Neutrophil Extracellular Traps (NETs) impair mouse sperm function and fertilization potential.

Fig. 1 Sperm triggered the formation of NETs. ( A ) The structure of NETs was co-located with DNA, citH3, and MPO. Sperm were co-cultured with PMNs(ratio: 1:1) for 4 h, with the use of DNase I. Incubation was carried out with specific antibodies, followed by final observation using fluorescence microscopy (citH3 and MPO represented in green, DNA in red). NETs are identified by white arrows. ( B ) Sperm were co-cultured with PMNs for 4 h. Pico Green was employed for the quantification of NETs. ( C ) The NADPH oxidase, ERK1/2, and p38 signaling pathways regulated sperm-induced NETs. Sperm were incubated with PMNs (1:1) for 4 h, with the use of DPI, U0126, and SB202190 as inhibitors. DNase I treatment confirmed the DNA nature of NETs. Data were presented as mean ± SD ( n  = 3). Results were considered significant at a p-value of < 0.05 (*** p  < 0.001, ** p  < 0.01, * p  0.05). Sperm triggered the formation of NETs. ( A ) The structure of NETs was co-located with DNA, citH3, and MPO. Sperm were co-cultured with PMNs(ratio: 1:1) for 4 h, with the use of DNase I. Incubation was carried out with specific antibodies, followed by final observation using fluorescence microscopy (citH3 and MPO represented in green, DNA in red). NETs are identified by white arrows. ( B ) Sperm were co-cultured with PMNs for 4 h. Pico Green was employed for the quantification of NETs. ( C ) The NADPH oxidase, ERK1/2, and p38 signaling pathways regulated sperm-induced NETs. Sperm were incubated with PMNs (1:1) for 4 h, with the use of DPI, U0126, and SB202190 as inhibitors. DNase I treatment confirmed the DNA nature of NETs. Data were presented as mean ± SD ( n  = 3). Results were considered significant at a p-value of < 0.05 (*** p  < 0.001, ** p  < 0.01, * p  0.05). Our results showed that mouse sperm elicited the generation of NETs, which were characterized by the presence of DNA (Red), citH3 (Green), and MPO (Green). Treatment with DNase I inhibited the formation of sperm-induced NETs, as shown in Fig.  1 A. Additionally, we quantified the NETs using the PicoGreen assay and observed a significant increase in sperm-induced NETs compared to PMN group, as depicted in Fig.  1 B. As a positive control, ZYM induced a more than three-fold increase in NETs. These results provide strong evidence supporting the induction of NETs by mouse sperm. To further investigate the mechanism underlying sperm-induced NETs, quantification of NETs was done both in presence and absence of inhibitors.Our data showed that specific inhibitors, including DPI, U0126, and SB202190, significantly attenuated sperm-induced NETs compared to PMN + sperm group, as depicted in Fig.  1 C. Additionally, the release of NETs triggered by mouse sperm were found to be susceptible to degradation by DNase I, as evidenced by the results shown in Fig.  1 C. Data were presented as mean ± SD ( n  = 3). Results were considered significant at a p-value of < 0.05 (*** p  < 0.001, ** p  < 0.01, * p   0.05). Fig. 2 Measurement of ROS, sperm motility and sperm acrosin. ( A ) Sperm triggered the formation of ROS. Sperm were co-cultured with PMNs for 4 h and DCFH-DA was employed for the quantification of ROS. ( B ) Effects of PMNs on mouse sperm motility. Sperm were co-cultured with PMNs(ratios: 1:1, 1:2 or 1:3) for 4 h, and subsequently, motility of the sperm were evaluated by CASA at 0, 2 and 4 h. ( C ) NETs reduced sperm acrosome enzyme activity. After co-culturing sperm with PMNs (1:1) for 4 h, the activity of acrosin was detected using ELISA assay. Measurement of ROS, sperm motility and sperm acrosin. ( A ) Sperm triggered the formation of ROS. Sperm were co-cultured with PMNs for 4 h and DCFH-DA was employed for the quantification of ROS. ( B ) Effects of PMNs on mouse sperm motility. Sperm