Effect of normal seminal plasma replacement on cryopreservation of sperm in patients with non-liquefied semen

Effect of normal seminal plasma replacement on cryopreservation of sperm in patients with non-liquefied semen | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of normal seminal plasma replacement on cryopreservation of sperm in patients with non-liquefied semen Shuai Shao, Mei Jiang, Cespuglio Raymond, Nianping Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4485698/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective : To explore the effects of normal seminal plasma replacement on cryopreservation of human non-liquefied spermatozoa. Methods : Sixty Semen samples (30 samples of normal semen and 30 samples of non-liquefied semen) were collected from infertile male patients in the Reproductive Medicine Center of Jingmen People's Hospital. After centrifugation, spermatozoa were divided into 5 aliquots, of which 2 aliquots were added with non-liquefied and normal seminal plasma, respectively. Computer-assisted semen analysis system (CASA) measured sperm motility after various periods of incubation (0, 0.5, 1 and 2 h). The remaining 3 aliquots of sperm were employed as follows for cryopreservation: a seminal plasma-free group in which sperm was preserved by adding sperm cryoprotectants; a non-liquefied semen-derived seminal plasma group for which sperm was added with non-liquefied plasma; a normal seminal plasma group for which sperm was added with normal seminal plasma. After the freezing-thawing process, sperm parameters, DNA integrity and ability to resist oxidative stress damage were again examined for these 3 groups. Results: Incubation of normal semen plasma, without liquefaction, can improve the sperm motility. After cryopreservation, sperm parameters were significantly lower versus those of the sample not cryopreserved. Normal semen plasma can reasonably protect the sperm of unliquefied semen and maintain the sperm parameters. It can also maintain the DNA integrity and prevent oxidative stress injury. Conclusion : Normal seminal plasma can maintain the semen key parameters after cryopreservation of non-liquefied semen. Protective effects observed with this normal seminal liquid might be due to the antioxidants remaining within the seminal liquid. Cryopreservation human sperm non-liquefied semen seminal plasma Figures Figure 1 Figure 2 Introduction With the rapid development of human assisted reproductive technology, human sperm cryopreservation has become more and more common. At present, this technology is mainly used for sperm preservation in cancer patients requiring radiotherapy and chemotherapy. However, it is also applicable to men of childbearing age who do not want to procreate, and to men engaged in high-risk occupations that may damage fertility. Finally, above technology is also employed for infertile patients who have the need for an assisted reproduction. The freezing-thawing process can cause damage to the DNA, organelles, and motility of sperm cells due to ice crystal formation, cold shock, and osmotic stress [ 1 ]. These factors are known for inducing the production of reactive oxygen species (ROS), which can negatively influence the stability of sperm genetic material and lead to strand breakage. ROS are also closely associated with cell apoptosis, membrane lipid peroxidation, sperm membrane damage, ultrastructural changes in sperm morphology, and mitochondrial dysfunction [ 2 – 3 ]. Parameters such as sperm cells viability and survival after freezing, examined in the laboratory, are considered as important potential indicators of in vitro fertilization [ 4 ]. Seminal plasma is the main component of semen cells. It contains water, amino acids, various ions, lipids, sugars and various hydrolytic enzymes, which provide nutritive and energetic substances for spermatozoa [ 5 ]. The presence of amino acids in seminal plasma stabilizes the dynamic balance of nitrogen in semen cells and plays an important role in the preservation of spermatozoids motility and metabolism. In addition, in the seminal plasma fluid there also exists enzymatic antioxidants including superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). Non-enzymatic antioxidants such as glutathione, albumin and polyunsaturated fatty acids coexist in the seminal fluid where they play a crucial role in sperm viability and motility [ 6 – 7 ]. However, the presence of abnormal spermatoplasmic components in abnormal semen resulted in a decreased sperm motility and a poor cryopreservation of the semen, such as non-liquefaction. The normal semen samples of men, at room temperature, undergoes a typical liquefaction within 10 to 30 minutes after ejaculation. If it fails to liquefy, partly or fully, for more than 60 minutes, it is then referred to as a non-liquefied semen [ 9 ]. It is to be noticed that an abnormal semen liquefaction is one of the common causes of male infertility. According to literature reports, its incidence accounts for 2.51–42.65% of male infertility [ 9 ]. Non-liquefaction of the semen adversely affects the physical and biochemical properties of semen (sperm viability and morphology), as well as the effectiveness of cryopreservation. Presently, the need for a more favorable cryopreservation of spermatozoa with a non-liquefied semen becomes a pressing issue. In the present study, according to the fact that non-liquefied semen could not fully support the induced damages of cryopreservation. Thus, the objective of our study was to evaluate whether normal semen plasma in fertile men could improve the cryotolerance of sperm in patients with non-liquefied semen. After cryopreservation, the sperm parameters, DNA integrity, and ability to resist oxidative stress damage were examined. Materials and Methods General information - This study was approved by the Medical Ethics Committee of Jingmen People's Hospital (KY-2024030601). All participants signed the informed consent. Male infertility patients treated in our hospital from January 2023 to June 2023 were selected and divided into two groups: 30 cases of infertile men with non-liquefied semen and 30 cases of infertile men with normal liquefied semen. The patients ranged from 20 to 45 years old with an average age of 31.1 ± 6.2 years. All the patients had no abnormality of sexual characteristics and genitalia, and no obvious abnormality of testicle sizes. Males with a normal physical examination were aged from 20 to 45 years, with an average age of 30.4 ± 5.8 years. Inclusion criteria - According to the World Health Organization(WHO) regulations, the inclusion criteria for non-liquefied semen indicate that: fresh semen liquefies within 60 minutes at room temperature; if it still contains non-liquefied clots or mucus filaments after 60 minutes, it is then defined as semen non-liquefied. Inclusion criteria for normal semen group is fresh semen samples liquefying within 30 minutes at room temperature. Exclusion criteria - Severe oligospermia, Semen volume < 1 mL, infectious disease, sexually transmitted disease, bilateral testicular volume < 8 mL, abnormality in the chromosomes of peripheral blood and normal microdeletion of chromosome Y. Methods Specimen collection and sperm quality analysis - Patients of both groups were operated according to the standard procedure of the Department of Reproductive Medicine. They were submitted to a sex abstinence for 2–7 days and masturbation was achieved in the sperm retrieval room. The complete semen was collected in a sterile sperm retrieval cup. Afterwards it was sent to the andrology laboratory in a water bath maintained at 37°C. Semen was observed every 10 min to check whether non-liquefied clots or mucus filaments take place. Semen analysis was performed on all liquefied samples according to WHO guidelines [ 22 ]. The sperms in normal or non-liquefied semen were separated from seminal plasma by high-speed centrifugation, and the upper plasma layer was retained. Spermatozoa from non-liquefied semen were divided into 5 equal aliquots, of which two aliquots were added respectively with 0.3 ml of normal semen plasma and 0.3 ml of non-liquefied semen-derived seminal plasma. CASA system analyzed the sperm mobility after 0.0, 0.5, 1 and 2 h. The remaining 3 sperm samples were cryopreserved. These groups were as it follows: a seminal plasma-free group added with 0.3 ml G-IVF PLUS and 0.3 ml of sperm cryoprotectant; a semen group with non-liquefied seminal plasma added with 0.3 ml of non-liquefied semen seminal plasma and 0.3 ml of sperm cryoprotectant; a normal semen group added 0.3 ml of normal seminal plasma and 0.3 ml of sperm cryoprotectant. Three semen groups were frozen for 7 days and thawed to observe the vitality, survival rate, morphology, DNA integrity and oxidative stress. Separation of seminal plasma - Simple seminal plasma components were isolated from semen specimens by high-speed centrifugation (3,000 g). After 5 min, upper seminal plasma, about 1–3 mL, was obtained. Then, a microscopic examination confirmed that there were no spermatozoa present in the seminal plasma. The above procedures to obtain sperm-free seminal plasma are consistent with literature [ 6 ]. Sperm preparation - Enriched spermatozoa were obtained by density gradient centrifugation. Semen specimens with obvious clots were blown several times with a syringe to promote liquefaction. The liquefied semen was then added to a gradient centrifuge medium with the upper layer of 40% solution and the lower layer of 80% solution (SAGE), and the mixture was centrifuged at 300 g for 16 min to obtain sperm precipitation. Subsequently, 3ml of G-IVF (VItrolife) was added to the sperm precipitation. The sperm