP4. Nethertheless, immunochemistry did not revealed differences in localization for both proteins between Groups A and B. Moreover, sperm morphology was not modified. Our results suggest a proteolysis of AKAP4 and hexokinase 1 in group B independently of sperm morphology. Proteolysis is a known to be as physiological phenomenon in spermatozoa. Ubiquitin dependent proteolysis is one of most important pathways implied in spermatogenesis, sperm quality control and fertilization (Sutovsky 2003; Sutovsky 2011). Moreover, proteasome mediated proteolysis implied in acrosomal reaction and digestion of vitelline membrane (Zimmerman and Sutovsky 2009; Rawe et al. 2008; Kong, Diaz, and Morales 2009). After fertilization, some proteins get an ubiquitin-tag as prohibitin, to be eliminated by oocyte (Thompson 2003). Proteasome pathways could be implied in proteolysis of AKAP4 and hexokinase 1.

(59) After analysis with ImageJ software, a quality index was determined and its relationship with sperm parameters was evaluated by biostatistical analysis. Two homogenous groups were constituted. Group A presents a quality index comprised between 1 to 2 corresponding to an homogeneous sperm protein profile in every molecular weight. Group B contained samples characterized by sperm proteome with a disappearance of bands from high molecular weight translated by a quality index lower/over than 1 or 2. Average from each sperm parameters considered as fertility markers (count of cells, motility and morphology of spermatozoa) were compared in both groups. Progressive motility appears to be strongly significantly different in group A compared with group B. Together, these results suggest that SPQI are a marker of progressive motility of spermatozoa and in relationship with a modification of AKAP4 and Hexokinase 1 expression.

(60) In conclusion, in the present invention, the inventors suggest that the sperm proteome quality index may represent a good in vitro assay to evaluate sperm quality. This SPQI relies on the integrity of two major sperm proteins AKAP4 and Hexokinase 1. Their proteolysis may serve as an important indicator of lower progressive motility and more widely of sperm quality and used to understand mechanism of unknown sperm dysfunction. Actually, WHO criteria 2010 recommend a decrease of normal value for sperm parameters particularly for motility and morphology. Our results suggest that better sperm parameters are correlated with our biochemical quality index. Moreover, our preliminary data with regards to the success at birth in between group A and group B using ART suggest a general higher success rate for men of group A and even more obviously for IVFc and IUI technics while may recommend IUI for group B men since the rate of success is very similar to that observed in group A. Altogether, our data suggest that the sperm quality index could really useful to associated with current laboratory sperm analysis in order to further predict the chance of conceive success in ART.

Example 2

Comparison of IIU Outcome Variables According to the Groups A and B

(61) The proteolyzed profile (group B) is indicative of a bad prognosis and bad sperm quality. The group B is associated with bad indicators such as higher number of biochemical pregnancy, abortion (miscarriage) than group A (Table 5).

(62) TABLE-US-00004 TABLE 4 Sperm parameters of semen samples according to the groups A (1D-SDS PAGE, non-proteolyzed proteome) and B (proteolyzed proteome) Group A Group B WHO 2010 (mean ± SD) (mean ± SD) recomman- n = 37 n = 38 p dations Volume (ml) 3.5 ± 1.1 3.6 ± 1.sup.  NS >1.5 Sperm count  110 ± 60.2 108.1 ± 74.6  NS >15 (×10.sup.6/mL) Sperm count 369.8 399.4 NS >39 (×10.sup.6/ejac) Ratio sperm count  0.3  0.3 NS / (mL/ejac) Progressive motility 54.2 ± 9.7  49.1 ± 11.3 0.03 >32 (a + b)(%) Normal morphology 36.9 ± 14.sup.  34.4 ± 12.2 NS 15 (%) MAI 1.6 ± 0.1 1.6 ± 0.2 NS <1.6 mL, millilitter; ejac, ejaculation; MAI, multiple abnormalities index; NS, non significant; WHO, world health organization