were co-cultured with PMNs(ratios: 1:1, 1:2 or 1:3) for 4 h, and subsequently, motility of the sperm were evaluated by CASA at 0, 2 and 4 h. ( C ) NETs reduced sperm acrosome enzyme activity. After co-culturing sperm with PMNs (1:1) for 4 h, the activity of acrosin was detected using ELISA assay. As shown in Fig.  2 A, co-culture of PMNs with sperm resulted in a significant, dose-dependent increase in ROS levels compared to PMNs alone, with peak production observed at a 1:2 sperm-to-PMN ratio. These results suggest that ROS play a key role in sperm-induced NETs formation. Table 1 Sperm parameters of mouse sperm co-cultured with PMN (1:1) for 4 h. VCL: curvilinear velocity; VSL: straight-line velocity; VAP: average path velocity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; STR: straightness; LIN: linearity. Compared with the control, * represents significant difference (* p  < 0.05). Parameters Control PMN + sperm PMN + sperm + DNase I CASA estimates  Motility (%) 57.14 ± 9.76 31.55 ± 7.86* 53.53 ± 6.68  Progressive (%) 44.37 ± 5.54 16.15 ± 4.32* 33.25 ± 4.57* Sperm motion variables  VCL (µm/s) 106.62 ± 26.08 61.8 ± 14.47* 87.42 ± 26.43  VSL (µm/s) 28.47 ± 9.41 15.9 ± 3.35* 23.53 ± 8.58  VAP (µm/s) 78.90 ± 12.70 44.52 ± 11.24* 63.63 ± 9.86  ALH (µm/s) 5.37 ± 0.83 2.97 ± 0.43* 4.63 ± 0.48  BCF (Hz) 4. 00 ± 0.99 2.95 ± 0.51 3.41 ± 0.70  STR (%) 43.58 ± 26.96 51.3 ± 23.21 47.22 ± 20.24  LIN (%) 30.32 ± 8.37 35.21 ± 12.59 29.13 ± 13.48 Sperm parameters of mouse sperm co-cultured with PMN (1:1) for 4 h. VCL: curvilinear velocity; VSL: straight-line velocity; VAP: average path velocity; ALH: amplitude of lateral head displacement; BCF: beat cross frequency; STR: straightness; LIN: linearity. Compared with the control, * represents significant difference (* p  < 0.05). As shown in Fig.  2 B, co-incubation with PMNs accelerated the time-dependent decline in sperm motility. Exposure to a low dose of PMNs (ratio 1:3) resulted in only a slight, non-significant change in sperm motility, whereas a high dose (ratio 1:2 or 1:1) led to a significant decrease compared to the sperm-only group. Acrosin activity, a marker of acrosomal integrity, was measured using an ELISA assay. Co-incubation with PMNs significantly reduced acrosin activity compared to controls (Fig.  2 C). Notably, DNase I treatment did not rescue this effect, suggesting that PMNs may impair acrosomal function through mechanisms independent of NETs, such as phagocytosis or soluble factors. Detailed CASA parameters are presented in Table  1 . Compared to the control group, sperm co-cultured with PMNs exhibited significantly reduced VCL, VSL, VAP and ALH while other parameters were not significantly altered. DNase I treatment partially restored these motility parameters, further supporting the role of NETs in sperm motility impairment. Fig. 3 Co-incubation with PMN reduced IVF fertilization rate. ( A ) Representative bright-field images of 2-cell embryos and blastocysts from different experimental groups. Abbreviations: 2-cell, 2-cell embryo; BL, blastocyst. ( B ) Fertilization rates assessed by 2-cell embryo formation. ( C ) Blastocyst formation rates. ( D ) NETs released during IVF were indicated by white arrows. ( E ) NETs released during IVF capture oocyte, represented by white arrows. ( F ) Quantitative analysis of NETs using DNA determination in supernatant. Data were presented as mean ± SD ( n  = 3).When the p-value was less than 0.05 (*** p  < 0.001, ** p  < 0.01, * p  < 0.05), the result is considered significant. Co-incubation with PMN reduced IVF fertilization rate. ( A ) Representative bright-field images of 2-cell embryos and blastocysts from different experimental groups. Abbreviations: 2-cell, 2-cell embryo; BL, blastocyst. ( B ) Fertilization rates assessed by 2-cell embryo formation. ( C ) Blastocyst formation rates. ( D ) NETs released