was resuspended, and the sperm suspension was centrifuged at 250 g for 6 min. The excess G-IVF from the upper layer was discarded and 0.5 mL of the liquid was retained. Enriched spermatozoa were thus obtained. Routine semen examination – 10 µL of liquefied semen was dropped into a Makler® cell counting chamber (10 µm deep), then covered with glass and analyzed by the Shanghai Beion Color Sperm Quality Analyzer. At least 400 sperms were analyzed in each specimen. The semen volume, sperm concentration, and sperm viability, including the three levels of the forward-motile sperm ratio (PR), the non-forward-motile sperm ratio (NP), and the immobile sperm (IM) ratio, were calculated. Sperm survival rates − 50 µL of eosin-aniline black staining solution and semen were mixed in a 1:1 ratio for 30 s. 10 µL of this mixed solution were taken to analyze the sperm survival rate under light microscope. Of about 200 sperms in each group, the stained (dead) and unstained (living) sperms were respectively counted and the survival rate was determined. Sperm morphology - The semen specimens were prepared and dried for at least 4 h and stained with modified Pap smear. Afterward, at least 200 spermatozoa were screened with a oil microscope under a 100 x lens magnitude, according to WHO Laboratory Manual for the Examination and Processing of Human Semen (Fifth Edition). The percentage of spermatozoa with a normal morphology was calculated. In this way, the percentage of sperm with normal morphology greater than or equal to 4% is considered as normal, and less than 4% is considered as abnormal.. Sperm acrosome integrity rate - Sperm acrosome integrity rate was analyzed by Pap staining. Normal acrosome was identified according to Menkveld's method [ 10 ]. The acrosomal region accounted for 40–70% of the sperm head area; there were no large vacuoles in the acrosome region and no more than two small vacuoles. The size of vacuoles was not more than 20% of the sperm head. At least 200 spermatozoa were examined, and the acrosome integrity rate was determined. Sperm plasma Membrane Integrity - The hypotonic swelling test was used to detect sperm plasma membrane integrity. About 20 µL of semen was added to 200 µL of hypotonic reagent (containing fructose and citric acid). After 30 min of incubation, 15 µL of semen was smeared on a glass slide, and the number of spermatozoa with curved tails was observed under a light microscope. Sperm DNA fragmentation index (DFI) - Sperm chromatin diffusion (SCD) was used to detect DFI (The kit was purchased from Shenzhen Boreal Biotechnology Co). The presence or absence of sperm head halo and its size can reflect the sperm DNA integrity. Normal sperm DNA is capable of producing diffused halo, while damaged sperm DNA produces little or no halo. The sperm head halo was examined under a 400x magnitude light microscope. Criteria for determining the presence of sperm DNA fragmentation: the sperm head produces only a small halo or no halo, and the thickness of the unilateral halo does not exceed the 1/3 of the minimum diameter of the sperm head. At least 500 spermatozoa were examined and the DFI was calculated. Enzyme-linked immunosorbent assay (ELISA) – After the semen liquefaction, the supernatant was centrifuged at 3,500 r/min for 10 min, and the enzyme-linked immunosorbent assay (ELISA) was used to measure the SOD, CAT and MDA contents according to the instructions of the kit. Statistical processing - SPSS 23.0 statistical software was used for data analysis and processing. Measurement data conforming to normal distribution were expressed as x¯± s, one-way ANOVA was used for comparison between multiple groups, and LSD-t test was used for two-by-two comparisons between multiple groups. Counted data were expressed as the number of cases or percentage, and χ2 test was used for comparison between groups. Differences were considered statistically significant at P < 0.05. Results Effect of normal seminal plasma on the motility of spermatozoa derived from non-liquefied semen - As shown in Table 1, with the prolongation of the placement time, the sperm motility was decreased in both groups. There was no difference in the motility between the two groups at 0.5 h. However, the sperm motility of the normal seminal plasma replacement group at 1 h was significantly higher than that of the non-liquefied semen group (p < 0.05). Remarkably, at 2 h, the sperm motility of the normal seminal plasma replacement group was even more superior to that of the non-liquefied semen group (p < 0.01). Effect of normal semen plasma on the motility of spermatozoa after cryopreservation of non-liquefied semen - As shown in Table 2, the motility of spermatozoa after cryopreservation was significantly lower than before cryopreservation in all three groups studied (all p < 0.01). There was a significant difference in spermatozoon motility after cryopreservation in the three groups. The motility in the normal seminal plasma (NSP) group was significantly higher than that in the seminal plasma-free (SPF) group and semen not liquefied seminal plasma group (SNLSP) group (p < 0.01). Effect of normal seminal plasma on survival rate of cryopreserved spermatozoa derived from non-liquefied semen - As shown in Table 3, the survival rates of spermatozoa after cryopreservation in the three groups were all significantly lower than those before cryopreservation. A significant difference in sperm survival rates among the three groups was observed after cryopreservation. The NSP group had a significantly higher sperm survival rate than that in the SNLSP group and SPF group. The survival rate in SNLSP group did not exhibit difference in comparison with that in the SPF group. Effect of normal seminal plasma on the morphology of cryopreserved spermatozoa derived from non-liquefied semen - The data concerning sperm with a normal morphology are presented in Fig. 1 . The normal morphology rate of sperm after freezing was significantly lower than before freezing in all three groups. There was a significant difference in the normal morphology of spermatozoa after freezing among the three groups. The normal morphology of spermatozoa in the NSP group was significantly higher than that in the SPF group and SNLSP group (p < 0.01). Effect of normal seminal plasma on the plasma membrane integrity of cryopreserved spermatozoa derived from non-liquefied semen - In each group, the membrane integrity rate of spermatozoa after freezing was significantly lower than that before freezing. The addition of SNLSP did not significantly protect the sperm plasma membrane integrity during cryopreservation, and there was no difference between SNLSP group and SPF group in terms of membrane integrity rate of spermatozoa. However, the sperm plasma membrane integrity rate of the NSP group after sperm freezing was significantly higher than that of the other two groups (p < 0.01, as illustrated in Table 4). Effect of normal seminal plasma on the acrosome integrity of cryopreserved spermatozoa derived from non-liquefied semen- The data concerning the acrosome integrity rate of spermatozoa are shown in Table 5. The acrosome integrity rate of spermatozoa after freezing was significantly lower than that before freezing in all three groups. There was a significant difference in the acrosome integrity rate of spermatozoa after freezing among the three groups, and the acrosome integrity rate in the NSP group after sperm freezing was significantly higher than that in the other two groups (p < 0.01). Effect of normal seminal plasma on the DNA integrity of cryopreserved spermatozoa derived from non-liquefied semen - The addition of SNLSP did not have a significant protective effect on the DNA integrity of spermatozoa during cryopreservation. There was no difference between SNLSP group and SPF group in terms of sperm DNA fragmentation index. However, the addition of normal semen plasma was able to induce a significant protective effect on the DNA integrity of the spermatozoa during cryopreservation. The sperm DNA fragmentation index of NSP group was significantly lower than that of the SPF group and SNLSP group. (p < 0.01). (As shown in Fig. 2 B) Effect of normal seminal plasma on antioxidative defenses in cryopreserved spermatozoa derived from non-liquefied semen - The results of oxidative stress-related indicators showed that the content of MDA significantly increased after sperm cryopreservation. The contents of SOD and GSH-Px were significantly decreased after sperm freezing in all the 3 groups (P < 0.01). The normal seminal plasma was able to exert an antioxidant protection on spermatozoa during the course of cryopreservation. This addition of normal plasma reduced the MDA content and increased the expression of SOD and GSH-Px compared with the other two groups. There was no difference in the contents of MDA, SOD and GSH-Px between the SNLSP group and the SPF group, as demonstrated in Table 6. Discussion Sperm cryopreservation has been widely used in men needing an assisted reproductive technology. In addition to sperm cryopreservation for infertile male patients, fertility preservation and banking for male patients with a certain type of cancer are commonly practiced in reproductive medicine [ 11 ]. In 1953, Bunge and Sherman reported a protocol for the preservation of human spermatozoa using 10% glycerol solution and dry ice. With this method, a 67%-sperm survival rate was obtained after cryopreservation. These authors also reported three cases of pregnancies following fertilization of frozen spermatozoa [ 12 ]. Later, Sherman [ 13 ] et al. successfully preserved semen samples in liquid nitrogen. Despite decades of development and improvement, the quality