(63) TABLE-US-00005 TABLE 5 Comparison of IIU outcome variables according to the groups A and B Group A Group B (mean ± SD) (mean ± SD) n = 37 n = 38 p Patient age (yrs) 33.8 ± 4.5 35.6 ± 5.6 NS BMI patient 27.1 ± 3.sup.  25.2 ± 3.3 NS BMI partner 23.6 ± 3.9 24.9 ± 5.3 NS Tobacco patient (cig/d) 4.6  5.4 NS Tobacco partner (cig/d) 1.1  0.8 NS Duration of infertility (yrs)  3.5 ± 2.1  5.1 ± 3.2 NS Total cycles (n) n = 116 n = 106 Cycles  3.1 ± 1.8  2.8 ± 1.5 NS Biochemical pregnancy (%) 9.5 20.7 0.02 Clinical pregnancy (%) 6.9 10.4 NS Delivery (%) 6   10.4 NS Abortion (%) 3.5 10.3 0.04 SD, standard deviation; yrs, years; BMI, Body mass index; cig/d, cigarettes/day; n, number; NS, non significant

Example 3

Comparison of IVF/ICSI Outcome Variables According to the Groups A and B

(64) The group A is significantly associated with a higher embryo quality, embryo implantation, pregnancy and delivery than the group B (Table 7).

(65) TABLE-US-00006 TABLE 6 Sperm parameters of semen samples according to the groups A and B Group A Group B WHO 2010 (mean ± SD) (mean ± SD) recomman- n = 38 n = 35 p dations Volume (mL) .sup. 4 ± 1.8 3.7 ± 1.sup.  NS >1.5 Sperm count 105.4 ± 75.5  92.8 ± 67.2 NS >15 (×10.sup.6/mL) Sperm count .sup. 404 ± 352.4 .sup. 335 ± 258.3 NS >39 (×10.sup.6/ejac) Ratio sperm count 0.3 ± 0.1 0.3 ± 0.1 NS / (mL/ejac) Progressive motility 52.5 ± 10.sup.  47.7 ± 15.6 NS >32 (a + b) (%) Normal morphology 35.2 ± 11.3 32.4 ± 15.4 NS 15 (%) MAI 1.6 ± 0.1 1.6 ± 0.2 NS <1.6 mL, millilitter; ejac, ejaculation; MAI, multiple abnormalities index; NS, non significant; WHO, world health organization

(66) TABLE-US-00007 TABLE 7 Comparison of IVF/ICSI outcome variables according to the groups A and B Group A Group B (mean ± SD) (mean ± SD) n = 38 n = 35 p Patient age (yrs) 34.1 ± 4.8  36.2 ± 6   NS Partner age (yrs) 30.3 ± 3.8  33.1 ± 4.4  NS BMI patient 27.4 ± 1.5  23.8 ± 1.7  NS BMI partner 22.8 ± 3    23 ± 4.5 NS Tobacco patient (cig/d) 6 8 NS Tobacco partner (cig/d) 4 3 NS Duration of infertility (yrs) 4.2 ± 1.4 4.6 ± 2.sup.  NS Total cycles (n) n = 63  n = 64  Cycles 1.7 ± 0.8 1.8 ± 1.sup.  NS Total oocytes retrieved 9.2 ± 5.5 7.5 ± 3.6 NS Atretic oocytes .sup. 1 ± 1.6 0.6 ± 1.1 NS Immature oocytes .sup. 1 ± 1.9 1.1 ± 1.3 NS Injected oocytes .sup. 7 ± 4.4 5.8 ± 3.2 NS Pronuclear assessment 1PN 0.2 ± 0.5 0.2 ± 0.6 NS 2PN 5.4 ± 3.6 4.4 ± 2.8 NS Polyploid 0.3 ± 0.6 0.3 ± 0.6 NS Fertilization rate (%) 79 ± 20 77 ± 22 NS Early clivage (%) 48 ± 39  52 ± 0.4 NS Total embryos produced (n) n = 321 n = 269 Embryos 5.1 ± 3.3 4.2 ± 2.7 NS Embryo quality A 1.6 ± 1.8 0.9 ± 1.1 0.017 B 0.2 ± 0.5 0.2 ± 0.5 NS C 3.2 ± 2.7 3.1 ± 2.3 NS Freezed embryos 1.8 ± 2.sup.  .sup. 1 ± 1.7 0.01  Total embryos tranfered 1.2 ± 1.9 1.1 ± 1.9 NS Clinical pregnancy (%) 44 ± 50 19 ± 39 0.002 Delivery (%) 39 ± 49 19 ± 39 0.01  Implantation (%) 27 ± 42 12 ± 32 0.006 Abortion (%) 10 ± 30  6 ± 24 NS SD, standard deviation; yrs, years; BMI, body mass index; cig/d, cigarettes/day; n, number; NS, non significant; PN, pronucleus

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