during IVF were indicated by white arrows. ( E ) NETs released during IVF capture oocyte, represented by white arrows. ( F ) Quantitative analysis of NETs using DNA determination in supernatant. Data were presented as mean ± SD ( n  = 3).When the p-value was less than 0.05 (*** p  < 0.001, ** p  < 0.01, * p  < 0.05), the result is considered significant. To further explore the influence of NETs on sperm, we conducted IVF experiments. The statistical analysis of the IVF rate was based on the assessment of 2-cell embryos. The results described in Fig.  3 (A, B) showed that co-incubation with PMNs led to a decrease in IVF rate, while the introduction of DNase I restored this rate by inhibiting the generation of NETs. As shown in Fig.  3 C, the two-cell and blastocyst rates were identical, indicating that all fertilized embryos developed to the blastocyst stage. However, a striking difference was observed in the kinetics of blastocyst development. Half of embryos from the PMN groups exhibited a significant delay (6–8 h) in reaching the blastocyst stage compared to controls (SI Fig. 1). This suggests that NETs did not compromise the developmental competence of fertilized embryos but rather caused a significant developmental delay. In vitro fluorescence staining revealed that PMN-released NETs contributed to the decline in IVF rate by capturing sperm (Fig.  3 D). Notably, during the IVF experiment, we observed that NETs could capture oocyte, as shown in Fig.  3 E. However, we were uncertain about whether these NETs were induced by sperm or by oocyte. Additionally, the quantification of NETs in the PMN group’s supernatant was significantly higher than that in the control group (Fig.  3 F). Fig. 4 Vital staining of embryo entangled in the release of NETs. Mouse PMNs were co-cultured with 2-cell embryos (ratio 100:1) at 37 °C for 4 h ( n  = 3). Then the samples were observed by scanning confocal microscope. Optical image showed PMNs were congregated around embryos. Hoechst33342 (nucleus) and Sytox Green (NETs) were used for immunofluorescence staining. White arrows showed NETs around the embryo. Three separate experiments were carried out to observe the embryo entangled in the release of NETs. Vital staining of embryo entangled in the release of NETs. Mouse PMNs were co-cultured with 2-cell embryos (ratio 100:1) at 37 °C for 4 h ( n  = 3). Then the samples were observed by scanning confocal microscope. Optical image showed PMNs were congregated around embryos. Hoechst33342 (nucleus) and Sytox Green (NETs) were used for immunofluorescence staining. White arrows showed NETs around the embryo. Three separate experiments were carried out to observe the embryo entangled in the release of NETs. Two-cell mouse embryos were successfully acquired using IVF technique, and the adjacent sperm were completely eliminated. PMNs were observed to accumulate around the embryos, as shown in Fig.  4 . Vital microscopy analysis showed that PMNs exhibited Hoechst staining (blue), while deceased PMNs and filamentous DNA structures were observed to exhibit Sytox Green staining (green) on the embryo. White arrows showed NETs-like structure around the embryo.

All the reagents were purchased from Sigma unless otherwise noted. Six-week-old female ICR mice and three-month-old male ICR mice were purchased from Weitonglihua Experimental Animal Technology Co., Ltd (Beijing, China). Mice were housed in specific pathogen-free (SPF) conditions on a 12-h light and 12-h dark cycle at constant temperature and under controlled humidity at the Experimental Animal Centre, First Hospital of Jilin University. All research procedures followed the guidelines of Institutional Animal Care and Use Committee of the First Hospital of Jilin University (Approval ID: SYXK2019-0012). All methods and experimental protocols were performed in accordance with the relevant