Characterization of the Sperm Proteome and Reproductive Outcomes with in Vitro, Fertilization after a Reduction in Male Ejaculatory Abstinence Period

Semen samples from men after a short ejaculatory abstinence show improved sperm quality and result in increased pregnancy rates, but the underlying mechanisms remain unclear. Herein, we report that ejaculates from short (1–3 h) compared with long (3–7 days) periods of abstinence showed increases in motile sperm count, sperm vitality, normal sperm morphology, acrosome reaction capacity, total antioxidant capacity, sperm mitochondrial membrane potential, high DNA stainability, and a decrease in the sperm DNA fragmentation index (p, < 0.05). Sperm proteomic analysis showed 322 differentially expressed proteins (minimal fold change of ±1.5 or greater and p, < 0.05), with 224 upregulated and 98 downregulated. These differentially expressed proteins are profoundly involved in specific cellular processes, such as motility and capacitation, oxidative stress, and metabolism. Interestingly, protein trimethyllysine modification was increased, and butyryllysine, propionyllysine, and malonyllysine modifications were decreased in ejaculates from a short versus, long abstinence (p, < 0.05). Finally, the rates of implantation, clinical pregnancy, and live births from in vitro, fertilization treatments were significantly increased in semen samples after a short abstinence. Our study provides preliminary mechanistic insights into improved sperm quality and pregnancy outcomes associated with spermatozoa retrieved after a short ejaculatory abstinence. Semen samples from men after a short ejaculatory abstinence show improved sperm quality and result in increased pregnancy rates, but the underlying mechanisms remain unclear. Herein, we report that ejaculates from short (1–3 h) compared with long (3–7 days) periods of abstinence showed increases in motile sperm count, sperm vitality, normal sperm morphology, acrosome reaction capacity, total antioxidant capacity, sperm mitochondrial membrane potential, high DNA stainability, and a decrease in the sperm DNA fragmentation index (p, < 0.05). Sperm proteomic analysis showed 322 differentially expressed proteins (minimal fold change of ±1.5 or greater and p, < 0.05), with 224 upregulated and 98 downregulated. These differentially expressed proteins are profoundly involved in specific cellular processes, such as motility and capacitation, oxidative stress, and metabolism. Interestingly, protein trimethyllysine modification was increased, and butyryllysine, propionyllysine, and malonyllysine modifications were decreased in ejaculates from a short versus, long abstinence (p, < 0.05). Finally, the rates of implantation, clinical pregnancy, and live births from in vitro, fertilization treatments were significantly increased in semen samples after a short abstinence. Our study provides preliminary mechanistic insights into improved sperm quality and pregnancy outcomes associated with spermatozoa retrieved after a short ejaculatory abstinence. In 1952, MacLeod and Gold surveyed fertile men and suggested that the most motile spermatozoa were found among samples from men with fewer than 4 days of ejaculatory abstinence (1.Macleod J. Gold R.Z. The male factor in fertility and infertility. V. Effect of continence on semen quality.Fertil. Steril. 1952; 3: 297-315Abstract Full Text PDF PubMed Google Scholar). In 1979, Schwartz et al., using multivariate statistical techniques, were the first to evaluate the within-subject variability for semen characteristics in normal subjects who maintained an approximately normal ejaculatory frequency. It has been shown that there is very large within-subject variability in semen characteristics, but certainly one influential factor is the period of abstinence (2.Schwartz D. Laplanche A. Jouannet P. David G. Within-subject variability of human semen in regard to sperm count, volume, total number of spermatozoa and length of abstinence.J. Reprod. Fertil. 1979; 57: 391-395Crossref PubMed Scopus (145) Google Scholar). Recently, Alipour et al., using standardized semen analysis, characterized the intra-individual differences in semen samples of normozoospermic men collected after 2 h versus, 4–7 days of abstinence. A higher percentage of motile spermatozoa with higher velocity and progressive motility was detected in the 2-hour semen samples (3.Alipour H. Van Der Horst G. Christiansen O.B. Dardmeh F. Jorgensen N. Nielsen H.I. Hnida C. Improved sperm kinematics in semen samples collected after 2 h versus 4–7 days of ejaculation abstinence.Hum. Reprod. 2017; 32: 1364-1372Crossref PubMed Scopus (34) Google Scholar). Likewise, ejaculates from men with oligozoospermia exhibited a significant improvement in sperm motility, progression, and morphology when a second ejaculate was produced within only 40 min of the first (4.Bahadur G. Almossawi O. Zeirideen Zaid R. Ilahibuccus A. Al-Habib A. Muneer A. Okolo S. Semen characteristics in consecutive ejaculates with short abstinence in subfertile males.Reprod. Biomed. Online. 