of sperm cryopreservation remains unsatisfactory. In particular, when non-liquefied semen is preserved using cryogenic method, the quality of spermatozoa decreases. This is due to the non-liquefaction of the seminal plasma as well as to the lack of related substance conducing to a reduced sperm viability. Normal semen seminal plasma contains endogenous antioxidants and non-enzymatic antioxidants that play a protective role in sperm cryopreservation [ 14 ]. Therefore, normal seminal plasma was employed to replace non-liquefied seminal plasma in the current study, when non-liquefied semen was preserved at low temperature. We found that normal seminal plasma was able to maintain the viability of spermatozoa derived from non-liquefied semen. When the semen was left for a longer period of time, normal seminal plasma is better able to maintain spermatozoon viability than non-liquefied seminal plasma. It has been reported that spermatozoa protein Ⅰ (Semenogelin Ⅰ, Sg-I) covered the surface of spermatozoa by binding with Epididymis protease inhibitor (Eppin) to form Sg-I-Eppin complex. Thereby inhibiting the forward motion of sperm. [ 15 ]. Normal seminal plasma provides nutrients for spermatozoon activity, and participates in the de-capacitation and capacitation of sperm. In this way, spermatozoa can maintain a good vitality. Zinc in the seminal plasma slows down the lipid oxidation of the cell membrane, thereby maintaining the permeability and stability of the cell membrane, which is necessary for the good viability of spermatozoa. Fructose is able to provide energy for spermatozoa. The citrate in seminal plasma maintains the osmotic pressure, appropriate pH and good buffering capacity of the semen. The above substances are conducive to the survival of spermatozoa, and meet well their acticity needs [ 16 , 17 ]. Normal seminal plasma employed for cryopreservation can play a cryoprotective role in sperm. In the study conducted by Montoya Páez et al. [ 18 ], it is reported that 10% homologous seminal plasma provides protection for sperm freezing, improves the sperm viability, and maintains the membrane integrity and morphology of sperm. Zoca et al. [ 19 ] also reported that seminal plasma provides an increased acrosomal integrity and a reduced remodeling of the F-actin cytoskeleton. Similar to these studies, our results show that during cryopreservation of non-liquefied semen-derived sperm, the addition of normal seminal plasma reduces the sperm cryoinjury, increases the viability and survival of sperm, and maintains the sperm morphology and plasma membrane integrity.. The oxidative stress is one of the most important factors conducing to an impaired sperm function during cryopreservation. Spermatozoa produce appropriate amounts of Reactive Oxygen Species (ROS), a byproduct of redox, which regulates the physiological functions of spermatozoa at physiological concentration, including energy acquisition, hyper-activation, acrosome reaction, and binding to zona pellucida [ 20 ]. During cryopreservation, antioxidant-rich seminal plasma is diluted and the antioxidant activity of sperm is reduced. Sperm damage, dead spermatozoa and leukocytes caused by cryopreservation also produce peroxides in excess [21. The balance between ROS production and the antioxidant capacity of the biological milieu is then disrupted and the excessive ROS produces oxidative stress damages. The ROS in excess, at first, results in the lipid peroxidation (LPO) of the sperm plasma membrane. After then, this peroxidation conduces to the decarboxylation of unsaturated fatty acids, which gives rise to malondialdehyde (MDA), a compound capable of impairing the permeability and fluidity of the sperm plasma membrane [ 22 ]. The above processes also cause damages to the proteins, mitochondria, and DNA of sperm [ 23 ]. For sperm proteins, the oxidation of the thiol groups makes the sperm cells susceptible to leukocyte attack. An excessive ROS production affects the phosphorylation and glycosylation of protein, and production of mitochondrial ATP, which subsequently affects the sperm fertilization [ 24 ]. The present study also confirms that sperm freezing leads to an oxidative stress with an increased expression of MDA and a decreased expression of SOD and GSH-Px in seminal plasma. The addition of normal seminal plasma not only enhances the anti-oxidative stress ability of sperm, but also exerts a certain cryoprotective effect, which reduces the expression of MDA and increases the expression of SOD and GSH-Px. This is consistent with the fact that normal semen seminal plasma contains superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, as well as non-enzymatic antioxidants such as glutathione, ascorbic acid, vitamin E, albumin, and taurine [ 6 – 7 ]. These compounds are capable of counteracting the oxidative stress damages caused by ROS. Under normal conditions, endogenous antioxidants such as CAT, SOD and GSH-Px in seminal plasma can protect sperm from oxidative stress injury [ 25 ]. However, during the freezing process of oligo-zoosperm and non-liquefied semen, the dynamic balance of the antioxidant system is disrupted. In such a situation, the endogenous antioxidants themselves cannot fully protect sperm cells from the oxidative stress damage. The resort to exogenous antioxidants is then necessary to resist the oxidative stress damage [ 26 ]. In this way, the addition of the exogenous antioxidant melatonin into the semen was able to improve sperm motility after thawing. It also protects the acrosome and plasma membrane integrities, and increases the activities of T-AOC, T-SOD, GSH-Px and CAT, whereas reduces the MDA and 4-HNE activities [ 27 ]. At the same time, the excessive ROS causes DNA damage in spermatozoa, with a high frequency of single- and double-stranded DNA breaks [ 28 ]. Disruption of DNA integrity in the sperm nucleus is the underlying cause of irreversible sperm damage. Human sperm DNA fragmentation suggests sperm damage and is a factor of male infertility. The percentage of sperm DNA damages correlates with the fertilization rate and the unfavorable outcomes in embryo development [ 29 – 30 ]. Sperm DNA fragment Index (DFI) is the main indicator of sperm DNA integrity. In the current study, we also found that the DFI increased after sperm freezing, and neither non-liquefied seminal plasma nor no-seminal plasma exerted a good protection of DNA integrity. The normal semen plasma may contain antioxidants that exert a more convenient DNA integrity protection. This suggests that normal seminal plasma plays an important role in antioxidant protection, which protects spermatozoa from freezing damages. In conclusion, our results demonstrate that the seminal plasma of non-liquefied semen could not resist to the damage of cryopreservation, which may lead to the occurrence of abnormal frozen sperm. The normal seminal plasma, however, containing antioxidants, fatty acids and nutrients, can exert a protective effect against oxidative species and resist cryopreservation risks. The addition of normal seminal plasma into the sperm extracted from non-liquefied semen improves the viability, survival rate and plasma membrane integrity of sperm, maintains DNA integrity and normal morphology of cryopreserved sperm, and reduces oxidative stress damage. Declarations Acknowledgements - T he Jingmen Science and Technology Program (2022YFYB006) supported our work. We would like to express our gratitude to the staff of the Reproductive Medicine Center at Jingmen People's Hospital as well as to the experimental technicians involved in the study. Authors’ contributions - Shuai Shao and Mei Jiang conducted all experiments and completed the paper. Nianping Zhang participated in the experimental design and statistical analysis, and also revised the manuscript. PR Raymond CESPUGLIO contributed to the manuscript improvement. Funding - Jingmen Science and Technology Program (2022YFYB006). Availability of data and materials - The samples used in this study and the data sets collected were provided by the Reproductive Medicine Center of Jingmen People's Hospital. Declarations - Ethics approval and consent to participate. The Ethic committee of Jingmen People’s Hospital reviewed this study. Patients with an informed consent approved all samples. Consent for publication - Not applicable. Our manuscript does not contain data from individual person. Competing interests - The authors declare that they have no competing interests in this section. Author details - 1 Reproductive Medicine Center, Jingmen People's Hospital (Central Hospital Affiliated to Jingchu Institute of Technology); 2 Neuroscience Research Center of Lyon, Beliv plateau, Claude Bernard Lyon1 University & RUDN University; 3 Postdoctoral Mobile station, Shandong University of Traditional Chinese Medicine; 4 Experimental Center, Shandong University of Traditional Chinese Medicine. References Hungerford A, Bakos HW, Aitken RJ. Sperm cryopreservation: current status and future developments. Reprod Fertil Dev, 2023, 35(3): 265-281. Gouhier C, Pons-Rejraji H, Dollet S et al. Freezing does not alter sperm telomere length despite increasing DNA oxidation and fragmentation. Genes (Basel), 2023, 14(5): 1039. Hosseinmardi M, Siadat F, Sharafi M et al. Protective effect of cerium oxide nanoparticles on human sperm function during cryopreservation. Biopreserv Biobank, 2022, 20(1): 24-30. Lewin J, Lukaszewski T, Sangster P et al. Reproductive outcomes after surgical sperm retrieval in couples with male factor subfertility: a 10-year retrospective national cohort. Fertil Steril, 2023, 119(4): 589-595. Takahara T, Amemiya Y, Sugiyama R, et al . Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes. J Biomed Sci, 2020, 27(1): 87. Martínez-Soto JC, Landeras J, Gadea J. Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success. Andrology, 2013, 1(3): 365–375. Davalieva K, Kiprijanovska S, Noveski P, et al. Proteomic analysis of seminal plasma in men with different spermatogenic impairment. Andrologia, 2012, 44(4): 256–264. Björndahl L, Kirkman Brown J; other Editorial Board Members of the WHO Laboratory Manual for the Examination and Processing of Human Semen. The sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen: ensuring quality and standardization in basic examination of human ejaculates. Fertil Steril, 2022, 117(2): 246-251. Eisenberg ML, Lathi RB, Baker VL et al. Frequency of the male infertility evaluation: data from the national survey of family growth. J Urol, 2013, 189(3): 1030-1034. Menkveld R, Rhemrev JP, Franken DR, et al. Acrosomal morphology as a novel criterion for male fertility diagnosis: relation with acrosin activity, morphology (strict criteria), and fertilization in vitro. Fertil Steril, 1996, 65: 637-644. Li Y, Qin S, Cui W et al. Progress on the roles of zinc in sperm cryopreservation. Theriogenology, 2023, 211: 134-141. Bunge R. G., Sherman J. K. Fertilizing capacity of frozen human spermatozoa. Nature, 1953, 172: 767-768. Sherman J. K. Improved Methods of Preservation of Human Spermatozoa by Freezing and Freeze-Drying [J]. Fertility and Sterility, 1963,14: 49-64. Eini F, Kutenaei MA, Shirzeyli MH, et al. Normal seminal plasma could preserve human spermatozoa against cryopreservation damages in Oligozoospermic patients. BMC Mol Cell Biol, 2021, 22(1): 50. Terai K, Yoshida K, Yoshiike M, et al. Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men. Urol Int, 2010, 85(2): 209-215. Gruhl SL, Ho LM, Sim MYX, et al. Seminal biomarkers and their correlations to semen parameters in subfertile men. Eur J Obstet Gynecol Reprod Biol, 2023,19: 100229. Huang D, Cai J, Zhang C, et al. Semen quality and seminal plasma metabolites in male rabbits (Oryctolagus cuniculus) under heat stress. PeerJ, 2023, 11: e15112. Montoya Páez JD, Úsuga Suarez A, Restrepo Betancur G. Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma. Reprod Domest Anim. 2023, 58(4): 486-495. Zoca GB, Celeghini ECC, Pugliesi G et al. Influence of seminal plasma during different stages of bovine sperm cryopreservation. Reprod Domest Anim, 2021, 56(6): 872-883. Asadi A, Ghahremani R, Abdolmaleki A et al. Role of sperm apoptosis and oxidative stress in male infertility: a narrative review. International Journal of Reproductive Biomedicine, 2021, 19(6): 493-504. Sieme H, Oldenhof H. Sperm cleanup and centrifugation processing for cryopreservation. Methods in Molecular Biology, 2015, 1257(3): 43-52. Woelders H, Chaveiro A. Theoretical prediction of ‘optimal’ freezing programmes. Cryobiology, 2004, 49(3): 58-71. Brochier C, Langley B. Chromatin modifications associated with DNA double-strand breaks repair as potential targets for neurological diseases. Neurotherapeutics, 2013, 10(4): 817- 830. Nekoonam S, Nashtaei M S, Naji M et al. Effect of Trolox on sperm quality in normozospermia and oligozospermia during cryopreservation. Cryobiology, 2016,72(2): 106-111. Gadea J, Molla M, Selles E, et al. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology, 2011, 62(1): 40-46. Zhang W, Yi K, Chen C et al. Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa. Anim Reprod Sci, 2012, 132(3-4): 123-128. Li C, Ren C, Chen Y, et al. Changes on proteomic and metabolomic profiling of cryopreserved sperm effected by melatonin. J Proteomics, 2023, 273: 104791. [28] Thomson L K, Fleming S D, Aitken R J et al. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Human Reproduction, 2009, 24(9): 2061-2070. Cankut S, Dinc T, Cincik M et al. Evaluation of Sperm DNA Fragmentation via Halosperm Technique and TUNEL Assay Before and After Cryopreservation. Reprod Sci, 2019, 26(12): 1575-1581. Shaliutina-Loginova A, Loginov DS. Oxidative stress and DNA fragmentation in frozen/thawed common Carp Cyprinus carpio sperm with and without supplemental proteins. Anim Reprod Sci. 2023, 251: 107213. Tables Tables 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.pdf Table2.pdf Table3.pdf Table4.pdf Table5.pdf Table6.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4485698","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310910703,"identity":"7c990c8d-c9ed-470c-abde-8e47cc5eefbd","order_by":0,"name":"Shuai Shao","email":"","orcid":"","institution":"Jing men People’s hospital (Jingchu university of technology affiliated center hospital)","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Shao","suffix":""},{"id":310910704,"identity":"ad0c147f-10de-4d17-90bc-9c9fce54a1ca","order_by":1,"name":"Mei Jiang","email":"","orcid":"","institution":"Jing men People’s hospital (Jingchu university of technology affiliated center hospital)","correspondingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Jiang","suffix":""},{"id":310910705,"identity":"7e70209d-080d-4648-a947-3737c1f326ed","order_by":2,"name":"Cespuglio Raymond","email":"","orcid":"","institution":"Neuroscience Research Center of Lyon, Claude Bernard Lyon1 University","correspondingAuthor":false,"prefix":"","firstName":"Cespuglio","middleName":"","lastName":"Raymond","suffix":""},{"id":310910706,"identity":"bc427f2b-6436-4fd3-9ac3-35ec1f45368a","order_by":3,"name":"Nianping Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACfmbG9h8JBjZ2/AwMBlCxBPxaJNubGyQ+VKQlSzYAtRwgRotBz/EGyRlnDjFuOEC0FonEBmPetgPMxucPb5P+ULONgZ89x4Dh5w7cWsyBWpJ52+7wmd1IK5M4cOw2g2TPGwPG3jO4tVjOSGw4zNv2jNnsBo+ZxMGG2wwGN3IMmBnb8DjsRmJjM2/bYcbN/WcgWuwJajlzsJlxxpnDjBsYcqC2SBDQItne2MYACmSJG2nFFmeO3eaROPOs4GAvHi38zOzPGMBR2X94442Kmtty/O3JGx/8xKMFA/CAiAMkaBgFo2AUjIJRgAUAAHA8Wf0mCm9AAAAAAElFTkSuQmCC","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Nianping","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-05-27 14:24:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4485698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4485698/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58244677,"identity":"fa859323-16e1-47ab-9e92-f7b382f50d48","added_by":"auto","created_at":"2024-06-13 02:14:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of normal seminal plasma on the morphology of cryopreserved spermatozoa derived from non-liquefied semen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Pap smear was performed to visualize sperm morphology in four groups: (1) Bef Cryop, (2)SPF group, (3) SNLSP group, (4)NSP group; scar bar = 10μm.\u003c/p\u003e\n\u003cp\u003e(B) Normal sperm morphology rate.\u003c/p\u003e\n\u003cp\u003eStatistics -***P\u0026lt;0.01 compared with the situation before cryopreservation; **P\u0026lt;0.01 compared with SPF group or SNLSP group.\u003c/p\u003e\n\u003cp\u003eAbbreviations: Bef Cryop, Before cryopreservation; SPF, Seminal plasma-free group; SNLSP, Semen not liquefied seminal plasma group; NSP,Normal seminal plasma group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/1e4e773c4dfcf3da13ad4b9f.png"},{"id":58245435,"identity":"3dbfadac-c348-4c64-a2ca-fa37d99ebad4","added_by":"auto","created_at":"2024-06-13 02:22:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of normal seminal plasma on the DNA integrity of cryopreserved spermatozoa derived from non-liquefied semen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Sperm chromatin diffusion (SCD) was achieved to visualize sperm DNA fragmentation in four groups: (1) Bef Cryop, (2) SPF group, (3) SNLSP group, (4 )NSP group; scar bar = 10μm.\u003c/p\u003e\n\u003cp\u003eB. Sperm DNA fragmentation index.\u003c/p\u003e\n\u003cp\u003eStatistics -***P\u0026lt;0.01 compared with the situation before Cryopreservation; **P\u0026lt;0.01 compared with SPF group or SNLSP group.\u003c/p\u003e\n\u003cp\u003eAbbreviations: Bef Cryop, Before cryopreservation; SPF, Seminal plasma-free group; SNLSP, Semen not liquefied seminal plasma group; NSP,Normal seminal plasma group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/f942a405863973d87fd7d56a.png"},{"id":102942394,"identity":"b9c53a25-dd99-409a-a8f0-9a23917785d8","added_by":"auto","created_at":"2026-02-18 17:25:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1374918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/7a324d7a-a1c6-4de3-a0be-33e41dff6007.pdf"},{"id":58245449,"identity":"03898923-4c35-47f9-b150-f4ef94a70550","added_by":"auto","created_at":"2024-06-13 02:22:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":372223,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/efa32e867cc39713280baba7.pdf"},{"id":58244678,"identity":"0fce447f-0819-4d15-ab82-81579f188f06","added_by":"auto","created_at":"2024-06-13 02:14:09","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":588119,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/aefc73e16727b6d6ffb39f91.pdf"},{"id":58244682,"identity":"74beb5c4-ef3e-43c8-940e-55b07acb3322","added_by":"auto","created_at":"2024-06-13 02:14:09","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":583092,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/8243298023c558e1b0146175.pdf"},{"id":58244681,"identity":"fe9282ab-bcb2-4908-9eaf-14b9b16e6fe3","added_by":"auto","created_at":"2024-06-13 02:14:09","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":584635,"visible":true,"origin":"","legend":"","description":"","filename":"Table4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/ce9d7e506a4ec45d8bd05fe8.pdf"},{"id":58244683,"identity":"b668da67-a301-4be5-8140-0eae58686957","added_by":"auto","created_at":"2024-06-13 02:14:09","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":585501,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/ad1062d5efb74820933e0b10.pdf"},{"id":58245436,"identity":"4a4d2d4c-ef9f-423c-803b-117e90f0d740","added_by":"auto","created_at":"2024-06-13 02:22:09","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":432982,"visible":true,"origin":"","legend":"","description":"","filename":"Table6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485698/v1/323148e6380854a56d0fbcab.