guidelines and regulations approved by the committee of the First Hospital of Jilin University (Jilin, China). All experiments in this study were in accordance with ARRIVE guidelines. Six-week-old ICR mice were used to collect blood from their eyeballs into anticoagulant blood collection vessels. Subsequently, the collected blood was diluted twice using red blood cell sedimentation and the mouse peripheral blood PMN isolation kit was used (TianJin HaoYang Biological Manufacturing Co., China, LZS1100) for isolation. All PMNs in this study were isolated from female mice. To collect the sperm, cauda epididymis from 3-month-old male mice were carefully dissected and placed on filter paper. The surface fat, blood, and tissue fluid were removed by gentle washing. The cauda epididymis was then placed in a 20 µL droplet of Human Tubal Fluid (HTF) medium (Sigma, USA, MR-070-D) covered with paraffin oil. Using fine-tip forceps, the cauda epididymis was carefully torn to release the sperm. Female ICR mice (6 weeks old) were superovulated with 10 IU pregnant mare serum gonadotropin (PMSG) (Ningbo Second Hormone Factory, China, J-SP9970-1000) followed by 10 IU human chorionic gonadotropin (hCG) (Ningbo Second Hormone Factory, China, H44020673) 48 h later. The cumulus-oocyte complexes were collected from the oviducts 13–16 h after hCG injection and cumulus cells were removed with 1 mg/ml hyaluronidase. Then the oocytes were placed in 20 µL droplets of M2 medium (Sigma, USA, M7167) in paraffin oil at 37 °C under 5% CO 2 and 95% air. Mouse PMNs were co-cultured with mouse sperm at a 1:1 ratio in RPMI-1640 medium at 37 °C for 4 h, with PMNs cultured alone serving as the control. Samples were fixed with 4% (w/v) paraformaldehyde for 30 min, rinsed twice in phosphate buffered saline (PBS), and permeabilized with 0.1% Triton X-100 in PBS for 20 min. Samples were then blocked in 5% goat serum, and incubated with anti-citH3 (Abcam, UK, ab5103) and anti-MPO (Abcam, UK, ab208670) antibodies (1:200) at 4 °C for overnight. After two washes in PBS, PMNs were incubated with secondary goat anti-rabbit IgG-FITC (YEASEN, China, 33107ES60) antibody (1:200) for 2 h. PMNs were finally washed two times with PBS, stained with 5 µM Sytox Orange (Invitrogen, USA, S11368) and observed using a fluorescence confocal microscope (Olympus, Fluo View FV1000, Japan). We used Pico Green ® (Invitrogen, USA, P7589) to quantify NETs induced by mouse sperm following a 4 h co-incubation (at sperm: PMN ratios of 1:1, 1:1.5, 1:2, or 1:3) in phenol red-free RPMI (Gibco, USA, 11835055) at 37 °C. PMNs cultured alone served as the control. Briefly, the PMNs (2 × 10 5 ) and sperm were seeded into 96-well plates in RPMI-1640 medium (phenol-red-free). PMNs were pretreated for 30 min with the following inhibitors: DPI (NADPH oxidase inhibitor, 10 µmol/L, Sigma, USA, D2926), U0126 (MEK/ERK inhibitor, 50 µmol/L, Sigma, USA, 662009), and SB202190 (p38 MAPK inhibitor, 10 µmol/L, Sigma, USA, 559397), and then co-incubated with sperm (at a ratio of 1:1) for 4 h. In addition, PMNs treated only with 1 mg/mL Zymosan (ZYM, Solarbio, China, 58856-93-2) for 4 h was set as a positive control. After incubation, the fluorescence was measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm by an Infiniti M200 ® fluorescence plate reader (Tecan, Austria). Furthermore, DNase I (90 U, Solarbio, China, 622J037) was used to confirm the DNA nature of NETs. The level of ROS in PMNs was determined with DCFH-DA (Sigma, USA, 35845-1G). Briefly, the PMNs were incubated with mouse sperm (ratio: 1:1, 1:1.5, 1:2 or 1:3) for 4 h. PMNs cultured alone served as the control. Next, DCFH-DA (10 µM) was added to each well for 30 min. The fluorescence of the PMNs was detected with an excitation wavelength of 485 nm and an emission wavelength of 525 nm by an Infiniti M200 ® fluorescence plate reader (Tecan, Austria). PMNs (2 × 10 5 ) were spread on a 24-well culture plates for 30 min to allow for cell attachment. Sperm cultured in HTF medium without PMNs served as the motility control. Once the PMNs adhered, the RPMI medium was removed and substituted with HTF. The previously collected sperm were then added to the plate. PMNs were co-cultured with mouse sperm (ratios: 1:1, 1:2 or 1:3) for 4 h. The examination of sperm motility was performed at 0, 2 and 4 h. Sperm quality parameters were measured at 4 h. DNase I (90 U/mL) was added as the treatment group. All co-culture experiments were conducted in 24-well, plates with a final working volume of 200 µL per well at 37 °C under 5% CO 2 and 95% air. The culture chamber maintains a relative humidity of ≥ 95%.The motility of sperm was measured using a computer- assisted sperm analysis (CASA; Hamilton, TOX IVOS, USA) under HTF medium conditions. A 20 µL aliquot was taken from each co-culture well and loaded into a 20 μm depth counting chamber. The chamber was then placed on the microscope stage equipped with a heated plate (maintained at 37 °C) and connected to the CASA system. A minimum of 200 spermatozoa were analyzed per PMN/spermatozoa co-culture sample. Acrosomal enzyme activity was quantified using a mouse acrosome enzyme ELISA kit (Shanghai Bohu Biotechnology Co, China, BH-E5327) The absorbance was measured at a wavelength of 450 nm by Infiniti M200 ® fluorescence plate reader (Tecan, Austria) and the acrosomal enzyme quantificationin the mouse samples was determined by calculating it based on the standard curve. PMNs (2 × 10 5 ) were evenly spread on a 24-well culture plates for 30 min to allow for cell attachment. Sperm were capacitated in HTF for 1 h. The culture medium in the 24-well plate was replaced with HTF. Then, sperm (1 × 10⁶) and oocytes (20) were added to the 24-well plate. The cells were co-cultured in a final volume of 500 µL in the culture plates. The duration of the IVF process was 6 h. Oocytes cultured without PMNs served as negative controls for fertilization. PMN pre-incubating control group: Sperm pre-incubated with PMNs for 2 h, followed by addition of oocytes for IVF (without PMNs). The release of NETs during the IVF process was observed by Sytox Green (Invitrogen, USA, S7020) staining. The oocytes were then transferred into a clean culture dish and washed three times before being transferred to KSOM (Merck, Germany, MR-101-D). To evaluate fertilization and early embryonic development, the 2-cell embryo rate and blastocyst rate were recorded. Mouse PMN were co-cultured with embryos (ratio: 100:1) in HTF medium at 37 °C for 4 h on confocal dish. Then, Hoechst 33,342 (Solarbio, China, C003010) and Sytox Green (Invitrogen, USA, S7020) were added for staining for 20 min in the culture solution. Finally, the examination of the specimen was observed by using confocal microscope (Olympus FluoView FV1000). Data were analyzed using GraphPad Prism 9.5.1 (GraphPad Software, San Diego, CA, USA) and are presented as the mean ± standard deviation (SD) unless otherwise specified. One-way ANOVA followed by Tukey’s multiple comparison test was used for comparisons among groups. Differences in sperm motility were evaluated using two-way ANOVA with Bonferroni post-test. A p-values < 0.05 was considered statistically significant.

Our results demonstrate that mouse sperm induces NETs formation through ERK1/2, p38, and NADPH oxidase signaling pathways. NETs can impair mouse sperm motility and reduce IVF efficiency, while DNase I effectively degrades NETs and efficiently counteract the effects. Notably, we demonstrate for the first time that NETs like structure physically entrap embryos in vitro. These findings offer novel therapeutic targets for clinical intervention in human infertility, with particular relevance for immune-mediated reproductive dysfunction.