2016; 32: 323-328Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition, it is noteworthy that sperm deoxyribonucleic acid (DNA) fragmentation can be significantly reduced by short-term recurrent ejaculation (5.Gosalvez J. Gonzalez-Martinez M. Lopez-Fernandez C. Fernandez J.L. Sanchez-Martin P. Shorter abstinence decreases sperm deoxyribonucleic acid fragmentation in ejaculate.Fertil. Steril. 2011; 96: 1083-1086Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Higher pregnancy rates following intrauterine insemination have also been observed with semen obtained after an abstinence of less than 2 days (6.Marshburn P.B. Alanis M. Matthews M.L. Usadi R. Papadakis M.H. Kullstam S. Hurst B.S. A short period of ejaculatory abstinence before intrauterine insemination is associated with higher pregnancy rates.Fertil. Steril. 2010; 93: 286-288Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Prolonged abstinence decreases pregnancy rates after intrauterine insemination, independent of sperm motility parameters, suggesting that abstinence intervals should be controlled in studies of pregnancy outcomes after using assisted reproductive technology (7.Jurema M.W. Vieira A.D. Bankowski B. Petrella C. Zhao Y. Wallach E. Zacur H. Effect of ejaculatory abstinence period on the pregnancy rate after intrauterine insemination.Fertil. Steril. 2005; 84: 678-681Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, whether semen samples after a short abstinence improve rates of implantation, pregnancy, and live births, and what associated molecular mechanisms are involved, remains largely unknown. Notably, proteomic-based approaches are being applied to the study of cellular and developmental processes of gamete cells, and this method is currently being used to study sperm maturation and function because of the low level of transcriptional and translational activity in spermatozoa and the fact that sperm functions are principally controlled at the protein level (8.Brohi R.D. Huo L.J. Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.OMICS. 2017; 21: 245-256Crossref PubMed Scopus (48) Google Scholar, 9.Zhou T. Xia X. Liu J. Wang G. Guo Y. Guo X. Wang X. Sha J. Beyond single modification: Reanalysis of the acetylproteome of human sperm reveals widespread multiple modifications.J. Proteomics. 2015; 126: 296-302Crossref PubMed Scopus (11) Google Scholar). In the present study, we first compared the sperm characteristics and outcomes of in vitro, fertilization (IVF) 1The abbreviations used are: IVF, In vitro, fertilization; GO, gene ontology; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine Iodide; KEGG, kyoto encyclopedia of genes and genomes; MMP, mitochondrial membrane potential; PBS, phosphate buffered saline; PCA, principal components analysis; PSA, pisum sativum agglutinin; ROS, reactive oxygen species; TAC, total antioxidant capacity; WHO, World Health Organization.1The abbreviations used are: IVF, In vitro, fertilization; GO, gene ontology; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine Iodide; KEGG, kyoto encyclopedia of genes and genomes; MMP, mitochondrial membrane potential; PBS, phosphate buffered saline; PCA, principal components analysis; PSA, pisum sativum agglutinin; ROS, reactive oxygen species; TAC, total antioxidant capacity; WHO, World Health Organization. using semen samples collected following short (1–3 h) or long (3–7 days) periods of abstinence. We then used a tandem mass tag (TMT)-based quantitative proteomic approach to investigate proteomic changes in spermatozoa after reduced male ejaculatory abstinence. Our results suggest that the molecular events occurring in sperm proteins may play an important part in sperm quality and reproductive potential after reduced periods of male ejaculatory abstinence. The study was conducted in accordance with ethical standards and the Helsinki Declaration of 1975. This study was approved by the Institutional Review Board at China Medical University and informed written consent was obtained from all participants prior to the initiation of the study. The participants were divided into 3 groups. Group I was a control group of 361 couples and an experimental group of 167 couples who underwent their first round of IVF. Men in the control group provided a semen sample after 3–7 days of abstinence, while in the experimental group, men were told the possible benefits of producing a consecutive ejaculate and provided a semen sample after 3–7 days of such abstinence followed by another sample after only 1–3 h. All the women included in the study had normal ovarian reserve (designated as an anti-Müllerian hormone concentration of ≥ 1.1 ng/ml), and normal serum thyroid-stimulating hormone and prolactin concentrations. Clinical