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of normal seminal plasma replacement on cryopreservation of sperm in patients with non-liquefied semen","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWith the rapid development of human assisted reproductive technology, human sperm cryopreservation has become more and more common. At present, this technology is mainly used for sperm preservation in cancer patients requiring radiotherapy and chemotherapy. However, it is also applicable to men of childbearing age who do not want to procreate, and to men engaged in high-risk occupations that may damage fertility. Finally, above technology is also employed for infertile patients who have the need for an assisted reproduction.\u003c/p\u003e \u003cp\u003eThe freezing-thawing process can cause damage to the DNA, organelles, and motility of sperm cells due to ice crystal formation, cold shock, and osmotic stress [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These factors are known for inducing the production of reactive oxygen species (ROS), which can negatively influence the stability of sperm genetic material and lead to strand breakage. ROS are also closely associated with cell apoptosis, membrane lipid peroxidation, sperm membrane damage, ultrastructural changes in sperm morphology, and mitochondrial dysfunction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Parameters such as sperm cells viability and survival after freezing, examined in the laboratory, are considered as important potential indicators of \u003cem\u003ein vitro\u003c/em\u003e fertilization [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeminal plasma is the main component of semen cells. It contains water, amino acids, various ions, lipids, sugars and various hydrolytic enzymes, which provide nutritive and energetic substances for spermatozoa [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The presence of amino acids in seminal plasma stabilizes the dynamic balance of nitrogen in semen cells and plays an important role in the preservation of spermatozoids motility and metabolism. In addition, in the seminal plasma fluid there also exists enzymatic antioxidants including superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx). Non-enzymatic antioxidants such as glutathione, albumin and polyunsaturated fatty acids coexist in the seminal fluid where they play a crucial role in sperm viability and motility [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the presence of abnormal spermatoplasmic components in abnormal semen resulted in a decreased sperm motility and a poor cryopreservation of the semen, such as non-liquefaction. The normal semen samples of men, at room temperature, undergoes a typical liquefaction within 10 to 30 minutes after ejaculation. If it fails to liquefy, partly or fully, for more than 60 minutes, it is then referred to as a non-liquefied semen [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is to be noticed that an abnormal semen liquefaction is one of the common causes of male infertility. According to literature reports, its incidence accounts for 2.51\u0026ndash;42.65% of male infertility [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Non-liquefaction of the semen adversely affects the physical and biochemical properties of semen (sperm viability and morphology), as well as the effectiveness of cryopreservation. Presently, the need for a more favorable cryopreservation of spermatozoa with a non-liquefied semen becomes a pressing issue.\u003c/p\u003e \u003cp\u003eIn the present study, according to the fact that non-liquefied semen could not fully support the induced damages of cryopreservation. Thus, the objective of our study was to evaluate whether normal semen plasma in fertile men could improve the cryotolerance of sperm in patients with non-liquefied semen. After cryopreservation, the sperm parameters, DNA integrity, and ability to resist oxidative stress damage were examined.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eGeneral information -\u003c/b\u003e This study was approved by the Medical Ethics Committee of Jingmen People's Hospital (KY-2024030601). All participants signed the informed consent. Male infertility patients treated in our hospital from January 2023 to June 2023 were selected and divided into two groups: 30 cases of infertile men with non-liquefied semen and 30 cases of infertile men with normal liquefied semen. The patients ranged from 20 to 45 years old with an average age of 31.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 years. All the patients had no abnormality of sexual characteristics and genitalia, and no obvious abnormality of testicle sizes. Males with a normal physical examination were aged from 20 to 45 years, with an average age of 30.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 years.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInclusion criteria -\u003c/b\u003e According to the World Health Organization(WHO) regulations, the inclusion criteria for non-liquefied semen indicate that: fresh semen liquefies within 60 minutes at room temperature; if it still contains non-liquefied clots or mucus filaments after 60 minutes, it is then defined as semen non-liquefied. Inclusion criteria for normal semen group is fresh semen samples liquefying within 30 minutes at room temperature.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExclusion criteria -\u003c/b\u003e Severe oligospermia, Semen volume\u0026thinsp;\u0026lt;\u0026thinsp;1 mL, infectious disease, sexually transmitted disease, bilateral testicular volume\u0026thinsp;\u0026lt;\u0026thinsp;8 mL, abnormality in the chromosomes of peripheral blood and normal microdeletion of chromosome Y.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e\u003cb\u003eSpecimen collection and sperm quality analysis\u003c/b\u003e - Patients of both groups were operated according to the standard procedure of the Department of Reproductive Medicine. They were submitted to a sex abstinence for 2\u0026ndash;7 days and masturbation was achieved in the sperm retrieval room. The complete semen was collected in a sterile sperm retrieval cup. Afterwards it was sent to the andrology laboratory in a water bath maintained at 37\u0026deg;C. Semen was observed every 10 min to check whether non-liquefied clots or mucus filaments take place. Semen analysis was performed on all liquefied samples according to WHO guidelines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The sperms in normal or non-liquefied semen were separated from seminal plasma by high-speed centrifugation, and the upper plasma layer was retained. Spermatozoa from non-liquefied semen were divided into 5 equal aliquots, of which two aliquots were added respectively with 0.3 ml of normal semen plasma and 0.3 ml of non-liquefied semen-derived seminal plasma. CASA system analyzed the sperm mobility after 0.0, 0.5, 1 and 2 h. The remaining 3 sperm samples were cryopreserved. These groups were as it follows: a seminal plasma-free group added with 0.3 ml G-IVF PLUS and 0.3 ml of sperm cryoprotectant; a semen group with non-liquefied seminal plasma added with 0.3 ml of non-liquefied semen seminal plasma and 0.3 ml of sperm cryoprotectant; a normal semen group added 0.3 ml of normal seminal plasma and 0.3 ml of sperm cryoprotectant. Three semen groups were frozen for 7 days and thawed to observe the vitality, survival rate, morphology, DNA integrity and oxidative stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSeparation of seminal plasma\u003c/b\u003e - Simple seminal plasma components were isolated from semen specimens by high-speed centrifugation (3,000 g). After 5 min, upper seminal plasma, about 1\u0026ndash;3 mL, was obtained. Then, a microscopic examination confirmed that there were no spermatozoa present in the seminal plasma. The above procedures to obtain sperm-free seminal plasma are consistent with literature [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eSperm preparation\u003c/b\u003e - Enriched spermatozoa were obtained by density gradient centrifugation. Semen specimens with obvious clots were blown several times with a syringe to promote liquefaction. The liquefied semen was then added to a gradient centrifuge medium with the upper layer of 40% solution and the lower layer of 80% solution (SAGE), and the mixture was centrifuged at 300 g for 16 min to obtain sperm precipitation. Subsequently, 3ml of G-IVF (VItrolife) was added to the sperm precipitation. The sperm was resuspended, and the sperm suspension was centrifuged at 250 g for 6 min. The excess G-IVF from the upper layer was discarded and 0.5 mL of the liquid was retained. Enriched spermatozoa were thus obtained.