Previous research suggests that PMNs can capture sperm not only through phagocytosis, but also through NETs 19 , 20 . Our data show that mouse sperm can directly activate PMNs to form NETs. This finding is consistent with reports in several other species, such as horses 21 , humans 10 , cattle 22 and donkeys 23 . The structure of NETs includes both nuclear and granular constituents 24 . These NETs not only entrap sperm and impede their movement, but also reduce the success rate of conception by producing proteins that may impair sperm motility 25 . Our data show that sperm-induced NETs come into contact with sperm, resulting in a significant decrease in motility, preventing of sperm dissemination and substantially reducing IVF rates. Additionally, we observed the protective effect of DNase I on sperm affected by NETs. Our findings support previous hypotheses regarding natural mating or artificial insemination that the presence of vaginal PMNs may contribute to infertility. In terms of the formation of NETs, previous studies have reported that PMN preferentially eliminate sperm through phagocytosis in the female genital tract 26 . The interaction between PMN and sperm via phagocytosis can be blocked by CD11B antibodies and PI3 kinase inhibitors, providing evidence that supports the involvement of phagocytosis 27 , 28 . However, a single PMN cannot engulf an entire sperm due to its larger size. Other research has shown that NETs can affect sperm motility through PMN. In horses, sperm have been shown to induce NET release and extensive entrapment of sperm by neutrophils. This process is time-dependent and similar to bacterial activation of NETs. 21 . In swine, after co-incubation with PMNs for 2 h, NETs are formed; furthermore, the release of NETs increases with increasing amounts of sperm 11 . Interestingly in donkeys, seminal plasma rather than sperm elicits the release of NETs 29 . The central framework of these predominantly DNA-based structures consists mainly of antimicrobial proteins such as histones, elastase, and myeloperoxidase (MPO) which were prominently associated with their structure 30 . Using fluorescence confocal microscope analysis to examine the morphology of mouse-sperm-induced-NETs revealed concurrent labeling with DNA citH3 and MPO. PicoGreen assay was used to quantify NETs 31 . A quantitative approach was employed to assess the dose-dependent increase in mouse-sperm-induced-NET-release upon stimulation. These observations indicate that mouse sperm triggers the release of NETs by mouse-PMNs establishing an environment conducive to trapping sperm within them. The activation NADPH oxidase machinery is another crucial element responsible for triggering respiratory burst resulting in ROS production leading towards highly reactive superoxide anions capable enough to cause damage at multiple levels 32 . Neutrophils employ these functions to both intracellular vesicles and extracellular targets through degranulation. It has been extensively documented that PMNs generate NETs via NADPH oxidase-dependent as well as NADPH oxidase-independent pathways 33 . The results obtained in this study provide evidence that mouse sperm induce the production of NETs in PMNs through NADPH oxidase-dependent pathways. However, it should be noted that although sperm have a very limited capacity to release ROS compared to neutrophils, the ROS we detected in the culture medium may still include a contribution from sperm. Regarding the underlying NETs formation mechanism, studies have highlighted the involvement of ERK1/2 and p38 signaling pathways in regulating NETs release. The NADPH oxidase inhibitor DPI has been shown to impede ETs release, while the ERK1/2 inhibitor U0126 inhibits the ERK1/2 signaling pathway during cadmium chloride-induced ETs 34 . Similarly, SB202190, a p38 inhibitor, has been observed to inhibit the p38 signaling pathway during cadmium chloride-induced NETs 35 . To gain deeper insights into the mechanisms underlying sperm-induced NET formation, we employed inhibitors targeting NADPH oxidase (DPI), ERK1/2 (U0126), and p38 (SB202190) signaling pathways. Our results showed that all these inhibitors effectively downregulated sperm-induced NETs production, highlighting the pivotal involvement of NADPH oxidase, ERK1/2, and p38 signaling pathways in this process. These discoveries underscore the potential therapeutic utility of targeting these pathways for rescue and treatment purposes. It has been established that exogenous DNase I treatment significantly improved sperm motility in 83% of hyper-viscous sperm samples 36 . DNase I possesses the enzymatic capability to degrade DNA present in extracellular traps (ETs), thereby disrupting the overall structure of the NETs and compromising their ability to resist foreign microorganisms 37 . In this study, we initially examined the effect of DNase I on mouse sperm and separately evaluated its impact on sperm motility and acrosin. The statistical analysis of the data revealed that DNase I exhibited a protective effect by prolonging sperm motility. To further investigate the influence of NETs on sperm, we conducted IVF experiments and employed DNase I to eliminate NETs. Our results indicate that co-incubation with PMNs reduces the IVF rate, while treatment with DNase I restores it by inhibiting NETs generation. Oxidative stress is known to induce sperm DNA damage and subsequently impair embryonic development 38 . To further observe the effect of NETs on embryonic development, we cultured the embryos from the 2-cell embryo to the blastocyst stage. Almost all two-cell embryos successfully progressed to the blastocyst stage (Fig.  3 A and C). But an intriguing finding of this study is that half of embryos from the PMN and PMN-pre groups required approximately 