pregnancy was diagnosed by ultrasonographic evidence of intrauterine fetal heart beat at 7 weeks. Implantation, clinical pregnancy, early miscarriage, and live birth rates in fresh IVF or freeze-all cycles were compared. The subjects' characteristics are given in Table I. Group II was an experimental group of 20 normal men who provided a semen sample after 3–7 days of abstinence followed by another sample after only 1–3 h, which were then used for physiologic and biochemical analyses of spermatozoa. Group III was an experimental group of 5 normal men who provided a semen sample after 3–7 days of abstinence followed by another sample after only 1–3 h, which were used for sperm proteomic and protein modification analyses. The characteristics of the study groups are shown in Fig. 1. None of the included men had a medical history of orchitis, unilateral orchiectomy, vasectomy, ejaculatory disorders, genetic diseases, or other urinary system diseases; and were asked to maintain 3–7 days of ejaculatory abstinence before sample collection. The abstinence time was calculated by how long it took men to provide the consecutive ejaculate. Participants with a possible pituitary lesion on magnetic resonance imaging or with karyotype abnormalities were excluded.Table IBaseline characteristics of participantsEjaculate 1: 3–7 days of abstinenceEjaculate 2: 1–3 hours of abstinenceNumber of fresh cycles361167Age of women (S.D.)31.34 (3.82)30.73 (3.45)BMI of women (S.D.)23.46 (3.80)22.59 (3.74)Age of men (S.D.)32.40 (4.47)32.06 (4.85)BMI of men (S.D.)24.79 (3.51)24.41 (3.53)Primary infertility (%)232 (64.3)112 (67.1)Duration of infertility (S.D.)3.91 (2.86)3.56 (2.31)Percentage of ICSI (%)28 (7.8)12 (7.2)Retrieved oocyte number (S.D.)11.38 (6.89)11.06 (5.56)IVF protocol (%)Standard long GnRH agonist249 (69.0)113 (67.7)GnRH antagonist112 (31.0)54 (32.3)BMI, body mass index; ICSI, intracytoplasmic sperm injection; IVF, in vitro, fertilization; GnRH, gonadotropin-releasing hormone. Open table in a new tab BMI, body mass index; ICSI, intracytoplasmic sperm injection; IVF, in vitro, fertilization; GnRH, gonadotropin-releasing hormone. Two semen samples were obtained in a private room by masturbation into a sterile wide-mouthed plastic container after the recommended 3–7 days of abstinence, followed by 1–3 h of further abstinence. After liquefaction at 37 °C for 30 min, conventional semen analysis was conducted in accordance with guidelines from the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen (10.WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th edn. WHO Press, World Health Organization, Geneva, Switzerland2010Google Scholar), including semen volume and sperm concentration, count, motility, morphology (Papanicolaou staining), and viability (eosin-nigrosin staining). Sperm motion parameters were evaluated using a CASA system (WLJY 9000, Weili New Century Science and Tech Dev, Beijing, China). The percentage of motile spermatozoa was defined using WHO grades: grade A, rapid progressive motility with a velocity ≥ 25 μm/s at 37 °C; grade B, slow/sluggish progressive motility with a velocity ≥ 5 μm/s, but < 25 μm/s at 37 °C; grade C, nonprogressive motility with a velocity < 5 μm/s at 37 °C; and grade D, immotile spermatozoa at 37 °C (10.WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th edn. WHO Press, World Health Organization, Geneva, Switzerland2010Google Scholar). Grade A and B spermatozoa were defined as motile spermatozoa, and grade C spermatozoa were excluded from this analysis. Parameters for each semen sample were measured twice in succession by 2 well-trained technicians. The TAC of spermatozoa was evaluated by using a Total Antioxidant Capacity Assay Kit with the ferric-reducing ability of plasma method (Beyotime Institute of Biotechnology, Shanghai, China). ROS of spermatozoa was evaluated by using an Oxidative Stress Detection Kit (Haling Biotech Shanghai, China). the is to the and oxidative it that can be detected by a The of sperm was detected by using the which the method Science China). Two spermatozoa were with the of spermatozoa with and the percentage of as sperm The sperm DNA fragmentation index and high DNA were detected by using the Sperm Assay Biotech China). of sperm with to evaluate the of and Sperm was with a which the from to The of DNA was then expressed as the DNA fragmentation In addition, the of to for DNA is to spermatozoa with The sperm acrosome reaction was detected using the Sperm Biotech were with and and was measured with a Spermatozoa the acrosome reaction had a of PSA, and most of the sperm was observed to be Sperm was detected by using a 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine Biotech is a that is by all it and which can be measured with a The were normal semen parameters total motility, and to the WHO Laboratory Manual for the Examination and Processing of Human Semen (10.WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th edn. WHO Press, World Health Organization, Geneva, Switzerland2010Google diseases; used in the and a pregnancy sperm were as M. S. T. A. S. S. R. Human Spermatozoa and Proteomics. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). semen was at for 20 min at 4 °C to spermatozoa from The was then 3 with and was to 30 of protein was by and to The were in and for min at room and with to malonyllysine trimethyllysine and at 4 The were with followed by at room with and detected using samples of spermatozoa from ejaculates following 3–7 days and 1–3 h of abstinence were by in were by and the were human protein using the mass were the human and with a was as to 2 The mass for was at and for The of a on was as a and on was as a The protein rate was to < J. M. high rates, mass and protein PubMed Scopus Google Scholar). A was only used for it the of a and only proteins at p, < were The mass were in the the A. N. J. I. G. Y. F. T. Wang R. H. of the and 2016; PubMed Scopus Google with the A of the is given in the and the on proteins and is given in and components analysis was to and the in sperm proteins in ejaculate and ejaculate 2 by using and We and for 2 and 3 principal components Fig. The results showed that each group and among After the the approach was to the differentially expressed genes by using the expressed proteins were by an change of and p, < the differentially expressed proteins is in Table We then analysis on the differentially expressed and all differentially expressed proteins were to their cellular and molecular for differentially expressed the of and were and the was used for The was used to the in the and was used to the among of analyses are given in the we compared sperm proteins and 322 differentially expressed proteins with 4 male reproductive and using the The for all 4 was from The Human The and consecutive ejaculates were as and the was used when parameters the 2 The was used to for The are as differences in the were evaluated by or as and were significant at p, < Notably, as shown in Table the implantation, clinical pregnancy, and live birth rates were significantly increased by and from ejaculates after 1–3 h of abstinence compared with 3–7 days of abstinence in In addition, the live birth rate was also higher from ejaculates after 1–3 h of abstinence to 3–7 days of abstinence in fresh IVF and the statistical (p, 1: 3–7 days of abstinenceEjaculate 2: 1–3 hours of was used to pregnancy birth to the first in a freeze-all was pregnancy was birth was to the first in a freeze-all The was used to was Open table in a new tab the semen volume and total sperm were significantly the sperm concentration and motile sperm were significantly increased in ejaculates after 1–3 h of abstinence compared with 3–7 days of abstinence. was significant in immotile sperm 1–3 h and 3–7 days of abstinence Sperm vitality, normal sperm morphology, acrosome reaction capacity, and total antioxidant were in ejaculates after 1–3 h of abstinence compared with 3–7 days of abstinence was also an in sperm and DNA in ejaculates after 1–3 h of abstinence compared with 3–7 days of abstinence and the sperm DNA fragmentation index was significantly decreased after reduced male abstinence there was significant in sperm or ROS 1–3 h and 3–7 days of abstinence and investigate the of spermatozoa in ejaculates 3–7 days versus, 1–3 h of abstinence, we quantitative proteomic studies using and analysis A total of proteins were from sperm among which proteins were expressed proteins were by an change of and p, < by analysis Table A total of 322 proteins as differentially including 224 upregulated and 98 proteins and The differentially expressed proteins were using and the to further investigate their These proteins were found to be involved in specific cellular processes such as sperm motility and sperm and antioxidant and and and of with analysis also that there was a of among proteins Fig. These suggested that capacitation, and gene were in spermatozoa after a reduced period of male abstinence. the proteomic analysis, of protein modifications of spermatozoa were and sperm butyryllysine, propionyllysine, and malonyllysine modifications were significantly decreased However, trimethyllysine modification was increased after reduced male abstinence. was significant in sperm or modifications 1–3 h and 3–7 days of abstinence there are studies on the of a short period of male ejaculatory abstinence (1–3 h) on reproductive We for the first time that the and clinical pregnancy the live birth significantly increased using a semen sample obtained after 1–3 h of abstinence in a than in a fresh IVF We from the that reproductive outcomes can be significantly improved in cycles compared with fresh IVF cycles because of the of the after controlled ovarian J. T. L.J. versus clinical with and clinical Reprod. PubMed Scopus Google Scholar, M. M. F. M. S. fresh Steril. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). It that cycles an are to show improved quality and reproductive outcomes after IVF. we used proteomic to the potential molecular of spermatozoa in ejaculates after 3–7 days and 1–3 h of ejaculatory abstinence. We compared sperm proteins and 322 differentially expressed proteins with 4 male reproductive and and we observed a the in of proteins Fig. The is the male reproductive and is for the of spermatozoa as as It that may play an important in the reproductive potential of spermatozoa after reduced periods of ejaculatory abstinence. It is significant that differentially expressed proteins were found to be involved in sperm motility and capacitation, and that the acrosome reaction of spermatozoa was after 1–3 h of abstinence. These results suggested that spermatozoa may a greater motility and ability after a reduced period of male abstinence. evidence that the ROS and antioxidant in plasma may result in male infertility oxidative M. N. M. A. Oxidative and sperm DNA fragmentation in fertile and 2017; Scopus Google Scholar). However, it should be that semen is an of from the and the and the from each may ROS and P.B. A. S. Matthews M.L. Usadi N. Hurst B.S. of ejaculatory abstinence on total antioxidant and sperm membrane Steril. Full Text Full Text PDF PubMed Scopus Google Scholar). we measured sperm ROS and TAC in the present study. Interestingly, ROS change sperm TAC was significantly improved after reduced male and in this spermatozoa after a abstinence may antioxidant ROS in In addition, evidence has suggested a possible among ROS, DNA MMP, and reproductive including a significant in with high ROS decreased mitochondrial DNA and low O. G. P. R. N. G. E. R. Sperm of with show molecular and 2016; PubMed Scopus (48) Google mitochondrial which is associated with increased ROS and DNA fragmentation F. P. R. increases reactive oxygen and DNA fragmentation in human Reprod. 2015; PubMed Scopus (49) Google sperm DNA fragmentation and MMP, which when may be to semen parameters for the of S. B. A. M. B. Sperm DNA fragmentation and mitochondrial membrane potential are for than sperm Steril. 2016; Full Text Full Text PDF PubMed Scopus Google and sperm DNA which is associated with male recurrent pregnancy and a high of S. R. Oxidative in in sperm DNA and 2017; PubMed Scopus Google Scholar). In with that sperm TAC was that the DNA fragmentation index was and that was increased after a period of abstinence as short as 1–3 h. This with provided evidence of a potential for abstinence periods in IVF or the that how proteins in N. I. Posttranslational modifications of on 2017; PubMed Scopus Google Scholar), in spermatozoa, as their transcriptional and translational processes are sperm maturation (8.Brohi R.D. Huo L.J. Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.OMICS. 2017; 21: 245-256Crossref PubMed Scopus (48) Google Scholar, 9.Zhou T. Xia X. Liu J. Wang G. Guo Y. Guo X. Wang X. Sha J. Beyond single modification: Reanalysis of the acetylproteome of human sperm reveals widespread multiple modifications.J. Proteomics. 2015; 126: 296-302Crossref PubMed Scopus (11) Google Scholar, of modifications of 2016; PubMed Scopus Google Scholar). is evidence that (8.Brohi R.D. Huo L.J. Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.OMICS. 2017; 21: 245-256Crossref PubMed Scopus (48) Google Scholar, D. Zhao Y. of gene 2017; PubMed Scopus Google Scholar), of modifications of 2016; PubMed Scopus Google Scholar, H. B. B. Y. Y. Wang J. X. F. by of modifications in 2016; Full Text Full Text PDF PubMed Scopus Google Scholar), (8.Brohi R.D. Huo L.J. Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.OMICS. 2017; 21: 245-256Crossref PubMed Scopus (48) Google Scholar, P. Wang X. F. Zhao S. M. Y. Y. H. D. J. Liu in human male infertility by 2017; Full Text Full Text PDF PubMed Scopus Google Scholar), and (8.Brohi R.D. Huo L.J. Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.OMICS. 2017; 21: 245-256Crossref PubMed Scopus (48) Google Scholar, P. as for and their in PubMed Google in proteins play in sperm and the fertilization we evaluated of protein modifications in spermatozoa after reduced male abstinence. that modifications in sperm butyryllysine, propionyllysine, and malonyllysine were significantly and that trimethyllysine modification was increased after reduced male abstinence. We the of butyryllysine, propionyllysine, and trimethyllysine modifications in sperm and new in events with to sperm This study provides preliminary evidence of a significant in sperm gene molecular and clinical after a reduced male ejaculatory abstinence and a potential of using spermatozoa obtained after 1–3 h of abstinence in IVF Our may improve of the molecular underlying a reproductive potential of spermatozoa. The mass have been to the the with the The can be We the for with