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoutine semen examination \u0026ndash;\u003c/b\u003e 10 \u0026micro;L of liquefied semen was dropped into a Makler\u0026reg; cell counting chamber (10 \u0026micro;m deep), then covered with glass and analyzed by the Shanghai Beion Color Sperm Quality Analyzer. At least 400 sperms were analyzed in each specimen. The semen volume, sperm concentration, and sperm viability, including the three levels of the forward-motile sperm ratio (PR), the non-forward-motile sperm ratio (NP), and the immobile sperm (IM) ratio, were calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSperm survival rates \u0026minus;\u003c/b\u003e\u0026thinsp;50 \u0026micro;L of eosin-aniline black staining solution and semen were mixed in a 1:1 ratio for 30 s. 10 \u0026micro;L of this mixed solution were taken to analyze the sperm survival rate under light microscope. Of about 200 sperms in each group, the stained (dead) and unstained (living) sperms were respectively counted and the survival rate was determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSperm morphology -\u003c/b\u003e The semen specimens were prepared and dried for at least 4 h and stained with modified Pap smear. Afterward, at least 200 spermatozoa were screened with a oil microscope under a 100 x lens magnitude, according to WHO Laboratory Manual for the Examination and Processing of Human Semen (Fifth Edition). The percentage of spermatozoa with a normal morphology was calculated. In this way, the percentage of sperm with normal morphology greater than or equal to 4% is considered as normal, and less than 4% is considered as abnormal..\u003c/p\u003e \u003cp\u003e \u003cb\u003eSperm acrosome integrity rate -\u003c/b\u003e Sperm acrosome integrity rate was analyzed by Pap staining. Normal acrosome was identified according to Menkveld's method [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The acrosomal region accounted for 40\u0026ndash;70% of the sperm head area; there were no large vacuoles in the acrosome region and no more than two small vacuoles. The size of vacuoles was not more than 20% of the sperm head. At least 200 spermatozoa were examined, and the acrosome integrity rate was determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSperm plasma Membrane Integrity\u003c/b\u003e - The hypotonic swelling test was used to detect sperm plasma membrane integrity. About 20 \u0026micro;L of semen was added to 200 \u0026micro;L of hypotonic reagent (containing fructose and citric acid). After 30 min of incubation, 15 \u0026micro;L of semen was smeared on a glass slide, and the number of spermatozoa with curved tails was observed under a light microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSperm DNA fragmentation index (DFI)\u003c/b\u003e - Sperm chromatin diffusion (SCD) was used to detect DFI (The kit was purchased from Shenzhen Boreal Biotechnology Co). The presence or absence of sperm head halo and its size can reflect the sperm DNA integrity. Normal sperm DNA is capable of producing diffused halo, while damaged sperm DNA produces little or no halo. The sperm head halo was examined under a 400x magnitude light microscope. Criteria for determining the presence of sperm DNA fragmentation: the sperm head produces only a small halo or no halo, and the thickness of the unilateral halo does not exceed the 1/3 of the minimum diameter of the sperm head. At least 500 spermatozoa were examined and the DFI was calculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/b\u003e \u0026ndash; After the semen liquefaction, the supernatant was centrifuged at 3,500 r/min for 10 min, and the enzyme-linked immunosorbent assay (ELISA) was used to measure the SOD, CAT and MDA contents according to the instructions of the kit.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical processing\u003c/b\u003e - SPSS 23.0 statistical software was used for data analysis and processing. Measurement data conforming to normal distribution were expressed as x\u0026macr;\u0026plusmn; s, one-way ANOVA was used for comparison between multiple groups, and LSD-t test was used for two-by-two comparisons between multiple groups. Counted data were expressed as the number of cases or percentage, and χ2 test was used for comparison between groups. Differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on the motility of spermatozoa derived from non-liquefied semen\u003c/b\u003e - As shown in Table\u0026nbsp;1, with the prolongation of the placement time, the sperm motility was decreased in both groups. There was no difference in the motility between the two groups at 0.5 h. However, the sperm motility of the normal seminal plasma replacement group at 1 h was significantly higher than that of the non-liquefied semen group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Remarkably, at 2 h, the sperm motility of the normal seminal plasma replacement group was even more superior to that of the non-liquefied semen group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal semen plasma on the motility of spermatozoa after cryopreservation of non-liquefied semen\u003c/b\u003e - As shown in Table\u0026nbsp;2, the motility of spermatozoa after cryopreservation was significantly lower than before cryopreservation in all three groups studied (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). There was a significant difference in spermatozoon motility after cryopreservation in the three groups. The motility in the normal seminal plasma (NSP) group was significantly higher than that in the seminal plasma-free (SPF) group and semen not liquefied seminal plasma group (SNLSP) group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on survival rate of cryopreserved spermatozoa derived from non-liquefied semen\u003c/b\u003e - As shown in Table\u0026nbsp;3, the survival rates of spermatozoa after cryopreservation in the three groups were all significantly lower than those before cryopreservation. A significant difference in sperm survival rates among the three groups was observed after cryopreservation. The NSP group had a significantly higher sperm survival rate than that in the SNLSP group and SPF group. The survival rate in SNLSP group did not exhibit difference in comparison with that in the SPF group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on the morphology of cryopreserved spermatozoa derived from non-liquefied semen\u003c/b\u003e - The data concerning sperm with a normal morphology are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The normal morphology rate of sperm after freezing was significantly lower than before freezing in all three groups. There was a significant difference in the normal morphology of spermatozoa after freezing among the three groups. The normal morphology of spermatozoa in the NSP group was significantly higher than that in the SPF group and SNLSP group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on the plasma membrane integrity of cryopreserved spermatozoa derived from non-liquefied semen\u003c/b\u003e - In each group, the membrane integrity rate of spermatozoa after freezing was significantly lower than that before freezing. The addition of SNLSP did not significantly protect the sperm plasma membrane integrity during cryopreservation, and there was no difference between SNLSP group and SPF group in terms of membrane integrity rate of spermatozoa. However, the sperm plasma membrane integrity rate of the NSP group after sperm freezing was significantly higher than that of the other two groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, as illustrated in Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on the acrosome integrity of cryopreserved spermatozoa derived from non-liquefied semen-\u003c/b\u003e The data concerning the acrosome integrity rate of spermatozoa are shown in Table\u0026nbsp;5. The acrosome integrity rate of spermatozoa after freezing was significantly lower than that before freezing in all three groups. There was a significant difference in the acrosome integrity rate of spermatozoa after freezing among the three groups, and the acrosome integrity rate in the NSP group after sperm freezing was significantly higher than that in the other two groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on the DNA integrity of cryopreserved spermatozoa derived from non-liquefied semen\u003c/b\u003e - The addition of SNLSP did not have a significant protective effect on the DNA integrity of spermatozoa during cryopreservation. There was no difference between SNLSP group and SPF group in terms of sperm DNA fragmentation index. However, the addition of normal semen plasma was able to induce a significant protective effect on the DNA integrity of the spermatozoa during cryopreservation. The sperm DNA fragmentation index of NSP group was significantly lower than that of the SPF group and SNLSP group. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). (As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of normal seminal plasma on antioxidative defenses in cryopreserved spermatozoa derived from non-liquefied semen\u003c/b\u003e - The results of oxidative stress-related indicators showed that the content of MDA significantly increased after sperm cryopreservation. The contents of SOD and GSH-Px were significantly decreased after sperm freezing in all the 3 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The normal seminal plasma was able to exert an antioxidant protection on spermatozoa during the course of cryopreservation. This addition of normal plasma reduced the MDA content and increased the expression of SOD and GSH-Px compared with the other two groups. There was no difference in the contents of MDA, SOD and GSH-Px between the SNLSP group and the SPF group, as demonstrated in Table\u0026nbsp;6.