6–8 additional hours to reach the blastocyst stage compared to controls (Supplementary Figure S1). These findings suggest that oxidative stress associated with NETs leads to a developmental delay rather than permanent developmental failure. Interestingly, NETs were also found to capture embryos during the IVF process. Previous studies have shown that embryos obtained through IVF can be captured by NETs due to sperm attachment on their surface 11 . To determine whether mouse embryo is directly captured by NETs, we co-incubated embryos without any attached sperm with PMN and monitored for release of NETs. Consistent with previous studies, our experiments showed that embryos directly stimulate the release of NETs and become entangled within them, suggesting a potential role for NETs in embryo implantation. Researchers combined immunofluorescence staining with antisperm membrane antibody–based fluorescence in situ hybridization (ASM FISH) technology and observed a significant increase in neutrophil numbers at early pregnancy implantation sites in mice 39 . Depletion of neutrophils was achieved through anti-Gr1 injection, resulting in a 50% decrease in blastocyst implantation rates among mice 40 . Importantly, it remains to be determined whether increased neutrophils during implantation affect embryo implantation through involvement with NETs. Our study provides the first systematic investigation of how PMN-derived NETs impair mouse sperm motility and compromise IVF outcomes, while simultaneously elucidating the molecular mechanisms underlying sperm-induced NETs. The mouse model was selected based on its well-documented homology with human reproductive physiology, providing ethical and practical advantages for mechanistic studies. However, the simplified culture system cannot fully replicate the complex physiological microenvironment of the reproductive tract. The co-culture with PMNs increases the overall cell density, which may adversely affect sperm quality by altering gas exchange or nutrient availability, even though we scaled up the co-culture system in the experiments. DNase I holds translational promise for managing immune-related infertility. In principle, the targeted degradation of NETs by exogenous DNase I could mitigate their detrimental effects on sperm function and embryo viability within the female reproductive tract. While our data provide preclinical proof-of-concept in a murine model, future studies are warranted to evaluate the safety, optimal delivery methods, and efficacy of DNase I in human reproductive contexts. More in vivo experiments are needed to confirm the experimental results and potential mechanisms. This research has demonstrated that NETs like structure can directly entangle embryos. But the impact and mechanism of NETs on embryo implantation are still unclear. Increased NET formation or impaired NET clearance is associated with various inflammatory conditions of the reproductive tract, such as endometriosis, chronic endometritis, and unexplained recurrent implantation failure 20 . NETs present in the uterine microenvironment may similarly impair embryonic development or adversely affect implantation.

The incidence rate of infertility has been on the rise in recent years, with approximately one in six people have experienced infertility at some stage in their lives 1 . Infectious diseases causing chaotic inflammatory reactions in the female reproductive tract can lead to a decline in sperm quality and contribute significantly to fertility loss, making it one of the primary causes of infertility 2 , 3 . Following insemination, a significant number of polymorphonuclear neutrophils (PMNs) enter the reproductive tract, potentially triggering or exacerbating inflammatory reactions 4 . PMNs play a crucial role as part of the host innate immune system’s first line of defense by producing reactive oxygen species (ROS), chemokines, phagocytosis, and degranulation to combat bacterial infections. Extensive researche have reported that PMNs can impact sperm function and result in fertilization failure 5 – 7 . However, the precise mechanism through which PMNs affect sperm remains a subject of debate. In 2004, Brinkmann et al.. made the groundbreaking discovery of NETs, elucidating the mechanism by which activated neutrophils release granular proteins and chromatin to form NETs 8 . The protein components of NETs, such as neutrophil elastase (NE), MPO, and peptidoglycan binding proteins, exhibit potent sterilization capabilities 8 , 9 . Recent investigations have verified the formation of NETs in humans, swine, and bovine during the interaction between PMNs and spermatozoa 10 – 13 . These studies have explored the deleterious effects of NETs on sperm function including entrapment and impaired motility. Fonseca et al.. indicate that Entamoeba histolytica induces the formation of NETs through the Raf/MEK/ERK signaling pathway 14 . Notably, NADPH oxidase-independent NETs formation typically leads to attenuate activation of the ERK 1/2 and p38 MAPK signaling pathways 15 . Due to the high genetic similarity to humans, short reproductive cycle, ease of operation and management, and better animal welfare and ethical considerations, mice have become the commonly used model organism for research on the human reproductive system 16 – 18 . However, whether similar phenomenon and molecular mechanisms occur in mice remains unexplored. The objective of this study is to investigate the direct effects of NETs on mouse sperm and their impact on IVF. Additionally, we aim to elucidate the molecular mechanism underlying NET formation induced by mouse sperm, and systematically evaluate the impact of mouse PMNs on early embryonic development in vitro.”

Supplementary Material 1 Supplementary Material 1