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSperm cryopreservation has been widely used in men needing an assisted reproductive technology. In addition to sperm cryopreservation for infertile male patients, fertility preservation and banking for male patients with a certain type of cancer are commonly practiced in reproductive medicine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In 1953, Bunge and Sherman reported a protocol for the preservation of human spermatozoa using 10% glycerol solution and dry ice. With this method, a 67%-sperm survival rate was obtained after cryopreservation. These authors also reported three cases of pregnancies following fertilization of frozen spermatozoa [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Later, Sherman [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] et al. successfully preserved semen samples in liquid nitrogen. Despite decades of development and improvement, the quality of sperm cryopreservation remains unsatisfactory. In particular, when non-liquefied semen is preserved using cryogenic method, the quality of spermatozoa decreases. This is due to the non-liquefaction of the seminal plasma as well as to the lack of related substance conducing to a reduced sperm viability. Normal semen seminal plasma contains endogenous antioxidants and non-enzymatic antioxidants that play a protective role in sperm cryopreservation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, normal seminal plasma was employed to replace non-liquefied seminal plasma in the current study, when non-liquefied semen was preserved at low temperature.\u003c/p\u003e \u003cp\u003eWe found that normal seminal plasma was able to maintain the viability of spermatozoa derived from non-liquefied semen. When the semen was left for a longer period of time, normal seminal plasma is better able to maintain spermatozoon viability than non-liquefied seminal plasma. It has been reported that spermatozoa protein Ⅰ (Semenogelin Ⅰ, Sg-I) covered the surface of spermatozoa by binding with Epididymis protease inhibitor (Eppin) to form Sg-I-Eppin complex. Thereby inhibiting the forward motion of sperm. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Normal seminal plasma provides nutrients for spermatozoon activity, and participates in the de-capacitation and capacitation of sperm. In this way, spermatozoa can maintain a good vitality. Zinc in the seminal plasma slows down the lipid oxidation of the cell membrane, thereby maintaining the permeability and stability of the cell membrane, which is necessary for the good viability of spermatozoa. Fructose is able to provide energy for spermatozoa. The citrate in seminal plasma maintains the osmotic pressure, appropriate pH and good buffering capacity of the semen. The above substances are conducive to the survival of spermatozoa, and meet well their acticity needs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Normal seminal plasma employed for cryopreservation can play a cryoprotective role in sperm. In the study conducted by Montoya P\u0026aacute;ez et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], it is reported that 10% homologous seminal plasma provides protection for sperm freezing, improves the sperm viability, and maintains the membrane integrity and morphology of sperm. Zoca et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] also reported that seminal plasma provides an increased acrosomal integrity and a reduced remodeling of the F-actin cytoskeleton. Similar to these studies, our results show that during cryopreservation of non-liquefied semen-derived sperm, the addition of normal seminal plasma reduces the sperm cryoinjury, increases the viability and survival of sperm, and maintains the sperm morphology and plasma membrane integrity..\u003c/p\u003e \u003cp\u003eThe oxidative stress is one of the most important factors conducing to an impaired sperm function during cryopreservation. Spermatozoa produce appropriate amounts of Reactive Oxygen Species (ROS), a byproduct of redox, which regulates the physiological functions of spermatozoa at physiological concentration, including energy acquisition, hyper-activation, acrosome reaction, and binding to \u003cem\u003ezona pellucida\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. During cryopreservation, antioxidant-rich seminal plasma is diluted and the antioxidant activity of sperm is reduced. Sperm damage, dead spermatozoa and leukocytes caused by cryopreservation also produce peroxides in excess [21. The balance between ROS production and the antioxidant capacity of the biological milieu is then disrupted and the excessive ROS produces oxidative stress damages. The ROS in excess, at first, results in the lipid peroxidation (LPO) of the sperm plasma membrane. After then, this peroxidation conduces to the decarboxylation of unsaturated fatty acids, which gives rise to malondialdehyde (MDA), a compound capable of impairing the permeability and fluidity of the sperm plasma membrane [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The above processes also cause damages to the proteins, mitochondria, and DNA of sperm [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For sperm proteins, the oxidation of the thiol groups makes the sperm cells susceptible to leukocyte attack. An excessive ROS production affects the phosphorylation and glycosylation of protein, and production of mitochondrial ATP, which subsequently affects the sperm fertilization [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The present study also confirms that sperm freezing leads to an oxidative stress with an increased expression of MDA and a decreased expression of SOD and GSH-Px in seminal plasma. The addition of normal seminal plasma not only enhances the anti-oxidative stress ability of sperm, but also exerts a certain cryoprotective effect, which reduces the expression of MDA and increases the expression of SOD and GSH-Px. This is consistent with the fact that normal semen seminal plasma contains superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, as well as non-enzymatic antioxidants such as glutathione, ascorbic acid, vitamin E, albumin, and taurine [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These compounds are capable of counteracting the oxidative stress damages caused by ROS. Under normal conditions, endogenous antioxidants such as CAT, SOD and GSH-Px in seminal plasma can protect sperm from oxidative stress injury [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, during the freezing process of oligo-zoosperm and non-liquefied semen, the dynamic balance of the antioxidant system is disrupted. In such a situation, the endogenous antioxidants themselves cannot fully protect sperm cells from the oxidative stress damage. The resort to exogenous antioxidants is then necessary to resist the oxidative stress damage [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this way, the addition of the exogenous antioxidant melatonin into the semen was able to improve sperm motility after thawing. It also protects the acrosome and plasma membrane integrities, and increases the activities of T-AOC, T-SOD, GSH-Px and CAT, whereas reduces the MDA and 4-HNE activities [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt the same time, the excessive ROS causes DNA damage in spermatozoa, with a high frequency of single- and double-stranded DNA breaks [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Disruption of DNA integrity in the sperm nucleus is the underlying cause of irreversible sperm damage. Human sperm DNA fragmentation suggests sperm damage and is a factor of male infertility. The percentage of sperm DNA damages correlates with the fertilization rate and the unfavorable outcomes in embryo development [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Sperm DNA fragment Index (DFI) is the main indicator of sperm DNA integrity. In the current study, we also found that the DFI increased after sperm freezing, and neither non-liquefied seminal plasma nor no-seminal plasma exerted a good protection of DNA integrity. The normal semen plasma may contain antioxidants that exert a more convenient DNA integrity protection. This suggests that normal seminal plasma plays an important role in antioxidant protection, which protects spermatozoa from freezing damages.\u003c/p\u003e \u003cp\u003eIn conclusion, our results demonstrate that the seminal plasma of non-liquefied semen could not resist to the damage of cryopreservation, which may lead to the occurrence of abnormal frozen sperm. The normal seminal plasma, however, containing antioxidants, fatty acids and nutrients, can exert a protective effect against oxidative species and resist cryopreservation risks. The addition of normal seminal plasma into the sperm extracted from non-liquefied semen improves the viability, survival rate and plasma membrane integrity of sperm, maintains DNA integrity and normal morphology of cryopreserved sperm, and reduces oxidative stress damage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements - T\u003c/strong\u003ehe Jingmen Science and Technology Program (2022YFYB006) supported our work. We would like to express our gratitude to the staff of the Reproductive Medicine Center at Jingmen People\u0026apos;s Hospital as well as to the experimental technicians involved in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions -\u0026nbsp;\u003c/strong\u003eShuai Shao and Mei Jiang conducted all experiments and completed the paper. Nianping Zhang participated in the experimental design and statistical analysis, and also revised the manuscript. PR Raymond CESPUGLIO contributed to the manuscript improvement. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e- Jingmen Science and Technology Program (2022YFYB006). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e - The samples used in this study and the data sets collected were provided by the Reproductive Medicine Center of Jingmen People\u0026apos;s Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e - Ethics approval and consent to participate. The Ethic committee of Jingmen People\u0026rsquo;s Hospital reviewed this study. Patients with an informed consent approved all samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e - Not applicable. Our manuscript does not contain data from individual person. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests -\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests in this section.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e - \u003csup\u003e1\u003c/sup\u003eReproductive Medicine Center, Jingmen People\u0026apos;s Hospital (Central Hospital Affiliated to Jingchu Institute of Technology); \u003csup\u003e2\u003c/sup\u003eNeuroscience Research Center of Lyon, Beliv plateau, Claude Bernard Lyon1 University \u0026amp; RUDN University;\u003csup\u003e3\u003c/sup\u003ePostdoctoral Mobile station, Shandong University of Traditional Chinese Medicine; \u003csup\u003e4\u003c/sup\u003eExperimental Center, Shandong University of Traditional Chinese Medicine.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHungerford A, Bakos HW, Aitken RJ. Sperm cryopreservation: current status and future developments. Reprod Fertil Dev, 2023, 35(3): 265-281.\u003c/li\u003e\n\u003cli\u003eGouhier C, Pons-Rejraji H, Dollet S et al. Freezing does not alter sperm telomere length despite increasing DNA oxidation and fragmentation. Genes (Basel), 2023, 14(5): 1039.\u003c/li\u003e\n\u003cli\u003eHosseinmardi M, Siadat F, Sharafi M et al. Protective effect of cerium oxide nanoparticles on human sperm function during cryopreservation. Biopreserv Biobank, 2022, 20(1): 24-30.\u003c/li\u003e\n\u003cli\u003eLewin J, Lukaszewski T, Sangster P et al. Reproductive outcomes after surgical sperm retrieval in couples with male factor subfertility: a 10-year retrospective national cohort. Fertil Steril, 2023, 119(4): 589-595.\u003c/li\u003e\n\u003cli\u003eTakahara T, Amemiya Y, Sugiyama R, et al . Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes. J Biomed Sci, 2020, 27(1): 87.\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-Soto JC, Landeras J, Gadea J. Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success. Andrology, 2013, 1(3): 365\u0026ndash;375.\u003c/li\u003e\n\u003cli\u003eDavalieva K, Kiprijanovska S, Noveski P, et al. Proteomic analysis of seminal plasma in men with different spermatogenic impairment. Andrologia, 2012, 44(4): 256\u0026ndash;264.\u003c/li\u003e\n\u003cli\u003eBj\u0026ouml;rndahl L, Kirkman Brown J; other Editorial Board Members of the WHO Laboratory Manual for the Examination and Processing of Human Semen. The sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen: ensuring quality and standardization in basic examination of human ejaculates. Fertil Steril, 2022, 117(2): 246-251. \u003c/li\u003e\n\u003cli\u003eEisenberg ML, Lathi RB, Baker VL et al. Frequency of the male infertility evaluation: data from the national survey of family growth. J Urol, 2013, 189(3): 1030-1034.\u003c/li\u003e\n\u003cli\u003eMenkveld R, Rhemrev JP, Franken DR, et al. Acrosomal morphology as a novel criterion for male fertility diagnosis: relation with acrosin activity, morphology (strict criteria), and fertilization in vitro. Fertil Steril, 1996, 65: 637-644.\u003c/li\u003e\n\u003cli\u003eLi Y, Qin S, Cui W et al. Progress on the roles of zinc in sperm cryopreservation. Theriogenology, 2023, 211: 134-141.\u003c/li\u003e\n\u003cli\u003eBunge R. G., Sherman J. K. Fertilizing capacity of frozen human spermatozoa. Nature, 1953, 172: 767-768. \u003c/li\u003e\n\u003cli\u003eSherman J. K. Improved Methods of Preservation of Human Spermatozoa by Freezing and Freeze-Drying [J]. Fertility and Sterility, 1963,14: 49-64.\u003c/li\u003e\n\u003cli\u003eEini F, Kutenaei MA, Shirzeyli MH, et al. Normal seminal plasma could preserve human spermatozoa against cryopreservation damages in Oligozoospermic patients. BMC Mol Cell Biol, 2021, 22(1): 50.\u003c/li\u003e\n\u003cli\u003eTerai K, Yoshida K, Yoshiike M, et al. Association of seminal plasma motility inhibitors/semenogelins with sperm in asthenozoospermia-infertile men. Urol Int, 2010, 85(2): 209-215.\u003c/li\u003e\n\u003cli\u003eGruhl SL, Ho LM, Sim MYX, et al. Seminal biomarkers and their correlations to semen parameters in subfertile men. Eur J Obstet Gynecol Reprod Biol, 2023,19: 100229.\u003c/li\u003e\n\u003cli\u003eHuang D, Cai J, Zhang C, et al. Semen quality and seminal plasma metabolites in male rabbits (Oryctolagus cuniculus) under heat stress. PeerJ, 2023, 11: e15112.\u003c/li\u003e\n\u003cli\u003eMontoya P\u0026aacute;ez JD, \u0026Uacute;suga Suarez A, Restrepo Betancur G. Donkey semen cryopreservation: Alternatives with permeable, non-permeable cryoprotectants and seminal plasma. Reprod Domest Anim. 2023, 58(4): 486-495.\u003c/li\u003e\n\u003cli\u003eZoca GB, Celeghini ECC, Pugliesi G et al. Influence of seminal plasma during different stages of bovine sperm cryopreservation. Reprod Domest Anim, 2021, 56(6): 872-883.\u003c/li\u003e\n\u003cli\u003eAsadi A, Ghahremani R, Abdolmaleki A et al. Role of sperm apoptosis and oxidative stress in male infertility: a narrative review. International Journal of Reproductive Biomedicine, 2021, 19(6): 493-504. \u003c/li\u003e\n\u003cli\u003eSieme H, Oldenhof H. Sperm cleanup and centrifugation processing for cryopreservation. Methods in Molecular Biology, 2015, 1257(3): 43-52. \u003c/li\u003e\n\u003cli\u003eWoelders H, Chaveiro A. Theoretical prediction of \u0026lsquo;optimal\u0026rsquo; freezing programmes. Cryobiology, 2004, 49(3): 58-71.\u003c/li\u003e\n\u003cli\u003eBrochier C, Langley B. Chromatin modifications associated with DNA double-strand breaks repair as potential targets for neurological diseases. Neurotherapeutics, 2013, 10(4): 817- 830.\u003c/li\u003e\n\u003cli\u003eNekoonam S, Nashtaei M S, Naji M et al. Effect of Trolox on sperm quality in normozospermia and oligozospermia during cryopreservation. Cryobiology, 2016,72(2): 106-111. \u003c/li\u003e\n\u003cli\u003eGadea J, Molla M, Selles E, et al. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology, 2011, 62(1): 40-46.\u003c/li\u003e\n\u003cli\u003eZhang W, Yi K, Chen C et al. Application of antioxidants and centrifugation for cryopreservation of boar spermatozoa. Anim Reprod Sci, 2012, 132(3-4): 123-128.\u003c/li\u003e\n\u003cli\u003eLi C, Ren C, Chen Y, et al. Changes on proteomic and metabolomic profiling of cryopreserved sperm effected by melatonin. J Proteomics, 2023, 273: 104791.\u003c/li\u003e\n\u003cli\u003e[28] Thomson L K, Fleming S D, Aitken R J et al. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Human Reproduction, 2009, 24(9): 2061-2070. \u003c/li\u003e\n\u003cli\u003eCankut S, Dinc T, Cincik M et al. Evaluation of Sperm DNA Fragmentation via Halosperm Technique and TUNEL Assay Before and After Cryopreservation. Reprod Sci, 2019, 26(12): 1575-1581.\u003c/li\u003e\n\u003cli\u003eShaliutina-Loginova A, Loginov DS. Oxidative stress and DNA fragmentation in frozen/thawed common Carp Cyprinus carpio sperm with and without supplemental proteins. Anim Reprod Sci. 2023, 251: 107213. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cryopreservation, human sperm, non-liquefied semen, seminal plasma","lastPublishedDoi":"10.21203/rs.3.rs-4485698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4485698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: To explore the effects of normal seminal plasma replacement on cryopreservation of human non-liquefied spermatozoa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Sixty Semen samples (30 samples of normal semen and 30 samples of non-liquefied semen) were collected from infertile male patients in the Reproductive Medicine Center of Jingmen People's Hospital. After centrifugation, spermatozoa were divided into 5 aliquots, of which 2 aliquots were added with non-liquefied and normal seminal plasma, respectively. Computer-assisted semen analysis system (CASA) measured sperm motility after various periods of incubation (0, 0.5, 1 and 2 h). The remaining 3 aliquots of sperm were employed as follows for cryopreservation: a seminal plasma-free group in which sperm was preserved by adding sperm cryoprotectants; a non-liquefied semen-derived seminal plasma group for which sperm was added with non-liquefied plasma; a normal seminal plasma group for which sperm was added with normal seminal plasma. After the freezing-thawing process, sperm parameters, DNA integrity and ability to resist oxidative stress damage were again examined for these 3 groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Incubation of normal semen plasma, without liquefaction, can improve the sperm motility. After cryopreservation, sperm parameters were significantly lower versus those of the sample not cryopreserved. Normal semen plasma can reasonably protect the sperm of unliquefied semen and maintain the sperm parameters. It can also maintain the DNA integrity and prevent oxidative stress injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Normal seminal plasma can maintain the semen key parameters after cryopreservation of non-liquefied semen. Protective effects observed with this normal seminal liquid might be due to the antioxidants remaining within the seminal liquid.\u003c/p\u003e","manuscriptTitle":"Effect of normal seminal plasma replacement on cryopreservation of sperm in patients with non-liquefied semen","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 02:14:04","doi":"10.21203/rs.3.rs-4485698/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0ef26cd-58d4-4e13-85a0-4103826ac7ef","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-18T17:25:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-13 02:14:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4485698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4485698","identity":"rs-4485698","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}